method

Abstract

The disclosure provides methods of providing antigen-specific T cells, which method involves stimulating the sample with an antigen (or an immunogen comprising the same) to enrich T cells specific for that antigen before expanding an optimized memory (antigen- specific) an T cell-enriched product. The disclosure further provides T cells obtainable by the disclosed methods and their use in therapy.

Description

[0001] METHOD

[0002] FIELD

[0003] The disclosure provides methods of making antigen specific T cells, antigen specific T cells obtainable by such methods, compositions, therapeutic methods and uses exploiting the same.

[0004] BACKGROUND

[0005] Decades of research and clinical trials have shown that a strong cellular immune response can restore immune control over viral infections and cancers. However, for a variety of reasons, current cell therapies have largely failed to achieve their aims. For example, current approaches tend to generate T cells of low yield (quantity), low quality (function) and low diversity (the T cells being restricted to a few or single antigenic epitopes). Moreover, prior art approaches generate heterogeneous T cell products without defined target homing abilities. These problems contribute to the relatively high failure rate associated with T cell therapies.

[0006] Accordingly, there is a need for an improved method of generating antigen-specific T cells.

[0007] SUMMARY

[0008] Disclosed are methods of providing antigen-specific T cells. The methods use an antigen (or an immunogen comprising the same) to stimulate and enrich T cells specific for that antigen before expanding an optimized memory (antigen-specific) T cell-enriched product.

[0009] The antigen may be provided in the form of an immunogen. For example, the immunogen

[0010] ) , 2. 1. ) 2). , 1 ) . 1. . 2 1. . ‘) 2). ’ . ) . 21. ) . 2 which is itself antigenic and capable of giving rise to a T cell with specificity for that antigen, or an immunogen which comprises the antigen. As such, and for convenience, the term ‘) 2). ’ ) . 1. . 2 . + ) , . 5 ) ) 2). 2 ■ 2 + ) 5 2 0. B121 comprise such antigenic entities.

[0011] Without wishing to be bound by theory, it is submitted that in contrast to prior art methods, the methods described herein are rapid, efficient and yield cells with numerous advantageous properties. For example, the antigen-specific T-cells provided or generated by the methods of this disclosure have expanded in response to a recent immunizing event or events in vivo and may be regarded as of high quality, non-exhausted memory T cells (CD8+ve and CD4+ve). . 3> B121 ) ■ ‘ - Cl) . ’ exhibit a limited expression of exhaustion markers. This can be defined as a low percentage of cells expressing two or more known markers of T cell exhaustion, including but not limited to the PD-1 , LAG-3, Tim- 3, TIGIT markers. In one teaching, the methods of this disclosure may yield a product comprising a population of cells, ) ‘5 B . ) 0. ’ of which co-express any two of the disclosed , , 3>. Cl ) 2 ) . 1. . ‘5 B . ) 0. ’ may mean that none of the cells (of the product or population generated by the methods disclosed herein) exhibit coexpression of any two of the disclosed markers or that no more than about 30% of the cells (of the product or population generated by the methods disclosed herein) exhibit such coexpression. By way of example, <30% of the cells (of the product or population generated by the methods disclosed herein) may exhibit co-expression (of any two of the markers disclosed herein), <15% of the cells (of the product or population generated by the methods disclosed herein) may exhibit co-expression (of any two of the markers disclosed herein), <10% of the cells (of the product or population generated by the methods disclosed herein) may exhibit co-expression (of any two of the markers disclosed herein) or <5% of the cells (of the product or population generated by the methods disclosed herein) may exhibit coexpression (of any two of the markers disclosed herein). A cell product or population which is ‘ -. Cl ) . ’ ) , 2. ) , population in which fewer than about 30%, 29%,

[0012] 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21 %, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or about 1 % of the cells of said product or population express at least two of the T cell exhaustion markers described herein (where 1. . ■) + ’ ■ ) ±

[0013] The product of the methods described herein may be further characterised by the absence (or substantial absence) of regulatory T cells (Tregs) - a specialized subpopulation of T cells that act to suppress the immune response; such cells and are an undesired subset in T cell therapy products. By way of example, the product of the methods described may comprise no Tregs or no more than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1 % Tregs (the percentages being a percentage of the total number of cells in the product).

[0014] The methods of this disclosure may be used to manufacture antigen-specific T cells for use in therapy. A therapy of this type may otherwise be referred to as an enhanced autologous T cell-based therapeutic which is specific for an antigen. As such, in one teaching, the antigenspecific T cells generated (or obtained or obtainable) by any of the methods described herein, may be used in therapy, in medicine and / or as a medicament. The therapeutic utility of the disclosed antigen-specific T cells is enhanced by the fact that cells generated by the methods of this disclosure exhibit better diversity and embrace multiple antigen-specific clones which are reactive to individual antigens. This reduces the risk of immune escape (a problem associated with T cell therapies with low clonal diversity).

[0015] Moreover, the antigen-specific T cells generated by methods according to this disclosure are able to home to relevant in vivo antigenic depot sites - for example as expressed in a tumour or on a pathogen. Additionally, the antigen-specific T cells are generated in significant numbers and as dense populations of T cells that have affinity / specificity for the antigen(s) of interest.

[0016] Accordingly, the disclosure provides a method of providing an antigen-specific T-cell, said method comprising: purging a sample comprising antigen-specific T cells, of unwanted cell types; contacting the purged sample with an antigen or a representation thereof to activate the antigen-specific T-cells; expanding the activated antigen-specific T-cells; and harvesting the expanded antigen-specific T-cells.

[0017] As stated in more detail below, the sample may be purged of Naive T cells, terminally differentiated memory T cells, NK cells and B cells.

[0018] It should be noted that 1. . “, 2. ” “, 220” ) “, 2. ” 2 ) . . feature or features. It should be understood that this / these terms may also encompass ) . , ) . + 2 . B121 “, 2 . . ® / ” “, 2 / ” 1. . 5 ) I. ) . or features. Moreover, the entire contents of all references are to be regarded as incorporated herein by reference.

[0019] The term “antigen-specific T-, . 35 embraces a T-cell with specificity for an entire antigen or a fragment, component or epitope thereof.

[0020] 1. . “antigen-specific T-, . 35 / 1. . + ) , . ) -cell with a specificity for an antigen. Moreover, one of skill will appreciate that ) ‘) 2). -specific T-, . S> / 12 2 , 5 ) be specific for the antigenic part of an immunogen. In such cases, an immunogen may be processed (for example, by an antigen presenting cell) into a fragment, which fragment comprises an antigenic component of the immunogen, this antigenic component may then be presented by the antigen presenting cell (via surface expressed MHC) whereupon it interacts with a naive T cell having specificity for the presented antigen.

[0021] In one embodiment, an antigen-specific T-cell of this disclosure is an MHC-restricted T-cell, that is a T-cell which recognises, binds and responds to (i.e. is activated by) an antigen when it is complexed with an MHC molecule (either Class I or Class II). As such, the term ‘antigen’ may include fragments (including fragments of an immunogen) which are processed by an antigen presenting cell and presented on the cell surface via an MHC molecule (Class I or II). As such, an antigen-specific T cell of this disclosure may further have a specificity for, or an ability to bind to, an MHC-presented / bound antigen and / or a fragment of an immunogen.

[0022] The antigen-specific T-cells generated by the methods of this disclosure may comprise T cells which all recognise the same part, fragment or epitope. Alternatively, the methods of this disclosure may yield a population of clonally diverse antigen-specific T cells. A population of clonally diverse antigen-specific T cells may comprise T cells which all bind to or have specificity for an antigen or an antigenic component of a specific immunogen, but which further comprises sub-populations that bind to different parts, fragments and / or epitopes of that antigen or antigenic component. As such, the methods of this disclosure generate clonally diverse antigen-specific T cell populations.

[0023] Antigen-specific T-cells may comprise memory T cells (Tmcells). Such cells may be generated in a host following exposure to a particular antigen and remain after said antigen (and / or, the associated infection) has been cleared. Memory T cells may be activated following re-exposure to the relevant antigen. The . ‘m, . S ’ ) + ) , . , . ) 5 memory T cells (Tcmcells) and effector memory T cells (Temcells) these terms are nonexclusive as T cell memory subsets may be subdivided into a number of fractions with distinct functions.

[0024] The antigen-specific T cells of this disclosure may be characterised by expression of CD45RO (in other words the T cells may be CD45RO+ve T cells). A proportion of the T cells may also have low or absent CD45RO expression and some re-expression of CD45RA, comprising terminally-differentiated effector memory CD45RA positive T cells (TEMRA).

[0025] The term antigen (or immunogen) may embrace any molecule or combination of molecules capable of inducing an immune response in a host. An ‘2 0. ’ may comprise an antigen (referred to herein as the “antigenic component” of an immunogen). Within the context of this disclosure, an antigen may comprise a protein, a peptide, a polysaccharide, a lipid, a hapten bound to a carrier protein, a virus and any combination thereof.

[0026] It should be noted that th. . ‘immunogen’ may take the form of a DNA or RNA vaccine, which vaccine results in the expression of one or more antigen(s) in vivo.

[0027] 1. . ‘) 2). ’ ) ) 5 comprise expressed by pathogens such as viruses. In one teaching a pathogen may express an immunogen which comprises one or more antigens and in such cases, the immunogen may be fragmented or otherwise processed (for example by an antigen presenting cell or via some protein degradation pathway) to yield the one or more presented antigens.

[0028] The term ‘) 2). ’ may comprise a synthetic or recombinant molecule. The term ‘) 2). ’ may embrace molecules (for example antigens) of microbial origin. For example, an antigen may comprise a viral antigen, a fungal antigen and / or a bacterial antigen.

[0029] The term antigen may comprise a tumour antigen.

[0030] The term antigen may comprise a vaccine component. For example, the term antigen may comprise a vaccinate - that is the antigenic component of a vaccine.

[0031] As stated, the disclosure further relates to immunogens which comprise any one or more of the disclosed antigens.

[0032] A ‘representation’ of any of the antigens (or immunogens) described herein may comprise a molecule which is immunologically identical or similar to said antigen. For example and within the context of this disclosure, a representation may induce the same immune response in a host as the original antigen.

[0033] A representation of an antigen may induce a memory or recall response among the relevant antigen-specific T cells in the sample. In other words, whereas an antigen may induce an initial immune response in a host (which immune response comprises the generation of T cells with specificity for the antigen), if the step of activating those cells comprises contacting with a representation of that antigen, then the representation will activate those antigen specific T cells by inducing a memory or recall response.

[0034] The term immunogen may further include the combination of an antigen (as defined above) given with an adjuvant. By way of example, a sample may be provided or obtained from a subject administered a combination of an antigen and an adjuvant. In such circumstances, the contacting step of a method of this disclosure may comprise contacting the purged sample with the same antigen / adjuvant combination or, as stated a combination which includes a representation of the antigen and / or the adjuvant.

[0035] 1. . ‘) 3 ) ’ . + ) , . ) , 2 3 . . 1) , . 1. immune response in a host to a specific immunogen. 1. . ‘) 3 A) ’ ) . + ) , . ) type of adjuvant and by way of example (but without limitation), the adjuvant may comprise delivery vehicles such as metal salts, microparticles, lipid emulsions and nanoparticles such as ISCOMs, or immune potentiators such as growth factors including GM-CSF or G-CSF, or TLR agonists such as poly-IC (Polyinosinic-polycytidylic acid), flagellin, MPL or C-poly G (CpG). 1. . ‘ ) 5 ’ ) . + ) , . ) ) 5 , 220 (or providing a source of) any of the antigen-specific T-cells described herein. The sample may be provided by or obtained from any human or animal subject.

[0036] The antigen-specific cell content of the sample may be sufficient to yield a product which comprises a suitable or acceptable number of antigen-specific T cells. By way of example, the antigen-specific cell content of the sample at least about 0.02% (in other words, about one in in every 5000 T cells may be specific for a predetermined or particular antigenic component of the immunogen). In one teaching, the antigen-specific cell content of the sample may be at least about 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1 %.

[0037] 12 , . C 1. . ‘) + ’ . ) ±

[0038] The sample may comprise peripheral blood from a subject. The sample may comprise blood (whole blood or a fraction thereof), leukapheresis, lymph, lymphoid tissue, a tumour (including a tumour biopsy) or any other leukocyte containing tissues.

[0039] The subject may have been immunologically primed by, for example, repeated exposure to or immunisation or vaccination with any of the antigens (optionally in combination with an adjuvant) described herein. One of skill will appreciate that such a repeated priming, exposure, immunisation or vaccination (against a pathogen or cancer / tumour type) or the like) event may activate a naive T cell and lead to the generation of a cohort of antigenspecific T cells. For convenience, the initial priming, exposure or immunisation event generating the antigen specific T cells, may be . / . . ) ) ‘ 2 20 . A ’ The methods of this disclosure may then be used to selectively activate, enhance and enrich those antigen-specific T cells. The advantages and / or benefits associated with the antigen-specific T cells generated by the methods of this disclosure result, in part, from the fact that the methods are to be executed at a specific, predetermined or chosen time after the priming event. By way of example, a method of this disclosure may be performed within about one month to about 12 months of a priming event, wherein the priming event results in the formation of antigen-specific T cells. For example, a method of this disclosure may be performed on a sample obtained from a subject repeatedly primed, exposed to or vaccinated / immunised (e.g. against a pathogen or a cancer / tumour type), with an antigen about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months or about 12 m 1 2 1. . ‘) + ’ ) . ) - 2 weeks.

[0040] In one teaching, a blood sample from a donor or patient may be screened (e.g. prescreened) to determine the frequency of antigen-specific T cells generated by the priming event. This may help ensure successful T cell manufacturing. This pre-screen may comprise tests to confirm the quantity and quality of the antigen-specific T cell response in the sample. The results of such tests can be used as a reliable predictor of success, irrespective of the timings of the immunization events. A useful pre-screen may involve the use of FACS analysis to determine the cellular profile of a sample.

[0041] The methods of this disclosure are suitable for use with samples obtained from or provided by subjects that have been repeatedly exposed to or vaccinated, immunised or primed with any form of antigen or immunogen, irrespective of format or therapeutic field. By way of example subjects may be suffering from specific disease or conditions - in which case they have likely been exposed to a number of antigens / from the relevant disease-causing entity or pathogen, which exposure event will have generated T cells specific for one or more of those antigens. By way of example, the subject may have received (as an antigen) any one or more of the following:

[0042] (i) A personalised vaccine e.g. a neoantigen, “ +5 ” “ ) , ^ . ” ) 2). vaccine in cancer using H LA-restricted antigens personal to the patient (optionally combined with an adjuvant);

[0043] (ii) A non-personalised vaccine in cancer, viral and other infectious diseases for individual or mass use based on predicted antigenic sequences (optionally combined with an adjuvant).

[0044] (iii) Short (9-20mer) peptides singly or in a cocktail (optionally combined with an adjuvant).

[0045] (iv) A long chain peptide (up to 500mer) singly, in an overlapping peptide pool representation or in a cocktail of peptides (optionally combined with an adjuvant).

[0046] (v) A recombinant protein (optionally combined with an adjuvant or a hapten bound to a carrier protein).

[0047] (vi) A naturally occurring protein (optionally combined with an adjuvant)

[0048] (vii) A lysate antigen - including but not limited to, vi rally-infected cell lysate, autologous tumour materials, allogeneic tumour materials including in vitro-cultured cells

[0049] (viii) An apoptotic / Necrotic cell antigen

[0050] (ix) A RNA-based vaccine

[0051] (x) A DNA-based vaccine

[0052] (xi) A vector-based vaccine (examples include lentivirus, adenovirus, AAV) (xii) An autologous or allogeneic antigen presenting cell (APC)-based immunization (for example monocytes, B cells, monocyte-derived Dendritic Cells (DC), naturally occurring DC, stem-cell derived DC)

[0053] (xiii) An autologous or allogeneic cell-based vaccines including but not limited to, vi rally-infected cell lysate, autologous tumour materials, allogeneic tumour materials including in vitro-cultured cells.

[0054] As such, where a subject has received an immunogen according to any one of the options listed as (i)-(xiii) above, the contacting step of the methods described herein may comprise either the same immunogen or a representation thereof.

[0055] As an example, the subject may have been administered a cancer vaccine, that is a vaccine comprising a cancer or tumour antigen designed to elicit an immune response against that antigen. That immune response may comprise the generation of T cells with specificity and / or affinity for the relevant cancer / tumour antigen, which T cells may help the host clear and / or resolve the cancer / tumour. The methods of this disclosure may be applied to samples obtained from any subject administered a cancer / tumour vaccine and used to generate an enhanced (autologous) cohort of the cancer / tumour antigen specific T cells, which enhanced cohort is therapeutically useful in the treatment of the relevant cancer / tumour.

[0056] One of skill will appreciate that there are numerous different cancer vaccines available with many more in research and development. The vaccines take many different forms and may include cell-based vaccines, peptide-based vaccines, mRNA vaccines, viral vaccines and combinations thereof.

[0057] Moreover, a similar approach could be taken with subjects suffering from a disease or condition caused or contributed to by any type of pathogen. In such cases, samples from such subjects (which samples comprise cohorts of antigen-specific T cells, the antigen being an antigen derived from the pathogen) may be subject to a method of this disclosure (i.e. contacted with the relevant pathogen-derived antigen (or a representation thereof) so as to activate the antigen-specific T cells), enhancing and enriching the antigen-specific T cells and subsequently using those cells in the treatment of the disease and / or condition.

[0058] 1 . . ‘ ) 5 ’ ) , 2 . ) +2502 ) 5 / 52 / . Q 5 +5 ) / ) , 2 1. . / such as but not limited to a whole white cell fraction or buffy coat. The sample may comprise serum or plasma.

[0059] 1. . ‘ ) 5 ’ may comprise peripheral blood mononuclear cells (PBMCs). Without being bound by theory, these mononuclear cells will include the relevant (or target) antigenspecific T cells, but also other ‘) 2). - . . 20’ , . 3> , 1 ) , . ) , . 3> . These antigen presenting cells can take up an immunogen or an antigen, process it and present the antigen (or the antigenic component of an immunogen) on their surfaces in an MHC-restricted context. The target antigen-specific T cells will interact with the antigen- presenting cells (and the surface presented antigen) and become activated through binding of their T cell receptor (TCR) with the MHC-bound antigen. This binding event may also engage other costimulatory receptors such as CD28 as co-stimulating factors which activate the target antigen-specific T cells, causing them to become activated and to proliferate / express cytokines etc.

[0060] 1. ‘ ) 5 ’ ) +. . 2\ / ) ) 1. . 2 5 ) 1. . 2

[0061] The sample may be purged of unwanted cells or cell types or of certain (or specific) cells or cell types. In other words, certain (or specific) cells or cell types may be purged, removed or depleted (or substantially purged or removed or depleted) from the sample.

[0062] Without wishing to be bound by theory, this may passively enrich the antigen-specific T-cell content and contributes to the overall quality of the T cell product generated by the methods of this disclosure.

[0063] The sample may be purged of cells which express a certain marker or markers and / or an amount of that marker (or those markers) which exceeds a threshold level.

[0064] In one teaching, the sample may be purged of any one or more of the cell types:

[0065] (i) naive alpha / beta (CD4+ and CD8+); and / or

[0066] (ii) Terminally Differentiated Effector Memory T cells (TEMRA) CD4 and CD8 T cells; and / or

[0067] (iii) subsets of NK cells; and / or

[0068] (iv) B cells.

[0069] Additionally, or alternatively any one or more of the cell types listed as (i)-(iv) above, may be removed or substantially removed from the sample.

[0070] One of skill will appreciate that techniques such as flow cytometry may be used to identify populations of any of these cell types in a sample. Further, techniques such as fluorescence- activated cell sorting may be used to sort and / or remove (or purge) cells from a sample.

[0071] The abovementioned cells (any one or more of which are to be removed or purged from a sample) may express the following markers (see Table 1 ):

[0072] Table 1 : cell surface markers of cells that may be removed or purged from a sample.

[0073] One or more (or all) of the markers listed in the table above may be used to identify or detect (e.g. by flow cytometry) cells for removal from a sample.

[0074] Purging (or substantially purging) a sample of B cells is advantageous as it avoids this cell type becoming a contaminant in expansion cultures.

[0075] By purging a sample of any one or more of the cells listed as (i)-(iv) above, one may enrich the sample in, for example monocytes (this is advantageous as these cells act as antigen- presenting cells (e.g. in the neoantigen stimulations)).

[0076] A sample may be contacted with agents which bind any of the markers identified in Table 1 . The binding agent may be conjugated, labelled, bound, joined or linked to an optically detectable moiety. The optically detectable moiety may comprise a fluorescent moiety, e.g. a fluorophore. The binding agent may comprise an antibody, for example a monoclonal antibody. An antibody for use (which antibody may optionally be conjugated to an optically detectable moiety) may have an affinity for, or selectively bind to, any of the markers listed in Table 1 and may be used in the detection of a specific cell type.

[0077] A sample may comprise cells (for example T cells or CD3+ve T cells) which express either or both of the CD45RA and CD45RO surface markers. Cells which express the CD45RA marker may be referred to as CD45RA+ve cells. Cells which express the CD45RO marker may be referred to as CD45RO+ve cells. Some cells may may be referred to as CD45RAhigh cells and some cells may be referred to as CD45ROhigh cells. Cells which are either CD45RAhigh or CD45ROhigh are easily identifiable to one of skill using, for example, flow cytometry. The term CD45RAhigh may refer to cells which one of skill recognises as including naive T cells.

[0078] Other cells may express far less of the CD45RA and / or CD45RO marker(s) - these cells may be referred to as CD45RAIow or CD45ROIow cells. As stated, cells which are CD45RA+ve and in particular the subset of cells which may be graded as being CD45RAhigh, include naive T cells, B cells and some NK cells.

[0079] In view of the above, the CD45RAhigh cells may be removed (or purged) from the sample..

[0080] Cells which are CD45RAhigh (namely those cells which express high or the highest levels of CD45RA) may be purged from a sample by probing that sample to identify those cells which, relative to others in the sample, express a high level of CD45RA, and then removing or purging those cells from the sample.

[0081] CD45RAhigh cells may be identified and subsequently purged or removed from a sample by various methods known in the art. Without wishing to be bound by theory, flow cytometry, magnetic beads and / or fluorescence-activated cell sorting may be used to identify, isolate and / or remove a specific cell type, population of cells and / or subpopulation of cells from a mixed population. For example, a combination of flow cytometry, magnetic beads and / or fluorescence-activated cell sorting may be used to remove CD45RA high and / or CD45RO low cells from a sample.

[0082] Where flow cytometry, magnetic beads and / or fluorescence-activated cell sorting are used to identify, isolate and / or remove a specific cell type from a sample (e.g. any of the cells listed in Table 1 , mean fluorescence intensity (MFI) may be used as a measure of the relative abundance and / or expression of one or more specific marker (e.g. cell surface markers like CD45RA and / or CD45RO). The MFI may be used to delineate specific cell types and / or (sub)populations in a sample. For example, MFI may be used to delineate those cells which are CD45RAhigh from those cells which are CD45RAIow. It should be noted that the precise value or measurement obtained from a flow cytometer may vary depending on a number of factors, such as the sample (e.g. its source and / or composition), the cytometer detector settings and / or the type of (fluorescence) label used etc.

[0083] By way of example only, Figures 1 , 2 and 3 (see especially panels 1C, 2B and 2D and 3B) show how a technique like flow cytometry may be applied to a sample in order to identify any CD45RAhigh cells. In this example, the cells of the sample are labelled with fluorescent antibodies for CD45RA and CD45RO and the data is expressed as a flow cytometry plot of fluorescence (one of skill will appreciate that the amount of fluorescence will be directly proportional to the amount of CD45RA / RO expression on the cell surface). In this case, the sample is shown to comprise a population of T cells which may be segregated into two main populations: a CD45RAhigh / CD45ROIow population (CD45RAhigh / CD45ROIow cells which are the undesirable cells which are to be removed or purged from the sample) and a CD45ROhigh / CD45RAIow population (CD45Rhigh / CD45RAIow cells - the cells (the desired memory T cells) that are to be retained). After analysis using flow cytometry, the sample may be subject to the disclosed purging step so as to remove the CD45RAhigh / CD45ROIow cells. Additionally or alternatively, the sample is purged of one or more of the cells referred to or classified as:

[0084] (i) naive alpha / beta T-cells (CD4+ and CD8+);

[0085] (ii) Terminally Differentiated Effector Memory T cells (TEMRA) CD4 and CD8 T cells;

[0086] (iii) subsets of NK cells;

[0087] (iv) B cells.

[0088] In one teaching, the purging step may not remove (or may leave a portion of) the early memory T cells, such as, for example, the cells characterised as CD45RAmid CD45ROmid (see Figure 3F).

[0089] Without wishing to be bound by any particular method or theory, the number of cells that need to be purged (or are desirably / preferably purged) from the sample may be present in very variable proportions. In other words, there may be more of one cell type to be purged than in another. Moreover, even within the same sample, certain cells to be purged may be presented in higher numbers than others, but the relative proportions of all these cells may vary from sample to sample. The purging step of this disclosure homogenizes or standardises (in terms of cell content) the starting material. This is especially important where the sample has been obtained from, or provided by, a cancer patient (or someone exposed to a vaccine).

[0090] The purged sample may then be contacted with an antigen or a representation thereof. The purpose of the contacting step is to activate the antigen-specific T cells which have been generated by the initial priming event (e.g. an antigen exposure, vaccination or immunisation event). As such, the antigen (or its representation) used in the contacting step, should be the same antigen as the antigen-specific T cells (generated by the priming event) are specific for). The activation which results from the contacting step itself results in the expression of activation markers (which include but are not limited to CD25, CD57, CD69, CD137 and CD154), cellular proliferation and cytokines such as interferon-gamma, TNF-alpha and IL-2 of any and all clones of the T cell with specificity for the antigen used in the contacting step.

[0091] The purged sample may be contacted with the antigen (or a representation thereof) at a concentration of 0.01 pg / ml - 1000pg / mL One of skill will appreciate that the amount of antigen or a representation thereof used in the contacting step may be sufficient to achieve activation of the antigen specific T cells. By way of example, the purged sample may be contacted with the antigen (or a representation thereof) at a concentration of between 0.05 pg / ml and about 5 pg / ml, (e.g. about 0.1 pg / ml, about 0.5 pg / ml, about 1 pg / ml, about 2 pg / ml, about 3 pg / ml, about 4 pg / ml), between 0.05 pg / ml and about 500 pg / ml, between about 100 pg / ml and 400 pg / ml, between about 200 pg / ml-300 pg / ml. For example, the antigen may be contacted at a concentration of about 1 pg / ml. In this context, the term about may mean ± 0.05 pg / ml.

[0092] After the contacting step, and before the expansion step, the activated antigen-specific T cells may be harvested and / or enriched. One of skill will understand how to harvest and / or enrich the activated antigen-specific T cells and by way of a non-limiting example, the activated antigen-specific T cells could be harvested by methods comprising immunomagnetic and / or flow-cytometry-based techniques. Such techniques may exploit hapten, fluorochrome or magnetic bead labelled antibodies specific for CD25, CD137, antihapten or fluorochrome magnetic beads, and / or Cytokine Capture Systems. The step of harvesting / enrichment will yield a negative fraction - that is a fraction which lacks or has been depleted of (or substantially lacks or has been substantially depleted of) the activated antigen-specific T cells.

[0093] The harvested and / or enriched / activated antigen-specific T cells may be expanded. The step of expansion may comprise contacting (or maintaining) the harvested and / or enriched activated antigen-specific T cells with feeder cells and / or cytokines.

[0094] In terms of cytokines, the expansion step may exploit IL-2, IL-4, IL-7, IL-15 and / or IL-23. The cytokines may each, independently, be used at a concentration of anywhere between 1 - 1000IU / ml. The cytokines may aid proliferation, survival, potency and / or differentiation - as such, each cytokine may be used in an amount sufficient to achieve one or more of these aims.

[0095] In terms of feeder cells, any suitable source of feeder cells may suffice, however, in one teaching, the feeder cells may be autologous and . 2k I 1. ‘ ) 5 ’ / B121 1 - antigen-specific T cells are derived or obtained.

[0096] The feeder cells may be derived from an un-manipulated portion of the sample or a portion of the sample which has been manipulated and / or subject to enrichment protocols including any of the methods, or sections / steps thereof, described herein).

[0097] The feeder cells may be derived from the purged sample. By way of example, once the sample has been purged (again, as described herein) at least some of the purged sample may be used to prepare feeder cells for use. In a further example, the feeder cells may be derived from the abovementioned negative fraction (generated during the harvesting of the antigen-specific T cells after the activation step). In one teaching, the feeder cells may be derived from the sample by:

[0098] (i) stimulating the sample with an antigen or a representation thereof (wherein the feeder cells are thereafter derived from an activated fraction of the sample); and / or

[0099] (ii) depleting the sample of CD45RA+ cells (wherein the feeder cells are derived from a CD45RA+ depleted fraction of the sample); and / or

[0100] (iii) depleting the sample of CD3+ cells (wherein the feeder cells are derived from a CD3+ depleted fraction of the sample).

[0101] (iv) inactivating the feeders using irradiation or compounds such as mitomycin C

[0102] The activated and enriched antigen-specific T cells are combined with feeder cells in a ratio of about 1 :5 to about 1 :1000, for example about 1 :5 to about 1 :500, about 1 :5 to about 1 :200 (antigen-specific T cells: feeder cells). Moreover, the antigen specific T cells and the feeder cells may be combined or mixed so that the total seeding cell density is, or is in the region of, 0.5x106-1x107cells per cm2.

[0103] The expansion step may further comprise the use of autologous serum and / or plasma. For example, the expansion step may be supplemented with up to about 20% (by volume of the culture) autologous serum and / or plasma.

[0104] The expansion step may comprise contacting the harvested and activated antigen-specific T cells with feeder cells and / or cytokines and appropriate culture medium for about 7- 21 days, for example about 14 days.

[0105] The step of expansion may further exploit the use of the antigen or its representation and / or T cell mitogens. By way of example, the T cell mitogens may comprise CD3 and / or CD23 specific antibodies.

[0106] The step of expansion may use a GMP compliant medium.

[0107] The expansion step may be repeated one or more times. For each repeat expansion step, the expanded cells are harvested and then reformulated in a medium, for example a GMP compliant medium. As stated, the medium may be further supplemented by the antigen (or a representation thereof) and / or T cell mitogens (e.g. CD3 and / or CD28 specific antibodies. Each repeat expansion step may take place over a period of about 7-21 days, for example about 7 days. After the expansion step(s), the expanded antigen-specific T cells may be harvested. This may be done by aseptic transfer and buffer exchange to remove culture medium ready for formulation as a final drug product.

[0108] In a further teaching, the disclosure provides antigen-specific T cells obtainable (or obtained) by any of the methods of this disclosure. A method of this disclosure may yield a population of cells, wherein >5%, >10%, >15%, >20%, >25%, >30%, >35%, >40%, >45%, >50% of the cells are T cells specific for a particular antigen (namely the antigen involved in the initial priming event and the antigen used in the later contacting step). In one teaching, the product of the disclosed methods may 0.1 x108and 10 x 109antigen-specific T cells.

[0109] The disclosure further provides antigen-specific T cells, including those obtainable (or obtained) by any of the methods of this disclosure, for use in medicine or for use as a medicament.

[0110] The disclosure also provides antigen-specific T cells, including those obtainable by any of the methods of this disclosure, for use in the treatment or prevention of any of the diseases or conditions described herein, including for example, cancer and infectious diseases.

[0111] Also disclosed are therapeutic methods, wherein said method comprises administering to a subject in need thereof antigen-specific T cells obtainable by any of the methods of this disclosure. Such therapeutic methods may be for the treatment or stabilisation of any of the diseases or conditions described herein, including, for example, cancer and infectious diseases.

[0112] Therapeutic uses of the antigen-specific T cells generated by the methods of this disclosure include uses to enhance the treatment of, or immune response in, a subject that has been immunologically primed by some antigen exposure, vaccination or immunisation event.

[0113] In one teaching, antigen-specific T cells generated (or obtainable) by the methods of this disclosure are for use in the subject providing the sample, or from whom the sample was obtained.

[0114] In another teaching, antigen-specific T cells generated (or obtainable) by the methods of this disclosure are for use in T- cell therapy or for use in a method of T-cell therapy. In some examples, the subject of the T-cell therapy may be the subject providing the sample, or from whom the sample was obtained.

[0115] As such, a method treating a disease or condition may comprise, obtaining or providing a sample from a subject, which sample comprises antigen specific T cells, wherein the antigen specific T cells are specific to an antigen involved in the disease or condition, or expressed by a tissue or pathogen associated with the disease or condition; subjecting the sample to any of the methods described herein to generate an expanded cohort of the antigen-specific T cells, and then using the expanded antigen-specific T cells, to treat the disease or condition in the subject.

[0116] Antigen-specific T cells of this disclosure, including those obtainable by any method disclosed herein, may be administered or dosed to a subject in combination with one or more other therapeutic options, including for example a vaccine, chemotherapy, check point blockage, co stimulatory antibodies, T cell specific stimulatory cytokines (including IL-2, IL-7 and IL-15 therapy), radiotherapy and / or monoclonal antibodies.

[0117] A combined administration or dosing regime may comprise concurrent administration of the full combination or sequential administration where an antigen-specific T cell-based therapy of this disclosure is administered before or after any other therapeutic option.

[0118] An antigen-specific T cell-based therapy of this disclosure may be dosed or administered to a subject in need thereof once or on multiple occasions per day, per week or per month.

[0119] An antigen-specific T cell-based therapy of this disclosure may be used to treat clonal neoplastic processes that can be considered premalignant. These may include, monoclonal gammopathy of undetermined significance (MGUS), leucoplakia, erythroplakia, lichen planus, submucous fibrosis, carcinoma in situ 2 , 5 20 B. ’ 2. ) . and colonic polyp.

[0120] The disclosure further provides compositions comprising any of the antigen-specific T cells described herein, including, for example the antigen-specific T cells obtainable (or obtained) by any of the methods of this disclosure. Such compositions may comprise buffers, diluents, carriers and / or excipients.

[0121] A composition of this disclosure may be a sterile and / or pharmaceutical composition and may further comprise pharmaceutically acceptable diluents, carriers, excipients and / or buffers.

[0122] The antigen-specific T cells of this disclosure may be formulated for administration by IV infusion.

[0123] Accordingly, the disclosure provides a composition for IV infusion, said composition comprising antigen-specific T cells of this disclosure including those obtainable by any of the methods described herein.

[0124] A composition for IV infusion to a subject, may comprise autologous antigen-specific T cells obtainable or obtained by a method of this disclosure. Such a composition may further comprise a suitable excipient buffer such as, for example, saline, crystalloid buffers (e.g. plasmalyte), cryoprotectant (eg Cryostor) and serum products (eg AB serum).

[0125] DETAILED DESCRIPTION

[0126] The present disclosure will now be described in detail with reference to the following Figures which show:

[0127] Figure 1. Exemplary flow cytometry plot of start material.

[0128] Assessment of mononuclear cells in start material (donor leukapheresis) indicating the required populations (A) of CD14+ monocytes as antigen presenting cell (APC) function and CD3+ T cells. The T cells are classified as CD4 or CD8 (B) and expressing high CD45RA and low CD45RO (naive and TEMRA) or low CD45RA and high CD45RO (memory T cells). The other cells present include B cells, NK cells, residual neutrophils (E), NKT and gammadelta T cells (F). Depletion of CD45RAhigh T cells will remove the undesired naive and TEMRA T cells (F), leaving the required CD45ROhigh T cells for antigen stimulation and culture.

[0129] Figure 2. Donor variability in T cell compartment.

[0130] Exemplar flow cytometry plots from different cancer patients demonstrates that there is variability in the percentages of T cell subsets, both in CD4: CD8 ratios (A+C) and in CD45RAIow / CD45ROhigh memory versus naive / TEMRA CD45RAhigh / CD45ROIow T cell compartments (B+D). The method used seeks to correct and balance these differences.

[0131] Figure 3. Optimised CD45RA purging enhances early memory T cell retention.

[0132] Exemplar flow cytometry plots showing the CD4:CD8 ratio (A) and CD45RA I CD45RO ratio

[0133] (B) of CD3 T cells in start material. The cells have been labelled with fluorescent antibodies to surface CD45RA and CD45RO markers and visualised as plots of fluorescent signal. Naive and other unwanted T cells are characterized as having high mean fluorescence intensity (MFI) for CD45RA and Low MFI for CD45RO (Blue box, 96,600 and 1 ,760 respectively). Early memory T cells have mid MFI for CD45RA and RO (red box, 8,798 and 20,346 respectively). Memory T cells have low MFI for CD45RA and high MFI for CD45RO (3,046 and 38,361 respectively, black box). These MFI values are specific for this donor and the flow cytometer settings and type used here. Nevertheless, in any given sample, the cells that are to be removed are those which express relatively high (e.g. versus other cells (e.g. other CD3 T cells) in the sample) amounts of the cell surface marker, CDR45RA.

[0134] Using a standard CD45RA depletion approach there is a slight increase in CD8 percentage

[0135] (C) after purging, and cells exhibiting a high MFI for CD45RA are removed, but the early memory T cell percentage is low (1.6%, D). However the novel purge approach described here leads to a higher retention of the key CD8 T cell compartment (E) and increases the early memory T cell compartment retention by 5.6 fold in comparison to the standard approach (F), while still removing the CD45RA high population.

[0136] Note that MFI values for CD45RA and CD45RO will vary between different flow cytometer platforms which use different scales and detector settings, however the difference between the values (Delta MFI) should be similar irrespective of the scaling system used. Delta MFI values in this example are: Early memory T cells have a delta MFI of -11 for CD45RA and +11.5 compared to naive T cells. Memory cells have a delta MFI of -31 .7 for CD45RA and +21.8 compared to naive T cells.

[0137] Figure 4. Optimised CD45RA purging results in less cell loss.

[0138] Exemplar flow cytometry plots demonstrating changes in key cell populations after CD45RA purging by our process. The start material (A) contains the key T cell and monocyte material and optimised CD45RA depletion (B) there is a large cell loss, resulting in only 58% viable Total Nucleated Cells (TNC) retained, whereas our optimised CD45RA purge process results in reduced cell loss and a yield of 72% viable TNC from the start material. Both methods preserve the critical cell types, but the optimised method yields 24% more T cells than the standard.

[0139] Figure 5. Optimised CD45RA purging removes unwanted components and enriches desired components in addition to enriching desired memory T cells. Flow cytometry plots showing optimized CD45RAhigh cell depletion from a cancer patient peripheral blood sample. Panel A shows the desired cell types (CD8 T cells, CD4 T cells and APCs) and the undesired cell types (NKT cells, NK / B cells / other and lymphocytes). Panels B and C show the cell profile after optimised CD45RA purge (Panel B: CD45RAmid / low; Depleted -CD4 and CD8 T cells; CD4+ FSC-High APC used for further manufacturing: Panel C: CD45RA high CD4 and CD8 cells; NKT / NK / B cells; other lymphocytes discarded from manufacturing process).

[0140] Figure 6: Donor samples (n=7, 3x HD leukapheresis, 4x Cancer patient peripheral blood) were characterised pre- and post-CD45RA depletion using the process of this disclosure. A) There is a slight increase in mean T cell content post-depletion and a slight trend to increase in CD4 with a concomitant slight decrease in CD8 although there is variation from donation to donation. There is a significant decrease in some NK cell populations but no change in gdT cells. Monocytes are not significantly affected by CD45RA depletion which is an advantage as they act as antigen-presenting cells in the neoantigen stimulations. However, B cells are significantly reduced which provides a further advantage as these can remain as a contaminating cell in expansion cultures.

[0141] Figure 7. Optimised CD45RA purging removes unwanted components

[0142] In addition to the removal of CD45RA high naive T cells and terminally-differentiated Temra T cells, the optimised purge process also reduces other leukocyte populations. In the innate lymphocyte compartment the NK fraction of the innate lymphocyte compartment is modified by purging (A). The number and percentage of NK are reduced and isolates with higher NK numbers are depleted more substantially (B).

[0143] Monocyte percentage and absolute numbers are unaffected by CD45RA purging which ensures that the product retains appropriate APC function in the feeder cells (C). B cells are reduced by purging predominantly in isolates with high CD19 levels which ensures that the process is unlikely to retain CD19 cells in the final product.

[0144] Figure 8. Optimised CD45RA purging enhances percentage of target cells after antigen recall.

[0145] Exemplar flow cytometry plots from a healthy donor in response to Epstein-Barr virus (EBV) antigen recall. In unpurged leukapheresis start material stimulated overnight with EBV- specific peptides, the EBV-specific T cells reacted by expressing the activation marker CD137 on CD4 (A) and CD8 (B) T cells. The same antigen recall process done on start material purged of CD45RA cells in our process demonstrated a 73% increase in the number of antigen-responsive T cells detected in the CD4 (C) and CD8 (D) T cells. The purging process generated a viable TNC of 72% of the starting material and resulted in a net gain of 52% more antigen-specific T cells returned than without purging.

[0146] Figure 9. Optimised CD45RA purging generates substantially larger final T cell products.

[0147] Quantitative assessment of cell numbers and cytokine response in a demonstrator donor. CD137+ T cells were isolated and cultured from a donor with (Purged) or without an initial optimised CD45RA purging step (Unpurged). The cells were then cultured in appropriate culture medium alone (NoTA) or supplemented with GMP-compliant T cell activation reagent (TA - Transact CD3 / CD28 reagent). After 18 days expansion the cells were harvested and counted (A) demonstrating that CD45RA purging resulted in a 40.6 to 68.5 fold increase in cell numbers over equivalent unpurged T cell products. In addition, the purging process resulted in a 49.7-fold increase in antigen-specific IFN-g producing T cells and a 59.3-fold increase in TNF-a producing T cells.

[0148] Materials and methodology Materials for use in process.

[0149] Equipment

[0150] Microbiological Safety Cabinet (MSC)

[0151] Fridge (2-8°C)

[0152] Centrifuge

[0153] Incubator (37°C, 5% CO2)

[0154] Flow Cytometer (e.g. Thermo Attune or Miltenyi MACSQuant 10)

[0155] Gatherex cell harvester (Wilson Wolf)

[0156] CliniMACS cell processor (Miltenyi Biotech)

[0157] Lovo Cell Processor (Scale Ready)

[0158] Calibrated pipettes and pipette controllers

[0159] Consumables

[0160] TS and LS-TS CliniMACS tubing kits (Miltenyi Biotech)

[0161] Sterile 600ml transfer bags

[0162] Sterile 50ml cryobags (Miltenyi)

[0163] GMP-compatible culture medium (eg TexMACS, Miltenyi Biotech)

[0164] G-Rex M100CS closed-process sterile culture flasks

[0165] Human platelet lysate (Life Science Production Ltd)

[0166] Human AB serum (Life Science Production Ltd) I Serum Replacement (Thermo)

[0167] GMP-compliant recombinant human IL-2 (eg Miltenyi)

[0168] GMP CliniMACS buffer (Miltenyi)

[0169] Human serum albumin (20%)

[0170] Crystalloid solution (eg Plasmalyte A or 148 solution)

[0171] Cryostor 10 cryopreservation reagent (BioLife Ltd)

[0172] Phosphate-buffered saline

[0173] Sterile Pipette Tips (1 Oul 120ul 1200ul 1 10OOul)

[0174] Sterile 0.5ml and 1.5ml eppendorfs

[0175] Sterile cryotubes (1.8ml)

[0176] Azo Spray (70% isopropanol)

[0177] Method for generation of T-Swarm product

[0178] Day -7

[0179] Prepare CliniMACS 500ml buffer bags with 12.5ml Human Serum Albumin 20% in a cleanroom using a syringe (final concentration 0.5% HAS) . 2) ) . ) “ B) 1 + / / . ”

[0180] In addition a bag of GMP TexMACS medium is supplemented with 50ml human plasma lysate using a syringe to give a final concentration of 2.5%. This is . 2) ) . ) “ . 2 ” In addition a GMP Medium bag is supplemented with IL-2 to a final concentration of 400IU / ml ( . 2) ) . “ C 5 . . 2 ” 12 B25+. E B21 1. edium during processing to give a final culture concentration of 200IU / ml IL-2. Check sterility and store at 4oC till use.

[0181] Day 0

[0182] Starting Material Preparation

[0183] The donor blood sample in the form of a leukapheresis product is supplied and a cell count is performed using a flow cytometer with a validated cell counting program. Mononuclear cells are prepared by washing a suitable volume of leukapheresis (calculated from cell count) twice in GMP Wash Buffer in order to ensure removal of collection buffer, reduction in residual platelets, red blood cells and debris, and re-formulation for purge. This process is done by placing the cells in a suitable transfer bag and washing by either manual process (centrifugation in transfer bag in suitable bucket centrifuge at 300g for 15 minutes, then the supernatant removed via plasma expresser) or by cell processor such as Lovo device. The bag is attached to a A +20 . / 3> B20 ) / ) , . ’ 2 , 2 and run through the Lovo device using a platelet wash and buffer exchange. In either case the cells are eluted in 90ml final volume wash buffer.

[0184] PBMC feeder stock preparation

[0185] A suitable number of washed PBMC are taken into transfer bag in cleanroom then resuspending cells in one-third volume crystalloid (Plasmalyte) then adding two-thirds volume Cryostor CS10. Mix thoroughly and then transfer sterile to transfer bags (in 10ml aliquots) plus 1.8ml cryovials (1 ml I 1 x107cells per vial). Cryopreserve in an appropriate controlled rate freezer and store at -80oC or vapour phase liquid nitrogen. The bags are for use as feeder layers for manufacturing.

[0186] The remaining leukapheresis is held overnight at controlled room temperature.

[0187] CliniMACS CD45RA labelling and depletion (Start Material Purge)

[0188] AIM - Depletion of CD45RAhigh T cells and unwanted leukocytes from starting material

[0189] Addition of CliniMACS CD45RA reagent (Miltenyi Biotech). Reagent is added by syringe to cell suspension transfer bag and then incubating for 30 minutes on a controlled RT gentle rocking (rocker platform 30 rpm). Dock on buffer bag and make volume up to 500ml with wash buffer then wash either by manual process (centrifuge in 600ml transfer bag at 300g for 15 minutes and remove supernatant using plasma expresser) or by cell processor such as Lovo 1 . +) 0 2 ) ) , 1 . ) A +20 . / S B2 0 ) / ) , . ’ 2 , 2 ) through the Lovo device. The cells are resuspended in wash buffer then the bag is connected to a Miltenyi LS-TS tubing set. Perform custom depletion of CD45RAhigh T cells using CliniMACS Plus instrument and the Depletion 2.1 program.

[0190] The CD45RA-depleted purged cell product is transferred to a new transfer bag and washed with GMP-, $ . 2 5 1 ) § . 5 5 ) . 1 “ . 2 ”). The wash is performed as before on either a manual process or through a cell processor such as the Lovo. A well-mixed sample is taken aseptically for cell counting, then GMP medium added to adjust cell count to 2x107cells / ml.

[0191] Activation with antigen (Ag)

[0192] Aseptically connect cell bag to G-Rex M100CS culture vessel and add cell suspension to give 2x107cells per ml. Add GMP medium supplemented with antigen or peptide mixture at 2x final concentration to give final volume of 1x Antigen at 107cells / ml. Plate into a single G-Rex- CS100M flask (equivalent to 107cells / cm2) then incubate overnight in tissue culture incubator at 37oC, 5% CO2.

[0193] Day 1

[0194] Cell harvest

[0195] Harvest cells from G-Rex flask by closed process using Gatherex device into transfer bag. Adjust volume to 500ml with GMP Wash buffer then wash by centrifugation at 300g for 15 minutes and remove supernatant via plasma expresser or alternatively wash using Lovo as ) / ) , . ’ 2 , 2 . . . S 2 . 2 ) 5B) 1 + / / . ) A 5 . ) . up to 90ml with GMP Wash buffer.

[0196] CD137 Labelling

[0197] Add up to 3.75ml CliniMACS CD137-Biotin reagent by syringe to cell suspension transfer bag then incubate for 30 minutes controlled RT gentle rocking (rocker platform 30 rpm). Dock on buffer bag and volume made up to 500ml with wash buffer and centrifuge in Transfer bag at 300g for 15 minutes then remove supernatant using plasma expresser or through cell processor as before. The cell pellet is resuspended in residual buffer and volume made up to 90ml with wash buffer. Add up to 3.75ml CliniMACS Anti-biotin reagent by syringe to cell suspension transfer bag then incubate for 30 minutes on controlled RT gentle rocking (rocker platform 30 rpm). Dock on wash buffer bag and make volume up to 500ml with wash buffer then wash by centrifugation of 600ml transfer bag at 300g for 15 minutes followed by supernatant removal using plasma expresser or via cell processor as before.

[0198] Enrichment of CD137+ T cells

[0199] Enrichment of CD137+ T cells is made using the CliniMACS Plus instrument with the TS tubing . ) 20 1 - 0 ) ) . ) / ) , . ’ 2 , 2 S et positive and negative fraction bags for further processing.

[0200] Formulate fractions

[0201] The CD137 positive fraction is assessed using a cell count using a well-mixed sample taken aseptically. The cell volume is adjusted to 2x105cells / ml using GMP Medium (providing the dilution results in >50% of final volume as GMP medium).

[0202] The CD137 negative fraction is transferred to a suitable irradiator (X-ray I sealed source etc) and cells irradiated to 25Gy. The cells are then washed by manual or cell processor approach as described before. The cells are resuspended in residual buffer and GMP medium added in to 100ml. The bag is mixed thoroughly and a sample aseptically removed for cell counting. Add GMP medium to adjust cell count to 2x107 / ml.

[0203] Initiate Expansion Culture

[0204] Aseptically connect positive fraction cell bag to G-Rex M100CS culture vessel and add 50ml of cell suspension to give 105cells per cm2. Subsequently aseptically connect the negative fraction cell bag to G-Rex M100CS culture vessel and add 50ml of cell suspension to give 107cells per cm2at a 100:1 feedentarget ratio and a seeding density of >2x106cells / cm2. Alternatively, ensure negative fraction and target cells are at >100:1 ratio, transfer to 600ml transfer bag and connect to Lovo tubing set and run buffer exchange and volume reduction process program. Elute combined cells in 100ml GMP medium. Transfer aseptically to a G- Rex flask at a minimum of 2-4x108total cells per flask (seeding density of 2-4x106cells / cm2). Add 100ml of GMP medium supplemented with 400IU / ml IL-2 (final concentration 200IU / ml). Repeat for subsequent G-Rex flasks if required and incubate at 37oC, 5% CO2.

[0205] Day 4 Aseptically connect GMP medium bag to G-Rex M100CS culture vessel and add 100ml of GMP medium plus 400IU / ml IL-2 to give a total volume of 300ml. Repeat for subsequent G- Rex flasks and continue incubation at 37°C, 5% CO2.

[0206] Day 7

[0207] Aseptically connect medium bag to G-rex M100CS culture vessel and add 100ml of medium plus 400IU / ml IL-2 to give a total volume of 400mL Repeat for subsequent G-Rex flasks and incubate at 37°C, 5% CO2.

[0208] Day 11

[0209] Harvest cells from G-Rex flasks using Gatherex device (up to 4 G-Rex connected in single harvesting step) into transfer bags. Determine volume by weight, then aseptically remove 1 - 2ml medium from the thoroughly mixed bag and perform a cell count. Adjust sample to 4x106cells / ml using GMP medium - this may require a volume reduction using the Lovo cell processor appropriate program.

[0210] Medium only density reset

[0211] Aseptically connect cell bag to G-rex M100CS culture vessel and add sufficient GMP medium to give 4x106cells / cm2. Aseptically connect medium bag to G-rex M100CS culture vessel and add an equivalent volume of medium supplemented with 400IU / ml IL-2 to give a total cell density of 2x106cells / cm2or as near as possible. Repeat for subsequent G-Rex flasks and incubate at 37oC, 5% CO2.

[0212] Feeder Cell Repeat culture

[0213] Thaw frozen PBMC vials stored from leukapheresis on day 10 and culture overnight in appropriate flask. Harvest cells, wash and then formulate feeder cells to a density of 4x10(7) in medium plus 400IU / ml IL-2. Add 1 ug / ml / peptide to the cells and mix thoroughly. Aseptically connect cell bag to G-rex M100CS culture vessel and add 50ml to give 2x106cells / cm2. Repeat for subsequent G-Rex flasks then incubate at 37oC, 5% CO2.

[0214] Transact Stimulation

[0215] GMP TransAct reagent is added aseptically at a volume of 1 :20 to the G-Rex flasks, though the optimal concentration is still under investigation.

[0216] Day 14 Aseptically connect medium bag to G-rex M100CS culture vessel and add 100ml of medium plus 400IU / ml IL-2 to give a total volume of 200ml. Repeat for subsequent G-Rex flasks and incubate at 37oC, 5% CO2. After mixing of medium and cells, take a 1 ml sample for cell count. If the density of T cells exceeds 109cells total (107cells / cm2) then split to a second G-Rex flask.

[0217] Day 18

[0218] Harvest cells from G-Rex using Gatherex device (up to 4 G-Rex connected in single harvesting step) by removing spent medium I supernate and concentrating the cells in the bottom fraction of the flask. Transfer cells into transfer bag and make up to 500ml with wash buffer then wash by manual or cell processor approach as described previously.

[0219] The cell pellet is resuspended in residual buffer and made up to 50ml with suitable buffer precryopreservation - e.g. saline for injection plus AB serum I Human Serum Albumin or with crystalloid e.g. Plasmalyte plus AB serum I Human Serum Albumin). A cell count is taken from a well-mixed sample taken aseptically. This is the bulk drug substance and further sample will be used for a suite of QC assays to determine phenotype, viability and function and to provide formal assessment that the product meets the release criteria.

[0220] Adjust the volume of the cell product using the GMP buffers outlined in the previous section to provide a cell concentration of 3-4.5x107cells per ml. Aseptically dock CS10 container and add 2x volume to achieve a final concentration of 1-1.5x107cells / ml and a DMSO concentration of 6.66%. Dispense into appropriate 50ml cryobags (e.g. CryoMACS) at 10- 15ml per bag (tbc) using Cryodock system or appropriate fill-and-finish device (eg ScaleReady Cue or Sexton Ct-5) if available.

[0221] In addition a specific number of 1.8ml cryovials will be filled (1 ml / vial) to be used for stability studies and identity testing on drug product. All bags I vials are labelled appropriately with patient code, date, cell number and all other data required for tracking and analysis, then transferred to an appropriate controlled rate freezing device before transfer to storage vapour phase LN2 (<-150oC). Preparation of cells for flow cytometric analysis

[0222] This example protocol outlines a process for the washing, blocking and antibody labelling of cells with antibodies for analysis with a flow cytometer.

[0223] Procedure

[0224] 1. Prepare a buffer, e.g. PES buffer (500ml PBS plus 2.5mM EDTA and 0.5% Immune Serum Replacement or suitable alternative - AB serum or Human Serum Albumin); 2. Prepare sufficient antibody mastermix for all samples with the antibodies for the required panel. Allow 1 -2 ul extra antibody to ensure there is sufficient after pipetting.

[0225] 3. Prepare Fc receptor (FcR) block solution for all samples by adding 5p I of FcR block to 10OpI of PES per sample. a. For example, for 8 samples add 40pl of FcR block to 800 p I of PES.

[0226] 4. Count the cells to be labelled and calculate the volume of cell suspension required for 1x106cells per tube.

[0227] 5. Transfer 1-2x106cells to a flow cytometry tube and add 2-3ml of PES.

[0228] 6. Centrifuge the cells at 300g for 5 minutes.

[0229] 7. Discard the supernatant and resuspend the cell pellets in 105pl of prepared FcR block. Incubate at RT for 60 seconds before adding antibody mastermix.

[0230] 8. Add the appropriate volume of antibody mastermix to each sample and incubate in the fridge for 15-20 minutes.

[0231] 9. Add 2-3ml of PES to the cells and wash by centrifugation at 300g for 5 minutes.

[0232] 10. Discard the supernatant and resuspend the cells in 300pl PES.

[0233] 11 . Just before analysing on the flow cytometer, add 1 pl of DRAQ7 to the cells and mix.

[0234] 12. Acquire the sample on the flow cytometer and analyse results appropriately.

[0235] Cell analysis

[0236] 1. The initial detector settings for each channel used are set up on the flow cytometer using appropriate beads (e.g. UltraComp beads, Thermo-Fisher) labelled with individual antibodies from the specific panel. The detector thresholds are then set to ensure that each channel is at an optimal level of fluorescence. The individual labelled beads are then combined to run compensation for each channel. Once complete the template settings are then fixed to ensure that the appropriate level of fluorescence and compensation is set for each channel in the panel.

[0237] 2. This is incorporated as part of a specific gating process for each panel, which includes forward scatter versus side scatter (FSC / SSC) for general cell assessment (removes debris), SSC area versus height for singlet cell assessment, and CD45 for leukocytes and dead cell discriminator (eg DRAQ7) for live cells. This initial screening is used prior to assessment of individual populations on the analysis template to ensure that appropriate cells are examined.

[0238] 3. Beads, unlabelled cells and then cells labelled with one or more of the fluorescent antibodies in each panel are used to confirm the settings and compensations. Individual populations of positive cells are identified using a Fluorescence Minus One approach (FMO), where all labelled antibodies in the panel are added except for the antibody target. The flow plots are then compared between the FMO and the full panel to identify where the box or quandrant should be placed to accurately capture the positive or high population. This protocol is applicable across all QC panels, which are outlined in Appendix 1 below. Appendix 1

[0239] 1 . Cell counting by flow cytometry

[0240] 2. QC flow cytometry panel for assessment of CD45RA depletion of start material

Claims

Claims1. A method of providing antigen-specific T-cells, said method comprising: purging a sample comprising antigen-specific T cells, of unwanted cell types; contacting the purged sample with an antigen or a representation thereof to activate the antigen-specific T-cells; expanding the activated antigen-specific T-cells; and harvesting the expanded antigen-specific T-cells.

2. The method of claim 1 , wherein the sample is purged of naive T cells and / or CD45RAhigh cells and / or cells with the following phenotypic marker profile: CD3+, CD4+ and / or CD8+, CD45RAhigh, CD45ROIow, CD62L+; optionally, wherein the sample is further or additionally purged of any one or more of the following cell types:(i) terminally-differentiated T cells; and / or(ii) cells with the following phenotypic marker profile: CD3+, CD4+ and / or CD8+, CD45RAhigh, CD45RO, CD62L-; and / or(iii) NK cells; and / or(iv) cells with the following phenotypic marker profile: CD3-, CD56+, CD16+; and / or(v) B cells; and / or(vi) cells with the following phenotypic marker profile: CD3-, CD19+.

3. The method of any preceding claim, wherein the antigen-specific T-cells are for therapeutic use.

4. The method of any preceding claim, wherein the method provides enriched antigenspecific T-cells for use in therapy.

5. The method of any preceding claim, wherein the sample comprises a blood sample or fraction thereof.

6. The method of any preceding claim, wherein the sample comprises peripheral blood mononuclear cells (PBMCs).

7. The method of any preceding claim, wherein the sample comprises >0.02% antigenspecific T cells with no upper limit.

8. The method of any preceding claim, wherein the sample is provided by or obtained from a subject exposed to or immunised or vaccinated with an antigen.

9. The method of any preceding claim, wherein the antigen is a cancer / tumour antigen, an antigen from a pathogen, a bacterial antigen, a viral antigen or a fungal antigen.

10. The method of any preceding claim, wherein the subject is a human or animal subject.

11. The method of claim 8, wherein the exposure, immunisation and / or vaccination protocol has induced an immune response to the antigen.

12. The method of claim 11 , wherein the sample is obtained or provided after induction of the immune response.

13. The method of claims 11 or 12, wherein the antigen specific T cells are specific for said antigen.

14. The method of any preceding claim wherein the purging step preserves the early memory T cells such as those expressing CD45RAmid and CD45ROmid in the sample.

15. The method of any preceding claim, wherein the purging step enriches the antigenspecific T-cell content.

16. The method of any preceding claim, wherein the purged sample is contacted with said antigen or a representation thereof for 12-40 hours.

17. The method of any preceding claim, wherein the purged sample is further or additionally contacted with autologous serum or plasma.

18. The method of claim 16 or 17, wherein the purged sample is further or additionally contacted with up to 20% autologous serum or plasma.

19. The method of any preceding claim, wherein the purged sample is contacted with the antigen or a representation thereof at a concentration of 0.01 - 1000pg / mL20. The method of any preceding claim, wherein the step of contacting the purged sample with said antigen or a representation thereof activates and enriches the antigenspecific T cells within the sample.21 . The method of claim 20, wherein prior to the expansion step, the activated and enriched antigen-specific T cells are isolated from the other cells.

22. The method of any preceding claim, wherein the step of expansion comprises contacting the activated and enriched antigen-specific T cells with feeder cells and / or cytokines.

23. The method of claim 22, wherein the cytokines are one or more selected from the group consisting of: IL-2; IL-4; IL-7; IL-15 and IL-23.

24. The method of claim 22, wherein the activated and enriched antigen-specific T cells are combined with feeder cells in a ratio of 1 :5-1 :500 antigen-specific T cells: feeder cells.

25. The method of claim 22-24, wherein the total cell density of antigen-specific T cells and feeder cells at initial seeding is of 0.5x106-1x107cells per cm2.

26. The method of claim 22 or 24-25, wherein the feeder cells are modified by CD3 depletion or inactivated by irradiation or cytotoxic treatment.

27. The method of claim 26, wherein the feeder cells are derived from the same patient sample.

28. The method of any one of claims 26 or 27, wherein the feeder cells are derived from an un-manipulated portion of the sample or a portion of the sample which has been manipulated and / or subject to enrichment protocols.

29. The method of any one of claims 26-28, wherein the feeder cells are derived from the sample by:(i) stimulating the sample with the antigen or a representation thereof; and / or(ii) depleting the sample of CD3+ cells and / or(iii) inactivating the sample with irradiation or mitomycin c.

30. The method of any preceding claim, wherein the step of expansion comprises contacting the activated T-cells with feeder cells for up to 14 days.31 . The method of claim 30, wherein the step of expansion further comprises expansion in the presence of T cell mitogens and / or the antigen or a representation thereof.

32. The method of claim 31 , wherein the T cell mitogens comprise CD3 and / or CD28 specific antibodies.

33. The method of any preceding claim, wherein the cells are expanded in GMP- compliant medium for up to 21 days.

34. The method of any preceding claim, wherein prior to harvesting, the method comprises a further expansion step.

35. The method of claim 34, wherein the further expansion step comprises expansion in the presence of feeder cells and / or cytokines.

36. The method of claim 35, wherein the feeder cells are as defined in any one of claims 25-28.

37. The method of any one of claims 34-36, wherein the further expansion step comprises expansion in the presence of T-cell mitogens and / or the antigen or a representation thereof.

38. The method of claim 37, wherein the T-cell mitogens comprise CD3 and / or CD28 specific antibodies.

39. The method of claims 34-38, wherein the cells are expanded in GMP-compliant medium for up to 21 days.

40. The method of any one of claims 34-39, wherein the activated, enriched and expanded antigen-specific T cells are combined with feeder cells in a ratio of 1 :5-1 :500 antigen-specific T cells: feeder cells.41 . The method of any one of claims 34-40, wherein the total cell density of antigenspecific T cells and feeder cells at initial seeding is of 0.5x106-1x107cells per cm2.

42. The method of any one of claims 34-41 , wherein further expansion step comprises the use of one or more cytokines selected from the group consisting of IL-2; IL-4; IL-7; IL-15 and / or IL-23.

43. The method of any preceding claim, wherein the harvested cells are washed.

44. The method of any preceding claim, wherein the harvested cells are subject to a quality control procedure.

45. The method of any preceding claim, wherein the harvested cells are transferred to a suitable excipient containing cryoprotectant and subsequently frozen.

46. The method of any preceding claim, wherein the harvested cells are prepared and / or formulated for administration to a subject.

47. The method of any preceding claim, wherein the sample is obtained or provided by a subject and the harvested cells are prepared and / or formulated for administration to the same subject.

48. A T-cell therapy obtained or obtainable from a method according to any preceding claim.

49. The T-cell therapy of claim 48, for use in medicine or for use as a medicament.

50. The T-cell therapy of claims 48-49, for use in the treatment or prevention of cancer.51 . The T-cell therapy of claims 35-36 or 38, for combined use with cancer therapy, a check point inhibitor treatment, a co-stimulatory antibody, T-cell specific stimulatory cytokine, an IL-2 therapy, an IL-7 therapy, an IL-15 therapy, chemotherapy, radiotherapy and / or another monoclonal antibody-based therapy.

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