Cancer immunotherapy

Abstract

The invention relates to antagonists for use in methods of treating cancer, wherein the cancer comprises an immunosuppressive tumour microenvironment comprising cancer cells and one or more types of non-cancerous cells. The invention also relates to bispecific agents useful in these methods, wherein the bispecific agents comprise a first antigen-binding domain that specifically binds CD115 or CD112 and a second antigen-binding domain that specifically binds CD3.

Description

[0001] CANCER IMMUNOTHERAPY

[0002] Field of invention

[0003] The invention relates to antagonists and their use in methods for treating cancers comprising an immunosuppressive tumour microenvironment. The invention also relates to methods of predicting whether or not a subject having cancer will respond to treatment with the antagonists.

[0004] Background to the invention

[0005] Tumours comprise not only cancer cells but also a heterogeneous and dynamic collection of non-cancerous infiltrating and resident host cells, an extracellular matrix and secreted factors that maintain and drive the progression of the tumour. The complex ecosystem of the tumour and its cellular composition collectively form the tumour microenvironment (TME), which is closely associated with cancer metastasis, recurrence and worse prognosis. In particular, TMEs of various cancers share the common characteristic of being immunosuppressive, which very often upregulates certain surface proteins or ligands to further suppress anti-tumour immune functions. As it sequesters drugs and prevents immune cells from infiltrating to target the cancer cells, and the TME has been a major challenge of cancer immunotherapy. However, despite being one of the main drivers of cancer recurrence and poor treatment response, most current anti-cancer therapies do not target the TME.

[0006] CD 155 and CD112 are pan-TME markers ubiquitously expressed on multiple cell types within the TME but not in healthy tissues, and thus are promising targets for novel anti-cancer therapies that target the TME. CD155, also known as poliovirus receptor (PVR), belongs to the nectin-like family of proteins. It is involved in many cellular processes such as cell adhesion, migration, proliferation and survival by interacting with growth factor receptors and integrins in cis or nectin-3 in trans to activate Ras and RAP1 signalling. The expression of CD 155 in most healthy tissue is very low, and overexpression of CD155 is predominantly observed in different malignancies. It promotes cancer aggressiveness and metastasis, and is correlated with worse prognosis and shorter overall survival in cancer patients.

[0007] More recently, the immune regulatory functions of CD 155 gained significant traction due to the emergence of cancer immunotherapy. Apart from mediating cell motility, CD 155 serves as a ligand that interacts with the inhibitory receptor TIGIT and the activating receptor CD226. The bifunctional CD155 maintains the balance of inhibitory and activating signals in healthy individuals. The balance is disrupted in cancer patients, as overexpression of CD155 not only suppresses T cells and NK cells via the CD155 / TIGIT axis but also downregulates CD226 expression. CD155 also exhibits a higher binding affinity to TIGIT than CD226, further reinforcing its inhibitory functions.

[0008] Immunotherapies targeting the CD155 / TIGIT signalling axis have shown some success, especially with the combination of PD1 / PD-L1 blockade. A syngeneic mouse model showed that TIGIT blockade induced a similar level of IL2, IFNg and TNFa production in CD8+ T cells as PD1 blockade, while the combination potentiated the CD8+ T cell response and tumour regression. This augmented effect may be explained by the suppression of CD226 signalling by PD1 signalling. Different combinations of TIGIT blockade and PD1 blockade are now being evaluated in clinical trials. In contrast, very few therapeutics targeting CD155 have been explored. NTX-1088, an anti-CD155 monoclonal antibody, has shown robust inhibition of tumour growth with an induction of infiltrating CD137+, CD226+ and CD8+ T cells in humanised mouse models.

[0009] CD112, also known as nectin-2 or PVR-related protein 2 (PVRL2), belongs to the nectin family of protein. It is highly expressed on cancer cells as compared to healthy tissues. It is believed that CD112 can form homodimers or heterodimers with other family members contributing to cell-to-cell adhesion, and overexpression of CD112 has been associated with cancer growth, metastasis and angiogenesis.

[0010] Although CD112 can bind to inhibitory CD112 receptor (CD112R) and TIGIT as well as activatory CD226, binding affinity to CD112R is higher. CD112R is expressed on T cells and NK cells, and binding to CD112 is found to attenuate the effector functions, paving the identification of CD112 as a novel checkpoint marker. In addition, CD112 is detected on CD14+cells, most likely antigen-presenting cells and dendritic cells, and the level is upregulated upon maturation and activation, highlighting a regulatory mechanism on T cell and NK cells response.

[0011] Given the expression of CD112R on NK cells, CD112R blockade can enhance the degranulation and production of IFNy in NK cells triggered by trastuzumab-induced antibodydependent cellular cytotoxicity (ADCC). The addition of TIGIT blockade can further enhance the NK cell functions.

[0012] Both CD112 and CD 155 share the same activatory ligand CD226, yet their predominant inhibitory ligands are different. TIGIT is the predominant ligand of CD155, while CD112R preferentially binds to CD112R but also TIGIT with low affinity, perhaps suggesting two non- redundant pathways in suppressing T cell and NK cells.

[0013] Summary of the invention

[0014] The inventors have discovered that CD 155 and CD112 are ubiquitously expressed in the TME and that human malignant peritoneal ascites fluid or immunosuppressive medium upregulates CD155 and CD112 expression. Building upon these discoveries, the inventors have, for the first time, developed bispecific T cell engagers (BiTEs) that bind CD155 or CD112 as a pan-TME target. These BiTEs exhibit immunostimulatory effect.

[0015] The BiTEs have several advantages over prior art anti-cancer therapies. Firstly, unlike prior art BiTEs that only target one specific cell type, the anti-CD155 and anti-CDl 12 BiTEs were found to be capable of killing multiple cell types of the TME, and thus have potential to deplete the TME of most cancer types completely. As shown in the Examples, the BiTEs trigger T cell activation and killing against both cancer cells and non-cancerous cells in the tumour microenvironment, such as cancer-associated fibroblasts, macrophages and myeloid-derived suppressor cells. Secondly, the BiTEs were found to perform better in immunosuppressive environments that would otherwise reduce the efficacy of most current immunotherapies, and hence are capable of exploiting the immunosuppressive nature of the TME. Indeed, the efficacy of the BiTEs was surprisingly potentiated in immunosuppressive conditions, which may be due to the higher density of target CD155 or CD112 in cancers comprising a more immunosuppressive tumour microenvironment. Thirdly, unlike most anti-cancer biologies that target CD 155 or CD112, the small size of the BiTEs gives the BiTEs the potential to more effectively penetrate into the TME of multiple cancer types. Fourthly, compared to many prior art anti-cancer therapies, the BiTEs are expected to have fewer off-target effects, given the minimal expression of CD155 or CD112 on healthy, non-TME cells and the small size of the BiTEs, which enables rapid removal by renal clearance even if the BiTEs leach into circulation.

[0016] CD 155 and CD112 are TIGIT ligands. Accordingly, the exemplified BiTEs are TIGIT ligand-binding agents.

[0017] Without wishing to be bound by theory, the therapeutic activity of the BiTEs may be exerted at least in part by preventing the interaction of CD 155 or CD112 with TIGIT, a co- inhibitory receptor with immunosuppressive activity expressed on several types of lymphocytes. Reduction of the immunosuppressive activity of TIGIT in addition to the BiTE-induced T cell activation and direct killing of cancer cells and non-cancerous cells in the tumour microenvironment may lead to the depletion of the tumour microenvironment as described in the Examples. The depletion of the tumour microenvironment may occur from the periphery of the tumour microenvironment, even if the BiTEs cannot initially infiltrate into the tumour core. Depletion of the tumour microenvironment may further facilitate infiltration of immune cells and anti-cancer agents into the tumour, and so could improve response to co-therapy using other anticancer treatments.

[0018] Accordingly, the invention relates to TIGIT ligand-binding agents, in particular CD155- or CD112-binding agents, and their uses. The invention further provides a TIGIT antagonist for use in a method of treating cancer, wherein the cancer comprises an immunosuppressive tumour microenvironment comprising cancer cells and one or more types of non-cancerous cells. The invention also provides a method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a TIGIT antagonist, wherein the cancer comprises an immunosuppressive tumour microenvironment comprising cancer cells and one or more types of non-cancerous cells. The invention also provides use of a TIGIT antagonist in the manufacture of a medicament for treating cancer in a subject, wherein the cancer comprises an immunosuppressive tumour microenvironment comprising cancer cells and one or more types of non-cancerous cells.

[0019] The invention further provides a bispecific agent, wherein the bispecific agent is a bispecific T cell engager (BiTE) comprising a first single-chain variable fragment (scFv) and a second scFv, wherein the first scFv specifically binds to CD 155 and the second scFv specifically binds to CD3.

[0020] The invention further provides a bispecific agent comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain specifically binds to CD112 and the second antigen-binding domain specifically binds to CD3.

[0021] The invention further provides a nucleic acid encoding the bispecific agent of the invention, a vector comprising one or more nucleic acids of the invention, and a host cell comprising one or more vectors of the invention.

[0022] The invention further provides a method of predicting whether or not a subject having cancer will respond to treatment with a TIGIT antagonist, wherein the method comprises determining whether or not the cancer comprises an immunosuppressive tumour microenvironment and thereby predicting whether or not the subject will respond to the treatment.The invention will now be described in more detail, by way of example and not limitation, and by reference to the accompanying drawings. 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 are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the scope of the invention. All documents cited herein, whether supra or infra, are expressly incorporated by reference in their entirety. The present invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or is stated to be expressly avoided. Section headings are used herein are for convenience only and are not to be construed as limiting in any way.

[0023] General definitions

[0024] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this disclosure belongs.

[0025] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a TGIT antagonist” includes “TIGIT antagonists”, reference to “a BiTE” includes “BiTEs”, reference to “a nucleic acid” includes “nucleic acids”, reference to “a vector” includes “vectors”, reference to “a cell” includes “cells”, reference to “a clinical disease” includes “clinical diseases”, and the like.

[0026] In general, the term “comprising” is intended to mean including but not limited to. For example, the phrase “the tumour microenvironment comprises cancer-associated fibroblasts” should be interpreted to mean that the tumour microenvironment contains cancer-associated fibroblasts, but that the tumour microenvironment may contain one or more additional components, such as one or more other cell types. As a further example, the phrase “the method comprises determining whether or not the cancer comprises an immunosuppressive tumour microenvironment and thereby predicting whether or not the subject will respond to treatment with a TIGIT antagonist” should be interpreted to mean that the method contains the step of determining whether or not the cancer comprises an immunosuppressive tumour microenvironment and thereby predicting whether or not the subject will respond to treatment with a TIGIT antagonist, but that the method may comprise one or more additional steps, such as administering the TIGIT antagonist to a subject predicted to respond to the treatment.

[0027] In some aspects of the disclosure, the word “comprising” is replaced with the phrase “consisting of’. The term “consisting of’ is intended to be limiting. For example, the phrase “the BiTE consists of a first antibody, or antigen-binding fragment thereof, and a second antibody, or antigen-binding fragment thereof’ should be understood to mean that the BiTE contains a first antibody, or antigen-binding fragment thereof, and a second antibody, or antigen-binding fragment thereof, and no additional components.

[0028] The terms “protein” and “polypeptide” are used interchangeably herein, and are intended to refer to a polymeric chain of amino acids of any length.

[0029] For the purpose of this disclosure, in order to determine the percent identity of two sequences (e.g., two polypeptide sequences or two polynucleotide sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in a first sequence for optimal alignment with a second sequence). The nucleotide residues at nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide residue as the corresponding position in the second sequence, then the nucleotides are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions in the reference sequence x 100).

[0030] Typically, the sequence comparison is carried out over the length of the reference sequence. For example, if the user wished to determine whether a given (“test”) sequence has a certain percentage identity to SEQ ID NO: X, SEQ ID NO: X would be the reference sequence. To assess whether a sequence is, for instance, at least 80% identical to SEQ ID NO: X (an example of a reference sequence), the skilled person would carry out an alignment over the length of SEQ ID NO: X, and identify how many positions in the test sequence were identical to those of SEQ ID NO: X. If at least 80% of the positions are identical, the test sequence is at least 80% identical to SEQ ID NO: X. If the sequence is shorter than SEQ ID NO: X, the gaps or missing positions should be considered to be non-identical positions.

[0031] The skilled person is aware of different computer programmes that are available to determine the homology or identity between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. The UWGCG Package provides the BESTFIT programme which can be used to calculate homology, for example used on its default settings (Devereux et al. (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (such as identifying equivalent residues or corresponding sequences (typically on their default settings)), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S.F et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0032] Description of the figures

[0033] Figure 1 - CD155 is highly expressed on cancer cell lines derived from different tissues. (A) The expression of CD 155 on cancer cell lines derived from colorectal, pancreatic, lung, brain and ovarian cancer was measured by flow cytometry. (B) Summary of the CD 155 expression on the tested cell lines.

[0034] Figure 2 - CD155 is differentially upregulated on NHDF cells cultured in conditioned medium or malignant peritoneal ascites fluid. NHDF cells were cultured in DMEM supplemented with 5% FBS (normal medium) or DLD-1 cell-conditioned medium (cancer- conditioned medium) or ascites fluid mixed (50% v / v) with DMEM (ascites-conditioned medium) for 72 hours before measuring the expression of CD 155 by flow cytometry. (B) Summary of CD155 expression on the NHDF cells cultured in different conditions. NM: normal medium, CM: cancer-conditioned medium, AS: ascites-conditioned medium.

[0035] Figure 3 - CD155 is differentially upregulated on human cancer tissues but not the adjacent healthy tissues. A solid biopsy of colorectal cancer liver metastasis was first separated into malignant and healthy tissues before dissociating them into single cells. The expression of CD155 on cancer tissue (A) and the adjacent healthy liver tissue (B) was measured by flow cytometry.

[0036] Figure 4 - CD155 is highly expressed on multiple cell types in human solid cancer biopsies. Solid biopsies of colorectal cancer liver metastasis were dissociated into single cells, followed by the measurement of CD155 expression on cancer cells (EpCAM+) (A), cancer- associated fibroblasts (FAP+) (B), M2-macrophages (CDl lb+CD206+) (C), and myeloid- derived suppressor cells (CD1 lb+CD33+) (D) by flow cytometry. Figure 5 - BiTEs are produced by transfected 293 A cells and secreted into the medium. (A) Schematic representation of the structure of the CD155 scFvl BiTE, CD155 scFv2 BiTE and FHA BiTE. (B) Plasmids containing the BiTE were transfected into HEK293 A for 72 hours. The supernatants were collected and analysed by western blotting. VL, variable fragment light chain; VH, variable fragment light chain, SP, signal peptide; L, linker; FLAG, FLAG-tag.

[0037] Figure 6 - CD 155 BiTEs triggered T cell activation and cytotoxicity in a dose-dependent manner. PBMC-derived T cells were cocultured with human colorectal carcinoma HCT116 cells and CD155 BiTE or Control BiTE of different doses for 72 hours, followed by the measurement of CD25 (A) and CD69 (B) by flow cytometry. (C) Cytotoxicity of the BiTE-mediated killing of HCT116 cells was measured by XTT assay. Data shown as singlicate.

[0038] Figure 7 - CD 155 BiTE induces the activation and degranulation of CD4+ and CD8+ T cell subsets. (A-B) PBMC-derived T cells were cocultured with CD155 BiTE or Control BiTE or anti-CD3 / CD28 Dynabeads in the presence or absence of Panel cells for 72 hours, followed by the measurement of CD25 (A) and CD69 (B) by flow cytometry. (C-H) PBMC-derived T cells were cocultured with Panel cells and CD155 BiTE or Control BiTE or anti-CD3 / CD28 Dynabeads for 72 hours, followed by measuring the expression of CD25 (C), CD69 (D), CD 107a (E), granzyme B (F), perforin (G), and IFNy (H) on CD4+ and CD8+ T cells, respectively, by flow cytometry.

[0039] Figure 8 - CD155 BiTE triggers T cell activation and cytotoxicity against cancer cell lines derived from different tissues. PBMC-derived T cells were cocultured with the indicated cancer cell lines and CD 155 BiTE or Control BiTE for 72 hours, followed by the measurement of CD25 (A) and CD69 (B) by flow cytometry, and cytotoxicity (C) by XTT assay.

[0040] Figure 9 - The expression of CD155 is upregulated on activated T cells. PBMC-derived T cells were cocultured with CD155 BiTE or Control BiTE or anti-CD3 / CD28 Dynabeads in the presence or absence of Panel cells for 72 hours, followed by measuring the expression of CD25 (A) and CD69 (B) by flow cytometry.

[0041] Figure 10 - CD155 BiTE does not induce fratricide of activated T cells. PBMC-derived T cells were cocultured with CD155 BiTE or Control BiTE or anti-CD3 / CD28 Dynabeads in the presence or absence of Panel cells for 72 hours, followed by counting the number of T cells by flow cytometry. Fold-change of CD3+ T cells (A), CD4+ T cells (B) and CD8+ T cells (C) after the treatment was determined by normalising with the corresponding untreated control. The proportion of live CD3+ T cells (D), CD4+ T cells (E) and CD8+ T cells (F) were determined by flow cytometry.

[0042] Figure 11 - Activation and cytotoxicity induced by CD155 BiTE are CD155-specific. PBMC-derived T cells were cocultured with CHO cells and CD155 BiTE or Control BiTE for 72 hours, followed by measuring the expression of CD25 on CD3+ T cells (A), and CD4+ and CD8+ T cell subsets (B).

[0043] Figure 12 - CD155 BiTE induces T cell activation against NHDF cells cultured in the ascites fluid. NHDF cells were pre-treated with the normal medium or ascites fluid (50% v / v) mixed with normal medium for 72 hours before coculturing with PBMC-derived T cells and CD155 BiTE or Control BiTE for another 48 hours. The expression of CD69 on CD3+ T cells (A), CD4+ T cells (B), and CD8+ T cells (C) was measured by flow cytometry.

[0044] Figure 13 - CD155 BiTE triggers T cell killing against cancer-associated fibroblasts. NHDF cells were cultured in DLD-1 -conditioned medium (cancer-conditioned medium) or ascites-conditioned medium for 72 hours, before coculturing with PBMC-derived T cells and CD 155 BiTE or Control BiTE for another 72 hours. Cytotoxicity was measured by XTT assay. CM: cancer-conditioned medium, AS: ascites-conditioned medium.

[0045] Figure 14 - CD155 BiTE induces T cell activation and killing against tumour-associated endothelial cells. HUVEC cells were cultured in the DLD-1 cell-conditioned medium (cancer- conditioned medium) or ascites fluid (50% v / v) mixed with normal medium (ascites-conditioned medium) for 72 hours before coculturing with PBMC-derived T cells and CD155 BiTE or Control BiTE for another 72 hours. The expression of CD25 (A) and CD69 (B) on CD3+ T cells was measured by flow cytometry. (C) Cytotoxicity was measured by XTT assay. CM: cancer- conditioned medium, AS: ascites-conditioned medium.

[0046] Figure 15 - CD155 BiTE induces T cell activation and killing against M2 macrophages. M2 macrophages were cocultured with PBMC-derived T cells and CD155 BiTE or Control BiTE. The expression of CD25 (A-B) and CD69 (C-D) on CD4+ and CD8+ T cells were measured by flow cytometry. (E) Cytotoxicity was measured by XTT assay.

[0047] Figure 16 - CD155 BiTE augments degranulation and activation of NK cells against HLA class I-negative cancer cells. PBMC-derived NK cells were cocultured with HLA I- negative K562 cells in the presence or absence of CD155 BiTE or PMA / ion for 48 hours. The expression of the degranulation marker CD 107 (A) and activation marker TIGIT (B) on NK cells were measured by flow cytometry. PMA / ion: Phorbol myristate acetate / ionomycin. Figure 17 - The CD155 BiTE synergises the activation of tumour-reactive Jurkat cells.

[0048] Panel cells were plated in the presence or absence of NY-ESO-1 for 24 hours before coculturing with CD8+ and 1G4 TCR-expressing Jurkat cells and CD155 BiTE or Control BiTE for another 24 hours. The ratio of Jurkat cells and Panel cells was 1 : 1 (A) and 5: 1 (B), respectively. The expression of CD69 on Jurkat was measured by flow cytometry. Bliss independence score (BIS) was calculated using the CD69 gMFI to determine the presence of synergy of different combination treatments (BIS > 0, synergy; BIS = 0, independent; BIS < 0, antagonistic).

[0049] Figure 18 - CD155 BiTE targets both cancer and stromal cells. CD155 BiTE or Control BiTE were cultured with ascites cells in the normal medium or autologous ascites fluid (50% v / v) for 120 hours. The number of CD155+cells (A), EpCAM+cells (B), FAP+cells (C), CD1 lb+CD64+CD206+cells (D), CD1 lb+CD64+FRp+cells (E) and CD1 lb+CD33+cells (F) remaining was measured by flow cytometry.

[0050] Figure 19 - CD155 BiTE triggers the activation and proliferation of endogenous T cells in ascites. CD155 BiTE or Control BiTE was cultured with ascites cells in normal medium or autologous ascites fluid (50% v / v) for 120 hours. The expression of CD25 on T cells (A) and the number of T cells, calculated as fold change (B), were measured by flow cytometry.

[0051] Figure 20 - Ascites fluid upregulates CD155 expression on cancer cells. DLD-1 cells (A) and Panel cells (B) were cultured in ascites fluid (50% v / v) mixed with normal medium for 72 hours, followed by the measurement of CD 155 expression by flow cytometry.

[0052] Figure 21 - CD155 BiTE overcomes the immunosuppression of ascites fluid to induce killing against cancer cells. PBMC-derived T cells were cocultured with Panel cells and CD155 BiTE or Control BiTE for 72 hours. (A) T cells expressing both CD25 and CD69 as measured by flow cytometry. The expression of CD25 (B) and CD69 (C) on T cells was measured by flow cytometry. (D) The cytotoxicity of CD 155 BiTE was determined by XTT assay.

[0053] Figure 22 - CD155 BiTE triggers fFNy release and killing in solid cancer biopsies. Thin slices of human colorectal cancer liver metastasis biopsies were incubated with CD155 BiTE or Control BiTE for 6 days. Supernatants were collected on day 3 and day 6. The concentration of IFNy in the supernatants on day 3 (A) and day 6 (B) was measured by ELISA. (C) Cytotoxicity of the BiTEs was determined by measuring the released LDH, normalised to the corresponding day 0 controls. Data show the mean ± SD of duplicate.

[0054] Figure 23 - CD155 BiTE triggers the activation of resident T cells in solid cancer biopsies. Thin slices of matched human solid biopsies were incubated with the CD155 BiTE or Control BiTE for 6 days, before fixation and staining of the CD25 expression. IHC images of colorectal cancer liver metastasis (A) and the matched normal adjacent liver tissue (B). The top panel is the IHC images at IX magnification. The zoomed-in images of the rectangular boxes were shown in the bottom panel at a 5X magnification.

[0055] Figure 24 - CD112 is highly expressed on cancer cell lines derived from different tissues. (A) The expression of CD112 on cancer cell lines derived from colorectal, pancreatic, lung, brain and ovarian cancer was measured by flow cytometry. (B) Summary of the CD112 expression on the tested cell lines.

[0056] Figure 25 - CD112 is differentially upregulated on human cancer tissues but not the adjacent healthy tissues. A solid biopsy of colorectal cancer liver metastasis was first separated into malignant and healthy tissues before dissociating them into single cells. The solid biopsy of healthy colon tissue was also dissociated. The expression of CD112 on cancer tissue (A), the adjacent healthy liver tissue (B), and healthy colon tissues (C) was measured by flow cytometry.

[0057] Figure 26 - CD112 is highly expressed on multiple cell types in human solid cancer biopsies. Solid biopsies of primary colorectal cancer and colorectal cancer liver metastasis were dissociated into single cells, followed by the measurement of CD112 expression on cancer cells (EpCAMT) (A), cancer-associated fibroblasts (FAP+) (B), M2-macrophages (CDl lb+CD206+) (C), and myeloid-derived suppressor cells (CD1 lb+CD33+) (D) by flow cytometry.

[0058] Figure 27 - Schematic representation of the structure of the CD112 scFv7 BiTE, CD112 scFvl 1 BiTE and FHA BiTE. VL, variable fragment light chain; VH, variable fragment light chain, SP, signal peptide; L, linker; FLAG, FLAG-tag.

[0059] Figure 28 - CD112 BiTEs triggers T cell activation and cytotoxicity in a dose-dependent manner. PBMC-derived T cells were cocultured with human colorectal carcinoma HCT116 cells and CD112 BiTE or Control BiTE of different doses for 72 hours, followed by the measurement of CD25 (A) and CD69 (B) by flow cytometry. (C) Cytotoxicity of the BiTE-mediated killing of HCT116 cells was measured by XTT assay. Data shown as singlicate.

[0060] Figure 29 - CD112 BiTE induces the activation and degranulation of CD4+ and CD8+ T cell subsets. (A-B) PBMC-derived T cells were cocultured with CD112 BiTE or Control BiTE or anti-CD3 / CD28 Dynabeads in the presence or absence of Panel cells for 72 hours, followed by the measurement of CD25 (A) and CD69 (B) by flow cytometry. (C-H) PBMC-derived T cells were cocultured with Panel cells and CD112 BiTE or Control BiTE or anti-CD3 / CD28 Dynabeads for 72 hours, followed by measuring the expression of CD25 (C), CD69 (D), CD 107a (E), granzyme B (F), perforin (G), and IFNy (H) on CD4+ and CD8+ T cells, respectively, by flow cytometry.

[0061] Figure 30 - CD112 BiTE triggers T cell activation and cytotoxicity against cancer cell lines derived from different tissues. PBMC-derived T cells were cocultured with the indicated cancer cell lines and CD112 BiTE or Control BiTE for 72 hours, followed by the measurement of CD25 (A) and CD69 (B) by flow cytometry, and cytotoxicity (C) by XTT assay.

[0062] Figure 31 - The expression of CD112 is upregulated on activated T cells. PBMC- derived T cells were cocultured with CD112 BiTE or Control BiTE or anti-CD3 / CD28 Dynabeads in the presence or absence of Panel cells for 72 hours, followed by measuring the expression of CD25 (A) and CD69 (B) by flow cytometry.

[0063] Figure 32 - CD112 BiTE does not induce fratricide of activated T cells. PBMC-derived T cells were cocultured with CD112 BiTE or Control BiTE or anti-CD3 / CD28 Dynabeads in the presence or absence of Panel cells for 72 hours, followed by counting the number of T cells by flow cytometry. Fold-change of CD3+ T cells (A), CD4+ T cells (B) and CD8+ T cells (C) after the treatment was determined by normalising with the corresponding untreated control. The proportion of live CD3+ T cells (D), CD4+ T cells (E) and CD8+ T cells (F) were determined by flow cytometry.

[0064] Figure 33 - CD112 BiTE preferentially induces T cell activation against NHDF cells cultured in the ascites fluid. NHDF cells were pre-treated with the normal medium or ascites fluid (50% v / v) mixed with normal medium (ascites-conditioned medium) for 72 hours before coculturing with PBMC-derived T cells and CD112 BiTE or Control BiTE for another 48 hours. The expression of CD69 on CD3+ T cells (A), CD4+ T cells (B), and CD8+ T cells (C) was measured by flow cytometry. NM: normal medium, AS: ascites-conditioned medium.

[0065] Figure 34 - CD112 BiTE triggers T cell killing against cancer-associated fibroblasts. NHDF cells were cultured in DLD-1 -conditioned medium for 72 hours, before coculturing with PBMC-derived T cells and CD112 BiTE or Control BiTE for another 72 hours. Cytotoxicity was measured by XTT assay.

[0066] Figure 35 - CD112 BiTE induces T cell activation and killing against tumour-associated endothelial cells. HUVEC cells were cultured in the DLD-1 cell-conditioned medium (cancer- conditioned medium) or ascites fluid (50% v / v) mixed with normal medium (ascites-conditioned medium) for 72 hours before coculturing with PBMC-derived T cells and CD112 BiTE or Control BiTE for another 72 hours. The expression of CD25 (A) and CD69 (B) on CD3+ T cells was measured by flow cytometry. (C) Cytotoxicity was measured by XTT assay. CM: cancer- conditioned medium, AS: ascites-conditioned medium.

[0067] Figure 36 - CD112 BiTE induces T cell activation and killing against M2 macrophages. M2 macrophages were cocultured with PBMC-derived T cells and CD112 BiTE or Control BiTE. The expression of CD25 (A) and CD69 (B) on CD4+ and CD8+ T cells were measured by flow cytometry. (C) Cytotoxicity was measured by XTT assay.

[0068] Figure 37 - CD112 BiTE augments degranulation and activation of NK cells against HLA class I-negative cancer cells. PBMC-derived NK cells were cocultured with HLA I- negative K562 cells in the presence or absence of CD112 BiTE or PMA / ion for 48 hours. The expression of the degranulation marker CD 107 (A) and activation marker TIGIT (B) on NK cells were measured by flow cytometry. PMA / ion: Phorbol myristate acetate / ionomycin.

[0069] Figure 38 - CD112 BiTE targets both cancer and stromal cells. CD112 BiTE or Control BiTE were cultured with ascites cells in the normal medium or autologous ascites fluid (50% v / v) for 120 hours. The number of CD112+cells (A), EpCAM+cells (B), FAP+cells (C), CD1 lb+CD64+CD206+cells (D), CD1 lb+CD64+FRp+cells (E) and CD1 lb+CD33+cells (F) remaining was measured by flow cytometry.

[0070] Figure 39 - CD112 BiTE triggers the activation and proliferation of endogenous T cells in ascites. CD112 BiTE or Control BiTE was cultured with ascites cells in normal medium or autologous ascites fluid (50% v / v) for 120 hours. The expression of CD25 on T cells (A) and the number of T cells, calculated as fold-change (B), were measured by flow cytometry.

[0071] Figure 40 - Ascites fluid upregulates CD112 expression on cancer cells. DLD-1 cells were cultured in ascites fluid (50% v / v) mixed with normal medium for 72 hours, followed by the measurement of CD112 expression by flow cytometry.

[0072] Figure 41 - CD112 BiTE overcomes the immunosuppression of ascites fluid to induce killing against cancer cells. PBMC-derived T cells were cocultured with Panel cells and CD112 BiTE or Control BiTE or anti-CD3 / CD28 Dynabeads for 72 hours. (A) T cells expressing both CD25 and CD69 as measured by flow cytometry. The expression of CD25 (B) and CD69 (C) on T cells was measured by flow cytometry. (D) The cytotoxicity of CD112 BiTE was determined by XTT assay.

[0073] Figure 42 - CD112 BiTE triggers fFNy release and killing in solid cancer biopsies. Thin slices of human colorectal cancer liver metastasis biopsies were incubated with CD112 BiTE or Control BiTE for 6 days. Supernatants were collected on day 3 and day 6. The concentration of IFNy in the supernatants on day 3 (A) and day 6 (B) was measured by ELISA. (C) Cytotoxicity of the BiTEs was determined by measuring the released LDH, normalised to the corresponding day 0 controls. Data show the mean ± SD of duplicate.

[0074] Figure 43 - CD112 BiTE triggers the activation of resident T cells in solid cancer biopsies. Thin slices of matched human solid biopsies were incubated with the CD112 BiTE or Control BiTE for 6 days, before fixation and staining of the CD25 expression. IHC images of colorectal cancer liver metastasis (A) and the matched normal adjacent liver tissue (B). The top panel is the IHC images at IX magnification. The zoomed-in images of the rectangular boxes were shown in the bottom panel at a 5X magnification.

[0075] Description of the sequence listing

[0076] SEQ ID NO: 1 - HCDR1 sequence of anti-CD155 scFv of “anti-CD155 scFvl BiTE” SEQ ID NO: 2 - HCDR2 sequence of anti-CD155 scFv of “anti-CD155 scFvl BiTE” SEQ ID NO: 3 - HCDR3 sequence of anti-CD155 scFv of “anti-CD155 scFvl BiTE”

[0077] SEQ ID NO: 4 - LCDR1 sequence of anti-CD155 scFv of “anti-CD155 scFvl BiTE”

[0078] SEQ ID NO: 5 - LCDR2 sequence of anti-CD155 scFv of “anti-CD155 scFvl BiTE”

[0079] SEQ ID NO: 6 - LCDR3 sequence of anti-CD155 scFv of “anti-CD155 scFvl BiTE”

[0080] SEQ ID NO: 7 - heavy chain variable region sequence of anti-CD155 scFv of “anti¬

[0081] CD 155 scFvl BiTE”

[0082] SEQ ID NO: 8 - light chain variable region sequence of anti-CD155 scFv of “antiCD 155 scFvl BiTE”

[0083] SEQ ID NO: 9 - amino acid sequence of anti-CD155 scFv of “anti-CD155 scFvl BiTE” SEQ ID NO: 10 - amino acid sequence of mature “anti-CD155 scFvl BiTE”

[0084] SEQ ID NO: 11 - HCDR1 sequence of anti-CD155 scFv of “anti-CD155 scFv2 BiTE” SEQ ID NO: 12 - HCDR2 sequence of anti-CD155 scFv of “anti-CD155 scFv2 BiTE” SEQ ID NO: 13 - HCDR3 sequence of anti-CD155 scFv of “anti-CD155 scFv2 BiTE” SEQ ID NO: 14 - LCDR1 sequence of anti-CD155 scFv of “anti-CD155 scFv2 BiTE” SEQ ID NO: 15 - LCDR2 sequence of anti-CD155 scFv of “anti-CD155 scFv2 BiTE” SEQ ID NO: 16 - LCDR3 sequence of anti-CD155 scFv of “anti-CD155 scFv2 BiTE” SEQ ID NO: 17 - heavy chain variable region sequence of anti-CD155 scFv of “anti¬

[0085] CD 155 scFv2 BiTE” SEQ ID NO: 18 - light chain variable region sequence of anti-CD155 scFv of “antiCD 155 scFv2 BiTE”

[0086] SEQ ID NO: 19 - amino acid sequence of anti-CD155 scFv of “anti-CD155 scFv2 BiTE”

[0087] SEQ ID NO: 20 - amino acid sequence of mature “anti-CD155 scFv2 BiTE”

[0088] SEQ ID NO: 21 - HCDR1 sequence of anti-CDl 12 scFv of “anti-CDl 12 scFv7 BiTE” SEQ ID NO: 22 - HCDR2 sequence of anti-CDl 12 scFv of “anti-CDl 12 scFv7 BiTE” SEQ ID NO: 23 - HCDR3 sequence of anti-CDl 12 scFv of “anti-CDl 12 scFv7 BiTE” SEQ ID NO: 24 - LCDR1 sequence of anti-CDl 12 scFv of “anti-CDl 12 scFv7 BiTE” SEQ ID NO: 25 - LCDR2 sequence of anti-CDl 12 scFv of “anti-CDl 12 scFv7 BiTE” SEQ ID NO: 26 - LCDR3 sequence of anti-CDl 12 scFv of “anti-CDl 12 scFv7 BiTE” SEQ ID NO: 27 - heavy chain variable region sequence of anti-CDl 12 scFv of “antiCD 112 scFv7 BiTE”

[0089] SEQ ID NO: 28 - light chain variable region sequence of anti-CDl 12 scFv of “antiCD 112 scFv7 BiTE”

[0090] SEQ ID NO: 29 - amino acid sequence of anti-CDl 12 scFv of “anti-CDl 12 scFv7 BiTE”

[0091] SEQ ID NO: 30 - amino acid sequence of mature “anti-CDl 12 scFv7 BiTE”

[0092] SEQ ID NO: 31 - HCDR1 sequence of anti-CDl 12 scFv of “anti-CDl 12 scFvl 1 BiTE” SEQ ID NO: 32 - HCDR2 sequence of anti-CDl 12 scFv of “anti-CDl 12 scFvl 1 BiTE” SEQ ID NO: 33 - HCDR3 sequence of anti-CDl 12 scFv of “anti-CDl 12 scFvl 1 BiTE” SEQ ID NO: 34 - LCDR1 sequence of anti-CDl 12 scFv of “anti-CDl 12 scFvl 1 BiTE” SEQ ID NO: 35 - LCDR2 sequence of anti-CDl 12 scFv of “anti-CDl 12 scFvl 1 BiTE” SEQ ID NO: 36 - LCDR3 sequence of anti-CDl 12 scFv of “anti-CDl 12 scFvl 1 BiTE” SEQ ID NO: 37 - heavy chain variable region sequence of anti-CDl 12 scFv of “antiCD 112 scFvl 1 BiTE”

[0093] SEQ ID NO: 38 - light chain variable region sequence of anti-CDl 12 scFv of “antiCD 112 scFvl 1 BiTE”

[0094] SEQ ID NO: 39 - amino acid sequence of anti-CDl 12 scFv of “anti-CDl 12 scFvl 1 BiTE”

[0095] SEQ ID NO: 40 - amino acid sequence of mature “anti-CDl 12 scFvl 1 BiTE”

[0096] SEQ ID NO: 41 - HCDR1 sequence of anti-CD3 scFv SEQ ID NO: 42 - HCDR2 sequence of anti-CD3 scFv

[0097] SEQ ID NO: 43 - HCDR3 sequence of anti-CD3 scFv

[0098] SEQ ID NO: 44 - LCDR1 sequence of anti-CD3 scFv

[0099] SEQ ID NO: 45 - LCDR2 sequence of anti-CD3 scFv

[0100] SEQ ID NO: 46 - LCDR3 sequence of anti-CD3 scFv

[0101] SEQ ID NO: 47 - heavy chain variable region sequence of anti-CD3 scFv

[0102] SEQ ID NO: 48 - light chain variable region sequence of anti-CD3 scFv

[0103] SEQ ID NO: 49 - amino acid sequence of anti-CD3 scFv

[0104] SEQ ID NO: 50 - G4S linker

[0105] SEQ ID NO: 51 - G4TG2S linker

[0106] SEQ ID NO: 52 - SG4linker

[0107] SEQ ID NO: 53 - G4SG4linker

[0108] SEQ ID NO: 54 - G2SG2 linker

[0109] SEQ ID NO: 55 - nucleic acid sequence of mature “anti-CD155 scFvl BiTE”

[0110] SEQ ID NO: 56 - nucleic acid sequence of mature “anti-CD155 scFv2 BiTE”

[0111] SEQ ID NO: 57 - nucleic acid sequence of mature “anti-CDl 12 scFv7 BiTE”

[0112] SEQ ID NO: 58 - nucleic acid sequence of mature “anti-CDl 12 scFvl 1 BiTE”

[0113] SEQ ID NO: 59 - amino acid sequence of C-terminal FLAG-tag

[0114] SEQ ID NO: 60 - amino acid sequence of N-terminal signal peptide

[0115] SEQ ID NO: 61 - nucleic acid sequence of C-terminal FLAG-tag

[0116] SEQ ID NO: 62 - nucleic acid sequence of N-terminal signal peptide

[0117] Detailed description of the invention

[0118] It is to be understood that different applications of the disclosed methods, products and uses may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the disclosure only, and is not intended to be limiting.

[0119] Therapeutic methods and uses

[0120] The present invention provides a TIGIT antagonist for use in a method of treating cancer, wherein the cancer comprises an immunosuppressive tumour microenvironment comprising cancer cells and one or more types of non-cancerous cells. Antagonists

[0121] TIGIT is an inhibitory immune receptor that exerts an immunosuppressive effect when activated by its ligands, in particular CD155 and CD112. A TIGIT antagonist is any agent that is directed against TIGIT or antagonises TIGIT indirectly or directly. In particular, the TIGIT antagonist may be an agent that reduces TIGIT function. The antagonist may decrease the function of TIGIT by any therapeutically or prophylactically effective amount. For instance, the function may be decreased as appropriate by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%. The antagonist may abolish the function of TIGIT (i.e. the function is decreased by 100%). TIGIT function may be measured by any suitable technique.

[0122] The antagonist may reduce TIGIT function by reducing the activity or expression of TIGIT. The antagonist may decrease the amount of TIGIT by decreasing the production or expression of, or increasing the degradation of, TIGIT. The antagonist may decrease the presentation of TIGIT on the surface of cells. The antagonist may be capable of neutralising or removing TIGIT from the surface of cells. The antagonist may inhibit or prevent effective binding of TIGIT to one or more of its ligands, such as CD155 and / or CD112. The antagonist may decrease the transcription of TIGIT. The antagonist may disrupt the DNA of TIGIT by sitespecific mutagenesis using methods such as Zinc-finger nucleases. The antagonist may decrease the mRNA level of TIGIT, or interfere with the processing of TIGIT mRNA, for instance by antisense RNA or RNA interference. The antagonist may increase protein degradation of TIGIT. The antagonist may increase the level of natural inhibitors of TIGIT. The antagonist may decrease the function of TIGIT by post-translational modification, such as phosphorylation, ubiquitylation, SUMOylation or the like.

[0123] Alternatively, or in addition, the antagonist may reduce TIGIT function by binding to, or antagonising ligands of, TIGIT, such as CD155 and / or CD112. The antagonist may reduce the immunosuppressive function of CD155 and / or CD112. The immunosuppressive function may comprise binding and / or activating TIGIT. Where the antagonist reduces the immunosuppressive function of CD155, the immunosuppressive function may comprise binding and / or activating CD96. Where the antagonist reduces the immunosuppressive function of CD112, the immunosuppressive function may comprise binding and / or activating CD112R. The antagonist may increase the immunostimulatory function of CD155 and / or CD112. The immunostimulatory function may comprise binding and / or activating CD226.

[0124] Preferably, the antagonist reduces TIGIT function by reducing the immunosuppressive function of CD155 and / or CD112. The antagonist may decrease the immunosuppressive function of CD155 and / or CD112 by any therapeutically or prophylactically effective amount. For instance, the immunosuppressive function may be decreased as appropriate by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%. The antagonist may abolish the immunosuppressive function of CD155 and / or CD112 (i.e. the function is decreased by 100%). The immunosuppressive function of CD155 and / or CD112 may be measured by any suitable technique.

[0125] Alternatively, or in addition, the antagonist may reduce TIGIT function by increasing the immunostimulatory function of CD155 and / or CD112. The immunostimulatory function may comprise binding and activating CD226. The antagonist may increase the immunostimulatory function of CD155 and / or CD112 by any therapeutically or prophylactically effective amount. For instance, the immunostimulatory function may be increased as appropriate by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400% or at least 500%. The immunostimulatory function of CD155 and / or CD112 may be measured by any suitable technique.

[0126] The antagonist may reduce the immunosuppressive function of CD155 and / or CD112 by reducing the activity or expression of CD 155 and / or CD112. The immunosuppressive activity may comprise binding and / or activating TIGIT. Where the antagonist reduces the immunosuppressive function of CD155, the immunosuppressive function may comprise binding and / or activating CD96. Where the antagonist reduces the immunosuppressive function of CD112, the immunosuppressive activity may comprise binding and / or activating CD112R. The antagonist may increase the immunostimulatory function of CD155 and / or CD112 by increasing the immunostimulatory activity of CD155 and / or CD112. The immunostimulatory activity may comprise binding and / or activating CD226.

[0127] Preferably, the antagonist reduces the immunosuppressive function of CD155 and / or CD112 by reducing the immunosuppressive activity or expression of CD155 and / or CD112. More preferably, the antagonist reduces the immunosuppressive activity or expression of CD155 and / or CD112 without affecting or reducing the immunostimulatory activity of CD155 and / or CD112. The antagonist may decrease the amount of CD 155 and / or CD112 by decreasing the production, or expression of, or increasing the degradation of, CD155 and / or CD112. The antagonist may decrease the presentation of CD155 and / or CD112 on the surface of cells. The antagonist may be capable of neutralising or removing CD155 and / or CD112 from the surface of cells. For example, the antagonist may be capable of neutralising the immunosuppressive activity of CD155 and / or CD112 without affecting or reducing the immunostimulatory activity of CD 155 and / or CD112. The antagonist may inhibit or prevent effective binding of CD 155 and / or CD112 to their ligands. For example, the antagonist may inhibit or prevent effective binding to immunosuppressive ligands of CD155 and / or CD112, such as TIGIT, CD96 and / or CD112R, without affecting or reducing effective binding to immunostimulatory ligands of CD155 and / or CD112, such as CD226. The antagonist may decrease the transcription of CD155 and / or CD112. The antagonist may disrupt DNA encoding for CD 155 and / or CD112, for example by site-specific mutagenesis using methods such as Zinc-finger nucleases. The antagonist may decrease the mRNA level of CD155 and / or CD112, or interfere with the processing of CD155 and / or CD112 mRNA, for instance by antisense RNA or RNA interference. The antagonist may increase protein degradation of CD155 and / or CD112. The antagonist may increase the level of natural inhibitors of CD155 and / or CD112. For example, the antagonist may increase the level of natural inhibitors of the immunosuppressive activity of CD155 and / or CD112, such as natural inhibitors of TIGIT binding, CD96 binding and / or CD112R binding, without affecting or increasing the level of natural inhibitors of the immunostimulatory activity of CD155 and / or CD112, such as natural inhibitors of CD226 binding. The antagonist may decrease the activity of CD155 and / or CD112 by post-translational modification, such as phosphorylation, ubiquitylation, SUMOylation or the like. For example, the antagonist may decrease the immunosuppressive activity of CD 155 and / or CD112 by post- translational modification without affecting or reducing the immunostimulatory activity of CD155 and / or CD112.

[0128] Alternatively, or in addition, the antagonist may increase the immunostimulatory function of CD155 and / or CD112 by increasing the immunostimulatory activity of CD155 and / or CD112. Preferably, the antagonist increases the immunostimulatory activity of CD155 and / or CD112 without affecting or increasing the immunosuppressive activity of CD155 and / or CD112. The antagonist may increase effective binding of CD155 and / or CD112 to their immunostimulatory ligands. For example, the antagonist may increase effective binding to immunostimulatory ligands, such as CD226, without affecting or increasing effective binding to immunosuppressive ligands, such as TIGIT, CD96 and / or CD112R. The antagonist may reduce the level of natural inhibitors of the immunostimulatory activity of CD155 and / or CD112, such as natural inhibitors of CD226 binding, without affecting or reducing the level of natural inhibitors of the immunosuppressive activity of CD155 and / or CD112, such as natural inhibitors of TIGIT binding, CD96 binding and / or CD112R binding. The antagonist may increase the immunostimulatory activity of CD155 and / or CD112 by post-translational modification. For example, the antagonist may increase the immunostimulatory activity of CD155 and / or CD112 by post-translational modification without affecting or increasing the immunosuppressive activity of CD 155 and / or CD112.

[0129] The TIGIT antagonist may be specific to CD155 or CD112. That is, the antagonist may act predominantly or exclusively on CD 155 or CD112, or act on CD 155 or CD112 in preference to other molecules. Preferably, the antagonist is specific to CD155 or CD112. Such antagonists may be useful for treating cancers comprising an immunosuppressive tumour microenvironment by virtue of their ability to reduce CD 155- or CD112-induced activation of TIGIT, and optionally CD112-induced activation of CD112R and / or CD155-induced activation of CD96. As demonstrated in the Examples, CD155 and CD112 are also ubiquitously expressed in multiple cell types in the tumour microenvironment. Therefore, CD155 and / or CD112 antagonists may also be capable of targeting multiple cell types in the tumour microenvironment, and may be capable of effectively depleting an immunosuppressive tumour microenvironment.

[0130] The antagonist may be a small molecule, a peptide, a peptidomimetic, a protein, an antibody or an antigen-binding fragment thereof, a CAR T cell, a CAR NK cell, a CAR macrophage, a polynucleotide, an oligonucleotide, an antisense molecule (e.g. an antisense RNA or morpholino), an interfering RNA (such as a small interfering RNA (siRNA), a small hairpin RNA (shRNA), or a modified RNAi therapeutic prodrug, such as a short interfering ribonucleic neutral (siRNN)).

[0131] Preferably, the TIGIT antagonist comprises an antibody or an antigen-binding fragment thereof. The antibody may be polyclonal or monoclonal. The antibody may be produced by any suitable method known in the art. For example, polyclonal antibodies may be obtained by immunising a mammal, typically a rabbit or a mouse, with the target antigen under suitable conditions and isolating antibody molecules from, for example, the serum of the mammal. Monoclonal antibodies may be obtained by hybridoma or recombinant methods. The antibody is preferably a mammalian antibody, such as a primate, human, rodent (e.g. mouse or rat), rabbit, ovine, porcine, equine or camel antibody. The antibody may be a camelid antibody or shark antibody. The antibody may be any class or isotype of antibody (e.g. IgM or IgG). The antibody may be a chimeric antibody comprising sequences from different natural antibodies, such as a humanised antibody. The antibody may be a synthetic antigen-binding scaffold or synthetic antibody, for example an Alphabody, Affibody, Affitin, Anticalin, Monobody or Adnectin. The antibody may be a whole antibody,

[0132] An antibody comprises an antigen-binding domain. An antigen-binding domain comprises one or more immunoglobulin variable domains. Each immunoglobulin variable domain typically comprises three complementarity determining regions (CDRs). The CDRs typically are responsible for antigen specificity (e.g. by making direct interactions with the antigen). Exemplary conventions that can be used to identify the boundaries of CDRs include the Kabat definition, the Chothia definition and the IMGT definition (see, for example, Kabat, Elvin Abraham. Sequences of proteins of immunological interest. No. 91. US Department of Health and Human Services, Public Health Service, National Institutes of Health, 1991; Lefranc, Marie-Paule, et al. "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains." Developmental & Comparative Immunology 27.1 (2003): 55-77). The immunoglobulin variable domains may also comprise framework regions, which provide the immunoglobulin-like structure of the domain and typically do not make direct interactions with an antigen. The immunoglobulin variable domains may be selected from the immunoglobulin variable domain from an scFv domain, an antibody domain (e.g. VH and / or VL), an Fab, an Fab’, an F(ab’)2 fragment, a VHH domain and a VNAR domain.

[0133] An scFv domain comprises a VH domain and VL domain of an immunoglobulin. Whilst an scFv is typically arranged VH-VLin an N-terminal to C-terminal orientation, the scFv may also be arranged VL-VH in an N-terminal to C-terminal orientation. The scFv may be derived from a human immunoglobulin. The scFv may be derived from a murine immunoglobulin. Two scFv domains may be connected by a short linker peptide. Any linker may be used to link the scFv domains.

[0134] The antigen-binding domain may be any such domain that is able to specifically bind to a target antigen (e.g. CD155 or CD112). For example, the antigen-binding domain may be a nanobody (i.e. a single domain antibody, sdAb or VHH) or an Fab’ fragment, in isolation or complexed, or may comprise a modified Fc region (e.g. an Fcab), in isolation or combined with specific Fab domains against alternative targets as a bi-specific therapeutic agent (mAb2).

[0135] An antigen-binding fragment of an antibody comprises an antigen-binding site (e.g. Fab or F(ab)2 fragments or scFv). The whole antibody, or fragment thereof, may be an isolated antibody, or fragment thereof, or may be associated with or complexed with other moieties or may be in the form of a fusion protein.

[0136] The antibody may be a monospecific antibody or a multi-specific antibody. The multispecific antibody may be a bispecific antibody. The bispecific antibody may be a bispecific T cell engager (BiTE) or a trifunctional antibody.

[0137] Particularly preferably, the TIGIT antagonist is a BiTE. In this case, the BiTE comprises a first antigen-binding domain and a second antigen-binding domain. The antigen-binding domains may be selected from any such domains discussed above.

[0138] Accordingly, the BiTE may comprise for example a first scFv and a second scFv. One antigen-binding domain (such as a first scFv) may bind to an antigen or receptor of a T cell, and the other antigen-binding domain (such as a second scFv) may bind to an antigen or receptor of a target cell. Thus, a BiTE may be capable of linking a T cell with a target cell and causing the T cell to exert its cytotoxic activity against the target cell independently of the presence of MHC I or co-stimulatory molecules. The T cell may be a CD8+ T cell or cytotoxic T cell. The T cell may be a CD4-CD8+ T cell. The T cell may be a CD4+ T cell or helper T cell (TH cell), such as a TH1, TH2, TH3, TH17, TH9, or TFH cell. The T cell may be a cytotoxic T cell (e.g. a CD8+ T cell). Where the T cell is a CD8+ T cell or CD4+ T cell, one scFv may specifically bind to an antigen or receptor of the T cell (e.g. CD3). The target cell may be a cancer cell or a non- cancerous cell (e.g. a non-cancer cell comprised in the tumour microenvironment of the target cancer). Where the target cell is a cancer cell or a non-cancerous cell, the other scFv may specifically bind to an antigen or receptor of the cancer cell or non-cancerous cell.

[0139] A preferred BiTE is an anti-CD155 or anti-CDl 12 BiTE. In preferred embodiments, the TIGIT antagonist used in the method of treating cancer is a BiTE comprising a first scFv and a second scFv, wherein the first scFv specifically binds to CD 155 or CD112 and the second scFv specifically binds to CD3. In these embodiments, the BiTE is capable of crosslinking cytotoxic T cells comprising CD3 with target cells comprising CD155 or CD112. Clustering of CD3 triggers the release of cytotoxic granules from the T cells and the killing of the CD155- or CD112-expressing target cells. As demonstrated in the Examples, CD155 and CD112 are ubiquitously expressed on multiple cell types in the tumour microenvironment, and are upregulated in immunosuppressive tumour microenvironments. Therefore, an anti-CD155 or anti-CDl 12 BiTE may be particularly useful for treating cancers comprising immunosuppressive tumour microenvironments by inducing T cell activation and killing of multiple cell types in the tumour microenvironment, and hence depleting the whole tumour microenvironment. As also shown in the Examples, the BiTEs described herein may induce T cell activation and degranulation, and cytotoxicity against different cancer cell types and non-cancerous cell types in the tumour microenvironment. Therefore, the BiTEs may be useful for treating the cancer by depleting the immunosuppressive tumour microenvironment of the cancer. The BiTEs may also be capable of depleting the tumour microenvironment at the periphery even if they are unable to initially penetrate into the tumour core. Depletion of the immunosuppressive tumour microenvironment may at least partially be due to the ability of the BiTEs to reduce CD155- or CD112-induced activation of TIGIT, without being bound by theory.

[0140] All references to a TIGIT antagonist herein may be replaced by references to a TIGIT ligand-binding agent. For example, references to TIGIT antagonists which are specific to CD155 or CD112 may be replaced by references to CD155- or CD112-binding agents. A TIGIT ligand-binding agent is an agent which specifically binds to a TIGIT ligand. TIGIT ligands are known in the art and are described herein. Preferably, the TIGIT ligand is CD155 or CD112. That is, a TIGIT ligand-binding agent is preferably a CD155- or CD112-binding agent.

[0141] Cancers

[0142] A cancer or tumour targeted in accordance with the antagonists of the invention may be any cancer or tumour. In particular aspects, the cancer or tumour comprises an immunosuppressive tumour microenvironment (TME). The TME is characterised by comprising cancer cells and one or more types of non-cancerous cells. The cancer or tumour may comprise cancer cells, together with non-cancerous infiltrating and resident host cells, an extracellular matrix and secreted factors. The tumour microenvironment of the cancer typically maintains and drives the progression of the tumour, at least by promoting tumour growth and suppressing immune functions.

[0143] An immunosuppressive tumour microenvironment typically promotes or favours immune escape of cancer cells comprised in the tumour microenvironment. The cancer cells comprised in the immunosuppressive tumour microenvironment may have low immunogenicity. The low immunogenicity may be due to a reduced level or lack of neoantigens, a reduced level or lack of HLA molecules and / or presentation of tumour antigens to HLA molecules.

[0144] The immunosuppressive tumour microenvironment may exhibit reduced dendritic cell activity, wherein the dendritic cell activity may comprise dendritic cell recruitment, activation, maturation, antigen cross-presentation and / or T cell priming. The reduced dendritic cell activity may result in the cancer or tumour exhibiting antigen presentation deficiency.

[0145] The immunosuppressive tumour microenvironment may exhibit impaired T cell trafficking. The impaired T cell trafficking may be due to the tumour microenvironment comprising reduced, a lack of or insufficient chemokine secretion, circulating T cells having low chemotactic ability, abnormal vasculature, collagen-rich stroma and / or fibroblast-rich stroma. Abnormal vasculature, collagen -rich stroma and / or fibroblast-rich stroma may inhibit T cell infiltration.

[0146] The immunosuppressive tumour microenvironment may exhibit increased T cell dysfunction and / or T cell death.

[0147] The immunosuppressive tumour microenvironment may be hypoxic. The immunosuppressive tumour microenvironment may comprise an increased level of lactate, fatty acid, and / or potassium ions. As noted above, the tumour microenvironment typically comprises cancer cells and one or more types of non-cancerous cells. The one or more types of non- cancerous cells types may, for example, be cancer-associated fibroblasts, stellate cells, mesenchymal stem cells, pericytes, tumour endothelial cells, cancer-associated adipocytes and / or immune cells (e.g. macrophages, myeloid-derived suppressor cells, regulatory T cells and / or neutrophils).

[0148] Cancer-associated fibroblasts and stellate cells, which may be derived from resident mesenchymal stem cells, are major regulators of the extracellular matrix of the tumour microenvironment and may contribute to tumour non-response to anti-cancer therapies. Indeed, cancer-associated fibroblasts and stellate cells can secrete cytokines, chemokines and angiogenic factors, the release of which is responsible for the formation of connective tissue observed in the tumour microenvironment of the majority of pancreatic cancers. Thus, in the treatments described herein, the cancer may comprise an immunosuppressive tumour microenvironment comprising cancer cells and cancer-associated fibroblasts and / or stellate cells.

[0149] Immune cells also play a key role in maintaining the tumour microenvironment. For example, the tumour microenvironment may comprise tumour-associated macrophages, which may be immunosuppressive (supporting cancer immune escape), metastasis-associated (supporting metastasis), perivascular (supporting intravasation) or invasive (supporting tumour cell invasion). The levels of tumour-associated macrophages are correlated with poor prognosis. By way of further example, the tumour microenvironment may comprise myeloid-derived suppressor cells, which negatively regulate immune responses by inhibiting CD8+ T cell responses and innate immune cells. By way of further example, the tumour microenvironment may comprise regulatory T cells, which may contribute to immunosuppression and maintaining an immunosuppressive tumour microenvironment. By way of further example, the tumour microenvironment may comprise neutrophils, which may promote angiogenesis, immunosuppression and / or cancer metastasis. Thus, the cancer may comprise an immunosuppressive tumour microenvironment comprising cancer cells and one or more types of non-cancerous cells, wherein the one or more types of non-cancerous cells are immune cells, optionally wherein the immune cells are macrophages, myeloid-derived suppressor cells, regulatory T cells and / or neutrophils.

[0150] The tumour microenvironment of the cancer or tumour may comprise a higher level of any one or more of the non-cancerous cells described herein relative to the tumour microenvironment of a control cancer, such as a cancer comprising a non-immunosuppressive tumour microenvironment or a less immunosuppressive tumour microenvironment (e.g. melanoma). Methods for determining the presence of a specific cell type or combination of cell types in the tumour microenvironment of a given cancer sample, and methods for determining the density of each cell type in the tumour microenvironment of a given cancer sample are known to the skilled person. For example, flow cytometry, immunohistochemistry, multiplex immunofluorescence, in situ hybridisation, single cell transcriptomics and spatial transcriptomics may be used.

[0151] The presence of cancer-associated fibroblasts, mesenchymal stem cells, pericytes, tumour endothelial cells, macrophages, myeloid-derived suppressor cells, regulatory T cells and / or neutrophils may be determined by detecting the presence of a specific marker or combinations of markers associated with each cell type. In particular, detecting the presence or expression of fibroblast activation protein-a (FAP) and / or a-smooth muscle actin (aSMA) may indicate the presence of cancer-associated fibroblasts; detecting the presence or expression of CD73, CD90 and / or CD 105 may indicate the presence of mesenchymal stem cells; detecting the presence or expression of nerve / glial-antigen 2 (NG2) and / or platelet-derived growth factor receptor p (PDGFRP) may indicate the presence of pericytes; detecting the presence or expression of CD31, VEGFR1, VEGFR2, endoglin and / or endosialin may indicate the presence of tumour endothelial cells; detecting the presence or expression of CD163, CD206 and / or folate receptor p (FRP) may indicate the presence of macrophages; detecting the presence of CD33 may indicate the presence of myeloid-derived suppressor cells; detecting the presence or expression of forkhead box P3 (FoxP3) may indicate the presence of regulatory T cells; and detecting the presence of CD 15 and / or CD16 (i.e. FCYRIII) may indicate the presence of neutrophil s. The presence of neutrophils may also be determined by detecting the absence of CD14 or low expression of CD14 relative to the tumour microenvironment of a control cancer that does not comprise neutrophils.

[0152] The tumour microenvironment may comprise a higher level of any one or more of FAP, aSMA, CD73, CD90, CD105, NG2, PDGFRP, CD31, VEGFR1, VEGFR2, endoglin, endosialin, CD163, CD206, FRp, CD33, FoxP3, CD15 and / or CD16 (i.e. FcyRIII), relative to the tumour microenvironment of a control cancer, such as a cancer comprising a non-immunosuppressive tumour microenvironment or a less immunosuppressive tumour microenvironment (e.g. melanoma). Methods for identifying the presence or expression of a specific marker or combination of markers in the tumour microenvironment of a given cancer sample, and the density of each marker in the tumour microenvironment of a given cancer sample are known to the skilled person. For example, flow cytometry, immunohistochemistry, multiplex immunofluorescence, in situ hybridisation, single cell transcriptomics and spatial transcriptomics may be used.

[0153] Both the cancer cells and non-cancerous cells in the tumour microenvironment may express CD155 and / or CD112. As demonstrated in the Examples, the CD155 and CD112 antagonists described herein are capable of depleting the cancer cells and or the one or more types of non-cancerous cells expressing or comprising CD155 or CD112, respectively. Thus, the antagonist described herein may deplete cancer cells and / or one or more types of non-cancerous cells. As the tumour microenvironment may be regulated and / or maintained by the cancer cells and / or the one or more types of non-cancerous cells, depleting the cancer cells and / or the one or more types of non-cancerous cells may cause the depletion of the immunosuppressive tumour microenvironment.

[0154] As noted above, an immunosuppressive tumour microenvironment may contribute to cancer cell immune escape. In particular, the cancer comprising an immunosuppressive tumour microenvironment described herein may be a cancer comprising a tumour microenvironment that comprises an immune checkpoint molecule. For example, the tumour microenvironment may comprise expression of one or more of CD111, CD112, CD112R, CD113, CD114, CD155, CD96, TIGIT, PD-L1, PD-L2, PD1, gelectin-9, adenosine, adenosine A2a receptor, IDO, TDO, CEACAM1, CD47, CD200, CD200R, BTLA, CD 160 and / or SIRP alpha. The tumour microenvironment may comprise a higher level of CD111, CD112, CD112R, CD113, CD114, CD155, CD96, TIGIT, PD-L1, PD-L2, gelectin-9, adenosine, adenosine A2a receptor, IDO, TDO, CEACAM1, CD47, CD200, CD200R, BTLA, CD 160 and / or SIRP alpha relative to the tumour microenvironment of a control cancer, such as a cancer comprising a nonimmunosuppressive tumour microenvironment or a less immunosuppressive tumour microenvironment (e.g. melanoma). Alternatively, the tumour microenvironment may comprise a higher level of expression of CDl l l, CD112, CD112R, CD113, CD114, CD155, CD96, TIGIT, PD-L1, PD-L2, gelectin-9, adenosine, adenosine A2a receptor, IDO, TDO, CEACAM1, CD47, CD200, CD200R, BTLA, CD 160 and / or SIRP alpha relative to the level of expression of healthy tissue (e.g. healthy tissue of the same tissue type as the cancer, such as healthy tissue of the same tissue type adjacent to the cancer). Typically, the healthy tissue is non-cancerous tissue that is not comprised in the cancer or tumour microenvironment. Healthy tissue adjacent to a cancer may be separated from the cancer by peritumoral tissue. Peritumoral tissue is typically inflammatory, whilst the healthy tissue is typically non-inflammatory.

[0155] Typically, the tumour microenvironment comprises a higher level of CD112 and / or CD155 relative to healthy tissues. The tumour microenvironment may comprise a level of CD112 and / or CD155 that is higher than the level of CD112 and / or CD155 of healthy tissue, for example at least 20%, 30%, 40%, 50%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450% or 500% higher. The healthy tissue is typically of the same tissue type as the cancer of interest. That is, if the cancer is colon cancer, then the tumour microenvironment may comprise a level of CD112 and / or CD155 that is higher than the level of CD112 and / or CD112 in healthy colon tissue, such as healthy colon tissue adjacent to the cancer. By way of further example, if the cancer is liver cancer, then the tumour microenvironment may comprise a level of CD112 and / or CD155 that is higher than the level of CD112 and / or CD155 in healthy liver tissue, such as healthy liver tissue adjacent to the cancer. In some cases, the healthy tissue may be of a different tissue type to the cancer of interest. Indeed, a cancer may be a metastatic cancer and may be at a metastatic site. For example, breast cancer may metastasise at the site of lung tissues. In such cases, the healthy tissue may be healthy tissue adjacent to the breast cancer at a metastatic site, such as healthy lung tissue adjacent to breast cancer that has metastasised to the lung.

[0156] Methods for identifying the presence or expression of an immune checkpoint molecule or combinations of immune checkpoint molecules in the tumour microenvironment or cancer cells comprised in the tumour microenvironment of a given cancer sample, and the density of each immune checkpoint in the tumour microenvironment or cancer cells comprised in the tumour microenvironment of a given cancer sample are known to the skilled person. For example, flow cytometry, immunohistochemistry, multiplex immunofluorescence, in situ hybridisation, single cell transcriptomics and spatial transcriptomics may be used.

[0157] Co-stimulatory molecules are molecules that enhance the level of immunological response against cancer cells. Therefore, in further aspects, the cancer described herein may not comprise expression of one or more co-stimulatory molecules. Alternatively, the cancer may comprise a lower level of expression of the one or more co-stimulatory molecules relative to the level of expression in a control cancer, such as a cancer comprising a non-immunosuppressive tumour microenvironment or a less immunosuppressive tumour microenvironment (e.g. melanoma), or relative to the level of expression of healthy tissue (e.g. healthy tissue of the same tissue type as the cancer, such as healthy tissue of the same tissue type adjacent to the cancer). That is, the tumour microenvironment may not comprise expression of, or comprise a lower level of expression of, one or more of CD226, CD80, CD86, HVEM, ICOS ligand, 4-1BBL, GITRL, CD48, TL1A, CD28, ICOS, 4-1BB, LIGHT, 0X40, CD27, GITR and DR3.

[0158] Methods for identifying the presence or expression of a co-stimulatory molecule or combinations of co-stimulatory molecules in a cancer, such as in the tumour microenvironment or on the cancer cells comprised in the tumour microenvironment of a given cancer sample, are known to the skilled person. Methods for identifying the density of each co-stimulatory molecule in a cancer, such as in the tumour microenvironment or on the cancer cells comprised in the tumour microenvironment of a given cancer sample, are also known to the skilled person. For example, flow cytometry, immunohistochemistry, multiplex immunofluorescence, in situ hybridisation, single cell transcriptomics and spatial transcriptomics may be used. In further aspects, the cancer described herein may be a cancer comprising a defective HLA class I pathway. For example, the cancer may comprise loss-of-function in one or more of the genes selected from the group consisting of an interferon response pathway gene (e.g. JAK1, JAK2, STAT1, STAT2, TYK2 or IRF9), an antigen presentation gene (e.g. 02 microglobulin (B2M), TAPI, TAPBPL, CALR, PSMB9, PSMB10, ERAP1, PDIA3, NLRC5, RFX5, PSME1, PSME2, PSME3, CIITA, HSP90AB1, HSP90AA1, HSP90B1, TAP2, TAPBP, PDIA3, ERAD, HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ or HLA-DP), an HLA class I gene (e g. HLA-A, HLA-B or HLA-C) and an HLA class II gene (e g. HLA-DR, HLA-DQ or HLA-DP). The loss-of- function may be the result of a loss-of-function mutation in the one or more genes (e.g. a loss-of- function genetic or epigenetic mutation), and / or transcriptional, post-transcriptional and / or translational modification in the gene product of the one or more genes.

[0159] The cancer described herein may be a cancer comprising a low mutational burden, such as a cancer comprising less than 20 mutations per megabase, 15 mutations per megabase, 10 mutations per megabase, 9 mutations per megabase, 8 mutations per megabase, 7 mutations per megabase, 6 mutations per megabase, 5 mutations per megabase, 4 mutations per megabase, 3 mutations per megabase, 2 mutations per megabase or 1 mutation per megabase. Typically, the cancer comprises equal to or less than 10 mutations per megabase. Alternatively, the cancer cancer comprising an immunosuppressive tumour microenvironment described herein may be a cancer comprising a high mutational burden, such as a cancer comprising more than 20 mutations per megabase, 25 mutations per megabase, 30 mutations per megabase, 35 mutations per megabase, 40 mutations per megabase, 50 mutations per megabase, 60 mutations per megabase, 70 mutations per megabase, 80 mutations per megabase, 90 mutations per megabase, 100 mutations per megabase, 150 mutations per megabase, 200 mutations per megabase, 250 mutations per megabase, 300 mutations per megabase, 350 mutations per megabase or 400 mutations per megabase.

[0160] The cancer described herein may be a primary cancer or a metastatic cancer. The cancer described herein may be stage 1 cancer, stage 2 cancer, stage 3 cancer or stage 4 cancer. Preferably, the cancer is stage 3 or 4 cancer, more preferably stage 4 cancer. Later-stage cancers are known to comprise a more immunosuppressive tumour microenvironment than earlier-stage cancers. Later-stage cancers are also expected to comprise a tumour microenvironment comprising a higher density of CD155 and / or CD112. The TIGIT antagonists described herein are therefore expected to be particularly useful for treating late-stage cancers, such as stage 4 cancer.

[0161] The cancer described herein may be pancreatic cancer, oesophageal cancer, colorectal cancer, mesothelioma, prostate cancer, breast cancer, head and neck cancer, bladder cancer, peritoneal cancer, lung cancer, gastric cancer, liver cancer, cervical cancer, thyroid cancer, kidney cancer, ovarian cancer, gallbladder and biliary cancer, lymphoma, leukaemia, myeloma, brain and CNS cancer, melanoma or sarcoma. A particularly preferred cancer is pancreatic cancer. The aforementioned cancers are known to comprise an immunosuppressive tumour microenvironment, which prohibits the infiltration of immune cells and many therapeutic agents. As demonstrated in the Examples, the TIGIT antagonists described herein, such as CD155 or CD112 antagonists, are capable of depleting the tumour microenvironment and hence is particularly useful for treating these cancers.

[0162] By virtue of the ability of the TIGIT antagonist to deplete the tumour microenvironment and promote the infiltration of immune cells and therapeutic agents, it may be useful to administer the TIGIT antagonist in the method of treatment described herein as a co-therapy. That is, the method of treatment may comprise administering the TIGIT antagonist and an additional therapy. The additional therapy may comprise administering a second TIGIT antagonist. For example, the method of treatment may comprise co-administering the antiCD 155 BiTE described herein and the anti-CDl 12 BiTE described herein. Alternatively, the additional therapy may comprise administering a chemotherapeutic agent, an immunotherapeutic agent or an antibody-drug conjugate. The additional therapy may comprise administering radiotherapy.

[0163] The invention also generally relates to use of a CD155 or CD112 antagonist in any of the uses and cancers described herein. The CD155 or CD112 antagonist may be selected from any classes of antagonists described herein. While it is considered that depletion of the immunosuppressive tumour microenvironment shown with the CD 155 or CD112 BITEs exemplified herein may at least partially be achieved by the ability of the BiTEs to reduce CD155- or CD112-induced activation of TIGIT, and optionally CD112-induced activation of CD112R and / or CD155-induced activation of CD96, the effects may more generally be described as based on antagonism of CD155 or CD112. Thus, the invention relates generally to CD155 or CD112 antagonists, without being limited to TIGIT antagonists. Accordingly, the invention further provides a CD155 or CD112 antagonist for use in a method of treating cancer, wherein the cancer comprises an immunosuppressive tumour microenvironment comprising cancer cells and one or more types of non-cancerous cells. The TME and non-cancerous cells may be any TME and non-cancerous cells as described above in relation to TIGIT antagonists.

[0164] The invention further provides a method of treating a cancer described herein in a subject, comprising administering to the subject a therapeutically effective amount of the TIGIT antagonist (or CD155 or CD112 antagonist) described herein. The invention also provides use of the TIGIT (or CD155 or CD112 antagonist) antagonist described herein in the manufacture of a medicament for treating the cancer described herein in a subject. Any of the relevant features described in connection with the antagonist that may be used in the method of treatment described above and / or the cancer that may be treated are also applicable to the use of the antagonist in the manufacture of the medicament.

[0165] Prediction methods

[0166] The invention further provides a method of predicting whether or not a subject having cancer will respond to treatment with an antagonist described herein, wherein the method comprises determining whether or not the cancer comprises an immunosuppressive tumour microenvironment and thereby predicting whether or not the subject will respond to the treatment.

[0167] Predicting whether or not a subject will respond to treatment with the antagonist includes determining the likelihood that the subject will respond and / or predicting the extent to which the subject will respond, for example, the extent to which the subject’s symptoms will be alleviated by the treatment.

[0168] Predicted responsiveness in a subject individual to treatment with the antagonist means that the subject is expected to derive benefit, or a sufficient extent of benefit, from receiving the treatment. Predicted non-responsiveness in an individual to treatment with the antagonist means that the individual is not expected to derive benefit, or a sufficient extent of benefit, from receiving the treatment. The method for predicting the response may be carried out before administration of the treatment. The prediction may then be considered when selecting or recommending a suitable treatment for the subject. Alternatively, the method may be carried out after treatment with the antagonist and used to monitor and predict the subject’s response to the treatment. Typically, the method is for predicting whether or not the subject will have a primary response to treatment with an antagonist (i.e. whether or not the subject will respond when first receiving the treatment). In some cases, the method is for predicting secondary non- responsiveness, i.e. whether or not a subject who initially responds to treatment will later stop responding to treatment or will respond less well to the treatment. The method comprises determining whether or not the cancer comprises an immunosuppressive tumour microenvironment. In some cases, determining whether or not the cancer comprises an immunosuppressive tumour microenvironment comprises determining whether or not the tumour microenvironment comprises one or more specific cell types. In these cases, determining whether or not the cancer comprises an immunosuppressive tumour microenvironment may further comprise measuring the density of the one or more specific cell types in the tumour microenvironment and comparing the density with a reference or control (e.g. a nonimmunosuppressive tumour microenvironment or a less immunosuppressive tumour microenvironment, such as the tumour microenvironment of melanoma). The cell type may comprise cancer-associated fibroblasts, mesenchymal stem cells, pericytes, tumour endothelial cells, macrophages, myeloid-derived suppressor cells, regulatory T cells and / or neutrophils. The presence or increased density of one or more of these cell types, as compared with a reference or control (e.g. a non-immunosuppressive tumour microenvironment or a less immunosuppressive tumour microenvironment, such as the tumour microenvironment of melanoma), may indicate that the subject will respond to the treatment. Conversely, the absence or reduced density of one or more of these cell types, as compared with a reference or control, may indicate that the subject will not respond to the treatment.

[0169] In some cases, determining whether or not the cancer comprises an immunosuppressive tumour microenvironment comprises determining whether or not the tumour microenvironment comprises one or more specific biomarkers. In these cases, determining whether or not the cancer comprises an immunosuppressive tumour microenvironment comprises measuring the expression level of the one or more biomarkers in the tumour microenvironment and comparing the expression level with a reference or control, such as the expression level of the one or more biomarkers in the tumour microenvironment of melanoma or the expression level of the one or more biomarkers in healthy tissue (e.g. colon tissue or liver tissue). Typically, the healthy tissue is non-cancerous tissue that is not comprised in the cancer or tumour microenvironment. Healthy tissue adjacent to a cancer may be separated from the cancer by peritumoral tissue. Peritumoral tissue is typically inflammatory, whilst the healthy tissue is typically noninflammatory. The healthy tissue may be from the subject having cancer or from a different subject, such as a subject not having cancer. The healthy tissue is typically of the same tissue type as the cancer of interest. That is, if the cancer is colon cancer, then determining whether or not the cancer comprises an immunosuppressive tumour microenvironment may comprise measuring the expression level of the one or more biomarkers in the tumour microenvironment and comparing the expression level with the expression level of the one or more biomarkers in healthy colon tissue from the subject having cancer or from a different subject, such as a subject not having colon cancer. By way of further example, if the cancer is liver cancer, then determining whether or not the cancer comprises an immunosuppressive tumour microenvironment may comprise measuring the expression level of the one or more biomarkers in the tumour microenvironment and comparing the expression level with the expression level of the one or more biomarkers in healthy liver tissue from the subject having cancer or from a different subject, such as a subject not having liver cancer.

[0170] The biomarker(s) may comprise activation protein-a (FAP), a-smooth muscle actin, CD73, CD90, CD105, nerve / glial-antigen 2 (NG2), platelet-derived growth factor receptor p (PDGFRP), CD31, VEGFR1, VEGFR2, endoglin, endosialin, CD163, CD206, folate receptor p (FRP), CD33, forkhead box P3 (FoxP3), CD15, CD16 (i.e. FCYRIII), CD111, CD112, CD112R, CD113, CD114, CD155, CD96, TIGIT, CD226, PD-L1, PD-L2, PD1, CD80, CD86, B7-H7, HVEM, gelectin-9, adenosine, adenosine A2a receptor, IDO, TDO, CEACAM1, CD47, CD200, CD200R, CTLA-4, TMIGD2, BTLA, CD160, LAG3, TIM3, CEACAM1, SIRP alpha, ICOS ligand, 4-1BBL, GITRL, CD48, BTN2A1, TL1A, CD28, ICOS, 4-1BB, LIGHT, 0X40, CD27, GITR, 2B4, DC-SIGN and / or DR3. Typically, determining whether or not the cancer comprises an immunosuppressive tumour microenvironment comprises measuring the expression level of CD112 and / or CD155 and comparing the expression level with the expression level of healthy tissues, such as healthy tissues of the same tissue type as the cancer (e.g. healthy tissue adjacent to the cancer).

[0171] The presence or increased level of one or more of the biomarkers, as compared with the reference or control, may indicate that the subject will respond to the treatment. Typically, a level that is at least 20% higher than the level or density of the reference or control indicates that the subject will respond to the treatment. For example, the level may be at least 20%, 30%, 40%, 50%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450% or 500% higher. Conversely, the absence or reduced density of one or more of these biomarkers, as compared with the reference or control, may indicate that the subject will not respond to the treatment. Typically, a level that is less than 20% higher than the level of the reference or control indicates that the subject will not respond to the treatment. For example, the level may be less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% higher.

[0172] In some cases, determining whether or not the cancer comprises an immunosuppressive tumour microenvironment comprises determining whether or not the cancer comprises a defective HLA class I pathway. In such cases, determining whether or not the cancer comprises an immunosuppressive tumour microenvironment comprises determining whether or not the cancer comprises loss-of-function in one or more of the genes selected from the group consisting of an interferon response pathway gene (e.g. JAK1, JAK2, STAT1, STAT2, TYK2 or IRF9), an antigen presentation gene (e.g. P2 microglobulin (B2M), TAPI, TAPBPL, CALR, PSMB9, PSMB10, ERAP1, PDIA3, NLRC5, RFX5, PSME1, PSME2, PSME3, CIITA, HSP90AB1, HSP90AA1, HSP90B1, TAP2, TAPBP, PDIA3, ERAD, HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ or HLA-DP), an HLA class I gene (e.g. HLA-A, HLA-B or HLA-C) and an HLA class II gene (e.g. HLA-DR, HLA-DQ or HLA-DP). In these cases, determining whether or not the cancer comprises loss-of-function in one or more of the genes may comprises determining whether or not the cancer comprises one or more loss-of-function mutations (e.g. a loss-of- function genetic or epigenetic mutation) in the one or more genes, and / or transcriptional, post- transcriptional and / or translational modifications in the gene product of the one or more genes. The presence of loss-of-function of one or more of these genes may indicate that the subject will respond to the treatment.

[0173] The cancer may be a stage 1, 2, 3 or 4 cancer, preferably a late stage cancer, such as stage 4 cancer. The cancer may be pancreatic cancer, oesophageal cancer, colorectal cancer, mesothelioma, prostate cancer, breast cancer, head and neck cancer, bladder cancer, peritoneal cancer, lung cancer, gastric cancer, liver cancer, cervical cancer, thyroid cancer, kidney cancer, ovarian cancer, gallbladder and biliary cancer, lymphoma, leukaemia, myeloma, brain and CNS cancer, melanoma or sarcoma.

[0174] Determining whether or not the cancer comprises an immunosuppressive tumour microenvironment may comprise determining the presence or measuring the density of one or more specific cell types, determining the presence or measuring the level of the biomarkers, and / or determining the presence of the loss-of-function in the genes described above in vitro in a biological sample obtained from the subject. The sample may comprise a tissue sample (e.g. a biopsy). Typically, the tissue sample is from a part of the body that is affected by the disease or condition. For example, the tissue sample may be a surgical resection sample. The sample may be processed prior to being assayed, for example by centrifugation or extraction of DNA, RNA or protein. The sample may also be stored prior to assay, preferably below -10°C, below -20°C, below -30°C, below -40°C, below -60°C or below -70°C.

[0175] Standard methods known in the art, including the methods described herein, may be used to determine the presence or measure the density of the cells described above, determine the presence or measure the level of the biomarkers described above, and / or determine the presence of the loss-of-function in the genes described above. The methods may involve using an agent that binds to or reacts with the relevant target. The agent may be contacted with the sample from the subject, and complex formation or a reaction between the agent and the relevant target may be measured. A variety of protocols for competitive binding or immunoradiometric assays to determine the specific binding capability of compounds, such as antibodies or antibody constructs and oligonucleotides are well known in the art (see, for example, Maddox et al., J. Exp. Med. 158, 1211-1226, 1993). Methods to assess protein levels include antigen-capture dipstick assays and Enzyme-linked Immunosorbant Assay (ELISA). ELISA is typically carried out using the sandwich technique or the competitive technique, which are known to those skilled in the art. The methods may also employ antibodies to target proteins in direct sensing techniques including, but not limited to, those based upon surface plasmon resonance, surface acoustic wave, quartz crystal microbalance, microcalorimetry or electrochemical impedance spectroscopy. A specific mAb could be used in a monoclonal antibody-based immunochromatographic strip test for the detection of levels of target proteins. A modified oligonucleotide Aptamer could be used as part of a multiplex analyte detection system using the Somalogic Platform. Protein expression levels could be determined by, for example, flow cytometry or by quantitative immunohistochemistry analysis on histological sections of tissues obtained from the subject. mRNA levels can be accurately quantified by RNA analysis methods including qRT-PCR and next-generation sequencing.

[0176] The method of prediction may further comprise administering the antagonist to a subject predicted to respond to the treatment. If the subject is predicted not to respond to the treatment, then the treatment may not be administered to the subject.

[0177] The subject concerned is typically a mammal, for example a primate, rodent (including mice and rats), or other common laboratory, domestic or agricultural animal, including but not limited to rabbits, dogs, cats, horses, cows, sheep, goats or pigs. Typically, the subject is a human.

[0178] Any of the relevant features in connection with the antagonist that may be used in the method of treatment described above and / or the cancer that may be treated in the method of treatment described above may also apply to the method of prediction.

[0179] Anti-CD155 / CD3 bispecific T cell engager The invention further provides a bispecific T cell engager (BiTE) comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain specifically binds to CD 155 and the second antigen-binding domain specifically binds to CD3. Thus, the BiTE is capable of crosslinking T cells comprising CD3 with target cells comprising CD155. Clustering of CD3 may accordingly trigger the release of cytotoxic granules from the T cells and the killing of the CD155-expressing target cells. As demonstrated in the Examples, CD155 is ubiquitously expressed on multiple cell types in the tumour microenvironment, and is upregulated in immunosuppressive tumour microenvironments. Therefore, the BiTE is particularly useful for treating cancers comprising immunosuppressive tumour microenvironments by inducing T cell activation and killing of multiple cell types in the tumour microenvironment, and hence depleting the whole tumour microenvironment. As also shown in the Examples, the BiTE can induce T cell activation and degranulation, and cytotoxicity against different cancer cell types and non-cancerous cell types in the tumour microenvironment. Therefore, the BiTE is useful for treating the cancer by depleting the immunosuppressive tumour microenvironment of the cancer. Even if the BiTEs cannot initially penetrate into the tumour core, the BiTEs may be capable of depleting the tumour microenvironment at the periphery. Here, depletion of the immunosuppressive tumour microenvironment would be expected to at least partially be due to the ability of the BiTEs to reduce CD155-induced activation of TIGIT.

[0180] Preferably, the first antigen-binding domain is a first single-chain variable fragment (scFv) and the second antigen-binding domain is a second scFv, wherein the first scFv specifically binds to CD 155 and the second scFv specifically binds to CD3. Each antigenbinding domain of the BiTE typically comprises six complementarity determining regions (CDRs). In some cases, the first antigen-binding domain comprises heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 1 to 6, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto; and the second antigen-binding domain comprises heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 41 to 46, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto. In some cases, the first antigen-binding domain comprises heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 11 to 16, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto; and the second antigen-binding domain comprises heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 41 to 46, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto.

[0181] Each antigen-binding domain of the BiTE may comprise a heavy chain variable region and a light chain variable region sequence. In some cases, the first antigen-binding domain comprises heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 7 and 8, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto; and the second antigen-binding domain comprises heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 47 and 48, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto. In some cases, the first antigen-binding domain comprises heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 17 and 18, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto; and the second antigenbinding domain comprises heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 47 and 48, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto.

[0182] In some cases, the first antigen-binding domain comprises or consists of the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto; and the second antigen-binding domain comprises or consists of the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto. In some cases, the first antigen-binding domain comprises or consists of the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto; and the second antigen-binding domain comprises or consists of the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto. The first antigen-binding domain and the second antigen-binding domain, such as a first scFv and a second scFv, may be linked via a linker sequence. Any linker may be used to link the domains. The linker is preferably non-immunogenic. Suitable linker peptides include a linker sequence comprising or consisting of G4S (SEQ ID NO: 50), G4TG2S (SEQ ID NO: 51), SG4 (SEQ ID NO: 52), G4SG4 (SEQ ID NO: 53), G2SG2, (SEQ ID NO: 54), an amino acid sequence with at least 80% identity thereto. Typically, the linker sequence comprises or consists of the amino acid sequence of SEQ ID NO: 50, or an amino acid sequence with at least 80% identity thereto.

[0183] In some cases, the BiTE comprises or consists of the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto. In some cases, the BiTE comprises or consists the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto.

[0184] In preferred cases, the BiTEs comprise scFvs. Whilst an scFv is typically arranged VH- VLin an N-terminal to C-terminal orientation, the scFv may also be arranged VL-VH in an N- terminal to C-terminal orientation. The scFv may be derived from a human immunoglobulin. The scFv may be derived from a murine immunoglobulin.

[0185] The BiTE may comprise a C-terminal tag. The C-terminal tag may be a FLAG-tag comprising or consisting of the amino acid sequence of SEQ ID NO: 59. The C-terminal tag may be a His-tag (e.g. a His-tag consisting of or comprising 4, 5, 6, 7, 8, 9, 10, 11 or 12 histidine residues).

[0186] The BiTE is typically mature. That is, the BiTE does not comprise a signal peptide. Alternatively, the BiTE may be an immature BiTE. That is, the BiTE may comprise an N- terminal signal peptide. The N-terminal signal peptide may comprise or consist of the amino acid sequence of SEQ ID NO: 60.

[0187] Anti-CDl 12 / CD 3 bispecific agent

[0188] The invention also provides a bispecific agent comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain specifically binds to CD112 and the second antigen-binding domain specifically binds to CD3.

[0189] The bispecific agent may be a bispecific antibody. The bispecific antibody may be a bi specific T cell engager (BiTE) or a trifunctional antibody. Preferably, the bispecific agent is a BiTE. In this case, the BiTE comprises a first antigen-binding domain and a second antigen-binding domain. Each antigen-binding domain comprises one or more immunoglobulin variable domains. Each immunoglobulin variable domain typically comprises three complementarity determining regions (CDRs). The CDRs typically are responsible for antigen specificity (e.g. by making direct interactions with the antigen). Exemplary conventions that can be used to identify the boundaries of CDRs include the Kabat definition, the Chothia definition and the IMGT definition as described above. The immunoglobulin variable domains also comprise framework regions, which provide the immunoglobulin-like structure of the domain and typically do not make direct interactions with an antigen. The immunoglobulin variable domains may be selected from the immunoglobulin variable domain from an scFv domain, an antibody domain (e.g. VH and / or VL), an Fab, an Fab’, an F(ab’)2 fragment, a VHH domain and a VNAR domain.

[0190] An scFv domain comprises a VH domain and VL domain of an immunoglobulin. Whilst an scFv is typically arranged VH-VLin an N-terminal to C-terminal orientation, the scFv may also be arranged VL-VH in an N-terminal to C-terminal orientation. The scFv may be derived from a human immunoglobulin. The scFv may be derived from a murine immunoglobulin. Two scFv domains may be connected by a short linker peptide. Any linker may be used to link the scFv domains.

[0191] Accordingly, the BiTE may comprise a first antigen-binding domain and a second antigen-binding domain. Where the BiTE comprises a first antigen-binding domain and a second antigen-binding domain, the first antigen-binding domain specifically binds to CD112 and the second antigen-binding domain specifically binds to CD3. In this case, the BiTE may be capable of crosslinking T cells comprising CD3 with target cells comprising CD112. Clustering of CD3 can trigger the release of cytotoxic granules from T cells and the killing of the CD112- expressing target cells. As demonstrated in the Examples, CD112 is ubiquitously expressed on multiple cell types in the tumour microenvironment, and is upregulated in immunosuppressive tumour microenvironments. Therefore, the BiTE is particularly useful for treating cancers comprising immunosuppressive tumour microenvironments by inducing T cell activation and killing of multiple cell types in the tumour microenvironment, and hence depleting the whole tumour microenvironment. As also shown in the Examples, the BiTE induces T cell activation and degranulation, and cytotoxicity against different cancer cell types and non-cancerous cell types in the tumour microenvironment. Therefore, the BiTE is useful for treating the cancer by depleting the immunosuppressive tumour microenvironment of the cancer. Even if the BiTE cannot initially penetrate into the tumour core, the BiTE may be capable of depleting the tumour microenvironment at the periphery. Here, depletion of the immunosuppressive tumour microenvironment would be expected to at least partially be due to the ability of the BiTEs to reduce CD112-induced activation of TIGIT, and optionally CD112-induced activation of CD112R.

[0192] Each antigen-binding domain of the BiTE typically comprises six complementarity determining regions (CDRs). In some cases, the first antigen-binding domain comprises heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 21 to 26, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto; and the second antigen-binding domain comprises heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 41 to 46, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto. In some cases, the first antigen-binding domain comprises heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 31 to 36, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto; and the second antigen-binding domain comprises heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 41 to 46, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto.

[0193] Each antigen-binding domain of the BiTE may comprises a heavy chain variable region and a light chain variable region sequence. In some cases, the first antigen-binding domain comprises heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 27 and 28, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto; and the second antigen-binding domain comprises heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 47 and 48, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto. In some cases, the first antigen-binding domain comprises heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 37 and 38, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto; and the second antigenbinding domain comprises heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 47 and 48, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto.

[0194] In some cases, the first antigen-binding domain comprises or consists of the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto; and the second antigen-binding domain comprises or consists of the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto. In some cases, the first antigen-binding domain comprises or consists of the amino acid sequence of SEQ ID NO: 39, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto; and the second antigen-binding domain comprises or consists of the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto.

[0195] The first antigen-binding domain and the second antigen-binding domain, such as a first scFv and a second scFv, may be linked via a linker sequence. Any linker may be used to link the domains. The linker is preferably non-immunogenic. Suitable linker peptides include a linker sequence comprising or consisting of G4S (SEQ ID NO: 50), G4TG2S (SEQ ID NO: 51), SG4 (SEQ ID NO: 52), G4SG4 (SEQ ID NO: 53), G2SG2, (SEQ ID NO: 54), an amino acid sequence with at least 80% identity thereto. Typically, the linker sequence comprises or consists of the amino acid sequence of SEQ ID NO: 50, or an amino acid sequence with at least 80% identity thereto.

[0196] In some cases, the BiTE comprises or consists of the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto. In some cases, the BiTE comprises or consists the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto.

[0197] The BiTE may comprise a C-terminal tag. The C-terminal tag may be a FLAG-tag comprising or consisting of the amino acid sequence of SEQ ID NO: 59. The C-terminal tag may be a His-tag (e.g. a His-tag consisting of or comprising 4, 5, 6, 7, 8, 9, 10, 11 or 12 histidine residues). The BiTE is typically mature. That is, the BiTE does not comprise a signal peptide. Alternatively, the BiTE may be an immature BiTE. That is, the BiTE may comprise an N- terminal signal peptide. The N-terminal signal peptide may comprise or consist of the amino acid sequence of SEQ ID NO: 60.

[0198] Nucleic acids

[0199] The invention also provides one or more isolated nucleic acids (i.e. polynucleotides) encoding the bispecific agent or BiTE described herein. In some cases, the encoding nucleic acid sequence may be provided by more than one nucleic acid sequence, optionally present on more than one nucleic acid molecule, but collectively together they are able to encode the bispecific agent or BiTE.

[0200] Nucleic acids which encode a bispecific agent or BiTE can be obtained by methods well known to those skilled in the art. For example, DNA sequences coding for part or all of the heavy and light chains may be synthesised as desired from the corresponding amino acid sequences.

[0201] The nucleic acid may be a DNA sequence. The nucleic acid may be an RNA sequence, such as mRNA. The nucleic acid may comprise or consist of the nucleic acid sequence of any one of SEQ ID NOs: 55 to 58, or a nucleic acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto. Whilst SEQ ID NOs: 55 to 58 are provided as a DNA sequence, the corresponding RNA sequence (replacing ‘T’ with ‘U’) is also encompassed.

[0202] The nucleic acid sequence may comprise a nucleic acid sequence encoding a C-terminal tag, such as a C-terminal FLAG-tag or a C-terminal His-tag (e.g. a His-tag consisting of or comprising 4, 5, 6, 7, 8, 9, 10, 11 or 12 histidine residues) . The nucleic acid sequence encoding the C-terminal FLAG-tag may comprise or consist of the amino acid sequence of SEQ ID NO: 61.

[0203] The nucleic acid sequence typically encodes a mature BiTE. That is, the nucleic acid sequence may encode a BiTE that does not comprise a signal peptide. Alternatively, the nucleic acid sequence may encode an immature BiTE. That is, the nucleic acid sequence may encode a BiTE comprising an N-terminal signal peptide. The nucleic acid sequence encoding the N- terminal signal peptide may comprise or consist of the amino acid sequence of SEQ ID NO: 62.

[0204] The nucleic acid sequence typically comprises a stop codon. Typically, the stop codon comprises the nucleic acid sequence TGA. A nucleic acid may be provided in the form of an expression cassette, which includes control sequences operably linked to the inserted sequence, thus allowing for expression of the bispecific agent or BiTE described herein in vivo. Hence, the nucleic acid may be comprised in one or more expression cassettes encoding the one or more nucleic acids that encode a bispecific agent or BiTE described herein. These expression cassettes, in turn, are typically provided within vectors (e.g. plasmids or recombinant viral vectors).

[0205] Vectors

[0206] A vector may comprise the nucleic acid or the expression cassette comprising the nucleic acid. Accordingly, the invention also provides a vector comprising one or more nucleic acids described herein. The vector may be a viral vector. Conventional viral based expression systems could include retroviral, alpha-retroviral, lentiviral, adenoviral, adeno-associated virus (AAV), herpes simplex virus (HSV), Maraba virus, vesicular stomatitis virus and vaccinia virus vectors for gene transfer. Non-viral transduction vectors include transposon-based systems including PiggyBac and Sleeping Beauty systems. Methods for producing and purifying such vectors are known in the art.

[0207] The vector may be a cloning vector or an expression vector. A suitable vector may be any vector which is capable of carrying a sufficient amount of genetic information, and allowing expression of a bispecific agent or BiTE described herein.

[0208] The vector is preferably an RNA vector. Suitable RNA vectors include the RNA vectors described in Schutsky, Keith, etal., Oncotarget 6.30 (2015): 28911 and Beatty, Gregory L., et al., Gastroenterology 155.1 (2018): 29-32.

[0209] General methods by which the vectors may be constructed, transfection methods and culture methods are well known to those skilled in the art. In this respect, reference is made to “Current Protocols in Molecular Biology”, 1999, F. M. Ausubel (ed), Wiley Interscience, New York and the Maniatis Manual produced by Cold Spring Harbor Publishing.

[0210] The vector may be a human artificial chromosome. Human artificial chromosomes are described in e.g. Kazuki et al., Mol. Ther. 19(9): 1591-1601 (2011), and Kouprina et al., Expert Opinion on Drug Delivery 11(4): 517-535 (2014).

[0211] The vector may be a non-viral delivery system, such as DNA plasmids, naked nucleic acid (e.g. naked RNA), and nucleic acid complexed with a delivery vehicle, such as a liposome or a nanoparticle. Host cells

[0212] The nucleic acids, expression cassettes or vectors described herein may be introduced into a host cell, e.g. by transfection. Hence, the invention also provides a host cell comprising the one or more nucleic acids, expression cassettes or vectors described herein. The nucleic acids, expression cassettes or vectors described herein may be introduced transiently or permanently into the host cell, allowing expression of an antibody from the one or more nucleic acids, expression cassettes or vectors. Such host cells include transient, or preferably stable higher eukaryotic cell lines, such as mammalian cells or insect cells, lower eukaryotic cells, such as yeast, or prokaryotic cells, such as bacteria cells. Particular examples of mammalian cells include HEK293 (such as HEK293F, HEK293T, HEK293S), HEK Expi293F, CHO, HeLa, NSO and COS cells. Particular examples of insect cells, include Sf9 and Sf21. A particular example of a bacterial cell is Escherichia coli. Preferably, the nucleic acids, expression cassettes or vectors described herein are introduced transiently into the host cell.

[0213] Pharmaceutical compositions and modes of administration

[0214] The antagonist, bispecific agent or BiTE described herein may be formulated in a pharmaceutical composition. The composition may comprise, in addition to the therapeutically active ingredient(s), a pharmaceutically acceptable excipient, pharmaceutically acceptable salt, carrier, diluent, buffer, stabiliser 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 active ingredient. The pharmaceutically acceptable salt may be, for example, an acid addition salt or a base addition salt. The pharmaceutical carrier or diluent may be, for example, an isotonic solution.

[0215] The precise nature of the carrier or other material may depend on the route of administration (e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular and intraperitoneal routes). Examples of suitable compositions and methods of administration are provided in Esseku and Adeyeye (2011) and Van den Mooter G. (2006). For example, solid oral forms may contain, together with the active substance, diluents (e.g. lactose, dextrose, saccharose, cellulose, corn starch and / or potato starch), lubricants (e.g. silica, talc, stearic acid, magnesium or calcium stearate, and / or polyethylene glycols), binding agents (e.g. starches, gum arabic, gelatin, methylcellulose, carboxymethylcellulose and / or polyvinyl pyrrolidone), disaggregating agents (e.g. starch, alginic acid, alginates and / or sodium starch glycolate), effervescing mixtures, dyestuffs, sweeteners, wetting agents (e.g. lecithin, polysorbates and / or laurylsulphates), and / or, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations. Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film-coating processes.

[0216] Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% of active ingredient, preferably 25% to 70%. Where the pharmaceutical composition is lyophilised, the lyophilised material may be reconstituted prior to administration (e.g. a suspension). Reconstitution is preferably effected in buffer.

[0217] Capsules, tablets and pills for oral administration to an individual may be provided with an enteric coating comprising, for example, Eudragit “S”, Eudragit “L”, cellulose acetate, cellulose acetate phthalate and / or hydroxypropylmethyl cellulose.

[0218] Liquid dispersions for oral administration may be syrups, emulsions or suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and / or mannitol and / or sorbitol.

[0219] Suspensions and emulsions may contain as carrier (e.g. a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, and / or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active substance, a pharmaceutically acceptable carrier (e.g. sterile water, olive oil, ethyl oleate, glycols such as propylene glycol) and, if desired, a suitable amount of lidocaine hydrochloride.

[0220] Solutions for intravenous administration or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.

[0221] For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%.

[0222] Polynucleotide or oligonucleotides may be naked nucleotide sequences or be in combination with cationic lipids, polymers or targeting systems. They may be delivered by any available technique. For example, the polynucleotide or oligonucleotide may be introduced by needle injection, preferably intradermally, subcutaneously or intramuscularly. Alternatively, the polynucleotide or oligonucleotide may be delivered directly across the skin using a delivery device such as particle-mediated gene delivery. The polynucleotide or oligonucleotide may be administered topically to the skin, or to mucosal surfaces, by intranasal, oral, or intrarectal administration.

[0223] Uptake of polynucleotide or oligonucleotide constructs may be enhanced by several known transfection techniques, such as those including the use of transfection agents. Examples of these agents include cationic agents, such as calcium phosphate and DEAE-Dextran, and lipofectants, such as lipofectam and transfectam. The dosage of the polynucleotide or oligonucleotide to be administered can be altered.

[0224] Administration is typically in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual (e.g. an effective amount to prevent or delay onset of the disease or condition, to ameliorate one or more symptoms, to induce or prolong remission, or to delay relapse or recurrence).

[0225] The dose may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the individual to be treated; the route of administration; and the required regimen. A physician will be able to determine the required route of administration and dosage for any particular individual. A typical daily dose is from about 0.1 to 50 mg per kg of body weight dependent on the conditions mentioned above. The dose may be provided as a single dose or may be provided as multiple doses, for example, taken at regular intervals (e.g. 2, 3 or 4 doses administered hourly). Typically, polynucleotide or oligonucleotides are administered in the range of 1 pg to 1 mg, preferably 1 pg to 10 pg nucleic acid for particle mediated delivery and 10 pg to 1 mg for other routes.

[0226] Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

[0227] A composition may be administered alone or in combination with an additional therapeutic agent or treatment, for example as adjunct therapy. By virtue of the ability of the described antagonists to deplete the tumour microenvironment and promote the infiltration of immune cells and therapeutic agents, it may be useful to co-administer the composition and the additional therapeutic agent in the method of treatment described herein. The additional therapeutic agent or treatment may be one or more of those described herein, and may be administered either simultaneously or sequentially with the composition or treatment described herein. The additional therapeutic agent may be a chemotherapeutic agent.

[0228] Examples

[0229] Materials and methods

[0230] Cell lines

[0231] A549, A172, DLD-1, HCT116, HEK293A, NHDF, Panel, PC9, U-87 MG and SKOV3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich) supplemented with 10% heat-inactivated foetal bovine serum (FBS, Gibco). PSN-1 were cultured in Roswell Park Memorial Institute Medium (RPMI, Sigma-Aldrich) supplemented with 10% heat-inactivated FBS. The aforementioned cell lines were purchased from ATCC. Jurkat cells with CD4-knockout, CD8-knockin and 1G4 TCR-knockin were cultured in RPMI supplemented with 10% heat-inactivated FBS, 1% sodium pyruvate (Gibco), 1% glutamine (Gibco) and 1% penicillin-streptomycin (Gibco). Cell lines were kept in a humidified incubator at 37°C with 5% CO2 unless otherwise stated. Cell lines tested negative for mycoplasma (MycoAlert Mycoplasma Detection kit, Lonza).

[0232] Engineering and production of BiTE

[0233] BiTE targeting CD155 or CD112 was constructed by linking an anti-CD155 scFv or anti- CD112 scFv to an anti-CD3s scFv via a flexible glycine-serine linker (G4S). Control BiTE was generated similarly and contained an scFv recognising an irrelevant antigen - filamentous hemagglutinin adhesin of Bordetella pertussis. An N-terminal signal peptide and C-terminal DYKDDDDK-tag (FLAG-tag, SEQ ID NO: 59) were added for mammalian section and detection. Plasmid DNA encoding the BiTE was transfected in HEK293 A cells using Lipofectamine 2000 (Invitrogen) for 72 hours. The serum-free supernatants were harvested, concentrated using 10,000 MWCO Amicon Ultra-15 Filter Units (Millipore), and stored at - 80°C.

[0234] Processing of human PBMCs and isolation of T cells

[0235] PBMCs were isolated from human leukocyte cones from consented healthy volunteers (NHS, Blood and Transfusion Service, Oxford, UK) by density-gradient centrifugation using Ficoll-Paque Plus (GE Healthcare). CD3+ T cells were isolated from PBMCs using the Pan T cell isolation kit (Miltenyi Biotech).

[0236] Processing of clinical liquid biopsy samples

[0237] Primary human malignant ascites samples were obtained from Churchill Hospital (Oxford, UK), followed by immediate separation into cells and fluids by two rounds of centrifugation (400 x g). The cellular fractions were treated with red blood cell lysis buffer (Qiagen).

[0238] Processing of clinical solid biopsy samples

[0239] Primary colorectal cancer liver metastasis and primary colorectal cancer were obtained from John Radcliffe Hospital (Oxford, UK). For the colorectal cancer liver metastasis biopsy, the tissues were embedded in UltraPure low melting-point agarose (4% w / v, Thermo) and sectioned into 300 pm slices using a vibratome (Leica Microsystem). For the colorectal cancer biopsy, they are dissociated enzymatically and mechanically with the tumour dissociation kit (Miltenyi Biotech) and gentleMACS Dissociator (Miltenyi Biotech), followed by passing the mixture through a 70 pm cell strainer (Coming) to obtains single cells for subsequent experiments.

[0240] Flow cytometry

[0241] Cells were plated in V-bottom 96 well plates (Corning) in MACS staining buffer, followed by two-round of washing in MACS buffer before resuspending in human FcR blocking reagent (Miltenyi Biotech) for 15 min at RT. For surface detection of CD3, CD4, CD8, CD1 lb, CD25, CD33, CD45, CD64, CD69, CD107a, CD112, CD155, CD163, CD206, EpCAM, FAP, cells were incubated with appropriate antibodies at 1 :200 dilution in MACS buffer. The stained cells were then fixed with 10% formalin. When intracellular staining is necessary, the cells were permeabilised and washed in intracellular staining permeabilisation wash buffer (Biolegned) before incubating with the appropriate antibodies JFNy, granzyme B and perforin at 1 : 100 dilution. Target expression was quantified by flow cytometry using Attune NxT Flow Cytometer (Thermo).

[0242] Coculture experiment in vitro Isolated CD3+ T cells were isolated and cocultured with the indicated cell lines at a ratio of 5: 1 in RPMI supplemented with 2% FBS, together with the BiTE (0.4 nM). Where appropriate, CD3 / CD28 Dynabeads (Thermo) were included as positive controls for T cell activation. T cells were harvested after 72 hours (free BiTE) on day 3, and stained with fluorescently labelled antibodies. The stained cells were analysed by flow cytometry in Attune NxT Flow Cytometer (Thermo). Target cell cytotoxicity was measured by XTT cell proliferation assay (II) (Roche).

[0243] Human coculture experiment ex vivo

[0244] Total ascites cells were cultured in either RPMI supplemented with 2% FBS and 1% penicillin-streptomycin or autologous ascites fluids (50% v / v) together with the BiTE (0.4 nM) for 120 hours. The cells were then harvested with cell dissociation buffer for T cell activation. To determine the viability of specific cell types, the residual number of viable target cells was measured by flow cytometry using an amine-reactive fluorescence live-dead stain.

[0245] For slices of solid cancer biopsies, the samples were cultured in 24-well plates containing RPMI medium supplemented with 10% FBS, GlutaMAX (Thermo), insulin-transferrin-selenium (Gibco) and 1% penicillin-streptomycin. The next day, 200 pL supernatants were removed, stored at -20°C and replaced with fresh medium containing the BiTE. On day 3 and day 6, 200 pL and the rest of the supernatants were, respectively, collected and stored at -20°C for future analysis.

[0246] IHC

[0247] The biopsy slices were first fixed in paraformaldehyde (4%) and embedded in paraffin, before sectioning into thinner slices for automated staining using Leica BOND-MAX autostainer (Leica Microsystems). Antigen retrieval was performed at 100°C using Epitope Retrieval Solution 2 (Leica Biosystems), followed by incubation with antibodies for CD25 (Atlas Antibodies). Detection was performed using the BOND Polymer Refine Detection System (Leica Biosystems), followed by staining with hematoxylin before scanning with Aperio CS2 slice scanner (Leica Microsystems).

[0248] Ethics for clinical biopsy Primary human ascites were received from the Churchill Hospital, Oxford University Hospitals with informed consent from patients with different indications of advanced carcinoma. Human solid colorectal biopsies and the adjuvant healthy tissues were received from the John Radcliffe Hospital, Oxford University Hospitals (Oxford, UK) with informed consent from patients undergoing surgical resection. Both works were approved by the research ethics committee of the Oxford Centre for Histopathology Research (Reference 09 / H0606 / 5+5).

[0249] Statistics

[0250] Different statistical analyses were used to evaluate the significance between datasets. The one-way Analysis of Variance (ANOVA) test was used with Tukey’s post hoc analysis when there are more than two variables to compare. The two-way ANOVA test was used with Bonferroni post hoc analysis for grouped datasets with multiple variables. Data are presented as means ± s.d. unless otherwise indicated. In all figures *P < 0.05; **P < 0.01; ***P < 0.001; **** < 0.0001; NS: not significant. To assess synergy between treatments, the Bliss independence score was calculatedl, using the following formula: EInd=Ea+Eb~EaEb,- E= EObs-EIncb where EInd is the expected relative cytotoxicity, calculated as a function of the cytotoxicity from the two treatments, Ea and Eb used in isolation. EInd is compared to the actual observed value of cytotoxicity (EObs) when Ea and Eb are used in combination. If the difference (AE) between the observed and expected values is a positive value, then the relationship is deemed to be synergistic. If the difference is 0, then the variables are independent, whereas if the difference is a negative value, then the variables have an antagonistic relationship. All experiments were performed in triplicate, unless otherwise stated.

[0251] Example 1 - CD155 is highly expressed on cancer cells and cancer-associated fibroblasts but not on healthy tissues

[0252] To assess the potential of CD155 as a therapeutic target for cancer immunotherapy, its expression was first studied in multiple cancer cell lines. Flow cytometry showed that CD155 was highly expressed, from 80% - 98% in all the tested cell lines derived from colorectal, pancreatic, lung, brain and ovarian cancer (Figures 1 A-B).

[0253] One of the reasons behind selection of CD 155 as a BiTE target is because its expression in both tumour and non-tumour cells within the TME could allow depletion of stromal cells alongside cytotoxicity against cancer cells. Accordingly, the expression of CD155 was evaluated in fibroblasts - arguably the most important and most abundant cellular component of the TME. Given there is no representative cell line derived from CAFs, normal human dermal fibroblast (NHDF) cells were used as a source of fibroblasts. They were cultured and ‘activated’ in cancer cell-conditioned medium or malignant peritoneal ascites fluid, which mimicked the biological conditions of the TME, to confer the NHDF cells with a cancer-associated phenotype. Interestingly the expression of CD155 was very low on NHDF cells cultured in their normal medium but increased significantly in the counterparts that were cultured in cancer-conditioned medium or 50%(v / v) ascites fluid (Figures 2A-B). This suggests the TME may contain cytokines that could upregulate CD 155 on cancer-associated fibroblasts and potentially other stromal cell populations too.

[0254] A good target for cancer immunotherapy should be abundantly expressed on cancer cells and ideally on cellular components of the TME but not significantly expressed in healthy tissues. The best way to assess this in a translational context is to use freshly-resected clinically- relevant biopsies. These materials contain cells that precisely reflect the real clinical challenge, maintained within their pathologic architecture. Hence, they could give clinically-predictive data outputs. With the help of Mr Mike Silva, Ms Katherine Gordon-Quayle and under ethics permission from the institutional review board and research ethics committee of the Oxford Centre for Histopathology Research, a fresh biopsy of colorectal cancer liver metastasis was obtained from a cancer patient undergoing surgery. The biopsy was first dissected into tumour and healthy tissue, which were each then enzymatically and mechanically dissociated into single cells for CD155 staining. Flow cytometry showed that CD155 was only expressed on cells within the tumour but not the adjacent healthy tissue, highlighting the differential expression of CD155 on malignant and healthy tissues (Figure 3).

[0255] Example 2 - CD155 is expressed on multiple cell types in the tumour microenvironment

[0256] TME is typically composed of many different cell types that support tumour growth and suppress immune functions. The most noticeable players are the cancer cells themselves, CAFs and immune cells. To further understand the expression of CD 155 on different stromal cell subsets, cancer biopsies from five different patients were dissociated and stained for CD155 expression. CD155 was expressed on multiple cell types, including cancer cells (Figure 4A), CAFs (Figure 4B), anti-inflammatory (“M2”) macrophages (Figure 4C), and MDSCs (Figure 4D). Though the expression varied among patients, all the stromal subsets examined were found to express CD155, suggesting a fairly ubiquitous expression pattern of CD155 across different cell types within the TME. This further reinforces its promise as a BiTE target, allowing T cell cytotoxicity towards both tumour and stromal cells.

[0257] Example 3 - Bispecific T cell engagers targeting CD155 induce T cell activation and killing

[0258] To create BiTEs targeting CD155 (CD155 BiTEs), anti-CD3s scFv was engineered as a fusion protein to anti-CD155 scFvl or anti-CD155 scFv2. The two BiTEs produced were designated CD 155 scFvl BiTE and CD155 scFv2 BiTE (Figure 5 A). In each case the two scFv were joined by a glycine-serine linker. The N-terminal of each BiTE contained a signal peptide for secretion and the C-terminal contained a DYKDDDDK-tag (FLAG-tag) for detection and quantification. An scFv targeting filamentous hemagglutinin (FHA), an irrelevant antigen, was fused to antiCD3 scFv and used as a control BiTE.

[0259] Plasmids encoding the BiTEs were then transfected into HEK293 A cells for 72 hours. Supernatants were harvested and all three BiTEs - CD 155 scFvl BiTE, CD 155 scFv 2 BiTE and FHA BiTE i.e. control BiTE could be detected in the supernatants by western blotting with an anti-FLAG antibody (Figure 5B).

[0260] To compare the activity of the two CD155 BiTEs in inducing T cell activation and killing, human colorectal cancer cells HCT116 cells and human PBMC-derived T cells were cocultured with various doses of BiTEs. T cell activation was evaluated after 72 hours by the expression of CD25 and CD69, late and early T cell activation markers, respectively, while the T cell-mediated killing of the HCT116 cells was measured by XTT cell viability assay. From the dose-response curves, it is found that CD155 scFvl BiTE induced a more robust T cell activation i.e. a higher level of CD25 and CD69 expression, and killing than CD155 scFv2 BiTE (Figures 6A-C). Meanwhile, the control BiTE did not trigger any T cell activation. Since CD155 scFvl BiTE was a more potent BiTE, it would be prioritised for use in the rest of the study.

[0261] Example 4 - CD155 BiTE triggers T cell activation and degranulation, and killing against different cancer cell types

[0262] To confirm the observed BiTE-mediated T cell activation is dependent on the presence of the cancer cells, T cells were cocultured with BiTE in the presence or absence of Panel cells. T cells activated by anti-CD3 / CD28 Dynabeads were used as a positive control. In the presence of Panel cells, T cells activated by the CD155 showed a significant increase in CD25 and CD69 expression (Figures 7A-B), while in the absence of Panel cells, T cells showed no sign of activation. Moreover, the control BiTE did not activate T cells regardless of the presence of cancer cells.

[0263] In addition, CD4+ and CD8+ T cells activated by CD155 BiTE expressed a higher level of CD25 and CD69 (Figures 7C-D). Both of the subsets showed an increase in degranulation, as indicated by the expression of CD 107a, with increased production of cytotoxic granules, granzyme B and perforin, and proinflammatory cytokine IFNg (Figures 7E-H). CD8+ T cells demonstrated a more degranulated phenotype than CD4+ T cells, perhaps reflecting its role as a cytotoxic killer cell in the immune system.

[0264] Furthermore, CD 155 BiTE was able to trigger T cell activation and killing against a wide range of human cancer cells, including carcinomas and glioma, highlighting the potential therapeutic value of CD155 BiTE in treating different cancer cell types (Figures 8A-C).

[0265] Example 5 - CD155 BiTE does not induce fratricide of activated T cells

[0266] Since CD155 is an immunoregulatory ligand, T cells may express a certain level of CD155. In the resting state (untreated group), T cells did not express CD155. However, when T cells were activated by the CD155 BiTE or Dynabeads, there was a significant increase in CD 155 expression on both CD4+ and CD8+ T cells. Since both BiTE and Dynabeads upregulated CD 155 expression, its upregulation was a biological consequence of the T cell activation but not specifically mediated by the BiTE-induced activation (Figures 9A-B).

[0267] To investigate whether the CD155 BiTE affected the T cell number after the treatment, T cells were cocultured with CD155 BiTE in the presence or absence of Panel cells. Dynabeads was included as a positive control. In the presence of Panel cells, the CD155 BiTE triggered an overall increase in T cell numbers with a 1.5-fold increase in CD4+ T cells and 2.5-fold in CD8+ T cells, while T cell number was not affected by the control BiTE (Figures 10A-C).

[0268] Given T cell expression of CD 155 it was possible that a level of fratricide was occurring despite an increase in the total T cell number. Dynabeads were included as a positive control to account for the activation-induced cell death (AICD). In the presence of Panel cells, CD3+ T cells activated by the CD155 BiTE or Dynabeads showed a comparable level of viability (CD155 BiTE: 89%, Dynabeads: 84%), though their viability was slightly lower than that of the untreated T cells (99%) or T cells cocultured with the control BiTE (99%) (Figure 10D). A decrease in viability of T cells treated with CD155 BiTE may be explained by the AICD, which was also observed in T cells treated with Dynabead. In addition, the viability of CD8+ T cells activated by the CD155 BiTE and Dynabeads was 89% and 85% while the viability of CD4+ T cells activated by the CD155 BiTE and Dynabeads was 95% and 93%, suggesting that CD8+ T cells were more prone to AICD than CD4+ T cells (Figures 10E-F). Meanwhile, in the absence of Panel cells, the viability of CD3+ T cells, CD4+ T cell, and CD8+ T cells across all treatment groups were very similar.

[0269] Example 6 - Activity of CD155 BiTE is CD155-dependent

[0270] Chinese Hamster Ovary (CHO) cells do not express human CD155, and were used as CD 155-negative cells. When CHO cells were cocultured with human PBMC T cells, CD 155 BiTE did not induce a significant increase in CD25 expression as compared to the control BiTE, suggesting that the activity of the CD155 BiTE was dependent on CD155 (Figures 11 A-B).

[0271] Example 7 - CD155 BiTE preferentially activates T cells against cancer-associated fibroblasts

[0272] As previously demonstrated, NHDF cells cultured in ascites fluid had a higher expression of CD155 (Figure 3). In agreement with this, T cells became more activated by the CD155 BiTE when they were cocultured with ascites fluid-conditioned NHDF cells as compared to untreated fibroblasts (Figures 12A-C). The expression of CD69 was increased from 28% to 62% in CD3+ T cells and the increase was observed in both CD4+ and CD8+ T cell subsets.

[0273] Example 8 - CD155 BiTE kills cancer-associated fibroblasts

[0274] NHFH cells were first differentiated in DLD-1 cell-conditioned medium or ascites fluid- conditioned medium to acquire the phenotypes of cancer-associated fibroblasts. The addition of CD155 BiTE and T cells triggered robust depletion of the fibroblasts (Figure 13).

[0275] Example 9 - CD155 BiTE kills tumour-associated endothelial cells

[0276] HUVEC cells were either cultured in DLD-1 cells-conditioned medium or ascites fluids to acquire the phenotypes of the tumour-associated endothelial cells before coculturing with T cells. CD155 BiTE was able to activate T cells to kill both conditioned medium- and ascites- induced HUVEC cells (Figures 14A-C).

[0277] Example 10 - CD155 BiTE kills anti-inflammatory M2 macrophages Anti-inflammatory (M2-like) macrophages suppress antitumour response. To explore the ability of CD155 in killing the M2 macrophages, PBMC-derived monocytes were first polarised to M2 phenotype with IL10 before coculturing with CD155 BiTE autologous T cells. A robust activation was observed on both CD4+ and CD8+ T cells, and more than 90% of M2 macrophages were depleted after the treatment (Figures 15A-E).

[0278] Example 11 - CD155 augments degranulation and activation ofNK cells

[0279] Activation of NK cells is triggered when they encounter cancer cells that lack the expression of HLA class I molecules. To explore the potential CD155 BiTE in augmenting NK cell activation, we cocultured PBMC-derived NK cells with HLA class I-negative K562 cells. In resting state, NK cells alone expressed a low level of CD 107a (degranulation marker) and TIGIT (NK cell activation marker). Expression of CD 107a and TIGIT was higher when NK cells were cocultured with K562 cells (Figures 16A-B). The level was further increased in the presence of CD155 BiTE, albeit relatively lower as compared to the NK cells activated by phorbol myristate acetate / ionomycin (PMA / ion).

[0280] Example 12 - CD155 BiTE synergises the activation of tumour-reactive T cells

[0281] The ability of CD155 BiTE to activate naive T cells was clearly established in previous experiments. Nonetheless, naive T cells are not commonly found in the TME. Instead, many tumour-infiltrating T cells are thought to have specificity for tumour antigens. The ability of BiTE to augment the activation of T cells with intrinsic specificity for tumour antigens was assessed using CD4-knockout, CD8-knockin Jurkat cells, transduced with 1G4 T cell receptors (Jurkat-1G4), which recognise the HLA-A2 / NY-ESO-1 complex. Jurkat-IG4 cells were cocultured with HLA-A2 -positive Panel cells (ratio 1 : 1) in the presence or absence of NY-ESO- 1 peptide and CD155 BiTE for 24 hours. In the absence of NY-ESO-1, the CD69 gMFI of Jurkat cells was 8, and the addition of CD155 increased the CD69 gMFI to 96 (Figure 17A). In the presence of NY-ESO-1, the CD69 gMFI of Jurkat cells was 76, and the addition of CD155 increased the CD69 gMFI to 260. The Bliss independence test confirmed the CD155 BiTE synergised the activation of tumour-reactive T cells (Figure 17 A). A higher ratio of Jurkat-IG4 cells with Panel cells (5: 1) also demonstrated a similar synergistic effect on CD69 expression (Figure 17B). Example 13 - CD155 BiTE activates endogenous T cells to deplete both cancer cells and cancer- associated fibroblasts in human malignant ascites

[0282] Ascites is fluid accumulated in the peritoneal cavity of many cancer patients, and drainage of ascites is a common palliative management. Ascites often contains cancer cells, fibroblasts, lymphocytes and myeloid cells, making it a fully-controlled near-clinical model that represents many components of the heterogenous TME. Total cells were harvested from the ascites and incubated with CD155 BiTE and the respective control. The cells were suspended in the normal cell culture medium or in the presence of autologous fluid (50% v / v) for 120 hours before analysing the cell populations that remained after the treatment. CD 155 BiTE, but not the control BiTE was able to trigger a robust depletion of CD155+ cells in both the normal medium and ascites fluid (Figure 18A). Meanwhile, around 10%-30% of cancer cells, as categorised by the EpCAM+ cells, were left after the treatment, perhaps reflecting that some cancer cells may not express CD155 on their surface (Figure 18B). In addition, there was a complete eradication of CAFs, as categorised by the FAP+ cells, after the treatment (Figure 18C). The addition of CD155 BiTE depleted most of the anti-inflammatory CD206+M2 macrophages (Figure 18D). Another subset of M2 macrophages, as denoted by folate receptor p (FRP), was also significantly reduced (Figure 18E). More than 50% of MDSCs were depleted, with one sample showing completed eradication after the treatment (Figure 18F). These highlight the ability of the CD155 BiTE in depleting multiple cell types within the TME.

[0283] In addition, the activation of endogenous ascites T cells by the CD155 BiTE, as measured by CD25 expression, in ascites fluid (40% to 90%) was more robust than that in normal medium (33% to 71%) (Figure 19A). T cells activated by the BiTE also showed a greater proliferation in ascites fluid (1.4-fold to 4.5-fold) as compared to that in normal medium (0.5-fold to 2.1-fold) (Figure 19B).

[0284] To understand the increase in CD 155 BiTE-mediated T cell activation in ascites fluid, Panel cells and DLD-1 cells were cultured in normal medium or ascites fluids (50% v / v) collected from seven different patients for 72 hours. Use of ascites fluids significantly upregulated CD155 expression, though the level of upregulation was more robust in DLD-1 cells (Figure 20A) than that in Panel cells (Figure 20B). The upregulation of CD155 expression by ascites fluid may explain the more activated phenotypes of T cells when they were cultured in ascites fluid with the CD 155 BiTE. Example 14 - CD155 BiTE overcomes the immunosuppressive ascites to trigger T cell activation and killing

[0285] The composition of ascites fluid varies among patients as they contain different cytokines, chemokines and growth factors to modulate immune functions. To understand the effect of ascites fluid on T cell activation, PBMC-derived T cells were stimulated with Dynabeads in normal medium or different ascites fluids (50% v / v). Marked evaluation of CD25+ and CD69+ co-expression was observed in T cells cultured in normal medium, while at least a 1-fold reduction in CD25+ and CD69+ co-expression was observed in T cells cultured in ascites fluid (Figure 21 A), suggesting an immunosuppressive characteristic in all the tested ascites.

[0286] To explore whether the CD 155 BiTE could overcome the immunosuppressive ascites, Panel cells and T cells were cocultured with CD155 BiTE in the normal medium or ascites fluid. The level of CD25 and CD69 on T cells activated by the CD155 BiTE in ascites was comparable to the T cells activated in normal medium , and the BiTE outperformed the Dynabeads in triggering T cell activation (Figure 21B-C). Similarly, the cytotoxicity of the CD 155 BiTE- mediated killing in ascites was comparable to that in the normal medium (Figure 2 ID).

[0287] Example 15 - CD155 BiTE triggers the activation of resident T cells to mediate killing in human solid cancer biopsies

[0288] Human solid cancer biopsy preserves the heterogeneity and complex architecture of the TME, providing a good model to study the efficacy of therapeutics ex vivo. Five biopsies of colorectal cancer liver metastasis were obtained from different patients and sectioned into thin slices before culturing them with the BiTEs for 6 days. On day 3 and day 6, the supernatants of the culture were collected for measuring the level of fFNy and LDH. There was a significant increase in fFNy secreted from the slices treated with CD 155 BiTE across all samples on both day 3 and day 6 (Figures 22A-B). In addition, slices treated with the CD 155 BiTE showed a substantial release of LDH on day 3 and day 6 as compared to the respective controls, suggesting a continuous killing against the biopsies over the treatment period (Figure 22C).

[0289] To confirm that the CD155 BiTE was capable to activate resident T cells, the thin tissue slices were ethanol-fixed and paraffin-embedded before staining for CD25 expression. Tumour slices treated with the CD 155 BiTE showed a strong CD25 staining, while the staining was barely detectable in the tumour slices treated with the control BiTE (Figure 23 A). In addition, some colorectal cancer liver metastasis biopsies came with adjacent apparently-healthy liver tissues. In these cases, the matched adjacent healthy tissue was also cultured with the CD155 BiTE and control BiTE. Both slices showed mild staining of CD25, perhaps reflecting that the T cells are in a relatively ‘active’ state in the healthy tissues (Figure 23B). No strong CD25 staining was observed in the slices treated with the CD155 BiTE, supporting the notion that CD 155 was basally expressed on healthy cells, and the CD 155 BiTE can be a safe therapeutic approach for depleting the TME without causing much ‘on-target off- tumour’ effect.

[0290] Example 16 - Discussion (CD155)

[0291] The following conclusions can be drawn from the experimental data set out above: (1) CD 155 is highly expressed on cancer cells and TME as compared to normal healthy tissues; (2) immunosuppressive ascites can induce upregulation of CD155 expression on cancer cells and fibroblasts; (3) CD155 BiTE activates endogenous T cells to deplete cancer cells and CAFs in ascites; and (4) CD155 BiTE activates resident T cells in cancer biopsies but not in healthy tissues.

[0292] Though many studies reported the association of CD 155 with cancer aggressiveness and poor prognosis, the expression of CD155 on cells in the TME has been largely overlooked. Here CD155 was found to be highly expressed on cancer cells derived from different tissues (Figure 1). More importantly, CD155 was ubiquitously expressed on multiple cell types within the TME, including CAFs, macrophages and MDSCs (Figure 4). In addition, using ascites fluid to mimic the immunosuppressive environment, it is demonstrated that ascites could upregulate CD155 expression on not only cancer cells but also NHDF cells, conferring them an immunosuppressive phenotype (Figures 2 and 20). Furthermore, the expression of CD155 was localised in the TME but not the healthy tissue, highlighting the potential of targeting CD155 as a therapeutic approach (Figure 3).

[0293] Given the role of CD 155 as an inhibitory ligand and its differential expression in cancer, a BiTE targeting CD155 was developed for cancer immunotherapy, particularly for depleting the TME. The CD 155 BiTE was capable to trigger T cell activation and degranulation in both CD4+and CD8+T cell subsets, and induce T cell-mediated killing against a wide range of cancer cells. With the use of different clinically-relevant models such as primary ascites and solid cancer biopsies, CD155 BiTE targeted both cancer cells and CAFs. Importantly, it could exploit the ascites-induced CD155 upregulation to trigger a more robust T cell activation and proliferation in immunosuppressive conditions. Though many BiTEs have been studied nowadays, most of them only targeted one specific cell type. For example, EpCAM BiTE or EGRF BiTE targeted cancer cells, FAP BiTE targeted CAFs, and folate receptor-P (FRP) BiTE targeted M2 macrophages. The ability of CD155 BiTE to target multiple cell types gives it great potential to be a powerful therapeutic against the TME.

[0294] In addition to the TME, expression of CD155 was upregulated on activated T cells regardless of whether the activation was mediated by the BiTE or Dynabeads. Though this observation was not previously reported, it likely reflects the role of CD155 in balancing immune activation and inhibition. Despite an induction of CD 155 expression, no cell death caused by fratricide was observed (Figure 10). The exact mechanism of how T cell escapes fratricide is still unclear, but the presence of granzyme B inhibitors in lymphocytes may be one of the explanations. Similarly, PD-L1 was upregulated on activated T cells, but again T cell fratricide was not observed in the presence of the PD-L1 BiTE.

[0295] Although another group has previously reported the use of the CD155 x CD3 bispecific antibody to target prostate and bladder cancer, the bispecific antibody they produced was formed by chemically crosslinking anti-CD155 IgG with anti-CD3 IgG with the help of sulfo-SMCC and Traut' s reagent. Their bispecific antibody structure was completely different from the CD155 BiTE that was used here, which is a single fusion protein. The CD155 BiTE contained antiCD 155 scFv fused to anti-CD3 scFv using a very short glycine-serine linker, intended to bring T cells and target cells in very close proximity to form tighter synapses for better killing.

[0296] According to the kinetic-segregation model, a tighter synapse is thought to exclude the bulky CD45 phosphatase from dephosphorylating the CD3 and suppressing T cell functions. The BiTE could also be expressed from one single plasmid and produced from mammalian cells without the need of any chemical conjugation.

[0297] In addition, previous workers did not test the activity of the CD155 x CD3 bispecific antibody by simply adding it to T cells and cancer cells. Instead, the bispecific antibody was first tethered to the pre-activated T cells by binding the anti-CD3 IgG to the CD3 on T cells. All the subsequent experiments were performed using the tethered pre-activated T cells. For this project, the CD155 BiTE was not tethered with any pre-activated T cells or immune cells, rather it was simply added exogenously as a single fusion antibody. There are three advantages of this approach: (i) T cells are not required to be expanded ex vivo to allow tethering of the bispecific antibodies before reinfusing back to the body, rather the BiTE binds to and repurposes endogenous T cells; (ii) BiTEs are much smaller in size than T cells, which makes tumourinfiltration more efficient for the simple fusion protein than it would be for a pre-conjugated T cells; and (iii) the BiTE can be expressed by controlled genetic means, providing a major pharmacokinetic advantage by avoiding exposure to sites of potential systemic toxicity. Therefore, CD155 BiTE is a novel and powerful approach for targeting the TME in cancer immunotherapy.

[0298] Example 17 - CD112 is highly expressed on cancer cells and cancer-associated fibroblasts but not on healthy tissues

[0299] To assess the potential of CD112 as a therapeutic target for cancer immunotherapy, its expression was first studied in multiple cancer cell lines. Flow cytometry showed that CD112 was highly expressed, from 76% to 98% in all the tested cell lines derived from colorectal, pancreatic, lung, brain and ovarian cancer (Figures 24A-B).

[0300] A good target for cancer immunotherapy should be abundantly expressed on cancer cells and ideally on cellular components of the TME but not significantly expressed in healthy tissues. The best way to assess this in a translational context is to use freshly-resected clinically-relevant biopsies. These materials contain cells that precisely reflect the real clinical challenge, maintained within their pathologic architecture. Hence, they could give clinically-predictive data outputs. Under ethics permission from the institutional review board and research ethics committee of the Oxford Centre for Histopathology Research, fresh biopsies of colorectal cancer liver metastasis and healthy colon tissue were obtained from cancer patients undergoing surgery. The cancer biopsy was first dissected into tumour and healthy tissue, which were each then enzymatically and mechanically dissociated into single cells for CD112 staining. Flow cytometry showed that CD112 was only expressed on cells within the colorectal cancer liver metastatic lesion but not the adjacent healthy liver tissue nor the healthy colon tissue, highlighting the differential expression of CD112 on malignant and healthy tissues (Figure 25).

[0301] Example 18 - CD112 is expressed on multiple cell types in the tumour microenvironment

[0302] TME is typically composed of many different cell types that support tumour growth and suppress immune functions. The most noticeable players are the cancer cells themselves, CAFs and immune cells. To further understand the expression of CD112 on different stromal cell subsets, biopsies of primary colorectal cancer and colorectal cancer liver metastasis were obtained from two different patients, and dissociated and stained for CD112 expression. It is found that CD112 was expressed on multiple cell types, including cancer cells (Figure 26 A), CAFs (Figure 26B), anti-inflammatory (“M2”) macrophages (Figure 26C), and MDSCs (Figure 26D). Collectively, both primary colorectal cancer and colorectal cancer liver metastasis demonstrated a fairly ubiquitous expression pattern of CD112 across different cell types within the TME. This further reinforces its promise as a BiTE target, allowing T cell cytotoxicity towards both tumour and stromal cells.

[0303] Example 19 - Bispecific T cell engagers targeting CD112 induce T cell activation and killing

[0304] To create BiTEs targeting CD112 (CD112 BiTEs), anti-CD3s scFv was engineered as a fusion protein to anti-CDl 12 scFv7 or anti-CDl 12 scFvl 1. The two BiTEs produced were designated CD112 scFv7 BiTE and CD112 scFv7 BiTE (Figure 27). In each case the two scFv were joined by a glycine-serine linker. The N-terminal of each BiTE contained a signal peptide for secretion and the C-terminal contained a DYKDDDDK-tag (FLAG-tag) for detection and quantification. An scFv targeting filamentous hemagglutinin (FHA), an irrelevant antigen, was fused to anti-CD3 scFv and used as a control BiTE.

[0305] To compare the activity of the two CD112 BiTEs in inducing T cell activation and killing, human colorectal cancer cells HCT116 cells and human PBMC-derived T cells were cocultured with various doses of BiTEs. T cell activation was evaluated after 72 hours by the expression of CD25 and CD69, late and early T cell activation markers, respectively, while the T cell-mediated killing of the HCT116 cells was measured by XTT cell viability assay. From the dose-response curves, it is found that CD112 scFv7 BiTE and CD112 scFvl 1 BiTE induced a comparable level of T cell activation as well as T cell-mediated cytotoxicity (Figures 28A-C). Since they are similar in potency, CD112 scFv7 BiTE would be selected for use in the rest of the study.

[0306] Example 20 - CD112 BiTE triggers T cell activation and degranulation, and killing against different cancer cell types

[0307] To confirm the observed BiTE-mediated T cell activation is dependent on the presence of the cancer cells, T cells were cocultured with BiTE in the presence or absence of Panel cells. T cells activated by anti-CD3 / CD28 Dynabeads were used as a positive control. In the presence of Panel cells, T cells activated by the CD112 showed a significant increase in CD25 and CD69 expression (Figures 29A-B), while in the absence of Panel cells, T cells showed no sign of activation. Moreover, the control BiTE did not activate T cells regardless of the presence of cancer cells.

[0308] In addition, CD4+ and CD8+ T cells activated by CD112 BiTE expressed a high level of CD25 and CD69 (Figures 29C-D). Both of the subsets showed an increase in degranulation, as indicated by the expression of CD 107a, with increased production of cytotoxic granules, granzyme B and perforin, and proinflammatory cytokine IFNy (Figure 29E-H). CD8+ T cells demonstrated a more degranulated phenotype than CD4+ T cells, perhaps reflecting its role as a cytotoxic killer cell in the immune system.

[0309] Furthermore, CD112 BiTE was able to trigger T cell activation and killing against a wide range of human cancer cells, including carcinomas and glioma, highlighting the potential therapeutic value of CD112 BiTE in treating different cancer cell types (Figure 30A-C).

[0310] Example 21 - CD112 BiTE does not induce fratricide of activated T cells

[0311] Since CD112 is an immunoregulatory ligand, T cells may express a certain level of CD112. In the resting state (untreated group), T cells did not express CD112. However, when T cells were activated by the CD112 BiTE or Dynabeads, there was a significant increase in CD112 expression on both CD4+ and CD8+ T cells. Since both BiTE and Dynabeads upregulated CD112 expression, its upregulation was a biological consequence of the T cell activation but not specifically mediated by the BiTE-induced activation (Figures 31 A-B).

[0312] To investigate whether the CD112 BiTE affected the T cell number after the treatment, T cells were cocultured with CD112 BiTE in the presence or absence of Panel cells. Dynabeads was included as a positive control. In the presence of Panel cells, the CD112 BiTE triggered an overall increase in T cell numbers with a 1.8-fold increase in CD4+ T cells and 3.0-fold in CD8+ T cells, while T cell number was not affected by the control BiTE (Figures 32A-C).

[0313] Given T cell expression of CD112, it was possible that a level of fratricide was occurring despite an increase in the total T cell number. Dynabeads were included as a positive control to account for the activation-induced cell death (AICD). In the presence of Panel cells, CD3+ T cells activated by the CD112 BiTE or Dynabeads showed a comparable level of viability (CD112 BiTE: 89%, Dynabeads: 84%), though their viability was slightly lower than that of the untreated T cells (99%) or T cells cocultured with the control BiTE (99%) (Figure 32D). A decrease in viability of T cells treated with CD112 BiTE may be explained by the AICD, which was also observed in T cells treated with Dynabead. In addition, the viability of CD8+ T cells activated by the CD112 BiTE and Dynabeads was 89% and 85% while the viability of CD4+ T cells activated by the CD112 BiTE and Dynabeads was 95% and 93%, suggesting that CD8+ T cells were more prone to AICD than CD4+ T cells (Figures 32E-F). Meanwhile, in the absence of Panel cells, the viability of CD3+ T cells, CD4+ T cells, and CD8+ T cells across all treatment groups were very similar.

[0314] Example 22 - CD112 BiTE preferentially activates T cells against cancer-associated fibroblasts T cells became more activated by the CD112 BiTE when they were cocultured with ascites fluid-conditioned NHDF cells as compared to untreated NHDF cells (Figures 33 A-C). The expression of CD69 was increased from 26% to 63% in CD3+ T cells and the increase was observed in both CD4+ and CD8+ T cell subsets.

[0315] Example 23 - CD112 BiTE kills cancer-associated fibroblasts

[0316] NHFH cells were first differentiated in DLD-1 cell-conditioned medium to acquire the phenotypes of cancer-associated fibroblasts. The addition of CD112 BiTE and T cells triggered robust depletion of the fibroblasts (Figure 34).

[0317] Example 24 - CD112 BiTE kills tumour-associated endothelial cells

[0318] HUVEC cells were either cultured in DLD-1 cells-conditioned medium or ascites fluids to acquire the phenotypes of the tumour-associated endothelial cells before coculturing with T cells. CD112 BiTE was able to activate T cells to kill both conditioned medium- and ascites- induced HUVEC cells (Figures 35 A-C).

[0319] Example 25 - CD112 BiTE kills anti-inflammatory M2 macrophages

[0320] Anti-inflammatory (M2-like) macrophages suppress antitumour response. To explore the ability of CD112 in killing the M2 macrophages, PBMC-derived monocytes were first polarised to M2 phenotype with IL10 before coculturing with CD112 BiTE autologous T cells. A robust activation was observed on both CD4+ and CD8+ T cells, and more than 90% of M2 macrophages were depleted after the treatment (Figures 36A-C). Example 26 - CD112 augments degranulation and activation of NK cells

[0321] Activation of NK cells is triggered when they encounter cancer cells that lack the expression of HLA class I molecules. To explore the potential CD112 BiTE in augmenting NK cell activation, we cocultured PBMC-derived NK cells with HLA class I-negative K562 cells. In resting state, NK cells alone expressed a low level of CD 107a (degranulation marker) and TIGIT (NK cell activation marker). Expression of CD 107a and TIGIT was higher when NK cells were cocultured with K562 cells (Figures 37A-B). The level was further increased in the presence of CD112 BiTE, albeit relatively lower as compared to the NK cells activated by phorbol myristate acetate / ionomycin (PMA / ion).

[0322] Example 27- CD112 BiTE activates endogenous T cells to deplete both cancer cells and cancer- associated fibroblasts in human malignant ascites

[0323] Ascites is fluid accumulated in the peritoneal cavity of many cancer patients, and drainage of ascites is a common palliative management. Ascites often contains cancer cells, fibroblasts, lymphocytes and myeloid cells, making it a fully-controlled near-clinical model that represents many components of the heterogenous TME. Total cells were harvested from the ascites and incubated with CD112 BiTE and the respective control. The cells were suspended in the normal cell culture medium or in the presence of autologous fluid (50% v / v) for 120 hours before analysing the cell populations that remained after the treatment. CD112 BiTE, but not the control BiTE, was able to trigger a robust (65%) depletion of CD112+ cells in the normal medium, and the effect was more robust in ascites fluid, where 80% of CD112+ cells were removed (Figure 38 A). Meanwhile, around 28% of cancer cells, as categorised by the EpCAM+ cells, were left after the treatment, perhaps reflecting that some cancer cells may not express CD112 on their surface (Figure 38B). The addition of CD112 BiTE depleted most of the immunosuppressive myeloid cells including anti-inflammatory CD206+M2 macrophages (Figure 38D), folate receptor P+(FRP+) M2 macrophages (Figure 38E) and MDSCs (Figure 38F).

[0324] In addition, there was a significant eradication of CAFs, as categorised by the FAP+ cells, after the treatment (Figure 38C). These highlight the ability of the CD112 BiTE in depleting multiple cell types within the TME, particularly conferring a greater cytotoxicity in ascites as compared to that in normal medium. In addition, the activation of endogenous ascites T cells by the CD112 BiTE, as measured by CD25 expression, in ascites fluid (60% to 85%) was more robust than that in normal medium (33% to 81%) (Figure 39A). T cells activated by the BiTE also showed a greater proliferation in ascites fluid (1.8-fold to 4.8-fold) as compared to that in normal medium (0.7-fold to 2.7-fold) (Figure 39B).

[0325] To understand the increase in CD112 BiTE-mediated T cell activation in ascites fluid, DLD-1 cells were cultured in normal medium or ascites fluids (50% v / v) collected from seven different patients for 72 hours. Use of ascites fluids significantly upregulated CD112 expression (Figure 40). The upregulation of CD112 expression by ascites fluid may explain the more activated phenotypes of T cells when they were cultured in ascites fluid with the CD112 BiTE.

[0326] Example 28 - CD112 BiTE overcomes the immunosuppressive ascites to trigger T cell activation and killing

[0327] The composition of ascites fluid varies among patients as they contain different cytokines, chemokines and growth factors to modulate immune functions. To understand the effect of ascites fluid on T cell activation, PBMC-derived T cells were stimulated with Dynabeads in normal medium or different ascites fluids (50% v / v). Marked evaluation of CD25+ and CD69+ co-expression was observed in T cells cultured in normal medium, while at least a 1-fold reduction in CD25+ and CD69+ co-expression was observed in T cells cultured in ascites fluid (Figure 41 A), suggesting an immunosuppressive characteristic in all the tested ascites fluids.

[0328] To explore whether the CD112 BiTE could overcome the immunosuppressive ascites, Panel cells and T cells were cocultured with CD112 BiTE in the normal medium or ascites fluid. T cell activation triggered by the CD112 BiTE was not suppressed in ascites, and the BiTE outperformed the Dynabeads in activating the T cells (Figures 24B-C). The cytotoxicity of the CD112 BiTE-mediated killing in ascites was comparable to that in the normal medium (Figure 41D).

[0329] Example 29 - CD112 BiTE triggers the activation of resident T cells to mediate killing in human solid cancer biopsies

[0330] Human solid cancer biopsy preserves the heterogeneity and complex architecture of the TME, providing a good model to study the efficacy of therapeutics ex vivo. Three biopsies of colorectal cancer liver metastasis were obtained from different patients and sectioned into thin slices before culturing them with the BiTEs for 6 days. On day 3 and day 6, the supernatants of the culture were collected for measuring the level of fFNy and LDH. There was a significant increase in IFNy secreted from the slices treated with CD112 BiTE across all samples on both day 3 and day 6 (Figures 42A-B). In addition, slices treated with the CD112 BiTE showed a substantial release of LDH on day 3 and day 6 as compared to the respective controls, suggesting a continuous killing against the biopsies over the treatment period (Figure 42C).

[0331] To confirm that the CD112 BiTE was capable to activate resident T cells, the thin tissue slices were ethanol-fixed and paraffin-embedded before staining for CD25 expression. Tumour slices treated with the CD112 BiTE showed a strong CD25 staining, while the staining was barely detectable in the tumour slices treated with the control BiTE (Figure 43 A).

[0332] In addition, some colorectal cancer liver metastasis biopsies came with adjacent apparently-healthy liver tissues. In these cases, the matched adjacent healthy tissue was also cultured with the CD112 BiTE and control BiTE. Both slices showed mild staining of CD25, perhaps reflecting that the T cells are in a relatively ‘active’ state in the healthy tissues (Figure 43B).

[0333] Example 30 - Discussion (CD112)

[0334] The following conclusions can be drawn from the experimental data set out above: (1) CD112 is highly expressed on cancer cells and TME as compared to normal healthy tissues; (2) immunosuppressive ascites can induce upregulation of CD112 expression on cancer cells; (3) CD112 BiTE activates endogenous T cells to deplete cancer cells and CAFs in ascites; and (4) CD112 BiTE activates resident T cells in cancer biopsies but not in healthy tissues.

[0335] Though many studies reported the association of CD112 with cancer aggressiveness and poor prognosis, the expression of CD112 on cells in the TME has been largely overlooked. Here CD112 was found to be highly expressed on cancer cells derived from different tissues (Figure 24). More importantly, CD112 was ubiquitously expressed on multiple cell types within the TME, including CAFs, macrophages and MDSCs (Figure 26). In addition, using ascites fluid to mimic the immunosuppressive environment, it is demonstrated that ascites could upregulate CD112 expression on cancer cells, conferring them a more immunosuppressive phenotype (Figure 40). Furthermore, the expression of CD112 was localised in the TME but not the healthy tissue, highlighting the potential of targeting CD112 as a therapeutic approach (Figure 24). Given the role of CD112 as an inhibitory ligand and its differential expression in cancer, a BiTE targeting CD112 was developed for cancer immunotherapy, particularly for depleting the TME. The CD112 BiTE was capable to trigger T cell activation and degranulation in both CD4+ and CD8+ T cell subsets, and induce T cell-mediated killing against a wide range of cancer cells. With the use of different clinically-relevant models such as primary ascites and solid cancer biopsies, CD112 BiTE targeted both cancer cells and CAFs. Importantly, it could exploit the ascites-induced CD112 upregulation to trigger a more robust T cell activation and proliferation in immunosuppressive conditions. Though many BiTEs have been studied nowadays, most of them only targeted one specific cell type. For example, EpCAM BiTE9 or EGRF BiTElO targeted cancer cells, FAP BiTEl 1 targeted CAFs, and folate receptor-P (FRP) BiTE12 targeted M2 macrophages. The ability of CD112 BiTE to target multiple cell types gives it great potential to be a powerful therapeutic against the TME.

[0336] In addition to the TME, expression of CD112 was upregulated on activated T cells regardless of whether the activation was mediated by the BiTE or Dynabeads. Though this observation was not previously reported, it likely reflects the role of CD112 in balancing immune activation and inhibition. Despite an induction of CD112 expression, no cell death caused by fratricide was observed (Figure 32). The exact mechanism of how T cell escapes fratricide is still unclear, but the presence of granzyme B inhibitors in lymphocytes may be one of the explanations. Similarly, PD-L1 was upregulated on activated T cells, but again T cell fratricide was not observed in the presence of the PD-L1 BiTE14.

[0337] To our knowledge, there has not been any T cell engager targeting CD112 reported. Given its ubiquitous expression on different stromal subsets and its minimal expression on healthy tissues, our data demonstrated CD112 BiTE is a novel and powerful approach for targeting the TME in cancer immunotherapy.

[0338] Numbered further embodiments

[0339] 1. A TIGIT antagonist for use in a method of treating cancer, wherein the cancer comprises an immunosuppressive tumour microenvironment comprising cancer cells and one or more types of non-cancerous cells.

[0340] 2. The TIGIT antagonist for use according to embodiment 1, wherein the one or more types of non-cancerous cells comprise cancer-associated fibroblasts, mesenchymal stem cells, pericytes, tumour endothelial cells, macrophages, myeloid-derived suppressor cells, regulatory T cells and / or neutrophils.

[0341] 3. The TIGIT antagonist for use according to embodiment 2, wherein the one or more types of non-cancerous cells comprise cancer-associated fibroblasts.

[0342] 4. The TIGIT antagonist for use according to any one of embodiments 1 to 3, wherein the cancer cells and / or the one or more types of non-cancerous cells express CD155 and / or CD112.

[0343] 5. The TIGIT antagonist for use according to any one of embodiments 1 to 4, wherein the cancer is a stage 1 cancer, stage 2 cancer, stage 3 cancer or stage 4 cancer.

[0344] 6. The TIGIT antagonist for use according to embodiment 5, wherein the cancer is a stage 4 cancer.

[0345] 7. The TIGIT antagonist for use according to any one of embodiments 1 to 6, wherein the cancer is pancreatic cancer, oesophageal cancer, colorectal cancer, mesothelioma, prostate cancer, breast cancer, head and neck cancer, bladder cancer or peritoneal cancer.

[0346] 8. The TIGIT antagonist for use according to any one of embodiments 1 to 7, wherein the antagonist depletes the cancer cells and / or the one or more types of non-cancerous cells.

[0347] 9. The TIGIT antagonist for use according to any one of embodiments 1 to 8, wherein the antagonist depletes the immunosuppressive tumour microenvironment. 10. The TIGIT antagonist for use according to any one of embodiments 1 to 9, wherein the antagonist binds to CD 155 and / or CD112.

[0348] 11. The TIGIT antagonist for use according to any one of embodiments 1 to 10, wherein the antagonist is a small molecule, a peptide, a peptidomimetic, a protein, an antibody, or antigenbinding fragment thereof, a CAR T cell, a CAR NK cell, or a CAR macrophage.

[0349] 12. The TIGIT antagonist for use according to embodiment 11, wherein the antibody is a monospecific antibody or multi-specific antibody.

[0350] 13. The TIGIT antagonist for use according to embodiment 12, wherein the multi-specific antibody is a bispecific antibody or trispecific antibody.

[0351] 14. The TIGIT antagonist for use according to embodiment 13, wherein the bispecific antibody is a bispecific T-cell engager (BiTE) or a trifunctional antibody.

[0352] 15. The TIGIT antagonist for use according to embodiment 14, wherein the BiTE comprises a first antigen-binding domain and a second antigen-binding domain.

[0353] 16. The TIGT antagonist for use according to embodiment 15, wherein the first antigenbinding domain is a single-chain variable fragment (scFv) and the second antigen-binding domain is an scFv.

[0354] 17. The TIGIT antagonist for use according to embodiment 15 or 16, wherein the first anti gen -binding domain specifically binds to CD 155 and the second antigen-binding domain specifically binds to CD3.

[0355] 18. The TIGIT antagonist for use according to embodiment 17, wherein the first antigenbinding domain comprises:

[0356] (a) heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 1 to 6, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto; or

[0357] (b) heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 11 to 16, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto.

[0358] 19. The TIGIT antagonist for use according to embodiment 18, wherein the first antigenbinding domain comprises:

[0359] (a) heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 7 and 8, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto; or

[0360] (b) heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 17 and 18, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto.

[0361] 20. The TIGIT antagonist for use according to embodiment 19, wherein the first antigenbinding domain comprises the amino acid sequence of SEQ ID NO: 9 or 19, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto.

[0362] 21. The TIGIT antagonist for use according to embodiment 15, wherein the first antigenbinding domain specifically binds to CD112 and the second antigen-binding domain specifically binds to CD3.

[0363] 22. The TIGIT antagonist for use according to embodiment 21, wherein the first antigenbinding domain comprises:

[0364] (a) heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 21 to 26, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto; or

[0365] (b) heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 31 to 36, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto.

[0366] 23. The TIGIT antagonist for use according to embodiment 22, wherein the first antigenbinding domain comprises:

[0367] (a) heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 27 and 28, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto; or

[0368] (b) heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 37 and 38, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto.

[0369] 24. The TIGIT antagonist for use according to embodiment 23, wherein the first antigenbinding domain comprises the amino acid sequence of SEQ ID NO: 29 or 39, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto.

[0370] 25. The TIGIT antagonist for use according to any one of embodiments 15 to 24, wherein the second antigen-binding domain comprises heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 41 to 46, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto.

[0371] 26. The TIGIT antagonist for use according to embodiment 25, wherein the second antigenbinding domain comprises heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 47 and 48, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto.

[0372] 27. The TIGIT antagonist for use according to embodiment 26, wherein the second antigenbinding domain comprises the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto. 28. The TIGIT antagonist for use according to any one of embodiments 15 to 27, wherein the first and second antigen-binding domains are scFvs and are linked via a linker sequence, optionally wherein the linker sequence comprises the amino acid sequence of SEQ ID NO: 50, or an amino acid sequence with at least 80% identity thereto.

[0373] 29. A bispecific agent, wherein the bispecific agent is a bispecific T cell engager (BiTE) comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first anti gen -binding domain specifically binds to CD 155 and the second antigen-binding domain specifically binds to CD3.

[0374] 30. The bispecific agent according to embodiment 29, wherein the first antigen-binding domain is a single-chain variable fragment (scFv) and the second antigen-binding domain is an scFv.

[0375] 31. The bispecific agent according to embodiment 29 or 30, wherein:

[0376] (a) the first antigen-binding domain comprises: heavy chain complementarity determining region heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 1 to 6, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto; or heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 11 to 16, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto; and

[0377] (b) the second antigen-binding domain comprises: heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 41 to 46, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto.

[0378] 32. The bispecific agent according to embodiment 31, wherein:

[0379] (a) the first antigen-binding domain comprises: heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 7 and 8, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto; or heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 17 and 18, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto; and

[0380] (b) the second antigen-binding domain comprises: heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 47 and 48, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto.

[0381] 33. The bispecific agent according to embodiment 32, wherein:

[0382] (a) the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 9 or 19, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto; and

[0383] (b) the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto.

[0384] 34. The bispecific agent according to any one of embodiments 29 to 33, wherein the first antigen-binding domain and the second antigen-binding domain are scFvs and linked via a linker sequence, optionally wherein the linker sequence comprises the amino acid sequence of SEQ ID NO: 50, or an amino acid sequence with at least 80% identity thereto. 35. The bispecific according to embodiment 34, wherein the BiTE comprises the amino acid sequence of SEQ ID NO: 10 or 20.

[0385] 36. A bispecific agent comprising a first antigen-binding domain and a second antigenbinding domain, wherein the first antigen-binding domain specifically binds to CD112 and the second antigen-binding domain specifically binds to CD3.

[0386] 37. The bispecific agent according to embodiment 36, wherein the bispecific agent is a bi specific antibody or a trifunctional antibody.

[0387] 38. The bispecific agent according to embodiment 37, wherein the bispecific antibody is a bispecific T cell engager (BiTE).

[0388] 39. The bispecific agent according to any one of embodiments 36 to 38, wherein the first antigen-binding domain is a first single-chain variable fragment (scFv) and the second antigenbinding domain is a second scFv.

[0389] 40. The bispecific agent according to any one of embodiments 36 to 39, wherein:

[0390] (a) the first antigen-binding domain comprises: heavy chain complementarity determining region heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 21 to 26, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto; or heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 31 to 36, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto; and

[0391] (b) the second antigen-binding domain comprises: heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 41 to 46, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto.

[0392] 41. The bispecific agent according to embodiment 40, wherein:

[0393] (a) the first antigen-binding domain comprises: heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 27 and 28, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto; or heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 37 and 38, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto; and

[0394] (b) the second antigen-binding domain comprises: heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 47 and 48, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto.

[0395] 42. The bispecific agent according to embodiment 41, wherein:

[0396] (a) the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 29 or 39, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto; and

[0397] (b) the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto.

[0398] 43. The bispecific agent according to any one of embodiments 36 to 42, wherein the first antigen-binding domain and the second antigen-binding domain are scFvs and linked via a linker sequence, optionally wherein the linker sequence comprises the amino acid sequence of SEQ ID NO: 50, or an amino acid sequence with at least 80% identity thereto. 44. The bispecific agent according to embodiment 43, wherein the BiTE comprises the amino acid sequence of SEQ ID NO: 30 or 40.

[0399] 45. A nucleic acid encoding the bispecific agent according to any one of embodiments 29 to 44.

[0400] 46. The nucleic acid according to embodiment 45, wherein the nucleic acid is DNA or RNA.

[0401] 47. The nucleic acid according to embodiment 45 or 46, wherein the nucleic acid comprises the nucleic acid sequence of any one of SEQ ID NOs: 55 to 58.

[0402] 48. A vector comprising one or more nucleic acids according to any one of embodiments 45 to 47.

[0403] 49. The vector according to embodiment 48, wherein the vector is a viral vector, an RNA vector, liposomal vector or lipid nanoparticle vector.

[0404] 50. The vector according to embodiment 49, wherein the viral vector is a lentiviral vector, adenoviral vector, retroviral vector, adeno-associated virus vector, herpes virus vector, Maraba virus vector, vesicular stomatitis virus vector or vaccinia virus vector.

[0405] 51. A host cell comprising one or more vectors according to any one of embodiments 48 to 50.

[0406] 52. The host cell according to embodiment 51, wherein the cell is a mammalian cell, an insect cell or a bacterial cell, optionally wherein the mammalian cell is an HEK293 cell, the insect cell is an Sf9 cell or Sf21 cell, and the bacterial cell is an E.coli cell.

[0407] 53. A method of predicting whether or not a subject having cancer will respond to treatment with a TIGIT antagonist, wherein the method comprises determining whether or not the cancer comprises an immunosuppressive tumour microenvironment comprising cancer cells and one or more types of non-cancerous cells, and thereby predicting whether or not the subject will respond to the treatment.

[0408] 54. The method according to embodiment 53, wherein determining whether or not the cancer comprises an immunosuppressive tumour microenvironment comprises:

[0409] (a) measuring the expression level of a biomarker in the tumour microenvironment; and

[0410] (b) comparing the expression level with a reference level.

[0411] 55. The method according to embodiment 54, wherein the one or more types of non- cancerous cells comprise cancer-associated fibroblasts, mesenchymal stem cells, pericytes, tumour endothelial cells, macrophages, myeloid-derived suppressor cells, regulatory T cells and / or neutrophils, optionally wherein the biomarker comprises CD155, CD112, fibroblast activation protein-a (FAP), a-smooth muscle actin (aSMA), CD163, CD206, folate receptor 0 (FR0), CD33, CD31, VEGFR1, VEGFR2, endoglin and / or endosialin.

[0412] 56. The method according to any one of embodiments 53 to 55, wherein the method further comprises administering the TIGIT antagonist to a subject predicted to respond to the treatment.

[0413] Sequence Listing

[0414] SEQ ID NO: 1 - DHEMH

[0415] SEQ ID NO: 2 - TIHPGSGVTAYNQKFKG

[0416] SEQ ID NO: 3 - LWLRRD

[0417] SEQ ID NO: 4 - KASQNVATNVV

[0418] SEQ ID NO: 5 - SASYRYS

[0419] SEQ ID NO: 6 - QQYNNYPLT

[0420] SEQ ID NO: 7 -

[0421] EVOLOQSGAELVRPGTSVKLSCKALGYTFTDHEMHWVKOTPVHGLEWIGTIHPGSGVTAYNOK

[0422] FKGKATLTADKSSSTAYMELSTLTSEDSAVYYCTPLWLRRDWGOGTTLTVST

[0423] SEQ ID NO: 8 -

[0424] DIQMTQTPKFMSTSVGDRVSVTCKASONVATNVVWFQQKSGQSPKALIYSASYRYSGVPDRFTG

[0425] SGSGTDFTLTISNVQSEDLAEYFCOQYNNYPLTFGAGTKLELK

[0426] SEQ ID NO: 9 -

[0427] EVOLOQSGAELVRPGTSVKLSCKALGYTFTDHEMHWVKOTPVHGLEWIGTIHPGSGVTAYNOK

[0428] FKGKATLTADKSSSTAYMELSTLTSEDSAVYYCTPLWLRRDWGQGTTLTVSTGGGGSGGGGSG

[0429] GGGSALDIOMTOTPKFMSTSVGDRVSVTCKASQNVATNVVWFOQKSGOSPKALIYSASYRYSG

[0430] VPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNNYPLTFGAGTKLELKAAAGAPVPYPDPLEP

[0431] RGAASA

[0432] SEQ ID NO: 10 -

[0433] EVOLOQSGAELVRPGTSVKLSCKALGYTFTDHEMHWVKOTPVHGLEWIGTIHPGSGVTAYNOK

[0434] FKGKATLTADKSSSTAYMELSTLTSEDSAVYYCTPLWLRRDWGQGTTLTVSTGGGGSGGGGSG

[0435] GGGSALDIQMTQTPKFMSTSVGDRVSVTCKASONVATNVVWFQQKSGQSPKALIYSASYRYSG

[0436] VPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNNYPLTFGAGTKLELKAAAGAPVPYPDPLEP

[0437] RGAASAGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYIN

[0438] PSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTL

[0439] TVSSVEGGSGGSGGSGGSGGVDDIOLTOSPAIMSASPGEKVTMTCRASSSVSYMNWYOQKSGTS

[0440] PKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCOOWSSNPLTFGAGTKLELK

[0441] SEQ ID NO: 11 - EYTMH

[0442] SEQ ID NO: 12 - GIHPNNGDTSYNQRFKG

[0443] SEQ ID NO: 13 - WTGDFDY

[0444] SEQ ID NO: 14 - KASQNVGTNVA

[0445] SEQ ID NO: 15 - SASYRYS SEQ ID NO: 16 - QQYNSYPYT

[0446] SEQ ID NO: 17 -

[0447] OLOQSGPELVKPGASVKISCKTSGYTFTEYTMHWVKOSHGKSLEWIGGIHPNNGDTSYNORFKG

[0448] KATLTVDKSSSTAYMELRSLTSEDSAVYYCARWTGDFDYWGQGTTLTVST

[0449] SEQ ID NO: 18 -

[0450] DIVMTOSPKFMSTSVGDRVSVTCKASQNVGTNVAWYOOKPGOSPKALIYSASYRYSGVPDRFT

[0451] GSGSGTDFTLTISNVOSEDLAEYFCOOYNSYPYTFGGGTKLEIK

[0452] SEQ ID NO: 19 -

[0453] QLQQSGPELVKPGASVKISCKTSGYTFTEYTMHWVKQSHGKSLEWIGGIHPNNGDTSYNORFKG

[0454] KATLTVDKSSSTAYMELRSLTSEDSAVYYCARWTGDFPYWGQGTTLTVSTGGGGSGGGGSGG

[0455] GGSALDIVMTQSPKFMSTSVGDRVSVTCKASONVGTNVAWYQQKPGQSPKALIYSASYRYSGV

[0456] PDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPYTFGGGTKLEIKAAAGAPVPYPDPLEPRG

[0457] AASA

[0458] SEQ ID NO: 20 -

[0459] OLOQSGPELVKPGASVKISCKTSGYTFTEYTMHWVKOSHGKSLEWIGGIHPNNGDTSYNORFKG

[0460] KATLTVDKSSSTAYMELRSLTSEDSAVYYCARWTGDFDYWGQGTTLTVSTGGGGSGGGGSGG

[0461] GGSALDIVMTOSPKFMSTSVGDRVSVTCKASQNVGTNVAWYOQKPGOSPKALIYSASYRYSGV

[0462] PDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPYTFGGGTKLEIKAAAGAPVPYPDPLEPRG

[0463] AASAGGGGSDIKLOOSGAELARPGASVKMSCKTSGYTFTRYTMHWVKORPGOGLEWIGYINPS

[0464] RGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLT

[0465] VSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSP

[0466] KRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCOQWSSNPLTFGAGTKLELK

[0467] SEQ ID NO: 21 - RFTMS

[0468] SEQ ID NO: 22 - TISSGGSYTYYPDSVKG

[0469] SEQ ID NO: 23 - DRDFYGPYYAMDY

[0470] SEQ ID NO: 24 - KSSQSLLNSGNQKNYLA

[0471] SEQ ID NO: 25 - FASTRES

[0472] SEQ ID NO: 26 - QQHYTTPLT

[0473] SEQ ID NO: 27 -

[0474] DVNLVESGGGLVKPGGSLKLSCAASGFTFSRFTMSWVROTPEKTLDWVATISSGGSYTYYPDSV

[0475] KGRFTISRDNAKNTLYLOMSSLKSEDTAMYYCTRDRDFYGPYYAMDYWGOGTSVTVSS

[0476] SEQ ID NO: 28 -

[0477] DIVMTQSPSSLAISVGQKVTMSCKSSOSLLNSGNOKNYLAWYQOKPGQSPKLLVHFASTRESGV

[0478] PDRFIGSGSGTDFTLTISSVQAEDLADYFCOQHYTTPLTFGAGTKLELK SEQ ID NO: 29 -

[0479] DVNLVESGGGLVKPGGSLKLSCAASGFTFSRFTMSWVROTPEKTLDWVATISSGGSYTYYPDSV

[0480] KGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCTRDRDFYGPYYAMDYWGQGTSVTVSSGGGG

[0481] SGGGGSGGGGSDIVMTOSPSSLAISVGOKVTMSCKSSOSLLNSGNOKNYLAWYOOKPGOSPKLL

[0482] VHFASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCOQHYTTPLTFGAGTKLELK

[0483] SEQ ID NO: 30 -

[0484] DVNLVESGGGLVKPGGSLKLSCAASGFTFSRFTMSWVROTPEKTLDWVATISSGGSYTYYPDSV

[0485] KGRFTISRDNAKNTLYLOMSSLKSEDTAMYYCTRDRDFYGPYYAMDYWGOGTSVTVSSGGGG

[0486] SGGGGSGGGGSDIVMTOSPSSLAISVGOKVTMSCKSSOSLLNSGNOKNYLAWYOOKPGOSPKLL

[0487] VHFASTRESGVPDRFIGSGSGTDFTLTISSVOAEDLADYFCOOHYTTPLTFGAGTKLELKGGGGS

[0488] DIKLOQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKORPGOGLEWIGYINPSRGYTNYNOK

[0489] FKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGOGTTLTVSSVEGGSGG

[0490] SGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKV

[0491] ASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCOQWSSNPLTFGAGTKLELK

[0492] SEQ ID NO: 31 - SYWIH

[0493] SEQ ID NO: 32 - AVYPGNSDSNYNQKFKA

[0494] SEQ ID NO: 33 - LVGTFDY

[0495] SEQ ID NO: 34 - KASQNVGINVV

[0496] SEQ ID NO: 35 - SASYRYS

[0497] SEQ ID NO: 36 - QQYNTNPFT

[0498] SEQ ID NO: 37 -

[0499] EVOLOQSGTVLTRPGASVKMSCKASGYIFTSYWIHWVKORPGOGLEWIGAVYPGNSDSNYNQK

[0500] FKAKAKLTAVTSTSTAYMELSSLTSEDSAVYYCTKLVGTFDYWGOGTTLTVSS

[0501] SEQ ID NO: 38 -

[0502] DIVMTOSOKFMSSSIGDRVSVTCKASONVGINVVWYOORAGOSPKTLIYSASYRYSGVPDRFTG

[0503] SGSGTDFTLTISNVQSEDLAEYFCOQYNTNPFTFGSGTKLEIK

[0504] SEQ ID NO: 39 -

[0505] EVOLOQSGTVLTRPGASVKMSCKASGYIFTSYWIHWVKORPGOGLEWIGAVYPGNSDSNYNQK

[0506] FKAKAKLTAVTSTSTAYMELSSLTSEDSAVYYCTKLVGTFDYWGOGTTLTVSSGGGGSGGGGS

[0507] GGGGSDIVMTOSOKFMSSSIGDRVSVTCKASQNVGINVVWYOQRAGOSPKTLIYSASYRYSGVP

[0508] DRFTGSGSGTDFTLTISNVOSEDLAEYFCOOYNTNPFTFGSGTKLEIK

[0509] SEQ ID NO: 40 -

[0510] EVQLQQSGTVLTRPGASVKMSCKASGYIFTSYWIHWVKQRPGQGLEWIGAVYPGNSDSNYNOK

[0511] FKAKAKLTAVTSTSTAYMELSSLTSEDSAVYYCTKLVGTFDYWGOGTTLTVSSGGGGSGGGGS GGGGSDIVMTOSOKFMSSSIGDRVSVTCKASONVGINVVWYOQRAGOSPKTLIYSASYRYSGVP

[0512] DRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNTNPFTFGSGTKLEIKGGGGSDIKLQQSGAELAR

[0513] PGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSS

[0514] STAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGOGTTLTVSSVEGGSGGSGGSGGSGGVDDI

[0515] QLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGS

[0516] GTSYSLTISSMEAEDAATYYCOQWSSNPLTFGAGTKLELK

[0517] SEQ ID NO: 41 - RYTMH

[0518] SEQ ID NO: 42 - YINPSRGYTNYNQKFKD

[0519] SEQ ID NO: 43 - YYDDHYCLDY

[0520] SEQ ID NO: 44 - RASSSVSYMN

[0521] SEQ ID NO: 45 - DTSKVAS

[0522] SEQ ID NO: 46 - QQWSSNPLT

[0523] SEQ ID NO: 47 -

[0524] DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQK

[0525] FKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGOGTTLTVSS

[0526] SEQ ID NO: 48 -

[0527] DIOLTOSPAIMSASPGEKVTMTCRASSSVSYMNWYOQKSGTSPKRWIYDTSKVASGVPYRFSGS

[0528] GSGTSYSLTISSMEAEDAATYYCOQWSSNPLTFGAGTKLELK

[0529] SEQ ID NO: 49 -

[0530] DIKLOQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKORPGOGLEWIGYINPSRGYTNYNOK

[0531] FKDKATLTTDKSSSTAYMOLSSLTSEDSAVYYCARYYDDHYCLDYWGOGTTLTVSSVEGGSGG

[0532] SGGSGGSGGVDDIOLTOSPAIMSASPGEKVTMTCRASSSVSYMNWYOQKSGTSPKRWIYDTSKV

[0533] ASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCOOWSSNPLTFGAGTKLELK

[0534] SEQ ID NO: 50 - GGGGS

[0535] SEQ ID NO: 51 - GGGGTGGS

[0536] SEQ ID NO: 52 - SGGGG

[0537] SEQ ID NO: 53 - GGGGSGGGG

[0538] SEQ ID NO: 54 - GGSGG

[0539] SEQ ID NO: 55 -

[0540] GAGGTTCAATTGCAACAGAGCGGGGCGGAACTTGTACGCCCAGGTACCTCCGTTAAACTTAG

[0541] CTGCAAAGCACTTGGATATACGTTCACAGATCACGAGATGCACTGGGTCAAGCAAACGCCA

[0542] GTGCACGGTCTTGAATGGATCGGCACAATCCATCCCGGATCAGGAGTAACAGCGTACAATC

[0543] AGAAGTTTAAAGGGAAAGCCACCTTGACGGCGGATAAATCCAGTAGTACGGCATACATGGA

[0544] GTTGAGCACCTTGACCTCAGAAGATTCTGCGGTTTATTACTGTACCCCACTTTGGTTGCGCCG AGATTGGGGCCAGGGCACCACTTTGACGGTATCTACGGGCGGAGGCGGATCCGGAGGTGGC

[0545] GGAAGTGGTGGAGGCGGGTCAGCGCTCGATATTCAAATGACACAGACTCCCAAGTTCATGA

[0546] GCACATCAGTGGGTGATCGGGTGAGTGTGACTTGTAAAGCTTCACAAAATGTAGCCACTAAC

[0547] GTAGTCTGGTTCCAGCAAAAATCCGGACAGAGTCCTAAGGCCCTTATTTATTCTGCTAGCTA

[0548] TCGATACAGCGGGGTTCCTGATCGGTTCACCGGATCTGGATCCGGCACAGATTTTACGCTTA

[0549] CGATTTCAAATGTCCAGTCTGAAGACTTGGCTGAATATTTTTGTCAGCAATATAACAATTATC

[0550] CGCTCACCTTTGGTGCCGGGACGAAACTGGAATTGAAGGCAGCTGCTGGGGCCCCTGTACCG

[0551] TACCCTGATCCGCTTGAGCCTCGAGGGGCGGCAAGCGCCGGTGGCGGGGGGAGTGACATAA

[0552] AACTCCAGCAATCTGGGGCTGAACTTGCACGACCCGGGGCTAGCGTAAAAATGTCCTGTAA

[0553] AACATCAGGGTACACCTTCACCCGATATACCATGCATTGGGTGAAGCAACGGCCTGGACAG

[0554] GGGCTTGAGTGGATCGGTTATATAAACCCGTCACGGGGGTACACCAATTATAACCAGAAATT

[0555] CAAGGACAAAGCTACCTTGACCACGGACAAGTCAAGTTCTACGGCATACATGCAGCTCTCCA

[0556] GCTTGACTTCTGAGGACTCAGCCGTCTATTACTGTGCGCGATATTACGATGACCACTACTGTC

[0557] TGGATTATTGGGGACAGGGAACTACCTTGACAGTGTCCTCCGTCGAAGGTGGCAGTGGTGGC

[0558] TCTGGAGGCTCAGGCGGATCCGGCGGGGTGGATGACATTCAGCTTACGCAATCACCGGCTAT

[0559] TATGTCCGCGAGTCCAGGGGAGAAAGTAACGATGACCTGTCGGGCATCAAGCTCCGTAAGC

[0560] TACATGAACTGGTACCAACAGAAAAGTGGAACAAGTCCAAAAAGGTGGATCTATGATACGA

[0561] GCAAGGTAGCCTCAGGTGTACCATACAGGTTCTCCGGCTCCGGCTCAGGTACATCATATTCC

[0562] CTTACGATTTCTTCTATGGAGGCCGAAGACGCCGCAACGTACTACTGTCAGCAATGGAGTTC

[0563] AAACCCACTTACCTTTGGAGCTGGGACGAAACTGGAATTGAAG

[0564] SEQ ID NO: 56 -

[0565] CAGTTGCAGCAGTCAGGTCCAGAGCTTGTCAAACCCGGAGCTTCTGTAAAAATCTCATGTAA

[0566] AACATCTGGCTATACCTTCACTGAATATACGATGCATTGGGTTAAACAAAGCCACGGGAAGT

[0567] CTTTGGAGTGGATCGGAGGAATACATCCTAACAATGGGGATACGTCCTATAATCAACGGTTC

[0568] AAAGGAAAGGCTACCCTCACGGTAGACAAAAGCAGCTCAACCGCGTACATGGAACTTAGAT

[0569] CTCTGACCTCAGAAGACAGCGCAGTCTATTACTGTGCCAGATGGACAGGTGACTTCGATTAT

[0570] TGGGGGCAAGGCACTACGTTGACCGTGTCTACCGGGGGAGGGGGATCCGGTGGAGGTGGAT

[0571] CCGGAGGAGGAGGAAGTGCTCTGGACATTGTCATGACCCAGTCTCCTAAGTTTATGTCTACA

[0572] TCCGTCGGAGACAGAGTATCTGTCACCTGTAAAGCCTCTCAGAATGTGGGCACTAATGTCGC

[0573] ATGGTATCAACAAAAACCGGGCCAAAGTCCCAAGGCTCTTATCTACAGCGCTTCCTACCGAT

[0574] ATTCCGGAGTTCCCGATAGGTTTACAGGAAGTGGAAGTGGTACTGATTTCACATTGACGATT

[0575] TCTAACGTACAATCAGAAGACCTCGCGGAATACTTTTGCCAACAGTACAACTCTTATCCATA

[0576] CACTTTTGGAGGTGGAACCAAATTGGAAATAAAAGCCGCAGCCGGCGCACCCGTTCCGTAC

[0577] CCTGACCCTCTCGAACCACGAGGCGCTGCCAGTGCAGGTGGCGGGGGGAGTGACATAAAAC

[0578] TCCAGCAATCTGGGGCTGAACTTGCACGACCCGGGGCTAGCGTAAAAATGTCCTGTAAAAC ATCAGGGTACACCTTCACCCGATATACCATGCATTGGGTGAAGCAACGGCCTGGACAGGGG

[0579] CTTGAGTGGATCGGTTATATAAACCCGTCACGGGGGTACACCAATTATAACCAGAAATTCAA

[0580] GGACAAAGCTACCTTGACCACGGACAAGTCAAGTTCTACGGCATACATGCAGCTCTCCAGCT

[0581] TGACTTCTGAGGACTCAGCCGTCTATTACTGTGCGCGATATTACGATGACCACTACTGTCTGG

[0582] ATTATTGGGGACAGGGAACTACCTTGACAGTGTCCTCCGTCGAAGGTGGCAGTGGTGGCTCT

[0583] GGAGGCTCAGGCGGATCCGGCGGGGTGGATGACATTCAGCTTACGCAATCACCGGCTATTAT

[0584] GTCCGCGAGTCCAGGGGAGAAAGTAACGATGACCTGTCGGGCATCAAGCTCCGTAAGCTAC

[0585] ATGAACTGGTACCAACAGAAAAGTGGAACAAGTCCAAAAAGGTGGATCTATGATACGAGCA

[0586] AGGTAGCCTCAGGTGTACCATACAGGTTCTCCGGCTCCGGCTCAGGTACATCATATTCCCTT

[0587] ACGATTTCTTCTATGGAGGCCGAAGACGCCGCAACGTACTACTGTCAGCAATGGAGTTCAAA

[0588] CCCACTTACCTTTGGAGCTGGGACGAAACTGGAATTGAAG

[0589] SEQ ID NO: 57 -

[0590] GACGTTAATCTCGTTGAAAGTGGCGGTGGTCTTGTTAAACCCGGAGGTTCTCTTAAGCTTTCC

[0591] TGCGCAGCAAGCGGCTTTACCTTCTCAAGATTCACTATGAGTTGGGTTAGGCAGACCCCCGA

[0592] AAAAACACTCGACTGGGTAGCCACCATATCTTCAGGTGGCAGCTACACGTACTATCCGGATA

[0593] GTGTGAAAGGGCGATTCACCATTAGCAGGGATAATGCAAAGAATACACTCTATCTTCAAATG

[0594] AGCAGCTTGAAATCAGAGGATACGGCAATGTACTACTGCACCCGGGACCGCGACTTTTATGG

[0595] GCCCTACTATGCGATGGATTACTGGGGACAGGGGACCTCTGTAACCGTCAGCTCCGGTGGAG

[0596] GGGGATCCGGAGGGGGAGGAAGCGGTGGTGGGGGAAGCGATATAGTTATGACGCAGAGTC

[0597] CTAGCTCCCTTGCGATCAGTGTCGGACAGAAAGTGACTATGAGTTGTAAGAGCTCCCAATCC

[0598] CTCTTGAATTCAGGAAATCAAAAAAACTACCTCGCGTGGTACCAACAGAAGCCAGGCCAAA

[0599] GTCCAAAACTGTTGGTTCATTTTGCTTCAACCCGGGAATCTGGTGTCCCAGACCGGTTCATAG

[0600] GGAGCGGTTCCGGAACTGACTTCACTTTGACAATCAGCTCTGTACAAGCAGAGGATCTGGCG

[0601] GACTATTTCTGTCAGCAACACTACACTACACCCCTTACTTTCGGAGCGGGGACAAAGTTGGA

[0602] ATTGAAAGGTGGCGGGGGGAGTGACATAAAACTCCAGCAATCTGGGGCTGAACTTGCACGA

[0603] CCCGGGGCTAGCGTAAAAATGTCCTGTAAAACATCAGGGTACACCTTCACCCGATATACCAT

[0604] GCATTGGGTGAAGCAACGGCCTGGACAGGGGCTTGAGTGGATCGGTTATATAAACCCGTCA

[0605] CGGGGGTACACCAATTATAACCAGAAATTCAAGGACAAAGCTACCTTGACCACGGACAAGT

[0606] CAAGTTCTACGGCATACATGCAGCTCTCCAGCTTGACTTCTGAGGACTCAGCCGTCTATTACT

[0607] GTGCGCGATATTACGATGACCACTACTGTCTGGATTATTGGGGACAGGGAACTACCTTGACA

[0608] GTGTCCTCCGTCGAAGGTGGCAGTGGTGGCTCTGGAGGCTCAGGCGGATCCGGCGGGGTGG

[0609] ATGACATTCAGCTTACGCAATCACCGGCTATTATGTCCGCGAGTCCAGGGGAGAAAGTAACG

[0610] ATGACCTGTCGGGCATCAAGCTCCGTAAGCTACATGAACTGGTACCAACAGAAAAGTGGAA

[0611] CAAGTCCAAAAAGGTGGATCTATGATACGAGCAAGGTAGCCTCAGGTGTACCATACAGGTT

[0612] CTCCGGCTCCGGCTCAGGTACATCATATTCCCTTACGATTTCTTCTATGGAGGCCGAAGACGC CGCAACGTACTACTGTCAGCAATGGAGTTCAAACCCACTTACCTTTGGAGCTGGGACGAAAC

[0613] TGGAATTGAAG

[0614] SEQ ID NO: 58 -

[0615] GAGGTACAACTCCAGCAGTCTGGTACCGTTCTTACTAGGCCTGGAGCGAGTGTTAAGATGTC

[0616] ATGTAAAGCGTCTGGATATATTTTCACGAGCTACTGGATTCACTGGGTGAAGCAAAGACCAG

[0617] GTCAAGGTCTGGAGTGGATTGGGGCAGTTTATCCCGGAAACAGCGACTCAAATTACAACCA

[0618] AAAATTCAAAGCTAAGGCAAAACTTACGGCAGTAACATCTACGTCTACTGCGTACATGGAG

[0619] CTGTCTAGCCTCACAAGCGAGGACAGCGCTGTCTATTATTGTACCAAGCTCGTTGGTACCTTT

[0620] GATTATTGGGGGCAAGGCACAACTTTGACGGTTTCTAGCGGGGGCGGCGGATCCGGAGGGG

[0621] GAGGGTCTGGAGGCGGGGGAAGTGACATCGTCATGACACAATCCCAAAAGTTTATGAGTAG

[0622] TAGTATTGGGGATAGGGTTTCCGTAACGTGCAAGGCATCTCAGAACGTGGGGATTAACGTTG

[0623] TTTGGTACCAACAGAGAGCCGGACAGAGCCCAAAAACATTGATTTACAGCGCGAGTTACAG

[0624] GTACTCCGGCGTACCAGATCGGTTCACTGGAAGCGGATCTGGAACTGATTTTACCCTGACAA

[0625] TATCTAATGTCCAATCTGAAGATCTTGCCGAATATTTCTGCCAGCAGTACAATACGAACCCG

[0626] TTCACTTTTGGGTCCGGTACAAAACTGGAGATAAAAGGTGGCGGGGGGAGTGACATAAAAC

[0627] TCCAGCAATCTGGGGCTGAACTTGCACGACCCGGGGCTAGCGTAAAAATGTCCTGTAAAAC

[0628] ATCAGGGTACACCTTCACCCGATATACCATGCATTGGGTGAAGCAACGGCCTGGACAGGGG

[0629] CTTGAGTGGATCGGTTATATAAACCCGTCACGGGGGTACACCAATTATAACCAGAAATTCAA

[0630] GGACAAAGCTACCTTGACCACGGACAAGTCAAGTTCTACGGCATACATGCAGCTCTCCAGCT

[0631] TGACTTCTGAGGACTCAGCCGTCTATTACTGTGCGCGATATTACGATGACCACTACTGTCTGG

[0632] ATTATTGGGGACAGGGAACTACCTTGACAGTGTCCTCCGTCGAAGGTGGCAGTGGTGGCTCT

[0633] GGAGGCTCAGGCGGATCCGGCGGGGTGGATGACATTCAGCTTACGCAATCACCGGCTATTAT

[0634] GTCCGCGAGTCCAGGGGAGAAAGTAACGATGACCTGTCGGGCATCAAGCTCCGTAAGCTAC

[0635] ATGAACTGGTACCAACAGAAAAGTGGAACAAGTCCAAAAAGGTGGATCTATGATACGAGCA

[0636] AGGTAGCCTCAGGTGTACCATACAGGTTCTCCGGCTCCGGCTCAGGTACATCATATTCCCTT

[0637] ACGATTTCTTCTATGGAGGCCGAAGACGCCGCAACGTACTACTGTCAGCAATGGAGTTCAAA

[0638] CCCACTTACCTTTGGAGCTGGGACGAAACTGGAATTGAAG

[0639] SEQ ID NO: 59 - DYKDDDDK

[0640] SEQ ID NO: 60 - MGWSCIILFLVATATGVHS

[0641] SEQ ID NO: 61 - GACTACAAAGACGACGATGACAAG

[0642] SEQ ID NO: 62 -

[0643] ATGGGCTGGTCCTGCATAATACTCTTCCTTGTGGCCACCGCTACAGGGGTGCATAGT

Claims

Claims1. A bispecific agent, wherein the bispecific agent is a bispecific T cell engager (BiTE) comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first anti gen -binding domain specifically binds to CD 155 and the second antigen-binding domain specifically binds to CD3.

2. The bispecific agent according to claim 1, wherein the first antigen-binding domain is an scFv and the second antigen-binding domain is an scFv.

3. The bispecific agent according to claim 1 or 2, wherein:(a) the first antigen-binding domain comprises: heavy chain complementarity determining region heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 1 to 6, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto, optionally wherein the first antigen-binding domain comprises heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 7 and 8, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto, further optionally wherein the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto; or heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 11 to 16, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto, optionally wherein the first antigen-binding domain comprises heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 17 and 18, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identitythereto, further optionally wherein the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto; and(b) the second antigen-binding domain comprises: heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 41 to 46, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto, optionally wherein the second antigen-binding domain comprises heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 47 and 48, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto, further optionally wherein the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto.

4. The bispecific agent according to any one of claims 1 to 3, wherein the first antigenbinding domain and the second antigen-binding domain are scFvs and are linked via a linker sequence, optionally wherein the linker sequence comprises the amino acid sequence of SEQ ID NO: 50, or an amino acid sequence with at least 80% identity thereto, optionally wherein the BiTE comprises the amino acid sequence of SEQ ID NO: 10 or 20.

5. A bispecific agent comprising a first antigen-binding domain and a second antigenbinding domain, wherein the first antigen-binding domain specifically binds to CD112 and the second antigen-binding domain specifically binds to CD3.

6. The bispecific agent according to claim 5, wherein:(a) the bispecific agent is a bispecific antibody or a trifunctional antibody, optionally wherein the bispecific antibody is a bispecific T cell engager (BiTE); and / or(b) the first antigen-binding domain is a first single-chain variable fragment (scFv) and the second antigen-binding domain is a second scFv.The bispecific agent according to claim 5 or 6, wherein:(a) the first antigen-binding domain comprises: heavy chain complementarity determining region heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 21 to 26, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto, optionally wherein the first antigen-binding domain comprises heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 27 and 28, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto, further optionally wherein the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto; or heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 31 to 36, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto, optionally wherein the first antigen-binding domain comprises heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 37 and 38, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto, further optionally wherein the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 39, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto; and(b) the second antigen-binding domain comprises: heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 41 to 46, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto, optionally wherein the second antigen-binding domain comprises heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 47 and 48, respectively, oran amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto, further optionally wherein the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto.

8. The bispecific agent according to any one of claims 5 to 7, wherein the first antigenbinding domain and the second antigen-binding domain are scFvs and linked via a linker sequence, optionally wherein the linker sequence comprises the amino acid sequence of SEQ ID NO: 50, or an amino acid sequence with at least 80% identity thereto, optionally wherein the BiTE comprises the amino acid sequence of SEQ ID NO: 30 or 40.

9. A nucleic acid encoding the bispecific agent according to any one of claims 1 to 8.

10. The nucleic acid according to claim 9, wherein:(a) the nucleic acid is DNA or RNA; and / or(b) the nucleic acid comprises the nucleic acid sequence of any one of SEQ ID NOs: 55 to 58.

11. A vector comprising one or more nucleic acids according to claim 9 or 10.

12. The vector according to claim 11, wherein the vector is a viral vector, an RNA vector, liposomal vector or lipid nanoparticle vector, optionally wherein the viral vector is a lentiviral vector, adenoviral vector, retroviral vector, adeno-associated virus vector, herpes virus vector, Maraba virus vector, vesicular stomatitis virus vector or vaccinia virus vector.

13. A host cell comprising one or more vectors according to claim 11 or 12.

14. The host cell according to claim 13, wherein the cell is a mammalian cell, an insect cell or a bacterial cell, optionally wherein the mammalian cell is an HEK293 cell or CHO cell, the insect cell is an Sf9 cell or Sf21 cell, and the bacterial cell is an E.coli cell.

15. A TIGIT ligand-binding agent for use in a method of treating cancer, wherein the cancer comprises an immunosuppressive tumour microenvironment comprising cancer cells and one or more types of non-cancerous cells.

16. The TIGIT ligand-binding agent for use according to claim 15, wherein:(a) the one or more types of non-cancerous cells comprise cancer-associated fibroblasts, mesenchymal stem cells, pericytes, tumour endothelial cells, macrophages, myeloid- derived suppressor cells, regulatory T cells and / or neutrophils, optionally wherein the one or more types of non-cancerous cells comprise cancer-associated fibroblasts;(b) the cancer cells and / or the one or more types of non-cancerous cells express CD155 and / or CD112; and / or(c) the cancer is: a stage 1 cancer, stage 2 cancer, stage 3 cancer or stage 4 cancer, optionally wherein the cancer is a stage 4 cancer; and / or metastatic cancer, pancreatic cancer, oesophageal cancer, colorectal cancer, mesothelioma, prostate cancer, breast cancer, head and neck cancer, bladder cancer, peritoneal cancer, lung cancer, gastric cancer, liver cancer, cervical cancer, thyroid cancer, kidney cancer, ovarian cancer, gallbladder and biliary cancer, lymphoma, leukaemia, myeloma, brain and CNS cancer, melanoma or sarcoma.

17. The TIGIT ligand-binding agent for use according to claim 15 or 16, wherein:(a) the TIGIT ligand-binding agent depletes: the cancer cells and / or the one or more types of non-cancerous cells; and / or the immunosuppressive tumour microenvironment; and / or(b) the TIGIT ligand-binding agent is a small molecule, a peptide, a peptidomimetic, a protein, an antibody, or antigen-binding fragment thereof, a CAR T cell, a CAR NK cell, or a CAR macrophage, optionally wherein the antibody is a monospecific antibody or multi-specific antibody, further optionally wherein the multi-specific antibody is a bispecific antibody or trispecific antibody, further optionally wherein the bispecific antibody is a bispecific T-cell engager (BiTE) or a trifunctional antibody.

18. The TIGIT ligand-binding agent for use according to claim 17, which comprises a first anti gen -binding domain and a second antigen-binding domain, optionally wherein the TIGIT ligand-binding agent is a BiTE comprising a first antigen-binding domain and a second antigenbinding domain, further optionally wherein the first antigen-binding domain is a single-chain variable fragment (scFv) and the second antigen-binding domain is an scFv.

19. The TIGIT ligand-binding agent for use according to claim 18, wherein the first antigenbinding domain specifically binds to CD155 and the second antigen-binding domain specifically binds to CD3, optionally wherein the first antigen-binding domain comprises:(a) heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 1 to 6, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto, optionally wherein the first antigen-binding domain comprises heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 7 and 8, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto, further optionally wherein the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto; or(b) heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 11 to 16, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto, optionally wherein the first antigen-binding domain comprises heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 17 and 18, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto, further optionally wherein the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto.

20. The TIGIT ligand-binding agent for use according to claim 18 or 19, wherein the second antigen-binding domain comprises heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 41 to 46, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto, optionally wherein the second antigen-binding domain comprises heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 47 and 48, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto, further optionally wherein the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto.

21. The TIGIT ligand-binding agent for use according to any one of claims 18 to 20, which is a BiTE comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain and the second antigen-binding domain are scFvs and are linked via a linker sequence, optionally wherein the linker sequence comprises the amino acid sequence of SEQ ID NO: 50, or an amino acid sequence with at least 80% identity thereto, further optionally wherein the BiTE comprises the amino acid sequence of SEQ ID NO: 10 or 20.

22. The TIGIT ligand-binding agent for use according to claim 18, wherein the first antigenbinding domain specifically binds to CD112 and the second antigen-binding domain specifically binds to CD3, optionally wherein the first antigen-binding domain comprises:(a) heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 21 to 26, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto, optionally wherein the first antigen-binding domain comprises heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 27 and 28, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto, further optionally wherein the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto; or(b) heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 31 to 36, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto, optionally wherein the first antigen-binding domain comprises heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 37 and 38, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto, further optionally wherein the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 39, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto.

23. The TIGIT ligand-binding agent for use according to claim 22, wherein the second antigen-binding domain comprises heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3 and light chain complementarity determining region LCDR1, LCDR2 and LCDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 41 to 46, respectively, or amino acid sequences with at least 80%, 85%, 90%, 95% or 99% identity thereto, optionally wherein the second antigen-binding domain comprises heavy chain variable region and light chain variable region sequences comprising the amino acid sequences of SEQ ID NOs: 47 and 48, respectively, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto, further optionally wherein the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto.

24. The TIGIT ligand-binding agent for use according to any one of claims 18, 22 and 23, which is a BiTE comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain and the second antigen-binding domain are scFvs and are linked via a linker sequence, optionally wherein the linker sequence comprises the amino acid sequence of SEQ ID NO: 50, or an amino acid sequence with at least 80% identity thereto, further optionally wherein the BiTE comprises the amino acid sequence of SEQ ID NO: 30 or 40.

25. A method of predicting whether or not a subject having cancer will respond to treatment with a TIGIT ligand-binding agent, wherein the method comprises determining whether or not the cancer comprises an immunosuppressive tumour microenvironment comprising cancer cells and one or more types of non-cancerous cells, and thereby predicting whether or not the subject will respond to the treatment, optionally wherein the TIGIT ligand-binding agent is as defined in any one of claims 17 to 24.

26. The method according to claim 25, wherein determining whether or not the cancer comprises an immunosuppressive tumour microenvironment comprises:(a) measuring the expression level of a biomarker in the tumour microenvironment; and(b) comparing the expression level with a reference level, optionally wherein the one or more types of non-cancerous cells comprise cancer- associated fibroblasts, mesenchymal stem cells, pericytes, tumour endothelial cells, macrophages, myeloid-derived suppressor cells, regulatory T cells and / or neutrophils, further optionally wherein the biomarker comprises CD155, CD112, fibroblast activation protein- a (FAP), a-smooth muscle actin (aSMA), CD163, CD206, folate receptor 0 (FR0), CD33, CD31, VEGFR1, VEGFR2, endoglin and / or endosialin.

27. The method according to claim 25 or 26, wherein the method further comprises administering the TIGIT ligand-binding agent to a subject predicted to respond to the treatment.

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