WO2013175237A1

WO2013175237A1 - Composition comprising a cd2 ligation agent and a nkg2d ligation agent

Info

Publication number: WO2013175237A1
Application number: PCT/GB2013/051387
Authority: WO
Inventors: Mark Lowdell
Original assignee: UCL Business Ltd
Current assignee: UCL Business Ltd
Priority date: 2012-05-24
Filing date: 2013-05-24
Publication date: 2013-11-28
Anticipated expiration: 2014-11-24

Description

COMPOSITION COMPRISING A CD2 LIGATION AGENT AND A NKG2D LIGATION AGENT

FIELD OF THE INVENTION

The present invention relates to a composition for priming a human Natural Killer (NK) cell. Following priming, the NK cell may have the capacity to lyse an NK-resistant cancer cell. BACKGROUND TO THE INVENTION

A number of cancers are, at present, incurable. For others, chemotherapy is only partially effective and a significant proportion of patients relapse following treatment. Some haematological malignancies are treatable by hematopoietic stem cell transplantation (HSCT), but fewer than 30% of patients requiring HSCT have a suitable donor and are the requisite age.

Natural Killer (NK) cells are a subset of peripheral blood lymphocytes which can spontaneously lyse certain tumour cells. The use of NK cells in adoptive tumour immunotherapy has been proposed, and there has been interest in the in vitro or ex vivo stimulation of NK cells to increase their capacity to lyse tumour cells.

The discovery of interleukin-2 (IL-2) and its role in NK-cell activation in the 1980's led to considerable interest in the use of lymphokine-activated killer (LAK) cells in tumour immunotherapy. The results of these trials were, however, largely disappointing. In a study investigating the effect of administering autologous LAK cells to patients along with IL-2, fewer than 20% of patients responded (Rosenburg et al (1987) N. Engl. J. Med. 316: 889-897). In studies using daily IL-2 administrations to cancer patients along with chemotherapy and autologous HCT, it was shown that, although IL-2 significantly expanded the number of circulating NK cells in vivo, the cells are not maximally cytotoxic according to an in vitro assay (Miller et al (1997) Biol. Blood Marrow Transplant. 3: 34-44).

Resting human NK cells require at least two activating signals before commitment to cytokine secretion and/or target cell lysis. It has been shown that these two signals can be divided into discrete "priming" and "triggering" events, with the priming signal being provided either by an activating cytokine, such as IL-2, or conjugation to a tumour cell expressing an appropriate intensity and combination of signals (North et al (2007) J. Immunol. 178:85-94).

WO 2006/097743 describes a method for activating an NK cell by contacting the NK cell in vitro with a preparation of the tumour cell line CTV-1 , which primes resting NK cells but fails to trigger lysis.

Various other leukemia cell lines have been shown which prime NK cells but are resistant to lysis, including MV-411 and SEM (Sabry et al (201 1) J. Immunol. 187:6227-6234).

It is therefore possible to activate resting human NK (rNK) cells in vitro by bringing the rNK cells into contact with such tumour cells, or a cell membrane preparation thereof. In order to produce such an "activating tumour cell preparation" (ATCP) it is necessary to grow and purify the tumour cells (such as CTV-1 , MV-411 or SEM) and optionally lyse them to provide a cell membrane preparation.

Membrane preparations have the advantage over preparations comprising intact tumour cells as they avoid the risk of transferring potentially malignant tumour cells to the patient. However, there are still safety issues associated with the use of a tumour cell membrane preparation and problems with accurately quantifying the preparation.

There is thus a need for an alternative ATCP which is simple to prepare and standardise and does not give rise to the safety issues mentioned above.

DESCRIPTION OF THE FIGURES

Figure 1 - Investigating the optimum concentrations of the anti-CD2 and anti-NKG2D antibodies for bead coating.

Figure 2 - Investigating the Optimum ratio of anti-CD2 beads to anti-NKG2D beads to resting human NK cells. SUMMARY OF ASPECTS OF THE I NVENTION

It has been shown that CD2-ligation is one of the mechanisms whereby resting human N K (rN K) cells are primed to activate. CTV-1 causes CD2 ligation through expression of CD15, which can bind CD2 (Sabry et al (201 1 ) J . Immunol. 187:6227- 6234).

However, CD15 alone is insufficient to prime rNK since normal human myeloid cells which constitutively express CD15 do not prime rNK.

The present inventors have now found that it is possible to prime rNK cells by co- ligating both CD2 and NKG2D.

Thus in a first aspect, the invention provides a human natural killer (NK) cell-priming composition which comprises (i) a CD2 ligation agent; and (ii) an N KG2D ligation agent.

The CD2 ligation agent may comprise the CD2 ligand from CD15. The CD2 ligation agent may be CD15 with its associated carbohydrate structure.

Alternatively, the CD2 ligation agent may be an anti-CD2 antibody.

The NKG2D ligation agent may be an anti-NKG2D antibody. The N KG2D ligation agent may comprise the N KG2D binding site from an N KG2D ligand, such as MICA, MICB or ULBPs. The NKG2D ligation agent may comprise all or a part of MICA, MICB or a ULBP.

In a second aspect, the invention provides a human NK-cell priming substrate or vesicle which comprises a CD2 ligation agent and an N KG2 D l igation agent as defined in connection with the first aspect of the invention.

The substrate may, for example, be a two-dimensional surface (e.g. filter, plate, well, flask, bag, or the like) or a three-dimensional surface (e.g., bead, nanoparticle or the like), coated with a CD2 ligation agent and an NKG2D ligation agent. The vesicle may be a liposome which expresses a CD2 ligation agent and an NKG2D ligation agent at the surface.

The liposome may also contain one or more cytokines involved in NK function/survival//proliferation, such that the cytokine(s) are released when NK cell priming occurs. The cytokine(s) may, for example, be IL2, IL12 and/or IL15.

In a third aspect, the present invention provides a method for activating a human Natural Killer (NK) cell, which comprises the step of contacting the NK cell in vitro with:

a CD2 ligation agent and an NKG2D ligation agent as defined in connection with the first aspect of the invention;

a human NK-cell priming composition according to the first aspect of the invention; or

a human NK-cell priming substrate or vesicle according to the second aspect of the invention.

In a fourth aspect, the present invention provides an activated NK cell produced by a method according to the third aspect of the invention.

The activated NK cell may maintain its activated state following removal of the NK-cell priming composition, substrate or vesicle.

The activated NK cell may maintain its activated state following cryopreservation.

In a fifth aspect, the present invention provides a pharmaceutical composition comprising a plurality of activated NK cells according to the fourth aspect of the invention for treating a subject in need of same. I n a sixth aspect, the present invention provides the use of a pharmaceutical composition according to the fifth aspect of the invention in the manufacture of a medicament for the treatment of cancer.

In a seventh aspect, the present invention provides a method for treating cancer or infection which comprises the step of administering a pharmaceutical composition according to the fifth aspect of the invention to a subject. I n an eighth aspect, the present invention provides a human N K-cell priming composition according to the first aspect of the invention, or an N K-cell priming substrate or vesicle according to the second aspect of the invention for use in the activation of an NK cell in vivo.

In a ninth aspect, the invention provides a human NK-cell priming composition according to the first aspect of the invention, or an NK-cell priming substrate or vesicle according to the second aspect of the invention for use in the treatment of cancer or infection.

In a tenth aspect, the present invention provides a method for activating a human NK cell in vivo which comprises the step of administering a NK-cell priming composition according to the first aspect of the invention, or an NK-cell priming substrate or vesicle according to the second aspect of the invention to a subject.

In an eleventh aspect, the present invention provides a method for treating cancer or infection in a subject in need of same which comprises which comprises the step of administering an NK-cell priming composition according to the first aspect of the invention, or an NK-cell priming substrate or vesicle according to the second aspect of the invention to the subject.

In a twelfth aspect, the invention provides a kit for preparing a composition according to the first aspect of the invention, a substrate or vesicle according to the second aspect of the invention and/or for use in a method according to the third aspect of the invention which kit comprises (i) a CD2 ligation agent; and (ii) an NKG2D ligation agent.

In a thirteenth aspect, the present invention provides a method for making a substrate according to the second aspect of the invention which comprises the step of attaching a CD2 ligation agent and an NKG2D ligation agent to a substrate.

In a fourteenth aspect, the present invention provides a method of making a vesicle according to the present invention which comprises the step of incorporating or causing the expression of a CD2 ligation agent and an NKG2D ligation agent at the vesicle surface. Because the method of the present invention causes activation of an NK cell via ligation of two cell surface receptors by ligation agents, the method is entirely "synthetic" and does not rely on the presence of an activating tumour cell or cell membrane preparation thereof. The invention replaces CTV-1 with an artifical NK priming reagent using a solid phase presentation system (such as a filter, plate, well, flask, bag or other two dimensional surface, beads, nanoparticles or liposomes). The method of the invention therefore overcomes the disadvantages associates with the tumour cell activation method, as outlined in the previous section.

DETAILED DESCRIPTION

NATURAL KILLER (NK) CELL H uman N K cells are a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3). They recognise and kill transformed cell lines without priming, in an MHC-unrestricted fashion. NK cells represent the predominant lymphoid cell in the peripheral blood for many months after clinical allogeneic or autologous stem cell transplant and they have a primary role in immunity to pathogens during this period (Reittie et al (1989) Blood 73: 1351-1358; Lowdell et al (1998) Bone Marrow Transplant 21 : 679-686). The role of NK cells in engraftment, graft-versus-host disease, anti-leukemia activity and post- transplant infection is reviewed in Lowdell (2003) Transfusion Medicine 13:399-404.

Human NK cells mediate the lysis of tumour cells and virus-infected cells via natural cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC). Human NK are controlled by positive and negative cytolytic signals. Negative (inhibitory) signals are transduced by C-lectin domain containing receptors CD94/NKG2A and by some Killer Immunoglobulin-like Receptors (KIRs). The regulation of NK lysis by inhibitory signals is known as the "missing self" hypothesis in which specific HLA-class I alleles expressed on the target cell surface ligate inhibitory receptors on NK cells. The down-regulation of HLA molecules on tumor cells and some virally infected cells (e.g. CMV) lowers this inhibition below a target threshold and the target cells becomes susceptible to NK cell-mediated lysis. I nhibitory receptors fall into two groups, those of the Ig-superfamily called Killer Immunoglobulin-like Receptors (KIRs) and those of the lectin family, the NKG2, which form dimers with CD94 at the cell surface. KIRs have a 2- or 3-domain extracellular structure and bind to HLA-A, -B or -C. The NKG2/CD94 complexes ligate HLA-E.

Inhibitory KIRs have up to 4 intracellular domains which contain ITIMs and the best characterized are KIR2DL1 , KIR2DL2 and KIR2DL3 which are known to bind HLA-C molecules. KIR2DL2 and KIR2DL3 bind the group 1 HLA-C alleles whilst KIR2DL1 binds to group 2 alleles. Certain leukemia/lymphoma cells express both group 1 and 2 HLA-C alleles and are known to be resistant to NK-mediated cell lysis

As regards positive activating signals, ADCC is mediated via CD16 and a number triggering receptors involved in natural cytotoxicity have been identified, including CD2, CD38, CD69, NKRP-1 , CD40, B7-2, NK-TR, NKp46, N Kp30 and N Kp44. In addition, several KIR molecules with short intracytoplasmic tails are also stimulatory. These KIRs (KIR2DS1 , KIR2DS2 and KIR2DS4) are known to bind to HLA-C; their extracellular domains being identical to their related inhibitory KIRs. The activatory KIRs lack the ITIMs and instead associate with DAP12 leading to NK cell activation. The mechanism of control of expression of inhibitory versus activatory KIRs remains unknown.

The NK cells activated by the method of the present invention may be autologous or allogeneic NK cells.

"Autologous" NK cells are cells derived from the patient. "Allogeneic" NK cells are derived from another individual, having non-identical gene at one or more loci. If the NK cells are derived from an identical twin, they may be termed "syngeneic". Donor NK cells may be HLA-KI R matched or mismatched. The present inventors have shown that the degree of matching between the NK cells and target tumour cells is of no significance.

PRIMING

Resting human NK cells require a two-stage activation process, involving a "priming" and a "triggering" step. NK-sensitive tumours provide both priming and triggering signals, leading to lysis. NK-resistant tumours evade lysis, mostly by failure to prime. However, there are some tumour cells, such as the tumour cell line CTV-1 , which have the capacity to prime tumour cells but fail to trigger lysis. It has previously been shown that such cells can be used to prime or activate NK cells, such that it can go on to lyse a target cell which is resistant to lysis by an equivalent unstimulated NK cell.

The terms "priming", "activating" and "stimulating" NK cells are used synonymously in this document to mean rendering a resting NK into a state such that can lyse a target cell which is normally resistant to NK cell lysis. The target cell may be an NK resistant tumour cell. Raji and Daudi cell lines are useful models for NK-resistant tumours. The composition of the present invention is capable of priming an NK cell in the same way as treatment with an activating tumour cell preparation of, for example, CTV-1 cells.

Primed or activated NK cells have a characteristic phenotype, which distinguishes them from resting NK cells.

Tumour-mediated NK priming occurs by a different mechanism than IL-2 stimulation. Tumour-mediated NK priming involves activation of the ΰϋ3ζ-Ι_ΑΤ-8ί3ί5 pathway (see below) whereas IL-2 is known to activate NK cells via MAPK1/extracellular signal-related protein kinase. With tumour-mediated NK activation, upregulation of CD69 and IFNy synthesis is observed within 4 hours, whereas it takes a minimum of 48 hours following activation by IL-2 (Sabry et al (201 1) as above). Upregulation of CD25 expression following IL-2 stimulation is also significantly slower than that induced by CTV-1 , and the proportion of activating NK cells is smaller.

Tumour-mediated NK activation is also stable after removal of the priming signal. Whereas IL-2 activated NK cells rapidly return to the non-activated state after removal of IL-2, CTV-1 activated NK cells retain their capacity for Raji-cell lysis even after many months of cryopreservation.

In preferred embodiments, incubation of NK cells with the subject ligation agents as described herein causes rapid upregulation of CD69 on the NK cells. In addition to CD69, the I L-2 receptor, CD25, is also upregulated. Accordingly, in a preferred embodiment the subject ligation agents produce an activated NK cell population that is CD69+ and/or CD25+. In a further embodiment, contact with appropriate ligation agents may also result in the transfer of CD15 to the activated NK cells (e.g., activated NK cells gain CD15), and the reduction of CD16 expression from the NK cell after activation. Accordingly, in further embodiments, the activating agents contemplated for use herein may further produce an activated NK cell population that is also CD15+ and/or CD16low.

LIGATION AGENTS

The composition of the present invention comprises at least two ligation agents: a CD2 ligation agent, and an NKG2D ligation agent.

A ligation agent is an entity which binds and activates a receptor.

A receptor is a cell-associated protein that binds to a bioactive molecule (the "ligand") and mediates the effect of the ligand on the cell. Binding of ligand to receptor results in a change in the receptor (and, in some cases, receptor multimerization, i. e. , association of identical or different receptor subunits) that causes interactions between the effector domain (s) of the receptor and other molecule (s) in the cell. These interactions in turn lead to alterations in the metabolism of the cell. The ligation agent may be a natural or synthetic ligand for the receptor. The ligation agent may trigger the same intracellular effect as the natural ligand. The ligation agent may be a binding agent such as an antibody.

The ligation agent may be an agonist for the receptor, or an analog or derivative thereof including, e.g., a fusion of the agonist with another protein or compound.

The ligation agent may be an antibody or an antigen-binding fragment thereof, including, e.g., a monoclonal or polyclonal antibody, a tetrabody, a nanobody, a chimeric antibody, a deimmunized antibody, a humanized antibody or a human antibody. In particular embodiments of the present invention the antigen binding fragment is selected from the group consisting of F(ab)2, F(ab')2, Fab, Fab', Fd, Fv, single-chain Fv, and disulfide-linked Fvs (dsFv). The term "antibody" includes antibody-like molecules with alternative scaffolds such as DARPins and other repeat protein scaffolds and domain antibodies (d(Ab)s).

The ligation agents can be modified, e.g., by the covalent attachment of any type of molecule as long as such covalent attachment permits the agonist or antibody to retain its activation of the receptor. For example, but not by way of limitation, suitable derivatives and analogs of the ligation agents include those that have been further modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a substrate or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, etc. Additionally, the analog or derivative can contain one or more unnatural amino acids, or have a modification (e.g., substitutions, deletions or additions) in amino acid residues that interact with Fc receptors.

CD2 LIGATION AGENT

CD2 is a cell adhesion molecule found on the surface of T cells and NK cells. It acts as a costimulatory molecule.

It has been shown that, during tumour mediated NK priming, CD2 on the NK cell binds to a ligand within CD15 on the tumour cell (Sabry et al (201 1) J. Immunol. 187:6227-6234). Blockade of CD15 on tumour cells has been shown to significantly inhibit priming of NK cells. Also, NK-resistant Raji cells become susceptible to NK lysis following transfection and expression of CD15.

The CD2 ligation agent may comprise the CD2L site of CD15. The ligand for CD2 of CD15 is a carbohydrate structure which is closely associated with, yet distinct from CD15 (Warren et al (1996) J. Immunol. 156:2866-2873). The carbohydrate structure is Gal^1-4 GlcNAc a1-3Fuc. The ligation agent may comprise this carbohydrate structure optionally in association with all or a part of CD15.

Various anti-human CD2 antibodies are known, such as clone RPA-2.10 (Bryceson et al (2006) Blood 107:159-166), and OKT11 (Sabry et al (201 1 as above).

CD2 ligation activates the Οϋ3ζ-Ι_ΑΤ-8ί3ί5 pathway. The cytoplasmic Οϋ16/Οϋ3ζ complex interacts with the intracellular domain of CD2, leading to the phosphorylation of linker for activation of T cells (LAT). Ligation of CD2 leads to phosphorylation of Stat5 and upregulation of CD25 and CD69.

The CD2 ligation agent may cause activation of the Οϋ3ζ-Ι_ΑΤ-8ί3ί5 pathway. This may be detected by monitoring phosphorylation of LAT and/or Stat5; by examining the levels of CD25 and/or CD69; or by investigating the production of IFNy.

NKG2D LIGATION AGENT NKG2D is an activating receptor found on the surface of NK cells and CD8+ T cells.

NKG2D consists of two disulphide-linked type II transmembrane proteins with short intracellular proteins which are incapable of transducing signals. NKG2D therefore requires an adaptor protein in order to transduce signals. Two adaptor proteins exist, DAP10 and DAP12, which associate as homodimers to the receptor. The entire receptor complex therefore appears as a hexamer.

DAP10, the signalling subunit, carries a phosphatidylinositol-3-kinase binding motif. Ligands for NKG2D are induced during times of cellular stress, either as a result of infection or genomic stress such as in cancer, which renders the cell susceptible to NK cell mediated lysis.

Currently known ligands for NKG2D include MICA, MICB, ULBP1 , ULBP2, ULBP3 and ULBP4-6.

The NKG2D ligation agent used in the composition of the present invention may comprise all or part of a NKG2D ligand. The ligand may also comprise a portion of an antibody, for example the Fc portion, giving MICA-Fc and ULBP-Fc.

The NKG2D ligation agent used in the composition of the present invention may comprise all or part of an antibody. Commercial anti-human NKG2D antibodies are known, such as clone 149810 from R&D systems, Minneapolis, and 1 D1 1 from eBioscience.

SUBSTRATE The second aspect of the present invention provides a substrate which comprises a CD2 ligation agent and an NKG2D ligation agent. The ligation agents may be attached to the surface of the substrate. The substrate may be used to activate rN K cells, by bringing the rN K cells into contact with the substrate such that ligation of CD2 and NKG2D on the NK cells occurs.

One of the advantages of using a solid substrate is it facilitates removal of the activating agents (namely the CD2 and NKG2D ligation agents) following NK cell activation. Once the substrate is removed, the resulting preparation should comprise activated NK cells in a relatively pure form (i.e. without the CD2 and NKG2D ligation agents). Where the ligation agents are antibodies, it is possible to use substrates coated with a "second-layer" antibody (i.e. an antibody reactive with the ligation agent) in order to attach the ligation agents.

The substrate may present a two dimensional surface, such as a filter, plate, well, flask, roller bottle, capillary, bag and the like; or a three-dimensional surface, such as a bead or microparticle. Magnetic/paramagnetic beads or particles may be used to aid separation. The substrate material employed in the present invention may be of any suitable material and may be a porous or a non-porous support. Preferably, the substrate is a solid support, particle or bead. In one embodiment, the substrate is comprised of a cross-linked carbohydrate material, such as agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan, alginate etc. The substrate may easily be prepared according to standard methods, or is a commercially available product, such as DYNABEADS™ (Life Technologies, California, USA) or SEPHADEX™ or SEPHAROSE™ FF (Amersham Biosciences AB, Uppsala, Sweden). In an alternative embodiment, the substrate is comprised of cross-linked synthetic polymers, such as styrene or styrene derivatives, divinylbenzene, acrylamides, acrylate esters, methacrylate esters, vinyl esters, vinyl amides, or may be an inorganic material such as glass or silica. Preferably, the polymer is selected from the group consisting of polystyrene, polypropylene, polyvinyltoluene, polyacrylamide, polyacrylonitrile and polycarbonate. Solid supports of such polymers are easily produced according to standard methods, see e.g. "Styrene based polymer supports developed by suspension polymerization" (Arshady, R., Chimica e L'lndustria, (1988), 70(9), 70-75).

The invention also provides a method for making a substrate according to the second aspect of the invention which comprises the step of attaching a CD2 ligation agent and an NKG2D ligation agent to a substrate. The agents may be directly attached, or attached via another entity, such as an antibody or linker.

VESICLE

The invention also provides a vesicle which comprises a CD2 ligation agent and an NKG2D ligation agent. The ligation agents are displayed on the surface of the vesicle such that they can interact with CD2 and NKG2D on the surface of rNK cells. The vesicle may be a membranous vesicle. A liposome is an artificial vesicle composed of a lipid bilayer.

The vesicle may comprise one or more cytokines. The cytokine(s) may be associated with NK cell activation, survival and/or proliferation. The cytokine(s) are available to the NK cell when NK cell priming occurs.

The vesicle may, for example, comprise IL2, IL12 or IL15. The cytokines may be released from the vesicle at the site of NK cell activation or taken up by the NK cell within the vesicle.

The vesicle may be a cell-like structure but may not be a continuously growing tumour cell line. Where the vesicle is a cell, the CD2 ligation agent and an NKG2D ligation agent should not be endogenous to the cell. The cell may be a non-tumorous cell or a primary tumour within or isolated from a patient induced to express these agents artificially (i.e. by recombinant means).

The invention also provides a method of making a vesicle according to the present invention which comprises the step of incorporating or causing the expression of a CD2 ligation agent and an NKG2D ligation agent at the vesicle surface.

Where the vesicle comprises a membrane, the CD2 ligation agent and NKG2D ligation agent may be, comprise, or be attached to an entity which is capable of being included in the membrane. For example, the agent(s) may be attached to a transmembrane protein.

Methods for including membrane proteins in the lipid bilayer of liposomes are known in the art and include: methods involving the use of an organic solvent, methods involving the use of the mechanical means, methods involving the use of detergents and direct incorporation of the protein into the preformed liposomes.

It is also possible to express proteins within liposomes by providing the liposomes with the necessary machinery to transcribe and translate the relevant gene.

ACTIVATION METHOD

The present invention also provides a method for activating a human Natural Killer (NK) cell, which comprises the step of contacting the NK cell in vitro with:

a CD2 ligation agent and an NKG2D ligation agent;

an NK-cell priming composition according to the present invention; or an NK-cell priming substrate or vesicle according to the present invention. In accordance with the present invention, NK cells may be activated by the one of the above methods alone, or in combination with another NK cell activation technique.

For example, US7435596 describes the expression of chimeric receptors on NK cells to enhance their capacity to kill target cells. The NK cells of the present invention may express a chimeric receptor comprising an anti-CD19 receptor and a signalling domain. The signalling domain may, for example be ΟΌ3ζ or DAP10.

The chimeric receptor may also comprise the costimulatory molecule 4-1 BB. The resting NK cell may be autologous or allogeneic.

Allogenic NK cells may be obtained from peripheral blood from a donor individual. Allogeneic peripheral blood mononuclear cells may be collected by standard techniques (e.g. conventional apheresis). To minimize the possibility of graft versus host disease and immune mediated aplasia, allogeneic cells may be depleted of T cells. For example, the cell preparation may be depleted of CD3+ T-cells using microbeads conjugated with monoclonal mouse anti-human CD3 antibody and a cell selection device (such as the Miltenyi Biotec CliniMACS® cell selection device).

However, NK cells produced by such "negative selection" procedures alone do not have a high degree of purity and may be contaminated with T and B cells.

In order to reduce contamination, it is possible to obtain an NK cell preparation by direct immunomagnetic separation, for example on the basis of CD56 expression. To further reduce T cell contamination, the product may be depleted for CD3+ cells (for example using CD3 FITC and anti-FITC beads).

Prior to activation by the method of the invention, the NK cell preparation may comprise at least 80%, at least 90%, at least 95% or at least 98% CD56+ cells. Prior to activation by the method of the invention, the N K cell preparation may comprise less than 15%, less than 10%, less than 5% or less than 3% CD3+ cells.

ACTIVATED NK CELL The present invention also provides an NK cell activated by a method according to the present invention.

PHARMACEUTICAL COMPOSITION The present invention also provides a pharmaceutical composition comprising NK cells activated by a method of the invention.

The composition may comprise or consist essentially of autologous and/or allogeneic NK cells.

Allogeneic NK cells may be HLA mismatched.

The composition may also comprise the CD2 ligation agent and/or NKG2D ligation agent; or a substrate or vesicle comprising the CD2 ligation agent and/or NKG2D ligation agent. The CD2 ligation/ NKG2D ligation may be the only activation the NK cells receive, or there may be further activation steps. The N K cells may or may not also be non- specifically activated by I L-2 (for example by incubation of the cells in medium supplemented with IL-2). Alternatively, the cells may be activated in the absence of IL-2, but IL-2 may be used for the ex vivo expansion of stimulated cells.

METHOD OF TREATMENT

The composition of the present invention may be used in medicine. For example, the composition may be used to treat or prevent cancer or infection in a subject.

The composition comprising activated NK cells may be manufacture of a medicament for the treatment of cancer or infection. The composition may be administered to the subject by any suitable method known in the art, for example, intravenous infusion.

The present invention also provides a method for treating a subject in need of same, which comprises the following steps:

(i) activating a resting NK cell in vitro by a method according to the present invention; and

(ii) administering the activated NK cell to the subject.

The method may be for treating a disease in the subject. The disease may be cancer or an infection.

The composition may be used to treat a subject in need of same. The procedure is low-risk and particularly suitable for cancer patients for whom intensive cancer treatments are precluded (for example, elderly patients). It also provides an alternative for patients (with, for example, lymphoma, myeloma or AML) who lack a suitable donor for allogeneic stem cell transplantation.

Prior to treatment with the composition, the patient may receive some pre-treatment, for example, to de-bulk the tumour and /or immunosuppress the patient. This may be achieved, for example, by chemotherapy, radiotherapy or a combination thereof.

It is possible to obtain primary tumour cells from patients at time of diagnosis and to cryopreserve these as viable single cell suspensions. It is thus possible for a composition according to the invention to be tested in vitro against patient blasts. This could be done before embarking on a treatment regime, to gauge the suitability of the approach. The correlation of the results of the in vitro study and the corresponding clinical response to treatment may also be investigated.

DISEASE

The activated NK cell composition may be used to treat or prevent a disease or medical condition.

The disease may be a cancer. There are about 200 different types of cancer. Lists of types of cancer are available (for example, see http://www.acor.org/types.html or http://www.cancerresearch uk.org). Some more common cancers include leukaemia (acute and chronic), bladder cancer, bone cancer (osteosarcoma), Bowel (colorectal cancer), brain cancer, breast cancer, cervical cancer, oesophageal cancer, Hodgkin's lymphoma, kidney cancer, liver cancer, lung cancer, mesothelioma, multiple myeloma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, skin cancer (melanoma and non-melanoma) soft tissue carcinoma, gastric cancer, testicular cancer, thyroid cancer and endometrial cancer.

The activated NK cell composition produced by the method of the present invention may be useful to treat any cancer which is accessible to NK cells.

In particular the cancer may be a haematological malignancy, such as leukaemia (AML); myeloma; Lymphoma.

Myeloma is an incurable and fatal malignancy. NK activity against myeloma plasma cells is documented in vitro and enhanced NK activity against autologous myeloma cells has been shown to correlate with response to treatment with Thalidomide derivatives. Myeloma patients are generally young and fit enough to undergo autologous haematopoietic stem cell transplantation and could readily undergo a less invasive procedure such as the one provided by the present invention.

Post-transplant lymphoproliferative disease (PTLD) is a serious and relatively common complication after solid organ transplantation and T cell immunotherapy is currently under trial but with little success. Therapy using NK cells activated according to the present invention therapy would be easy and safe in this group of patients. In addition the composition may be used to treat solid tumours such as breast cancer.

The procedure is particularly suitable to treat "NK-resistant" tumours. Normal NK cells can spontaeously lyse some human tumours, but many other tumours are NK- resistant. "NK-resistant" as used herein, therefore, indicates tumour cells resistant to lysis by normal N K cells which have not been stimulated with a ATCP or by the method according to the present invention.

As explained above, inhibition of NK-mediated lysis is controlled by expression of specific MHC class I molecules on the target cell surface, particularly HLA-C. There are two distinct groups of HLA-C alleles with regard to N K cell recognition. Some tumours express both types of HLA-C allele, which is thought to make them resistant to NK-mediated lysis. "NK resistant" cells may, therefore express both groups of class I allele. Some leukemia/lymphoma-derived cell lines, such as Raji and Daudi express both types of HLA-C allele, making them useful models for NK-resistant tumour cells in vivo.

Common infections that may be treated with a cellular composition comprising the activated NK cells as described herein include viral infections such as, e.g., hepatitis type A virus, hepatitis type B virus, hepatitis type C virus, etc.; parvoviruses, such as adeno-associated virus and cytomegalovirus; papovaviruses such as papilloma virus, polyoma viruses, and SV40; adenoviruses; herpes viruses such as herpes simplex type I (HSV-I), herpes simplex type II (HSV-II), and Epstein-Barr virus; poxviruses, such as variola (smallpox) and vaccinia virus; RNA viruses, including but not limited to human immunodeficiency virus type I (HIV-I), human immunodeficiency virus type II (HIV-II), human T-cell lymphotropic virus type I (HTLV-I), and human T-cell lymphotropic virus type II (HTLV-II); influenza virus; measles virus; rabies virus; Sendai virus; picornaviruses such as poliomyelitis virus; coxsackieviruses; rhinoviruses; reoviruses; togaviruses such as rubella virus (German measles) and Semliki forest virus; arboviruses; rinderpest; echovirus; rotavirus; respiratory syncytial virus; echinovirus; huntavirus; mumps virus; measles virus; rubella virus; polio virus; coronavirus; and combinations thereof. KIT

The present invention also provides a kit for preparing a NK cell activating composition according to the present invention.

The present invention also provides a kit for use in a method for activating an NK cell according to the method of present invention.

The kit may comprise (i) a CD2 ligation agent; and (ii) an NKG2D ligation agent or a precursor thereof. For example, where the CD2 ligation agent and/or NKG2D ligation agent is/are a protein, the kit may comprise a protein-encoding gene.

The present invention also provides a method for preparing a composition according to the present invention, which comprises the step of combining (i) a CD2 ligation agent; and (ii) an NKG2D ligation agent.

The present invention also provides the use of the kit in a method for preparing a substrate according to the present invention, which method comprises the step of attaching or immobilising (i) a CD2 ligation agent; and (ii) an NKG2D ligation agent on a substrate.

The present invention also provides the use of a kit in a method for preparing a vesicle according to the present invention, which method comprises the step of causing (i) a CD2 ligation agent; and (ii) an NKG2D ligation agent to be expressed or present on the surface of a vesicle.

The kit may also comprise instructions for use.

The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention. EXAMPLES

Example 1 - Preparation of activating microbeads The optimal concentrations of each mAb to label 3-D microbeads is determined by co-incubating aliquots of 5x10⁵ Dynal goat anti-sheep 450uM beads with increasing amounts of

a) anti-CD2 (OKT11) from 0.1 ug to 10.0ug detected with FITC conjugated Goat- anti-mouse by flow cytometry

b) anti-NKG2D (MEM158 - Serotec U K) directly conjugated with FITC from 0.1 ug to 10ug and detected by flow cytometry

The results are shown in Figure 1. It was determined that the optimal bead loading to achieve saturation was as follows:

3μg anti-NKG2D

5μg Anti-CD2 (OKT11 clone) - per 4x10⁷ Dynal goat-anti-sheep beads.

Example 2 - Selection of a ligand for NKG2D

The cognate ligand(s) for NKG2D may be MICA, MICB or ULBP. CTV-1 cells are immunophenotyped for MICA, MICB and ULBPs to determine which are expressed and their relative densities compared to each other and to CD15. Blocking of CTV-1 -mediated NK priming is tested with saturating concentrations of anti-MICA, anti-MICB and available mAbs to ULBPs to determine the most important NKG2D ligand for NK priming induced by CTV-1.

Example 3 - Activation of Resting NK cells

Resting human NK (rNK) cells are directly isolated from healthy volunteer donors by immunomagnetic selection with anti-CD56 PE and anti-PE microbeads (Miltenyi Biotec UK) and resuspended in culture medium supplemented with clinical-grade FBS (10%) at 10⁶/ml. The results from Example 1 above is used to provide an appropriate ratio of anti-CD2 and anti-NKGD2 coated beads.

The ratios indicated below provide an example of the method:

Al iq uots of 1 0⁶ purified NK cells (1 ml) are co-incubated with the following combinations of M450 bead pre-loaded with saturating concentrations of anti-CD2 and anti-NKG2D:

a) NK alone Neg control

b) NK + CTV1 1 :2 ratio Pos control

c) NK + 5x10⁵ anti-CD2 beads + 5x10⁵ anti-NKG2D beads

d) NK + 10⁶ anti-CD2 beads + 5x10⁵ anti-NKG2D beads

e) NK + 10⁶ anti-CD2 beads + 10⁶ anti-NKG2D beads

f) N K + 5 x 1 0⁵ anti-CD2 beads + 10⁶ anti-NKG2D beads The cell/bead suspensions are incubated overnight at 37°C. The following day the bead:cell conjugates are disrupted by vortex mixer and the beads removed by magnetic separation (Dynal - Invtrogen). The NK cells are recovered and tested for activation (CD25 and CD69 expression by flow cytometry). The results are shown in Figure 2. It was found that a 1 : 1 ratio of each bead plus 16:1 mixed beads to NK cell ratio induces NK cell priming (as measured by CD69 upregulation) in 16 hours to 30% of the level achieved with whole CTV-1 and 80% of the level achieved with CTV-1 lysate (data for lysate not shown in Figure 2) Example 4 - Preparation of a 2D surface for use in synthetic priming method

A 2-D surface, such as a 96 well ELISA plate is coated with an appropriate concentration of each mAb selected following the information provided in Example 1 , and the activation experiment outlined in Example 3 is repeated. This allows a) easier analysis of the temporal dynamics of NK priming by co-ligation of CD2/NKG2D; b) a scale-up production process for commercialisation as there is no bead removal step.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular and/or cell biology or related fields are intended to be within the scope of the following claims.

Claims

1. A natural killer (N K) cell-priming composition which comprises (i) a CD2 ligation agent; and (ii) an NKG2D ligation agent.

2. An NK-cell priming composition according to claim 1 , wherein the CD2 ligation agent comprises the CD2 ligand from CD15.

3. An NK-cell priming composition according to claim 1 , wherein the CD2 ligation agent comprises CD15 with its associated carbohydrate structure.

4. An NK-cell priming composition according to claim 1 , wherein the CD2 ligation agent is an anti-CD2 antibody.

5. An NK-cell priming composition according to any preceding claim, wherein the NKG2D ligation agent is an anti-NKG2D antibody.

6. An NK-cell priming composition according to any of claims 1 to 4, wherein the NKG2D ligation agent comprises the NKG2D binding site from an NKG2D ligand.

7. An NK-cell priming composition according to claim 6, wherein the NKG2D ligation agent comprises the NKG2D binding site from MICA, MICB or ULBPs.

8. An NK-cell priming composition according to claim 7, wherein the NKG2D ligation agent comprises MICA, MICB or ULBPs.

9. An NK-cell priming substrate or vesicle which comprises a CD2 ligation agent and an NKG2D ligation agent as defined in any preceding claim.

10. An NK-cell priming substrate according to claim 9 which comprises a two- dimensional surface, bead or nanoparticle coated with a CD2 ligation agent and an NKG2D ligation agent.

1 1. An NK-cell priming vesicle according to claim 10, which is a liposome expressing a CD2 ligation agent and an NKG2D ligation agent on the surface of the liposome.

12. An NK priming liposome according to claim 11 , which comprises one or more NK-activating cytokines within the liposome.

13. An N K-priming liposome according to claim 12, which comprises IL2, IL12 and/or IL15.

14. A method for activating a resting Natural Killer (NK) cell, which comprises the step of contacting the NK cell in vitro with:

a CD2 ligation agent and an NKG2D ligation agent as defined in any of claims 1 to 8;

an NK-cell priming composition according to any of claims 1 to 8; or an NK-cell priming substrate or vesicle according to any of claims 9 to 13.

15. A method according to claim 14, in which the resting NK cell is obtained from a patient.

16. A method for treating a subject in need of same, which comprises the following steps:

(i) activating a resting NK cell in vitro by a method according to claim 14; and (ii) administering the activated NK cell to the subject.

17. A method according to claim 16, wherein the resting NK cell is obtained from the subject to be treated.

18. An activated NK cell produced by a method according to claim 14 or 15.

19. An activated NK cell according to claim 18, which maintains its activated state following removal of the NK-cell priming composition, substrate or vesicle.

20. An activated NK cell according to claim 18, which maintains its activated state following cryopreservation.

21. A pharmaceutical composition comprising a plurality of activated NK cells according to any of claims 18 to 20 for treating a subject in need of same.

22. The use of a pharmaceutical composition according to claim 21 in the manufacture of a medicament for the treatment of cancer.

23. A method for treating cancer which comprises the step of administering a pharmaceutical composition according to claim 21 to a subject.

24. A NK-cell priming composition according to any of claims 1 to 8, or an NK-cell priming substrate or vesicle according to any of claims 9 to 13 for use in the activation of an NK cell in vivo.

25. A NK-cell priming composition according to any of claims 1 to 8, or an NK-cell priming substrate or vesicle according to any of claims 9 to 13 for use in the treatment of cancer.

26. A method for activating an NK cell in vivo which comprises the step of administering a NK-cell priming composition according to any of claims 1 to 8, or an NK-cell priming substrate or vesicle according to any of claims 9 to 13 to a subject.

27. A method for treating cancer in a subject in need of same which comprises which comprises the step of administering a NK-cell priming composition according to any of claims 1 to 8, or an NK-cell priming substrate or vesicle according to any of claims 9 to 13 to the subject.

28. A kit for preparing a composition according to any of claims 1 to 8 and/or for use in a method according to claim 14 or 15 which comprises (i) a CD2 ligation agent; and (ii) an NKG2D ligation agent.

29. A method for making a substrate according to claim 9 or 10 which comprises the step of attaching a CD2 ligation agent and an NKG2D ligation agent to a substrate.

30. A method of making a vesicle according to claim 9 or 1 1 which comprises the step of incorporating or causing the expression of a CD2 ligation agent and an NKG2D ligation agent at the vesicle surface.