NZ740820B2 - Chimeric antigen receptors and uses thereof - Google Patents

Abstract

The present invention relates to chimeric antigen receptors (CARs) directed to cells expressing a dysfunctional or non-functional P2X purinoceptor 7 receptor. Further provided are methods of targeting neoplastic cells and tumours expressing a dysfunctional or non- functional P2X purinoceptor 7 receptor and methods of treating and preventing cancer is a subject. tor and methods of treating and preventing cancer is a subject.

Description

CHIMERIC ANTIGEN RECEPTORS AND USES F PRIORITY

Claims (1)

1. CLAIM This application claims priority from Australian provisional patent ation number 2015903719 filed on 11 September 2015, the contents of which are to be taken as incorporated herein by this referenceFIELD OF THE INVENTION The present invention relates to chimeric antigen receptors, T cells expressing chimeric antigen receptors and s of using chimeric antigen receptors for the prevention and/or treatment of cancerBACKGROUND OF THE INVENTION The immune system has highly d and specific mechanisms that protect us from a range of pathologiesAmongst these pathologies is the detection and elimination of unwanted pathogens such as bacterial infections, virally infected cells, and importantly, mutated cells that may cause malignant neoplasia (cancer)The ability for the immune system to prevent the formation and growth of cancers is dependent on the y of the cells of the immune system to distinguish between a ‘healthy’ cell and a ‘diseased’ (e.gneoplastic or pre-neoplastic) cellThis is achieved by recognition of cell markers (antigens) that are indicative of the transition in a cell from a healthy state to a diseased stateThere have been many attempts to develop immunotherapeutic approaches to treat cancer by manipulating or directing the immune system to target cells expressing cancer cell antigensImmunotherapeutic ches have largely centred on either exploiting the humoral immune system by utilising isolated or ered antibodies or, more recently, the cellular arm of the immune systemEarly ts to utilise cellular immunotherapy for the treatment of cancer utilised T lymphocytes isolated from tumours and expanded ex vivoWhilst this approach has provided some initial promise in early igations, there are many technical challenges associated with this approachThe ability to isolate and expand T cell tions to clinically relevant numbers is technically challenging and the poorly controlled nature of the ion s in a final T cell population that is distinctly heterogeneous, and may contain only a small number of cancer antigen-specific T cellsAs a result, the efficacy of this method is unpredictable and variableW0 20172’041143 In order to address some of the shortfalls related to the use of ex vivo expanded tumour-isolated T cells, chimeric antigen receptors (CARS or artificial T cell receptors) began to be developed in the late 19803Chimeric antigen receptors are created by linking an ellular region that is specific for a desired antigen to a signalling region, ing in an antigen-specific receptor that can induce T cell functionTransformation of isolated T cells with CARs results in a population of T cells that are specific for a given antigenAs a result, large populations of n-specific T cells can be generated and used for immunotherapyInitial clinical trials of CAR-transformed T cells specific for tumour ated antigens were promisingHowever, the efficacy of the CAR-transformed T cells led to significant hypercytokinemia, and ultimately death in some patientsThese adverse effects are largely believed to be induced by on-target, but off-tumour activity of the ansformed T cells induced as a result of endogenous expression of the cognate antigen for the CAR on healthy, non-cancerous, cell populationsIt is therefore apparent that there is a need for the pment of a CAR that targets a tumour-associated antigen which is selectively expressed by cancerous cells but not endogenously expressed on non-cancerous cellsThe sion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present inventionIt is not suggested or represented that any or all of these matters formed part of the prior art base or were common general dge in the field relevant to the present invention as it existed before the priority date of each claim of this applicationSUMMARY OF THE INVENTION The present invention is predicated in part on the recognition that due to the significant ‘on-target’ but ‘off-tumour’ ty of CAR-expressing immune cells, there is a need for the development of a CAR, and a genetically modified cell expressing the same, which targets a marker specifically associated with a range of neoplastic (cancerous) or pre- stic (pre-cancerous) cellsThe ors have ised that a dysfunctional P2X7 receptor is a suitable marker for targeting with a CARW0 2017;041143 _ 3 _ Accordingly, in a first aspect, the present invention provides a chimeric antigen receptor including an antigen-recognition domain and a signalling domain, wherein the antigen-recognition domain ises a dysfunctional P2X7 receptorIn some embodiments, the antigen-recognition domain ises an epitope associated with an ine triphosphate (ATP)-binding site of the dysfunctional P2X7 receptorIn some embodiments, the dysfunctional P2X7 receptor has a reduced capacity to bind ATP at the ATP-binding site compared to an ATP-binding capacity of a wild-type (functional) P2X7 orIn some embodiments the ctional P2X7 receptor cannot bind ATP at the ATP-binding siteIn some embodiments, the dysfunctional P2X7 receptor has a conformational change that renders the receptor dysfunctionalIn some embodiments, the conformational change is a change of an amino acid from the trans-conformation to the cis-conformationIn some embodiments, the amino acid that has changed from a trans-conformation to a cis- conformation is proline at amino acid position 210 of the ctional P2X7 receptorIn some embodiments, the antigen-recognition domain recognises an epitope that includes the proline at amino acid on 210 of the dysfunctional P2X7 receptorIn some embodiments, the antigen-recognition domain recognises an epitope that includes one or more amino acid residues spanning from e at amino acid position 200 to cysteine at amino acid position 216, inclusive, of the dysfunctional P2X7 receptorThe antigen-recognition domain of the CAR can be any suitable molecule that can interact with and specifically recognise a dysfunctional P2X7 receptorHowever, in some embodiments, the antigen-recognition domain includes amino acid sequence homology to the amino acid sequence of an antibody, or a fragment thereof, that binds to the ctional P2X7 receptorIn some ments, the antigen-recognition domain includes amino acid sequence homology to the amino acid sequence of a fragment-antigen binding (Fab) portion of an antibody that binds to a dysfunctional P2X7 receptorIn some embodiments, the antibody is a humanised antibodyIn some embodiments, the antigen-recognition domain includes amino acid sequence homology to the amino acid ce of a -chain variable fragment (scFv) or a multivalent scFv that binds to a dysfunctional P2X7 receptorIn some embodiments, the multivalent scFv is a di-valent or tri-valent scFvW0 2017;041143 _ 4 _ In some embodiments, the antigen-recognition domain includes amino acid sequence homology to a single-antibody domain (sdAb) that binds to a dysfunctional P2X7 receptorIn some ments, the antigen-recognition domain includes a binding peptide that es amino acid sequence homology to one or more CDR regions of an dy that binds to a dysfunctional P2X7 receptorIn some embodiments, the biding peptide includes amino acid sequence homology to the CDR1, 2 and 3 domains of the VH andior VL chain of an antibody that binds to a dysfunctional P2X7 receptorIn some embodiments, the antigen recognition domain includes one or more amino acid sequences which are at least 50%, 60%, 70%, 80%, 90% or 94% identical to any one of the regions spanning positions 30 to 35, 50 to 67 or 98 to 108 of the sequences set forth in SEQ ID NOS: 10, 32, 33 or 34In some ments, the n recognition domain includes one or more of the sequences spanning positions 30 to 35, 50 to 67 or 98 to 108 of the sequences set forth in SEQ ID NOS: 10, 32, 33 or 34In some embodiments, the antigen recognition domain includes one or more of the sequences set forth in SEQ ID NOS: 10, 32, 33 or 34In some embodiments, the signalling domain includes a portion derived from an tion orIn some embodiments, the activation receptor is a member of the CD3 co- receptor complex or is an Fc receptorIn some embodiments, the portion derived from the CD3 co-receptor complex is CD3-CIn some embodiments, the portion derived from the Fc receptor is chRl or chRIIn some embodiments, the signalling domain includes a portion derived from a co- stimulatory receptorIn some ments, the signalling domain includes a portion derived from an activation receptor and a portion derived from a co-stimulatory orIn some embodiments, the co-stimulatory receptor is selected from the group consisting of CD27, CD28, CD30, CD40, DAP10, 0X40, 4-1BB (CD137) and ICOSIn a second aspect, the present invention provides a nucleic acid molecule including a nucleotide sequence encoding the chimeric antigen receptor according to the first aspect of theinvenfionIn a third aspect, the present invention provides a nucleic acid construct that includes a nucleic acid molecule according to the second aspect of the inventionIn some embodiments, expression of the c acid le is under the control of a transcriptional l sequenceIn some embodiments, the riptional control sequence may be a constitutive promoter or an inducible promoterW0 20172’041143 In some embodiments of the third aspect of the ion, the nucleic acid construct further includes an internal ribosome entry site (IRES) that allows for translation initiation within the mRNA once expressed from the nucleic acid constructIn some embodiments of the third aspect of the invention, the nucleic acid construct is a vector such as a viral vector, which can be used to orm a T cell to induce expression of the CARIn a fourth aspect, the present invention provides a genetically modified cell that includes a CAR according to the first aspect of the inventionIn some embodiments, the cell includes two or more different CARsIn a fifth aspect, the t invention provides a genetically modified cell that es a nucleic acid molecule according to the second aspect of the invention, or a nucleic acid construct according to the third aspect of the invention, or a cally integrated form of the uctIn some embodiments, the nucleic acid molecule or the nucleic acid construct encodes two or more different CARsIn some embodiments of the fourth and fifth aspects of the invention, the two or more different CARs have different signalling domainsIn some embodiments of the fourth and fifth aspects of the invention, the cell es a first CAR with a signalling domain including a portion d from an activation receptor and a second CAR with a signalling domain including a portion derived from a co-stimulatory receptorIn some embodiments, the tion receptor is a member of the CD3 co-receptor complex or is an Fc receptorIn some embodiments, the co-stimulatory receptor is selected from the group ting of CD27, CD28, CD30, CD40, DAP10, 0X40, 4-1BB (CD137) and ICOSIn some embodiments of the fourth and fifth aspects of the invention, the cell is further modified so as to constitutively express mulatory receptorsIn some embodiments, the cell is further modified so as to express ligands for the co-stimulatory receptors, thereby facilitating auto-stimulation of the cellW0 2017;041143 _ 6 _ In some embodiments of the fourth and fifth s of the invention, the cell is further ed to secrete cytokinesIn some embodiments, the cytokines are selected from the group consisting of IL-2, IL- 7, IL-12, IL-15, IL-17 and IL-21, or a combination thereofIn some embodiments of the fourth and fifth aspects of the invention, the cell is a leukocyteIn some embodiments, the cell is a Peripheral Blood Mononuclear Cell (PBMC), a lymphocyte, a T cell (including a CD4+ T cell or a CD8+ T cell), a natural killer cell, or a l killer T cellIn a sixth aspect, the present invention provides a method of killing a cell sing a ctional P2X7 receptor, the method including exposing the cell expressing a dysfunctional P2X7 receptor to a genetically modified cell having a chimeric antigen receptor, wherein the chimeric n receptor is directed against a dysfunctional P2X7 receptorIn some ments of the sixth aspect of the invention, the CAR directly recognises the dysfunctional P2X7 receptor, or recognises the dysfunctional P2X7 receptor via an intermediateIn some embodiments, the ediate is a probe that binds to a dysfunctional P2X7 receptor and the CAR recognises the probeIn some ments, the probe is an antibody or an aptamerIn some embodiments, the probe includes a tag and the CAR recognises the tagIn a seventh aspect, the present invention provides a method of killing a cell expressing a dysfunctional P2X7, the method including exposing the cell expressing a dysfunctional P2X7 receptor to a genetically modified cell according to the fourth or fifth aspects of the inventionIn some embodiments of the sixth and seventh aspects of the invention, the cell expressing a dysfunctional P2X7 receptor is exposed to the genetically ed cell together with an exogenous cytokineIn some embodiments, the genetically modified cell is a genetically modified cell, autologous to the cell expressing a dysfunctional P2X7 receptorIn some ments of the sixth and h aspects of the invention, the cell expressing a ctional P2X7 or is a cancer cellIn some embodiments the cancer is selected from the group consisting of; brain cancer, oesophageal cancer, mouth cancer, tongue cancer, thyroid cancer, lung cancer, stomach cancer, pancreatic cancer, kidney cancer, colon cancer, rectal cancer, prostate cancer, bladder cancer, cervical cancer, epithelial cell cancers, skin cancer, leukaemia, lymphoma, myeloma, breast cancer, ovarian , endometrial W0 2017;041143 _ 7 _ cancer and testicular cancerIn some embodiments the cancer is selected from the group consisting of; lung cancer, oesophageal cancer, stomach cancer, colon cancer, prostate cancer, bladder cancer, cervical cancer, vaginal cancers, epithelial cell cancers, skin cancer, blood-related cancers, breast cancer, endometrial cancer, uterine cancer and testicular cancerIn some embodiments of the sixth and seventh aspects of the invention, the cancer is metastaticIn some embodiments, the cancer is stage III cancer or is stage IV cancerIn an eighth aspect, the present invention provides a method of ing in vitro the genetically modified cell according to the fourth or fifth aspects of the invention, the method including the step of exposing the cell to an antigen for the CARIn some embodiments, the method includes the further step of exposing the cell to a cytokineIn a ninth aspect, the present invention provides a method of expanding in vitro the cally modified cell according to the fourth or fifth aspects of the invention, the method including the step of exposing the cell to an antigen for the CAR and simultaneously exposing the cell to a cytokineIn some embodiments of the eighth and ninth aspects of the invention, the cytokine is a member of the IL-2 subfamily, the eron subfamily, the |L-10 subfamily, the IL-1 subfamily, the |L-17 subfamily or the TGF-B subfamilyIn some embodiments of the eighth and ninth aspects of the invention, the cytokine is selected from the group consisting of IFN-v, IL-2, IL-5, IL- 7, IL-8, IL-10, |L-12, |L-13, |L-15, |L-17, |L-18, TNF-o, TGF-B‘l, TGF-BZ, TGF-BB and GM-CSF, or a combination thereofIn a tenth aspect, the present invention provides a method of expanding in vitro the cally ed cell according to the fourth or fifth aspects of the invention, the method including ng the cell to immobilised D3 and anti-CD28 diesIn some embodiments of the tenth aspect of the invention, the antibodies are immobilised on a beaded substrate (for example on “Human Activator” DynabeadsT'V')In some embodiments of the tenth aspect of the invention, the antibodies are lised on a surface of a tissue culture vessel such as a e of a culture flask, plate or bioreactorIn an eleventh , the present invention provides a pharmaceutical ition including a genetically modified cell according to the fourth or fifth aspects of the invention W0 2017;041143 _ 8 _ and a pharmaceutically acceptable carrierIn some ments, the pharmaceutical composition includes suitable adjuvants which may consist of cytokinesIn some embodiments, the pharmaceutical composition may also e an intermediate as described hereinBRIEF PTION OF THE