Invariant natural killer t cell

Abstract

The present invention relates to invariant natural killer T (iNKT) cells, and particularly, although not exclusively, to cord blood INKT cells, which have anti-cancer activity. The invention includes CD4-positive enriched or CD4-negative enriched cord blood INKT cells. The invention extends to novel methods for producing such enriched cord blood INKT cells, as well as pharmaceutical compositions comprising these iNKT cells, and to their use in therapy, as well as therapies and methods for treating, preventing, or ameliorating cancer, autoimmune, and alloimmune disease and infections.

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 cord blood INKT cells, which have anti-cancer activity. The invention includes CD4-positive enriched or CD4-negative enriched cord blood INKT cells. The invention extends to novel methods for producing such enriched cord blood INKT cells, as well as pharmaceutical compositions comprising these INKT cells, and to their use in therapy, as well as therapies and methods for treating, preventing, or ameliorating cancer, autoimmune, and alloimmune disease and infections.

[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 TCRVa24Jal8 pairing with a TCRVpi l chain1, and are restricted by the non-polymorphic, glycolipid-presenting, MHC class I-like molecule CDld2. Functionally, INKT cells have features of both innate and adaptive immunity, and possess effector, as well as immunoregulatory, activity3. Their notable direct and indirect anti-tumour activity is mediated by CDld-dependent and CDld-independent mechanisms, including direct killing of tumour cells, antigen presenting cell (APC) maturation, and activation of tumour-specific T and NK cells4'5.

[0004] In addition, while only the CDR2p region of the TCRVpil chain makes contact with the a2 helix of CDld, the CDR3p region sequence is important for the tuning of the overall affinity of the invariant TCR and, therefore, the avidity of iNKT cells in response to exogenous and, importantly, endogenous CDld-presented lipids, including those in CDld-expressing cancers cells. As such, a monoclonal TCR repertoire having a nonvaried affinity for endogenous, tumour-associated lipids limits the anti-tumour efficacy of iNKT-based immunotherapy. iNKT cells comprise CD4+ and CD4- subsets, with the former displaying a ThO / 2 phenotype while the latter display Thl polarisation, with higher expression of IFNy and cytolytic granules, and enhanced anti-tumour activity, with CD4-CD8+ iNKT cell displaying the highest cytotoxic potential6-8. Importantly, in pre-clinical models, INKT cells deplete immunosuppressive, CDld-expressing, tumour-associated myeloid cells9'10, while extensive pre-clinical and clinical data, including from the inventors' lab, show that allogeneic iNKT cells do not cause acute graft-versus-host disease (aGVHD), in fact they protect from it11'12.

[0005] The inventors have previously shown that CD4- INKT are more powerful than their CD4+ counterparts in suppressing T cell alloreactivity as assessed for example in mixed lymphocyte reaction assays (MLR)11. In line with this, a higher dose of CD4- iNKT in the donor graft is associated with lower risk of clinically significant aGVHD11; hence their development by the inventors as an 'off-the-shelf' immunotherapy platform.

[0006] The inventors have also previously shown that in experimental B non-Hodgkin lymphoma (B-NHL), peripheral blood (PB) CAR-iNKT outperform CAR-T, achieving improved survival and tumour free survival13. Notably, CAR-iNKT, but not CAR-T, could also eradicate brain lymphoma. The inventors have also shown that PB iNKT equipped with CAR targeting clonotypic TCRVP chains effectively control experimental T cell lymphoma14.

[0007] An ongoing phase I clinical trial has reported 60% CR in relapsed / refractory B-NHL; remarkably, without evidence of aGVHD or CAR-related cytokine release syndrome and neurotoxicity15. Similarly, treatment of patients with respiratory COVID-19 with hundreds of millions of allogeneic iNKT cells was not associated with any significant adverse effects16. Therefore, unlike allogeneic CAR-T, which require additional gene editing of their TCR to mitigate risk of aGVHD, there is no need for deletion of the iTCR in iNKT cells; on the contrary, as the inventors and others have previously shown, the iTCR contributes to the anti-tumour effect of CAR-iNKT cells directly or indirectly13'17.

[0008] There are, however, limitations to existing peripheral blood (PB) iNKT cells (and T cells), and thus PB CAR-iNKT cells, and their use in immunotherapy. For example, the majority (>80-90%) of healthy humans are carriers of latent DNA viruses such as EBV, CMV, HHV6 and HHV7, which can, during manufacturing, reactivate and infect previously uninfected iNKT cells. Indeed, while adult peripheral blood (PB) carries latent viruses in >80-90% of cases, latent viruses can be detected in only <2-3% of cord blood (CB) units.

[0009] In a survey of 352 CB units by PCR to detect viral DNA, the incidence of EBV, CMV, and HHV6, 7, and 8 was < 1%23. In another study, the incidence of CMV detection in CB was <0.4%24and of HHV8, 1.3%25. This contrasts with the incidence of these viruses in adult life, which is >80-90% each. Therefore, in terms of a virus-free source of iNKT cells, use of CB would be advantageous because, as described recently for autologous and allogeneic CAR-T cells, this could result in additional latent virus-related morbidity, such as neurotoxicity26'27.

[0010] By contrast, infection of cord blood by latent viruses is very low, thus making it a more readily available, virus-free, and safer source for iNKT than peripheral blood iNKT cells. Furthermore, the longer telomeres and greater telomerase activity in cord blood than peripheral blood T cells suggest higher proliferative potential in the former, including in cord blood iNKT cells28.

[0011] There is, therefore, a need to provide an improved platform technology for immunotherapy of cancer that overcomes the limitations associated with PB iNKT cells. There is also a need to increase the effectiveness of iNKT-based immunotherapy.

[0012] In order to address the above problems, the inventors explored the development of cord blood (CB) iNKT cells as an immunotherapy platform for cancer and compared it to PB iNKT cells.

[0013] Cord blood (CB) iNKT cells are memory T cells, derived from the umbilical cord and placenta, comprising mostly (>95%) CD4+ iNKT cells18'19. This contrasts with PB, in which iNKT CD4+ vs CD4- subsets are 40% vs 60%, although, in given healthy individuals, frequency of the CD4- subset can be as high as 90% of all iNKT cells. From a developmental perspective, while both CD4- and CD4+ iNKT cells are thought to be selected in the thymus by CDld-expressing CD4+CD8+ T cell thymocytes, CD4+ iNKT cells differentiate and mature in the thymus before they migrate to the periphery20'21. By contrast, CD4- iNKT cells migrate to the periphery, where they complete their differentiation and maturation, in a CDld-dependent manner22. The fact that PB iNKT cells have a considerably higher level of T-cell receptor excision circles (TREC) dilution than their CD4+ counterparts22suggests that they undergo considerable proliferative expansion early in life, so that in later life, in the majority of healthy individuals, they comprise the main iNKT cell subset.

[0014] As discussed, CD4- iNKT cells display Thl polarisation, with higher expression of IFNy and cytolytic granules, and enhanced anti-tumour activity, with CD4-CD8+ iNKT cell displaying the highest cytotoxic potential6-8. However, as CD4- iNKT cells18'19make up <5% of CB, it is challenging to simply isolate such cells for subsequent use in immunotherapy. On the other hand, there is need to be able to polarise the ThO / 2 cytokine profile of CD4+ CB iNKT cells towards a Thl profile in order to render this subset of CB iNKT cells suitable for anti-cancer therapies. The inventors have developed a bespoke and innovative method for the generation and expansion of CD4-negative enriched CB iNKT cells and CD4-positive enriched CB iNKT cells. Furthermore, the inventors have demonstrated that such cells have an inherently higher anti-cancer activity over both their PB CD4-negative and CD4+ CB counterparts, and that CD4-negative enriched CB iNKT cells suppress alloreactivity. As such, the inventors have discovered a novel, off-the-shelf immunotherapy, in the form of CD4- negative or CD4-positive enriched cord blood iNKT cells, which may be deployed as a platform technology in conjunction with module technologies, such as CAR, TCR, and / or iNKT cell engagers. Furthermore, the inventors have surprisingly shown that CB iNKT cells (either CD4-positive or CD4-negative) can be efficiently transduced with anti-CD19 and anti-BCMA CARs and subsequently expanded, and that such CB CAR iNKT cells are equally effective as their PB counterparts.

[0015] Thus, according to a first aspect of the invention, there is provided a population comprising either CD4-negative enriched cord blood invariant natural killer T (iNKT) cells or CD4-positive enriched cord blood invariant natural killer T (iNKT) cells.

[0016] The term "CD4-" or "CD4-negative" can mean that an invariant natural killer T (iNKT) cell lacks and / or does not express the cluster of differentiation 4 (CD4) glycoprotein. It will be appreciated that CD4 serves as a co-receptor for the T-cell receptor (TCR). It will also be appreciated that CD4-negative invariant natural killer T (iNKT) cells display Thl polarisation, and have higher expression of interferon-gamma (IFNy) and cytolytic granules than CD4-positive iNKT cells (i.e., invariant natural killer T (iNKT) cells which do express CD4). Typically, therefore, the CD4-negative enriched cord blood invariant natural killer T (iNKT) cells of the first aspect do not express CD4.

[0017] The term "CD4+" or "CD4-positive" can mean that an invariant natural killer T (iNKT) cell comprises and / or expresses the cluster of differentiation 4 (CD4) glycoprotein. It will be appreciated that CD4 serves as a co-receptor for the T-cell receptor (TCR). It will also be appreciated that CD4-positive invariant natural killer T (iNKT) cells display a ThO / 2 phenotype. Typically, therefore, the CD4-positive enriched cord blood invariant natural killer T (iNKT) cells of the first aspect do express CD4.

[0018] It will be appreciated that although only the CDR2p region of TCRVpil makes contact with the a2 helix of CDld, the CDR3p region sequence is considered important for the tuning of the overall affinity of the invariant TCR and, therefore, the avidity of iNKT cells in response to exogenous and, importantly, endogenous CDld-presented lipids, including those in CDld-expressing cancers cells. The inventors believe, therefore, that in contrast to a monoclonal, non-varied TCRpilCDR3 repertoire, using a highly diverse TCRpilCDR3 repertoire that includes multiple clones with varied affinity for endogenous, tumour-associated lipids, as described herein, would significantly enhance the antitumour efficacy of iNKT-based immunotherapy, while simultaneously preserving the low affinity 'auto-reactive' interaction of iNKT cells with physiological lipid ligands on CDld- expressing antigen presenting cells (APC). This iNKT-APC interaction provides the iNKT cells with beneficial survival signals. The inventors believe, therefore, that iNKT-based immunotherapy would be more effective if it included a diverse, polyclonal TCRpilCDR3 repertoire rather than a single TCRVpil CDR3 chain.

[0019] Accordingly, in an embodiment, the CD4-negative enriched cord blood invariant natural killer T (iNKT) cells or the CD4-positive enriched cord blood invariant natural killer T (iNKT) cells comprise a diverse TCRVp repertoire, optionally a diverse TCRVpi l repertoire.

[0020] Advantageously, having a diverse TCR repertoire results in the cell population exhibiting different affinities and cell survival signals resulting in enhanced immunotherapy.

[0021] The CD4-negative enriched cord blood invariant natural killer T (iNKT) cells or the CD4- positive enriched cord blood invariant natural killer T (iNKT) cells may comprise a diverse TCRVpil CDR3p chain.

[0022] The CD4-negative enriched cord blood invariant natural killer T (iNKT) cells or the CD4- positive enriched cord blood invariant natural killer T (iNKT) cells may comprise two or more TCRVp chain types, optionally two or more TCRVpil CDR3 chain types.

[0023] The term "chain type" can mean the sequence or species of TCRVpil. Thus, the CD4- negative enriched cord blood invariant natural killer T (iNKT) cells or the CD4-positive enriched cord blood invariant natural killer T (iNKT) cells may comprise a plurality of different TCRVp chains each having a different sequence, optionally a plurality of different TCRVpil CDR3 chains each having a different sequence.

[0024] The CD4-negative enriched cord blood invariant natural killer T (iNKT) cells or the CD4- positive enriched cord blood invariant natural killer T (iNKT) cells may comprise five, 10 or 15 or more TCRVp chain types, optionally five, 10 or 15 or more TCRVpil CDR3 chain types. The CD4-negative enriched cord blood invariant natural killer T (INKT) cells or the CD4- positive enriched cord blood invariant natural killer T (INKT) cells may comprise 50, 100, 200 or 500 or more TCRVp chain types, optionally 50, 100, 200 or 500 or more TCRVpil CDR3 chain types.

[0025] The CD4-negative enriched cord blood invariant natural killer T (INKT) cells or the CD4- positive enriched cord blood invariant natural killer T (INKT) cells may comprise 750, 800, 900 or 1000 or more TCRVp chain types, optionally 750, 800, 900 or 1000 or more TCRVpil CDR3 chain types.

[0026] The CD4-negative enriched cord blood invariant natural killer T (INKT) cells or the CD4- positive enriched cord blood invariant natural killer T (INKT) cells may comprise 1100, 1200, 1300 or 1400 or more TCRVp chain types, optionally 1100, 1200, 1300 or 1400 or more TCRVpi l CDR3 chain types.

[0027] The population may comprise polyclonal TCRVpil CD4-negative enriched cord blood invariant natural killer T (INKT) cells or polyclonal TCRVpil CD4-positive enriched cord blood invariant natural killer T (INKT) cells. The population may comprise either polyclonal TCRVpil (i.e. diverse CDR3p chain) CD4-negative enriched cord blood invariant natural killer T (INKT) cells or polyclonal TCRVpil (i.e. diverse CDR3p chain) CD4-positive enriched cord blood invariant natural killer T (INKT) cells.

[0028] The term "enriched" can mean isolated from a larger heterogeneous sample. In the same manner, the term "enriched" can mean purified cells of interest from a sample composed of a variety of different cell types. As such, the concentration of an "enriched" cell type of interest is increased, or elevated, from its former concentration in a sample.

[0029] Typically, therefore, the CD4-negative enriched cord blood invariant natural killer T (INKT) cells in the population of the first aspect are purified from a heterogeneous sample such to increase their concentration. Furthermore, typically, the CD4-positive enriched cord blood invariant natural killer T (INKT) cells in the population of the first aspect are purified from a heterogeneous sample so as to increase their concentration.

[0030] In one embodiment, enrichment of the cord blood INKT cells in the population is such that more than 3% of the INKT cells are CD4-negative INKT cells. Suitably, enrichment of the cord blood INKT cells in the population is such that more than 5%, 10%, 15% or 20% of the INKT cells are CD4-negative INKT cells. More suitably, enrichment of the cord blood INKT cells in the population is such that more than 25%, 30%, 35% or 40% of the INKT cells are CD4-negative INKT cells. Even more suitably, enrichment of the cord blood INKT cells in the population is such that more than 45%, 50%, 55% or 60% of the INKT cells are CD4-negative INKT cells. Typically, enrichment of the cord blood INKT cells in the population is such that more than 65%, 70%, 75% or 80% of the INKT cells are CD4-negative INKT cells. It will be appreciated that the level of enrichment of the CD4- negative INKT cells in the population can be determined by staining for INKT cell markers, CD4, CD8, and assessed by flow-cytometry.

[0031] In one embodiment, the enrichment of the cord blood INKT cells in the population is such that between 10 and 90% of all INKT cells are CD4-negative INKT cells. Typically, the enrichment of the cord blood INKT cells in the population is such that between 35% and 60% of all INKT cells are CD4-negative INKT cells.

[0032] It will be appreciated that the rest of the population comprising CD4-negative enriched cord blood invariant natural killer T (INKT) cells of the first aspect comprises CD4- positive cord blood INKT cells.

[0033] In one embodiment, therefore, the population comprising CD4-negative enriched cord blood invariant natural killer T (INKT) cells of the first aspect comprises CD4-positive cord blood INKT cells. Typically, the enrichment of the cord blood INKT cells in the population is such that between 35% and 60% of all INKT cells are CD4-negative INKT cells, and the rest of the population comprises CD4-positive cord blood INKT cells.

[0034] In another embodiment, enrichment of the cord blood INKT cells in the population is such that more than 15% of the INKT cells are CD4-positive INKT cells. Suitably, enrichment of the cord blood INKT cells in the population is such that more than 20%, 30%, 40% or 50% of the INKT cells are CD4-positive INKT cells. More suitably, enrichment of the cord blood INKT cells in the population is such that more than 60%, 65%, 70% or 75% of the INKT cells are CD4-positive INKT cells. Even more suitably, enrichment of the cord blood INKT cells in the population is such that more than 80%, 85%, 90% or 95% of the INKT cells are CD4-positive INKT cells.

[0035] Typically, enrichment of the cord blood INKT cells in the population is such that more than 96% of the INKT cells are CD4-positive INKT cells. More typically, enrichment of the cord blood INKT cells in the population is such that more than 97% of the INKT cells are CD4-positive INKT cells. Even more typically, enrichment of the cord blood INKT cells in the population is such that more than 98% of the INKT cells are CD4-positive INKT cells. Typically, enrichment of the cord blood INKT cells in the population is such that more than 99% of the INKT cells are CD4-positive INKT cells. It will be appreciated that the level of enrichment of the CD4-positive INKT cells in the population can be determined by staining for INKT cell markers, CD4, CD8, and assessed by flow-cytometry.

[0036] In one embodiment, the enrichment of the cord blood INKT cells in the population is such that between 10-100% of all INKT cells are CD4-positive INKT cells. Typically, the enrichment of the cord blood INKT cells in the population is such that between 80% and 99%, or between 95% and 99%, or between 96% and 99%, or between 97% and 99%, or between 98% and 99%, of all INKT cells are CD4-positive INKT cells.

[0037] It will be appreciated that the rest of the population comprising CD4-positive enriched cord blood invariant natural killer T (INKT) cells of the first aspect comprises CD4- negative cord blood INKT cells.

[0038] In one embodiment, therefore, the population comprising CD4-positive enriched cord blood invariant natural killer T (INKT) cells in the population of the first aspect comprises CD4-negative cord blood INKT cells. Typically, the enrichment of the cord blood INKT cells in the population is such that between 80% and 99% or between 95% and 99% of all INKT cells are CD4-positive INKT cells, and the rest of the population comprises CD4- negative cord blood INKT cells.

[0039] In one embodiment, the population comprising either CD4-negative enriched cord blood invariant natural killer T (INKT) cells or CD4-positive enriched cord blood INKT cells of the first aspect may be cell sorted subsequent to their enrichment.

[0040] It will be appreciated that cell sorting is the isolation of a particular cell population from a mixed population, which may be the enriched population. As such, a cell sorted population contains increased, or elevated, concentrations of a cell type of interest over its former concentration in a sample.

[0041] Typically, the cell sorting of the population comprises positive selection, depletion, and / or negative selection.

[0042] It will be appreciated that "positive selection" is when the cell type of interest is targeted by the removal mechanism, typically performed by targeting a cell surface marker with a monoclonal antibody. It will be appreciated that "depletion" is when a single unwanted cell type is targeted and removed from a biological sample. It will be appreciated that "negative selection", similar to depletion, is when several unwanted cell types are removed to leave one cell type of interest untouched.

[0043] More typically, the cell sorting comprises positive selection.

[0044] It will be appreciated that cell sorting positive selection approaches include fluorescence- activated cell sorting (FACS), magnetic-activated cell sorting (MACS™), immunomagnetic cell sorting, and buoyancy-activated cell sorting (BACS™).

[0045] Typically, therefore, the cell sorting of the population comprises fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS™), and / or buoyancy-activated cell sorting (BACS™).

[0046] Most typically, the method of sorting comprises immunomagnetic cell sorting or fluorescence-activated cell sorting (FACS).

[0047] Typically, therefore, the cell sorting of the population comprising CD4-negative enriched cord blood invariant natural killer T (iNKT) cells may achieve a concentration of the cord blood INKT cells that is between 36% and 100% of all INKT cells which are CD4-negative iNKT cells.

[0048] Typically, therefore, the cell sorting of the population comprising CD4-positive enriched cord blood invariant natural killer T (INKT) cells may achieve a concentration of the cord blood INKT cells that is between 81% and 100% of all INKT cells which are CD4-positive iNKT cells.

[0049] The term "cord blood" can mean blood derived from the umbilical cord and placenta. Typically, therefore, the CD4-negative enriched or CD4-positive enriched cord blood invariant natural killer T (INKT) cells in the population of the first aspect are derived from cord blood.

[0050] More typically, the CD4-negative enriched or CD4-positive enriched cord blood invariant natural killer T (INKT) cells in the population of the first aspect are enriched from a heterogeneous sample of cord blood.

[0051] In a second aspect of the invention, there is provided a method for producing a population of either CD4-negative enriched cord blood invariant natural killer T (INKT) cells or CD4-positive enriched cord blood INKT cells, the method comprising contacting cord blood iNKT cells with one or more cytokine, to thereby enrich either the CD4- negative iNKT cell population or the CD4-positive cord blood INKT cell population.

[0052] The method may be carried out in vitro, ex vivo, or in vivo. However, in an embodiment, the method is carried out in vitro. The level of enrichment of either the CD4-negative enriched cord blood invariant natural killer T (iNKT) cells or CD4-positive enriched cord blood iNKT cells may be as defined in the first aspect.

[0053] In one embodiment, the one or more cytokine may be selected from a group of cytokines consisting of: interleukin (IL)-2, IL-15, IL-7, IL-12, and IL-21.

[0054] In an embodiment, however, the method according to the second aspect comprises contacting cord blood iNKT cells with one or more cytokine to produce a population of CD4-negative enriched cord blood invariant natural killer T (iNKT) cells, wherein the one or more cytokine is IL-2 and / or IL-21. Typically, the method comprises contacting cord blood iNKT cells with one cytokine to produce a population of CD4-negative enriched cord blood invariant natural killer T (iNKT) cells, wherein the cytokine is either IL-2 or IL- 21. Typically, however, the method comprises contacting cord blood iNKT cells with more than one cytokine to produce a population of CD4-negative enriched cord blood invariant natural killer T (iNKT) cells, wherein the cytokines are IL-2 and IL-21.

[0055] In another embodiment, the method according to the second aspect comprises contacting cord blood iNKT cells with one or more cytokine to produce a population of CD4-positive enriched cord blood invariant natural killer T (iNKT) cells, wherein the cytokine is IL-15, or IL-2 and / or IL-21. Typically, however, the method according to the second aspect comprises contacting cord blood iNKT cells with one or more cytokine to produce a population of CD4-positive enriched cord blood invariant natural killer T (iNKT) cells, wherein the one or more cytokine is IL-15, or IL-2 and IL-21.

[0056] In one embodiment, the concentration of IL-15 is at least 200 ng / ml, or at least 400 ng / ml, or at least 600 ng / ml, or at least 700 ng / ml. Typically, the concentration of IL-15 is at least 720 ng / ml, or at least 735 ng / ml, or at least 750 ng / ml.

[0057] In one embodiment, the concentration of IL-2 is at least 200 ng / ml, or at least 400 ng / ml, or at least 600 ng / ml, or at least 700 ng / ml. Typically, the concentration of IL-15 is at least 720 ng / ml, or at least 735 ng / ml, or at least 750 ng / ml. In one embodiment, the concentration of IL-21 is at least 1 ng / ml, or at least 5 ng / ml, or at least 10 ng / ml, or at least 12 ng / ml. Typically, the concentration of IL-15 is at least 12.1 ng / ml, or at least 12.3 ng / ml, or at least 12.5 ng / ml.

[0058] In one embodiment, the concentration of IL-12 is at least 0.5 ng / ml, or at least 1 ng / ml, or at least 2 ng / ml, or at least 4 ng / ml. Typically, the concentration of IL-15 is at least 4.5 ng / ml, or at least 4.8 ng / ml, or at least 5 ng / ml.

[0059] In one embodiment, the ratio of IL-2 : IL-21 is between 20: 1 and 100 : 1, or between 30: 1 and 90: 1, or between 40: 1 and 80: 1, or between 50: 1 and 70: 1. Typically, the ratio of IL-2:IL-21 is 60: 1 ng / ml.

[0060] In one embodiment, the ratio of IL-2: IL- 15 is between 0.1 : 1 and 2: 1, or between 0.25: 1 and 1.75: 1, or between 0.5: 1 and 1.5: 1, or between 0.75: 1 and 1.25: 1. Typically, the ratio of IL-2 : IL-15 is 1 : 1 ng / ml.

[0061] In one embodiment, the ratio of IL- 15 : IL- 12 is between 80: 1 and 210: 1, or between 110: 1 and 190: 1, or between 130: 1 and 170: 1, or between 140: 1 and 160: 1. Typically, the ratio of IL- 15 : IL- 12 is 150: 1 ng / ml.

[0062] In one embodiment, the ratio of IL- 15 : IL-21 is between 20: 1 and 100: 1, or between 30: 1 and 90: 1, or between 40: 1 and 80: 1, or between 50: 1 and 70: 1. Typically, the ratio of IL- 15 : IL-21 is 60: 1 ng / ml.

[0063] In one embodiment, the method comprises contacting the CD4-negative enriched cord blood invariant natural killer T (iNKT) cells or CD4-positive enriched cord blood iNKT cells with a glycolipid to further enrich the CD4-negative iNKT cell population.

[0064] Typically, the glycolipid is alpha-Galactosylceramide (aGalCer).

[0065] It will be appreciated that a-galactosylceramide (aGalCer) is a synthetic glycolipid derived from structure-activity relationship studies of galactosylceramides isolated from the marine sponge Agelas mauritianus. A-galactosylceramide is a strong immunostimulant, and shows potent anti-tumour activity in many in vivo models. A- galactosylceramide is a potent activator of iNKT cells, and a model CDld antigen. The invariant T cell receptor of the INKT cell is able to bind the CD Id -g lycol i p id complex, leading to iNKT cell activation in both mice and humans. When in combination with a peptide antigen, a-galactosylceramide is able to stimulate a strong immune response against the epitope, and thus has adjuvant activity. The CDld-glycolipid-TCR interaction activates the iNKT cell, which can then activate dendritic cells. This causes the release of a range of cytokines, and licenses the dendritic cell to activate a peptide-specific T cell response. As such, this adjuvant acts through this described cellular interaction, rather than through classic pattern recognition receptor pathways.

[0066] In one embodiment, the method comprises pulsing the CD4-negative enriched cord blood invariant natural killer T (INKT) cells or CD4-positive enriched cord blood INKT cells with the glycolipid.

[0067] The term "pulsing" can mean adding a-galactosylceramide to live or irradiated CDld- expressing cells with antigen presenting features, e.g., peripheral blood or cord blood mononuclear cells, monocytes, macrophages, dendritic cells, or various cell lines expressing CDld (ClR-CDld), ideally for a few hours before the CDld-expressing cells are co-cultured with iNKT cells.

[0068] Typically, therefore, the method comprises expanding the CD4-negative enriched cord blood invariant natural killer T (INKT) cells or CD4-positive enriched cord blood INKT cells with the CDld-epxressing cells pulsed with glycolipid, preferably alpha- Galactosylceramide I.

[0069] In an embodiment, the population may further comprise CD4+ or CD4-positive iNKT cells.

[0070] In a third aspect of the invention, there is provided a population of either CD4-negative enriched cord blood INKT cells or CD4-positive enriched cord blood INKT cells obtained, or obtainable, by the method according to the second aspect.

[0071] It is possible to attach different modules to the CD4-negative or CD4-positive enriched cord blood invariant natural killer T (INKT) cells in the population of the first or third aspect. For example, the CD4-negative or CD4-positive enriched cord blood invariant natural killer T (INKT) cells in the population of the first or third aspect may comprise a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR).

[0072] In one embodiment, the CD4-negative or CD4-positive enriched cord blood invariant natural killer T (INKT) cells in the population of the first or third aspect may comprise a chimeric antigen receptor (CAR). Typically, the CAR is an anti-tumour antigen CAR. The tumour antigen to which the CAR is specific 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.

[0073] 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.

[0074] 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-13 O2, CD171, MUC16, PSMA, AFP, AXL, c-MET, DLL-3, DR5, EpHA2, FRa gplOO, MAGE-A1 / 3 / 4, LMP1, and others.

[0075] 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 of the CD4- negative or CD4-positive enriched cord blood CAR-INKT cells would be derived from an antibody that would specifically bind to the MHC class I / II-peptide complex.

[0076] Such MHC-resented peptides may be derived from:

[0077] (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);

[0078] (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;

[0079] (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);

[0080] (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);

[0081] (e) prostate-associated antigens, such as PAP, PSA, PSMA, PSH-PI, PSM-PI, PSM-P2, associated with e.g., prostate cancer; and / or

[0082] (f) immunoglobulin idiotypes (associated with myeloma and B cell lymphomas.

[0083] As described in the Examples, CD19 and BCMA were used as an antigen for proof-of- concept. Thus, in one embodiment, the CAR comprises an anti-CD19 CAR.

[0084] Accordingly, in one embodiment, the amino acid sequence of the anti-CD19 CAR may be represented herein as SEQ ID No: 1, as follows:

[0085] DIQMTQTTSSLSASLGDRVTI SCRASQDI SKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTI SNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD YGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTI IKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYA MDYWGQGTSVTVSS

[0086] [SEQ ID No: 1] In one embodiment, the anti-CD19 CAR is encoded by the nucleotide sequence represented herein as SEQ ID No: 2, as follows:

[0087] GACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCAGAGCC

[0088] AGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCAC

[0089] ACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATC

[0090] AGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCCTACACCTTCGGAGGC

[0091] GGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGCGGAGGCGGCTCTGAGGTGAAGCTGCAGGAG

[0092] TCTGGCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGAC

[0093] TACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACC

[0094] ACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAG

[0095] ATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTATGGCGGCAGCTACGCT ATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCG

[0096] [SEQ ID No: 2]

[0097] Therefore, in an embodiment, the CD4-negative or CD4-positive enriched cord blood invariant natural killer T (INKT) cells in the population may comprise a chimeric antigen receptor (CAR) which (i) comprises an amino acid sequence substantially as set out in SEQ ID No: 1 or a variant or fragment thereof, and / or (ii) is encoded by a nucleotide sequence substantially as set out in SEQ ID No: 2 or a variant or fragment thereof.

[0098] In another embodiment, the CAR comprises an anti-BCMA CAR. In one embodiment, the amino acid sequence of the anti-BCMA CAR may be represented herein as SEQ ID No: 3, as follows:

[0099] MALPVTALLLPLALLLHAARPDIVLTQSPPSLAMSLGKRATI SCRASESVTILGSHLIHWYQQKPGQPPTLLIQL

[0100] ASNVQTGVPARFSGSGSRTDFTLTI DPVEEDDVAVYYCLQSRTI PRTFGGGTKLEIKGGGGSGGGGSGGGGSQIQ

[0101] LVQSGPELKKPGETVKI SCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSAST

[0102] AYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVS SFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEA

[0103] CRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQ PYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0104] [SEQ ID No: 3]

[0105] In one embodiment, the anti-BMCA CAR is encoded by the nucleotide sequence represented herein as SEQ ID No: 4, as follows:

[0106] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGACATCGTGCTG

[0107] ACACAGTCTCCCCCTAGCCTGGCCATGAGCCTGGGCAAGAGAGCCACCATCAGCTGCCGGGCCTCTGAGAGCGTG

[0108] ACAATCCTGGGCAGCCACCTGATCCACTGGTACCAGCAGAAGCCAGGACAGCCACCCACCCTGCTGATCCAGCTG

[0109] GCCTCCAACGTGCAGACAGGAGTGCCAGCCCGCTTCTCCGGATCTGGCAGCCGGACCGACTTTACCCTGACAATC

[0110] GATCCCGTGGAGGAGGACGATGTGGCCGTGTACTATTGCCTGCAGTCCAGAACCATCCCTAGGACATTCGGCGGC

[0111] GGCACCAAGCTGGAGATCAAGGGAGGCGGAGGCTCTGGAGGCGGAGGCTCTGGAGGCGGAGGCTCTCAGATCCAG

[0112] CTGGTGCAGTCCGGACCAGAGCTGAAGAAGCCTGGCGAGACAGTGAAGATCTCCTGTAAGGCCTCTGGCTACACC

[0113] TTTACAGACTATTCTATCAACTGGGTGAAGAGGGCACCAGGCAAGGGCCTGAAGTGGATGGGCTGGATCAATACC

[0114] GAGACACGGGAGCCTGCCTACGCCTATGACTTCAGAGGCCGGTTCGCCTTTAGCCTGGAGACAAGCGCCTCTACA

[0115] GCCTATCTGCAGATCAACAATCTGAAGTACGAGGATACCGCCACATATTTTTGTGCCCTGGACTACTCTTATGCT ATGGACTATTGGGGCCAGGGCACCAGCGTGACAGTGAGCTCCTTTGTGCCGGTGTTTCTGCCGGCGAAACCGACC ACCACCCCGGCGCCGCGCCCGCCGACCCCGGCGCCGACCATTGCGAGCCAGCCGCTGAGCCTGCGCCCGGAAGCG TGCCGCCCGGCGGCGGGCGGCGCGGTGCATACCCGCGGCCTGGATTTTGCGTGCGATATTTATATTTGGGCTCCT CTTGCTGGTACTTGTGGTGTTCTTCTTCTTTCTCTTGTTATTACTCTGTATTGCAACCATCGCAACCGCAGCAAA CGCAGCCGCCTGCTGCATAGCGATTATATGAACATGACCCCGCGCCGCCCGGGCCCGACCCGCAAACATTATCAG CCGTATGCGCCGCCGCGCGATTTTGCGGCGTATCGCAGCCGCGTGAAGTTTAGCCGGTCCGCCGATGCCCCTGCC TACCAGCAGGGCCAGAATCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAG AGGAGGGGAAGAGATCCCGAGATGGGAGGCAAGCCACGGAGAAAGAACCCCCAGGAGGGCCTGTACAATGAGCTG CAGAAGGACAAGATGGCCGAGGCCTATTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGAT GGCCTGTACCAGGGCCTGAGCACCGCCACAAAGGACACCTATGATGCCCTGCACATGCAGGCCCTGCCACCAAGG

[0116] [SEQ ID No: 4]

[0117] Therefore, in another embodiment, the CD4-negative or CD4-positive enriched cord blood invariant natural killer T (INKT) cells in the population may comprise a chimeric antigen receptor (CAR) which (i) comprises an amino acid sequence substantially as set out in SEQ ID No: 3 or a variant or fragment thereof, and / or (ii) is encoded by a nucleotide sequence substantially as set out in SEQ ID No: 4 or a variant or fragment thereof.

[0118] In another embodiment, the CD4-negative enriched or CD4-positive enriched cord blood invariant natural killer T (INKT) cells in the population of the first or third aspect may comprise a T-Cell Receptor (TCR). In such an embodiment, the CD4-negative enriched or CD4-positive enriched cord blood invariant natural killer T (INKT) cells in the population of the first or third aspect may comprise an agT cell receptor (TCR) against MHC class I and II presented peptides derived from tumour antigens as outlined above. Alternatively, CD4-negative enriched or CD4-positive enriched cord blood invariant natural killer T (INKT) cells in the population may comprise or be engineered to express a tumour- reactive ySTCR.

[0119] TCR-engineered CD4-negative enriched or CD4-positive enriched cord blood invariant natural killer T (INKT) cells in the population could be used for treatment of cancer; in some cases, iNKT cells will be engineered to co-express an anti-cancer TCR, optionally along with one or more CARs.

[0120] In a fourth aspect of the invention, there is a pharmaceutical composition comprising a therapeutically effective amount of either the CD4-negative enriched cord blood INKT cells or the CD4-positive enriched cord blood INKT cells in the population of the first or third aspect, and a pharmaceutically acceptable vehicle.

[0121] CD4+ and CD4-negative enriched cord blood invariant natural killer T (INKT) cells in the population of the first or third aspect may also be engineered to express anti-cancer CARs or TCRs. The iNKT cells in the population of the first or third aspect may be adoptively transferred to recipients following immunodepleting chemotherapy. iNKT cell engagers targeting cancer antigen(s) of interest may be co-transferred either at the same time or soon after adoptive transfer of the iNKT cells.

[0122] Typically, the CD4-negative enriched or CD4-positive enriched cord blood invariant natural killer T (iNKT) cells in the population of the first or third aspect are configured to bind to at least one iNKT cell specific engager.

[0123] In a fifth aspect, there is provided either the CD4-negative enriched cord blood iNKT cells or the CD4-positive enriched cord blood iNKT cells in the population of the first or third aspect, or the pharmaceutical composition of the fourth aspect, for use in therapy or diagnosis.

[0124] In a sixth aspect, there is provided either the CD4-negative enriched cord blood iNKT cells or the CD4-positive enriched cord blood iNKT cells in the population of the first or 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 and alloimmune disease (e.g., acute graft- versus-host disease); or (iv) for treating, preventing or ameliorating any infectious disease, including viral disease, such as HIV infection.

[0125] 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 in a subject; (iii) for treating, preventing or ameliorating an autoimmune or alloimmune disease (e.g., acute graft-versus-host disease) in a subject; or (iv) for treating, preventing or ameliorating any infectious disease, including viral disease, such as HIV infection, in a subject the method comprising administering, or having administered, to a patient in need of such treatment, a therapeutically effective amount of either the CD4-negative enriched cord blood iNKT cells or the CD4-positive enriched cord blood iNKT cells in the population of the first or third aspect, or the pharmaceutical composition of the fourth aspect.

[0126] In one embodiment of the sixth or seventh aspect of the invention, the CD4-negative or CD4-positive enriched cord blood invariant natural killer T (iNKT) cells in the population of the first or third aspect, or the pharmaceutical composition of the fourth aspect, are for use in treating, preventing or ameliorating cancer. Typically, the cancer is a T-cell malignancy, which may be a solid tumour or a liquid tumour. Typically, the CD4-negative or CD4-positive enriched cord blood invariant natural killer T (iNKT) cells in the population of the first or third 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.

[0127] In other embodiments, the CD4-negative or CD4-positive enriched cord blood invariant natural killer T (iNKT) cells in the population of the first or 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.

[0128] In other embodiments, the CD4-negative or CD4-positive enriched cord blood invariant natural killer T (iNKT) cells in the population of the first or 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.

[0129] It will be appreciated that the CD4-negative or CD4-positive enriched cord blood invariant natural killer T (iNKT) cells, 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 CD4-negative enriched cord blood invariant natural killer T (iNKT) cells, or pharmaceutical composition alone), for therapy, typically use in (i) for treating, preventing or ameliorating an autoimmune disease in a subject; or (ii) for treating, preventing or ameliorating any infectious disease, including viral disease, such as HIV infection. Alternatively, the CD4-negative or CD4-positive enriched cord blood invariant natural killer T (iNKT) cells, or pharmaceutical composition according to the invention, may be used in combination with known immunotherapies or for treating disease caused by pathogenic B and T cells, as well as cancers or autoimmune disease. For example, the inventors believe that the CD4-negative or CD4-positive enriched cord blood invariant natural killer T (iNKT) cells in the population of the invention may be used in conjunction with other therapeutic modalities, such as CAR, TCR, and iNKT cell engagers, to enhance the modularity and the efficacy of the CD4-negative or CD4- positive enriched cord blood 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 of the present invention, preceded, or followed by, infusions of INKT cell engagers.

[0130] 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, typically 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.

[0131] 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.

[0132] It will be appreciated that the amount of the CD4-negative or CD4-positive enriched cord blood invariant natural killer T (iNKT) cells, 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.

[0133] 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 either the CD4-negative enriched cord blood invariant natural killer T (iNKT) cells or the CD4-positive enriched cord blood iNKT cells in the population according to the first aspect or third aspect, and a pharmaceutically acceptable vehicle.

[0134] A "subject" may be a vertebrate, mammal, or domestic animal. Typically, the subject is a human being.

[0135] A "therapeutically effective amount" of the CD4-negative or CD4-positive enriched cord blood invariant natural killer T (INKT) cells in the population, 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.

[0136] For example, the therapeutically effective amount of the CD4-negative enriched cord blood invariant natural killer T (iNKT) cells used may be at least 100, 1000, or 10,000 cells. Typically, at least 100,000, or at least 1,000,000 or at least 10,000,000 cells are used.

[0137] 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. Typically, for a successful therapy, the composition comprising the CAR-effector cell is prepared and then delivered as a cell suspension, preferably intravenously.

[0138] 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, typically 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 pressurised compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.

[0139] Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be utilised 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 another appropriate sterile injectable medium.

[0140] 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.

[0141] Suitably, the amino acid / polynucleotide / polypeptide sequence has a sequence identity which is greater than 65%, more suitably greater than 70%, even more suitably greater than 75%, and still more suitably greater than 80% sequence identity to any of the sequences referred to are also envisaged. Suitably, the amino acid / polynucleotide / polypeptide sequence has at least 85% identity with any of the sequences referred to, more suitably at least 90% identity, even more suitably at least 92% identity, even more suitably at least 95% identity, even more suitably at least 97% identity, even more suitably at least 98% identity and, most suitably at least 99% identity with any of the sequences referred to herein.

[0142] 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.

[0143] BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional form and constants.

[0144] 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.

[0145] 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.

[0146] Typically, 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. Typically, 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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 :-

[0152] Figure 1 illustrates the frequency of iNKT cells and their subsets in adult peripheral blood (PB) and cord blood (CB). A) Examples of iNKT frequency and pattern of CD4 and CD8 expression, as assessed by flow-cytometry. B) Overall frequency of iNKT cells in PB and CB T cells; higher frequency of iNKT cells in CB (n = 10 each, p = 0.04). C & D) Cumulative data on CD4 and CD8 iNKT subsets in PB and CB iNKT. Figure 2 illustrates cord blood (CB) vs peripheral blood (PB) CAR-iNKT. A) BCMA CAR transduction of CB iNKT and CAR expression over a period of 18 weeks, as assessed by L-protein staining and flow-cytometry. B & C) 4 hour (B) and 24 hour (C) cytotoxicity by CB and PB BCMA CAR-transduced and untransduced iNKT cells against the BCMA- expressing myeloma cell lines MM1S and H929. D) CD19 CAR transduction of PB and CB INKT cultured with the indicated cytokines. E) 4 hour and 24 hour cytotoxicity by CB and PB CD19 CAR-transduced iNKT cells against the CD19-expressing acute lymphoblastic leukaemia SEM cells.

[0153] Figure 3 illustrates the generation, expansion, and functional characterisation of CD4- negative enriched cord blood (CB) iNKT cells. A) Generation of expanded CD4-CD8+ iNKT from two cord blood units, as per the protocol described in the text, and from PB of a healthy donor. Cells were stained on day 28. B) The inventors' bespoke protocol allows expansion of CD4-negative enriched CB iNKT cells up to 6,000-fold. C) Cytotoxicity (4 hour and 16 hour) of CD4-negative enriched CB iNKT compared to PB counterparts against the B, T, and myeloid lineage ClR-CDld, Jurkat and K562-CDld cells respectively. D) Cytotoxicity of CD4-negative enriched CB iNKT compared with same donor CB CD4+ counterparts against ClR-CDld and K562-CDld cells.

[0154] Figure 4 illustrates the phenotypic characterisation of CD4-negative enriched cord blood (CB) iNKT cells. A) Highly pure iNKT were stained with CDld-aGalCer tetramer (top) or empty CDld (bottom) control tetramer. Both CD4+ and CD4-CD8+ subsets stain with the tetramer. B) CD4-CD8+ CB iNKT highly co-express NKG2D and CD62L.

[0155] Figure 5 illustrates that cord blood (CB) CD4-negative enriched CB iNKT suppress alloreactivity. Impact of CD4-negative iNKT cells from two CB units on PB T cell alloreactivity (two donors) in mixed lymphocyte reaction assays, as assessed by Incucyte zoom real time imaging. Using as responders and irradiated stimulators PBMC from two donors, the inventors used special iNKT from two cord blood units (C9 and CIO) as MLR suppressors against T cells from two donors stimulated by each other's irradiated PBMC. T-cells were isolated using negative selection beads and were stained with IncuCyte Green Dye. Responders and stimulators were mixed in 1 : 1 ratio and CD4-negative enriched CB iNKT were added in different ratios (10: 1, 5: 1, 1: 1) to this mixture of R 8i S at the same time. Images were captured every two hours for five days, and analysed using Cell-to-Cell analysis software which can differentiate between different population of cells. Examples

[0156] The inventors have demonstrated the generation and expansion of CD4-negative enriched or CD4-positive cord blood (CB) iNKT cells through the use of their innovative protocol. Furthermore, the inventors have shown that such cells have an inherently higher activity against the CDld-expressing myeloid B and T lineage cancer cells, used here as a proof-of-concept for cancer in general, over both their PB CD4-negative and CD4-positive CB counterparts, and that CD4-negative enriched CB iNKT cells suppress alloreactivity. The inventors have also shown that CB iNKT cells (either CD4-positive enriched or CD4-negative enriched) can be efficiently transduced with anti-CD19 and - BCMA CARs and subsequently expanded, and that such CB CAR iNKT cells are equally effective as their CD4-negative enriched PB counterparts.

[0157] Materials and Methods iNKT and TCRV011 cell isolation, expansion, and cell culture

[0158] Cord blood mononuclear cells (CBMCs) were isolated from cord blood using Lymphoprep (Stemcell Technologies; catalogue: 07811) and centrifugation at 800g for 37 minutes, 1 acceleration, 1 deceleration at 21°C. iNKT cells were purified from CBMCs using anti-iNKT MicroBeads, human (Miltenyi Biotech; catalogue: 130-094-842) with selection buffer (RPMI1640, 10% FBS, 1% Penicillin-Streptomycin, 0.5% human serum albumin) by passing through LS column (Miltenyi Biotech; catalogue: 130-042-401).

[0159] TCRV311 cells were isolated from CBMCs using anti-PE-Microbeads, human (Miltenyi Biotech; catalogue: 130-048-801) after staining the CBMCs with anti-TCRV[311 antibody conjugated with PE fluorochrome (Beckman Coulter; catalogue: IM2290) and by passing these PE stained and bead attached cells through LS column (Miltenyi Biotech; catalogue: 130-042-401).

[0160] These two cells, i.e.; human cord blood derived iNKT and TCRV311, were co-cultured in 1 : 10 ratio, with a minimum number of TCRV311 cells kept at 100,000 or more, in a 48- well tissue culture plate (Corning, catalogue: 3548), in R10 media (RPIM-1640, 10% Foetal Bovine Serum, 1% Pen / strep, 2% L-Glutamine, 1% sodium Pyruvate, 1% Non- essential Amino acids, 15mM HEPES buffer, 0.05mM Beta Mercaptoethanol) supplemented with IL-2 plus IL-21 cytokines (Premium grade - human IL2; catalogue 130-097-748 and Human IL21; catalogue 130-095-769) and 1:3 feeder cells (Gammairradiated (5Gy) CBMCs of the same donor which were loaded with 200ng / ml alpha- GalCer (KRN7000) five hours before irradiation and mixing with iNKT and TCRV[311 cells).

[0161] These cells were left to expand for 2 weeks in the incubator at 37 °C. Half media (supplemented with cytokines) was changed twice a week. After 2 weeks of expansion, these cells were stimulated with Gamma-irradiated (5Gy) K562-CDld Antigen Presenting Cells, which were loaded with 200 ng / ml alpha-GalCer (KRN7000) five hours before irradiation, in a ratio of 1: 5. These post-stimulated cells were left to expand for another 2 weeks, at which point the purity and CD4-CD8 expression was checked.

[0162] Method for obtaining enriched CD4 positive iNKTs

[0163] 1. Obtain cord blood mononuclear cells (CBMNC) by Ficoll or other type of gradient centrifugation;

[0164] 2. Incubate CBMNC with iNKT cell immunomagnetic beads and sort INKT cells by immunomagnetic bead separation; perform twice to obtain >90% iNKT cell purity of which >90-95% are CD4+ cells;

[0165] 3. Thereafter, CD4-positive INKT cells can be activated in the presence of 1 : 1 to 1 :4-5 irradiated, alpha-galactosylceramide (200ng / ml)-pulsed CBMNC alone, or in combination with CD3 / CD28 activating antibodies, and either IL-15 or IL-2 plus IL-21;

[0166] 4. On day 3, activated iNKT cells are subjected to lentiviral CAR (or TCR, or to introduce any other exogenous molecule that would modify the function of iNKT cells) transduction with cytokines added every 3 days. On day 8-10, lentivirally transduced iNKT are stimulated with alpha-galactosylceramide (100-200ng / ml)- pulsed, CDld expressing antigen presenting cell lines, such as ClR-CDld, Jurkat T cells, K562-CDld (irradiated post alpha-galactosylceramide-pulsing) or myeloid dendritic cells in 1 : 1 to 1 :3 ratio;

[0167] 5. Continue CAR / TCR or other -iNKT cell expansion with addition of cytokine(s) of choice every 3-4 days.

[0168] Flow cytometry analysis of iNKT surface receptors

[0169] Cell surface receptor expression was assessed by using multicolour immunofloresence cytometry. The following monoclonal antibodies were used: Allophycocyanin (APC) - anti-human TCRVpil (Miltnyei Biotech; catalogue: 130-108-733), Brilliant Violet 421 (BV 421) - anti-human TCR Va24-Jal8 (Biolegend ; catalogue: 342916 , APC / Fire™ 750 - anti-human CD3 (Biolegend ; catalogue: 300470), Phycoerythrin (PE) - anti-human CD4 (Biolegend; catalogue: 317410), Fluorescein isothiocyanate (FITC) - anti-human CD8 Biolegend ; catalogue: 344704 ), Brilliant Violet 510 (BV 510) - anti-human CXCR5 (BD Biosciences; catalogue: 563105 ), Brilliant Violet 711 (BV 711 ) - anti-human CD49d (Biolegend; catalogue: 304332), PE-CF594 - anti-human NKG2D (BD Biosciences; catalogue: 562498), Brilliant Violet 711 (BV 711) - anti-human CD62L (BD Biosciences; catalogue: 565040), 7-aminoactinomycin D (7-AAD) - viability staining solution (Biolegend; catalogue: 420404).

[0170] Cytotoxicity assays

[0171] Effector cells (INKT) were incubated with indicated concentrations of target cells. The target cells, such as ClRCDld, K562CDld, Jurkat Cells, myeloma cell line H929, and MM1.S cells, were stained with CellTrace™ Violet dye (Life technology; Catalogue number: C34557) followed by co-culturing with effector cells. After co-incubation of effector cells (INKT) and target cells (H929 or MM1.S) for 4 hours and 24 hours, the cells were stained with 7AAD (Cambridge Bioscience; catalogue number: ANA83201) and the cell death were analysed by flowcytometry.

[0172] IncuCyte Live Cell Analysis (Proliferation, Cytotoxicity and MLR)

[0173] IncuCyte system was used for live cell analysis for phase expansion, cytotoxicity and MLR (Mixed Lymphocyte Reaction). The staining protocol was optimised for INKT, T cells, and various target cells. The following reagents were used: IncuCyte® Annexin V Red Reagent for apoptosis (Sartorius; catalogue: 4641), Poly-L-ornithine (Sigma; catalogue: P4957), IncuCyte® Caspase-3 / 7 Red Apoptosis Assay Reagent (Sartorius; catalogue: 4704), IncuCyte® NucLight Rapid Red Reagent for live-cell nuclear labelling (Sartorius; catalogue: 4717), IncuCyte® Cytotox Green Reagent for counting dead cells (Sartorius ; catalogue: 4633).

[0174] For MLR experiments, untouched T-cells from two different donors were isolated using Pan T-cell selection Kit (Miltenyi Biotech; catalogue: 130-096-535). T-cells from Donor 1 were stained with Cytolight rapid Green Reagent (Sartorius; catalogue: 4705). Responder cells and the T-cells from Donor 2 were Gama irradiated (5 Gy)- Stimulator Cells. The responder and stimulator cells were mixed in a 1 : 1 ratio. iNKT cells (Suppressors) were added to this mix of cells in different ratios. This cell mix was plated in 96 well flat bottom TC plates (Corning; Catalogue: 359) in T-cell culture media (RPMI1640, 10% FBS, 1% Penicillin-Streptomycin). These plates were placed in IncuCyte Incubator, where live images were captured every 2 hours for five days. These images were then analysed using IncuCyte cell by cell analysis software, where stained cells (Responders) were counted and normalized to time 0. These experiments were repeated with different Responders, Stimulators and Suppressors. Data analysis and software

[0175] All the flow cytometry data was analysed using flow Jo version 10.9.0 software, and all the statistical analysis were performed using GraphPad prims software version 9.4.0. SX5 Cell by Cell analysis software was used was used to analyse IncuCyte live cell images and the MLR data generated from IncuCyte.

[0176] Example 1 - Frequency of Cord Blood INKT cells

[0177] The inventors found that INKT frequency is higher in CB than adult PB (mean 0.16% vs 0.09, p = 0.04), and with less variation, as shown in figure 1 A, and B. Importantly, while the mean frequency of CD4-CD8- INKT in PB was 75% of T cells, as previously reported, in CB it comprised only 2.5% of T cells, as shown in figure 1 A, C, and D.

[0178] Example 2 - CD4+ Cord Blood CAR-iNKT cells are as effective as their CD4- Peripheral Blood counterparts in vitro

[0179] The inventors then sought to investigate the feasibility of engineering CB INKT cells with anti-cancer CARs. Using their previously established protocol13, the inventors show that highly selected CB iNKT cells can be efficiently transduced with anti-CD19 and anti-BCMA CARs and subsequently expanded, as shown in Figure 2 A.

[0180] The inventors then compared the cytotoxic potential of such CD4+ CB CAR iNKT cells with that of PB CD4-negative enriched CAR-iNKT cells against BCMA-expressing myeloma or CD19-expressing B acute lymphoblastic leukaemia cells. In 4 hour and 24 hour cytotoxicity assays, the inventors found that CB CAR iNKT cells are equally effective as their PB counterparts, highlighting an inherently potent anti-cancer activity of CB iNKT cells, despite their being almost entirely CD4+, as shown in figure 2 B-E.

[0181] Example 3 - Protocol for preferential expansion of CD4-neaative Cord Blood INKT cells In vivo expansion of CD4-negative iNKT cells from a minority of <2.5% in CB to the dominant subset in adult PB is a process that happens over a matter of years. The inventors sought to emulate and accelerate this process in vitro. For this purpose, the inventors compared the impact of different cytokines and their combinations, as well as of CDld-expressing cell lines as APC.

[0182] Starting with CB mononuclear cells (CBMNC), and after obtaining highly pure CB iNKT cells using 6B11 mAb immunomagnetic bead selection, cells were plated in the presence of IL-2, IL-15, IL-7, IL-12 and IL-21 cytokines (singly or in combination), and alpha- Galactosylceramide (aGalCer)-pulsed, irradiated flow-through CBMNC at an iNKT:CBMNC ratio of 1:2 to 1 : 5. With cytokine(s) added every two to three days, expression pattern of CD4 and CD8 was assessed on days ten to 14. The inventors found that the IL-2+IL- 21 combination was the only one amongst those tested that was associated with expansion of CD4-negative iNKT cells. Additional activation of iNKT cells with aGalCer- pulsed K562-CDld or CDld+ Jurkat cells on days ten to 14 resulted in further expansion of CD4-negative CB iNKT cells, which by day 25 to 30 of the culture comprised up to 60% of all INKT cells, as shown in figure 3 A; remarkably, these CD4-negative CB INKT cells were almost entirely CD8hi. These CD4-CD8+-enriched CB INKT cells expanded up to 6,000-fold over 40 days, and their phenotype was stable for up to 100 days of continued culture, as shown in figure 3 B.

[0183] Example 4 - Cord Blood INKT enriched in CD4-neaative subset exert a higher anti-cancer activity than their Peripheral Blood counterparts

[0184] To investigate the inherent anti-cancer activity of CD4-negative enriched CB iNKT, the inventors compared their cytotoxic activity with CD4-negative enriched PB iNKT cells against cancer cell lines. In 4 hour and 24 hour assays, CD4-negative enriched CB iNKT were considerably more cytotoxic than their PB counterparts against the CDld- expressing myeloid (K562-CDld), B (ClR-CDld), and T (Jurkat) lineage cancer cells, as shown in figure 3 C.

[0185] The inventors further compared CD4-negative-enriched CB iNKT with their CD4+ counterparts from the same CB unit, and found that the former were considerably more cytotoxic against K562-CDld and ClR-CDld cancer cells, as shown in figure 3 D.

[0186] Further immunophenotypic characterisation showed that CD4-negative enriched CB iNKT cells express lower levels of invariant TCR than their CD4+ counterparts, as assessed by CDld / aGalCer tetramer staining23, and the majority express the activating NK cell receptor NKG2D24 and the secondary lymphoid organ homing marker CD62L25, as shown in figure 3 A, B, and C, both previously linked to enhanced anti-tumour potential of INKT cells.

[0187] The inventors conclude that the CD4-negative enriched CB iNKT cells generated by their bespoke and innovative protocol have an inherently higher anti-cancer activity than their PB CD4-negative or CD4+ CB counterparts, and therefore they can be deployed as a highly effective platform for CAR, TCR and bispecific engager-based allogeneic immunotherapeutic approaches.

[0188] Example 5 - Cord Blood INKT enriched in CD4-neaative subset suppress alloreactivitv

[0189] The inventors previously showed that, compared to their CD4+ counterparts, PB CD4- iNKT cells were more effective in suppressing T cell alloreactivity, as assessed by mixed lymphocyte reaction (MLR) assays11. The inventors therefore investigated whether CB iNKT enriched in CD4-negative cells also have MLR suppressive capacity.

[0190] Using as responders and irradiated stimulators PBMC from two donors, the inventors used CD4-negative enriched CB iNKT from two cord blood units (CB9 and CB10) as MLR suppressors against T cells from two donors, stimulated by each other's irradiated PBMC. T cells were isolated using negative selection beads, and were stained with IncuCyte Green Dye. Responders and stimulators were mixed in 1 : 1 ratio, and CD4-negative enriched CB iNKT were added in different ratios (10: 1, 5: 1, 1: 1) to this mixture of R & S at the same time. Images were captured every two hours for five days and analysed using Cell-to-Cell analysis software, which can differentiate between different population of cells.

[0191] The inventors found that, in all four MLR assays, CD4-negative enriched CB iNKT suppressed T cell alloreactivity in a dose-dependent manner, thus highlighting the potential of these cells for prevention and treatment of aGVHD in the context of allogeneic hematopoietic stem cell transplantation, as shown in figure 5.

[0192] Example 6 - Protocol for preferential expansion of CD4-oositive Cord Blood INKT cells The inventors sought to emulate and accelerate the process of expanding CD4-positive expansion and enrichment in vitro. The inventors compared the impact of different cytokines and their combinations, as well as of CDld-expressing cell lines as APC.

[0193] Firstly, cord blood mononuclear cells (CBMNC) were obtained from cord blood by Ficoll or other type of gradient centrifugation. Secondly, the CBMNC were incubated with iNKT cell immunomagnetic beads and iNKT cells were then sorted by immunomagnetic bead separation. This step was performed twice to obtain >90% iNKT cell purity of which >90- 95% were CD4+ cells. The CD4-positive iNKT cells can be activated in the presence of 1 : 1 to 1 :4-5 irradiated, alpha-galactosylceramide (200ng / ml)-pulsed CBMNC alone, or in combination with CD3 / CD28 activating antibodies, and either IL-15 or IL-2 plus IL-21.

[0194] On day 3, activated iNKT cells were subjected to a lentiviral CAR (or a TCR, or to introduce any other exogenous molecule that would modify the function of iNKT cells) transduction with cytokines added every 3 days. On day 8-10, lentivirally transduced iNKT cells were stimulated with alpha-galactosylceramide (100-200ng / ml)-pulsed, CDld expressing antigen presenting cell (APC) lines, such as ClR-CDld, Jurkat T cells, K562- CDld (irradiated post alpha-galactosylceramide-pulsing) or myeloid dendritic cells in 1: 1 to 1:3 ratio. CAR / TCR or other -iNKT cell expansion was continued with the addition of cytokine(s) of choice every 3-4 days.

[0195] Summary

[0196] The inventors have surprisingly demonstrated the generation and expansion of CD4- negative enriched CB iNKT cells through the use of their innovative method involving IL- 2 and IL-21. Such cells have been shown by the inventors to have an inherently higher anti-cancer activity over both their PB CD4-negative and CD4+ CB counterparts, and that CD4-negative enriched CB iNKT cells also suppress alloreactivity. In addition, the inventors have demonstrated the generation and expansion of CD4-positive enriched CB iNKT cells through the use of their innovative method involving either IL-15 or IL-2 plus IL-21. Thus, the inventors have shown that the ThO / 2 cytokine profile of CD4+ CB iNKT cells can be polarised towards a Th l profile in the presence of CDld-expressing antigen presenting cells pulsed with alpha-galactosyceramide or similar glycolipids thus making this subset of CB iNKT cells suitable for anti-cancer therapies.

[0197] Furthermore, the inventors have shown that CB iNKT cells (which are CD4-negative or CD4-positive) can be efficiently transduced with a CAR, for example anti-CD19 and anti- BCMA CARs, or a TCR, and subsequently expanded, and that such CB CAR iNKT cells are equally effective as their PB counterparts. As such, the inventors have discovered a novel, off-the-shelf immunotherapy, in the form of CD4-negative or CD4-positive enriched cord blood iNKT cells. The inventors envisage that these CD4-negative or CD4- positive enriched CB iNKT cells may be deployed as a platform technology in conjunction with therapeutic modalities, such as CAR, TCR, and / or iNKT cell engagers. The inventors also envisage that CB iNKT cells may be engineered / transduced with anti-cancer CARs.

[0198] References

[0199] 1. Dellabona P, Padovan E, Casorati G, Brockhaus M, Lanzavecchia A. An invariant V alpha 24- J 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.

[0200] 2. Kawano T, Cui J, Koezuka Y, et al. CDld-restricted and TCR-mediated activation of valphal4 NKT cells by glycosylceramides. Science. 1997;278(5343) : 1626-1629.

[0201] 3. Godfrey DI, Kronenberg M. Going both ways: immune regulation via CDld-dependent NKT cells. JCIinlnvest. 2004;114(10): 1379-1388.

[0202] 4. Salio M, Silk ID, Jones EY, Cerundolo V. Biology of CD1- and MRl-restricted T cells. Annu Rev Immunol. 2014;32:323-366.

[0203] 5. Fuji! S, Shimizu K, Okamoto Y, et al. NKT cells as an ideal anti-tumor immunotherapeutic. Front Immunol. 2013;4:409.

[0204] 6. 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. 7. Lee PT, Benlagha K, Teyton L, Bendelac A. Distinct functional lineages of human V(alpha)24 natural killer T cells. JExpMed. 2002;195(5):637-641.

[0205] 8. Lin H, Nieda M, Hutton JF, Rozenkov V, Nicol AT Comparative gene expression analysis of NKT cell subpopulations. J Leukoc Biol. 2006;80(l): 164-173.

[0206] 9. 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.

[0207] 10. 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.

[0208] 11. 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.

[0209] 12. 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.

[0210] 13. 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.

[0211] 14. 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.

[0212] 15. 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.

[0213] 16. Hammond TC, Purbhoo MA, Yigit B, et al. Administration of Novel Allogeneic iNKT Cell Infusion (agenT-797) in Patients With Severe COVID-19 Acute Respiratory Distress Syndrome (CARDS) Receiving Venovenous Extracorporeal Membrane Oxygenation (VV-ECMO) Support. American Thoracic Society Journal. 2023.

[0214] 17. Simonetta F, Lohmeyer JK, Hirai T, et al. Allogeneic CAR Invariant Natural Killer T Cells Exert Potent Antitumor Effects through Host CD8 T-Cell Cross-Priming. Clin Cancer Res. 2021;27(21):6054-6064.

[0215] 18. Kadowaki N, Antonenko S, Ho S, et al. Distinct cytokine profiles of neonatal natural killer T cells after expansion with subsets of dendritic cells. JExpMed. 2001;193(10): 1221-1226.

[0216] 19. Dec Vliet HJ, Nishi N, de Gruijl TD, et al. Human natural killer T cells acquire a memory-activated phenotype before birth. Blood. 2000;95(7) :2440-2442.

[0217] 20. Gapin L. Development of invariant natural killer T cells. Curr Opin Immunol. 2016;39:68-74.

[0218] 21. Mao AP, Constantinides MG, Mathew R, et al. Multiple layers of transcriptional regulation by PLZF in NKT-cell development. Proc Natl Acad Sci U S A. 2016;113(27):7602-7607.

[0219] 22. Baev DV, Peng XH, Song L, et al. Distinct homeostatic requirements of CD4+ and CD4- subsets of Valpha24-invariant natural killer T cells in humans. Blood. 2004;104(13):4150-4156.

[0220] 23. Weinberg A, Enomoto L, Li S, Shen D, Coll J, Shpall EJ. Risk of transmission of herpesviruses through cord blood transplantation. Biol Blood Marrow Transplant . 2005 Jan;ll(l) :35-8.

[0221] 24. Albano M, Ciubotariu R, Dobrila L, Tarnawski M, DeLeon M, Watanabe C, Krishnan S, Scaradavou A, Rubinstein P. Cytomegalovirus viral load in cord blood and impact of congenital infection on markers of hematopoietic progenitor cell potency. Transfusion. 2017 Nov;57(11):2768-2774.

[0222] 25. Golchin N , Kheirandish M , Sharifi Z , Samiee S , Kokhaei P , Pourpak Z. Quantification of viral genome in cord blood donors by real time PCR to investigate human herpesvirus type 8 active infection. Transfus Apher Sci. 2015 Dec;53(3):378-80.

[0223] 26. Robert R. Stickels RR, Verboon JM et al,. Latent human herpesvirus 6 is reactivated in CAR T cells Nature 2023 Nov;623(7987):608-615.

[0224] 27. Spanjaart AM, van der Valk FM, van Rooijen G, Brouwer MC, Kersten MJ. Confused about Confusion N Engl J Med 2022 Jan 6;386(l):80-87. Frumento G, Zheng Y, Aubert G et al, Cord Blood T Cells Retain Early Differentiation Phenotype Suitable for Immunotherapy After TCR Gene Transfer to Confer EBV Specificity. American Journal of Transplantation, Volume 13, Issue 1, January 2013, Pages 45-55.

Claims

Claims1. A population comprising either CD4-negative enriched cord blood invariant natural killer T (INKT) cells or CD4-positive enriched cord blood invariant natural killer T (INKT) cells.

2. The population according to claim 1, wherein the CD4-negative enriched cord blood invariant natural killer T (INKT) cells or the CD4-positive enriched cord blood invariant natural killer T (INKT) cells comprise a diverse TCRVp repertoire, optionally a diverse TCRVpil repertoire.

3. The population according to any preceding claim, wherein the CD4-negative enriched cord blood invariant natural killer T (INKT) cells or the CD4-positive enriched cord blood invariant natural killer T (INKT) cells comprise a diverse TCRVpil CDR3p chain.

4. The population according to any preceding claim, wherein the CD4-negative enriched cord blood invariant natural killer T (INKT) cells or the CD4-positive enriched cord blood invariant natural killer T (INKT) cells comprise two or more TCRVp chain types, optionally two or more TCRVpi 1 CDR3 chain types.

5. The population according to any preceding claim, wherein :(i) the CD4-negative enriched cord blood invariant natural killer T (INKT) cells or the CD4-positive enriched cord blood invariant natural killer T (INKT) cells comprise five, 10 or 15 or more TCRVp CDR chain types, optionally five, 10 or 15 or more TCRVpi l CDR chain types;(ii) the CD4-negative enriched cord blood invariant natural killer T (INKT) cells or the CD4-positive enriched cord blood invariant natural killer T (INKT) cells comprise 50, 100, 200 or 500 or more TCRVp chain types, optionally 50, 100, 200 or 500 or more TCRVpil CDR3 chain types;(iii) the CD4-negative enriched cord blood invariant natural killer T (INKT) cells or the CD4-positive enriched cord blood invariant natural killer T (INKT) cells comprise 750, 800, 900 or 1000 or more TCRVp chain types, optionally 750, 800, 900 or 1000 or more TCRVpil CDR3 chain types; and / or(iv) the CD4-negative enriched cord blood invariant natural killer T (INKT) cells or the CD4-positive enriched cord blood invariant natural killer T (INKT) cells comprise 1100, 1200, 1300 or 1400 or more TCRVp chain types, optionally 1100, 1200, 1300 or 1400 or more TCRVpil CDR3 chain types.

6. The population according to any preceding claim, wherein the population comprises polyclonal TCRVpil CD4-negative enriched cord blood invariant natural killer T (INKT) cells or polyclonal TCRVpil CD4-positive enriched cord blood invariant natural killer T (INKT) cells.

7. The population according to any preceding claim, wherein :(i) more than 3% of the INKT cells are CD4-negative INKT cells;(II) more than 5%, 10%, 15% or 20% of the INKT cells are CD4-negative INKT cells;(ill) more than 25%, 30%, 35% or 40% of the INKT cells are CD4-negative INKT cell;(iv) more than 45%, 50%, 55% or 60% of the INKT cells are CD4-negative INKT cells; and / or(v) more than 65%, 70%, 75% or 80% of the INKT cells are CD4-negative INKT cells.

8. The population according to any preceding claim, wherein the enrichment of the cord blood INKT cells in the population is such that between 10 and 90% of all INKT cells are CD4-negative INKT cells, or between 35% and 60% of all INKT cells are CD4-negative INKT cells.

9. The population according to any preceding claim, wherein the population comprising CD4-negative enriched cord blood invariant natural killer T (INKT) cells comprises CD4-positive cord blood INKT cells, and wherein the enrichment of the cord blood INKT cells in the population is such that between 35% and 60% of all INKT cells are CD4-negative INKT cells, and the rest of the population comprises CD4-positive cord blood INKT cells.

10. The population according to any preceding claim, wherein :(i) more than 15% of the iNKT cells are CD4-positive INKT cells;(II) more than 20%, 30%, 40% or 50% of the INKT cells are CD4-positive INKT cells;(ill) more than 60%, 65%, 70% or 75% of the INKT cells are CD4-positive INKT cells;(iv) more than 80%, 85%, 90% or 95% of the iNKT cells are CD4-positive INKT cells; and / or(v) more than 96%, 97%, 98% or 99% of the INKT cells are CD4-positive INKT cells.

11. The population according to any preceding claim, wherein between 10 and 100% of all INKT cells are CD4-positive INKT cells, or between 80% and 99% of all INKT cells are CD4-positive INKT cells.

12. The population according to any preceding claim, wherein the population comprising CD4-positive enriched cord blood invariant natural killer T (INKT) cells comprises CD4-negative cord blood INKT cells, and wherein enrichment of the cord blood INKT cells in the population is such that between 80% and 99% of all INKT cells are CD4-positive INKT cells, and the rest of the population comprises CD4-negative cord blood INKT cells.

13. The population according to any preceding claim, wherein the population comprising either CD4-negative enriched cord blood invariant natural killer T (INKT) cells or CD4-positive enriched cord blood INKT cells is cell sorted subsequent to enrichment.

14. The population according to claim 13, wherein the cell sorting of the population comprises positive selection, depletion, and / or negative selection.

15. The population according to either claim 13 or 14, wherein the cell sorting of the population comprises fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS™), immunomagnetic cell sorting, and / or buoyancy-activated cell sorting (BACS™).

16. The population according to any one of claims 13-15, wherein the cell sorting of the population comprising CD4-negative enriched cord blood invariant natural killer T (INKT) cells achieves a concentration of the cord blood INKT cells of between 36% and 100% of all INKT cells which are CD4-negative INKT cells.

17. The population according to any one of claims 13-16, wherein the cell sorting of the population comprising CD4-positive enriched cord blood invariant natural killer T (INKT) cells achieves a concentration of the cord blood INKT cells of between 81% and 100% of all INKT cells which are CD4-positive INKT cells.

18. A method for producing a population of either CD4-negative enriched cord blood invariant natural killer T (INKT) cells or CD4-positive enriched cord blood INKT cells, the method comprising contacting cord blood INKT cells with one or more cytokine, to thereby enrich either the CD4-negative INKT cell population or the CD4-positive cord blood INKT cell population.

19. The method according to claim 18, wherein the one or more cytokine is selected from a group of cytokines consisting of: interleukin (IL)-2, IL-15, IL- 7, IL-12, and IL-21.

20. The method according to either claim 18 or claim 19, wherein the method comprises contacting cord blood INKT cells with one or more cytokine to produce a population of CD4-negative enriched cord blood invariant natural killer T (INKT) cells, wherein the one or more cytokine is IL-2 and / or IL-21.

21. The method according to any one of claims 18-20, wherein the method comprises contacting cord blood INKT cells with more than one cytokine to produce a population of CD4-negative enriched cord blood invariant natural killer T (INKT) cells, wherein the cytokines are IL-2 and IL-21.

22. The method according to either claim 18 or claim 19, wherein the method comprises contacting cord blood INKT cells with one or more cytokine to produce a population of CD4-positive enriched cord blood invariant natural killer T (INKT) cells, wherein the cytokine is IL-15, or IL-2 and / or IL-21.

23. The method according to any one of claims 18 -22, wherein:(i) the concentration of IL-15 is at least 200 ng / ml, or at least 400 ng / ml, or at least 600 ng / ml, or at least 700 ng / ml;(II) the concentration of IL-2 is at least 200 ng / ml, or at least 400 ng / ml, or at least 600 ng / ml, or at least 700 ng / ml;(ill) the concentration of IL-21 is at least 1 ng / ml, or at least 5 ng / ml, or at least 10 ng / ml, or at least 12 ng / ml; and / or(iv) the concentration of IL-12 is at least 0.5 ng / ml, or at least 1 ng / ml, or at least 2 ng / ml, or at least 4 ng / ml.

24. The method according to any one of claims 18-23, wherein the method comprises contacting the CD4-negative enriched cord blood invariant natural killer T (iNKT) cells or the CD4-positive enriched cord blood iNKT cells with a glycolipid to further enrich the CD4-negative iNKT cell population or CD4- positive enriched cord blood INKT cell population.

25. The method according to claim 24, wherein the glycolipid is alpha- Galactosylceramide (aGalCer).

26. The method according to either claim 24 or claim 25, wherein the method comprises pulsing the CD4-negative enriched or CD4-positive enriched cord blood invariant natural killer T (INKT) cells with the glycolipid.

27. A population of either CD4-negative enriched cord blood INKT cells or CD4- positive enriched cord blood INKT cells obtained, or obtainable, by the method according to any one of claims 18-26.

28. The population according to any one of claims 1-17 or claim 27, wherein the CD4-negative enriched or CD4-positive enriched cord blood invariant natural killer T (INKT) cells in the population comprise a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR).

29. The population according to claim 28, wherein the CAR is an anti-tumour antigen CAR.

30. The population according to either claim 28 or claim 29, wherein the CAR comprises an anti-CD19 CAR, optionally wherein the CD4-negative enriched or CD4-positive enriched cord blood invariant natural killer T (INKT) cells in the population comprise a chimeric antigen receptor (CAR) which (i) comprises an amino acid sequence substantially as set out in SEQ ID No: 1 or a variant or fragment thereof, and / or (ii) is encoded by a nucleotide sequence substantially as set out in SEQ ID No: 2 or a variant or fragment thereof.

31. The population according to either claim 28 or claim 29, wherein the CAR comprises an anti-BCMA CAR, optionally wherein the CD4-negative enriched or CD4-positive enriched cord blood invariant natural killer T (INKT) cells in the population comprise a chimeric antigen receptor (CAR) which (i) comprises an amino acid sequence substantially as set out in SEQ ID No: 3 ora variant or fragment thereof, and / or (ii) is encoded by a nucleotide sequence substantially as set out in SEQ ID No: 4 or a variant or fragment thereof.

32. The population according to claim 28, wherein (i) the CD4-negative enriched or CD4-positive enriched cord blood invariant natural killer T (INKT) cells in the population comprise an a[3T cell receptor (TCR) against MHC class I and II presented peptides derived from a tumour antigen, or (ii) the CD4-negative or CD4-positive enriched cord blood invariant natural killer T (INKT) cells in the population comprise or are engineered to express a tumour- reactive ySTCR.

33. A pharmaceutical composition comprising a therapeutically effective amount of either the CD4-negative enriched cord blood INKT cells or the CD4-positive enriched cord blood INKT cells in the population according to any one of claims 1-17 or claim 27-32, and a pharmaceutically acceptable vehicle.

34. A population according to any one of claims 1-17 or claim 27-32, wherein the CD4-negative enriched or CD4-positive enriched cord blood invariant natural killer T (INKT) cells are configured to bind to at least one INKT cell specific engager, optionally wherein the CD4+ enriched and CD4-negative enriched cord blood invariant natural killer T (INKT) cells are engineered to express anti-cancer CARs or TCRs, optionally wherein the iNKT cells are adoptively transferred to recipients following immunodepleting chemotherapy.

35. A population according to any one of claims 1-17 or claim 27-32, or the pharmaceutical composition according to claim 33, for use in therapy or diagnosis.

36. A population according to any one of claims 1-17 or claim 27-32, or the pharmaceutical composition according to claim 33, for use in (i) immunotherapy; (ii) for treating, preventing or ameliorating cancer; (iii) for treating, preventing or ameliorating an autoimmune and alloimmune disease (e.g., acute graft-versus-host disease) ; or (iv) for treating, preventing or ameliorating any infectious disease, including viral disease, such as HIV infection.

37. A population or pharmaceutical composition, for use according to claim 36, for use in treating, preventing or ameliorating myeloid or lymphoid leukaemias, Bcell and T cell lymphomas, plasma cell dyscrasias and for solid tumours cancers of the brain, respiratory tract, head and neck, or skin etc.

38. A population or pharmaceutical composition, for use according to claim 36, for treating, preventing or ameliorating an autoimmune disease 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.

Images