Invariant natural killer t cell

Abstract

The invention relates to invariant natural killer T (iNKT) cells, and particularly, although not exclusively, to iNKT cell engagers or fusion proteins, which specifically bind to iNKT cells. The invention extends to nucleic acids encoding such iNKT cell engagers, as well as novel pharmaceutical compositions comprising these engagers, and to their use in therapy, as well as therapies and methods for treating, preventing, or ameliorating cancer.

Description

[0001] Invariant Natural Killer T cell

[0002] The present invention relates to invariant natural killer T (iNKT) cells, and particularly, although not exclusively, to iNKT cell engagers or fusion proteins, which specifically bind to iNKT cells. The invention extends to nucleic acids encoding such iNKT cell engagers, as well as novel pharmaceutical compositions comprising these engagers, and to their use in therapy, as well as therapies and methods for treating, preventing, or ameliorating cancer.

[0003] Natural killer T (NKT) cells are a heterogeneous group of T cells that share properties of both T cells and natural killer cells. NKT cells are classified into two groups based on differences in T cell receptor (TCR) usage. Type I NKT cells or invariant NKT cells have an invariant TCRa-chain and are readily detectable by a-galactosylceramide-loaded CDld tetramer, whereas Type II NKT cells have a more diverse TCR repertoire, and their direct identification is more difficult. Hence, INKT cells are a rare subset of T cells characterised by an invariant TCRVa24Ja l8 nearly always pairing with a TCRVpi l chain1, and are restricted by the non-polymorphic, glycolipid-presenting, MHC-like molecule CDld2. Functionally, INKT cells have features of both innate and adaptive immunity and possess effector, as well as immunoregulatory activity. Their notable direct and indirect antitumour activity is mediated by CDld-dependent and CDld-independent mechanisms, including direct killing of tumour cells, antigen presenting cell (APC) maturation and activation of NK cells and tumour-specific T3'4. iNKT cells comprise CD4+ and CD4- subsets, with the former displaying a ThO / 2 phenotype while the latter display Th l polarisation, with higher expression of IFNy and cytolytic granules5and enhanced anti-tumour activity, with CD4-CD8+ iNKT cells having the highest cytotoxic potential. Importantly, in pre-clinical models, INKT cells deplete immunosuppressive, CDld-expressing tumour-associated myeloid cells5'7while extensive data, including from the inventors' lab, show that allogeneic iNKT cells do not cause acute graft-versus-host disease (aGVHD)8-9, and hence their development as an 'off-the- shelf' immunotherapy platform without need for deletion of the endogenous TCR.

[0004] The inventors have previously shown that anti-CD19 chimeric antigen receptor (CAR) iNKT outperform CD19 CAR-T, achieving improved survival and tumour-free survival in experimental non-Hodgkin B cell lymphoma (NHL)10, and anti-TCRVP chain CAR-iNKT are effective against pre-clinical T-NHL11. An ongoing early clinical trial of CD19 CAR-iNKT has reported 60% complete response in relapsed / refractory B-NHL. Remarkably, there was no evidence of aGVHD or CAR-related cytokine release syndrome and neurotoxicity12. Immunotherapy of cancer often requires targeting more than one tumour antigen, whereas cellular immunotherapy is best served by using off-the-shelf treatments. The inventors' cellular immunotherapy platform is being developed as an off-the-shelf option that can be enhanced by equipping iNKT cells with anti-cancer modules, such as CARs and TCRs. However, a problem they face is that genetic engineering has capacity limitations and does not allow the expression of more than two or three therapeutic modules, e.g., CARs or TCRs.

[0005] There is, therefore, a need to provide improved cellular immunotherapy options which overcome this problem.

[0006] In order to address the above problem, the inventors have investigated enhancing the modularity of iNKT cells, and thus their anti-tumour activity, by developing iNKT cellspecific engagers that bring iNKT cells to the close proximity of tumour cells, thereby resulting in their killing. As an exemplar of the platform technology, the inventors developed a bispecific iNKT cell engager (known as "biNTe"), in which one arm engages the unique invariant TCR of iNKT cells, while the other arm engages one or more tumour antigen(s) of interest, such as the multiple myeloma antigen, BCMA. They then surprisingly demonstrated the feasibility, specificity and activity of these iNKT-specific engagers against multiple myeloma as a proof-of-concept for cancer in general, in which the anti-tumour activity of iNKT cells is enhanced by iNKT cell-specific engagers that bring iNKT cells to the proximity of tumour cells, resulting in their killing.

[0007] This approach provides a novel, off-the-self, and highly flexible therapeutic module, which, in conjunction with adoptively transferred CAR- or TCR-INKT cells, can be adapted to target additional cancer antigens.

[0008] Thus, according to a first aspect of the invention, there is provided an invariant natural killer T (INKT) cell specific engager.

[0009] Advantageously, while existing bispecific engagers bind conventional T cells and NK cells, the engager for INKT cells according to the invention will enhance the already inherent anti-cancer properties of iNKT and CAR-iNKT cells. Although T and NK cell engagers rely on patients' own cells in vivo, the INKT cell engager according to the invention can be delivered in conjunction with allogeneically sourced and adoptively transferred INKT cells, i.e., both biNTe and INKT will be off-the-shelf procured. Advantageously, and preferably, therefore, the INKT cell specific engager may be delivered together with the transfer of iNKT cells without the latter necessarily requiring genetic engineering with all of the logistical, cost and biological risks associated with it. iNKT cells are a subset of immunoregulatory and effector T cells representing less than 0.1 % of the total T cell numbers in humans. There are several structural and functional differences between INKT cells and conventional T cells. In particular, INKT cells express an invariant Va24Jal8 chain, which is almost always paired with the same TCRVgll diverse chain. Furthermore, iNKT cells are also restricted by the non-polymorphic HLA class I-like molecule CDld presenting endogenous or exogenous, glyco- or phospho-lipid ligands to ITCR. In contrast to conventional T-cells, which are restricted by highly polymorphic MHC presenting peptides, INKT cells require the expression of CDld on thymocytes in order to be selected and activated. Conversely, conventional T-cells require the expression of MHC molecules on epithelial thymic epithelial cells for their selection and activation.

[0010] INKT cells provide effective immune responses against infectious agents, tumour, allo- and auto-reactivity and atheromatosis. Several pre-clinical studies have demonstrated the ability of adoptively transferred donor iNKT cells to prevent or even abrogate established experimental acute graft-versus-host disease (aGVHD), an alloreactive phenomenon that occurs in the context of allogeneic haemopoietic stem cell transplantation. aGVHD is driven primarily by donor alloreactive T cells activated in response to major or minor histocompatibility antigen disparities between donor and recipient. In line with the pre-clinical evidence, several clinical observational studies have demonstrated that a higher dose or frequency of donor iNKT cells transferred to the recipient with the peripheral blood stem cell graft impart significant protection from aGVHD, without compromising the graft-versus-tumour effect.

[0011] In humans, iNKT cells are quantitatively and qualitatively altered in different types of tumours, including blood cancers such as multiple myeloma, while tumour bed infiltration by iNKT cells appears to confer favourable prognosis in colorectal cancer. Much of the anti-tumour effect of iNKT cells depends on their ability to be cytolytic directly, through perforin / granzymes and other cell death pathways against tumours that express CDld, or indirectly, through their secretion of copious amounts of (interferongamma) IFNy, and / or secondary activation of NK cells or conventional T cell-dependent anti-tumour responses.

[0012] Preferably, the iNKT cell specific engager is configured to specifically bind to at least one iNKT cell, preferably at least one antigen on the at least one iNKT cell. Preferably, the iNKT cell specific engager is configured to specifically bind to at least one tumour antigen on at least one tumour cell.

[0013] Preferably, the INKT cell specific engager comprises a fusion protein comprising a first antigen-binding domain that specifically binds to an antigen on the at least one iNKT cell, and a second antigen-binding domain that specifically binds to a tumour antigen on the at least one tumour cell. Preferably, the first antigen-binding domain specifically binds to one or more epitope on the antigen on the at least one iNKT cell. Preferably, the second antigen-binding domain specifically binds to one or more epitope on the tumour antigen on the at least one tumour cell.

[0014] Preferably, the iNKT cell specific engager (and preferably the fusion protein) specifically binds to an invariant T cell receptor (iTCR), or a component thereof, on the at least one iNKT cell.

[0015] The iNKT cell specific engager may specifically bind to a TCRVpil, TCRVa24, and / or the TCRVa24Jal8 CDR3 of the iTCR on the at least one iNKT cell.

[0016] Advantageously, as the iNKT cell specific engager binds to TCRVpi l and / or TCRVa24, it is able to bind to both iNKT subsets and also conventional T cells. Such combination binding means that the engager exhibits improved anti-tumour activity, because it allows tumour-targeting by two different T cell types, i.e. iNKT cells and conventional T cells, which enhances the overall anti-tumour effects.

[0017] In an embodiment, the iNKT cell specific engager specifically binds to a TCRVpil on the at least one iNKT cell. In one embodiment, the amino acid sequence of TCRVpil (e.g. TRBV11-1; TRBVll-l*01) may be represented herein as SEQ ID No: 1, as follows:

[0018] MSTRLLCWMALCLLGAELSEAEVAQSPRYKITEKSQAVAFWCDPISGHATLYWYRQILGQGPELLVQFQDESVVD D S Q L P KD R F S AE RL KGVD S T L KI Q PAE L G D SAMY L CAS S L

[0019] [SEQ ID No: 1]

[0020] In one embodiment, TCRVpil is encoded by the nucleotide sequence represented herein as SEQ ID No:2, as follows:

[0021] ATGAAAAAGCATCTGACGACCTTCTTGGTGATTTTGTGGCTTTATTTTTATAGGGGGAATGGCAAAAACCAAGTG

[0022] GAGCAGAGTCCTCAGTCCCTGATCATCCTGGAGGGAAAGAACTGCACTCTTCAATGCAATTATACAGTGAGCCCC

[0023] TTCAGCAACTTAAGGTGGTATAAGCAAGATACGGGGAGAGGTCCTGTTTCCCTGACAATCATGACTTTCAGTGAG AACACAAAGTCGAACGGAAGATATACAGCAACTCTGGATGCAGACACAAAGCAAAGCTCTCTGCACATCACAGCC

[0024] TCCCAGCTCAGCGATTCAGCCTCCTACATCTGTGTGGTGAGCG

[0025] [SEQ ID No: 2]

[0026] In an embodiment, the iNKT cell specific engager specifically binds to a TCRVa24 on the at least one iNKT cell. In one embodiment, the amino acid sequence of TCRVa24 may be represented herein as SEQ ID No: 3, as follows:

[0027] MKKHLTTFLVILWLYFYRGNGKNQVEQSPQSLIILEGKNCTLQCNYTVSPASNLRWYKQDTGRGPVSLTIMTFSE NTKSNGRYTATLDADTKQSSLHITASQLSDSASYICWS

[0028] [SEQ ID No: 3]

[0029] In one embodiment, TCRVa24 is encoded by the nucleotide sequence represented herein as SEQ ID No:4, as follows:

[0030] ATGAAAAAGCATCTGACGACCTTCTTGGTGATTTTGTGGCTTTATTTTTATAGGGGGAATGGCAAAAACCAAGTG GAGCAGAGTCCTCAGTCCCTGATCATCCTGGAGGGAAAGAACTGCACTCTTCAATGCAATTATACAGTGAGCCCC TTCAGCAACTTAAGGTGGTATAAGCAAGATACGGGGAGAGGTCCTGTTTCCCTGACAATCATGACTTTCAGTGAG AACACAAAGTCGAACGGAAGATATACAGCAACTCTGGATGCAGACACAAAGCAAAGCTCTCTGCACATCACAGCC TCCCAGCTCAGCGATTCAGCCTCCTACATCTGTGTGGTGAGCG

[0031] [SEQ ID No: 4]

[0032] In an embodiment, the iNKT cell specific engager specifically binds to a TCRVa24Jal8 CDR3 on the at least one iNKT cell. In one embodiment, the amino acid sequence of TCRVa24Jal8 CDR3 may be represented herein as SEQ ID No: 5, as follows:

[0033] GNGKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTIMTFSENTKSNGRYTATLDADTKQ SSLHITASQLSDSASYI CWS DRGSTLGRLYF

[0034] [SEQ ID No: 5]

[0035] It will be appreciated that although the amino acid sequence of TCRVa24Jal8 CDR3 is invariant, the CDR3 nucleotide sequence is very variable.

[0036] Therefore, preferably the iNKT cell specific engager specifically binds to an antigen on the at least one iNKT cell, wherein the antigen comprises an amino acid sequence substantially as set out in any of SEQ ID No: 1, 3 and / or 5, or a fragment or variant thereof, and / or wherein the antigen is encoded by a nucleotide sequence substantially as set out in any of SEQ ID No: 2 and / or 4, or a fragment or variant thereof. Preferably, however, the iNKT cell specific engager specifically binds to the TCRVpil of the at least one INKT cell. Accordingly, preferably the INKT cell specific engager specifically binds to an antigen on the at least one INKT cell, wherein the antigen comprises an amino acid sequence substantially as set out in SEQ ID No: 1, or a fragment or variant thereof, and / or wherein the antigen is encoded by a nucleotide sequence substantially as set out in SEQ ID No: 2, or a fragment or variant thereof.

[0037] Advantageously, in contrast to the TCRVa24Ja 18-based engagers, the TCRVbll- and TCRVa24-based engagers, as well as iNKT, can also engage the TCRVbll+ and TCRVa24+ (each <5% of all) conventional T cells, thus allowing tumour targeting by two different T cell types. This would enhance the overall anti-tumour effect. Furthermore, by not engaging the TCRVa24Jal8 part of the ITCR , TCRVbll- and TCRVa24-based engagers allow the ITCR to engage with CDld expressed on tumour cells, an interaction that enhances the anti-tumour effect and can be further enhanced in the presence of INKT glycolipid agonists presented by CDld. By contrast, a bispecific engager that binds the TCRVa24Jal8 part of the iTCR would block the iTCR-CDld interaction.

[0038] As such, in some embodiments, and advantageously, the iNKT cell specific engager is configured to allow iTCRs to engage with CDld, which allows iNKT cell activation. In addition, in some embodiments, this interaction may be enhanced by using activating, CDld-presented glycolipids, such as a-galactosylceramide.

[0039] The tumour antigen may comprise a polypeptide tumour antigen or a glycoprotein tumour antigen displayed on the surface of the at least one tumour cell. The tumour antigen may be (a) a full length molecule associated with cancer cells, (b) a homologue or modified form of the same, including a molecule with deleted, added and / or substituted portions, or (c) a fragment of the same.

[0040] The tumour antigen may be selected from a list of surface antigens expressed in tumours such as: a testis cancer, melanoma, lung cancer, head and neck cancer, lung cancer, breast cancer, gastrointestinal cancer, bladder cancer, colorectal cancer, pancreatic cancer, lymphoma, leukaemia, multiple myeloma, renal cancer, hepatic cancer, ovarian cancer, gastric cancer, brain cancer and prostate cancer.

[0041] Such tumour antigens include: BCMA, CS1, CD38, FCRL5, CD19, CD20, CD22, CD30, CD123, CLL1, CD33, FTL3, CD133, CLAUDIN 18.2, NKG2D ligands, TCRVbeta / alpha variable and constant chains, CD5, CD7, B7-H3, EGFR, HER2, EGFR806, Mesothelin, PSCA, MUC1, EpCAM, GD2, VEGFR2, AFP, Nectin4 / FA, PCEA, Lewis Y, Glypican-3, EGFRIII, IL-13Ro2, CD171, MUC16, PSMA, AFP, AXL, c-MET, DLL-3, DR5, EpHA2, FRa gplOO, MAGE-A1 / 3 / 4, LMP1 and others.

[0042] Suitable tumour antigens may also include intracellular proteins which are presented as class I-restricted antigens recognized by CD 8+ lymphocytes or class Il-restricted antigens recognized by CD4+ lymphocytes. In this case, the tumour-targeting domain or arm of the iNKT engager would be derived from an antibody that would specifically bind to the MHC class I / II-peptide complex.

[0043] Such MHC-resented peptides may be derived from:

[0044] (a) cancer-testis antigens, such as NY-ESO-I, SSX2, SCP-1, as well as RAGE, BAGE, GAGE and MAGE family polypeptides, for example, GAGE-I, GAGE-2, MAGE-I, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12 (which can be used, for example, to address melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, and bladder tumours);

[0045] (b) mutated antigens, for example, p53 (associated with various solid tumours, e.g., colorectal, lung, head and neck cancer), p21 / Ras (associated with, e.g., melanoma, pancreatic cancer and colorectal cancer), CDK4 (associated with, e.g., melanoma), MUM-1 (associated with, e.g., melanoma), caspase-8 (associated with, e.g., head and neck cancer), CIA 0205 (associated with, e.g., bladder cancer), HLA-A2-R1701, beta catenin (associated with, e.g., melanoma), TCR (associated with, e.g., T- cell non-Hodgkins lymphoma), BCR-abl (associated with, e.g., chronic myelogenous leukaemia), triosephosphate isomerase, KIA 0205, CDC-27, and LDLR-FUT;

[0046] (c) over-expressed antigens, for example, Galectin 4 (associated with, e.g., colorectal cancer), Galectin 9 (associated with, e.g., Hodgkin's disease), proteinase 3 (associated with, e.g., chronic myelogenous leukaemia), WT 1 (associated with, e.g., various leukaemias), carbonic anhydrase (associated with, e.g., renal cancer), aldolase A (associated with, e.g., lung cancer), PRAME (associated with, e.g., melanoma), HER-2 / neu (associated with, e.g., breast, colon, lung and ovarian cancer), alpha-fetoprotein (associated with, e.g., hepatoma), KSA (associated with, e.g., colorectal cancer), gastrin (associated with, e.g., pancreatic and gastric cancer), telomerase catalytic protein, MUC-I (associated with, e.g., breast and ovarian cancer), G-250 (associated with, e.g., renal cell carcinoma), p53 (associated with, e.g., breast, colon cancer), and carcinoembryonic antigen (associated with, e.g., breast cancer, lung cancer, and cancers of the gastrointestinal tract such as colorectal cancer); (d) shared antigens, for example, melanoma-melanocyte differentiation antigens, such as MART-l / Melan A, gplOO, MCIR, melanocyte-stimulating hormone receptor, tyrosinase, tyrosinase related protein- 1 / TRPI and tyrosinase related protein-2 / TRP2 (associated with, e.g., melanoma);

[0047] (e) prostate-associated antigens, such as PAP, PSA, PSMA, PSH-PI, PSM-PI, PSM-P2, associated with e.g., prostate cancer; and / or

[0048] (f) immunoglobulin idiotypes (associated with myeloma and B cell lymphomas.

[0049] Preferably, however, the tumour antigen is BCMA. As described in the Examples, BCMA was used as the antigen for proof-of-concept.

[0050] Preferably, the iNKT cell specific engager (and preferably the fusion protein) is polyspecific. The term "polyspecific" can mean that the engager is configured to specifically bind to one or more antigen on the iNKT cell, and one or more tumour antigen on the tumour cell. For example, the iNKT cell specific engager may be bispecific, trispecific, or tetraspecific, and so on, which are described in more detail below.

[0051] Preferably, the iNKT cell specific engager is bispecific. The term "bispecific" can mean that the engager is configured to specifically bind to one antigen on the iNKT cell and one tumour antigen on the tumour cell. Preferably, the iNKT cell specific engager comprises a fusion protein comprising a first antigen-binding domain that specifically binds to an antigen on the at least one iNKT cell, and a second antigen-binding domain that specifically binds to a tumour antigen on the at least one tumour cell.

[0052] Preferably, the iNKT cell specific engager is trispecific. The term "trispecific" can mean that the engager is configured to specifically bind to two different antigens on the iNKT cell and one tumour antigen on the tumour cell, or one antigen on the iNKT cell and two different tumour antigens on the tumour cell. Preferably, in this embodiment, the iNKT cell specific engager comprises a fusion protein comprising a first antigen-binding domain that specifically binds to a first antigen on the at least one iNKT cell, a second antigen-binding domain that specifically binds to a tumour antigen on the at least one tumour cell, and a third antigen-binding domain that specifically binds to a second, different antigen on the at least one iNKT cell. In one embodiment, the iNKT cell antigen may be an iTCR, which may be TCRVgll, TCRVa24 and / or the TCRVa24Jal8 CDR3. In another embodiment, the iNKT cell antigen may be a co-stimulatory or activating molecule on the iNKT cell. For example, a co-stimulatory molecule may include CD28, 4- 1BB, ICOS or 0X40. For example, an activating molecule may include NKG2D or CD161.

[0053] Preferably, the INKT cell specific engager is tetraspecific. The term "tetraspecific" can mean that the engager is configured to specifically bind to two different antigens on the iNKT cell and two different tumour antigens on the tumour cell. Preferably, in this embodiment, the iNKT cell specific engager comprises a fusion protein comprising a first antigen-binding domain that specifically binds to a first antigen on the at least one iNKT cell, a second antigen-binding domain that specifically binds to a first tumour antigen on the at least one tumour cell, a third antigen-binding domain that specifically binds to a second, different antigen on the at least one iNKT cell, and a fourth antigen-binding domain that binds to a second, different antigen on the at least one tumour cell.

[0054] Thus, advantageously, in view of the above, it will be appreciated that the polyspecific nature of the iNKT cell specific engager or fusion protein means that it is configured to specifically bind to at least one, two or three different species of antigen on the iNKT cell, and simultaneously at least one, two or three different species of tumour antigen on the tumour cell. Accordingly, the fusion protein acts like a molecular linker or "glue", which simultaneously brings together the iNKT cell and tumour cell, such that the iNKT cell can impart its therapeutic properties on the tumour cell, thereby killing it.

[0055] Preferably, the iNKT cell specific engager is bi-specific and monovalent. This embodiment is shown in Figure 1A. The term "monovalent" bi-specific can mean that the iNKT cell specific engager is configured such that one arm can bind to a single antigen on the iNKT cell and the other arm to a single tumour antigen on the tumour cell.

[0056] Preferably, the iNKT cell specific engager is bi-specific and bivalent. This embodiment is shown in Figure IB. The term "bivalent" bi-specific can mean that the iNKT cell specific engager is configured such that one arm can bind to two antigens on the iNKT cell (same or two different antigens) and the other arm to two tumour antigens (same or two different antigen) on the tumour cell. Advantageously, the engager of the invention comprises a tumour-engaging arm which can bind to any tumour antigen, and not solely to CDld.

[0057] Preferably, the iNKT cell specific engager is trivalent. The term "trivalent" can mean that the iNKT cell specific engager is configured to bind to three of the same antigens on the iNKT cell and / or three of the same tumour-associated antigens on the tumour cell. The iNKT cell specific engager comprises a fusion protein comprising at least two antigen-binding domains, wherein the first antigen-binding domain specifically binds to an antigen on the at least one iNKT cell, and the second antigen-binding domain specifically binds to a tumour antigen on the at least one tumour cell. The fusion protein may comprise further antigen-binding domains binding to the iNKT cell and / or tumour cell depending on whether the engager is trispecific, or tetraspecific, and so on.

[0058] However, in each case, the antigen-binding domain preferably comprises a hypervariable CDR, or a functional portion thereof. The term "functional portion" of a CDR can mean a sequence within the CDR which shows specific affinity for the target antigen, which may be on either the iNKT cell (preferably the iTCR) or the tumour cell.

[0059] The term "CDR" can mean a hypervariable region in the heavy and light variable chains of an antibody, for example, the TCRVgll antibody clone, C21. There may be one, two, three, or more CDRs in each of the heavy and light chains of the antibody. Normally, there are at least three CDRs on each chain which, when configured together, form the antigen-binding site, i.e., the three-dimensional combining site with which the antigen binds or specifically reacts.

[0060] The definition of CDR also includes overlapping or subsets of amino acid residues when compared against each other. The exact residue numbers which encompass a particular CDR, or a functional portion thereof, will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.

[0061] The amino acid sequence boundaries of a CDR can be determined by using any of a number of known numbering schemes, including those described by Kabat et al., supra ("Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 ("Chothia" numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262 :732-745 ("Contact" numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27: 55-77 ("IMGT" numbering scheme); and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657- 70 ("AHo" numbering scheme).

[0062] The antigen-binding domain may comprise any antibody fragments produced by protease digestion or reduction of a human monoclonal antibody and by recombinant DNA methods known to those skilled in the art. Human monoclonal antibody functional fragments include, for example, individual heavy or light chains and fragments thereof, such as VL, VH and Fd; monovalent fragments, such as Fv, Fab, and Fab', single domain Ab, bivalent fragments such as F(ab')2, single chain Fv (scFv), bivalent or trivalent scFvs, scFv-Fc-scFv, an IgG-scFv, an IgG-dAb, KIH-IgG, KX-BODY, KIH-FC Fab / scFv, tri / tetraspecific, KIH tri / tetra-specific, or a tandem scFv, bispecific antibody (BsAb), and Fc fragments. The Fc fragment of the antibody may be disabled by introducing amino acid substitutions into the Fc region, which silence or reduce the effector function of the antibody.

[0063] Thus, the antigen-binding domain may comprise a domain selected from the group consisting of: an antibody or antigen-binding fragment thereof, CDR, VL, VH and Fd; Fv, Fab, and Fab'; bivalent fragments such as F(ab')2; single chain Fv (scFv); bivalent or trivalent scFvs, scFv-Fc-scFv, an IgG-scFv, an IgG-dAb, KIH-IgG, KX-BODY, KIH-FC Fab / scFv, tri / tetraspecific, KIH tri / tetra-specific, or a tandem scFv, bispecific antibody (BsAb), and Fc fragments.

[0064] The term "VL fragment" can mean a fragment of the light chain of a human monoclonal antibody which includes all or part of the light chain variable region, including the CDRs. A VL fragment can further include light chain constant region sequences.

[0065] The term "VH fragment" can mean a fragment of the heavy chain of a human monoclonal antibody which includes all or part of the heavy chain variable region, including the CDRs.

[0066] The term "Fd fragment" can mean the heavy chain variable region coupled to the first heavy chain constant region, i.e. VH and CH-1. The "Fd fragment" does not include the light chain, or the second and third constant regions of the heavy chain.

[0067] The term "Fv fragment" can mean a monovalent antigen-binding fragment of a human monoclonal antibody, including all or part of the variable regions of the heavy and light chains, and absent of the constant regions of the heavy and light chains. The variable regions of the heavy and light chains include, for example, the CDRs. For example, an Fv fragment includes all or part of the amino terminal variable region of about 110 amino acids of both the heavy and light chains.

[0068] The term "Fab fragment" can mean a monovalent antigen-binding fragment of a human monoclonal antibody that is larger than an Fv fragment. For example, a Fab fragment includes the variable regions, and all or part of the first constant domain of the heavy and light chains. Thus, a Fab fragment additionally includes, for example, amino acid residues from about 110 to about 220 of the heavy and light chains. The term "Fab1fragment" can mean a monovalent antigen-binding fragment of a human monoclonal antibody that is larger than a Fab fragment. For example, a Fab' fragment includes all of the light chain, all of the variable region of the heavy chain, and all or part of the first and second constant domains of the heavy chain. For example, a Fab' fragment can additionally include some or all of amino acid residues 220 to 330 of the heavy chain.

[0069] The term "F(ab')2 fragment" can mean a bivalent antigen-binding fragment of a human monoclonal antibody. An F(ab')2 fragment includes, for example, all or part of the variable regions of two heavy chains-and two light chains, and can further include all or part of the first constant domains of two heavy chains and two light chains.

[0070] The term "single chain Fv (scFv)" can mean a fusion of the variable regions of the heavy (VH) and light chains (VL) connected with a short linker peptide.

[0071] The term "bispecific antibody (BsAb)" can mean a bispecific antibody comprising two scFv linked to each other by a shorter linked peptide.

[0072] In one embodiment, the antigen-binding domain may be a single domain antibody (sdAb) (also referred to as a nanobody). The skilled person would understand that an sdAb is an antibody fragment consisting of a single monomeric variable antibody domain (referred to as a VHH).

[0073] Alternatively, in another embodiment, the antigen-binding domain is a single-chain antibody, an intrabody, a peptide (e.g. a bicyclic peptide), or any other type of fragment or protein scaffold.

[0074] In one embodiment, the invariant natural killer T (iNKT) cell specific engager may comprise a scFv-Fc-scFv, an IgG-scFv, an IgG-dAb, KIH-IgG, K -BODY, KIH-FC Fab / scFv, tri / tetraspecific, KIH tri / tetra-specific, or a tandem scFv.

[0075] Preferably, the first antigen-binding domain that specifically binds to an antigen on the at least one iNKT cell comprises a single-chain variable fragment (scFv) domain of an anti-iNKT cell antibody, a Fab domain, or a single domain Ab. Preferably, the second antigen-binding domain that specifically binds to a tumour antigen on the at least one tumour cell comprises a single-chain variable fragment (scFv) domain of an anti-tumour cell antibody, a Fab domain or a single domain Ab.

[0076] The skilled person will be aware that an scFv domain is a fusion protein that comprises the variable regions of the heavy (VH) and light chains (VL) of a given antibody. In the context of the present invention, the antibody is one that is specific for an iNKT cell (e.g., via an iTCR) and / or a tumour cell (e.g., via a tumour antigen).

[0077] Referring now to Figure 1, the inventors have constructed two embodiments of the iNKT cell bispecific engager (or fusion protein) of the invention. However, it will be appreciated that polyspecific iNKT cell engagers are also envisaged, for example a bispecific or trispecific engager, and so on. As discussed herein, the iNKT engager may be configured to specifically engage with either the TCRVgll or the TCRVa24 variable segments of the invariant T cell Receptor (iTCR), or the TCRVa24Jal8 CDR3 idiotype, on the iNKT cell. As described herein, however, as a proof-of-concept, the inventors have constructed an iNKT cell engager against TCRVpi l, based on the TCRVpil antibody clone, C21. The tumour antigen-targeting arm of the engager was designed to bind to the clinically validated myeloma antigen, BCMA, and was based on the Clld5.3 scFv with modifications in the linker, as shown in Figure 1. It will be appreciated, however, that other tumour-associated antigens can be targeted, as discussed herein.

[0078] Thus, in one embodiment, as shown in Figure 1A, the iNKT cell bispecific engager (biNTe) or fusion protein may comprise a heterodimer. For example, the engager may comprise a heterodimeric, monovalent single chain variable fragment scFv-Fc human wild-type IgGl. The engager may comprise an Fc silent (L234A / L235A; LALA) and a key (or knob)-in-hole arrangement. The N-terminal anti-iTCR arm has a Fab configuration.

[0079] Preferably, therefore, the iNKT cell specific engager comprises first and second polypeptides, wherein the first and second polypeptides dimerise to generate the iNKT cell specific engager. Preferably, the iNKT cell engager is a heterodimer comprising two different polypeptides.

[0080] In a second embodiment, as shown in Figure IB, the iNKT cell bispecific engager (biNTe) or fusion protein may comprise a homodimer. For example, the engager may comprise a homodimeric, bivalent single chain variable fragment (scFv)-Fc human wild-type IgGl. The engager may comprise an Fc silent (L234A / L235A; LALA) changes. The N-terminal anti-BCMA arm has a Fab configuration, whereas the anti-TCRVpil arm (C-terminal) has an scFv configuration.

[0081] Preferably, the iNKT cell engager is a homodimer comprising two sets of the same polypeptide(s).

[0082] The tumour antigen-targeting arm of the exemplified engager was designed to bind to the clinically validated myeloma antigen, BCMA, and was based on the Clld5.3 scFv with modifications in the linker, as shown in Figure 1.

[0083] Preferably, the INKT cell specific engager comprises a single chain Fv (scFv) comprising a fusion of the variable regions of the heavy (VH) and light chains (VL) connected with a short linker peptide. Preferably, the engager comprises two scFv linked together. Each scFV may be defined as a monomer. Thus, in one embodiment, a first monomer may comprise the antigen-binding domain specific for the iNKT cell, and the second monomer may comprise the antigen-binding domain specific for the tumour cell. As such, this embodiment may be defined as a heterodimeric form.

[0084] Preferably, the anti-ITCR scFv comprises a C21 light chain variable region, a linker, and a C21 heavy chain variable region. In one embodiment, the amino acid sequence of the antigen-binding domain which specifically binds to TCRVgll (e.g. from a C21 clone) on the INKT cell, is represented herein as SEQ ID NO: 6, as follows:

[0085] DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKAGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTI SSLEYEDMGIYYCLQYDEFPFTFGGGTRLEIKGGGGSGGGGSGGGGSQVQLQQSGPEWRPGVSVKISCKGSGYR FTDSAMHWVKQSHAKSLEWIGVI SSYNGNTNYNQKFKGKATMTVDKSSSTAYMELARMTSEDSAIYYCARSRDAM DYWGQGTSVTVSS

[0086] [SEQ ID No: 6]

[0087] Preferably, therefore, the INKT cell engager comprises an antigen-binding domain comprising an amino acid sequence substantially set out as SEQ ID No: 6, or a variant or fragment thereof.

[0088] It will be appreciated that GGGGSGGGGSGGGGS [SEQ ID No: 7] is a linker, and that the length may vary. Preferably, the INKT cell engager comprises a linker disposed between the light and heavy chain regions, wherein the linker comprises an amino acid sequence substantially set out as SEQ ID No: 7, or a variant or fragment thereof. In one embodiment, the amino acid sequence of the light chain antigen-binding domain, which is derived from the original light chain of the TCRV[311 antibody clone, C21, and which specifically binds to TCRV[311 on the iNKT cell, is represented herein as SEQ ID NO: 8, as follows:

[0089] DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKAGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTI SSLEYEDMGI YYCLQYDEFPFTFGGGTRLEIK

[0090] [SEQ ID No: 8]

[0091] Preferably, therefore, the INKT cell engager comprises an antigen-binding domain comprising an amino acid sequence substantially set out as SEQ ID No: 8, or a variant or fragment thereof.

[0092] In one embodiment, the amino acid sequence of the heavy chain antigen-binding domain, which is derived from the original heavy chain of the TCRVpil antibody clone, C21, and which specifically binds to TCRVpil on the iNKT cell, is represented herein as SEQ ID NO: 9, as follows:

[0093] QVQLQQSGPEWRPGVSVKISCKGSGYRFTDSAMHWVKQSHAKSLEWIGVI SSYNGNTNYNQKFKGKATMTVDKS SSTAYMELARMTSEDSAIYYCARSRDAMDYWGQGTSVTVSS

[0094] [SEQ ID No: 9]

[0095] Preferably, therefore, the INKT cell engager comprises an antigen-binding domain comprising an amino acid sequence substantially set out as SEQ ID No: 9, or a variant or fragment thereof.

[0096] Preferably, the anti-BCMA scFv comprises a Clld5.3 scFv light chain variable region, a linker, and a Clld5.3 scFv heavy chain variable region. In one embodiment, the amino acid sequence of the antigen-binding domain which specifically binds to the tumour antigen (e.g. BCMA, from a Clld5.3 scFv), on the tumour cell, is represented herein as SEQ ID No: 10, as follows:

[0097] DIVLTQSPPSLAMSLGKRATI SCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDF TLTIDPVEEDDVAVYYCLQSRTI PRTFGGGTKLEIKGGGGSGGGGSGGGGSQIQLVQSGPELKKPGETVKISCKA SGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALD YSYAMDYWGQGTSVTVSS

[0098] [SEQ ID No: 10] Preferably, therefore, the iNKT cell engager comprises an antigen-binding domain comprising an amino acid sequence substantially set out as SEQ ID No: 10, or a variant or fragment thereof.

[0099] In one embodiment, the amino acid sequence of the light chain variable region of the antigen-binding domain, which is derived from the original light chain of the Clld5.3 scFv, and which specifically binds to the tumour antigen (e.g., BCMA) is provided herein as SEQ ID No: 11, as follows:

[0100] DIVLTQSPPSLAMSLGKRATI SCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDF TLTI DPVEEDDVAVYYCLQSRTI PRTFGGGTKLEIK

[0101] [SEQ ID No: 11]

[0102] Preferably, therefore, the iNKT cell engager comprises an antigen-binding domain comprising an amino acid sequence substantially set out as SEQ ID No: 11, or a variant or fragment thereof.

[0103] In one embodiment, the amino acid sequence of the heavy chain variable region of the antigen-binding domain, which is derived from the original heavy chain of the Clld5.3 scFv, and which specifically binds to the tumour antigen (e.g., BCMA) is provided herein as SEQ ID No: 12, as follows:

[0104] QIQLVQSGPELKKPGETVKI SCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETS ASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSS

[0105] [SEQ ID No: 12]

[0106] Preferably, therefore, the iNKT cell engager comprises an antigen-binding domain comprising an amino acid sequence substantially set out as SEQ ID No: 12, or a variant or fragment thereof.

[0107] It will be appreciated that there are two independent sequences which are inserted into mammalian cells for expression of the desired constructs, as discussed in detail in the Materials and Methods. These two sequences produce different polypeptides, one which comprises the anti-tumour antigen binding domain (e.g., Clld5.3 scFv), and the other which comprises the anti-iTCR antigen binding domain (e.g., from C21 clone). These two polypeptides then self-associate to generate a heterodimeric, monovalent biNTe. With regards to the iNKT cell engagers described in the Examples, each of the two polypeptides also contain IgGl heavy chain constant regions. These constant regions are largely identical; the only differences between them lie in the key (or knob)-in-hole parts of the CH3 domain of the IgGl heavy chains.

[0108] It will be appreciated that "key" or "knob" means that one small amino acid residue has been replaced by a bulky one in one polypeptide, and that "hole" means that one bulky amino acid residue has been replaced by a small one in the other polypeptide.

[0109] It will be appreciated that "bulky" amino acid residues include phenylalanine (F), tyrosine (Y), and tryptophan (W), and that "small" amino acids residues include glycine (G), alanine (A), serine (S), cysteine (C), aspartic acid (D), proline (P), asparagine (N), and threonine (T).

[0110] As such, the inventors have produced two independent sequences as part of their proof- of-concept monovalent biNTe, referred to herein as polypeptide 1 and polypeptide 2. Polypeptide 1 comprises the anti-tumour antigen binding domain (i.e., Clld5.3 scFv) and the "key" (or "knob"), IgGl heavy constant chain, where a bulky tryptophan (W) residue has replaced a small threonine (T) residue. Polypeptide 2 comprises the anti- iTCR antigen binding domain (i.e., anti-TCRVgll scFv from a C21 clone) and the "hole" IgGl heavy constant chain, where a bulky amino acid residue is replaced by a small amino acid residue.

[0111] In one embodiment, the amino acid sequence of polypeptide 1 (having the anti-tumour antigen binding domain), including its leader peptide, of the monovalent biNTe is provided herein as SEQ ID No: 13, as follows:

[0112] MPLLLLLPLLWAGALADIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQ TGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIKGGGGSGGGGSGGGGSQIQLVQSG PELKKPGETVKI SCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQI NNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPK PKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSR QQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0113] [SEQ ID No: 13]

[0114] Preferably, therefore, the iNKT cell engager comprises a polypeptide comprising an amino acid sequence substantially set out as SEQ ID No: 13, or a variant or fragment thereof. In one embodiment, the nucleotide sequence encoding SEQ ID No: 13 (i.e., polypeptide 1), including its leader peptide, of the monovalent biNTe is provided herein as SEQ ID No: 18, as follows:

[0115] ATGCCGCTGCTGCTACTGCTGCCCCTGCTGTGGGCAGGGGCGCTAGCTGACATCGTGCTGACACAGAGCCCCCCT

[0116] AGCCTGGCCATGAGCCTGGGCAAGAGAGCCACCATCAGCTGCAGAGCAAGCGAGAGCGTGACCATCCTGGGCAGC CACCTGATCCACTGGTATCAGCAGAAGCCTGGGCAGCCCCCCACCCTGCTGATTCAGCTGGCAAGCAACGTGCAG ACCGGCGTGCCCGCTAGATTCAGCGGCAGCGGCAGCAGAACCGACTTCACCCTGACCATCGACCCCGTGGAGGAG GACGACGTGGCCGTGTACTACTGCCTGCAGAGCAGAACCATCCCTAGAACCTTCGGCGGCGGCACCAAGTTAGAG ATCAAGGGCGGCGGGGGATCTGGCGGTGGTGGGAGTGGTGGGGGGGGTTCACAGATACAGCTGGTGCAGAGCGGC CCTGAGCTGAAGAAGCCCGGCGAGACCGTGAAGATCAGCTGCAAGGCAAGCGGCTACACCTTCACCGACTACAGC ATCAACTGGGTGAAGAGAGCCCCCGGCAAGGGCCTGAAGTGGATGGGCTGGATCAACACCGAGACAAGAGAGCCC GCCTACGCCTACGACTTCAGAGGCAGATTCGCCTTCAGCCTGGAGACAAGCGCAAGCACCGCCTACCTGCAGATC AACAACCTGAAGTACGAGGACACCGCCACCTACTTCTGCGCCCTGGACTACAGCTACGCCATGGACTACTGGGGC CAAGGCACAAGCGTGACCGTAAGCAGCGGCGGCGGAGGGAGCGGTGGGGGAGGTAGCGGAGGAGGGGGCTCAGAC AAGACACACACCTGCCCACCGTGTCCTGCCCCCGAAGCCGCTGGAGGGCCTAGCGTGTTCCTGTTCCCCCCCAAG CCCAAGGACACCCTGATGATCAGCAGAACCCCTGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCT GAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTAGAGAGGAGCAGTAC AACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAAGACTGGCTGAACGGCAAGGAGTACAAGTGC AAGGTGAGCAACAAGGCCCTGCCCGCCCCCATCGAGAAGACCATCAGCAAGGCCAAGGGGCAGCCTAGAGAGCCC CAAGTGTACACCCTGCCCCCCTGCAGAGACGAGCTGACCAAGAACCAAGTGAGCCTGTGGTGCCTGGTGAAGGGC TTCTACCCTAGCGACATCGCCGTGGAGTGGGAGAGCAACGGGCAGCCTGAGAACAACTACAAGACCACCCCCCCC GTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAAGGCAAC GTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACACAGAAGAGCCTGAGCCTGAGCCCCGGC

[0117] [SEQ ID No: 18]

[0118] Preferably, therefore, the INKT cell engager comprises a polypeptide comprising a nucleotide sequence substantially set out as SEQ ID No: 18, or a variant or fragment thereof.

[0119] In one embodiment, the amino acid sequence of polypeptide 2 (having the iTCR antigen binding domain), including its leader peptide, of the monovalent biNTe is provided herein as SEQ ID No: 14, as follows:

[0120] MPLLLLLPLLWAGALADIKMTQS PS SMYASLGERVTITCKASQDINSYLSWFQQKAGKS PKTLIYRANRLVDGVP SRFSGSGSGQDYSLTI S SLEYEDMGIYYCLQYDEFPFTFGGGTRLEI KGGGGSGGGGSGGGGSQVQLQQSGPEW RPGVSVKI SCKGSGYRFTDSA HWVKQSHAKSLEWI GVI S SYNGNTNYNQKFKGKATMTVDKSS STAY ELAR T SEDSAI YYCARSRDAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL MI SRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPAPI EKTI SKAKGQPREPQVYTLPPCRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GS FFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0121] [SEQ ID No: 14]

[0122] Preferably, therefore, the INKT cell engager comprises a polypeptide comprising an amino acid sequence substantially set out as SEQ ID No: 14, or a variant or fragment thereof.

[0123] In one embodiment, the nucleotide sequence encoding SEQ ID No: 14 (i.e., polypeptide 2), including its leader peptide, of the monovalent biNTe is provided herein as SEQ ID No: 19, as follows:

[0124] ATGCCGCTGCTGCTACTGCTGCCCCTGCTGTGGGCAGGGGCGCTAGCTGACATCGTGCTGACACAGAGCCCCCCT AGCCTGGCCATGAGCCTGGGCAAGAGAGCCACCATCAGCTGCAGAGCAAGCGAGAGCGTGACCATCCTGGGCAGC CACCTGATCCACTGGTATCAGCAGAAGCCTGGGCAGCCCCCCACCCTGCTGATTCAGCTGGCAAGCAACGTGCAG ACCGGCGTGCCCGCTAGATTCAGCGGCAGCGGCAGCAGAACCGACTTCACCCTGACCATCGACCCCGTGGAGGAG GACGACGTGGCCGTGTACTACTGCCTGCAGAGCAGAACCATCCCTAGAACCTTCGGCGGCGGCACCAAGTTAGAG ATCAAGGGCGGCGGGGGATCTGGCGGTGGTGGGAGTGGTGGGGGGGGTTCACAGATACAGCTGGTGCAGAGCGGC CCTGAGCTGAAGAAGCCCGGCGAGACCGTGAAGATCAGCTGCAAGGCAAGCGGCTACACCTTCACCGACTACAGC ATCAACTGGGTGAAGAGAGCCCCCGGCAAGGGCCTGAAGTGGATGGGCTGGATCAACACCGAGACAAGAGAGCCC GCCTACGCCTACGACTTCAGAGGCAGATTCGCCTTCAGCCTGGAGACAAGCGCAAGCACCGCCTACCTGCAGATC AACAACCTGAAGTACGAGGACACCGCCACCTACTTCTGCGCCCTGGACTACAGCTACGCCATGGACTACTGGGGC CAAGGCACAAGCGTGACCGTAAGCAGCGGCGGCGGAGGGAGCGGTGGGGGAGGTAGCGGAGGAGGGGGCTCAGAC AAGACACACACCTGCCCACCGTGTCCTGCCCCCGAAGCCGCTGGAGGGCCTAGCGTGTTCCTGTTCCCCCCCAAG CCCAAGGACACCCTGATGATCAGCAGAACCCCTGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCT GAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTAGAGAGGAGCAGTAC AACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAAGACTGGCTGAACGGCAAGGAGTACAAGTGC AAGGTGAGCAACAAGGCCCTGCCCGCCCCCATCGAGAAGACCATCAGCAAGGCCAAGGGGCAGCCTAGAGAGCCC CAAGTGTACACCCTGCCCCCCTGCAGAGACGAGCTGACCAAGAACCAAGTGAGCCTGACATGCCTGGTGAAGGGC TTCTACCCTAGCGACATCGCCGTGGAGTGGGAGAGCAACGGGCAGCCTGAGAACAACTACAAGACCACCCCCCCC GTGCTGGACAGCGACGGCAGCTTCTTCCTGACAAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAAGGCAAC GTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACACAGAAGAGCCTGAGCCTGAGCCCCGGC

[0125] [SEQ ID No: 19]

[0126] Preferably, therefore, the iNKT cell engager comprises a polypeptide comprising a nucleotide sequence substantially set out as SEQ ID No: 19, or a variant or fragment thereof. Therefore, in a preferred embodiment, the iNKT cell engager comprises (i) a first polypeptide comprising an amino acid sequence substantially set out as SEQ ID No: 13, or a variant or fragment thereof, and / or is encoded by a nucleotide sequence substantially set out as SEQ ID No: 18, or a variant or fragment thereof, and (ii) a second polypeptide comprising an amino acid sequence substantially set out as SEQ ID No: 14, or a variant or fragment thereof, and / or is encoded by a nucleotide sequence substantially set out as SEQ ID No: 19, or a variant or fragment thereof, preferably wherein the polypeptides dimerise to form a heterodimer.

[0127] As discussed above, the monovalent INKT cell engager described in the Examples comprises an Fc region, which may be derived from or comprise an IgG domain, for example a constant region of an antibody heavy chain. In one embodiment, therefore, the amino acid sequence of the Fc is provided herein as SEQ ID No: 15, as follows:

[0128] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G

[0129] [SEQ ID No: 15]

[0130] Preferably, therefore, the INKT cell engager comprises an Fc region comprising an amino acid sequence substantially set out as SEQ ID No: 15, or a variant or fragment thereof.

[0131] Preferably, the antigen-binding domain is connected to the Fc region by a linker, the length of which may vary. In one embodiment, the linker may be represented by GGGGSGGGGSGGGGS [SEQ ID No: 16].

[0132] SEQ ID No: x comprises a leader peptide MPLLLLLPLLWAGALA [SEQ ID No: 17].

[0133] Preferably, the INKT cell specific engager comprises two single chain Fv (scFv), specific for a first antigen, comprising a fusion of the variable regions of the heavy (VH) and light chains (VL) connected with a short linker peptide, and two Fab domains, specific for a second antigen, comprising a fusion of the variable regions of the heavy (VH) and light chains (VL). Each scFV linked to a Fab domain may be defined as a monomer. Thus, in one embodiment, each monomer may comprise one antigen-binding domain specific for the iNKT cell and one antigen binding domain specific for the tumour cell. As such, this embodiment may be defined as a homodimeric form. Preferably, the anti-ITCR scFv comprises a C21 light chain variable region, a linker, and a C21 heavy chain variable region. In one embodiment, the amino acid sequence of the antigen-binding domain which specifically binds to TCRVgll (e.g. from a C21 clone) on the iNKT cell, is represented herein as SEQ ID NO: 20, as follows:

[0134] DI KMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKAGKS PKTLIYRANRLVDGVPSRFSGSGSGQDYSLTI SSLEYEDMGIYYCLQYDEFPFTFGGGTRLEIKGGGGSGGGGSGGGGSQVQLQQSGPEWRPGVSVKI SCKGSGYR FTDSAMHWVKQSHAKSLEWIGVI SSYNGNTNYNQKFKGKATMTVDKS SSTAYMELARMTSEDSAIYYCARSRDAM DYWGQGTSVTVS S

[0135] [SEQ ID No: 20]

[0136] Preferably, therefore, the iNKT cell engager comprises an antigen-binding domain comprising an amino acid sequence substantially set out as SEQ ID No: 20, or a variant or fragment thereof.

[0137] It will be appreciated that GGGGSGGGGSGGGGS [SEQ ID No: 21] is a linker, and that the length may vary. Preferably, the iNKT cell engager comprises a linker disposed between the light and heavy chains, wherein the linker comprises an amino acid sequence substantially set out as SEQ ID No: 21, or a variant or fragment thereof.

[0138] In one embodiment, the amino acid sequence of the light chain antigen-binding domain, which is derived from the original light chain of the TCRVgll antibody clone, C21, and which specifically binds to TCRVgll on the iNKT cell, is represented herein as SEQ ID NO: 22, as follows:

[0139] DI KMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKAGKS PKTLIYRANRLVDGVPSRFSGSGSGQDYSLTI SSLEYEDMGI YYCLQYDEFPFTFGGGTRLEIK

[0140] [SEQ ID No: 22]

[0141] Preferably, therefore, the iNKT cell engager comprises an antigen-binding domain comprising an amino acid sequence substantially set out as SEQ ID No: 22, or a variant or fragment thereof.

[0142] In one embodiment, the amino acid sequence of the heavy chain antigen-binding domain, which is derived from the original heavy chain of the TCRVgll antibody clone, C21, and which specifically binds to TCRVgll on the iNKT cell, is represented herein as SEQ ID NO: 23, as follows: QVQLQQSGPEWRPGVSVKISCKGSGYRFTDSAMHWVKQSHAKSLEWIGVI SSYNGNTNYNQKFKGKATMTVDKS

[0143] SSTAYMELARMTSEDSAIYYCARSRDAMDYWGQGTSVTVSS

[0144] [SEQ ID No: 23]

[0145] Preferably, therefore, the INKT cell engager comprises an antigen-binding domain comprising an amino acid sequence substantially set out as SEQ ID No: 23, or a variant or fragment thereof.

[0146] Preferably, the anti-BCMA Clld5.3 Fab comprises a Clld5.3 heavy and light chain. In one embodiment, the amino acid sequence of the heavy chain antigen-binding domain, which specifically binds to the tumour antigen (e.g. BCMA, from a Clld5.3 scFv) on the tumour cell, is represented herein as SEQ ID No: 24, as follows:

[0147] QIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETS ASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSS

[0148] [SEQ ID No: 24]

[0149] Preferably, therefore, the iNKT cell engager comprises a heavy chain antigen-binding domain comprising an amino acid sequence substantially set out as SEQ ID No: 24, or a variant or fragment thereof.

[0150] In one embodiment, the amino acid sequence of the light chain antigen-binding domain, which specifically binds to the tumour antigen (e.g. BCMA, from a Clld5.3 scFv) on the tumour cell, is represented herein as SEQ ID No: 25, as follows:

[0151] DIVLTQSPPSLAMSLGKRATI SCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDF TLTIDPVEEDDVAVYYCLQSRTI PRTFGGGTKLEIK

[0152] [SEQ ID No: 25]

[0153] Preferably, therefore, the iNKT cell engager comprises an antigen-binding domain comprising an amino acid sequence substantially set out as SEQ ID No: 25, or a variant or fragment thereof.

[0154] It will be appreciated that there are two independent amino acid sequences which are inserted into the cells. These two sequences produce different polypeptides, one which contains the anti-BCMA heavy chain variable region, the IgGl heavy chain constant region (i.e., CHI, CH2 and CH3 with LALA mutations), the linker, and the anti-iTCR scFV (i.e. polypeptide 3), and the other which contains the anti-BCMA light chain variable region, and the light chain constant region (i.e. polypeptide 4). These two polypeptides then associate covalently in two instances; two copies of polypeptide 3 self-associate to generate a homodimer. In this homodimer, the anti-BCMA heavy chain variable-CHl heterodimerises with two copies of polypeptide 4, to generate the BCMA-engaging arms of the complete molecule.

[0155] In one embodiment, the amino acid sequence of polypeptide 3 (having an anti-BCMA heavy chain variable region, the IgGl heavy chain constant region, the linker, and the anti-iTCR scFV), not including the leader peptide, of the bivalent biNTe is provided herein as SEQ ID No: 26, as follows:

[0156] QIQLVQSGPELKKPGETVKI SCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETS ASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPS SSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RWSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGS GGGGSGGGGSDI KMTQS PS SMYASLGERVTITCKASQDINSYLSWFQQKAGKS PKTLIYRANRLVDGVPSRFSGS GSGQDYSLTI SSLEYEDMGIYYCLQYDEFPFTFGGGTRLEIKGGGGSGGGGSGGGGSQVQLQQSGPEWRPGVSV KI SCKGSGYRFTDSAMHWVKQSHAKSLEWIGVI S SYNGNTNYNQKFKGKATMTVDKSS STAYMELARMTSEDSAI YYCARS RDAMDYWGQGT SVTVS S

[0157] [SEQ ID No: 26]

[0158] Preferably, therefore, the iNKT cell engager comprises a polypeptide comprising an amino acid sequence substantially set out as SEQ ID No: 26, or a variant or fragment thereof.

[0159] In one embodiment, the nucleotide sequence encoding SEQ ID No: 26 (i.e., polypeptide 3), not including the leader peptide, of the monovalent biNTe is provided herein as SEQ ID No: 32, as follows:

[0160] CAGATTCAGCTGGTGCAAAGCGGACCCGAACTGAAGAAGCCCGGCGAGACCGTGAAGATCAGCTGTAAAGCCTCC GGATACACCTTCACCGACTACAGCATCAACTGGGTGAAGAGAGCCCCCGGCAAGGGCCTGAAGTGGATGGGCTGG ATCAACACCGAGACAAGAGAGCCCGCCTACGCCTACGACTTCAGAGGCAGATTCGCCTTCAGCCTGGAGACAAGC GCAAGCACCGCTTACCTGCAGATCAACAACCTGAAGTACGAGGACACCGCAACCTACTTCTGCGCCCTGGACTAC AGCTATGCTATGGACTACTGGGGGCAAGGCACAAGCGTTACTGTAAGCAGCGCGTCTACCAAGGGCCCCTCTGTT TTTCCCCTGGCTCCGAGCAGCAAGTCAACAAGCGGCGGCACCGCCGCACTGGGCTGTCTGGTGAAGGACTACTTC CCTGAACCCGTCACCGTTAGCTGGAACAGCGGTGCTCTGACTTCGGGCGTGCACACCTTCCCCGCCGTGCTGCAG

[0161] AGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCAAGCAGCAGCCTGGGCACACAGACCTACATCTGC

[0162] AACGTGAACCACAAGCCTAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAGACGCATACC TGTCCACCCTGCCCCGCCCCTGAGGCAGCCGGCGGCCCAAGCGTGTTTCTGTTCCCCCCCAAGCCCAAGGACACC CTGATGATCAGCAGAACCCCTGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCTGAGGTGAAGTTC AACTGGTACGTCGACGGCGTAGAGGTACACAACGCCAAGACCAAGCCTAGAGAGGAGCAGTACAACAGCACCTAC AGAGTGGTGAGCGTGCTGACCGTGCTGCACCAAGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAAC AAGGCCCTGCCCGCCCCCATCGAGAAGACCATCAGCAAGGCCAAGGGGCAGCCTAGAGAGCCCCAAGTGTACACC CTGCCCCCTAGCAGAGACGAGCTGACCAAGAACCAAGTGAGCCTGACCTGCTTAGTAAAGGGCTTCTACCCTAGC GACATCGCCGTGGAGTGGGAGAGCAACGGGCAGCCTGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGC GACGGCAGCTTCTTCCTGTACAGCAAGTTGACCGTGGATAAGAGCAGATGGCAGCAAGGCAACGTGTTCAGCTGC AGCGTGATGCACGAGGCCCTGCACAACCACTACACACAGAAGAGCTTGAGCCTTAGCCCCGGTGGCGGAGGATCT GGCGGTGGTGGTTCCGGCGGAGGCGGATCTGACATTAAGATGACACAGAGCCCTAGCAGCATGTACGCAAGCCTG GGCGAGAGAGTGACCATCACCTGTAAAGCAAGCCAAGACATCAACAGCTACCTGAGCTGGTTTCAGCAGAAGGCC GGCAAGAGCCCCAAGACCCTGATCTACAGAGCCAACAGACTGGTGGACGGTGTCCCAAGCAGATTTAGCGGCAGC GGCAGCGGCCAAGACTACAGCCTGACCATCAGCAGCCTGGAGTATGAGGACATGGGCATTTACTATTGCCTGCAG TACGACGAGTTCCCCTTCACCTTCGGGGGCGGCACCCGTCTCGAAATAAAGGGGGGAGGTGGCTCTGGTGGAGGC GGAAGCGGCGGCGGTGGCTCTCAAGTGCAGCTGCAGCAAAGCGGCCCGGAAGTGGTGAGACCCGGCGTGAGCGTG AAAATCAGCTGCAAGGGAAGCGGCTACAGATTCACCGACAGCGCCATGCACTGGGTGAAGCAGAGCCACGCCAAG AGCCTGGAGTGGATCGGCGTGATCAGCAGCTACAACGGCAACACCAACTACAATCAGAAGTTCAAGGGCAAGGCC ACCATGACCGTGGACAAGAGCAGCTCAACCGCCTACATGGAGCTGGCTAGAATGACAAGCGAGGACAGCGCCATT TACTACTGCGCGAGAAGCAGAGACGCCATGGATTATTGGGGTCAAGGTACAAGCGTCACCGTAAGCAGC

[0163] [SEQ ID No: 32]

[0164] Preferably, therefore, the INKT cell engager comprises a polypeptide comprising a nucleotide sequence substantially set out as SEQ ID No: 32, or a variant or fragment thereof.

[0165] In one embodiment, the amino acid sequence encoding polypeptide 4 (having the anti- BCMA light chain variable region, and the light chain constant region), not including the leader peptide, of the bivalent biNTe is provided herein as SEQ ID No: 27, as follows:

[0166] DIVLTQSPPSLAMSLGKRATI SCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDF TLTI DPVEEDDVAVYYCLQSRTI PRTFGGGTKLEIKRTVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC

[0167] [SEQ ID No: 27]

[0168] Preferably, therefore, the iNKT cell engager comprises a polypeptide comprising an amino acid sequence substantially set out as SEQ ID No: 27, or a variant or fragment thereof. In one embodiment, the nucleotide sequence encoding SEQ ID No: 27 (i.e., polypeptide 4), not including the leader peptide, of the monovalent biNTe is provided herein as SEQ ID No: 33, as follows:

[0169] GACATCGTGCTGACACAGAGCCCCCCTAGCCTGGCCATGAGCCTGGGCAAGAGAGCCACCATCAGCTGCAGAGCA AGCGAGAGCGTGACCATCCTGGGCAGCCACCTGATCCACTGGTATCAGCAGAAGCCTGGGCAGCCCCCCACCCTG CTGATTCAGCTGGCAAGCAACGTGCAGACCGGCGTGCCCGCTAGATTCAGCGGCAGCGGCAGCAGAACCGACTTC ACCCTGACCATCGACCCCGTGGAGGAGGACGACGTGGCCGTGTACTACTGCCTGCAGAGCAGAACCATCCCTAGA ACCTTCGGCGGCGGCACCAAGCTCGAGATCAAGAGAACTGTGGCCGCGCCGTCAGTGTTTATCTTCCCTCCATCG GATGAACAGCTTAAGTCCGGCACGGCGTCTGTGGTCTGCCTGCTCAATAACTTTTACCCTAGGGAAGCTAAAGTC CAATGGAAAGTGGATAACGCCCTGCAGTCAGGAAACAGCCAGGAATCGGTTACCGAACAGGACAGCAAGGACAGC ACTTACTCCTTGTCGTCGACTCTTACTCTGAGCAAGGCCGATTACGAGAAGCACAAGGTCTACGCCTGCGAGGTC ACCCATCAGGGACTCTCGTCCCCGGTGACCAAATCCTTCAATAGAGGCGAATGC

[0170] [SEQ ID No: 33]

[0171] Preferably, therefore, the INKT cell engager comprises a polypeptide comprising a nucleotide sequence substantially set out as SEQ ID No: 33, or a variant or fragment thereof.

[0172] Therefore, in a preferred embodiment, the iNKT cell engager comprises (i) at least one first polypeptide comprising an amino acid sequence substantially set out as SEQ ID No: 26, or a variant or fragment thereof, and / or is encoded by a nucleotide sequence substantially set out as SEQ ID No: 32, or a variant or fragment thereof, and (ii) at least one second polypeptide comprising an amino acid sequence substantially set out as SEQ ID No: 27, or a variant or fragment thereof, and / or is encoded by a nucleotide sequence substantially set out as SEQ ID No: 33, or a variant or fragment thereof, preferably wherein the at least two polypeptides dimerise to form a homodimer.

[0173] The iNKT cell engager comprises an Fc region, which may be derived from or comprise an IgG domain, an IGA domain, or an IGM domain, for example a constant region of an antibody heavy chain (preferably a IgGl heavy chain constant domain). It will be appreciated however that the Fc region may be derived from or comprise a constant region of any one of IgG2, IgG3 and / or IgG4.

[0174] In one embodiment, therefore, the amino acid sequence of the Fc is provided herein as SEQ ID No: 28, as follows:

[0175] ASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLS SWTVPS S S

[0176] LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI SRTPEVTCVWDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQ

[0177] PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW

[0178] QQGNVFSCSVMHEALHNHYTQKSLSLS P

[0179] [SEQ ID No: 28]

[0180] Preferably, therefore, the INKT cell engager comprises an Fc region comprising an amino acid sequence substantially set out as SEQ ID No: 28, or a variant or fragment thereof.

[0181] Preferably, the antigen-binding domain is connected to the Fc region by a linker, the length of which may vary. In one embodiment, the linker may be represented by GGGGSGGGGSGGGGS [SEQ ID No: 29].

[0182] In another embodiment, the iNKT cell engager comprises an Fc region, which may be derived from or comprise an IgG domain, for example a constant region of an antibody light chain (e.g., IgGl K light chain constant domain). It will be appreciated that the Fc region may be derived from or comprise a constant region of IgA and / or IgM.

[0183] In one embodiment, therefore, the amino acid sequence of the Fc is provided herein as SEQ ID No: 30, as follows:

[0184] RTVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSS PVTKSFNRGEC

[0185] [SEQ ID No: 30]

[0186] SEQ ID No: x comprises a leader peptide MPLLLLLPLLWAGALA [SEQ ID No: 31].

[0187] In a second aspect, there is provided a nucleic acid encoding the iNKT cell specific engager of the first aspect.

[0188] Preferably, the nucleic acid comprises a promoter operably linked to the sequence encoding the iNKT cell specific engager. The promoter drives expression of the engager in a host cell.

[0189] The promoter may be any suitable promoter, including a constitutive promoter, an activatable promoter, an inducible promoter, or a tissue-specific promoter.

[0190] Constitutive promoters allow heterologous genes (also referred to as transgenes) to be expressed constitutively in the host cells. Exemplary constitutive promoters contemplated herein include, but are not limited to, Cytomegalovirus (CMV) promoters, human elongation factors-1 alpha (hEFIa), ubiquitin C promoter (UbiC), phosphoglycerokinase promoter (PGK), simian virus 40 early promoter (SV40), and chicken P-Actin promoter coupled with CMV early enhancer (CAGG). Inducible promoters belong to the category of regulated promoters. The inducible promoter can be induced by one or more conditions, such as a physical condition, microenvironment of the engineered immune effector cell, or the physiological state of the engineered immune effector cell, an inducer (i.e., an inducing agent), or a combination thereof. In some embodiments, the inducing condition does not induce the expression of endogenous genes in the engineered mammalian cell, and / or in the subject that receives the pharmaceutical composition. In some embodiments, the inducing condition is selected from the group consisting of: inducer, irradiation (such as ionizing radiation, light), temperature (such as heat), redox state, tumor environment, and the activation state of the engineered mammalian cell.

[0191] In a preferred embodiment, the nucleic acid of the second aspect is selected from any of the nucleic acid sequences described herein. Most preferably, the nucleic acid comprises SEQ ID No: 18, 19, 32 and / or 33, or a fragment or variant thereof.

[0192] In a third aspect, there is provided an expression vector or a plasmid encoding the iNKT cell specific engager of the first aspect, or comprising the nucleic acid of the second aspect.

[0193] Preferably, the vector is recombinant. Preferably, the vector is a viral vector, more preferably a retroviral vector, even more preferably a lentivirus.

[0194] Preferably, the vector comprises left (i.e., N-term) and / or right (i.e., C-term) Long Terminal Repeat sequences (LTRs). Preferably, each LTR is disposed at the N-term and / or C-term of the construct.

[0195] In a fourth aspect, there is provided a pharmaceutical composition comprising a therapeutically effective amount of the iNKT cell specific engager of the first aspect, the nucleic acid of the second aspect, or the expression vector of the third aspect, and a pharmaceutically acceptable excipient.

[0196] Preferably, the pharmaceutical composition comprises a plurality of the engager of the invention. For example, the composition may comprise at least 50, 100, 1000, or 10,000 engagers. Preferably, the composition comprises at least 100,000, or at least 1,000,000 or at least 10,000,000 engagers, or at least 100,000,000 engagers. The pharmaceutical composition may comprise one or more iNKT or CAR-iNKT cell. Preferably, the one or more INKT or CAR-iNKT cell allogeneically sourced. Preferably, the one or more INKT or CAR-iNKT cell is adoptively transferred. For example, the composition may comprise at least 50, 100, 1000, or 10,000 INKT or CAR-iNKT cells.

[0197] Preferably, the composition comprises at least 100,000, or at least 1,000,000 or at least 10,000,000 or up to 1,000,000,000 INKT or CAR-iNKT cells.

[0198] The pharmaceutical composition may comprise one or more TCR-INKT cell. Preferably, the one or more TCR-INKT cell allogeneically sourced. Preferably, the one or more TCR- INKT cell is adoptively transferred. For example, the composition may comprise at least 50, 100, 1000, or 10,000 TCR-INKT cells. Preferably, the composition comprises at least 100,000, or at least 1,000,000 or at least 10,000,000 or up to 1,000,000,000 TCR-INKT cells.

[0199] In certain embodiments, the invention comprises transferring, to recipient, preexpanded, allogeneic or autologous INKT cells or CAR-iNKT cells.

[0200] In a fifth aspect, there is provided the INKT cell specific engager of the first aspect, the nucleic acid of the second aspect, the expression vector of the third aspect, or the pharmaceutical composition of the fourth aspect, for use in therapy or diagnosis.

[0201] In a sixth aspect, there is provided the INKT cell specific engager of the first aspect, the nucleic acid of the second aspect, the expression vector of the third aspect, or the pharmaceutical composition of the fourth aspect, for use in (i) immunotherapy; (ii) for treating, preventing or ameliorating cancer; (iii) for treating, preventing or ameliorating an autoimmune disease; or (iv) for treating, preventing or ameliorating any infectious disease, including viral disease, such as HIV infection.

[0202] In a seventh aspect, the invention provides a method of: (i) treating, preventing or ameliorating a disease in a subject with immunotherapy; (ii) treating, preventing or ameliorating cancer; (iii) for treating, preventing or ameliorating an autoimmune disease in a subject; or (iv) for treating, preventing or ameliorating any infectious disease, including viral disease, such as HIV infection, the method comprising administering, or having administered, to a patient in need of such treatment, a therapeutically effective amount of the iNKT cell specific engager of the first aspect, the nucleic acid of the second aspect, the expression vector of the third aspect, or the pharmaceutical composition of the fourth aspect. In one embodiment of the sixth or seventh aspect of the invention, the INKT cell specific engager of the first aspect, the nucleic acid of the second aspect, the expression vector of the third aspect, or the pharmaceutical composition of the fourth aspect, is for use in treating, preventing or ameliorating cancer. Preferably, the cancer is a malignancy, which may be a solid tumour or a liquid tumour.

[0203] Preferably, the INKT cell specific engager of the first aspect, the nucleic acid of the second aspect, the expression vector of the third aspect, or the pharmaceutical composition of the fourth aspect, is for use in treating, preventing or ameliorating myeloid or lymphoid leukaemias, B cell and T cell lymphomas, plasma cell dyscrasias and for solid tumours cancers of the brain, respiratory tract, head and neck, or skin etc.

[0204] In other embodiments, the iNKT cell specific engager of the first aspect, the nucleic acid of the second aspect, the expression vector of the third aspect, or the pharmaceutical composition of the fourth aspect, may be used in treating any disease caused by pathogenic B or T cells, such as autoimmune disease.

[0205] In other embodiments, the iNKT cell specific engager of the first aspect, the nucleic acid of the second aspect, the expression vector of the third aspect, or the pharmaceutical composition of the fourth aspect, may be used in treating any infectious disease, including viral disease, such as HIV infection.

[0206] In other embodiments, the iNKT cell specific engager of the first aspect, the nucleic acid of the second aspect, the expression vector of the third aspect, or the pharmaceutical composition of the fourth aspect, may be used in treating, preventing or ameliorating an autoimmune disease. The autoimmune disease may be caused by pathogenic autoreactive T or B cells, or selected from a group consisting of: Type 1 diabetes. Rheumatoid arthritis (RA), Psoriasis and psoriatic arthritis, Lupus (Systemic Lupus Erythematosus, or SLE), Multiple sclerosis, Graves' disease, Hashimoto's thyroiditis Ulcerative colitis, Crohn's disease, Addison's disease, Sjogren's syndrome, Myasthenia gravis. Autoimmune vasculitis, Celiac disease, Guillain-Barre syndrome, Chronic inflammatory demyelinating polyneuropathy (CIDP), Vitiligo, Autoimmune Hemolytic anaemia, Immune thrombocytopenia, anti-phospholipid syndrome, thrombotic thrombocytopenic purpura.

[0207] It will be appreciated that the iNKT cell specific engager, fusion protein, nucleic acid, vector, or pharmaceutical composition according to the invention (collectively referred to herein as "agents") may be used in a monotherapy (e.g. the use of the iNKT cell specific engager, fusion protein, nucleic acid, vector, or pharmaceutical composition alone), for therapy, preferably use in ((Hi) for treating, preventing or ameliorating an autoimmune disease in a subject; or (iv) for treating, preventing or ameliorating any infectious disease, including viral disease, such as HIV infection. Alternatively, the INKT cell specific engager, fusion protein, nucleic acid, vector, or pharmaceutical composition according to the invention may be used along with adoptively transferred INKT cells as an adjunct to, or in combination with, known immunotherapies or for treating disease caused by pathogenic B and T cells, as well as cancers or autoimmune disease.

[0208] For example, the inventors believe that the iNKT engager of the invention may be used in conjunction with other therapeutic modalities, such as CAR and TCR, to enhance the modularity and the efficacy of iNKT cells as a platform for the treatment of cancers, such as blood or solid tumours, for example myeloma. The clinical employment of these modular therapies will entail transfer to patients of autologous or allogeneic in vitro expanded iNKT or CAR / TCR-iNKT, preceded, or followed by, infusions of the iNKT cell engagers. The skilled person will appreciate that although TCRVa24Jal8 CDR3 idiotype engagers may exclusively bind to iNKT cells, TCRVpil- and TCRVa24 engagers will also bind to the minority conventional T cells (<5%) that express these TCR chains.

[0209] Accordingly, TCRVpil- and TCRVa24 engagers of the invention will continue to be active against cancer cells after the adoptively transferred INKT cells have declined and the endogenous TCRVP11+ and TCRVa24+ T cells have recovered.

[0210] The agents according to the invention may be combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used. Thus, for example, the composition may be in the form of a liquid, preferably delivered intravenously to a person in need of treatment. It will be appreciated that the vehicle of medicaments according to the invention should be one which is well-tolerated by the subject to whom it is given.

[0211] In a preferred embodiment, agents and medicaments according to the invention may be administered to a subject by injection into the blood stream or directly into a site requiring treatment. Injections may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion), or intradermal (bolus or infusion), or directly into tumours.

[0212] It will be appreciated that the amount of the iNKT cell specific engager, fusion protein, nucleic acid, vector, or pharmaceutical composition (i.e. agent) that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of the agent, and whether it is being used as a monotherapy or in a combined therapy. The frequency of administration will also be influenced by the in vivo persistence of the agent within the subject being treated. Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular agent in use, the strength of the pharmaceutical composition, the mode of administration, and the advancement of the disease being treated, for example cancer, or autoimmune disease. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.

[0213] The invention also provides in an eighth aspect, a process for making the pharmaceutical composition according to the fourth aspect, the process comprising combining a therapeutically effective amount of the iNKT cell specific engager according to the first aspect, the nucleic acid of the second aspect, or the expression vector of the third aspect, and a pharmaceutically acceptable vehicle.

[0214] A "subject" may be a vertebrate, mammal, or domestic animal. Most preferably, the subject is a human being.

[0215] A "therapeutically effective amount" of the iNKT cell specific engager, fusion protein, nucleic acid, vector, or pharmaceutical composition is any amount which, when administered to a subject, is the amount of agent that is needed to treat the disease being treated, for example cancer, or produce the desired effect.

[0216] For example, the therapeutically effective amount of the iNKT cell specific engager used may be at least 100, 1000, or 10,000 engagers. Preferably, at least 100,000, or at least 1,000,000 or at least 10,000,000 engagers is used.

[0217] A "pharmaceutically acceptable vehicle" as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions. Preferably, for a successful therapy, the composition comprising the CAR-effector cell is prepared and then delivered as a cell suspension, most preferably intravenously.

[0218] The pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution. Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active agent according to the invention may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). For parenteral administration, the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.

[0219] Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and particularly subcutaneous injection. The agent may be prepared as a sterile solid composition that may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.

[0220] It will be appreciated that the invention extends to any nucleic acid or peptide or variant, derivative or analogue thereof, which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including variants or fragments thereof. The terms "substantially the amino acid / nucleotide / peptide sequence", "variant" and "fragment", can be a sequence that has at least 40% sequence identity with the amino acid / nucleotide / peptide sequences of any one of the sequences referred to herein, for example 40% identity with the sequences identified herein, and so on.

[0221] Amino acid / polynucleotide / polypeptide sequences with a sequence identity which is greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged. Preferably, the amino acid / polynucleotide / polypeptide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity and, most preferably at least 99% identity with any of the sequences referred to herein. The skilled technician will appreciate how to calculate the percentage identity between two amino acid / polynucleotide / polypeptide sequences. In order to calculate the percentage identity between two amino acid / polynucleotide / polypeptide sequences, an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value. The percentage identity for two sequences may take different values depending on :- (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional form and constants.

[0222] Having made the alignment, there are many different ways of calculating percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (v) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.

[0223] Hence, it will be appreciated that the accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred way for generating multiple alignments of proteins or DNA in accordance with the invention. Suitable parameters for ClustalW may be as follows: For DNA alignments: Gap Open Penalty = 15.0, Gap Extension Penalty = 6.66, and Matrix = Identity. For protein alignments: Gap Open Penalty = 10.0, Gap Extension Penalty = 0.2, and Matrix = Gonnet. For DNA and Protein alignments: ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will be aware that it may be necessary to vary these and other parameters for optimal sequence alignment.

[0224] Preferably, calculation of percentage identities between two amino acid / polynucleotide / polypeptide sequences may then be calculated from such an alignment as (N / T)*100, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps and either including or excluding overhangs. Preferably, overhangs are included in the calculation. Hence, a most preferred method for calculating percentage identity between two sequences comprises (i) preparing a sequence alignment using the ClustalW program using a suitable set of parameters, for example, as set out above; and (II) inserting the values of N and T into the following formula :- Sequence Identity = (N / T)*100.

[0225] Alternative methods for identifying similar sequences will be known to those skilled in the art. For example, a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to DNA sequences or their complements under stringent conditions. By stringent conditions, the inventors mean the nucleotide hybridises to filter-bound DNA or RNA in 3x sodium chloride / sodium citrate (SSC) at approximately 45°C followed by at least one wash in 0.2x SSC / 0.1% SDS at approximately 20-65°C. Alternatively, a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from the sequences shown herein.

[0226] Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof. Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent (synonymous) change. Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change. For example, small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine. The polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. The positively charged (basic) amino acids include lysine, arginine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids.

[0227] All of the features described herein (including any accompanying claims, abstracts and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some features and / or steps are mutually exclusive. For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which :-

[0228] Figure 1 illustrates an embodiment of a bispecific invariant NKT cell engager (known as a "biNTe"). The biNTe (the "engager") is an scFv-Fc fusion protein which binds to two different epitopes. The first epitope is the TCRVbll chain of the TCRVa24Jal8Vbll heterodimer on an INKT cell, and the second epitope is a tumour antigen, such as BCMA in multiple myeloma plasma cells. 1A shows an embodiment of a monovalent binTe binding the invariant TCR on the iNKT cell, and also the BCMA antigen on a myeloma tumour cell. IB shows an embodiment of a bivalent binTe binding to two invariant TCRs on the iNKT cell, and also two BCMA antigens on the tumour cell.

[0229] Figure 2 illustrates the binding of biNTe to iNKT cells, but not to T cells. Binding of iNKT cells (left) and T cells (right), with monovalent biNTe followed by anti-Fc secondary staining. iNKT cells (but not T cells) are stained by biNTe in an antibody (Ab) concentration-dependent manner.

[0230] Figure 3 illustrates the specificity of biNTe for the TCRVgll chain on iNKT cells. Purified iNKT cells were stained with fluorescently labelled anti-TCRVpil Ab C21 (left) or costained with fluorescently labelled anti-TCRVpil Ab plus monovalent biNTe. Since the iNKT cell binding arm of biNTe comprises an scFv also derived from C21, as expected, binding of fluorescently labelled anti TCRVgll is inhibited (right).

[0231] Figure 4 illustrates INKT cell and BCMA binding of iNKT bi-specific Ab. The mono-valent biNTe was incubated with pure iNKT cells (left), biotinylated soluble BCMA and PE- streptavidin, followed by flow-cytometric analysis. Only in the presence of bio-BCMA did the biNTe stain iNKT cells, while in its absence, staining is similar to unstained iNKT cells.

[0232] Figure 5 illustrates the anti-myeloma activity of biNTe. A) Mono- or bi-valent biNTE at different concentrations were co-cultured with purified iNKT cells, and the BCMA- expressing myeloma MM1.S as targets at an INKT: myeloma ratio of 2: 1. Cytotoxicity of MM1.S cells was measured 16hrs later by flow-cytometry. Both biNTe kill myeloma cells in a dose-dependent manner. B) Phase contrast light microscopy of myeloma cells cultured with iNKT and biNTe. C) EC50 of the bivalent BCMA biNTE against MM1.S and H929 myeloma cells is estimated at 60 and 3.5 nM, respectively. Figure 6 illustrates that biNTe are effective against drug resistant myeloma cells. Left: BCMA expression in dexamethasone-sensitive MM1.S and dexamethasone-resistant MM1.R myeloma cells. Right: co-culture of the two myeloma cell lines at 1: 1 ratio with iNKT cells and varying concentrations of the BCMA bispecific biNTe, and assessment of cytotoxicity of myeloma cells after 20 hours by flow-cytometry.

[0233] Figure 7 illustrates that biNTe are specific for BCMA and iNKT cells. A) Co-culture of iNKT and H929 myeloma cells with the H929 EC50 of 3.5nM in the presence of varying concentrations of soluble BCMA. Cytotoxicity was inhibited in a BCMA dose-dependent manner. B) Left: flow-cytometric assessment of BCMA expression in WT and BCMA knock out H929 cells. Right: 20hr cytotoxicity of iNKT cells against BCMA-expressing and BCMA knock out H929 myeloma cells (E:T ratio 1: 1) at varying concentrations of the bivalent BCMA biNTe, and assessment of cytotoxicity against myeloma cells after 2hrs by flow-cytometry. C) 20hr cytotoxicity of T or iNKT cells against BCMA-expressing H929 myeloma cells (E:T ratio 1: 1) at varying concentrations of the bivalent BCMA biNTe, and assessment of cytotoxicity against myeloma cells after 2hrs by flow-cytometry.

[0234] Figure 8 illustrates that BCMA biNTe-iNKT cells have equivalent anti-myeloma activity as BCMA CAR-iNKT cells. Myeloma cell MM1.S were co-cultured with non-transduced iNKT (NT) plus bi-specific BCMA biNTe (Ing / ml) or BCMA CAR-iNKT at different E:T ratios. Cytotoxicity against MM1.S cells was assessed by flow cytometry 20hrs later.

[0235] Figure 9 illustrates that biNTe kill primary myeloma cells. The impact of varying concentrations of biNTe in the presence of iNKT cells against primary bone marrow myeloma plasma cells identified by expression of CD138 (E:T ratio 1 : 1).

[0236] Figure 10 illustrates the inhibition of MM1S myeloma tumor growth after BiNTe treatment. A) tumor volume as measured in MM1S tumor mouse model before and after treatment with iNKT cells alone or BiNTe alone or both as compared to untreated group. n=4-5 mice / group, standard error mean, * p<0.05. days were calculated based on MM1S cell tumor cells injection and day -1 was set as iNKT treatment day followed by day 0 as BiNTe treatment. B) picture of tumors excised from the mice upon scarification at end of the study.

[0237] Figure 11 illustrates the weight of the mice. Total body weight of each mouse was monitored for the compete study and plotted for each group, n=4-5 mice / group. Each line represents individual mouse of the corresponding groups. Examples

[0238] The inventors have demonstrated the feasibility and activity of iNKT-specific engagers against multiple myeloma as a proof-of-concept for cancer in general. Specifically, the inventors have designed two different embodiments of a bispecific iNKT engager (iNTe), one monovalent, the other bivalent, both of which target TCRVpil on iNKT cells and BCMA antigen on tumour cells. The inventors have shown that these biNTes bind specifically to their target antigens. The inventors have also shown that the biNTes display surprising anti-myeloma activity, and therefore provide evidence for both BCMA- dependent and iNKT cell-dependent tumour cell killing in the presence of biNTe.

[0239] Materials and Methods iNKT cell isolation and cell culture Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood from healthy donor using Lymphoprep (Stemcell Technologies; catalogue: 07811) and centrifugation at 800 g for 30minutes, 1 acceleration, 1 deceleration at 24°C. iNKT cells were purified from PBMCs using Anti-iNKT MicroBeads, human (Miltenyi Biotech; catalogue: 130-094-842) with selection buffer (IxPBS, 0.5% human serum albumin, 2 mM EDTA) by passing through LS column (Miltenyi Biotech; catalogue: 130-042-401). The iNKT cells were activated using 100 ng of anti-human CD3 and 100 ng of antihuman CD28 antibodies and 1 :2 feeder cells (iNKT: gamma-irradiated PBMCs of the same donor) and expanded in R10 media (RPIM-1640, 10% Foetal Bovine Serum, 1% Pen / strep, 2% L-Glutamine, 1% sodium Pyruvate, 1% Non-essential Amino acids, 15 mM HEPES buffer, 0.05 mM Beta Mercaptoethanol) supplemented with IL-15 cytokine. These iNKT cells were expanded by stimulating with 200 ng / ml alpha-GalCer (KRN7000)- loaded ClR-CDld after 5Gy gamma irradiation. T cells were activated and expanded by stimulating PBMCs with 100 ng of human anti-CD3 and 100 ng of human anti-CD28 antibodies for 3 days. Both iNKT and T-cells were cultured in R10 media supplemented with 150U IL-15 cytokine. Myeloma cell lines MM1.S, H929 cells were cultured in RPIM- 1640 media supplemented with 10% Foetal Bovine Serum, 1% Pen / strep, 1% L- Glutamine, 1% sodium Pyruvate, 1% Non-essential Amino acids.

[0240] BiNTe development

[0241] Genes for monovalent and bivalent antibody were designed, and codon optimized for expression in mammalian cells (HEK293). DNA sequences were sub-cloned into an appropriate cloning and expression vector to deliver the desired constructs. Cultures were harvested, and purification was performed using Protein A affinity chromatography. The protein was analysed for purity by SDS-PAGE and concentration determined by UV spectroscopy. BiNTe staining and flow cytometry assays

[0242] The BiNTe was diluted in FACS buffer (IxPBS supplemented with 0.5% human serum albumin, 2 mM EDTA) followed by staining the cells for 30 minutes at 4°C followed by washing twice with FACS buffer. The BiNTe stained cells (INKT or T-cells or myeloma cells) were stained with anti-Fc chimera antibody conjugated with FITC fluorochrome (Biologend; catalogue: 410719). After removing the unbound antibodies by washing, the cells were re-stained with anti-human iTCR VgH Antibody (clone: REA559) and analysed by flow cytometry. INKT cells were identified by anti-CD3 (Clone: UCHT1), anti-human TCR V24-J 18 (Clone: 6B11), anti-human CD45 (Clone: HI30), anti-human ITCR Vpil (clone: REA559) staining. T cells were identified by anti-human CD3 and anti-human CD45 antibodies. BCMA expression levels on myeloma cell surface were detected using anti-human BCMA antibody (clone: REA559).

[0243] Cytotoxicity assays

[0244] Effector cells (INKT or T cells) were incubated with indicated concentrations of BiNTe for 30 minutes at room temperature. The target cells such as myeloma cell line H929, MM1.S cells were stained with CellTrace™ Violet dye (Life technology; Catalogue number: C34557) followed by coculturing with effector cells. After co-incubation of effector cells (INKT or T-cells) with BiNTe and target cells (H929 or MM1.S) for 20 hours, the cells were stained with 7AAD (Cambridge Bioscience; catalogue number: ANA83201) and the cell death were analysed by flowcytometry. However, the myeloma patient-derived bone marrow cells were co-incubated with effector cells without staining, and the myeloma cells were identified by anti-human CD138 antibody. For blocking assays, the myeloma cells were incubated with recombinant BCMA protein (clone: 10620-H40H-B-SIB) followed by incubation with iNKT cells (1 : 1) and BINTes at EC50 dose. Cytotoxic efficiency of BiNTe compared with iNKT cells engineered to express anti- BCMA chimeric antigen receptor of the same sequences as BCMA arm of BiNTes.

[0245] Primary myeloma sample

[0246] Bone marrow of myeloma patients was received from Hammersmith Hospital and processed following ethical committee approval (REC n. 11 / H0308 / 9). The bone marrow samples were lysed with lx red cell lysis buffer (Biolegend; catalogue: 420301) diluted in distilled water. The myeloma cells were identified by staining with anti-human CD138 (Syndecan-1) (Clone: MI15) and anti-human CD38 (Clone: HIT2). For the cytotoxicity, the cells were thawed and directly co-incubated with BiNTes and iNKT cells. Data analysis and software

[0247] All the flow cytometry data were analysed using flow Jo version 10.9.0 software. A doseresponse curve was generated using the sigmoidal dose-response (variable slope) model and all the statistical analysis were performed using GraphPad prims software version 9.4.0.

[0248] In vivo studies

[0249] 5E6 multiple myeloma MM1S cells prepared with mixture of lOOpI Corning® Matrigel® Basement Membrane Matrix, LDEV-free (Coring; cat # 354234) and lx PBS (Sigma; cat # D8537) (1: 1 ratio) and subcutaneously injected into 6-8 old female NSG mice. The mice were observed for visible tumor growth and the tumor size were measured using vernier caliper and the volume was calculated using the formula V = 0.5 x L x W2. For the treatment, the mice were intravenously injected with 15E6 iNKT cells / mice that were expanded in vitro for 28 days following a-GalCer (cat # KRN7000) loaded-CIR-CDId cells-mediated stimulation and cytokine 150U human IL15 in RPMI-1640 media (Sigma; cat # R8758) supplemented with 10% Fetal Bovine Serum (Life technologies), 1% L- Glutamine (Sigma; cat # G7513), 1% Sodium pyruvate (Sigma; cat # S8636), 1% Non- essential Amino acids (Sigma; cat # M7145), 1% Pen / strep (Sigma; cat # P0781) 1 day prior to the antibody treatment. For the BiNTe treatment, the mice were intraperitonia I ly injected with 0.2mg / Kg BiNTe, twice weekly.

[0250] Example 1 - Design of bisoecific INKT cell engagers

[0251] To provide the proof-of-concept in support of iNKT cell engagers, the inventors have designed a bispecific invariant NK T cell engager referred to herein as "biNTe". However, it will be appreciated that polyspecific invariant NK T cell engagers are also envisaged, for example a bispecific or trispecific engager, and so on.

[0252] The iNKT engager can be designed to engage either the TCRV311 or the TCRVo24 variable segments of the invariant T cell Receptor (iTCR), or the TCRVo24Jol8 CDR3 idiotype, on the iNKT cell. As described herein, however, as a proof of concept, the inventors have constructed iNKT cell engager against TCRV311, based on the TCRVpil antibody clone, C21.

[0253] The tumour antigen-targeting arm of the biNTe engager was designed to bind to the clinically validated myeloma antigen, BCMA, and was based on the Clld5.3 scFv with modifications in the linker, as shown in Figure 1. It will be appreciated that other tumour-associated antigens can be targeted. In one embodiment, as shown in Figure 1A, the biNTe comprises a heterodimeric, monovalent single chain variable fragment scFv-Fc human wild-type IgGl and Fc silent (L234A / L235A; LALA) and a key-in-hole arrangement.

[0254] In a second embodiment, as shown in Figure IB, the biNTe comprises a homodimeric, bivalent single chain variable fragment scFv-Fc human wild-type IgGl and Fc silent (L234A / L235A; LALA) changes. The N-terminal anti-BCMA arm has a Fab configuration, whereas the anti-TCRVbll arm (C-terminal) has an scFv configuration.

[0255] After cloning of the constructs into an expression vector and their transfection into 293T cells, protein was purified from the culture supernatant using Protein A columns, and the biNTe engager was confirmed by SDS PAGE.

[0256] Example 2 - Specificity of biNTE binding to INKT cells

[0257] To demonstrate that the monovalent embodiment of biNTE shown in Figure 1A binds iNKT cells, but not T cells, the inventors stained purified cells with biNTe followed by secondary fluorescently anti-human IgGl Fc Ab. The inventors found that, compared to unstained and secondary Ab-stained cells, biNTe stained purified iNKT cells, but not T cells in a dose-dependent manner, as shown in Figure 2. Furthermore, incubating purified iNKT cells with fluorescently labelled anti-TCRVgll Ab in the presence of monovalent biNTe plus secondary Ab considerably reduced the intensity of the staining of iNKT cells, as shown in Figure 3.

[0258] These data demonstrate the surprisingly specific binding of the iNKT cell arm of the biNTe to the invariant TCR on an iNKT cell.

[0259] Example 3 - biNTE binding to soluble BCMA

[0260] To test the ability of the tumour binding arm of the biNTe to bind their intended target BCMA antigen on the tumour cell, the monovalent (Figure 1A) or bivalent (Figure IB) embodiments of biNTe were incubated with pure iNKT cells, soluble biotinylated BCMA, and PE-streptavidin, followed by flow-cytometric analysis. Only in the presence of bio- BCMA did the biNTe stain iNKT cells, while in its absence, staining is similar to unstained iNKT cells, as shown in Figure 4.

[0261] Example 4 - Anti-mveloma activity of biNTe

[0262] To demonstrate the ability of the biNTe engager to kill myeloma cells, purified iNKT cells were co-cultured with the BCMA-expressing myeloma cells MM1.S as targets at an INKT: myeloma ratio of 2: 1 in the presence of different concentrations of mono- or bi-valent biNTE.

[0263] Cytotoxicity assay performed 16 hours later showed both embodiments of the biNTe killed myeloma cells similarly and at a dose-dependent manner, as shown in Figure 5A. This was also evidenced by phase contrast light microscopy inspection of the myeloma cells cultured with iNKT cells and biNTe, as shown in Figure 5B. Using the same approach, the inventors then determined that the EC50 of the bivalent embodiment of the BCMA-binding biNTE against the BCMA-expressing MM1.S and H929 myeloma cells was at 60 and 3.5 nM, respectively, as shown in Figure 5C.

[0264] Surprisingly, the biNTe retained its ability to kill dexamethasone-resistant MM1R cells which expressed lower levels of BCMA than their parental MM1S counterparts, as shown in Figure 6.

[0265] Example 5 - BCMA- and INKT cell-dependent killing by biNTe

[0266] To demonstrate BCMA-dependent killing by the biNTe engager, the inventors co-cultured iNKT cells and H929 myeloma cells in the presence of biNTe at its EC50 concentration and the presence of varying concentrations of soluble BCMA. The inventors found that cytotoxicity was inhibited in a BCMA dose-dependent manner, as shown in Figure 7A.

[0267] Further supporting the BCMA-dependent killing by the biNTe engager, BCMA-expressing (but not BCMA knock-out H929 cells) were killed by iNKT cells in the presence of biNTe and in a dose-dependent manner, as shown in Figure 7B. Further, co-culture of purified T cells or iNKT cells against BCMA-expressing H929 myeloma cells (E:T ratio 1: 1) in the presence of varying concentrations of the bivalent embodiment of the BCMA biNTe (Figure IB) showed killing of myeloma cells only by iNKT cells, but not by T cells, as shown in Figure 7C.

[0268] Together, these results confirm BCMA- and iNKT cell-dependent killing of myeloma cells by the biNTe.

[0269] Example 6 - BCMA biNTe-INKT have equivalent anti-mveloma activity as BCMA CAR-iNKT The inventors next compared the anti-myeloma activity of BCMA biNTe with that of BCMA CAR-iNKT. For this purpose, the inventors used a CAR comprising the Clld5.3 scFv (i.e., the same as in the biNTe) with a (G4S)3 linker, CD8a-derived spacer and transmembrane domain and a CD28-CD3 endodomain. Myeloma MM1.S cells were cocultured with non-transduced iNKT or BCMA CAR-iNKT at different E:T ratios plus bi- valent BCMA biNTe (1 ng / ml). Cytotoxicity against MM1.S cells assessed by flow cytometry 20 hours later showed similar killing of targets cells, thereby confirming the surprising potency of the BCMA biNTe, as shown in Figure 8. Accordingly, they can be delivered together with the transfer of iNKT cells without the latter necessarily requiring genetic engineering with all of the logistical, cost and biological risks associated with it.

[0270] The inventors next co-cultured bone marrow cells from a patient with myeloma enriched in myeloma plasma cells with iNKT cells and varying concentrations of biNTe. The inventors found that while the frequency of myeloma plasma cells dramatically decreased in a biNTE dose-dependent manner, the frequency of non-myeloma bone marrow cells increased, thereby suggesting specific killing of the myeloma, but not of the non-myeloma cells, as shown in Figure 9.

[0271] Example 7 - BiNTe efficacy in killing BCMA+ myeloma cells in vivo

[0272] The anti-tumor efficacy of BiNTe was investigated in MM IS myeloma cells, which express high levels of BCMA antigen on the cell surface. The tumor mouse model comprised MM1S cells grown subcutaneously in NSG mice. Mice were grouped into untreated and treated groups upon measurable visible tumor establishment. iNKT cells were injected on day 13 post-tumor cell injection and one day prior to BiNTe treatment. Significant reduction in tumor size were observed after 2 days, post-treatment with BiNTe in the iNKT-treated group whereas neither iNKT alone nor BiNTe alone treatment groups showed any significant tumor growth inhibition as compared to untreated groups (Figures 10A 8i B).

[0273] In addition, the weights of the mice were monitored for the complete study and no significant total body weight loss was observed in any of the treated mice both iNKT cells alone or antibody alone groups and both (Figure 11).

[0274] Summary

[0275] The inventors have demonstrated the feasibility and activity of iNKT-specific engagers against multiple myeloma tumour cells as a proof-of-concept for cancer in general. There exists three iTCR constituents that can be targeted by the engager of the invention, namely the TCRVpil variable segment, the TCRVa24 variable segment, or the TCRVa24Jal8 CDR3 idiotype of the iTCR.

[0276] The inventors envisage that such iNKT engagers may be used in conjunction with other therapeutic modalities, such as CAR and TCR, to enhance the modularity and the efficacy of iNKT cells as a platform for the treatment of myeloma, as well as other blood or solid tumour cancers, autoimmune and infectious diseases. The clinical employment of these modular therapies will entail transfer to patients of autologous or allogeneic in vitro expanded iNKT or CAR / TCR-iNKT, preceded, or followed by, infusions of the iNKT cell engagers.

[0277] Of note, while TCRVa24Jal8 CDR3 idiotype engagers may exclusively bind to iNKT cells, TCRV311- and TCRVa24 engagers will also bind to the minority conventional T cells (<5%) that express these TCR chains. This raises the prospect that such engagers will continue to be active against cancer cells after the adoptively transferred INKT cells have declined and the endogenous TCRV311+ and TCRVa24+ T cells have recovered.

[0278] References

[0279] 1. Dellabona P, Padovan E, Casorati G, Brockhaus M, Lanzavecchia A. An invariant V alpha 24-1 alpha Q / V beta 11 T cell receptor is expressed in all individuals by clonally expanded CD4-8- T cells. J Exp Med. 1994;180(3): 1171-1176.

[0280] 2. Salio M, Silk ID, Jones EY, Cerundolo V. Biology of CD1- and MRl-restricted T cells. Annu Rev Immunol. 2014;32:323-366.

[0281] 3. Fuji! S, Shimizu K, Okamoto Y, et al. NKT cells as an ideal anti-tumor immunotherapeutic. Front Immunol. 2013;4:409.

[0282] 4. Terabe M, Berzofsky JA. NKT cells in immunoregulation of tumor immunity: a new immunoregulatory axis. Trends Immunol. 2007;28(ll):491-496.

[0283] 5. Gumperz JE, Miyake S, Yamamura T, Brenner MB. Functionally distinct subsets of CDld-restricted natural killer T cells revealed by CDld tetramer staining. JExpMed. 2002;195(5):625-636.

[0284] 6. Song L, Asgharzadeh S, Salo J, et al. Valpha24-invariant NKT cells mediate antitumor activity via killing of tumor-associated macrophages. J Clin Invest. 2009;119(6): 1524-1536.

[0285] 7. Cortesi F, Delfanti G, Grilli A, et al. Bimodal CD40 / Fas-Dependent Crosstalk between iNKT Cells and Tumor- Associated Macrophages Impairs Prostate Cancer Progression. Cell Rep. 2018;22(l l) :3006-3020.

[0286] 8. Chaidos A, Patterson S, Szydlo R, et al. Graft invariant natural killer T-cell dose predicts risk of acute graft-versus-host disease in allogeneic hematopoietic stem cell transplantation. Blood. 2012;119(21):5030- 5036.

[0287] 9. Mavers M, Maas-Bauer K, Negrin RS. Invariant Natural Killer T Cells As Suppressors of Graft-versus- Host Disease in Allogeneic Hematopoietic Stem Cell Transplantation. Front Immunol. 2017;8:900.

[0288] 10. Rotolo A, Caputo VS, Holubova M, et al. Enhanced Anti-lymphoma Activity of CAR19-iNKT Cells Underpinned by Dual CD19 and CDld Targeting. Cancer Cell. 2018;34(4):596-610 e511.

[0289] 11. Rowan AG, Ponnusamy K, Ren H, Taylor GP, Cook LBM, Karadimitris A. CAR-iNKT cells targeting clonal TCRVbeta chains as a precise strategy to treat T cell lymphoma. Front Immunol. 2023;14: 1118681.

[0290] 12. Ramos C, Courtney A, Robinson S, et al. Allogeneic NKT Cells Expressing a CD19-Specific CAR in Patients with Relapsed or Refractory B-Cell Malignancies: An Interim Analysis. Blood. 2021;138.

[0291] 13. Wei J, Yang Y, Wang G, Liu M. Current landscape and future directions of bispecific antibodies in cancer immunotherapy. Front Immunol. 2022; 13: 1035276.

[0292] 14. Carpenter RO, Evbuomwan MO, Pittaluga S, et al. B-cell maturation antigen is a promising target for adoptive T-cell therapy of multiple myeloma. Clin Cancer Res. 2013; 19(8):2048-2060.

[0293] This work was supported by: EPSRC - EP / X52556X / 1, MRC - MR / X502959 / 1, BBSRC - BB / X511055 / 1, and The Kay Kendall Leukaemia Fund (KKLF).

Claims

Claims1. An invariant natural killer T (iNKT) cell specific engager.

2. The iNKT cell specific engager according to claim 1, wherein the iNKT cell specific engager is configured to specifically bind to at least one INKT cell, preferably at least one antigen on the at least one INKT cell.

3. The INKT cell specific engager according to either claim 1 or claim 2, wherein the iNKT cell specific engager is configured to specifically bind to at least one tumour antigen on at least one tumour cell.

4. The INKT cell specific engager according to any preceding claim, wherein the iNKT cell specific engager comprises a fusion protein.

5. The iNKT cell specific engager according to any preceding claim, wherein the iNKT cell specific engager specifically binds to an invariant T cell receptor (ITCR) or a component thereof, on at least one INKT cell.

6. The INKT cell specific engager according to claim 5, wherein the iNKT cell specific engager specifically binds to TCRVpi l, TCRVa24 and / or TCRVa24Jal8 CDR3 of the iTCR on at least one INKT cell.

7. The INKT cell specific engager according to claim 5 or claim 6, wherein the iNKT cell specific engager specifically binds to TCRVpil and / or TCRVa24 of the iTCR on at least one INKT cell.

8. The INKT cell specific engager according to any preceding claim, wherein the iNKT cell specific engager specifically binds to an antigen on the at least one INKT cell, wherein the antigen comprises an amino acid sequence substantially as set out in any of SEQ ID No: 1, 3 and / or 5, or a fragment or variant thereof, and / or wherein the antigen is encoded by a nucleotide sequence substantially as set out in any of SEQ ID No: 2 and / or 4, or a fragment or variant thereof.

9. The iNKT cell specific engager according to any preceding claim, wherein the iNKT cell specific engager specifically binds to the TCRVpil of the at least one INKT cell, optionally wherein the iNKT cell specific engager specifically binds to an antigen on the at least one INKT cell, wherein the antigen comprises an amino acid sequence substantially as set out in SEQ ID No: 1, or a fragment or variantthereof, and / or wherein the antigen is encoded by a nucleotide sequence substantially as set out in SEQ ID No: 2, or a fragment or variant thereof.

10. The iNKT cell specific engager according to any one of claims 3-9, wherein the tumour antigen is selected from a list of surface antigens expressed in tumours such as: a testis cancer, melanoma, lung cancer, head and neck cancer, breast cancer, gastrointestinal cancer, bladder cancer, colorectal cancer, pancreatic cancer, lymphoma, leukaemia, multiple myeloma, renal cancer, hepatic cancer, ovarian cancer, gastric cancer, brain cancer and prostate cancer.

11. The iNKT cell specific engager according to any one of claims 3-10, wherein the tumour antigen is BCMA.

12. The iNKT cell specific engager according to any preceding claim, wherein the iNKT cell specific engager comprises at least two antigen-binding domains, wherein a first antigen-binding domain specifically binds to an antigen on the at least one INKT cell, and a second antigen-binding domain specifically binds to a tumour antigen on the at least one tumour cell.

13. The INKT cell specific engager according to claim 12, wherein the antigen-binding domain comprises a domain selected from the group consisting of: an antibody or antigen-binding fragment thereof, CDR, VL, VH and Fd; Fv, Fab, and Fab'; bivalent fragments such as F(ab')2; single chain Fv (scFv); bivalent or trivalent scFvs, scFv-Fc-scFv, an IgG-scFv, an IgG-dAb, KIH-IgG, K -BODY, KIH-FC Fab / scFv, tri / tetraspecific, KIH tri / tetra-specific, or a tandem scFv, bispecific antibody (BsAb), and Fc fragments.

14. The iNKT cell specific engager according to any preceding claim, wherein the iNKT cell specific engager is polyspecific, wherein the engager is configured to specifically bind to one or more antigen on the iNKT cell, and one or more tumour antigen on the tumour cell.

15. The INKT cell specific engager according to any preceding claim, wherein the iNKT cell specific engager comprises a first antigen-binding domain that specifically binds to an antigen on the at least one INKT cell, and a second antigen-binding domain that specifically binds to a tumour antigen on the at least one tumour cell.

16. The INKT cell specific engager according to any preceding claim, wherein the iNKT cell specific engager comprises a first antigen-binding domain thatspecifically binds to a first antigen on the at least one iNKT cell, a second antigen-binding domain that specifically binds to a tumour antigen on the at least one tumour cell, and a third antigen-binding domain that specifically binds to a second, different antigen on the at least one INKT cell.

17. The INKT cell specific engager according to any preceding claim, wherein the iNKT cell specific engager is bi-specific and monovalent, wherein the iNKT cell specific engager is configured such that one arm can bind to a single antigen on the iNKT cell and the other arm to a single tumour antigen on the tumour cell.

18. The INKT cell specific engager according to any preceding claim, wherein the iNKT cell specific engager is bi-specific and bivalent, wherein the engager is configured to bind such that one arm can bind to two antigens on the iNKT cell (same or two different antigens) and the other arm to two tumour antigens (same or two different antigen) on the tumour cell.

19. The INKT cell specific engager according to any one of claims 12-18, wherein the first antigen-binding domain that specifically binds to an antigen on the at least one INKT cell comprises a single-chain variable fragment (scFv) domain of an anti-iNKT cell antibody, a Fab domain or a single domain Ab and / or the second antigen-binding domain that specifically binds to a tumour antigen on the at least one tumour cell comprises a single-chain variable fragment (scFv) domain of an anti-tumour cell antibody, a Fab domain or a single domain Ab.

20. The iNKT cell specific engager according to any preceding claim, wherein the iNKT cell specific engager comprises a single chain Fv (scFv) comprising a fusion of the variable regions of the heavy (VH) and light chains (VL) connected with a short linker peptide, and wherein the engager comprises two scFv linked together.

21. The iNKT cell specific engager according to any one of claims 12-20, wherein the iNKT cell engager comprises an antigen-binding domain comprising an amino acid sequence substantially set out as SEQ ID No: 6, 8, or 9, or a variant or fragment thereof.

22. The iNKT cell specific engager according to any one of claims 12-21, wherein the iNKT cell engager comprises an antigen-binding domain comprising an amino acid sequence substantially set out as SEQ ID No: 10, 11, or 12, or a variant or fragment thereof.

23. The iNKT cell specific engager according to any preceding claim, wherein the iNKT cell engager comprises a polypeptide comprising an amino acid sequence substantially set out as SEQ ID No: 13, or a variant or fragment thereof, and / or is encoded by a nucleotide sequence substantially set out as SEQ ID No: 18, or a variant or fragment thereof.

24. The iNKT cell specific engager according to any preceding claim, wherein the iNKT cell engager comprises a polypeptide comprising an amino acid sequence substantially set out as SEQ ID No: 14, or a variant or fragment thereof, and / or is encoded by a nucleotide sequence substantially set out as SEQ ID No: 19, or a variant or fragment thereof.

25. The iNKT cell specific engager according to any preceding claim, wherein the iNKT cell engager comprises an Fc region comprising an amino acid sequence substantially set out as SEQ ID No: 15, or a variant or fragment thereof.

26. The iNKT cell specific engager according to claim 25, wherein the antigen-binding domain is connected to the Fc region by a linker, optionally wherein the linker comprises SEQ ID No: 16, or a fragment or variant thereof.

27. The iNKT cell specific engager according to any one of claims 22-26, wherein the iNKT cell engager comprises (i) a first polypeptide comprising an amino acid sequence substantially set out as SEQ ID No: 13, or a variant or fragment thereof, and / or is encoded by a nucleotide sequence substantially set out as SEQ ID No: 18, or a variant or fragment thereof, and (ii) a second polypeptide comprising an amino acid sequence substantially set out as SEQ ID No: 14, or a variant or fragment thereof, and / or is encoded by a nucleotide sequence substantially set out as SEQ ID No: 19, or a variant or fragment thereof, preferably wherein the polypeptides dimerise to form a heterodimer.

28. The iNKT cell specific engager according to any one of claims 1 to 19, wherein the iNKT cell specific engager comprises two single chain Fv (scFv), specific for a first antigen, comprising a fusion of the variable regions of the heavy (VH) and light chains (VL) connected with a short linker peptide, and two Fab domains, specific for a second antigen, comprising a fusion of the variable regions of the heavy (VH) and light chains (VL).

29. The iNKT cell specific engager according to claim 28, wherein the iNKT cell engager comprises an antigen-binding domain comprising an amino acidsequence substantially set out as SEQ ID No: 20, 22, or 23, or a variant or fragment thereof.

30. The INKT cell specific engager according to claim 28 or 29, wherein the iNKT cell engager comprises an antigen-binding domain comprising an amino acid sequence substantially set out as SEQ ID No: 24 or 25, or a variant or fragment thereof.

31. The iNKT cell specific engager according to any one of claims 28-30, wherein the iNKT cell engager comprises a polypeptide comprising an amino acid sequence substantially set out as SEQ ID No: 26, or a variant or fragment thereof, and / or is encoded by a nucleotide sequence substantially set out as SEQ ID No: 32, or a variant or fragment thereof.

32. The iNKT cell specific engager according to any one of claims 28-31, wherein the iNKT cell engager comprises a polypeptide comprising an amino acid sequence substantially set out as SEQ ID No: 27, or a variant or fragment thereof, and / or is encoded by a nucleotide sequence substantially set out as SEQ ID No: 33, or a variant or fragment thereof.

33. The iNKT cell specific engager according to any one of claims 28-32, wherein the iNKT cell engager comprises an Fc region, which may be derived from or comprise an IgG domain, an IGA domain, or an IGM domain.

34. The iNKT cell specific engager according to any claim 33, wherein the iNKT cell engager comprises an Fc region comprising an amino acid sequence substantially set out as SEQ ID No: 28 and / or 30, or a variant or fragment thereof.

35. The iNKT cell specific engager according to any one of claims 30-34, wherein the iNKT cell engager comprises (i) at least one polypeptide comprising an amino acid sequence substantially set out as SEQ ID No: 26, or a variant or fragment thereof, and / or is encoded by a nucleotide sequence substantially set out as SEQ ID No: 32, or a variant or fragment thereof, and (ii) at least one polypeptide comprising an amino acid sequence substantially set out as SEQ ID No: 27, or a variant or fragment thereof, and / or is encoded by a nucleotide sequence substantially set out as SEQ ID No: 33, or a variant or fragment thereof, preferably wherein the at least two polypeptides dimerise to form a homodimer.

36. A nucleic acid encoding the iNKT cell specific engager according to any one of claims 1-35.

37. An expression vector or a plasmid encoding the iNKT cell specific engager according to any one of claims 1-35, or comprising the nucleic acid according to claim 36.

38. A pharmaceutical composition comprising a therapeutically effective amount of the iNKT cell specific engager according to any one of claims 1-35, the nucleic acid according to claim 36, or the vector according to claim 37, and a pharmaceutically acceptable excipient.

39. The pharmaceutical composition according to claim 38, comprising one or more iNKT or CAR-iNKT cell, optionally wherein the one or more iNKT or CAR-iNKT cell is allogeneically sourced or adoptively transferred.

40. The pharmaceutical composition according to either claim 38 or claim 39, comprising one or more TCR-iNKT cell, optionally wherein the one or more TCR- iNKT cell is allogeneically sourced or adoptively transferred.

41. The INKT cell specific engager according to any one of claims 1-35, the nucleic acid according to claim 36, the vector according to claim 37, or the pharmaceutical composition according to any one of claims 38-40, for use in therapy or diagnosis.

42. The iNKT cell specific engager according to any one of claims 1-35, the nucleic acid according to claim 36, the vector according to claim 37, or the pharmaceutical composition according to any one of claims 38-40, for use in (i) immunotherapy; (ii) for treating, preventing or ameliorating cancer; (iii) for treating, preventing or ameliorating an autoimmune disease; or (iv) for treating, preventing or ameliorating any infectious disease, including viral disease, such as HIV infection.

43. The iNKT cell specific engager, nucleic acid, vector or pharmaceutical composition, for use according to claim 42, for treating, preventing or ameliorating :(i) a malignancy, which may be a solid tumour or a liquid tumour;(ii) myeloid or lymphoid leukaemias, B cell and T cell lymphomas, plasma cell dyscrasias and for solid tumours cancers of the brain, respiratory tract, head and neck, or skin etc.;(iii) any disease caused by pathogenic B or T cells, such as autoimmune disease; or(iv) any infectious disease, including viral disease, such as HIV infection.

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