WO2018234421A1 - Method of modulating immune cells - Google Patents
Method of modulating immune cells Download PDFInfo
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- WO2018234421A1 WO2018234421A1 PCT/EP2018/066497 EP2018066497W WO2018234421A1 WO 2018234421 A1 WO2018234421 A1 WO 2018234421A1 EP 2018066497 W EP2018066497 W EP 2018066497W WO 2018234421 A1 WO2018234421 A1 WO 2018234421A1
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- cell
- immune effector
- cells
- effector cell
- brd56491
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Classifications
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Definitions
- Immunotherapy may be passive or active. Passive cancer immunotherapy, i.e. the use of monoclonal antibodies to target cancer-associated antigens, is well known in the art. However, the primary target in the development of cancer immunotherapy is active immunotherapy, in which the body's own immune system (or potentially the immune system of another) is stimulated to actively attack and destroy cancer in an individual. This would not only permit specificity of cell killing in a manner unavailable to the more traditional, "blunt" methods of treatment, but would also, it is hoped, generate immunity to cancer in which the particular target cancer antigen is expressed. A number of potential cancer immunotherapies are reviewed in Farkona, S. et al. (2016), BMC Medicine 14:73.
- adoptive cell transfer techniques can be found in e.g. Rosenberg, S.A. et al. (2008), Nat. Rev. Cancer 8(4): 299-308, or Rosenberg, S.A. et al. (2015), Science Vol. 348, issue 6230, pp. 62-68.
- adoptive cell transfer has to date proven successful in treating cancers including melanoma, cervical cancer, lymphoma, leukaemia, bile duct cancer and neuroblastoma.
- the present invention addresses the above problem by providing a method by which the function of immune effector cells is enhanced.
- This is predicated on the surprising discovery that contacting an immune effector cell with a non-biguanide mitochondrial electron transport chain (ETC) inhibitor or BRD56491 enhances the function of the contacted call, in particular proliferation, cytolytic or cytotoxic activity (i.e. target cell killing) and specificity of cytolytic/cytotoxic activity.
- ETC mitochondrial electron transport chain
- the invention provides use of a non-biguanide mitochondrial electron transport chain inhibitor or BRD56491 for enhancing the function of an in vitro or ex vivo immune effector cell in preparation for adoptive cell transfer therapy.
- the invention provides the use of a non-biguanide mitochondrial electron transport chain inhibitor or BRD56491 for enhancing the function of a immune effector cell in vitro or ex vivo.
- the immune effector cells obtained from such uses exhibit enhanced effector function relative to an immune effector cell which has not been treated (contacted) with the inhibitor.
- the immune effector cell is targeted against a particular target cell, or type of target cell, but the "targeting" may be of greater or lesser specificity, for example it may be a cytotoxic cell with more general (or generic) cell-killing activity, such as an NK cell, or it may be specifically targeted against a target cell by expressing a receptor against an antigen on a target cell, e.g. it may be a T-cell.
- the receptor may be a natural or native receptor of the cell, or it may be a heterologous receptor introduced into the immune effector cell.
- an immune effector cell may be effective in the treatment of cancer by targeting a cancer cell specifically by recognising an antigen expressed on the cancer cell or less specifically (e.g. by targeting cells for killing more generally).
- control immune effector cell is specifically meant a negative control, i.e. an immune effector cell showing a baseline activity level for any given function, to which a contacted cell can be compared to identify any increase (or decrease) in function following contacting with a non-biguanide ETC inhibitor or BRD56491 .
- control immune effector cell is not contacted with an ETC inhibitor or BRD56491 .
- target cells of a particular T-cell correspond to cells which express the appropriate MHC component (in the case of a human cell, the appropriate human leukocyte antigen (HLA)) and contain or express the appropriate antigen to produce the MHC-antigen complex recognised by the TCR of the T-cell.
- Target cells for NK cells include, for instance, cells with low or no expression of MHC I complexes.
- immune effector cell includes not only mature or fully differentiated immune effector cells but also precursor (or progenitor) cells thereof, including stem cells (more particularly haematopoietic stem cells, HSC), or cells derived from HSC.
- An immune effector cell may accordingly be a T-cell, NK cell, NKT-cell, neutrophil, macrophage, or a cell derived from HSCs contained within the CD34+ population of cells derived from a haematopoietic tissue, e.g. from bone marrow, cord blood or blood, e.g. mobilised peripheral blood, which upon administration to a subject differentiate into mature immune effector cells.
- Primary cells e.g.
- the immune effector cell is from a cell line, i.e. the immune effector cell is not a primary immune effector cell.
- the immune effector cell is an NK cell, it is not a primary NK cell, e.g. it is from an NK cell line.
- T-cells are isolated from PBMCs.
- PBMCs may be isolated from buffy coats obtained by density gradient centrifugation of whole blood, for instance centrifugation through a LYMPHOPREPTM gradient, a PERCOLLTM gradient or a FICOLLTM gradient.
- T-cells may be isolated from PBMCs by depletion of the monocytes, for instance by using CD14 DYNABEADS®.
- red blood cells may be lysed prior to the density gradient centrifugation.
- the immune effector cell may be modified to express a heterologous TCR.
- TCRs are protein complexes which protrude from the cell membrane of a T-cell. Most TCRs comprise an a- and a ⁇ -chain, both of which consist of a variable region and a constant region. The variable region is located at the N-terminus of the chain, and is wholly extracellular; the constant region is located at the C-terminus of the chain, and consists of an extracellular domain, a transmembrane domain and a short cytoplasmic domain. TCR chains are encoded and synthesised in an immature form, with an N-terminal signal (or leader) sequence.
- the immune effector cell for use in the method of the invention is a T-cell redirected against a cancer antigen, e.g. a CD8+ T-cell redirected against a cancer antigen or a CD4+ T-cell redirected against a cancer antigen.
- a cancer antigen e.g. a CD8+ T-cell redirected against a cancer antigen or a CD4+ T-cell redirected against a cancer antigen.
- piericidin A are also preferred mitochondrial ETC inhibitors according to the invention.
- Antimycin A functions by binding to and blocking the Q, site of Complex III, thus preventing ubiquinone docking to Complex III and hence preventing electron transfer from Complex III to ubiquinone, blocking the Q cycle.
- Complex III inhibitors with the same mechanism of action as antimycin A are also preferred mitochondrial ETC inhibitors according to the invention.
- the immune effector cell is contacted with rotenone or antimycin A. It is preferred that the immune effector cell is contacted with only one of these two compounds, i.e. that it is not contacted with both rotenone and antimycin A.
- the contacting step, or the method does not include contacting with an oligomycin.
- the contacting step is performed in the absence of an oligomycin.
- an oligomycin is considered to be absent from the culture medium if it is present at a concentration of no more than about 1 nM. In particular embodiments, an oligomycin if present, is present in a concentration of less than 0.1 ⁇ .
- Harvesting does not require, but may if desired include, that the cells are removed from the container or vessel in which they were contacted.
- the method of the invention may further comprise causing the cell to differentiate.
- Such methods are known in the art.
- Methods by which such differentiation can be performed are known in the art.
- the cells may also be stored prior to transfer to the pharmaceutically-acceptable medium.
- the cells are frozen prior to transfer to a
- the virus may be any virus, but generally will be a pathogenic virus.
- the virus may be HIV, a hepatitis virus (e.g. HBV or HCV), HPV, CMV or EBV, HHV-8, HTLV-1 , SV40 or an enterovirus.
- HBV or HCV hepatitis virus
- HPV hepatitis virus
- CMV or EBV HHV-8
- HTLV-1 e.g. SV40
- enterovirus e.g., a virus that kill virus
- Other possible infective agents or pathogens include also bacteria, e.g.
- cytochrome c reductase thereby inhibiting the reduction of ubiquinone in the Qi site, disrupting the Q-cycle and partially inhibiting respiration (ubiquinone is still reduced at Complex I).
- a first killing assay was conducted in order to test several metabolic drugs and study their effect on the killing efficiency of NK cells and T-cells.
- the killer cells (NK-92) were pretreated for 16 hours with the drugs described above. After treatment killer cells were centrifuged and washed twice (1 :1 ,000,000 dilution of the drug). They were then incubated together with target cells (lymphoma BL41 cell line, stably transduced with luciferase construct) and the luminescence signal was followed over time.
- target cells lymphoma BL41 cell line, stably transduced with luciferase construct
- three drugs demonstrated their potential to enhance killing ability of NK-92 cells (antimycin A, myxothiazol and rotenone) in comparison to untreated NK-92 cells.
- myxothiazol was set aside due to its high toxicity to human cells, rendering it impractical for use in potential therapeutic applications.
- NK-92 cells were incubated in X-VIVOTM 10 (Lonza, Switzerland) complete medium
- T-cells employing Dynabeads CD3/CD28 essentially as previously described (Inderberg et at. (2017), Oncolmmunology 6(4): e1302631 ).
- PBMCs were isolated from buffy coats by density gradient centrifugation and cultured with Dynabeads (Dynabeads®
- T-cells were washed in staining buffer (SB) consisting of phosphate buffered saline (PBS) containing 0.1 % human serum albumin (HSA) and 0.1 % sodium azide before staining for 20 min at RT. The cells were then washed in SB and fixed in SB containing 1 %
- SB staining buffer
- PBS phosphate buffered saline
- HSA human serum albumin
- SB sodium azide
Abstract
Provided herein is a method of enhancing immune effector cells for use in adoptive transfer therapy, based on the surprising discovery that treating such cells with non-biguanide inhibitors of the mitochondrial electron transport chain (in particular antimycin-A and rotenone) causes an enhancement in cell functionality. Similar effects were demonstrated for immune cell treatment with BRD56491. The invention provides methods of enhancing immune effector cells for use in adoptive cell transfer therapy comprising contacting the immune effector cells with non- biguanide mitochondrial electron transport chain inhibitors or BRD56491.
Description
Method of Modulating Immune Cells
The present invention is directed to a method for enhancing the function of an immune effector cell in preparation for use in adoptive cell transfer therapy. An enhanced immune effector cell obtained or obtainable by the method of the invention is also provided, as is a pharmaceutical composition comprising such an immune effector cell. Use of the immune effector cell (or pharmaceutical composition) in therapy, particularly adoptive cell transfer therapy is also disclosed. Such therapy may in particular be for the treatment of cancer.
Due to the well-known limitations of traditional chemotherapeutic/radiotherapeutic methods of cancer treatment, new cancer therapies are constantly being sought. One relatively new form of treatment is immunotherapy, in which aspects of the immune system are harnessed and exploited in an attempt to destroy the cancer.
Immunotherapy may be passive or active. Passive cancer immunotherapy, i.e. the use of monoclonal antibodies to target cancer-associated antigens, is well known in the art. However, the primary target in the development of cancer immunotherapy is active immunotherapy, in which the body's own immune system (or potentially the immune system of another) is stimulated to actively attack and destroy cancer in an individual. This would not only permit specificity of cell killing in a manner unavailable to the more traditional, "blunt" methods of treatment, but would also, it is hoped, generate immunity to cancer in which the particular target cancer antigen is expressed. A number of potential cancer immunotherapies are reviewed in Farkona, S. et al. (2016), BMC Medicine 14:73.
One form of active cancer immunotherapy of particular current interest is adoptive cell transfer (ACT). Generally in ACT immune cells (most commonly T-cells) are isolated either from a donor or from the patient him/herself, redirected against an antigen of interest (i.e. genetically engineered to recognise a particular target antigen) then (re)introduced into the patient, in whom they are intended to launch an immune response against the target cancer cells, thus killing the cancer cells with high specificity. Alternatively, immune cells may be isolated from the patient, clones which recognise neoantigens expressed by the cancer identified, expanded and reintroduced into the patient (Zacharakis et al. (2018), Nat Mec/ doi:10.1038/s41591 -018-0040-8). Use of the patient's own immune cells (i.e.
autologous immune cells) is particularly advantageous, as the cells are not recognised as foreign when reintroduced into the patient, avoiding inducing any immune response to the cells by the patient.
A review of adoptive cell transfer techniques can be found in e.g. Rosenberg, S.A. et al. (2008), Nat. Rev. Cancer 8(4): 299-308, or Rosenberg, S.A. et al. (2015), Science Vol. 348, issue 6230, pp. 62-68. As detailed therein, adoptive cell transfer has to date proven
successful in treating cancers including melanoma, cervical cancer, lymphoma, leukaemia, bile duct cancer and neuroblastoma.
Despite the successes of ACT in cancer treatment, difficulties with the technique remain. One particular difficulty is low efficacy of the redirected cells upon (re)introduction to the patient. Generally, following isolation of the immune effector cells from the relevant individual, in order to prepare the cells for ACT, the cells must be genetically modified so as to express an appropriate receptor which binds to the target antigen, then expanded, in order to obtain a therapeutically-effective amount of cells. This process can leave the cells metabolically exhausted, unable to proliferate significantly further and with low killing activity, leading to poor efficacy of the cells in target killing following their introduction into the subject. Enhancement of the effector functions of immune effector cells for use in ACT would thus improve the efficacy of ACT, leading to improved treatment outcomes.
The present invention addresses the above problem by providing a method by which the function of immune effector cells is enhanced. This is predicated on the surprising discovery that contacting an immune effector cell with a non-biguanide mitochondrial electron transport chain (ETC) inhibitor or BRD56491 enhances the function of the contacted call, in particular proliferation, cytolytic or cytotoxic activity (i.e. target cell killing) and specificity of cytolytic/cytotoxic activity. Thus by contacting an immune effector cell with a non-biguanide mitochondrial ETC inhibitor or BRD56491 , the function of the immune effector cell is enhanced, providing a superior therapeutic effect when transfused into a subject.
The mitochondrial ETC, as known to the skilled person, is a series of protein complexes located in the inner mitochondrial membrane of eukaryotic cells. The ETC comprises four redox carrier complexes, known as Complexes l-IV, and Complex V
(otherwise known as the F-|F0-ATPase). In the Krebs Cycle, electrons reduce NAD+ and FAD to NADH and FADH2, respectively. NADH and FADH2 are then oxidised back to NAD+ and FAD, the released electrons being passed to Complex I and Complex II, respectively.
Electrons from Complexes I (predominantly) and II are transferred first to Complex III, and then on to Complex IV. Each complex in turn is more electronegative than the last, meaning that transport of an electron to each successive complex releases energy, which is used by Complexes I, III and IV to pump hydrogen ions (H+) across the membrane. Eventually, at Complex IV, the transported electrons are used to reduce molecular oxygen to water.
Meanwhile, the hydrogen ions pumped across the inner membrane produce a proton gradient, i.e. a proton-motive force. Protons move down the proton gradient, through the FoF-i ATP synthase in a controlled manner, driving the generation of ATP in the process of oxidative phosphorylation.
Inhibition of the mitochondrial ETC, at one of Complexes l-IV or the F0F-i ATP synthase (Complex V) blocks oxidative phosphorylation, either partially or fully, and leads to
up-regulation of glycolysis to support the cell's bioenergetic needs. As is commonly-known, glycolysis alone is much less efficient than aerobic respiration. Glycolysis alone produces a net 2 molecules of ATP per molecule of glucose, while aerobic respiration is estimated to produce approximately 30-32 molecules of ATP per molecule of glucose.
Cells in which oxidative phosphorylation has been inhibited using an ETC inhibitor had been expected to become anergic in order to save energy. It is most surprising, therefore, that contacting an immune effector cell with a non-biguanide ETC inhibitor leads to enhanced cellular function, particularly enhanced high-energy functions such as proliferation. There is no teaching in the prior art to suggest that such a phenomenon might occur. Indeed, some prior art would appear to offer opposite teaching. For instance, Kim, K. et al. (2009), PLOS ONE 4(1 1 ): e7738, demonstrate the contacting of murine T-cells with mitochondrial ETC inhibitors, and show that ETC inhibition appears to inhibit signalling from the TCR (T-cell receptor), reduce T-cell viability and inhibit T-cell proliferation. Similarly, Yi, J.S. et al. (2006), J. Immunol. 177: 852-862, demonstrate that ETC inhibition using the Complex I inhibitor rotenone inhibits division of CD8+ T-cells in a dose-dependent fashion, and also blocks TCR-mediated CD8+ T-cell activation. Angela, M. et al. (2016), Nat. Commun.
7:13683 (DOI: 10.1038/ncomms13683) demonstrates that the ETC inhibitors rotenone and antimycin A have the expected effects on the oxygen consumption rate of CD4+ T-cells, but otherwise provides no evidence as to the effect of the agents on the function of the cells. Similarly, there was no indication in the prior art that contacting an immune effector cell with the small molecule BRD56491 would result in enhanced functionality of the cell, as has been discovered by the present inventors.
Thus in a first aspect the invention provides a method of preparing an immune effector cell for use in adoptive cell transfer therapy, comprising:
i) contacting in vitro or ex vivo an immune effector cell with a non-biguanide mitochondrial electron transport chain inhibitor or BRD56491 , wherein the function of said immune effector cell following said contacting is enhanced relative to an immune effector cell which has not been contacted with said inhibitor or BRD56491 ; and
ii) formulating the enhanced immune effector cell in a sterile composition suitable for parenteral administration to a subject.
In another aspect the invention provides a method of preparing an immune effector cell for use in adoptive cell transfer therapy, comprising:
i) contacting in vitro or ex vivo an immune effector cell with rotenone, antimycin A or BRD56491 , wherein the function of said immune effector cell following said contacting is enhanced relative to an immune effector cell which has not been contacted with rotenone, antimycin A or BRD56491 ; and
ii) formulating the enhanced immune effector cell in a sterile composition suitable for parenteral administration to a subject.
Also provided is a method for enhancing the function of an immune effector cell, comprising contacting in vitro or ex vivo an immune effector cell with a non-biguanide mitochondrial electron transport chain inhibitor or BRD56491 , wherein the function of said immune effector cell following said contacting is enhanced relative to an immune effector cell which has not been contacted with said inhibitor or BRD56491 .
In a related aspect, the invention provides use of a non-biguanide mitochondrial electron transport chain inhibitor or BRD56491 for enhancing the function of an in vitro or ex vivo immune effector cell in preparation for adoptive cell transfer therapy. Similarly, the invention provides the use of a non-biguanide mitochondrial electron transport chain inhibitor or BRD56491 for enhancing the function of a immune effector cell in vitro or ex vivo. The immune effector cells obtained from such uses exhibit enhanced effector function relative to an immune effector cell which has not been treated (contacted) with the inhibitor.
The invention also provides a method of preparing an immune effector cell composition for use in adoptive cell transfer therapy, said method comprising formulating a population of enhanced immune effector cells obtained according to a method of the invention in a pharmaceutically-acceptable medium.
In another aspect, the invention provides an enhanced immune effector cell obtained or obtainable by a method of the invention, wherein the function of said enhanced immune effector cell is enhanced relative to an immune effector cell which has not been contacted with said inhibitor or BRD56491 .
The invention also provides a pharmaceutical composition comprising the enhanced immune effector cell of the invention and one or more pharmaceutically-acceptable diluents, carriers or excipients.
The invention also provides an enhanced immune effector cell of the invention, or a pharmaceutical composition of the invention, for use in therapy. This includes the provision of an enhanced immune effector cell of the invention or a pharmaceutical composition of the invention for use in adoptive cell transfer therapy.
The invention further provides an enhanced immune effector cell of the invention, or a pharmaceutical composition of the invention, for use in the treatment of cancer.
Further provided is a method of adoptive cell transfer therapy, or a method of treating cancer, comprising administering to a subject in need thereof an effective amount of enhanced immune effector cell of the invention or a pharmaceutical composition of the invention.
Another method of adoptive cell transfer therapy provided herein comprises:
(i) preparing immune effector cells for transfer by contacting the immune effector cells in vitro or ex vivo with a non-biguanide mitochondrial electron transport chain inhibitor or BRD56491 , allowing said cells to proliferate and harvesting the cells; and
(ii) administering the harvested cells to a subject in need thereof.
The invention also provides the use of an enhanced immune effector cell of the invention in the manufacture of a medicament for use in adoptive cell therapy, preferably in cancer therapy.
In preferred embodiments of the various aspects above the immune effector cell and the subject are human.
As discussed in more detail below, the enhanced immune effector cell may be used in the treatment of any condition which may benefit from adoptive cell transfer therapy. This may particularly involve the abrogation, e.g. killing or removal, of unwanted, undesirable, or deleterious target cells, e.g. cells infected with an intracellular pathogen or, most notably, cancer cells. The immune effector cell may thus be a cytotoxic, or cytolytic, cell as discussed further below, but may include other cells, such as T-helper cells, which may augment an immune response against a target cell. The immune effector cell is targeted against a particular target cell, or type of target cell, but the "targeting" may be of greater or lesser specificity, for example it may be a cytotoxic cell with more general (or generic) cell-killing activity, such as an NK cell, or it may be specifically targeted against a target cell by expressing a receptor against an antigen on a target cell, e.g. it may be a T-cell. The receptor may be a natural or native receptor of the cell, or it may be a heterologous receptor introduced into the immune effector cell. Thus, an immune effector cell may be effective in the treatment of cancer by targeting a cancer cell specifically by recognising an antigen expressed on the cancer cell or less specifically (e.g. by targeting cells for killing more generally).
As mentioned above, the invention provides a method of preparing an immune effector cell for use in adoptive cell transfer therapy, comprising:
i) contacting in vitro or ex vivo an immune effector cell with a non-biguanide mitochondrial electron transport chain inhibitor or BRD56491 , wherein the function of said immune effector cell following said contacting is enhanced relative to an immune effector cell which has not been contacted with said inhibitor or BRD56491 ; and
ii) formulating the enhanced immune effector cell in a sterile composition suitable for parenteral administration to a subject.
In a particular embodiment the method may comprise harvesting the cell, optionally washing the cell, and formulating the enhanced immune cell in a sterile composition suitable for parenteral administration.
The function of the immune effector cell which is enhanced may be any function or characteristic which is of interest, and which is improved by the contacting of the cell with a non-biguanide mitochondrial ETC inhibitor or BRD56491 . By "enhanced" is meant that the function or characteristic in question is altered in a manner which is desirable or useful to the skilled person. In particular, the function may be increased, relative (or compared) to the level of function in the absence of contact with said inhibitor or BRD56491 . The function may be any desired effect or activity of the cell. In other words, an effector function of the cell is enhanced.
The method of the invention enhances the function of an immune effector cell relative to an immune effector cell which has not been contacted with a non-biguanide ETC inhibitor or BRD56491. The skilled person is well able to identify a suitable uncontacted immune effector cell whose function may be compared to that of the contacted immune effector cell. In some embodiments the function-of-interest of the immune effector cell contacted with a non-biguanide ETC inhibitor or BRD56491 may be compared to the same function of the same immune effector cell prior to the contacting. In this case, if the particular function of the immune effector cell is enhanced relative to prior to contacting, the function may be considered enhanced as defined herein.
In a preferred embodiment, the function of the contacted immune effector cell is compared to that of a control immune effector cell. By "control immune effector cell" is specifically meant a negative control, i.e. an immune effector cell showing a baseline activity level for any given function, to which a contacted cell can be compared to identify any increase (or decrease) in function following contacting with a non-biguanide ETC inhibitor or BRD56491 . The skilled person is well able to identify a suitable control cell. A control immune effector cell is not contacted with an ETC inhibitor or BRD56491 . Generally, a control immune effector cell is identical to the immune effector cell which is contacted with the non-biguanide ETC inhibitor or BRD56491 , and is treated identically to the contacted cell, with the exception that it is not itself contacted with an ETC inhibitor or BRD56491 .
A control immune effector cell will be of the same cell type as the contacted cell, e.g. if a T-cell has been contacted with a non-biguanide ETC inhibitor or BRD56491 , the control cell will be a T-cell, and if a natural killer cell (NK cell) has been contacted with a non- biguanide ETC or BRD56491 , the control cell will be an NK cell. Preferably, the control cell will be genetically identical, or essentially genetically identical, to the contacted cell. For instance, if the immune effector cell to be contacted is of a cell line, an uncontacted cell of the same cell line should be used as a control cell by which to measure any enhancement of function of the contacted cell. E.g. if the contacted cell is a natural killer cell of the NK-92 cell line, the control cell should be an uncontacted cell of the NK-92 cell line.
If the contacted cell is an ex vivo primary immune effector cell, isolated from an individual, e.g. a donor or an individual to be treated by adoptive cell transfer, the control cell is preferably isolated from the same individual, most preferably a clone of the contacted cell. If the contacted cell is genetically modified prior to contacting with the non-biguanide ETC inhibitor or BRD56491 , e.g. if it is modified to express a T-cell receptor (TCR) or suchlike in order to redirect its activity towards a particular antigen, the control cell should be identically genetically modified. As previously mentioned, the skilled person is competent to design an experiment and is well able to identify an appropriate control immune effector cell.
The function of the immune effector cell which is enhanced by the current method may be any function which is of interest to the skilled person and is enhanced by contacting of the immune effector cell with a non-biguanide mitochondrial ETC inhibitor or BRD56491. One or more functions of the contacted immune effector cell may be enhanced. In particularly preferred embodiments the function which is enhanced is proliferation, target cell killing and/or specificity of target cell killing. As shown below in the Examples, contacting of immune effector cells with non-biguanide ETC inhibitors can enhance cell proliferation, target cell killing and specificity of killing; contacting of immune effector cells with BRD56491 can enhance target cell killing and specificity of killing.
An enhancement in proliferation of an immune effector cell means that when proliferation (i.e. cell division to increase cell number) of an immune effector cell is stimulated following contacting of the cell with a non-biguanide ETC inhibitor, more proliferation (i.e. a greater increase in cell number) occurs compared to when an immune effector cell which has not been contacted with an ETC inhibitor is equivalently stimulated to proliferate. In other words, contacting of a cell with a non-biguanide ETC inhibitor may result in an enhancement of proliferation, compared to an immune effector cell which has not been contacted with an ETC inhibitor. If the proliferation of an immune effector cell is enhanced by contacting with a non-biguanide ETC inhibitor, its rate of division is increased. This means that at a set time-point after stimulation of the contacted cell to proliferate, the number of live cells in the culture is higher than the number of cells at the same time-point following the stimulation of proliferation of an immune effector cell which has not been contacted with an ETC inhibitor. The set time-point may be determined by the skilled person, e.g. by trial and error to identify an appropriate time-point and/or based on the Examples below, but may for instance be 48, 72 or 96 hours after the stimulation of proliferation.
Notably, enhancement of proliferation of an immune effector cell following contacting with a non-biguanide ETC inhibitor means the total number of live cells generated in a particular time period is increased, compared to the number of live cells generated by the proliferation of an immune effector cell which has not been contacted with an ETC inhibitor (e.g. a control immune effector cell). This does not necessarily correspond to an increase in
the proportion of live cells in the proliferating culture. The proportion of live cells in a culture of proliferating cells which have been contacted with a non-biguanide ETC inhibitor may be increased relative to the proportion of live cells in a culture of proliferating cells which have not been contacted with an ETC inhibitor, but may equally be decreased or unchanged. Accordingly, in particular, proliferation is measured by the total number of live cells generated, and not the proportion of live cells in the culture.
Methods for stimulating and measuring proliferation are well-known to the skilled person and are described in the Examples below. Immune effector cell proliferation generally requires activation of the cell. T-cells may be activated and expanded (i.e. stimulated to proliferate) by contacting them with e.g. anti-CD3 and anti-CD28 antibodies, generally attached to a bead or other surface (for instance in the form of CD3/CD28 DYNABEADS®), in a culture medium supplemented with appropriate cytokines, such as IL-2. A bead with both anti-CD3 and anti-CD28 antibodies attached serves as a surrogate antigen presenting cell (APC). Immune effector cell proliferation may require the culturing of the cell in an appropriate medium, e.g. a medium designed for immune cell proliferation. Suitable media are known to the skilled person and include e.g. CellGro® (CellGenix, Germany). Media may in particular be supplemented with IL-2 to stimulate immune effector cell proliferation. Live cells may be counted using standard cell counting methods (e.g. using a haemocytometer) in conjunction with trypan blue exclusion to identify (and exclude) dead cells.
NK cell proliferation may be similarly stimulated, e.g. they may be grown in a suitable medium under standard conditions. The skilled person is aware of the components of a suitable medium. For instance, a suitable medium may comprise L-glutamine, human serum and IL-2.
If target cell killing by the immune effector cell contacted with a non-biguanide ETC inhibitor or BRD56491 is enhanced relative to an uncontacted cell, this means that when the immune effector cell contacted with a non-biguanide ETC inhibitor or BRD56491 is incubated with target cells, a greater proportion of the target cells are killed than are killed by an immune effector cell not contacted with an ETC inhibitor or BRD56491 (e.g. a control immune effector cell). The proportion of target cells killed is measured at a chosen time-point after the beginning of the incubation of the immune effector cells and target cells. Such a time-point may be determined by a skilled person without difficulty, but may be e.g. 12, 24, 36 or 48 hours. Such an assay is known as a killing assay.
Appropriate target cells for a given immune effector cell are known to the skilled person, and are exemplified in the Examples below. NK cells generally target, amongst others, cells lacking MHC I expression. For instance, NK-cells are able to target BL41 or K562 cells (a Burkitt's lymphoma and a chronic myelogenous leukaemia cell line, respectively) for killing. T-cell targets are dependent on their TCR specificities. TCR targets
can be identified using techniques known in the art, or if a T-cell to be contacted is redirected using a specific known TCR the target is also known and appropriate target cells can be identified or designed accordingly.
Killing assay protocols are also known to the skilled person, and are exemplified in the Examples below. One method by which cell killing can be quantified is by using a target cell line which is genetically modified to express the luciferase gene. Luciferase enzymes catalyse light-emitting (luminescent) reactions, and thus in such an assay, when luciferase substrate is included, living target cells produce luminescence. Luminescence only occurs in living cells, and thus upon death luminescence by any given cell halts. The proportion of target cells killed by immune effector cells can thus be quantified according to the reduction in luminescence observed during incubation of the target cells with the immune effector cells (e.g. a 50 % reduction in luminescence corresponds to a 50 % reduction in cell number, i.e. the killing of 50 % of target cells).
A "target cell" as referred to herein is a cell which, according to theory, ought to be targeted (e.g. killed) by an immune effector cell. It can alternatively be defined as the cell against which an immune effector cell is directed, or against which it is able to act. Different immune effector cells have different target cells, dependent on the immune effector cell type and (in the case of e.g. T-cells) the receptor expressed, and the skilled person is well able to identify target cells for any given immune effector cell. For instance, target cells of a particular T-cell correspond to cells which express the appropriate MHC component (in the case of a human cell, the appropriate human leukocyte antigen (HLA)) and contain or express the appropriate antigen to produce the MHC-antigen complex recognised by the TCR of the T-cell. Target cells for NK cells include, for instance, cells with low or no expression of MHC I complexes.
Killing of target cells by immune effector cells is known as "specific" killing activity.
Specific killing activity thus refers only to the killing activity of an immune effector cell correctly directed against cells which the immune effector cell ought to kill, according to theory. Immune effector cells often display off-target activity, in which non-target cells (i.e. cells which, according to theory, should not be targeted or killed by the immune effector cell) are targeted (e.g. killed). This includes for instance the killing by NK cells of healthy cells which express the MHC I complex, or the targeting by T-cells of cells which do not comprise or express the antigen recognised by the TCR of the T-cells. Killing or targeting of non-target cells by an immune effector cell is known as non-specific activity.
According to the method of the invention, contacting an immune effector cell of the invention with a non-biguanide ETC inhibitor or BRD56491 can improve its specificity of killing, relative to an immune effector cell which has not been contacted with an ETC inhibitor or BRD56491. An improvement in specificity of killing means that non-specific killing activity
by an immune effector cell contacted according to the method of the invention is reduced, while specific killing activity is improved, unchanged or reduced less than non-specific killing activity, relative to an immune effector cell which has not been contacted with an ETC inhibitor (e.g. a control cell). A reduction in non-specific killing activity by an immune effector cell means that when the killing activity of an immune effector cell, contacted with an ETC inhibitor according to the method of the invention, is measured in a mixed culture of both target and non-target cells, the proportion of total killing activity directed against target cells is increased (and the proportion directed against non-target cells decreased) compared to, e.g., a control immune effector cell.
Specificity of killing can be measured using a killing assay as defined above, in which both target and non-target cells are present in culture, each for instance displaying luminescence at a different wavelength, and quantifying the proportion of both target and non-target cells killed as described above. Alternatively, separate killing assays can be performed with target and non-target cells, and the proportion of target and non-target cells killed quantified as described above.
In the methods of the invention, the enhancement of function may be any detectable enhancement of function. Preferably the enhancement is statistically significant. In particular embodiments, the function in question (e.g. proliferation, target cell killing or specificity of killing) may be enhanced by e.g. at least 10, 20, 30 , 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500 %, i.e. in the methods of the invention, in which the function of an immune effector cell is enhanced by contacting the immune effector cell with a non- biguanide ETC inhibitor or BRD56491 , the function in question of the contacted immune effector cell may be enhanced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500 % relative to an immune effector cell which has not been contacted with an ETC inhibitor or BRD56491 (e.g. a control cell).
With regard to the functions of proliferation, target cell killing and specificity of killing, quantitative increases in function can be easily calculated. For instance, for proliferation, quantitation of function is based on the number of live cells produced during proliferation by the time-point in question. For instance, if the immune effector cell contacted with an ETC proliferates to generate twice as many live cells as the control cell, the function of proliferation can be seen to be increased by 100 %. Similarly, if the immune effector cell contacted with an ETC kills twice as high a proportion of target cells as does an uncontacted cell (e.g. 50 % compared to 25 % of target cells), the function of target cell killing can be seen to be increased by 100 %.
Specificity of killing can be quantified as the ratio between target cell killing activity and non-target cell killing activity. For instance, if an uncontacted immune effector cell kills 30 % of target cells and 15 % of non-target cells, this would correspond to a ratio of target to
non-target cell killing of 2:1. If an immune effector cell contacted with an ETC inhibitor kills 50 % of target cells and 10 % of non-target cells, this corresponds to a ratio of target to non- target cell killing of 5:1 , i.e. an increase in specificity of killing of 250 %.
In the assays described above, the function of an immune effector cell following contacting with a non-biguanide ETC inhibitor or BRD56491 is compared to the function of an immune effector cell which has not been contacted with an ETC inhibitor or BRD56491. By "following" means simply after the contacting has commenced. Thus the function may be measured after the contacting has been performed and completed, i.e. the cells may be contacted with a non-biguanide ETC inhibitor or BRD56491 , then removed from the ETC inhibitor or BRD56491 prior to their function being assayed, e.g. the cells may be isolated from culture by centrifugation and washed to remove the ETC inhibitor or BRD56491 .
Alternatively, the function of the immune effector cells may be measured while they are in contact with the non-biguanide ETC inhibitor or BRD56491.
An immune effector cell as defined herein is any immune cell capable of an immune response against a target cell, or capable of regulating such a response. More particularly, an "immune effector cell," is any cell of the immune system that has one or more effector functions (e.g. cytotoxic cell killing activity, secretion of cytokines, induction of antibody- dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC). Representative immune effector cells thus include T-cells, in particular cytotoxic T- cells (CTLs; CD8+ T-cells) and helper T-cells (HTLs; CD4+ T-cells, including Th1 , Th2, Th17 and follicular (TFH) helper T-cells). Other populations of T-cells are also useful herein, for example naive T-cells, regulatory T-cells (Tregs) and memory T-cells. Other immune effector cells include NK cells, NKT-cells, neutrophils and macrophages. As described below, immune effector cells also include progenitors of effector cells, wherein such progenitor cells can be induced to differentiate into an immune effector cells in vivo or in vitro. An immune effector cell population which is subjected to the method of the invention may contain more than one type of immune effector cell. It is not a requirement for a particular type of immune effector cell to be separated, and used in isolated form, although this is not precluded. For example, a preparation of NK cells (e.g. an NK cell line) may be used, or a preparation of T-cells which contains more than one T-cell type may be used.
Preferably, the immune effector cell for use in the method of the invention is capable of abrogating, damaging or deleting a target cell, i.e. of reducing, or inhibiting, the viability of a target cell, preferably killing a target cell (in other words rendering a target cell less or nonviable). The immune effector cell is thus preferably a cytotoxic immune effector cell. The term "cytolytic" refers herein to a cell capable of inducing cell death by lysis. The term
"cytotoxic" is used more broadly herein to refer to a cell capable of inducing cell death by any means, including by lysis, as well as by apoptosis or any other cell death pathway in a target
cell. T-cells, including CD4+ and CD8+ T-cells, and NK cells represent preferred immune effector cells according to the invention.
The term "immune effector cell" as used herein includes not only mature or fully differentiated immune effector cells but also precursor (or progenitor) cells thereof, including stem cells (more particularly haematopoietic stem cells, HSC), or cells derived from HSC. An immune effector cell may accordingly be a T-cell, NK cell, NKT-cell, neutrophil, macrophage, or a cell derived from HSCs contained within the CD34+ population of cells derived from a haematopoietic tissue, e.g. from bone marrow, cord blood or blood, e.g. mobilised peripheral blood, which upon administration to a subject differentiate into mature immune effector cells. Primary cells, e.g. cells isolated from a subject to be treated or from a donor subject may be used, optionally with an intervening cell culture step (e.g. to expand the cells) or other cultured cells or cell lines may be used (e.g. NK cell lines such as the NK-92 cell line). In a particular embodiment, the immune effector cell is from a cell line, i.e. the immune effector cell is not a primary immune effector cell. In a more particular embodiment, where the immune effector cell is an NK cell, it is not a primary NK cell, e.g. it is from an NK cell line.
The term "NK cell" refers to a large granular lymphocyte, being a cytotoxic lymphocyte derived from the common lymphoid progenitor which does not naturally comprise an antigen-specific receptor (e.g. a T-cell receptor or a B-cell receptor). NK cells may be characterised by their CD3-, CD56+ phenotype. The term as used herein thus includes any known NK cell or any NK-like cell or any cell having the characteristics of an NK cell. Thus primary NK cells may be used or, in an alternative embodiment, a NK cell known in the art that has previously been isolated and cultured may be used. Thus an NK cell-line may be used. A number of different NK cells are known and reported in the literature and any of these could be used, or a cell-line may be prepared from a primary NK cell, for example by viral transformation (Vogel et al. 2014, Leukemia 28:192-195). Suitable NK cells include (but are by no means limited to), in addition to NK-92, the NK-YS, NK-YT, MOTN-1 , NKL, KHYG-1 , HANK-1 , or NKG cell lines. In a preferred embodiment, the cell is an NK-92 cell (Gong et al. 1994, Leukemia 8:652-658), or a variant thereof. A number of different variants of the original NK-92 cells have been prepared and are described or available, including NK-92 variants which are non-immunogenic. Any such variants can be used and are included in the term "NK-92". The variant of the NK-92 cell line may be the UK-92 variant, which has been transfected such that it expresses CD3. Variants of other cell lines may also be used.
T-cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue and tumours. In certain embodiments, T-cells can be obtained from a unit of blood collected from a subject using
any number of techniques known to the skilled person, such as FICOLL™ separation. In one embodiment, cells from the circulating blood of a subject are obtained by apheresis. The apheresis product typically contains lymphocytes, including T-cells, monocytes,
granulocytes, B-cells, other nucleated white blood cells, red blood cells and platelets. In one embodiment, the cells collected by apheresis may be washed to remove the plasma fraction and placed in an appropriate buffer or media for subsequent processing. In one embodiment of the invention, the cells are washed with PBS. In an alternative embodiment, the washed solution lacks calcium and/or magnesium or may lack many if not all divalent cations. As would be appreciated by those of ordinary skill in the art, a washing step may be
accomplished by methods known to those in the art, such as by using a semi-automated flow-through centrifuge, for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the like. After washing, the cells may be resuspended in a variety of biocompatible buffers or other saline solution with or without buffer. In certain embodiments, the undesirable components of the apheresis sample may be removed and the cell directly resuspended in culture media.
In certain embodiments, T-cells are isolated from PBMCs. PBMCs may be isolated from buffy coats obtained by density gradient centrifugation of whole blood, for instance centrifugation through a LYMPHOPREP™ gradient, a PERCOLL™ gradient or a FICOLL™ gradient. T-cells may be isolated from PBMCs by depletion of the monocytes, for instance by using CD14 DYNABEADS®. In some embodiments, red blood cells may be lysed prior to the density gradient centrifugation.
A specific subpopulation of T-cells, such as CD28+, CD4+, CD8+, CD45RA+ or CD45RO+ T-cells, can, if desired, be further isolated by positive or negative selection techniques. For example, enrichment of a T-cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively-selected cells. One method for use herein is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDIIb, CD16, HLA-DR and CD8. Flow cytometry and cell sorting may also be used to isolate cell populations of interest for use in the present invention.
In certain embodiments, both cytotoxic and helper T-cells can be sorted into naive, memory, and effector T-cell sub-populations either before or after genetic modification and/or expansion. CD8+ T-cells can be obtained by using standard methods as described above. In some embodiments, CD8+ T-cells are further sorted into naive, central memory, and effector cells by identifying cell-surface antigens that are associated with each of those
types of CD8+ T-cells. Memory T-cells may be present in both CD62L+ and CD62L- subsets of CD8+ peripheral blood T-cells. T-cells are sorted into CD62L-/CD8+ and CD62L+/CD8+ fractions after staining with anti-CD8 and anti-CD62L antibodies. Phenotypic markers of central memory T-cells (TCM) may include expression of CD45RO, CD62L, CCR7, CD28, CD3 and CD127, and lack of expression of granzyme B. TCMs may be
CD45RO+/CD62L+/CD8+ T-cells. Effector T-cells may be negative for CD62L, CCR7, CD28, and CD127 expression, and positive for granzyme B and perforin expression. Naive CD8+ T-cells may be characterised by the expression of phenotypic markers of naive T-cells including CD62L, CCR7, CD28, CD3, CD127 and CD45RA.
The immune effector cell for use in the methods of the invention may be derived from any mammal. For instance, the immune effector cell may be derived from a species of domestic pet, such as a mouse, rat, gerbil, rabbit, guinea pig, hamster, cat or dog, or livestock, such as a goat, sheep, pig, cow or horse. Preferably, the immune effector cell is derived from a primate, such as a monkey, gibbon, gorilla, orangutan, chimpanzee or bonobo. As noted above, most preferably, however, the immune effector cell is derived from a human (i.e. is a human cell).
In an embodiment of the invention, the immune effector cell is redirected. By redirected is meant that the cell is modified to express a non-native antigen receptor on its cell surface. By "non-native" is meant an antigen receptor which is not encoded by the cell in its native state, i.e. it is heterologous, or non-endogenous. Thus a redirected immune effector cell recognises an antigen which it did not natively recognise. Expression of the heterologous antigen receptor can thus be seen to introduce a new antigen specificity to the immune effector cell, causing the cell to recognise and bind a previously unrecognised antigen. The antigen receptor may be isolated from any useful source.
Preferably, a redirected immune effector cell expresses only one antigen receptor, i.e. the heterologous antigen receptor. The heterologous antigen receptor may be a natural antigen receptor, i.e. it may be derived and isolated from a natural source and have an unaltered, native sequence. Alternatively, the heterologous antigen receptor may be an altered, or modified, antigen receptor. For instance the sequence of the receptor may be modified relative to a native sequence, or the receptor may be an unnatural (i.e. synthetic) type of receptor.
In a particular embodiment, the immune effector cell may be modified to express a heterologous TCR. TCRs are protein complexes which protrude from the cell membrane of a T-cell. Most TCRs comprise an a- and a β-chain, both of which consist of a variable region and a constant region. The variable region is located at the N-terminus of the chain, and is wholly extracellular; the constant region is located at the C-terminus of the chain, and consists of an extracellular domain, a transmembrane domain and a short cytoplasmic
domain. TCR chains are encoded and synthesised in an immature form, with an N-terminal signal (or leader) sequence. This sequence forms the N-terminus of the variable region of an a- or β- TCR chain when it is synthesised. Following synthesis of the TCR chain, the signal sequence is cleaved, and so is not present in a mature TCR located at the cell surface.
The variable region of an a- or β-chain comprises three hypervariable,
complementarity determining regions (CDRs). These CDRs determine the specificity of the TCR, with CDR3 (that is, the third CDR from the N-terminus) being the most important CDR in determining TCR specificity. The sections of the variable regions of TCR chains which do not form the CDRs are known as framework regions. A TCR variable region contains four such framework regions. Framework region 1 is N-terminal to CDR1 ; framework region 2 links CDR1 and CDR2; framework region 3 links CDR2 and CDR3; framework region 3 links CDR3 to the constant region of the TCR chain. These framework regions are much less variable than the CDRs, and form a scaffold for the CDRs. The sequence of the framework regions is important for TCR function, as they determine the overall structure of the variable region of a TCR chain. This structure must hold the CDRs in the correct orientations and relative positions for them to bind the target antigen.
TCRs recognise specific MHC-antigen complexes. Upon binding of a TCR to its cognate MHC-antigen complex, the T-cell is stimulated to proliferate and its effector functions are activated. Thus a T-cell can be easily redirected by modification to express a heterologous TCR. Many TCRs of medical interest are known and have been used in clinical trials and therapy. For instance, the Radium-1 TCR (disclosed in WO 2017/194555 and used in the Examples below) recognises an antigen produced by a frameshift mutation in TGF3RII in the context of HLA-A2. The frameshift mutation which produces the antigen recognised by the Radium-1 TCR is associated with colon cancer. Any appropriate TCR may be used.
A TCR may also be used to redirect NK cells, as detailed in WO 2016/1 16601 . By modifying an NK cell to express a TCR, such that the TCR is co-expressed with CD3 on the cell surface, i.e. in the plasma membrane, to form a functional TCR complex, an NK cell can be altered to become a targeted killer cell (CD3 is required in T-cells for localisation of the TCR to the cell surface and for activation of a T-cell upon contact with its target MHC- antigen complex). Whilst in some cases, the NK cell may be modified to express one or more chains of CD3, depending on the cell this may not be necessary as some NK cells and cell lines may natively express one or more CD3 chains. As explained in WO 2016/1 16601 , a chain of the TCR molecule may be expressed as a fusion with a CD3 chain as a chimeric receptor molecule.
Thus, in a particular embodiment, the immune effector cell used in the method of the present invention is redirected using a TCR.
Another antigen receptor which is useful in the invention is a chimeric antigen receptor (CAR). CARs are fusion proteins comprising an antigen-binding domain, typically derived from an antibody, linked to the signalling domain of the TcR complex. CARs can be used to direct T-cells or other immune effector cells (e.g. NK cells) against a target antigen if a suitable antigen-binding domain is selected.
The antigen-binding domain of a CAR is typically based on an scFv (single chain variable fragment) derived from an antibody. In addition to an N-terminal, extracellular antibody-binding domain, CARs typically comprise a hinge domain, which functions as a spacer to extend the antigen-binding domain away from the plasma membrane of the immune effector cell on which it is expressed, a transmembrane (TM) domain, an intracellular signalling domain (e.g. the signalling domain from the zeta chain of the CD3 molecule {Οϋ3ζ) of the TcR complex, or an equivalent) and optionally one or more co- stimulatory domains which may assist in signalling or functionality of the cell expressing the CAR.
Thus, in another embodiment, the immune effector cell used in the method of the present invention is redirected using a CAR.
CARs and TCRs have different advantages. TCRs are very useful because they are able to recognise both extracellular and intracellular antigens (both are presented by MHC molecules). CARs, having antibody-derived antigen-recognition domains, are generally able only to recognise extracellular or cell surface antigens. Thus TCRs are able to recognise a much wider variety of antigens. However, as mentioned above, TCRs are MHC-restricted, and only recognise antigens in the context of a complex with (in humans) an appropriate HLA molecule. TCRs are thus are only of use for treatment of individuals who carry the appropriate HLA allele. Although some TCRs are promiscuous and can recognise antigens in the context of a number of HLA alleles, this is not the case for all TCRs and even in the case of a promiscuous TCR it would not be of use for treatment of a proportion of individuals. CARs are not MHC-restricted and are thus useful for the treatment of any individual having a target antigen. Furthermore, while a TCR is only useful in the species from which it is derived, a CAR can carry an antigen-binding domain derived from any species. In the instance that an immune effector cell of the invention is redirected with a CAR which contains an antigen-binding domain derived from a non-human animal (e.g. a murine antigen-binding domain), it is preferred that the antigen-binding domain is humanised, to avoid or minimise any immune response to the CAR itself.
The skilled person is able to select an appropriate antigen receptor with which to redirect an immune effector cell to be used in the method of the invention, based on the target antigen, the availability of an antigen receptor and the advantages and disadvantages of the types of receptor, discussed above.
In a particular embodiment, the immune effector cell for use in the method of the invention is a redirected T-cell, e.g. a redirected CD8+ T-cell or a redirected CD4+ T-cell.
Methods by which immune effector cells can be genetically modified to express a heterologous antigen receptor are well known in the art. A nucleic acid molecule encoding the antigen receptor may be introduced into the cell in the form of e.g. a vector, or any other suitable nucleic acid construct. Vectors, and their required components, are well known in the art. Nucleic acid molecules encoding antigen receptors can be generated using any method known in the art, e.g. molecular cloning using PCR. Antigen receptor sequences can be modified using commonly-used methods, such as site-directed mutagenesis.
Vectors or constructs may be introduced into an immune effector cell by a variety of means, including chemical transfection agents (such as calcium phosphate, branched organic compounds, liposomes or cationic polymers), electroporation, cell squeezing, sonoporation, optical transfection, hydrodynamic delivery or viral transduction. Introduction of a vector or construct by viral transduction may allow for more persistent expression of the heterologous antigen receptor. However, in some situations, e.g. in clinical trials, or in some clinical situations, it may be desirable to have a more transient period of expression of the receptor. In such a situation it may be desirable to deliver the nucleic acid molecule to the immune effector cell as mRNA. mRNA expression vectors for production of mRNA may be prepared according to methods known in the art (e.g. using Gateway Technology) and are known in the art (e.g. pClpA102, Saeb0e-Larssen et al., 2002, J Immunol Methods 259, p191 -203; and pClpAI 20-G, Walchli et al. , 201 1 , PLOS ONE 6(1 1 ) e27930).
The mRNA can be produced in vitro by e.g. in vitro transcription. The mRNA may then be introduced into the immune effector cells, e.g. as naked mRNA, e.g. by
electroporation (as described for example in Almasbak et al., Cytotherapy 201 1 , 13, 629- 640; Rabinovich et al., Hum Gene Ther, 2009, 20, 51 -60; and Beatty et al., Cancer Immunol Res 2014, 2, 1 12-120). Alternatively, mRNA may be introduced by other means such as by liposomes or cationic molecules etc. Heterologous nucleic acid molecules introduced into a cell may be expressed episomally, or may be integrated into the genome of the cell at a suitable locus.
In preferred embodiments, the immune effector cell is redirected against a cancer antigen. This means that the immune effector cell is modified to express a heterologous antigen receptor, as described above, which recognises a cancer antigen. By "cancer antigen" is meant any antigen (i.e. a molecule capable of inducing an immune response) which is associated with cancer. An antigen as defined herein may be any type of molecule which induces an immune response, e.g. it may be a polysaccharide or a lipid, but most preferably it is a peptide (or protein).
Human cancer antigens may be human or human-derived. A cancer antigen may be a tumour-specific antigen, by which is meant an antigen which is not found in healthy cells. Tumour-specific antigens generally result from mutations, in particular frame-shift mutations which generate a wholly new amino acid sequence not found in the healthy human proteome. An example of such tumour-specific antigens are those produced by the -1 A and +1A mutations in TGF3RII (Saeterdal et al. (2001 ), Proc Nat Acad Sci U S A, Vol. 98(23): 13255-13260). The Radium-1 TCR described above, and used in the Examples below, recognises tumour-specific antigens generated by the -1A mutation in TGF3RII.
Cancer antigens also include tumour-associated antigens, which are antigens whose expression or production is associated with (but not limited to) tumour cells. Examples of tumour-associated antigens include for instance MAGEA1 (melanoma-associated antigen 1 ) and CTAG1 B (cancer/testis antigen 1 B), whose expression in healthy individuals is limited to male germ-line cells, but which are also expressed in various cancer types (e.g. MAGEA1 is commonly expressed in melanoma cells).
Cancer antigens may be non-human, in particular a cancer antigen may be a molecule, particularly a protein, produced by an oncovirus. Examples of oncoviruses include human papilloma virus (HPV), a causative agent of a number of cancer types including in particular cervical cancer; and Kaposi's sarcoma-associated herpesvirus (KSHV), which causes skin cancer in immuno-compromised individuals (particularly those suffering from HIV/AIDS). Any protein produced by an oncovirus may be considered a cancer antigen, e.g. HPV proteins E6 and E7 which inactivate the tumour suppressor genes p53 and pRB, respectively.
Large numbers of cancer antigens are well known in the art. The immune effector cell to be used in the method of the invention is preferably redirected against a cancer antigen, i.e. the immune effector cell is preferably modified to express a heterologous antigen receptor which recognises a cancer antigen.
In a particular embodiment, the immune effector cell for use in the method of the invention is a T-cell redirected against a cancer antigen, e.g. a CD8+ T-cell redirected against a cancer antigen or a CD4+ T-cell redirected against a cancer antigen.
In other embodiments, the immune effector cell may be redirected against an antigen associated with an infection, e.g. an antigen from a bacterium, virus, parasite or fungus. The immune effector cell may alternatively be redirected against an antigen associated with an autoimmune disease.
In the method of the invention an immune effector cell is contacted in vitro or ex vivo with a non-biguanide mitochondrial electron transport chain inhibitor or BRD56491 .
Alternatively, the method may be seen to comprise contacting an in vitro or ex vivo immune effector cell with a non-biguanide mitochondrial electron transport chain inhibitor or
BRD56491 . Thus the contacting does not occur in vivo, and the method of the invention does not comprise the administration of a mitochondrial ETC inhibitor or BRD56491 to a subject.
The immune effector cell may be contacted with one or more non-biguanide mitochondrial ETC inhibitors. By "mitochondrial ETC inhibitor" is meant a specific inhibitor of the mitochondrial electron transport chain, i.e. a species which specifically targets one or more of the proteins in the mitochondrial ETC. More particularly, the inhibitor may bind to one or more of the proteins and inhibit (e.g. reduce or entirely abrogate) the function of the bound protein. For instance, the mitochondrial ETC inhibitor may be a specific inhibitor of one of the five ETC complexes, i.e. the ETC inhibitor may be an inhibitor of Complex I
(NADH dehydrogenase); an inhibitor of Complex I I (succinate dehydrogenase); an inhibitor of Complex I I I (the cytochrome bci complex); an inhibitor of Complex IV (cytochrome c oxidase); or an inhibitor of Complex V (i.e. the F0F-i ATP synthase). In a particular embodiment the mitochondrial ETC inhibitor is an inhibitor of any one of Complexes l-IV, i.e. an inhibitor of Complex I , Complex I I, Complex II I or Complex IV.
Each of Complexes l-V comprises multiple subunits. An inhibitor of any one of the complexes may bind any one or more of the subunits which form the complex. Alternatively, or additionally, the mitochondrial ETC inhibitor may specifically bind one or more of the inter- complex electron-transferring molecules, e.g. the ETC inhibitor may bind coenzyme Qi0 (including oxidised coenzyme Qi0 (ubiquinone), partially reduced coenzyme Qi0
(semiquinone) and fully reduced coenzyme Qi0 (ubiquinol)) or cytochrome c. It is to be understood that an ETC inhibitor as defined herein does not include uncoupling agents, e.g. FCCP. Accordingly, in certain embodiments of the methods herein the immune effector cell is not contacted with an uncoupling agent (for instance the immune effector cell is not contacted with FCCP). The immune effector cell may be contacted with at least one ETC inhibitor, e.g. it may be contacted with a single ETC inhibitor, or it may be contacted with multiple (i.e. two or more) inhibitors of the same complex. Preferably the immune effector cell is not contacted with one or more inhibitors of multiple complexes.
As noted above, according to the invention an immune effector cell may be contacted with a non-biguanide ETC inhibitor. A biguanide ETC inhibitor is an ETC inhibitor derived from biguanide, and thus a non-biguanide ETC inhibitor is an ETC inhibitor which is not derived from biguanide (i.e. it does not have a biguanide-based structure). A number of biguanide derivatives are used in the treatment of diabetes, including metformin, phenformin and buformin. These drugs may also have some activity as ETC inhibitors (in particular in inhibition of Complex I). However, according to the invention where an immune effector cell is contacted with an ETC inhibitor (as opposed to BRD56491 ) it is required that it is contacted with an ETC inhibitor which is not a biguanide derivative. (In other words, it is a
requirement of the invention as claimed that where the contacting agent in step (i) of the method is an ETC inhibitor, the cell is contacted at least with a non-biguanide inhibitor). In particular, an immune effector cell may be contacted with an ETC inhibitor which is not metformin, phenformin or buformin. Other biguanide derivatives are used as anti-malarials, including proguanil and chlorproguanil, which may also have activity as ETC inhibitors. According to the invention, an immune effector cell may be contacted with an ETC inhibitor which is not proguanil or chlorproguanil.
In an embodiment, the ETC inhibitor is capable of increasing ROS levels in the contacted cell. That is, the ETC inhibitor is able to increase the level of at least one reactive oxygen species (ROS) molecule in an immune effector cell. Without wishing to be bound by theory, we believe that an increase in ROS levels may be responsible for, or at least contribute to, the functional effects which are observed (i.e. to the enhanced function of the immune effector cell).
Examples of non-biguanide inhibitors of Complex I include acetogenins (such as rolliniastatin-2, annonacin, dispalin, etc.), rotenone, deguelin, amobarbital, piericidin A and MPP+ (1 -methyl-4-phenylpyridinium, a metabolite of MPTP (1 -methyl-4-phenyl-1.2,3,6- tetrahydropyridine); examples of inhibitors of Complex II include malonate, atpenin A5, carboxin and thenoyltrifluoroacetone (TTFA), along with the TCA cycle inhibitors malate and oxaloacetate; examples of inhibitors of Complex III include antimycin A, myxothiazol, stigmatellin, atovaquone and strobilurin derivatives, e.g. strobilurin A-H, azoxystrobin and trifloxystrobin; examples of Complex IV inhibitors include cyanide, azide and carbon monoxide. Other ETC inhibitors include pyrrolnitrin and thiabendazole.
ETC inhibitors are well-known in the art and are widely commercially available from chemicals suppliers. Suppliers from which all or many of the above-listed inhibitors may be obtained include Sigma-Aldrich (USA), Abeam (UK), Cayman Chemical (USA), Thermo Fisher Scientific (USA), MP Biomedicals (USA), etc.
Any non-biguanide ETC inhibitor may be used in the method of the invention. The ETC inhibitor may be an inhibitor listed above, a known ETC inhibitor not listed above, or a currently unknown ETC inhibitor. ETC inhibitors may be identified by analysis of cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). If following contacting human cells with an unknown substance, the OCR falls and the ECAR increases, this may indicate that the unknown substance is an ETC inhibitor. Such an assay may be performed using e.g. a Seahorse XF Analyser (Agilent Technologies, USA). Notably an ETC inhibitor is a compound which binds a Complex or intermediary of the ETC and halts electron flow - an ETC inhibitor as defined herein does not include compounds which prevent electron transport by simply damaging or destroying the ETC or a component thereof. The skilled person is able to identify an ETC inhibitor by methods known in the art.
Whether an ETC inhibitor is or is not a biguanide derivative may be determined by structural analysis. Biguanide has the structure set forth below:
An ETC inhibitor which is derived from this structure is a biguanide derivative, while an ETC inhibitor not derived from this structure is a non-biguanide. Structural analysis may be performed by e.g. NMR or X-ray crystallography.
The non-biguanide ETC inhibitor may be synthetic or natural. For instance, natural ETC inhibitors include rotenone, which is derived from plants, particularly the Fabaceae family, antimycin A and oligomycins which are produced by bacterial species of the genus Streptomyces, and myxothiazol which is produced by the bacterium Myxococcus fulvus. Conversely, atpenin A5, malonate and thenoyltrifluoroacetone, for instance, are synthetic.
Preferably the non-biguanide ETC inhibitor is not highly toxic to human immune effector cells. For instance, myxothiazol and cyanide may be excessively toxic for use in the method of the invention for the treatment of humans. Excessive toxicity can only be determined empirically, using e.g. cell viability assays as described above and detailed in the Examples.
In preferred embodiments of the invention, the non-biguanide ETC inhibitor inhibits Complex I or Complex III. Most preferred ETC inhibitors are rotenone and antimycin Aand compounds or molecules which act on Complex I or Complex III in an analogous manner to rotenone or antimycin A (i.e. which act in the same way). Antimycin A has the structure set forth below in Formula I, and rotenone the structure set forth below in Formula II. Rotenone functions by binding to and blocking the ubiquinone binding site of Complex I, thus preventing ubiquinone docking to Complex I and hence preventing electron transfer from Complex I to ubiquinone. Complex I inhibitors with the same mechanism of action as rotenone (e.g. piericidin A) are also preferred mitochondrial ETC inhibitors according to the invention. Antimycin A functions by binding to and blocking the Q, site of Complex III, thus preventing ubiquinone docking to Complex III and hence preventing electron transfer from Complex III to ubiquinone, blocking the Q cycle. Complex III inhibitors with the same mechanism of action as antimycin A are also preferred mitochondrial ETC inhibitors according to the invention.
In a preferred embodiment, the immune effector cell is contacted with rotenone or antimycin A. It is preferred that the immune effector cell is contacted with only one of these two compounds, i.e. that it is not contacted with both rotenone and antimycin A.
In a particular embodiment, the term "rotenone" encompasses both rotenone itself (i.e. the compound with the structure presented in Formula II) and analogues of rotenone. An analogue of rotenone, as defined herein, is a derivative of rotenone having corresponding activity to rotenone (i.e. a derivative of rotenone which binds Complex I at the same location as rotenone and has activity as a mitochondrial ETC inhibitor). A derivative of rotenone is a compound based on rotenone but with a minor alteration which does not affect its activity, for instance such a derivative may comprise an additional methyl group relative to rotenone or may be an e.g. halogenated derivative of rotenone.
In another embodiment, the term "rotenone" means only rotenone itself, that is to say the compound with the structure presented in Formula II.
In a particular embodiment, the term "antimycin A" encompasses both antimycin A itself (i.e. the compound with the structure presented in Formula I) and analogues of antimycin A. An analogue of antimycin A, as defined herein, is a derivative of antimycin A having corresponding activity to antimycin A (i.e. a derivative of antimycin A which binds Complex III at the same location as antimycin A and has activity as a mitochondrial ETC inhibitor). A derivative of antimycin A is a compound based on antimycin A but with a minor alteration which does not affect its activity, for instance such a derivative may comprise an additional methyl group relative to antimycin A or may be an e.g. halogenated derivative of antimycin A.
In another embodiment, the term "antimycin A" means only antimycin A itself, that is to say the compound with the structure presented in Formula I.
According to the invention, the immune effector cell may alternatively be contacted with BRD56491 (2-(4-Methylphenyl)-4H-benzo[h]chromen-4-one). BRD56491 is a non-toxic enhancer of reactive oxygen species, disclosed in Adams et al. (ACS Chem Biol 8: 923-929, 2013). The structure of BRD56491 is set forth in Formula III, below. The compound may be obtained from standard commercial sources, e.g. Sigma-Aldrich and MedKoo Biosciences.
Formula III (BRD56491 )
In a particular embodiment, the term "BRD56491 " encompasses both BRD56491 itself (i.e. the compound with the structure presented in Formula III) and analogues of BRD56491 . An analogue of BRD56491 , as defined herein, is a derivative of BRD56491 having corresponding activity to BRD56491 (i.e. a derivative of BRD56491 which causes an increase in ROS production in a contacted cell). A derivative of BRD56491 is a compound based on BRD56491 but with a minor alteration which does not affect its activity, for instance such a derivative may comprise an additional methyl group relative to BRD56491 or may be an e.g. halogenated derivative of BRD56491 .
In another embodiment, the term "BRD56491 " means only BRD56491 itself, that is to say the compound with the structure presented in Formula III.
Notably, while, according to the invention, an immune effector cell is contacted with a non-biguanide ETC inhibitor or BRD56491 , this does not preclude that the immune effector cell may also be contacted with a biguanide ETC inhibitor. That is to say, the immune
effector cell may be contacted with a non-biguanide ETC inhibitor or BRD56491 and a biguanide ETC inhibitor. For instance, the immune effector cell may be contacted with a non- biguanide ETC inhibitor such as rotenone or antimycin A in combination with a biguanide ETC inhibitor such as metformin, phenformin or buformin. In such embodiments the immune effector cell may be contacted with the non-biguanide ETC inhibitor and biguanide ETC inhibitor concurrently (i.e. at the same time) or consecutively (i.e. at separate times, either firstly with the non-biguanide ETC inhibitor and then with the biguanide ETC inhibitor, or firstly with the biguanide ETC inhibitor and then with the non-biguanide ETC inhibitor).
The contacting of the immune effector cell with the non-biguanide mitochondrial ETC inhibitor or BRD56491 may be performed at any suitable concentration and for any appropriate length of time, as determined empirically by the skilled person.
In certain embodiments the immune effector cell is contacted with the non-biguanide mitochondrial ETC inhibitor or BRD56491 at a concentration of 10 nM to 10 μΜ, in particular 50 nM to 10 μΜ, or 100 nM to 10 μΜ, preferably 0.5 μΜ to 5 μΜ, particularly 1 to 5 μΜ, most preferably about 2 μΜ (i.e. 1 .5 to 2.5 μΜ). In particular, immune effector cells may be contacted with antimycin A at a concentration of 0.5 to 5 μΜ, preferably about 2 μΜ, and/or with rotenone at a concentration of 0.5 to 5 μΜ, preferably about 2 μΜ.
In certain embodiments the immune effector cell is contacted with the non-biguanide mitochondrial ETC inhibitor or BRD56491 for 1 to 96 hours, e.g. 2 to 72 hours, 3 to 48 hours, 4 to 36 hours or 8 to 24 hours. Preferably, the contacting is performed for at least 12 hours. Preferably the contacting is performed for less than 48 or 36 hours. For instance, the contacting may be performed for 12 to 48 hours, 24 to 48 hours, 12 to 24 hours or 12 to 18 hours. In some embodiments the contacting is performed for about 16 hours.
The contacting may be performed by culturing the immune effector cell in medium containing the non-biguanide ETC inhibitor or BRD56491. The contacting may be initiated by, for instance, the addition of a non-biguanide ETC inhibitor or BRD56491 to culture medium comprising the immune effector cell. Alternatively, immune effector cell culture medium lacking a non-biguanide mitochondrial ETC inhibitor or BRD56491 may be replaced with culture medium comprising a non-biguanide mitochondrial ETC inhibitor or BRD56491 , or immune effector cells in a culture not comprising a non-biguanide mitochondrial ETC inhibitor or BRD56491 may be harvested (e.g. by centrifugation) and resuspended in culture medium comprising a non-biguanide mitochondrial ETC inhibitor or BRD56491 .
The contacting step may be performed in any suitable culture medium comprising suitable supplements for immune effector cell culture. Suitable culture media are known to the skilled person, and include for instance CellGenix® GMP SCGM and CellGenix® GMP DC Medium (both CellGenix, Germany) and X-VIVO™ 10, X-VIVO™ 15 and X-VIVO™ 20 (all Lonza, Switzerland). In a particular embodiment, the contacting step is not performed in
RPMI 1640 medium (commonly abbreviated to RPMI medium). RPMI medium is commonly available and its composition is disclosed, for instance, in the Sigma-Aldrich product information sheet for RPMI 1640 medium (herein incorporated by reference). A number of RPMI 1640 variants are available (e.g. with or without HEPES or phenol red). As used herein, the term RPMI 1640 medium encompasses all varieties of RPMI 1640 medium.
In another particular embodiment the contacting step is not performed in an XF medium, which is the medium commonly used in the Seahorse assay (i.e. an assay using the Seahorse analyser as discussed above). Various variants of XF media are available from Agilent Technologies, USA, and all are included within the term "XF medium".
Suitable supplements are also known to the skilled person, and may vary depending on the type of immune effector cell contacted. Suitable supplements for T-cell and NK cell culture include in particular IL-2 and human serum. Suitable supplements for immune effector cell culture also include foetal calf serum (FCS) and bovine calf serum (BCS). In embodiments the contacting step is performed in a cell culture medium comprising serum and/or IL-2.
Antibiotics are commonly used during cell culture to maintain sterility. Penicillin and streptomycin are examples of commonly-used antibiotics. In a particular embodiment, the contacting step is performed in the absence of antibiotics, in particular in the absence of penicillin and streptomycin. As defined herein, "absence" of penicillin and streptomycin does not necessarily mean that not a single molecule of penicillin or streptomycin is present in the culture medium, though it is preferred that penicillin and streptomycin are undetectable in the culture medium. However, as defined herein penicillin is considered to be absent from culture medium if it is present at a concentration of no more than about 1 ng/ml. Similarly, streptomycin is considered to be absent from culture medium if it is present at a
concentration of no more than about 1 ng/ml.
In another particular embodiment, the contacting step is performed in the absence of gelatin. Similar to the above, it is preferred that absence of gelatin from culture medium means that gelatin is undetectable in the culture medium. However, as defined herein gelatin is considered to be absent from culture medium if it is present at a concentration of no more than about 1 ng/ml.
In another particular embodiment, the contacting step is performed in the absence of ATP. The absence of ATP refers to the absence of exogenous ATP from the culture medium - clearly ATP is present within the cultured cells. However, ATP may be absent from the culture medium. As above, it is preferred that absence of ATP from culture medium means that ATP is undetectable in the culture medium. However, as defined herein ATP is considered to be absent from culture medium if it is present at a concentration of no more
than about 1 ng/ml. In certain embodiments, any ATP present (e.g. added to, or included in) the culture medium is present in an amount less than 0.1 , 0.5, 1 .0 or 3.0 mM ATP.
As indicated above, in certain embodiments, the contacting step, or the method, does not include contacting with an uncoupling agent, e.g. FCCP. In an embodiment the contacting step is performed in the absence of an uncoupling agent, e.g. in the absence of FCCP. As defined herein an uncoupling agent (e.g. FCCP) is considered to be absent from the culture medium if it is present at a concentration of no more than about 1 nM. In particular embodiments, FCCP, if present, is present in a concentration of less than 0.1 μΜ.
In other embodiments, the contacting step, or the method, does not include contacting with an oligomycin. In an embodiment the contacting step is performed in the absence of an oligomycin. As defined herein an oligomycin is considered to be absent from the culture medium if it is present at a concentration of no more than about 1 nM. In particular embodiments, an oligomycin if present, is present in a concentration of less than 0.1 μΜ.
The contacting of the immune effector cell with a non-biguanide mitochondrial ETC inhibitor or BRD56491 is generally ended by harvesting the immune effector cell from the culture medium comprising the ETC inhibitor or BRD56491 and optionally washing the cell to remove trace ETC inhibitor/BRD56491 . Following contacting, the cell is formulated in a sterile composition suitable for therapeutic administration (in particular parenteral administration) to a subject, which may in particular comprise resuspending the cell in culture medium not comprising an ETC inhibitor or BRD56491 .
The term "harvesting" is used herein to mean any method of recovering or collecting cultured cells. Harvesting is preferably performed by centrifugation, and may be undertaken to separate the immune effector cell from the non-biguanide mitochondrial ETC inhibitor or BRD56491 . Thus harvesting of the immune effector cell takes place following the contacting of the immune effector cell with the non-biguanide ETC inhibitor or BRD56491 , and may function to terminate the contacting. Methods of harvesting cultured cells also include removal of the cells from medium by filtration (which may be enhanced by centrifugation or the use of a vacuum). Methods of filtration include microfiltration and depth filtration.
Harvesting does not require, but may if desired include, that the cells are removed from the container or vessel in which they were contacted.
Following harvesting, the immune effector cell may be washed, e.g. to remove any remaining ETC inhibitor or BRD56491 from the culture. Washing may be performed according to any suitable method, e.g. the harvested cell may be resuspended in clean medium (e.g. medium lacking the ETC inhibitor or BRD56491 ) or an isotonic solution, e.g. PBS, and then harvested again, e.g. by centrifugation, and the washing solution removed. Washing may be performed once or more than once, e.g. two or three times or more. In an embodiment the washing solution does not comprise an ETC inhibitor or BRD56491 . A
solution which does not comprise an ETC inhibitor or BRD 56491 preferably does not contain detectable levels of ETC inhibitor or BRD56491 , or the amount of ETC inhibitor or BRD56491 present may be negligible. Alternatively, the washing solution may be considered not to comprise an ETC inhibitor or BRD56491 if each agent is present at a concentration of less than 10 nM, preferably less than 1 nM.
The treated cells may be formulated in a sterile composition by resuspending the cells in, or transferring or adding the cells to, a suitable sterile liquid (e.g. a medium or solution). This is discussed further below, and the terms "resuspending", "transferring" and "adding" include adding the liquid to the cells or vice versa. The cells may be subjected to a further treatment step (e.g. expansion, contact with a further agent, freezing and/or genetic modification, e.g. to redirect the cells) before or after the formulation step, as further discussed below.
The method of the invention may further comprise expanding the immune effector cell. By "expanding" the immune effector cell is meant stimulating the immune effector cell to proliferate. Methods by which an immune effector cell may be stimulated to proliferate are discussed above and are known to the person skilled in the art. Methods for activating and expanding T-cells are described, for example, in US 6905874, US 6867041 , US 6797514, and WO 2012/079000. Methods for expanding NK cells are described, for example, in WO 2014/037422.
Expansion of the immune effector cell may be performed following the contacting of the immune effector cell with the non-biguanide mitochondrial ETC inhibitor or BRD56491 . As defined above, "following" the contacting of the immune effector cell with the ETC inhibitor or BRD56491 means after the contacting has commenced. The contacting may be ongoing during expansion, or the expansion may occur once the contacting has been ended by separating the cell and the ETC inhibitor/BRD56491 , e.g. by harvesting and washing the cell. Thus harvesting of the immune effector cell may take place before expansion or after expansion. Alternatively, expansion may take place prior to contacting the immune effector cell with the non-biguanide mitochondrial ETC inhibitor or BRD56491. In other words, an expanded population of immune effector cells may be contacted with a non-biguanide mitochondrial ETC inhibitor or BRD56491. In the methods of the invention, "contacting an immune effector cell with a non-biguanide mitochondrial electron transport chain inhibitor or BRD56491 " may refer to the contacting of a single immune effector cell, but more preferably refers to the contacting of a population of immune effector cells (i.e. "immune effector cell" according to the invention may be understood as a singular noun, but more preferably as the plural). The immune effector cell contacted with the non-biguanide mitochondrial ETC inhibitor or BRD56491 may be an already expanded immune effector cell (or population of immune effector cells).
Expanding the immune effector cell preferably comprises increasing the number of live cells by at least 50 %, e.g. doubling, trebling or more the number of live cells. Expansion of the immune effector cells takes place over a period of time which may be at least 12, 24, 36, 48, 72 or 96 hours. The expansion may take place for a period of time which is at most 12, 24, 36, 48, 72 or 96 hours.
In the instance that the immune effector cell is a progenitor, the method of the invention may further comprise causing the cell to differentiate. Such methods are known in the art. Methods by which such differentiation can be performed are known in the art.
Disclosed herein is a method for enhancing the function of an immune effector cell, comprising contacting in vitro or ex vivo an immune effector cell with a non-biguanide mitochondrial electron transport chain inhibitor or BRD56491 , wherein the function of said immune effector cell following said contacting is enhanced relative to an immune effector cell which has not been contacted with said inhibitor or BRD56491 . According to the invention, the method is used to generate an immune effector cell for use in adoptive cell transfer therapy.
The immune effector cell used in the method is preferably non-immunogenic in the subject to which it will be administered. The immune effector cell may thus be autologous to the subject to which it will be administered, i.e. isolated from the subject. Alternatively, the immune effector cell may be a donor cell or cell-line cell (i.e. a non-autologous cell) modified to render it non-immunogenic. By non-immunogenic is meant that the cell does not, when administered to a subject, generate an immune response which affects, interferes with, or prevents the use of the cells in therapy.
Non-autologous immune effector cells to be administered to a human subject may be naturally non-immunogenic if they are HLA-matched to the patient, i.e. they express the same HLA alleles. Non-autologous immune effector cells, including those which are not HLA-matched to the patient and would therefore be immunogenic, and those which are HLA-matched to the patient and may not be immunogenic, may be modified to eliminate expression of MHC molecules, or to only weakly express MHC molecules at their surface. Alternatively, such cells may be modified to express non-functional MHC molecules.
Thus an immune effector cell to be administered to a subject (i.e. to a recipient subject) for adoptive cell transfer therapy may be a donor cell, which may be obtained from a donor subject (i.e. a subject who is different from the recipient subject), or from a cell line. Such a donor cell may be allogeneic, syngeneic or xenogeneic with respect to the recipient subject, as discussed further below.
Any means by which the expression of a functional MHC molecule is disrupted is encompassed. Hence, this may include knocking out or knocking down a molecule of the
MHC complex, and/or it may include a modification which prevents appropriate transport to and/or correct expression of an MHC molecule, or of the whole complex, at the cell surface.
In particular, the expression of one or more functional MHC class-l proteins at the surface of an immune effector cell of the invention may be disrupted. In one embodiment the immune effector cells may be human cells which are HLA-negative, such as cells in which the expression of one or more HLA molecules is disrupted (e.g. knocked out), e.g. molecules of the HLA Class I MHC complex.
In an embodiment, disruption of Class-l MHC expression may be performed by knocking out the gene encoding 32-microglobulin (β2-η"ΐ), a component of the mature Class-l MHC complex. Expression of β2-Γτι may be eliminated through targeted disruption of the β2-Γτι gene, for instance by site-directed mutagenesis of the 32-m promoter (to inactivate the promoter), or within the gene encoding the β2-ΓΤΐ protein to introduce an inactivating mutation that prevents expression of the β2-ηι protein, e.g. a frame-shift mutation or premature 'STOP' codon within the gene. Alternatively, site-directed mutagenesis may be used to generate non-functional β2-ηι protein that is not capable of forming an active MHC protein at the cell surface. In this manner the β2-ηι protein or MHC may be retained intracellular^, or may be present but non-functional at the cell surface.
The immune effector cells of the invention may also be subject to modification in other ways (in addition to redirection), for example to alter or modify other aspects of cell function or behaviour, and/or to express other proteins. For instance, the cells may be modified to express a homing receptor, or localisation receptor, which acts to target or improve the localisation of the cells to a particular tissue or location in the body.
In order to prepare an immune effector cell for use in adoptive cell transfer therapy, the method of the invention further comprises formulating the enhanced immune effector cells obtained from the contacting step of method of the invention in a sterile composition suitable for parenteral administration to a subject, specifically for administration to a subject for therapy.
In particular, the method may comprise harvesting and washing the immune effector cell as described above, and transferring the enhanced immune effector cell to a
pharmaceutically-acceptable medium. The cell may be transferred to any pharmaceutically- acceptable medium, as is known to the person of skill in the art. The term "transferring" includes any way of adding cells to the medium or vice versa. By a "pharmaceutically- acceptable medium" is meant a medium suitable for pharmaceutical administration to a subject. The pharmaceutically-acceptable medium may thus be any infusion medium, i.e. any medium used or proposed in the art for administration of cells to a subject.
As detailed above, the immune effector cell may be expanded before being transferred to the pharmaceutically-acceptable medium. The enhanced cells may also or
alternatively be subjected to a further treatment step prior to transfer to the pharmaceutically- acceptable medium. If the cells are harvested, such a step may occur before or after harvesting. If the cells are washed, such a step may occur before or after washing. That is to say the further treatment step may occur before harvesting, between harvesting and washing or after washing. Treatment steps which may be applied to the enhanced cells are discussed above, e.g. genetic modification to render the cells non-immunogenic or for expression of an antigen receptor such as a TCR or CAR. In a particular embodiment the enhancer cells are irradiated.
The cells may also be stored prior to transfer to the pharmaceutically-acceptable medium. In a particular embodiment, the cells are frozen prior to transfer to a
pharmaceutically-acceptable medium. Storage (and particularly freezing) may take place at any time during the process, but preferably takes place following harvesting and washing of the enhanced cells. Storage (and particularly freezing) may take place before or after the cells are subjected to a further treatment step. In a particular embodiment the cells are frozen in a cryopreservation medium (or freezing medium). Such media are used as standard in the art for preserving frozen cells and tissue. Such a medium may for instance comprise DMSO or glycerol (for instance common media used for freezing comprise 90 % complete medium and 10 % glycerol or 10 % DMSO). Cells may be frozen using standard methods in the art, e.g. in a cryovial using a programmable cooler or within an insulated box in a freezer. A freezing medium is preferably sterile.
The pharmaceutically-acceptable medium is preferably a liquid medium, in particular an aqueous medium, and may comprise one or more pharmaceutically-acceptable diluents, carriers or excipients. Liquid pharmaceutical media, whether they be solutions, suspensions or other like form, may include one or more of the following: sterile diluents such as water, saline solution (preferably physiological saline), Ringer's solution, isotonic sodium chloride; buffers such as neutral buffered saline, phosphate buffered saline, acetates, citrates or phosphates and the like; fixed oils such as synthetic mono- or diglycerides (which may serve as the solvent or suspending medium), polyethylene glycols, glycerin, propylene glycol or other solvents; proteins or polypeptides e.g. serum albumin (for instance human serum albumin), or amino acids such as glycine; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA) or glutathione; carbohydrates such as glucose, mannose, sucrose, dextrans or mannitol; adjuvants (e.g., aluminium hydroxide); preservatives, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pharmaceutically-acceptable medium preferably does not comprise detectable levels of mitochondrial ETC inhibitor or BRD56491. The pharmaceutically-acceptable medium is sterile.
Media may be sterilised in a number of ways known in the art. For instance, many sterile media are commercially available for purchase. Non-sterile media may be sterilised using standard methods such a autoclaving, filter sterilisation, etc.
The product of the method of the invention is a sterile pharmaceutical composition, suitable for parenteral administration to a subject, preferably a human patient, in adoptive cell transfer therapy. The composition may be a liquid composition, but may be frozen for storage and preservation of the cell. The composition may be provided in any suitable container, e.g. ampoules, disposable syringes or multiple dose vials made of glass or plastic.
In a particular embodiment, the invention provides a method as described above comprising the specific steps of:
a) contacting a human immune effector cell with rotenone, Antimycin A or BRD56491 in a concentration in the range of 0.1 to 10 μΜ for at least 12 hours;
b) harvesting and optionally washing the cell; and
c) transferring the cell into a pharmaceutically-acceptable medium.
The rotenone, antimycin A or BRD56491 concentration may be as defined above, i.e. preferably in the range of 0.5 to 5 μΜ, most preferably about 2 μΜ. Preferably in this embodiment the cell is contacted with rotenone or antimycin A.
The method disclosed herein can alternatively be seen as the use of a non-biguanide mitochondrial electron transport chain inhibitor or BRD56491 for enhancing the function of an immune effector cell in vitro or ex vivo. Specifically, the method of the invention can be seen as the use of a non-biguanide mitochondrial electron transport chain inhibitor or BRD56491 for enhancing the function of an in vitro or ex vivo immune effector cell in preparation for adoptive cell transfer therapy. The mitochondrial ETC inhibitor, contacting, immune effector cell and function thereof may be as defined above in relation to the method of the invention.
Following the use of a non-biguanide mitochondrial electron transport chain inhibitor or BRD56491 to enhance the function of an immune effector cell as defined herein, the function of the immune effector cell is enhanced relative to the function of an immune effector cell which has not been contacted with an ETC inhibitor or BRD56491 . The immune effector cell which has not been contacted with an ETC inhibitor or BRD56491 is preferably as defined above, e.g. it may be the contacted immune effector cell prior to the contacting with the non-biguanide ETC inhibitor, or it may be a control cell.
The method of the invention may alternatively be seen as a method for preparing immune effector cells for use in adoptive cell transfer therapy, said method comprising contacting in vitro or ex vivo an immune effector cell with a non-biguanide mitochondrial electron transport chain inhibitor or BRD56491 , wherein the function of said immune effector
cell following said contacting is enhanced relative to an immune effector cell which has not been contacted with said inhibitor or BRD56491 .
In another alternative, the method of the invention may be seen as method for preparing immune effector cells for use in adoptive cell transfer therapy, said method comprising contacting in vitro or ex vivo an immune effector cell with a non-biguanide mitochondrial electron transport chain inhibitor or BRD56491 , wherein the function of said immune effector cell following said contacting is enhanced relative to an immune effector cell which has not been contacted with said inhibitor or BRD56491 . Preferably the adoptive cell transfer therapy is for cancer.
The invention can alternatively be seen as providing a method of preparing an immune effector cell composition for use in adoptive cell transfer therapy, said method comprising formulating a population of enhanced immune effector cells prepared according to the method described above in a pharmaceutically-acceptable medium suitable for parenteral administration to a subject. By formulating the immune effector cell population in a pharmaceutically-acceptable medium is meant that the immune effector cells are transferred into or suspended in such a medium. The product of such a method is a pharmaceutical composition, suitable for parenteral administration to a subject, preferably a human patient, in therapy, e.g. adoptive cell transfer therapy, as described above.
The invention further provides an enhanced immune effector cell obtained or obtainable by the method of the invention, i.e. obtained or obtainable by a method comprising contacting in vitro or ex vivo an immune effector cell with a non-biguanide mitochondrial electron transport chain inhibitor or BRD56491 , wherein the function of the enhanced immune effector cell is enhanced relative to an immune effector cell which has not been contacted with said inhibitor or BRD56491 .
The immune effector cell, non-biguanide mitochondrial ETC inhibitor and contacting may be as defined above with respect to the method of the invention. The function which is enhanced may in particular be proliferation, target cell killing and/or specificity of killing, as discussed above. The immune effector cell which has not been contacted with the ETC inhibitor or BRD56491 may be as defined above. In particular it may be a control immune effector cell.
The enhanced immune effector cell of the invention preferably comprises (or exhibits or displays) a level of reactive oxygen species (ROS) which is higher than the level in an immune effector cell which has not been contacted with a mitochondrial ETC inhibitor or BRD56491 (e.g. a control immune effector cell). The level of ROS in the enhanced immune effector cell is detectably increased relative to an immune effector cell which has not been contacted with a mitochondrial ETC inhibitor or BRD56491. Preferably the increase is statistically significant. For instance, the level of ROS in the enhanced immune effector cell
may be at least 2, 3, 5, 10, 15, 20, 25, 50, 75 or 100 or more times higher than the level in an immune effector cell which has not been contacted with a mitochondrial ETC inhibitor or BRD56491 .
Intracellular ROS levels can be measured as described in the Examples below. In particular, ROS levels can be quantified using a ROS-specific probe. Examples of ROS- specific probes include CellROX® probes, obtainable from Thermo Fisher Scientific (USA). CellROX® probes are cell-permeant and are applied to cells of interest in a non-fluorescent, reduced state. Upon oxidation (e.g. by a ROS) the probes exhibit strong fluorescence and remain localised within the cell. The level of ROS in the cell is proportionate to the level of probe fluorescence, which can be measured by any suitable method known in the art, e.g. microplate fluorometry or flow cytometry. Assays using CellROX® probes should be performed according to the manufacturer's instructions.
Any other method for ROS quantification known in the art, e.g. methods using 2',7'-dichlorodihydrofluorescein diacetate, may alternatively be used to compare ROS levels between enhanced immune effector cells according to the invention and immune effector cells which have not been contacted with a mitochondrial ETC inhibitor or BRD56491 (e.g. control cells).
The invention provides a pharmaceutical composition comprising the enhanced immune effector cell of the invention and one or more pharmaceutically-acceptable diluents, carriers or excipients. Liquid pharmaceutical compositions, whether they be solutions, suspensions or other like form, may include one or more of the following: sterile diluents such as water, saline solution (preferably physiological saline), Ringer's solution, isotonic sodium chloride; buffers such as neutral buffered saline, phosphate buffered saline, acetates, citrates or phosphates and the like; fixed oils such as synthetic mono- or diglycerides (which may serve as the solvent or suspending medium), polyethylene glycols, glycerin, propylene glycol or other solvents; proteins or polypeptides e.g. serum albumin (for instance human serum albumin), or amino acids such as glycine; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA) or glutathione;
carbohydrates such as glucose, mannose, sucrose, dextrans or mannitol; adjuvants (e.g., aluminium hydroxide); preservatives, and agents for the adjustment of tonicity such as sodium chloride or dextrose. Compositions of the present invention are preferably formulated for intravenous administration.
The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. An injectable pharmaceutical composition is preferably sterile.
In one embodiment, the present invention provides a pharmaceutical composition comprising enhanced immune effector cells displaying a level of reactive oxygen species (ROS) 50 to 100 times higher than the ROS level of control immune effector cells. The control immune effector cell may be an immune effector cell which has not been contacted with a mitochondrial electron transport chain inhibitor or BRD56491 . In particular, the enhanced immune effector cells are obtainable or obtained by the methods described herein.
The invention further provides an enhanced immune effector cell or pharmaceutical composition as defined herein for use in therapy. By therapy is meant the treatment of a subject. By "therapy" as used herein is meant the treatment of any medical condition. Such treatment may be prophylactic (i.e. preventative), curative (or treatment intended to be curative), or palliative (i.e. treatment designed merely to limit, relieve or improve the symptoms of a condition). A subject, as defined herein, refers to any mammal, e.g. a farm animal such as a cow, horse, sheep, pig or goat, a pet animal such as a rabbit, cat or dog, or a primate such as a monkey, chimpanzee, gorilla or human. Most preferably the subject is a human.
In particular, the invention provides an enhanced immune effector cell or
pharmaceutical composition as defined herein for use in adoptive cell transfer therapy. In another embodiment the invention provides a method of adoptive cell transfer therapy comprising administering to a subject in need thereof an enhanced immune effector cell or pharmaceutical composition of the invention. Adoptive cell transfer therapy is defined above. As detailed, in adoptive cell transfer immune effector cells are transfused into a patient in order to improve or redirect immune functionality in order to treat a disease or condition. In adoptive cell transfer therapy it is preferable that the immune effector cells are non- immunogenic to the subject to be treated, e.g. being autologous, HLA-matched or modified to reduce immunogenicity, as defined above.
The enhanced immune effector cells of the invention can be utilised in methods and compositions for adoptive cell transfer therapy in accordance with known techniques. In some embodiments, the cells are formulated by first harvesting them from their culture medium, and then washing and concentrating them in a medium and container system suitable for administration (a "pharmaceutically-acceptable" carrier) in a therapeutically- effective amount. Suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but also 5 % dextrose in water or Ringer's lactate can be utilised. The infusion medium can be
supplemented with human serum albumin.
A subject in need of adoptive cell transfer therapy with the enhanced immune effector cell or pharmaceutical composition of the invention can be identified by the skilled physician.
In particular, a subject suffering from cancer may be in need of adoptive cell transfer therapy according to the present invention. A subject suffering from an autoimmune disease may also benefit from adoptive cell transfer according to the present invention, particularly the transfer of regulatory T-cells. A subject according to the present invention is defined above.
In the methods of adoptive cell transfer as described herein, a therapeutically- effective amount of enhanced immune effector cells are transfused into the subject. By "therapeutically-effective amount" is meant an amount sufficient to show benefit to the condition of the subject. Whether an amount is sufficient to show benefit to the condition of the subject may be determined by the subject him/herself or a physician/veterinarian.
The cells and compositions of the invention may be administered to a subject by any suitable means. In particular, the cells and compositions may be administered to a subject parenterally, i.e. by parenteral administration, which as defined herein includes
subcutaneous, intramuscular, intravenous, intraperitoneal and intradermal administration. As mentioned above, the method of the invention provides a sterile cellular composition suitable for parenteral administration to a subject. Preferably the cells or compositions are
administered to a subject intravenously. The cells and compositions are formulated appropriately for the desired manner of administration.
In particular embodiments, a therapeutically-effective amount of enhanced immune effector cells is typically greater than 102 cells, preferably at least 106 cells, up to and including 108 or 109 cells and can be more than 1010 cells. The number of cells will depend upon the ultimate purpose for which the therapy is intended, as will the type of cells included therein. For uses provided herein, the cells are generally in a volume of a litre or less, 500 ml or less, even 250 ml or 100 ml or less. Hence the density of the desired cells is typically greater than 106 cells/ml and generally is greater than 107 cells/ml, generally 108 cells/ml or greater. The clinically-relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 105, 106, 107, 108, 109, 1010, 1011, or 1012 cells. For example, 2, 3, 4, 5, 6 or more separate infusions may be administered to a patient, at intervals of 24 or 48 hours, or every 3, 4, 5, 6 or 7 days. Infusions may also be spaced at weekly, fortnightly or monthly intervals, or intervals of 6 weeks or 2, 3, 4, 5, or 6 months. It is also possible that yearly infusions may be administered. If desired, the treatment may also include administration of mitogens (e.g., PHA) or lymphokines, cytokines, and/or
chemokines (e.g., IFN-γ, IL-2, IL-12, TNF-alpha, IL-18, and TNF-beta, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, ΜΓΡΤα, etc.) to enhance induction of the immune response.
Adoptive cell transfer therapy is not without risks, and severe side-effects to the treatment can occur. For instance, in one study, three out of nine cancer patients treated with autologous anti-MAGE-A3 TCR-engineered T-cells experienced severe neurological toxicity (which was lethal in two cases) due to cross-reactivity of the TCR (Morgan, R.A. et
a/. (2013), Journal of Immunotherapy, 36(2):133-151 ). A second study targeting MAGE-A3 in myeloma and melanoma patients with an HLA-A*01 restricted TCR demonstrated lethal cross-reactivity with myocardial damage (Linette, G.P. et al. (2013), Blood 122(6):863-871 ; Cameron, B.J. ei a/. (2013), Science Translational Medicine 5(197):197ra103). Thus the enhanced immune effector cells of the invention may be modified to allow targeted killing of the cells if a negative reaction is suffered by a patient following treatment.
For instance, if the enhanced immune effector cells of the invention are redirected with an exogenous antigen receptor, the receptor may be designed to express a targetable tag which is exposed at the cell surface. In the event that a negative reaction occurs to the adoptive cell therapy, targeted killing of the infused cells can be achieved using antibodies which recognise the introduced tag. Examples of suitable tags include a Myc-tag, a FLAG- tag, a polyhistidine-tag (His-tag), an HA-tag, a Strep-tag, an S-tag. Suitable tags are well- known in the art. Alternatively, the enhanced immune effector cells of the invention may be modified with a suicide gene, such as inducible caspase 9, which allows targeted killing of the cells by administration to the subject of the inducer.
In another embodiment, the invention provides a method of adoptive cell transfer therapy, said method comprising:
(i) preparing immune effector cells for transfer by contacting the immune effector cells in vitro or ex vivo with a non-biguanide mitochondrial electron transport chain inhibitor or BRD56491 , allowing said cells to proliferate and harvesting the cells; and
(ii) administering the harvested cells to a subject in need thereof.
In this method, the immune effector cells, mitochondrial ETC inhibitor, contacting, harvesting, administration and subject are as defined above. Allowing of the cells to proliferate may be achieved by expanding the immune effector cell as described above.
The invention also provides use of an enhanced immune effector cell as defined herein in the manufacture of a medicament for use in adoptive cell therapy.
The invention also provides an enhanced immune effector cell or pharmaceutical composition as defined herein for use in the treatment of cancer. The enhanced immune effector cell or pharmaceutical composition of the invention may in particular be used in adoptive cell transfer therapy for the treatment of cancer.
For use in the treatment of cancer, the enhanced immune effector cell of the invention is preferably redirected against a cancer antigen. In a particular embodiment, the enhanced immune effector cell is autologous to the subject to be treated for cancer, most preferably a human subject. On another embodiment, the enhanced immune effector cell is not autologous to the subject to be treated. In particular the enhanced immune effector cell may be derived from a donor. By "derived from a donor" is simply meant that the cell is derived from a different individual to the individual to whom it is to be administered.
The donor immune effector cell may be syngeneic to the subject to be treated. By "syngeneic" is meant genetically similar such that the cell is immunologically compatible, e.g. that the donor immune effector cell is MHC-matched to the subject. A syngeneic immune effector cell is derived from the same species as the subject to be treated.
The donor immune effector cell may be allogeneic to the subject to be treated. By
"allogeneic" is meant that the immune effector cell is genetically different to the subject to be treated, such that the cell is not immunologically compatible. An allogeneic immune effector cell is derived from the same species as the subject to be treated. The donor immune effector cell may be xenogeneic to the subject to be treated, i.e. it may be derived from a different species. If the donor immune effector cell is allogeneic or xenogeneic to the subject to the treated, it is preferable that the cell is modified to be non-immunogenic, as described above. Preferably the donor immune effector cell is from the same species as the subject to be treated.
The donor immune effector cell may be a primary immune effector cell, i.e. an immune effector cell which is isolated from the blood of a donor individual prior to
enhancement and optional modification. Alternatively, the donor immune effector cell may be from a cell line.
As mentioned above, the immune effector cell or pharmaceutical composition of the invention may be used in the treatment of cancer. Cancer is defined broadly herein to include any neoplastic condition, whether malignant, pre-malignant or non-malignant.
Generally, however, it may be a malignant condition. Both solid and non-solid tumours are included and the term "cancer cell" may be taken as synonymous with "tumour cell".
Any type of cancer is encompassed, including both solid and haematopoietic cancers. Representative cancers include Acute Lymphoblastic Leukaemia (ALL), Acute Myeloid Leukaemia (AML), Adrenocortical Carcinoma, AIDS-Related Cancer (e.g. Kaposi Sarcoma and Lymphoma), Anal Cancer, Appendix Cancer, Astrocytomas, Atypical
Teratoid/Rhabdoid Tumour, Basal Cell Carcinoma, Bile Duct Cancer, Extrahepatic Bladder Cancer, Bone Cancer (e.g. Ewing Sarcoma, Osteosarcoma and Malignant Fibrous
Histiocytoma), Brain Stem Glioma, Brain Cancer, Breast Cancer, Bronchial Tumours, Burkitt Lymphoma, Carcinoid Tumour, Cardiac (Heart) Tumours, Cancer of the Central Nervous System (including Atypical Teratoid/Rhabdoid Tumour, Embryonal Tumours, Germ Cell Tumour, Lymphoma), Cervical Cancer, Chordoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukaemia (CML), Chronic Myeloproliferative Disorder, Colon Cancer, Colorectal Cancer, Craniopharyngioma, Cutaneous T-Cell Lymphoma, Bile Duct Cancer, Extrahepatic Ductal Carcinoma In Situ (DCIS), Embryonal Tumours, Endometrial Cancer, Ependymoma, Esophageal Cancer, Esthesioneuroblastoma, Ewing Sarcoma, Extracranial Germ Cell Tumour, Extragonadal Germ Cell Tumour, Extrahepatic Bile Duct Cancer, Eye
Cancer (including Intraocular Melanoma and Retinoblastoma), Fibrous Histiocytoma of Bone, Gallbladder Cancer, Gastric (Stomach) Cancer, Gastrointestinal Carcinoid Tumour, Gastrointestinal Stromal Tumours (GIST), Germ Cell Tumor, Gestational Trophoblastic Disease, Glioma, Hairy Cell Leukaemia, Head and Neck Cancer, Heart Cancer,
Hepatocellular (Liver) Cancer, Histiocytosis, Langerhans Cell, Hodgkin Lymphoma,
Hypopharyngeal Cancer, Intraocular Melanoma, Islet Cell Tumours, Pancreatic
Neuroendocrine Tumours, Kaposi Sarcoma, Kidney Cancer (including Renal Cell and Wilms Tumour), Langerhans Cell Histiocytosis, Laryngeal Cancer, Leukaemia (including Acute Lymphoblastic (ALL), Acute Myeloid (AML), Chronic Lymphocytic (CLL), Chronic
Myelogenous (CML), Lip and Oral Cavity Cancer, Liver Cancer (Primary), Lobular
Carcinoma In Situ (LCIS), Lung Cancer, Lymphoma, Macroglobulinemia, Waldenstrom, Melanoma, Merkel Cell Carcinoma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Midline Tract Carcinoma Involving NUT Gene, Mouth Cancer, Multiple Endocrine Neoplasia Syndromes, Childhood, Multiple Myeloma/Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes, Myelodysplastic/Myeloproliferative
Neoplasms, Multiple Myeloma, Myeloproliferative Disorders, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Non- Small Cell Lung Cancer, Oral Cancer, Oral Cavity Cancer, Oropharyngeal Cancer,
Osteosarcoma, Ovarian Cancer, Pancreatic Cancer, Pancreatic Neuroendocrine Tumours (Islet Cell Tumors), Papillomatosis, Paraganglioma, Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, Pharyngeal Cancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Pleuropulmonary Blastoma, Pregnancy and Breast Cancer, Primary Central Nervous System (CNS) Lymphoma, Prostate Cancer, Rectal Cancer, Renal Cell (Kidney) Cancer, Renal Pelvis and Ureter, Transitional Cell Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer,
Sarcoma, Sezary Syndrome, Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Cell Carcinoma, Squamous Neck Cancer with Occult Primary, Metastatic, Stomach (Gastric) Cancer, T-Cell Lymphoma, Testicular Cancer, Throat Cancer, Thymoma and Thymic Carcinoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Urethral Cancer, Uterine Cancer, Endometrial, Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldenstrom Macroglobulinemia and Wilms Tumour.
As noted above, a number of cancers have been identified to express particular or specific cancer antigens, or to be characterised by expression of particular or specific cancer antigen. Thus an enhanced immune effector cell, modified to express an antigen receptor specific for an antigen associated with the target cancer, may be used in cancer treatment.
The enhanced immune effector cells of the invention may be used to treat cancer in combination with other therapies, e.g. surgery, radiotherapy, chemotherapy, immunotherapy,
hormone therapy, photodynamic therapy, etc. The enhanced immune effector cells of the invention may thus be administered to subjects in combination with one or more other therapeutic agents, e.g. antibiotics, cytokines (e.g. IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1 , IL-12, IL-13, IL-15 and IL-17), growth factors, steroids, NSAIDs, DMARDs, anti-inflammatories, analgesics, chemotherapeutics (e.g. monomethyl auristatin E, fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, oxaliplatin, mitomycin, dacarbazine, procarbazine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase and 5-fluorouracil), radiotherapeutics, immune checkpoint inhibitors (e.g. tremelimumab, ipilimumab, nivolumab, pembrolizumab (MK-3475), urelumab, bavituximab, atezolizumab (MPDL3280A) and durvalumab (MEDI4736)), small molecule inhibitors or other active and ancillary agents.
Although the treatment of cancer is one preferred embodiment of the present invention, the enhanced immune effector cells may be used in the treatment of any disease or condition that may benefit from adoptive cell transfer therapy, or more particularly from therapy targeting cells for killing or removal etc. As well as cancer cells, other cells that it may be clinically desirable to remove include infected cells, that is cells infected with any pathogen. Typically such cells will be virus-infected cells, but they may also be infected with any other pathogenic organism or disease-causing molecule, e.g. any microorganism, for example bacteria, fungi, mycoplasma and protozoa, or prions. Alternatively, the target cell may be apoptotic or pre-apoptotic, or be in a stressed state (i.e. express stress-related markers at their cell surface), or may be a mutant cell, e.g. expressing a particular mutation.
In representative embodiments of a target cell infected with a virus the virus may be any virus, but generally will be a pathogenic virus. By way of example, the virus may be HIV, a hepatitis virus (e.g. HBV or HCV), HPV, CMV or EBV, HHV-8, HTLV-1 , SV40 or an enterovirus. Other possible infective agents or pathogens include also bacteria, e.g.
Helicobacter pylori, Chlamydia pneumoniae, and parasites e.g. Schistosoma haematobium and the liver flukes, Opisthorchis viverrini, Clonorchis sinensis, and malaria. In still other embodiments, the target cell may be a cell involved in an unwanted immune response, e.g. in an autoimmune condition.
The present invention may be more fully understood from the non-limiting Examples below and in reference to the drawings, in which: Figures 1 and 2 demonstrate the killing of BL41 cells by NK-92 cells. "AntiA" and "AA" refer to antimycin A; "Rot" refers to rotenone; "myxo" refers to myxothiazol.
Figure 3 shows the half-times obtained for the NK-92 killing of BL41 cells shown in Figure 2.
Figure 4 demonstrates the killing of K562 cells by NK-92 cells. "AA" refers to antimycin A; "Rot" refers to rotenone.
Figure 5 shows the half-times obtained for the NK-92 killing of K562 cells shown in Figure 4.
Figure 6 demonstrates the killing of Granta cells loaded with the agonist peptide p621 by UK-92-TCR Rad-1 cells. "AA" refers to antimycin A; "Rot" refers to rotenone.
Figure 7 demonstrates the killing of Granta cells loaded with the antagonist Mart-1 peptide by UK-92-TCR Rad-1 cells. "AA" refers to antimycin A; "R" refers to rotenone. "0.5 AA" "indicates 0.5 μΜ antimycin A, etc. Figure 8 demonstrates the killing of Granta cells not loaded with a peptide by UK-92-TCR Rad-1 cells. "AA" refers to antimycin A; "R" refers to rotenone. "0.5 AA" "indicates 0.5 μΜ antimycin A, etc.
Figure 9 demonstrates the killing of Granta cells either loaded with the agonist peptide p621 or not loaded with a peptide by Rad-1 T-cells. "pep" indicates the p621 peptide; "no pep" indicates no peptide. "AA" refers to antimycin A; "R" refers to rotenone.
Figure 10 shows the results of a proliferation assay with NK-92 cells. Part A shows the total number of live NK-92 cells generated, and Part B shows the proportion of live cells out of all cells generated. "AA" refers to antimycin A; "R" refers to rotenone.
Figure 11 shows the spreading adhesion of NK-92 and UK-92-TCR Rad-1 cells to a synthetic surface coated with anti-CD3 antibody. "AA" refers to antimycin A; "Rot" refers to rotenone.
Figure 12 shows the relative levels of ROS in NK-92 and UK-92-Rad-1 NK cells with and without treatment with an ETC inhibitor. "AA" refers to antimycin A; "Rot" refers to rotenone. The effect on ROS levels of further treatment with an ROS scavenger (following treatment with an ETC inhibitor) is also shown. "MnTm" refers to MnTMPyP.
Figure 13 shows the effect on killing ability of further treating antimycin A-treated UK-92 cells with the ROS scavenger MnTMPyP. "AA" refers to antimycin A.
Figure 14 shows the effect on killing ability of further treating rotenone-treated UK-92 cells with the ROS scavenger MnTMPyP. "Rot" refers to rotenone. Figure 15 shows the effect on the expression of various surface markers by CD8+ T-cells (A) and CD4+ T-cells (B) of treatment with ETC inhibitors. "NT" indicates no treatment; "AA" refers to antimycin A; "R" refers to rotenone. The markers listed in the legend are referred to in the graphs in the same order (left to right for each T-cell culture) as in the legend (top to bottom).
Figure 16 shows on the expression of various surface markers by CD8+ T-cells (A) and CD4+ T-cells (B) of treatment with ETC inhibitors. "Mock" indicates T-cells were mock transfected; "Radium-1 " indicates T-cells were transfected to express the Radium-1 TCR. "NT" indicates no treatment; "AA" refers to antimycin A; "R" refers to rotenone. The markers listed in the legend are referred to in the graphs in the same order (left to right for each T-cell culture) as in the legend (top to bottom).
Figure 17 shows the effect on the ECAR of treating NK cells with ETC inhibitors. "AA" refers to antimycin A; "Rot" refers to rotenone. The "injection" line indicates the point at which the "injected" cultures were injected with ETC inhibitor.
Figure 18 shows the effect on the OCR of treating NK cells with ETC inhibitors. "AA" refers to antimycin A; "Rot" refers to rotenone. The "injection" line indicates the point at which the "injected" cultures were injected with ETC inhibitor.
Figure 19 demonstrates the effect of ETC inhibitor treatment on T-cell proliferation, as measured by confluence in the well of a 96-well plate. The values shown in the legend indicate the initial number of T-cells added to the well. Figure 20 shows a comparison of the killing capacity of untreated and ETC inhibitor-treated irradiated NK-92 cells with untreated and ETC inhibitor-treated non-irradiated NK-92 cells.
Figure 21 shows the results of CyTOF analysis of expression of a number of markers by untreated and ETC inhibitor-treated T-cells. The top panel shows the results of analysis of expression of, from left to right: CD45; CD20; ICOS; TIGIT; CD14; CD127; CD19; CD56;
PD-L1 ; and CD21 . The middle panel shows the results of analysis of expression of, from left to right: CD38; CCR6; CD45RA; CD1 1 b; CD4; CD25; CD279; CD137; CCR7; CD161 ;
NKG2D; and CD8a. The bottom panel shows the results of analysis of expression of, from left to right: CD33; CD3; PD-L2; HLA-DR; CTLA-4; IgD; LAG 3; CXCR3; CXCR5; CXCR4; CD28; and CD16. Figure 22 shows the results of CyTOF analysis of expression of a number of markers by untreated and ETC inhibitor-treated T-cells. The figure shows the results of analysis of expression of, from left to right: LAMP1 ; CD3; PD-1 ; IL-17A; TNFa; IL-5; IL-4; CD4; IFNv; CD19; CD8a; ΜΙΡ-1 β; and Granzyme B. Figure 23 shows the average tumour load of mice injected with human HCT1 16 colon cancer cells following the administration of untreated and ETC inhibitor-treated T-cells expressing a relevant or irrelevant TCR.
Figure 24 shows the survival times of the mice of Figure 23 which were injected with T-cells expressing a relevant TCR.
Figure 25 shows the percentage of CD3+ cells (i.e. tumour-infiltrating lymphocytes) in tumour tissue isolated from the mice of Figure 23. The columns from left to right correspond to the legend entries from top to bottom.
Figure 26 shows the effect on T-cell killing capacity of a number of ROS inducers, relative to the capacity of an untreated T-cell (which has the ration of "1 "). Thus ratios above 1 indicate enhanced killing capacity and ratios below 1 indicate reduced killing capacity. The columns from left to right correspond to the legend entries from top to bottom. AA = antimycin-A; Rot = rotenone; Myxo = myxothiazol; BRD = BRD56491 ; Cycl = cyclosporin A;
sinl = 3-morpholinosydnonimine; Pyo = pyocyanine; Piper = piperlongumine;
Gly = glybenclamide; Mem = Menadione; Am = amiodarone.
Examples
Example 1 - Effect of Mitochondrial ETC Inhibitors on Cell Killing
Materials and Methods
T-Cell Preparation
A mixed T-cell population was obtained from a healthy human donor. A frozen stock was prepared and transfected to express the Radium-1 TCR as follows:
• Stock was thawed and rinsed in complete CellGro® medium (CellGenix, Germany) without serum or IL2.
• Cells were incubated at 37°C in a controlled atmosphere with 5 % C02 for 2 hours.
• Cells were transfected with RNA encoding the Radium-1 TCR by electroporation, as follows:
> Electoporator was turned on to a setting of 500 V, 2 ms.
> 50 μΙ of RNA (concentration 1 μg/μl) was added to a volume of 550 μΙ of medium containing the cells (4 x 107 cells in total) in an Eppendorf, and mixed carefully. The mixture was transferred to a cuvette, avoiding the generation of bubbles as far as possible.
> The cells were pulsed, and immediately transferred to a T25 flask at a
concentration of 2 x 106 cells/ml in complete CellGro medium supplemented with 20 U/ml I L2.
NK Cell Preparation
• UK-92 cells (an NK-92 derivative which expresses CD3) were electroporated
following the same protocol as above with RNA encoding the Radium-1 TCR.
Target Cell Preparation
• Granta cells (modified to express the firefly Luciferase gene) were counted and
resuspended in RPMI medium without serum at 2 x 106 cells/ml with 10 μΜ peptide 621 (p621 , see WO 2017/194555).
Killer Cell Preparation
• NK cells (NK-92 and Radium-1 -transfected UK-92) and Radium-1 -transfected T-cells were pretreated for 16 hours with the following drugs at the indicated final concentrations:
> Antimycin A: 0.1 , 0.5, 1 , 2 and 5 μΜ. Antimycin A binds to the Qi site of
cytochrome c reductase, thereby inhibiting the reduction of ubiquinone in the Qi site, disrupting the Q-cycle and partially inhibiting respiration (ubiquinone is still reduced at Complex I).
> Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP): 100, 200 and 500 nM. FCCP is an uncoupling agent which disrupts ATP synthesis by transporting hydrogen ions through the inner mitochondrial membrane before they can be used to provide the energy for oxidative phosphorylation.
> Myxothiazol: 100 and 300 nM. Myxothiazol is an inhibitor of the mitochondrial cytochrome bci complex (coenzyme Q - cytochrome c reductase) at the Qo
site, preventing oxidation of ubiquinol and hence completely inhibiting respiration.
> Rotenone: 0.1 , 0.5, 1 , 2 and 5μΜ. Rotenone inhibits the transfer of electrons from iron-sulphur centres in Complex I to ubiquinone.
> 3-morpholinosydnonimine (SIN-1 ): 100 and 300 nM. SIN-1 acts by releasing
NO from the endothelial cells non-enzymatically and also generating the superoxide anion, possibly producing peroxynitrite.
Assay
Killer cells (NK-92, UK-92-TCR-Rad-1 and T-cell-TCR-Rad-1 ) were centrifuged at 500 x g for 5 min and resuspended in complete RPMI without red phenol twice. Target cells (BL41 , Granta and K562) were centrifuged at 500 x g for 5 min and resuspended in complete RPMI without red phenol twice.
30,000 BL41 , Granta and K562 cells in 100 μΙ were seeded in each well of a 96 well plate with 5 μg ml of luciferin.
300,000 NK-92, UK-92-TCR-Rad-1 and T-cell-TCR-Rad-1 cells treated with the different drugs in 100 μΙ were added to each well in hexaplicate.
In 6 wells, the killer cells were replaced with Triton™ X-100 in order to ensure complete lysis of the cells, as a positive control, (Max).
In 6 wells, the killer cells were replaced with medium in order to measure
spontaneous luminescence signal release, as a negative control, (Sp).
The plate was then placed in an incubator with controlled temperature and atmosphere.
Luminescence signal was measured regularly at different time points with a plate reader in order to monitor the killing of the target cells by the killer cells.
Killing percentage (K) was measured as follows: K = (— ) x 100.
Sf3
Results
Results were analysed using IGOR Pro 6.36 (WaveMetrics, US). Drug Identification
A first killing assay was conducted in order to test several metabolic drugs and study their effect on the killing efficiency of NK cells and T-cells. The killer cells (NK-92) were pretreated for 16 hours with the drugs described above.
After treatment killer cells were centrifuged and washed twice (1 :1 ,000,000 dilution of the drug). They were then incubated together with target cells (lymphoma BL41 cell line, stably transduced with luciferase construct) and the luminescence signal was followed over time. As can be seen in Figure 1 , three drugs demonstrated their potential to enhance killing ability of NK-92 cells (antimycin A, myxothiazol and rotenone) in comparison to untreated NK-92 cells. For the following experiments, myxothiazol was set aside due to its high toxicity to human cells, rendering it impractical for use in potential therapeutic applications.
Dose Scale
Another killing assay was conducted using the same killer and target cells (NK-92 and BL41 respectively) with rotenone and antimycin A. The NK-92 cells were incubated in the following concentrations of antimycin A or rotenone: 0.1 , 0.5, 1 , 2 and 5 μΜ. As can be seen in Figure 2, the results obtained were in accordance with those obtained previously. Data were then
LTTICIX
fitted using a sigmoid function (non-Hill equation) (equation: L(t) = L0 +
with standard deviation as weighting factor, base (L0) was held to 0 and max (Lmax) kept below 100. Parameters as well as fitting error are presented in Table 1. Half-time was plotted against drug concentration, showing that 2 μΜ seems to be the concentration with the best potential (Figure 3). The "half-time" as referred to herein is the time taken for the killer cells to kill half the target cells in a killing assay.
Drug Treatment Base Max t1/2 (h) Rate (h 1)
No Drugs 0 +/- 0 80.2 +/- 4.0 19.4 +/- 1.3 4.5 +/- 0.8
0.1 μΜ AA 0 +/- 0 90.3 +/- 2.3 17.3 +/- 1.5 2.8 +/- 0.7
0.5 μΜ AA 0 +/- 0 93.2 +/- 1.8 16.5 +/- 1.4 2.5 +/-0.5
1 μΜ AA 0 +/- 0 93.9 +/- 1.9 16.7 +/- 1.1 2.8 +/- 0.5
2 μΜ AA 0 +/- 0 95.1 +/- 1.6 15.6 +/- 1.0 2.8 +/- 0.3
5 μΜ AA 0 +/- 0 93.5 +/- 2.4 16.9 +/- 1.0 3.5 +/- 0.4
0.1 μΜ Rot 0 +/- 0 85.0 +/- 2.9 17.8 +/- 1.3 3.5 +/- 0.7
0.5 μΜ Rot 0 +/- 0 91.9 +/- 3.1 17.8 +/- 1.3 3.5 +/- 0.6
1 μΜ Rot 0 +/- 0 92.2 +/- 3.7 18.6 +/- 1.1 4.4 +/- 0.7
2 μΜ Rot 0 +/- 0 94.3 +/- 2.4 17.3 +/- 0.9 3.7 +/- 0.4
5 μΜ Rot 0 +/- 0 87.1 +/- 3.3 18.9 +/- 1.1 4.3 +/- 0.6
Table 1 : Fitting parameters obtained based on a sigmoid curve of data displayed in
Figure 2. AA = antimycin A; Rot = Rotenone.
Effect on Other Target Cells
A similar experiment to that described above was conducted against another target cell line: the myelogenous leukaemia cell line K562, in order to confirm that the enhancement in killing efficiency observed above is not dependent on the identity of the target cell. As can be seen in Figure 4, this cell line is killed very quickly by NK-92 cells independently of drug treatment. However, at the first time points, the cells treated with the drug performed better that those untreated. After sigmoid fitting, the same conclusions can be made as above (Table 2 and Figure 5), i.e. that drug treatment has an important effect, even when the killer cells are in the presence of highly agonistic target cells. Moreover, this experiment confirms that 2 μΜ is the concentration that seems to have the highest impact on killing efficiency.
Drug Treatment Base Max t1/2 (h) Rate (h1)
No Drugs 0 +/-0 99.4 +/- 2.6 4.6 +/- 0.2 1.1 +/-0.2
0.1 μΜ AA 0 +/-0 98.4 +/- 1.7 4.4 +/-0.1 1.1 +/-0.1
0.5 μΜ AA 0 +/-0 98.4 +/- 1.7 4.4 +/-0.1 1.1 +/-0.1
1 μΜ AA 0 +/-0 98.7 +/- 1.9 4.3 +/-0.1 1.4 +/-0.1
2 μΜ AA 0 +/-0 98.6 +/-1.8 3.8 +/-0.1 1.3 +/-0.1
5 μΜ AA 0 +/-0 99.2 +/- 1.6 5.3 +/-0.1 1.5+/- 0.1
0.1 μΜ Rot 0 +/-0 98.9 +/-2.1 5.8 +/-0.1 1.0 +/-0.1
0.5 μΜ Rot 0 +/-0 98.5 +/- 1.5 4.5+/- 0.1 1.1 +/-0.1
1 μΜ Rot 0 +/-0 98.5+/- 1.8 4.3 +/-0.1 1.2 +/-0.1
2 μΜ Rot 0 +/-0 98.6 +/- 1.5 3.6 +/-0.1 1.3 +/-0.1
5 μΜ Rot 0 +/-0 99.3 +/- 1.0 5.8 +/-0.1 1.2 +/-0.1
Table 2: Fitting parameters obtained based on a sigmoid curve of data displayed in
Figure 5. AA = antimycin A; Rot = Rotenone.
Effect on Other Killer Cells
In order to prove the versatility of the metabolic enhancers, additional killing assays were run using other killer cells. UK-92-TCR Rad1:
UK-92-TCR Rad1 are TCR-transduced NK-92 cells which have also been modified to express CD3, and have a behaviour comparable to that of T-cells. These cells were tested against the Granta cell line (B-cell lymphoma) loaded with an agonist peptide (p621), an antagonist peptide (Mart-126-35, see WO 2017/194555) or no peptide. The experiment was intended to determine if the drugs also have an effect on killing specificity. As can be seen in Figures 6, 7 and 8, UK-92-TCR Rad-1 cells killed the specific target (Granta cells loaded
with p621 ) better and the unspecific targets (Granta cells loaded with the Mart-1 peptide or no peptide) worse than did the untreated killer cells. This proves that the drug treatment enhances not only NK cell killing efficiency but also its specificity. T-Cells
The same experiment described for UK-92-TCR Rad-1 cells was performed for human primary T-cells. These T-cells were also modified to express the Radium-1 TCR, then put in the presence of Granta cells either loaded with the agonist peptide p621 or not loaded with a peptide. The results obtained (shown in Figure 9) demonstrate that the ability of the drugs to enhance killing is maintained for T-lymphocytes.
Example 2 - Effect of Mitochondrial ETC Inhibitors on Proliferation
NK-92 cells were incubated in X-VIVO™ 10 (Lonza, Switzerland) complete medium
(X-VIVO 10 supplemented with 10 % human serum and 500 U/ml IL-2) at 1 x 106 cells/ml. The cultures were supplemented with Antimycin A or rotenone at various concentrations (0.5, 2 or 5 μΜ) for 4 days. Cells were counted (total cells and live cells) every 24 hours using Trypan blue exclusion on an automatic cell counter. Briefly, 10 μ I of resuspended cell culture was diluted at 1 : 1 ratio with Trypan blue and allowed to rest for 30 s before being measured on a Countess™ II automated cell counter (Thermo Fisher Scientific).
As can be seen in Figure 10A, the drugs seemed to reduce the number of live cells during the first day but as soon as the cells were able to adapt their metabolism to the new conditions, they divided at a higher pace than the untreated cells. Even though the percentage of live cells is lower after drug treatment (Figure 10B), the total number of live cells indicates not only the innocuousness of the drug treatment but also its ability to enhance proliferation.
Example 3 - Effect of Mitochondrial ETC Inhibitors on Adhesion
An important aspect of killer cell-target cell interaction is the ability of the killer cell to adhere to its target, and also to recruit and reorganise its internal components in order to maximise its activation. NK-92 cells were pretreated with antimycin A and rotenone and allowed to adhere to a synthetic surface coated with anti-CD3 antibody. They were then marked using fluorescent phalloidin. As NK-92 cells lack the CD3 receptor, adherence parameters measured should be due solely to non-specific adhesion.
As can be seen in Figure 11 , spreading adhesion is enhanced for NK-92 cells when they are pretreated with antimycin A or rotenone, demonstrating the ability of these drugs to enhance cell-cell contact and by extension its stabilisation.
Example 4 - Effect of Mitochondrial ETC Inhibitors on ROS Production
Reactive Oxygen Species (ROS) have been shown to be a crucial intermediate in the transduction of the activation signal that goes through the TCR. An elevated level of ROS is thought to permit a quicker and better activation of T-lymphocytes. Using a set up similar to the one described in Example 3, we marked the cells using CellRox (a probe developed by Life Technologies, US) in order to assess the level of ROS inside the cells.
As can be seen in Figure 12, the level of ROS is 50 to 100 times higher in the cells that were treated with antimycin A and rotenone for both NK-92 and UK-92-TCR Rad-1 cells. Moreover, the difference observed between NK-92 cells treated with the drugs and UK-92- TCR Rad-1 is probably due to the activation mediated by the Anti-CD3 antibody coated on the surface, showing that the drugs are able to very effectively transduce and amplify the activation signal from the TCR. Upon treatment of the NK-92 and UK-92 cells with 40 μΜ MnTMPyP (manganese(lll) tetrakis(1 -methyl-4-pyridyl)porphyrin, a ROS scavenger), a decrease in ROS production was observed.
Example 5 - Effect of ROS Scavenger on Killing Assay
UK-92-TCR Rad-1 cells were pretreated with ROS scavenger (40μΜ MnTMPyP) for 30 minutes before adding 2 μΜ antimycin A or rotenone for 16 hours. Granta cells were loaded with peptide p621 as described previously. The cells were washed to remove the ETC inhibitors and the killing assay was performed as described previously. The killing assay was performed in the presence of 40 μΜ MnTMPyP.
As can be seen in Figures 13 and 14, MnTMPyP significantly reduces killing efficiency of UK-92 cells independently of the ETC inhibitor treatment. These results demonstrate that ROS production is required for efficient killing of target cells by lymphocytes. Impairing ROS production using a scavenger results in reduced target cell killing.
Example 6 - Phenotyping ETC Inhibitor-Treated T-Cells
In Vitro Expansion of Human T-Cells
T-cells from healthy donors were expanded using a protocol adapted for production of
T-cells employing Dynabeads CD3/CD28 essentially as previously described (Inderberg et at. (2017), Oncolmmunology 6(4): e1302631 ). In brief, PBMCs were isolated from buffy coats by density gradient centrifugation and cultured with Dynabeads (Dynabeads®
ClinExVivo™ CD3/CD28, Thermo Fisher Scientific) at a 3:1 ratio in complete CellGro DC Medium with 100 U/ml recombinant human interleukin-2 (IL-2) (Proleukin, Novartis
Healthcare) for 10 days. The cells were frozen and aliquots were thawed and rested in complete medium before transfection.
Electroporation of Expanded T-cells
Expanded T-cells were washed twice and resuspended in CellGro DC medium and resuspended to a concentration of 7 107 cells/ml. Radium-1 mRNA was mixed with the cell suspension at 100 μ g/m I , and electroporated in a 4-mm gap cuvette at 500 V and 2 ms using a BTX 830 Square Wave Electroporator (BTX Technologies Inc., Hawthorne, NY, USA). Immediately after transfection, T-cells were transferred to complete culture medium at 37°C in 5 % C02 overnight to allow TCR expression. Drug Treatment
Electroporated T-cells in complete culture medium were treated with antimycin A (AA) and rotenone (R) at 2 μΜ; control cells received no treatment (NT), overnight for 20 hours before being phenotyped by flow cytometry. Antibodies and Flow Cytometry
T-cells were washed in staining buffer (SB) consisting of phosphate buffered saline (PBS) containing 0.1 % human serum albumin (HSA) and 0.1 % sodium azide before staining for 20 min at RT. The cells were then washed in SB and fixed in SB containing 1 %
paraformaldehyde.
The following antibodies were used: CD3-BV605, CD4-BV421 , CD8-PE-Cy7, TIGIT-APC, PD-1 -PE, CD57-FITC, CD69-PE-Cy5.5 (BD Biosciences, USA) and HLA-DR-FITC (BD Biosciences, USA), CD137-PE (BD Biosciences, USA) . All antibodies were purchased from eBioscience, except where noted. Cells were acquired on a FACSCanto flow cytometer and the data analyzed using FlowJo software (Treestar Inc.).
The results are shown in Figures 15 and 16. As can be seen, only the activation marker HLA-DR decreases consistently after treatment with AA and R. All other markers are stable. As shown in Figure 16, there is no difference in the phenotype including activation markers of CD4+ or CD8+ T-cells (Figure 16A and 16B, respectively) treated with antimycin A and rotenone compared with non-treated cells (NT).
Example 7 - Effect of ETC Inhibitors on Respiration
A Seahorse Analyzer (Agilent) was used to assess the impact of antimycin A and rotenone on the metabolism of NK-cells. NK-92 or UK-92-TCR cells were incubated for 16 hours with 2 μΜ antimycin A or Rotenone then washed to remove the ETC inhibitor. As can be seen in
Figures 17 and 18, upon drug treatment, the glycolysis level (measured by the ECAR) is drastically increased whereas aerobic respiration (measured by OCR) is decreased.
By injecting the drugs during the experiment, an immediate decrease in aerobic respiration level and enhancement of glycolysis can be seen upon ETC inhibitor application. These results show that the ETC inhibitors are directly impacting cellular metabolism by forcing the cells to switch from aerobic respiration-based metabolism to glycolysis-based metabolism. An enhancement of glycolysis to the detriment of aerobic respiration could be advantageous in a cancer patient: the tumour environment is usually more favourable to cells performing glycolysis rather than aerobic respiration.
Example 8 - Effect of ETC Inhibitor Treatment on T-cell Growth Capacity
In order to confirm results obtained with NK-92 and UK-92-TCR cells using Trypan blue exclusion (see Example 2), T-cells were incubated in complete X-VIVO 15 medium at various starting concentrations in a 96 well plate, and the confluence monitored using an Incucyte S3 (Sartorius, Germany). As can be seen in Figure 19, antimycin A- and rotenone- treated T-cells outgrew the control (i.e. untreated) T-cells at all starting concentrations. The most striking differences were observed for 30,000 and 60,000 per well starting T-cell number. Example 9 - Duration of ETC Inhibitor Effect on OCR
In order to assess the duration of the effect of treatment with ETC inhibitors on the OCR of immune cells, UK-92-TCR cells were incubated with 2 μΜ rotenone or 2 μΜ antimycin-A for 24 hours, washed twice and then cultured in drug-free complete medium for various amounts of time (0, 24 or 72 hours). Control cells showed an OCR of around 300 pmol/min, a level greatly reduced in ETC inhibitor-treated cells. UK-92-TCR cells treated with antimycin-A returned to an OCR corresponding to the control level within about 72 hours following treatment. Roten one-treated cells did not obtain an OCR corresponding to the control level within the 72 hour duration of the experiment (data not shown). Example 10 - Effect of ETC Inhibitor Treatment on Irradiated NK-92 Cells
NK-92 is a lymphoblastic FDA-approved cell line. A prerequisite to its use in clinic is the irradiation of the cells to prevent uncontrolled proliferation in patients. This experiment was designed to study the impact of ETC inhibitor treatment on irradiated NK-92 cells. This experiment was performed in the same manner as that detailed in Example 1 , the results of which are presented in Figure 1 : briefly, NK-92 cells were incubated with 2 μΜ antimycin-A or 2 μΜ rotenone for 16 hours, washed twice, irradiated with 100 Gray and co-incubated with BL41 cells modified to express firefly luciferase. Target cell killing was monitored by
bioluminescence imaging. As can be seen in Figure 20, irradiation impaired NK-92 killing capacity, but ETC inhibitor-treated irradiated cells were still able to kill more efficiently than control cells. Example 11 - Effect of ETC Inhibitor Treatment on Lymphocyte Markers
To investigate further the results obtained using flow cytometry (Figures 15 and 16), a CyTOF (mass cytometry) experiment was performed. TCR-transfected T-cells (expressing the Radium-1 TCR) were incubated for 16 hours with 2 μΜ antimycin-A or 2 μΜ rotenone, washed and incubated for 22 hours with Granta-519 cells previously loaded with TGF3RII peptidei27-i45 (recognised by the Radium-1 TCR).
The next day, the cells were resuspended in Maxpar Cell Staining buffer (Fluidigm, USA) in the presence of 1X cisplatin (Fluidigm) and incubated for 5 min at room temperature (RT). Cells were washed with PBS and incubated with Fc-receptor blocking solution (an aggregation of γ-globulin) for 10 min and then incubated with extra-cellular target antibody mix (comprising antibodies which recognise CCR6, CCR7, CD127, CD137, CD14, CD1 1 b, CD161 , CD16, CD19, CD20, CD25, CD279 (PD1 ), CD28, CD3, CD33, CD38, CD21 , CD4, CD45RA, CD45RO, CD56, CD8, CTLA-4, CXCR3, CXCR4, CXCR5, HLA-DR, ICOS, LAG 3, NKG2D, PD-L1 , PD-L2, TIGIT and TIM3 (Fluidigm)) or intra-cellular target antibody mix (comprising antibodies which recognise LAMP-1 , CD3, PD-1 , IL-17A, TNFa, IL-5, IL-4, CD4, IFNg, CD8a, CD19, MlPl b and Granzyme B) for 30 min at RT. Samples were washed twice with PBS, fixed with 4 % PFA and permeabilised with methanol for 1 hour. After two washes in PBS, cells were resuspended in PBS containing 1X intercalator (Ir, Fluidigm) and incubated for 20 min at RT. Following one wash in PBS cells were pelleted until ready to run in CytOF.
Prior to sample acquisition cells were resuspended in water with 10 % calibration beads and filtered before being injected into the CyTOF 2 machine (Fluidigm). Analysis was performed using Cytobank Cytomass (http:// www.Cytobank.org). Typical gating strategy was applied as follows: 1 . EQ-140 vs. EQ-120 to exclude calibration beads. 2. Event length vs. lr-191 to gate singlets. 3. Cisplatin vs. CD458-89Y to gate live cells. 4. Event length vs. CD19 to exclude CD19+ cells (Granta-519). Gated events were then run using the viSNE algorithm with the following parameters: 1000 iterations, 30 perplexity, 0.5 theta. The results (Figure 21 (extra-cellular targets) and Figure 22 (intra-cellular targets)) show an absence of any statistical difference in the expression of a wide range of lymphocyte markers. These results confirmed those obtained by regular flow cytometry and permit the conclusion that treatment with antimycin-A or rotenone has no identifiable impact on the marker phenotype of T-cells.
Example 12 - Effect of ETC Inhibitor Treatment on Cytokine Secretion
TCR-transfected T-cells (expressing the Radium-1 TCR) were incubated for 16 hours with 2 μΜ antimycin-A or 2 μΜ rotenone, washed and incubated for 22 hours with Granta-519 cells previously loaded with TGF3RII peptidei27-i45 for 24 hours, at an effector cell-to-target cell ratio of 1 :2. For the control, unloaded Granta-519 cells were used. Culture supernatant was harvested and cytokine release measured using the Bioplex Pro™ Human Cytokine 17- plex Assay (Bio-Rad Laboratories Inc., Hercules, US) according to the manufacturer's instructions. The analysis was performed on the Bio-Plex 200 system instrument from Bio- Rad Laboratories. Each condition was performed in hexaplicate; measurements represent mean +/- standard deviation.
It was found that ETC inhibitor treatment does not affect the ability of T-cells to react to a specific target, in particular:
there is no statistical difference in basal cytokine secretion;
the increase in secreted cytokine mediated by the specific activation of T-cells is not modified (data not shown).
Example 13 - Effect of ETC Inhibitor Treatment on Killing Efficiency in vivo
The goal of this set of experiments was to confirm in vivo the boost to killing efficiency and specificity observed in vitro.
UK-92-TCR in vivo pilot experiment
The goal of this first experiment was to assess the potential toxicity of ETC inhibitor-treated lymphocytes in mice. NOD.Cg-Prkdcscid ll2rgtm1 Wjl /SzJ (NSG) mice were administered eight injections of 5x106 UK-92-TCR cells over a 22-day period. Prior to administration, the UK-92-TCR cells were incubated with 2μΜ antimycin-A or 2μΜ rotenone for 16 hours, then washed twice and finally resuspended in X-VIVO 10 medium. 10 mice were treated with untreated UK-92-TCR cells, 3 with antimycin-A-treated UK-92-TCR cells and 2 with roten one-treated UK-92-TCR cells. No mice exhibited any clinical signs indicating residual toxicity from the drugs.
Redirected T-cell in vivo experiment
A larger scale experiment using redirected T-cells instead of UK-92-TCR was then performed. NSG mice were intra-peritoneally injected with 1 x106 luciferase-expressing HCT1 16 cells (HCT1 16 is a human colon cancer cell line). Three injections of 1 x107 redirected T-cells transduced with a relevant or irrelevant TCR were administered to the peritoneum at days 3, 6 and 10 post-tumour cell injection. Prior to administration, the T-cells
were incubated with 2 μΜ antimycin-A or 2 μΜ rotenone for 16 hours, then washed twice and finally resuspended in RPMI-1640. Tumour size was monitored weekly using an in vivo imaging system (MS), and mice were sacrificed based on clinical grading system. 10 mice were treated with untreated T-cells (5 with relevant TCR, 5 with irrelevant TCR), 10 with antimycin-A-treated T-cells (5 with relevant TCR, 5 with irrelevant TCR), and 5 with roten one-treated T-cells (5 with relevant TCR, 5 with irrelevant TCR).
Once again, no mice developed any kind of clinical symptom that could have been imputed to the residual presence of an ETC inhibitor. This experiment thus permits the conclusion there is no toxicity from residual ETC inhibitor in treated T-cells.
Also, as can be seen in Figures 23 and 24, ETC inhibitor-treated T-cells transduced with a relevant TCR were able to outperform all other T-cells in terms of tumour load reduction and overall survival, demonstrating the boosting of T-cell killing capacity. It was not possible to distinguish any difference in tumour load or survival between mice administered treated and untreated T-cells transduced with an irrelevant TCR.
Example 14 - Effect of ETC Inhibitor Treatment on T-Cell Infiltration in Tumour Tissue
Tumours from the previous experiment were harvested once mice reached humane endpoints. Tumour tissues were then mashed, washed thrice and blocked with antiimmunoglobulins antibodies for 10 mins at room temperature. They were then incubated with fluorescent AF647 anti-CD3 for 30 min and passed through a flow cytometer. The gating strategy used was the following: SSC vs. FSC to exclude cell clusters, then GFP vs. AF647 to isolate AF647+ GFP- population (unlike T-cells, tumour tissues stably express GFP). Overall percentages of each population were then normalised against the control.
As can be seen in Figure 25, tumour tissue treated with T-cells transduced with a relevant TCR and incubated with antimycin-A or rotenone exhibited a higher proportion of tumour- infiltrating lymphocytes (TILs) compared with the control tissue. On the other hand, tumour tissue treated with T-cells transduced with an irrelevant TCR and incubated with antimycin-A or rotenone exhibited a lower proportion of TILs in comparison with the control tissue.
Tumour load and survival of mice did not permit the exhibition of any boost in terms of specificity of T-cell action, although analysis of the tumour tissue demonstrated that the proportion of TILs was in accordance with the hypothesis that ETC inhibitor-treated T-cells show a specific infiltration of the tumour tissues.
Example 15 - Effect of Other ROS Inducers on T-Cell Killing Capacity
Effect of ROS production mediated by antimycin-A and rotenone has been demonstrated in Figures 12, 13 and 14. Based on this, the role of ROS was hypothesised to be central to the boosting effects observed. More ROS inducers were then tested in a bioluminescence-
based killing assay (as described in Example 1 ) to test this hypothesis. The inducers were the following: Cyclosporin A, Glybenclamide, Amiodarone, BRD56491 , pyocyanine, Piperlongumine and Menadione. These drugs were tested both on NK-92 cells and redirected T-lymphocytes.
The results of this experiment (in combination with results of previous experiments using antimycin-A, rotenone and other ETC inhibitors) are presented in Figure 26. One can see that ROS-inducing drugs had variable effects on the killing capacity of the lymphocytes. Only 4 molecules showed potency: myxothiazol, rotenone, antimycin-A and BRD56491 (the immune cells in this assay contacted with BRD56491 were contacted with 2 μΜ BRD56491 ) This experiment suggests that ROS induction does not fully explain the observed boost to lymphocyte function.
Claims
1 . A method of preparing a human immune effector cell for use in adoptive cell transfer therapy, comprising:
i) contacting in vitro or ex vivo a human immune effector cell with a non-biguanide mitochondrial electron transport chain inhibitor or BRD56491 , wherein the function of said immune effector cell following said contacting is enhanced relative to an immune effector cell which has not been contacted with said inhibitor or BRD56491 ; and
ii) formulating the enhanced immune effector cell in a sterile composition suitable for parenteral administration to a subject.
2. The method of claim 1 wherein the function of said immune effector cell is enhanced relative to a control immune effector cell.
3. The method of claim 1 or claim 2, further comprising expanding said immune effector cell.
4. The method of any one of claims 1 to 3, comprising harvesting said immune effector cell.
5. The method of claim 4, further comprising washing said immune effector cell.
6. The method of any one of claims 1 to 5, wherein said function is proliferation, target cell killing and/or specificity of killing.
7. The method of any one of claims 1 to 6, wherein said immune effector cell is an NK cell.
8. The method of any one of claims 1 to 7, wherein said immune effector cell is a T-cell.
9. The method of claim 8, wherein the T-cell expresses CD4 and/or CD8 on its cell surface.
10. The method of claim 8, wherein the T-cell is a cytotoxic T-cell or a helper T-cell.
1 1 . The method of any one of claims 1 to 10, wherein said immune effector cell is redirected.
12 The method of claim 1 1 , wherein said immune effector cell is redirected against a cancer antigen.
13. The method of any one of claims 1 to 12 wherein in step (ii) the cell is harvested, washed, and transferred to a pharmaceutically-acceptable medium.
14. The method of claim 13, wherein the cell is expanded and/or subjected to a further treatment step and/or frozen before or after harvesting, or before or after washing, and before transfer to a pharmaceutically-acceptable medium.
15. The method of any one of claims 1 to 14, wherein in step (i) the cell is contacted with a mitochondrial electron transport chain inhibitor.
16. The method of claim 15, wherein said mitochondrial electron transport chain inhibitor is an inhibitor of Complex I or Complex III.
17. The method of claim 16, wherein said mitochondrial electron transport chain inhibitor causes an increase in ROS levels in said immune effector cell.
18. The method of claim 16 or 17, wherein said mitochondrial electron transport chain inhibitor is Antimycin-A, rotenone or myxothiazol.
19. The method of claim 18, wherein said mitochondrial electron transport chain inhibitor is Antimycin-A or rotenone.
20. The method of any one of claims 1 to 14, wherein in step (i) the cell is contacted with BRD56491 .
21 . The method of any one of claims 1 to 20, wherein the contacting step comprises incubating the cell with the mitochondrial electron transport chain inhibitor or BRD56491 for a period of at least 12 hours or at least 24 hours.
22. The method of any one of claims 1 to 21 , wherein the cell is contacted with the mitochondrial electron transport chain inhibitor or BRD56491 at a concentration of 0.1 to 10 μΜ.
23. The method of any one of claims 13-14 or 19-22, comprising the steps: a) contacting a human immune effector cell with rotenone, Antimycin A or BRD56491 at a concentration in the range of 0.1 to 10 μΜ for at least 12 hours;
b) harvesting and washing the cell; and
c) providing the cell in a sterile medium suitable for parenteral administration.
24. The method of claim 23, wherein said method comprises contacting a T-cell or NK cell with rotenone at a concentration in the range of 1 to 3 μΜ for 12 to 24 hours.
25. The method of claim 23, wherein said method comprises contacting a T-cell or NK cell with Antimycin-A at a concentration in the range of 1 to 3 μΜ for 12 to 24 hours.
26. The method of claim 23, wherein said method comprises contacting a T-cell or NK cell with BRD56491 at a concentration in the range of 1 to 3 μΜ for 12 to 24 hours.
27. An enhanced immune effector cell obtained or obtainable by the method of any one of claims 1 to 26, wherein the function of said enhanced immune effector cell is enhanced relative to an immune effector cell which has not been contacted with said inhibitor or BRD56491 .
28. The enhanced immune effector cell of claim 27, wherein said function is proliferation, target cell killing and/or specificity of killing.
29. The enhanced immune effector cell of claim 27 or 28, wherein the cell exhibits a level of ROS which is at least 2, 3, 5, or 10 times higher than the level in an immune effector cell which has not been contacted with said inhibitor.
30. A pharmaceutical composition comprising the enhanced immune effector cell of any one of claims 27 to 29 and one or more pharmaceutically-acceptable diluents, carriers or excipients.
31 . An enhanced immune effector cell as defined in any one of claims 27 to 29, or a pharmaceutical composition as defined in claim 30, for use in therapy.
32. The enhanced immune effector cell or pharmaceutical composition for use according to claim 31 , wherein said therapy is adoptive cell transfer therapy.
33. An enhanced immune effector cell as defined in any one of claims 27 to 29, or a pharmaceutical composition as defined in claim 28, for use in the treatment of cancer.
34. The enhanced immune effector cell or pharmaceutical composition for use according to claim 32 or 33, wherein said enhanced immune effector cell is autologous to the subject to be treated.
35. The enhanced immune effector cell or pharmaceutical composition for use according to claim 32 or 33, wherein the enhanced immune effector cell is a donor cell.
36. A method of adoptive cell transfer therapy comprising administering to a subject in need thereof an enhanced immune effector cell as defined in any one of claims 27 to 29 or a pharmaceutical composition as defined in claim 30.
37. A method of adoptive cell transfer therapy, said method comprising:
(i) preparing human immune effector cells for transfer by contacting the immune effector cells in vitro or ex vivo with a non-biguanide mitochondrial electron transport chain inhibitor or BRD56491 , allowing said cells to proliferate and harvesting the cells; and
(ii) administering the harvested cells to a subject in need thereof.
38. The method of claim 36 or 37, wherein said method is for treating cancer.
39. Use of an enhanced immune effector cell as defined in any one of claims 27 to 29 in the manufacture of a medicament for use in adoptive cell therapy, preferably in cancer therapy.
40. Use of a non-biguanide mitochondrial electron transport chain inhibitor or BRD56491 for enhancing the function of an in vitro or ex vivo human immune effector cell in preparation for adoptive cell transfer therapy.
41. The use according to claim 40, wherein said therapy is for cancer.
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US10704021B2 (en) | 2012-03-15 | 2020-07-07 | Flodesign Sonics, Inc. | Acoustic perfusion devices |
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