Immune cells expressing a universal chimeric antigen receptor for targeting multiple diverse antigens, a method for producing said immune cells, and use of said immune cells for the treatment of cancer, infectious diseases and autoimmune diseases

Abstract

To provide immune cell-based anti-cancer therapeutic agents and methods of using the therapeutic agents in the treatment of cancer.SOLUTION: Provided is an isolated nucleic acid encoding a universal chimeric antigen receptor, the receptor comprising three domains in which the first domain is a tag-binding domain, the second domain is a linking peptide chain including an extracellular hinge and a transmembrane domain, and the third domain is a signal transduction domain, and the tag-binding domain binding to a tag derived from any human nuclear protein.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to immune cell-based therapeutics and methods of using same in the treatment of cancer, infectious diseases and autoimmune diseases. [Background technology]

[0002] 2. Background of the Invention Chimeric antigen receptors (CARs) are artificial receptors that consist of a binding moiety that confers antigen specificity and one or several signaling chains derived from immune receptors (Cartellieri et al., J. Biomed. Biotechnol. doi:10.1155 / 2010 / 956304 (2010)). These two main CAR domains are linked by a connecting peptide chain that includes a transmembrane domain that anchors the CAR in the cell plasma membrane. Immune cells, particularly T and NK lymphocytes, can be genetically modified to express CARs inserted into their plasma membrane. When such CAR-modified immune cells encounter other cells or tissue structures that express or bear the appropriate target of the CAR binding moiety, the CAR-modified immune cells are crosslinked to the target as soon as the CAR binding moiety binds to the target antigen. Crosslinking causes the induction of signaling pathways through the CAR signaling chains, which result in a change in the biological properties of the immune cells to which the CAR is attached. For example, CAR triggering in CD4+ and CD8+ effector T cells activates typical effector functions such as secretion of lytic compounds and cytokines that ultimately lead to the killing of the respective target cells. Adoptive transfer of immune cells engineered with chimeric antigen receptors (CARs) has been considered in recent years as a highly promising therapeutic option for treating infectious or autoimmune diseases, even if they are incurable and malignant. The first clinical trials have demonstrated both the safety and feasibility of this therapeutic strategy (Lamers et al. J.Clin.Oncol.24 e20-e22 (2006), Kershaw et al. Clin.Cancer Res.12 6106-6115 (2006)).In recent ongoing trials, the majority of patients with advanced tumors of B cell origin showed a complete or at least partial response to treatment lasting several months with autologous T cells carrying a CD19-specific CAR (Brentjens et al. Blood. 118 4817-4828 (2011), Sci. Transl. Med. 20(5) doi:10.1126 / scitranslmed.3005930 (2013); Kalos et al. 2011 Sci. Transl. Med. 3(95) doi:10.1126 / scitranslmed.3002842; Grupp et al. N. Engl. J. Med. 368:1509-1518 (2013)).

[0003] However, conventional CAR technology is accompanied by many significant problems that need to be resolved before this therapeutic approach can be widely applied for clinical treatment. First of all, several safety issues need to be addressed. Until now, the immune response of conventional CAR-engineered T cells has been difficult to control after infusion into patients. Unexpected target gene expression, especially in healthy tissues, can induce rapid and severe immune reactions of the engineered T cells against healthy cells, which can cause severe side effects (Lamers et al. J.Clin.Oncol.24 e20-e22(2006); Morgan et al. Mol.Ther.18:p.843-851(2010)). Another drawback of conventional CAR technology is the limitation of retargeting engineered T cells to a single antigen. Such a monotherapy approach carries the risk of the development of tumor escape mutants that have lost the target antigen during treatment. The occurrence of tumor escape mutants after several months of conventional CAR T cell therapy has already been observed in clinical trials (Grupp et al. N. Engl. J. Med. 368:1509-1518 (2013)).

[0004] International Publication No. WO2012082841A2 discloses T cells expressing a universal anti-tag chimeric antigen receptor and methods for treating cell-related diseases, such as cancer.

[0005] Furthermore, International Publication No. WO2013044225A1 discloses a universal immune receptor expressed by T cells for targeting diverse multiple antigens.

[0006] Both of these methods describe the use of engineered T cells expressing a universal anti-tag immune receptor. These T cells can be redirected to disease-associated cell surface antigens by binding the surface antigen and applying additional modules with the respective tags. A problem arising from the methods is that redirection of genetically engineered T cells using exogenous tags may possibly be immunogenic, which may endanger the patient and negatively affect the efficacy of the treatment. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO2012082841A2 [Patent Document 2] WO2013044225A1 [Non-patent literature]

[0008] [Non-Patent Document 1] Cartellieri et al., J. Biomed.Biotechnol.doi:10.1155 / 2010 / 956304(2010) [Non-Patent Document 2] Lamers et al. J.Clin.Oncol.24 e20-e22(2006) [Non-Patent Document 3] Kershaw et al. Clin.Cancer Res.12 6106-6115(2006) [Non-Patent Document 4] Brentjens et al. Blood.118 4817-4828(2011) [Non-Patent Document 5] Sci.Transl.Med.20(5) doi:10.1126 / scitranslmed.3005930(2013) [Non-Patent Document 6] Kalos et al. 2011 Sci.Transl.Med.3(95) doi:10.1126 / scitranslmed.3002842 [Non-Patent Document 7] Grupp et al. N. Engl. J. Med. 368:1509-1518 (2013) [Non-Patent Document 8] Lamers et al. J.Clin.Oncol.24 e20-e22(2006) [Non-Patent Document 9] Morgan et al. Mol.Ther.18:p.843-851 (2010) [Non-Patent Document 10] Grupp et al. N. Engl. J. Med. 368:1509-1518 (2013) Summary of the Invention [Problem to be solved by the invention]

[0009] It is therefore an object of the present invention to provide genetically modified immune cells that allow safe and effective redirection against a variety of diseases using exogenous nuclear protein-based tags. It is a further object of the present invention to provide a method for treating a variety of cell-related diseases, where the length and intensity of treatment can be easily adjusted. [Means for solving the problem]

[0010] Summary of the Invention The present invention provides a universal, modular anti-tag chimeric antigen receptor (UniCAR) system that allows retargeting of UniCAR-conjugated immune cells to multiple antigens. The system uses a gene therapy platform to generate immune cells capable of recognizing a variety of antigens, which has broad and beneficial clinical impact for immune cell-based therapies, especially T cell- and NK cell-based therapies.

[0011] In a first aspect, the present invention provides an isolated nucleic acid sequence encoding a universal chimeric antigen receptor, the receptor comprising three domains, the first domain being a tag-binding domain, the second domain being a connecting peptide chain comprising an extracellular hinge and a transmembrane domain, and the third domain being a signaling domain, wherein the tag-binding domain binds to a tag derived from any human nuclear protein. Particularly suitable tags are peptide sequences derived from nuclear antigens, which cannot be accessed and bound by the corresponding tag-binding domain in the context of the native protein under physiological conditions. Furthermore, the peptide sequence should not be a target of autoantibodies in autoimmune patients, thus making the tag less likely to be immunogenic in the context of the universal chimeric receptor. The optional fourth domain is a short peptide linker in the extracellular portion of UniCAR, which constitutes a linear epitope for a monoclonal antibody (mab) that specifically binds to the fourth domain. This additional domain is not required for the functionality of the UniCAR system, but may provide an additional clinical benefit to the invention.Preferably, the present invention provides an isolated nucleic acid sequence encoding a universal chimeric antigen receptor according to the present invention, wherein said nucleic acid sequence encodes an artificial chimeric fusion protein, and wherein said nucleic acid sequence is provided as a cDNA.

[0012] In a further aspect, the present invention provides a targeting module consisting of a binding moiety specific for a particular human cell surface protein or protein complex and a tag, wherein the tag is derived from any human nuclear protein.

[0013] In a further aspect, the present invention provides a nucleic acid encoding a targeting module according to the invention. Preferably, the present invention provides an isolated nucleic acid sequence encoding a targeting module according to the invention, wherein the isolated nucleic acid is provided as a cDNA.

[0014] In a further aspect, the present invention provides a cell comprising a nucleic acid encoding a universal chimeric antigen receptor according to the present invention comprising three domains, wherein one domain is a tag binding domain, a second domain is a connecting peptide chain comprising an extracellular hinge and a transmembrane domain, and a third domain is a signaling domain, wherein said tag binding domain binds to a tag derived from any human nuclear protein.

[0015] In a further aspect, the present invention provides a vector comprising a nucleic acid encoding a universal chimeric antigen receptor according to the present invention, the universal chimeric antigen receptor comprising three domains, one domain being a tag-binding domain, a second domain being a connecting peptide chain comprising an extracellular hinge and a transmembrane domain, and a third domain being a signaling domain, wherein the tag-binding domain binds to a tag derived from any human nuclear protein.

[0016] In a further aspect, the present invention provides a kit comprising a universal chimeric antigen receptor according to the invention and a vector according to the invention comprising a nucleic acid sequence encoding a targeting module according to the invention and / or a vector encoding an isolated nucleic acid sequence encoding a targeting module according to the invention.

[0017] The invention further includes a pharmaceutical composition comprising a cell and targeting module according to the invention together with a pharma- ceutical acceptable diluent or carrier, preferably in a form suitable for intravenous administration.

[0018] Preferably, the composition comprises a cell comprising a nucleic acid encoding a universal chimeric antigen receptor according to the invention and a targeting module according to the invention.

[0019] The pharmaceutical composition according to the invention includes various dosage forms. The pharmaceutical composition is preferably administered parenterally, particularly preferably intravenously. In one embodiment of the present invention, the parenteral pharmaceutical composition is present in a dosage form suitable for injection. Thus, particularly preferred compositions are solutions, emulsions or suspensions of cells and target modules present in a pharma-ceutical acceptable diluent or carrier.

[0020] As carriers, preferably water, buffered water, 0.4% saline, 0.3% glycine and similar solvents are used. The solution is a sterile solution. The pharmaceutical composition is sterilized by conventional well-known techniques. The composition preferably contains pharma- ceutical acceptable excipients, such as excipients required to provide approximately physiological conditions and / or to enhance the stability of the targeting module, such as agents for adjusting the pH value and buffering agents, agents for adjusting toxicity, etc., preferably selected from sodium acetate, sodium chloride, potassium chloride, calcium chloride and sodium lactate. Depending on the application, the concentration of the targeting module according to the invention in these formulations varies. The concentration is preferably less than 0.01% by weight, preferably at least 0.1% by weight, more preferably between 1% and 5% by weight, and these concentrations are selected mainly based on the fluid volume, viscosity, etc., or depending on the respective mode of administration.

[0021] The pharmaceutical compositions must be sterile and stable under the conditions of manufacture and storage.The compositions can be formulated as solutions, microemulsions, dispersions, in liposomes, or other ordered structures known by artesians suitable for this purpose.

[0022] The cells and targeting modules according to the invention are preferably introduced into a composition suitable for parenteral administration. Preferably, the pharmaceutical composition is an injectable buffer solution containing between 0.001 mg / ml and 500 mg / ml of antibody, particularly preferably between 0.001 mg / ml and 250 mg / ml of targeting module, in particular together with a buffering agent of 1 millimol / l (mM) to 500 millimol / l (mM). The injectable solution may be in liquid form. The buffering agent may be histidine (preferably 1 mM to 50 mM, particularly preferably 5 mM to 10 mM), preferably at a pH value of 5.0 to 7.0 (particularly preferably at a pH of 6.0).

[0023] Other suitable buffers include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate. Preferably, 0 mM to 300 mM, particularly preferably 150 mM, of sodium chloride is used for liquid dosage forms. In liquid dosage forms, a stabilizer is preferably used, particularly preferably between 1 mM and 50 mM L-methionine (preferably between 5 mM and 10 mM).

[0024] 1m per day 2 Typical dosage rates delivered per day are between 1 μg and 1000 mg, preferably between 10 μg and 1 mg, with dosages being administered one or more times per day or week, or continuously for a period of several weeks.

[0025] In a further aspect, the present invention provides the use of a cell according to the present invention comprising a nucleic acid sequence encoding a universal chimeric antigen receptor according to the present invention and a targeting module according to the present invention for stimulating a universal chimeric antigen receptor-mediated immune response in a mammal. Preferably, the present invention provides the use of a cell according to the present invention comprising a nucleic acid sequence encoding a universal chimeric antigen receptor according to the present invention and a targeting module according to the present invention as a medicament, more preferably as a medicament for the treatment of cancer or autoimmune diseases. Autoimmune diseases result from an abnormal immune response of the body against substances and tissues normally present in the body (autoimmunity).

[0026] The present invention further comprises the use of the cells and targeting modules according to the invention for the manufacture of a drug for therapeutic and / or prophylactic use in the case of cancer or autoimmune diseases.

[0027] The invention also includes a method of treating a human with cancer or an autoimmune disease by administering cells and a targeting module according to the invention.

[0028] For therapeutic use, a sterile pharmaceutical composition containing a pharmacologically effective amount of the cells and targeting module according to the invention is administered to a patient for the treatment of the above mentioned diseases.

[0029] The present invention will be described in more detail with reference to the following drawings and embodiments, but the present invention is not limited thereto. [Brief description of the drawings]

[0030] [Figure 1] 1 shows a schematic diagram of the Universal Chimeric Antigen Receptor (UniCAR). [Diagram 2] FIG. 1 shows a schematic diagram of the Universal Chimeric Antigen Receptor (UniCAR) platform for retargeting antigen-specific immune cells. [Diagram 3] 1 shows a schematic map of the lentiviral vector pLVX-EF1α-IRES-ZsGreen1. [Figure 4] 1 shows a schematic map of the lentiviral packaging plasmid psPAX2. [Diagram 5] 1 shows a schematic map of the envelope plasmid pMD2.G. [Figure 6] Graph showing UniCAR surface expression detected by using a monoclonal antibody against any of the fourth domains. [Figure 7] 1 shows a graph of effector function of UniCAR engineered T cells against tumor cells expressing prostate stem cell antigen and prostate membrane antigen. [Figure 8] 1 shows graphs of concentration-response curves for various target modules. [Figure 9] 1 shows a graph of effector function of UniCAR engineered T cells against acute myeloid leukemia. [Figure 10] 1 shows a graph depicting redirection of UniCAR-conjugated T cells against two antigens simultaneously. [Figure 11] 1 shows a graph depicting the in vivo pharmacokinetics of the bispecific αCD123-CD33 targeting module. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Detailed Description of the Invention Effector cells The effector cells used in the method of the present invention can be autologous, syngeneic or allogeneic, and its selection depends on the disease to be treated and the means available for its treatment.Suitable populations of effector cells that can be used in the method include any immune cell with cytolytic activity, phagocytic activity or immunosuppressive activity, such as T cells, such as regulatory T cells, NK cells and macrophages.In one embodiment, the effector cells are derived from a certain HLA background and used in an autologous or allogeneic system.Effector cells can be isolated from any source, such as from tumor explants of the subject to be treated or from intratumoral cells of the subject to be treated.Hereinafter, the term "effector cells" refers to any kind of immune cells as mentioned above, which are genetically modified to express UniCAR on their cell surface.

[0032] Universal Chimeric Antigen Receptor (UniCAR) The UniCAR expressed by the effector cell used in the method of the present invention allows the modular, highly flexible and tightly controllable retargeting of the immune cell expressing UniCAR in an antigen-specific manner.All that is required for the UniCAR used in the method is that (i) the UniCAR has binding specificity for a specific tag that can be bound to a target module, and the target module also binds to a cell surface protein or extracellular structure, and (ii) the immune cell can be engineered to express UniCAR.

[0033] The UniCAR comprises three domains (Figure 1). The first domain is a tag-binding domain. This domain is generally present at the amino-terminal end of the polypeptide comprising the UniCAR. The placement of the tag-binding domain at the amino-terminus allows it unrestricted access to the tagged target module that is bound to the target cell. The tag-binding domain is generally, but not limited to, an antibody or an antigen-binding fragment thereof. The identity of the antibody or fragment is limited only by the identity of the tag of the tagged target module. The tag can be derived from any human nuclear protein against which antibodies or other binding domains can be obtained. The antibody can be obtained from any species of animal, but preferably from a mammal, such as a human, monkey, mouse, rat, rabbit, guinea pig, horse, cow, sheep, goat, pig, dog or cat. Preferably, the antibody is a human antibody or a humanized antibody. There is no limitation on the specific class of antibodies that can be used, including, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE antibodies. Antibody fragments include single chain variable fragments (scFv), single chain antibodies, F(ab')2 fragments, Fab fragments and fragments produced by a Fab expression library, the only limitation being that the antibody fragment maintains the ability to bind the selected tag. The antibody may be a polyclonal, monoclonal or chimeric antibody, for example an antibody in which the antigen-binding region (e.g., F(ab')2 or hypervariable region) of a non-human antibody has been transferred into a human antibody backbone by recombinant DNA techniques to produce an essentially human molecule. Antigen-binding fragments, such as scFvs, can be produced from said antibodies. Antibodies against a selected tag can be produced by immunizing a variety of hosts, including but not limited to goats, rabbits, rats, mice, and humans, by injection with the particular protein or any portion, fragment, or oligopeptide that retains the immunogenic properties of the protein.

[0034] Depending on the host species, various adjuvants can be used to enhance the immunological response. Such adjuvants include, but are not limited to, detoxified non-heat-stable toxin from E. coli, Freund's adjuvant, inorganic gels such as aluminum hydroxide and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol. BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum are also potentially useful adjuvants. Antibodies and fragments thereof can be produced using any technique that provides for the production of antibody molecules, for example, by continuous cell lines in culture for monoclonal antibody production. Such techniques include, but are not limited to, the hybridoma technique first described by Koehler and Milstein (Nature 256:495-497 (1975)), the human B cell hybridoma technique (Kosbor et al., Immunol Today 4:72 (1983); Cote et al., Proc Natl. Acad. Sci 80:2026-2030 (1983)), and the EBV hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss Inc, New York NY, pp77-96 (1985)). Techniques developed for the production of "chimeric antibodies" can also be used, i.e., splicing mouse antibody genes into human antibody genes to obtain molecules with appropriate antigen specificity and biological activity (Morrison et al., Proc Natl. Acad. Sci 81:6851-6855 (1984); Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)). Alternatively, techniques described for the production of single chain antibodies can be adapted to produce tag-specific single chain antibodies.

[0035] In one embodiment, the tag-binding domain is a single chain variable fragment (scFv). scFv is the variable region of an antibody heavy chain (VH) and light chain (VL), which are generally linked via a short peptide of 10 to about 25 amino acids. The linker may link the N-terminus of VH to the C-terminus of VL, or vice versa.

[0036] As noted above, the binding specificity of the tag binding domain will depend on the identity of the tag attached to the protein used to bind the target structure.

[0037] In a preferred embodiment, the tag is a short linear epitope (E5B9) derived from human nuclear La protein, and the tag-binding domain may constitute an antibody or an antigen-binding fragment derived from an antibody, such as a single-chain variable fragment (scFv) that binds to the respective La epitope (5B9). The use of the 5B9 anti-La epitope in the UniCAR system is advantageous because the anti-La epitope scFv does not interact with the native La protein that is bound under normal physiological conditions to the cell surface. In this way, there is no undesirable interaction between immune cells expressing UniCAR, such as T cells or NK cells, and the 5B9 epitope on the native La. This minimizes the risk of uncontrollable on-target off-site toxicity by immune cells expressing UniCAR, such as the release of toxic levels of cytokines, variously referred to as cytokine storm or cytokine release syndrome (CRS). Reactivity of the respective monoclonal antibodies with denatured La protein in Western blots was confirmed, but not with native La protein in immunoprecipitation experiments (Figure 3). Furthermore, although patients with Sjögren's syndrome and systemic lupus erythematosus make autoantibodies to various La epitopes, no autoantibodies have been identified against E5B9, suggesting that this epitope is not immunogenic (Yiannaki et al., Clin Exp Immunol., 112(1):152-8 (1998)).

[0038] The second domain of UniCAR is the extracellular hinge and transmembrane (TM) domain. The hinge domain allows UniCAR to protrude from the surface of effector cells for optimal binding to its specific tag. The transmembrane (TM) domain anchors UniCAR in the cell membrane of effector cells. Exemplary hinge and transmembrane (TM) domains include, but are not limited to, the hinge and transmembrane regions of human CD28 molecule, CD8a chain, NK cell receptor, such as natural killer group 2D (NKG2D), or the constant region of antibody, as well as various combinations of hinge and transmembrane (TM) domains.

[0039] The third domain, if present, is a signal transduction domain. This domain transmits a cellular signal into the effector cell bearing the UniCAR when the effector cell is crosslinked to a cell or extracellular structure. The crosslinking between the effector and the target cell (i) depends on and is dependent on the presence of a target module that binds to its specific binding site on the target cell or target extracellular structure and has a tag, and (ii) the UniCAR expressed on the surface of the effector can recognize and bind to the tag contained in the target module. The activation of the effector cell includes the induction of cytokines or chemokines and the activation of the cytolytic, phagocytic or suppressive activity of the effector cell. Exemplary effector cell signaling domains include, but are not limited to, the cytoplasmic regions of CD28, CD137 (4-1BB), CD134 (OX40), DAP10 and CD27, which act to enhance T cell survival and proliferation; inhibitory receptors such as programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte antigen 4 (CTLA-4), and the cytoplasmic regions of CD3 chain (e.g., CD3 zeta), DAP12 and Fc receptor, which induce T cell and NK cell activation. One or more signaling domains may be included in a UniCAR, including two, three, four or more immune cell activation or costimulatory domains.

[0040] In a further embodiment, the UniCAR comprises a fourth domain, which is a short peptide linker outside the UniCAR (Figure 1). This domain is required for its functionality, and the fourth domain forms a linear epitope that allows the binding of a specific monoclonal antibody with a reasonable affinity. One or more linear epitopes may be included in the fourth domain, and the epitope may be located as a linker in the tag-binding domain, between the tag-binding domain and the extracellular linker, or in an integral part of the extracellular hinge domain. With the help of the optional fourth domain, the UniCAR-conjugated immune cells may be specifically stimulated, so that the UniCAR-conjugated immune cells preferentially proliferate and persist longer in vitro and in vivo compared to non-conjugated immune cells. The fourth domain can also be used to purify the UniCAR-conjugated immune cells from a mixed cell population. The domain can also be used to suppress immune responses mediated by UniCAR-conjugated immune cells and to eliminate UniCAR-conjugated immune cells in vivo.

[0041] To allow expression on the cell surface of effector cells, a signal peptide (sometimes also called a signal sequence, targeting signal or leader peptide) is placed upstream of the tag-binding domain at the N-terminus of the UniCAR nucleic acid sequence. The signal peptide targets the protein to the secretory pathway, either co-translationally or post-translationally. For this purpose, signal peptides from various species of proteins can be utilized, but advantageously leader peptides from CD28, CD8 alpha, IL-2 or the heavy or light chains of antibodies of human origin are used to avoid immunogenic reactions.

[0042] Target Module The targeting module is composed of a binding moiety and a tag specific for a particular human cell surface protein or protein complex. The targeting module is administered to the subject before, simultaneously with, or after administration of the effector cells expressing UniCAR. Alternatively, the effector cells expressing UniCAR may be loaded with the targeting module before being injected into the recipient. The binding site of the targeting module may include, but is not limited to, surface antigens such as CD2, CD3, CD4, CD8, CD10, CD19, CD20, CD22, CD23, CD33, CD38, CD44, CD52, CD99, CD123, CD274 and TIM-3, members of the epidermal growth factor receptor family (erb1, erb2, erb3, erb4 and their mutants), members of the ephrin receptor family (EphA1-10, EphB1-6), so-called prostate specific antigens (e.g. prostate stem cell antigen PSCA, prostate specific membrane antigen PSMA), fetal antigens (e.g. carcinoembryonic antigen CEA, fetal acetylcholine receptor), members of the vascular endothelial growth factor family (VEGFR1-3). Antibodies or fragments thereof that bind to antigens including members of the epithelial cell adhesion molecule EpCAM, alpha fetoprotein AFP, members of the mucin protein family (e.g., MUC1, MUC16), follicle stimulating hormone receptor (FSHR), human high molecular weight melanoma associated antigen (HMW-MAA), folate binding protein FBP, folate receptor alpha, ligands for the NKG2D receptor, members of the epithelial glycoprotein family (e.g., EGP-2, EGP-4), disialogangliosides (e.g., GD2, GD3), members of the carbonic anhydrase family (e.g., CAIX), and members of the carbohydrate antigen family (Ley), as well as mutants of the listed proteins and protein families. Additionally, the binding portion of the targeting module includes antibodies or fragments thereof that bind to cytoplasmic or nuclear antigens, such as, but not limited to, La / SSB antigen, members of the Rho family of GTPases, members of high mobility groups, and the like.Similarly, the binding portion of the targeting module may be composed of the alpha and beta or gamma and delta chains of a T cell receptor (TCR) or fragments thereof. Such binding portions derived from TCRs recognize and bind to proteins presented by the human leukocyte antigen class (HLA) I and II protein complexes. Examples include, but are not limited to, TCRs specific for peptides derived from the EGFR family, survivin, the sry-like high mobility box (SOX) protein family, melanoma-associated antigens (e.g., autoimmunogenic cancer / testis antigen NY-ESO-1, members of melanoma antigen family A MAGEA, an antigen predominantly expressed in melanoma PRAME), and leukemia-associated antigens (e.g., Wilms tumor gene 1 WT1). The binding portion of the targeting module may also include ligands for proteins and protein complexes, also referred to as receptors. Such ligands may bind, but are not limited to, cytokine receptors (e.g., IL-13 receptor), ligands for NKG2D receptors, ligands for EGFR family members, or autoreactive TCRs.

[0043] The binding portion of the targeting module may comprise a single antigen specificity (monospecific), two, three or more antigen specificities (bispecific and multispecific). Examples for bispecific and multispecific antigens include, but are not limited to, targeting modules that bind PSCA and PSMA antigens, CD19 and CD20 antigens, CD19, CD20 and CD22 antigens, CD33 and CD123 antigens, CD33 and CD99 antigens, CD33 and TIM-3, erb-1 and -2, PSCA and erb-2, and further combinations. The binding portion of the targeting module may comprise not only monovalent binding sites, but also bivalent and multivalent binding sites. Examples for bivalent and multivalent targeting strategies include, but are not limited to, targeting modules that introduce two scFvs that recognize different epitopes of PSCA, CD19 and CD33, and targeting modules that introduce ligand-scFv combinations that recognize various epitopes of the erb1 receptor.

[0044] The targeting module may have additional ligands that are not involved in target antigen binding, which are also referred to as payloads. Such payloads may include, but are not limited to, costimulatory ligands or cytokines fused to the N-terminus or C-terminus of the targeting module, in particular the extracellular domains of CD28, CD137 (4-1BB), CD134 (OX40) and CD27, as well as IL-2, IL-7, IL-12, IL-15, IL-17 and IL-21, all of which stimulate different types of immune cells. Other payloads may be radionuclides or chemical compounds that induce cell death in the target cell and neighboring cells.

[0045] method 1. A method for stimulating a universal chimeric antigen receptor-mediated immune response in a mammal, comprising: - administering to a mammal an effective amount of effector cells genetically modified to express a universal chimeric antigen receptor, said universal chimeric antigen receptor comprising three domains, a first domain being a tag-binding domain, a second domain being an extracellular hinge and transmembrane domain, and a third domain being a signaling domain, wherein the tag-binding domain binds to a tag derived from any human nuclear protein. - administering a targeting module consisting of a binding moiety specific for a particular human cell surface protein or protein complex and a tag, said tag being derived from any human nuclear protein; wherein the targeting module is administered to the subject prior to, simultaneously with, or after administration of effector cells expressing the universal chimeric antigen receptor.

[0046] In a preferred embodiment, the effector cells and targeting modules are administered to a human.

[0047] Generation of UniCAR effector cells In one embodiment of the present invention, immune cells may be genetically modified by various methods to express UniCAR.Generally, a polynucleotide vector encoding UniCAR and all the elements required to ensure its expression in genetically engineered immune cells is introduced into the cells.The introduction of the vector can be carried out by, but is not limited to, electroporation or transfection of nucleic acid, or with the aid of a viral vector system, for example, but is not limited to, gene transfer by adenovirus, adeno-associated virus, retrovirus, foamy virus or lentivirus.

[0048] In a further embodiment, lentiviral gene transfer can be applied by first constructing a lentiviral vector encoding a selected UniCAR for stable expression of UniCAR in immune cells. Exemplary lentiviral vectors include, but are not limited to, the vector pLVX-EF1alpha UniCAR 28 / ζ (Clontech, Takara Bio Group) shown in Figure 3, in which the lentiviral portion of the vector is derived from human immunodeficiency virus (HIV).

[0049] For the application described, the MSC / IRES / ZxGreenI part was replaced by the UniCAR construct. With reference to Figure 3, the abbreviations used are as follows: 5'LTR: 5' long terminal repeat, PBS: primer binding site, Ψ: packaging signal, RRE: Rev response element, cPPT / CTS: central polypurine tract / central termination sequence, PEF1α: human elongation factor 1 alpha promoter, MCS: multiple cloning site, IRES: internal ribosome entry site, ZsGreen1: human codon optimized, WPRE: woodchuck hepatitis virus post-transcriptional regulatory element, 3'LTR: 3' long terminal repeat, pUC: origin of replication, Ampr: ampicillin resistance gene; β-lactamase.

[0050] Lentiviral particles are typically generated by transiently transfecting human embryonic kidney (HEK) 293T (ACC635) cells with a lentiviral vector plasmid encoding UniCAR and co-transfecting with a plasmid encoding group specific antigen (gag) and polymerase (pol) as shown in FIG. 4 (e.g., psPAX2, addgene plasmid 12260) and an envelope encoding plasmid as shown in FIG. 5 (e.g., pMD2.G, addgene plasmid 12259). After transfection, the packaging plasmid expresses HIV-1 Gag and Pol proteins. Abbreviations used in FIG. 4 are as follows: CMVenh: CMV enhancer and promoter, SD: splice donor, SA: splice acceptor, Gag: group specific antigen, Pro: precursor protein encoding protease protein, Pol: protein encoding reverse transcriptase and integrase, RRE: rev response element, Amp: ampicillin.

[0051] Plasmid MD2.G (Figure 5) encodes the glycoprotein of the vesicular stomatitis virus (VSV-G). The VSV-G protein is used in lentiviral vectors for transduction of a broad range of mammalian cells. Abbreviations used in Figure 5 are as follows: CMV: CMV enhancer and promoter, beta-globin intror: beta-globin intron, beta-globin pA: beta-globin polyadenosine tail.

[0052] Various envelopes from different virus species can be used for this purpose. Lentiviral vectors can be pseudotyped with, effectively but not limited to, the envelope glycoprotein (Env) of amphotropic murine leukemia virus (MLV) or the G protein of vesicular stomatitis virus (VSV-G), modified envelopes of prototype foamy virus (PFV) or chimeric envelope glycoprotein mutants from gibbon ape leukemia virus (GaLV) and MLV. Supernatants from transfected HEK293T cells can be harvested 24-96 hours after transfection and viral particles can be, but need not be, concentrated from the supernatant by ultracentrifugation or other methods. For lentiviral transduction of immune cells, various established protocols can be applied. In one embodiment, peripheral blood mononuclear cells (PBMCs) or isolated T cells can be activated with monoclonal antibodies specific for the CD3 complex, such as clones OKT3 or UCHT1, either in solution or coated on plastic cell culture dishes or magnetic beads. Activation of PBMCs or isolated T cells can be further enhanced by stimulating the costimulatory pathways by providing specific monoclonal antibodies or ligands for, but not limited to, CD27, CD28, CD134 or CD137, alone or in various combinations, and exogenous recombinant cytokines, such as but not limited to, interleukin (IL)-2, IL-7, IL-12, IL-15 and IL-21. Concentrated or unconcentrated viral particles are added to the PBMC or T cell culture 24-96 hours after the initial administration of activating CD3 antibodies and / or recombinant cytokines, either as a single dose or multiple doses. Stable transduction of T cells can be measured by flow cytometry from day 3 onwards after the last administration of viral supernatant after staining with a targeting module containing a tag for the surface expression of UniCAR or after staining with a monoclonal antibody against the fourth domain of UniCAR.UniCAR-transduced T cells can be expanded in vitro by culturing the cells supplying recombinant cytokines and activating anti-CD3 monoclonal antibodies.

[0053] In the case where the UniCAR has an optional fourth domain, i.e., a peptide sequence that forms a linear epitope for a monoclonal antibody, immune cells genetically modified to express UniCAR can be specifically propagated in vitro by coating a monoclonal antibody or antibody fragment thereof that binds to the fourth UniCAR domain onto the surface of a culture dish or onto any type of bead that is added to the cell culture at a defined ratio, including but not limited to, one bead for every 1 to 4 UniCAR-conjugated effector cells. Binding of the surface-coated monoclonal antibody to the UniCAR peptide domain induces cross-linking of the UniCAR expressed on the cell surface and the formation of an immune synapse that leads to the activation of a signaling pathway specifically triggered by the signaling domain of UniCAR. This can result in enhanced proliferation and persistent resistance to activation-induced cell death of immune cells bearing UniCAR, thus leading to enrichment of UniCAR genetically modified immune cells in a mixed population.

[0054] Any fourth domain, i.e., a peptide sequence that forms a linear epitope for a monoclonal antibody, can further be used to enrich and purify UniCAR-expressing immune cells from a mixed population. Enrichment and purification can be performed with the aid of a monoclonal antibody or antibody fragment thereof that binds to the fourth UniCAR domain, to mark the UniCAR-expressing cells for cell sorting, or to transiently bind the UniCAR-expressing immune cells to small particles that can be used for cell isolation. In one embodiment, the immune cells conjugated with UniCAR are incubated with a monoclonal antibody that recognizes the fourth domain. Then, magnetic beads are added that are coupled with antibodies or fragments thereof against the species-specific and isotype-specific heavy and light chains of the monoclonal antibody that binds to any fourth domain. Thus, the UniCAR-expressing immune cells and the magnetic beads are combined, and can be captured and separated from other immune cells in a magnetic field.

[0055] In a further embodiment of the present invention, the optional fourth domain can be used for detection of UniCAR surface expression as shown in Figure 6. Figure 6(A) shows that UniCAR surface expression can be detected by using a monoclonal antibody against the optional fourth domain, followed by staining with a fluorochrome-conjugated anti-species secondary antibody. By additionally using the optional fourth domain, UniCAR-conjugated T cells can be purified to a high degree of purity as shown in Figure 6) in UniCAR immune cell administration.

[0056] Administration of UniCAR-expressing immune cells The population of UniCAR-expressing immune cells may be formulated for administration to a subject using techniques known to those of skill in the art.

[0057] The formulation comprising a population of UniCAR-expressing immune cells may comprise one or more pharma-ceutical acceptable excipients. The excipients included in the formulation will have various purposes, depending on, for example, the nature of the tag-binding domain comprising UniCAR, the population of immune cells used, and the mode of administration. Examples of commonly used excipients include, but are not limited to, saline, buffered saline, dextrose, water-for-infection, glycerol, ethanol, and combinations thereof, stabilizers, solubilizers and surfactants, buffers and preservatives, isotonicity agents, bulking agents, and lubricants. The formulation comprising a population of UniCAR-expressing immune cells will generally be prepared and cultured in the absence of any non-human components, such as animal serum (e.g., bovine serum albumin).

[0058] The preparation may comprise one or more populations of UniCAR-expressing immune cells, for example, 2, 3, 4, 5, 6 or more populations. The different populations of immune cells conjugated with UniCAR may vary based on the identity of tag-binding domain, the identity of signaling domain, the identity of subpopulation, the mode of production and culture, or a combination thereof. For example, the preparation may comprise a population of UniCAR-expressing T cells and NK cells that recognize and bind to one or more, for example, 2, 3, 4, 5, 6 or more different tagged proteins.

[0059] The formulation comprising one or more populations of UniCAR immune cells can be administered to a subject using modes and techniques known to those skilled in the art. Exemplary modes include, but are not limited to, intravenous injection. Other modes include, but are not limited to, intratumoral, intradermal, subcutaneous (sc, sq, sub-Q, Hypo), intramuscular (im), intraperitoneal (ip), intraarterial, intramedullary, intracardiac, intraarticular (joint), intracerebrospinal (synovial area), intracranial, intrathecal and subarachnoid (spinal fluid). Any known device useful for parenteral injection or infusion of the formulation can be used to carry out such administration. Injection can be carried out as bulk injection or continuous flow injection.

[0060] The formulation containing one or more populations of UniCAR-expressing immune cells administered to a subject contains a number of UniCAR-expressing immune cells effective for the treatment and / or prevention of a particular indication or disease. Thus, a therapeutically effective population of UniCAR-expressing immune cells is administered to a subject when carrying out the method of the invention. The number of UniCAR-expressing immune cells administered to a subject will vary between wide limits depending on the location, origin, identity, extent and severity of the disease, age and condition of the individual to be treated, etc. Generally, about 1×10 4 From about 1×10 10 In most cases, the preparation contains between about 1×10 5 From about 1×10 9 UniCAR-expressing immune cells, approximately 5 × 10 5 From about 5 × 10 8 UniCAR-expressing immune cells, or approximately 1 x 10 6 From about 1×10 9 The individual may contain up to 10 ...

[0061] Fabrication of target module The target module contains two domains: a binding moiety specific for a particular human cell surface protein or protein complex, and a tag directed to the tag-binding domain of UniCAR. The target module can be produced by techniques known to those skilled in the art, including but not limited to recombinant expression in prokaryotic or eukaryotic cells or artificial synthesis of polypeptide chains.

[0062] In one embodiment, the target module can be expressed in Chinese Hamster Ovary (CHO, ACC-110) cells, which are suitable for synthesizing large amounts of recombinant protein in a biologically active form. The nucleic acid sequence encoding the target module can be introduced into the CHO cells by established genetic engineering techniques, such as, but not limited to, naked nucleic acid transfection, electroporation or viral gene transfer. Highly productive single cell clones can be selected from the parent line using, for example, the dihydrofolate reductase (DHFR) selection system. In this system, DHFR-deficient CHO cell mutants (e.g., CHO sublines DXB11 or DG44) are genetically modified by co-transfection with a functional copy of the DHFR gene in addition to the nucleic acid sequence encoding the target module. Clonal selection is then performed by growth in medium lacking glycine, hypoxanthine and thymidine. Highly productive clones can be further selected by culturing the cells in high concentrations of methotrexate (MTX), a folate analog that blocks DHFR activity. Since genetically modified cells must deal with a loss of DHFR activity, which cannot be rescued by the presence of a single copy of DHFR, clones with an amplified copy of the DHFR gene are preferred under these conditions. The genetic link between DHFR and the gene of interest ensures that the transgene is also amplified at the same time, thus enhancing the chances of obtaining a highly productive cell clone. The selected cell clones are advantageously grown under good production conditions in the absence of any animal serum. The target module can be isolated from the cell culture supernatant by established preparative protein purification methods, including preliminary steps such as precipitation or ultracentrifugation, and by various purification techniques such as size exclusion or ion exchange chromatography. In one embodiment, the nucleic acid sequence of the target module has a coding sequence for 6 to 8 consecutive histidine amino acids forming a polyhistidine tag. The polyhistidine binds strongly to divalent metal ions such as nickel and cobalt.The cell culture supernatant can be passed over a column containing immobilized nickel ions, which bind the polyhistidine tag, while all untagged proteins pass through the column. The target module can be eluted with imidazole, which competes with the polyhistidine for binding to the column, or by lowering the pH, which reduces the affinity of the tag for the resin.

[0063] Target Module Administration One targeting module or more than one, for example two, three, four or more targeting modules, can be formulated using techniques known to those of skill in the art for administration to a subject.

[0064] A formulation containing one or more targeting modules may include one or more pharma- ceutical acceptable excipients. The excipients included in the formulation will have various purposes, for example, depending on the nature of the targeting modules and the mode of administration. Examples of commonly used excipients include, but are not limited to, saline, buffered saline, dextrose, water-for-infection, glycerol, ethanol and combinations thereof, stabilizers, solubilizers and surfactants, buffers and preservatives, isotonicity agents, bulking agents and lubricants. Formulations containing targeting modules will generally be prepared and cultured in the absence of any non-human components, such as animal serum (e.g., bovine serum albumin).

[0065] The formulation may contain one targeting module or more than one, for example, 2, 3, 4, 5, 6 or more targeting modules. The targeting modules can vary based on the identity of the binding moiety, the identity of the tag, the manner of production, or a combination thereof. For example, the formulation may contain one or more targeting modules that recognize and bind to different human cell surface proteins, protein complexes, or extracellular matrix structures, for example, 2, 3, 4, 5, 6 or more.

[0066] The preparation containing one or more populations of UniCAR-expressing immune cells can be incubated ex vivo with the preparation containing one or more targeting modules to allow the UniCAR-expressing immune cells to be coated with the targeting modules before administration to the subject. Alternatively, the preparation containing one or more targeting modules can be administered directly to the subject, or a combination of both strategies can be selected. The route and dosage will vary between wide limits depending on the location, origin, individuality, extent and severity of the disease, age and condition of the individual to be treated, etc. The physician will ultimately determine the appropriate route of administration and dosage to be used.

[0067] The formulation containing the targeting module is administered to the subject in an amount effective for the treatment and / or prevention of a particular indication or disease. 2 Typical dosage rates delivered per day are between 1 μg and 1000 mg, preferably between 10 μg and 1 mg, with dosages administered one or more times per day or week, or continuously for a period of several weeks. However, the amount of targeting module in the formulation administered to the subject will vary between wide limits depending on the location, origin, identity, extent and severity of the cancer, age and condition of the individual to be treated, etc. The physician will ultimately determine the appropriate dosage to be used.

[0068] The present invention relates to a method of treating a subject having cancer, an infectious disease or an autoimmune disease, comprising administering to a subject in need of treatment one or more formulations of a targeting module that binds to cancer cells, and one or more therapeutically effective populations of UniCAR-expressing immune cells that bind to the targeting module and induce cell death.

[0069] The term "cancer" is intended to be broadly construed and includes all aspects of abnormal cell growth and / or cell division. Examples include carcinomas, including but not limited to adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, small cell carcinoma, and cancers of the skin, breast, prostate, bladder, vagina, cervix, uterus, liver, kidney, pancreas, spleen, lung, trachea, bronchus, colon, small intestine, stomach, esophagus, gallbladder; sarcomas, including but not limited to chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, soft tissue sarcoma, and cancers of bone, cartilage, fat, muscle, blood vessels, and hematopoietic tissue; lymphomas and leukemias, including but not limited to mature B-cell neoplasms, such as chronic lymphocytic leukemia, leukemia, and leukemia ... Leukemia / small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoma and plasma cell neoplasms, mature T-cell and mature killer (NK) cell neoplasms, such as T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, aggressive NK cell leukemia, and adult T-cell leukemia / lymphoma, Hodgkin's lymphoma, and immunodeficiency-associated lymphoproliferative disorders; germ cell tumors, such as but not limited to testicular and ovarian cancer; blastomas, such as but not limited to hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, and retinoblastoma. The term also includes benign tumors.

[0070] As used herein, the terms "treat", "treating" and "treatment" have their ordinary and customary meanings, including one or more of: preventing cancer in a subject, remission of cancer, or reducing the severity and / or frequency of symptoms of cancer, and / or inhibiting the growth, division, spread or proliferation of cancer cells or the progression of cancer (e.g., the development of new tumors) in a subject. Treating refers to the prevention, remission, reduction or inhibition of only about 1% to about 100% relative to subjects in which the method of the invention is not being performed. Preferably, the prevention, remission, reduction or inhibition is about 100%, 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or 1% relative to subjects in which the method of the invention is not being performed.

[0071] The frequency of administration of both the formulation comprising the population of UniCAR-expressing immune cells and the formulation of the targeting module can vary depending on factors including the disease being treated, the elements comprising the UniCAR-expressing immune cells and the targeting module, and the mode of administration. Each formulation may be independently administered four, three, two or once a day, every other day, every third day, every fourth day, every fifth day, every sixth day, once a week, every eighth day, every ninth day, every tenth day, every two weeks, every month, and every two months.

[0072] The duration of treatment will be based on the disease being treated and is best determined by the attending physician, however, the duration of treatment may be contemplated to continue for days, weeks, or months.

[0073] The present invention provides flexibility in the treatment method, such that one or more formulations of the targeting module and one or more populations of UniCAR-expressing immune cells may be administered to the subject in any order. Thus, one or more formulations of the targeting module may be administered to the subject before, after, or simultaneously with one or more populations of UniCAR-expressing immune cells. Alternatively, when one or more formulations of the targeting module and / or one or more populations of UniCAR-expressing immune cells are administered to the subject, the administration may be alternating. For example, a first formulation of the targeting module may be administered, followed by a first population of UniCAR-expressing immune cells, followed by a second formulation of the tagged protein, followed by a second population of UniCAR-expressing immune cells.

[0074] The invention also includes a method in which a population of UniCAR-expressing immune cells is coated with a targeting module prior to administration of the UniCAR-expressing immune cells to a subject.

[0075] In each of the embodiments of the invention, the subject to be treated is a human or non-human animal, such as a non-human primate, bird, horse, cow, goat, sheep, companion animal, such as a dog, cat or rodent, or other mammal. EXAMPLES

[0076] In one embodiment, UniCAR engineered T cells can be specifically redirected against tumor cells expressing PSCA and / or PSMA, which are frequently detected in biopsies, such as those from prostate, bladder, spleen and breast tumors (Figure 7). Human T cells were mock transduced (white bars) or transduced with lentiviral vectors encoding UniCAR, either containing the two signaling domains of CD28 / CD3 zeta (black bars) or lacking all signaling domains (shaded bars) or expressing only the EGFP marker protein (striped bars). Specific lysis of prostate tumor cells (PC3) engineered to express either prostate stem cell antigen (PC3-PSCA) or prostate membrane antigen (PC3-PSMA) in the presence of engineered human T cells and targeting modules was analyzed in a 51Cr release assay. T cells were incubated with 51Cr-loaded PC3 target cells at an effector-to-target ratio (e:t) of 5:1 or 1:1. Targeting modules (TM) specific for PSCA (αPSCA TM) or PSMA (αPSMA TM) were added at a concentration of 15 nMol. Target cell lysis (chromium release) was measured after 20 hours of culture. The plots show the mean and standard deviation from experiments with three individual T cell donors (Figure 7A). Similarly, the killing capacity of UniCAR-engineered T cells against LNCap-4-2B tumor stem cells expressing both PSCA and PSMA antigens was supported by 51Cr release assays in the presence of either PSCA TM or PSMA TM (1 nMol) at the indicated e:t ratios. The mean and standard deviation from experiments with three individual T cell donors are shown (Figure 7B). The experiment from Figure 7B was repeated at a lower e:t ratio of 1:2, adding either 1 nMol of PSCA-specific TM or PSMA-specific TM, or combining both TMs at an overall concentration of 1 nMol. Specific lysis, as measured by 51Cr release, was measured after 24 and 48 hours. Means and standard deviations from experiments with three separate healthy T cell donors are shown (Figure 7C).This experiment demonstrates that the combination of two different TMs improves the killing capacity of UniCAR-redirected T cells against tumor cells compared to a single antigen retargeting strategy, even though the total amount of both TMs in the dual-targeted sample is equal to the total amount of each TM in the single antigen retargeting control sample (Figure 7C). Furthermore, as soon as human T cells crosslink to antigen-expressing tumor cells in the presence of the corresponding TM, UniCAR-equipped human T cells secrete inflammatory and proliferative cytokines (Figure 7D). Human T cells were incubated with PC3 cells expressing either PSCA or PSMA antigens in the presence or absence of TMs specific for PSCA or PSMA. After 24 h of culture, cell-free supernatants were harvested and subsequently analyzed for T cell-specific cytokine release using commercially available ELISA kits. Statistical analysis for Figure 7C,D was performed using non-parametric one-way ANOVA (Kruskal-Wallis test) and post-hoc Dunn's multiple comparison test (. ** p<0.01).

[0077] In a further embodiment, FIG. 8 shows concentration-response curves for UniCAR genetically modified human T cells in the presence of targeting modules that bind to various antigens on the surface of tumor cells of different origins. Human T cells were transduced with a lentiviral vector encoding a UniCAR containing two signaling domains of CD28 / CD3 zeta. UniCAR-conjugated T cells were incubated with 51Cr-loaded PC3 target cells engineered to express either PSCA or PSMA antigens at an e:t ratio of 5:1. Increasing concentrations of targeting modules (TM) specific for PSCA (αPSCA TM) or targeting modules (TM) specific for PSMA (αPSMA TM) were added (FIG. 7A). The half maximal effective concentration (EC50) was measured to be about 12 pMol for both TMs. Additional experiments were performed similarly to FIG. 7A, but choosing an e:t ratio of 1:1 and using the acute myeloid leukemia cell line MOLM-13 as the target tumor cells. Increasing concentrations of TM specific for the CD33 antigen (αCD33 TM) or CD123 antigen (αCD123 TM) were added, and EC50 values ​​of 137 pMol for αCD33 TM and 45 pMol for αCD123 TM were measured. These experiments confirm that UniCAR-conjugated T cells are effective at very low concentrations of TM, 10- to 100-fold lower than the EC50 values ​​experimentally measured for monoclonal antibody drugs approved for cancer therapy (e.g., Herter et al. Mol Cancer Ther 12(10):2031-2042(2013)).

[0078] In a further embodiment, Figure 9 confirms that UniCAR engineered T cells can effectively kill acute myeloid leukemia (AML) blasts. Human T cells were mock transduced (wt, diamonds) or transduced with lentiviral vectors encoding U-CAR, either containing the two signaling domains of CD28 / CD3 zeta (CAR 28 / ζ, open and closed circles) or lacking all signaling domains (CAR Stop, up-pointing triangles) or expressing only the EGFP marker protein (vc, down-pointing triangles). T cells were cultured at 2x10 4Alexa eFluor 674-labeled target cells from three AML cell lines (MOLM-13, MV4-11, OCI-AML3) were incubated with 0.1 nMol of CD33-specific targeting module (TM) (αCD33 TM, upper panel) or CD123-specific targeting module (TM) (αCD123 TM, lower panel) at a 1:1 e:t ratio for 24 hours (Figure 9A). The number of viable target cells that were propidium iodide (PI) negative but Alexa eFluor 674 positive was measured by flow cytometry using a MACSQuant® analyzer. The number of viable leukemic target cells was normalized to a control sample with target cells but without any T cells. This experiment confirms that UniCAR-conjugated T cells effectively lyse ALM blasts independent of antigen density, which is high for CD33 in MOLM-13, intermediate for MV4-1,1, and low for OCI-AML3, while for CD123 the antigen density is in the reverse order for those three cell lineages, after crosslinking with the corresponding TM. Furthermore, AML blasts are eliminated by UniCAR-conjugated T cells even at low e:t ratios typically found in patient samples upon TM-mediated crosslinking (Figure 9B). The experimental setup was similar to Figure 9A, but a low e:t ratio of 1:5 was chosen. The number of PI-negative viable T cells and target cells was measured at the indicated time points by flow cytometry using a MACSQuant® analyzer. The number of viable leukemic target cells was normalized to a control sample with target cells but lacking T cells (Figure 9B). Upon activation via CAR-mediated signaling, UniCAR-conjugated T cells start to proliferate as shown in Figure 9C. The experimental setup was as described for Figure 9B and T cell numbers were measured by flow cytometry using a MACSQuant® analyzer. T cell proliferation was calculated as the ratio of T cells present in samples after 144 hours (d6) to the number seeded at the start of the experiment (d0).Statistical analysis for Figures 8A and 8B was performed using non-parametric one-way ANOVA (Kruskal-Wallis test) and post-hoc Dunn's multiple comparison test. For Figure 9A, statistically significant results are shown, and for Figure 9B, results are shown in the table below the figure (ns = not significant, . * p<0.05, ** p<0.01, *** p<0.001).

[0079] In a further embodiment, FIG. 10 shows the redirection of UniCAR-conjugated T cells against two antigens simultaneously. Due to its modular nature, UniCAR technology allows redirection of UniCAR-conjugated immune cells (e.g., T cells) against two antigens simultaneously or sequentially using two individual TMs (TM1+TM2, left side of FIG. 10A) or using combined bispecific TMs (TM1-2, right side of FIG. 10A) arranged as a bispecific single-chain tandem construct (FIG. 10A). The use of bispecific TMs targeting two antigens may be more effective than using a combination of TMs specific for two single antigens, as supported by the concentration-response curves for the combination-specific retargeting of UniCAR-conjugated T cells against AML cell lines with CD33 and CD123 (FIG. 10B). Human T cells were transduced with a lentiviral vector encoding a UniCAR containing the two signaling domains of CD28 / CD3 zeta. UniCAR-conjugated T cells were incubated with Cr51-labeled MOLM-13 (average from experiments with T cells from four different healthy human donors, triangles) and OCI-AML3 (average from experiments with T cells from two different healthy human donors, open circles) at a 1:1 e:t ratio for 24 h. Increasing concentrations of CD33-specific targeting modules (TM) (αCD33 TM), CD123-specific targeting modules (TM) (αCD123 TM) or bispecific CD33-CD123 TM (αCD123-CD33 TM) were added. EC50 values ​​were measured as follows: αCD33+αCD123 TM: EC50 MOLM-13=70.2 pMol, EC50 OCI-AML3=80.2 pMol, αCD33-αCD123 TM: EC50 MOLM-13=2.9 pMol, EC50 OCI-AML3=11.7 pMol. We could then confirm that UniCAR-conjugated T cells from both healthy human donors and AML patients could be effectively redirected against AML blasts isolated from patients in blast crisis as well as against AML cell lines (Figure 10C, D, E, F).Human T cells were mock transduced (black circles in Fig. 10C, white bars in Fig. 10E,F) or transduced with lentiviral vectors encoding UniCAR, containing the two signaling domains of CD28 / CD3 zeta (triangles in Fig. 10C,D, black bars in Fig. 10E,F) or lacking all signaling domains (diamonds in Fig. 10C,D, grey bars in Fig. 10C,F). Highly efficient elimination of AML cells was mediated by UniCAR-conjugated T cells in the presence of a combination of αCD33 TM and αCD123 TM or in the presence of dual-targeted αCD123-CD33 TM, but not in the absence of these TMs, again supporting the dispensability of TM-mediated cross-linking for antigen-specific redirection of UniCAR-conjugated T cells (Fig. 10C,D,E). T cells were cultured at 2 × 10 4 Alexa eFluor 674-labeled target cells from three AML cell lines (MOLM-13, MV4-11, OCI-AML3) were incubated with 100 pMol of TM in a total amount (+) or absence (-) at an e:t ratio of 1:5 for 144 hours. TM was renewed after 48 hours. The number of viable PI-negative but Alexa eFluor 674-positive target cells was measured by flow cytometry using a MACSQuant® analyzer and compared to a control sample with target cells but without any T cells (Figure 10C, open circles). T cell proliferation was calculated in Figure 10D as the ratio of T cells present in the sample after 144 hours (d6) to the number seeded at the beginning of the experiment (d0). Results from experiments with six donors are shown, with the mean and standard deviation shown. These results convincingly support that activation via UniCAR signaling upon TM-mediated cross-linking not only leads to target cell killing, but also that UniCAR-conjugated T cells receive proliferation stimulation from the combined UniCAR signaling chain of CD28 / CD3ζ and begin to divide. 4Alexa eFluor 674-labeled leukemia cells from AML patients were incubated at a 1:1 e:t ratio in the presence (+) or absence (-) of 0.5 nMol of TM (Figure 10E). The number of viable Alexa eFluor 674-positive target cells was measured after 24 hours (left panel) and 48 hours (right panel) by flow cytometry using a MACSQuant® analyzer. The number of viable leukemia target cells was normalized to a control sample with target cells but without any T cells. The results from the five sets of experiments in Figure 9E confirm that UniCAR-engineered T cells can lyse patient-derived AML blast cells over time. It could also be confirmed that UniCAR-engineered T cells from AML patients are able to attack and lyse AML cells by the addition of AML antigen-specific TM. The engineered T cells were incubated at 2 × 10 4 Alexa eFluor 674-labeled target cells from three AML cell lines (MOLM-13, MV4-11, OCI-AML3) were incubated at a 1:1 e:t ratio in the presence (+) or absence (-) of 0.5 nMol total amount of TM for 24 hours. The number of viable PI-negative but Alexa eFluor 674-positive target cells was measured by flow cytometry using a MACSQuant® Analyzer.

[0080] In a further embodiment, Figure 11 shows a graph depicting the in vivo pharmacokinetics of the bispecific αCD123-CD33 targeting module. NSG mice (NOD / SCID IL2Rγ- / -) were injected with 250 μg / g body weight of αCD123-CD33 TM either intravenously (iv in Figure 11A) or intraperitoneally (ip in Figure 11B) and serum samples were taken at the indicated time points. Capture ELISA was used to measure the concentration of TM in the samples. Results show the mean and standard deviation (n=3). Half-decay times were measured using an exponential one-phase decay model (GraphPad Prism software) in the case of intravenous injection.

[0081] In a further embodiment, an isolated nucleic acid sequence is provided encoding a universal chimeric antigen receptor according to SEQ ID NO: 1. The isolated nucleic acid sequence encodes a human IL-2m leader peptide according to SEQ ID NO: 2, a humanized anti-La 5B9 variable region heavy chain according to SEQ ID NO: 3, a humanized anti-La 5B9 variable region light chain according to SEQ ID NO: 4, a La 7B6 epitope according to SEQ ID NO: 5, human CD28 according to SEQ ID NOs: 6-8, for example a human CD28 extracellular portion with a mutated binding motif according to SEQ ID NO: 6, a CD28 transmembrane domain according to SEQ ID NO: 7 and a human CD28 intracellular portion comprising a mutated internalization motif according to SEQ ID NO: 8, and a human CD3 zeta intracellular domain according to SEQ ID NO: 9.

[0082] The product of protein expression of the isolated nucleic acid sequence set forth in SEQ ID NO: 1 can be obtained in SEQ ID NO: 27. The nucleic acid sequence of the humanized anti-La 5B9 variable region heavy chain set forth in SEQ ID NO: 3 encodes a protein according to SEQ ID NO: 33, whereas the humanized anti-La 5B9 variable region light chain set forth in SEQ ID NO: 4 encodes a protein set forth in SEQ ID NO: 34.

[0083] The nucleic acid sequence of the human La 7B6 epitope set forth in SEQ ID NO:5 encodes the protein domain set forth in SEQ ID NO:35.

[0084] In a further embodiment of the invention, an isolated nucleic acid sequence encoding a targeting module having a binding site for the prostate specific antibody PSCA is provided in SEQ ID NO: 10. The isolated nucleic acid sequence encodes the leader peptide IgG kappa as set forth in SEQ ID NO: 11, the humanized light chain of an anti-PSCA scFv as set forth in SEQ ID NO: 12, the humanized heavy chain of an anti-PSCA scFv as set forth in SEQ ID NO: 13, the La 5B9 epitope as set forth in SEQ ID NO: 14, a myc tag as set forth in SEQ ID NO: 15 and a his tag as set forth in SEQ ID NO: 16.

[0085] The product of protein expression of the nucleic acid set forth in SEQ ID NO: 10 can be obtained from SEQ ID NO: 28. The nucleic acid sequence of the humanized light chain of the anti-PSCA scFv set forth in SEQ ID NO: 12 encodes the protein domain set forth in SEQ ID NO: 36, whereas the humanized heavy chain of the anti-PSCA scFv set forth in SEQ ID NO: 13 encodes the protein domain set forth in SEQ ID NO: 37. The La 5B9 epitope set forth in SEQ ID NO: 14 encodes the protein set forth in SEQ ID NO: 44.

[0086] In a further embodiment of the invention, an isolated nucleic acid sequence encoding a targeting module having a binding site for the prostate specific antibody PSMA is provided in SEQ ID NO: 17. The isolated nucleic acid sequence encodes the leader peptide IgG kappa according to SEQ ID NO: 11, the humanized heavy chain of the anti-PSMA scFv according to SEQ ID NO: 18, the humanized light chain of the anti-PSMA scFv according to SEQ ID NO: 19, the La 5B9 epitope according to SEQ ID NO: 14, a myc tag according to SEQ ID NO: 15 and a his tag according to SEQ ID NO: 16.

[0087] The product of protein expression of the nucleic acid set forth in SEQ ID NO: 17 can be derived from SEQ ID NO: 29. The nucleic acid sequence of the humanized heavy chain of the anti-PSMA scFv set forth in SEQ ID NO: 18 encodes the protein domain set forth in SEQ ID NO: 38, whereas the humanized light chain of the anti-PSMA scFv set forth in SEQ ID NO: 19 encodes the protein domain set forth in SEQ ID NO: 39. The La 5B9 epitope set forth in SEQ ID NO: 14 encodes the protein set forth in SEQ ID NO: 44.

[0088] In a further embodiment of the invention, an isolated nucleic acid sequence encoding a targeting module having a binding site for the anti-CD33 antigen is provided in SEQ ID NO: 20. The isolated nucleic acid sequence encodes the leader peptide IgG kappa according to SEQ ID NO: 11, the humanized light chain of the anti-CD33 scFv according to SEQ ID NO: 21, the humanized heavy chain of the anti-CD33 scFv according to SEQ ID NO: 22, the La 5B9 epitope according to SEQ ID NO: 14, a myc tag according to SEQ ID NO: 15 and a his tag according to SEQ ID NO: 16.

[0089] The product of protein expression of the nucleic acid set forth in SEQ ID NO: 20 can be derived from SEQ ID NO: 30. The nucleic acid sequence of the humanized light chain of the anti-CD33 scFv set forth in SEQ ID NO: 21 encodes the protein domain set forth in SEQ ID NO: 40, whereas the humanized heavy chain of the anti-CD33 scFv set forth in SEQ ID NO: 22 encodes the protein domain set forth in SEQ ID NO: 41. The La 5B9 epitope set forth in SEQ ID NO: 14 encodes the protein set forth in SEQ ID NO: 44.

[0090] In a further embodiment of the invention, an isolated nucleic acid sequence encoding a targeting module having a binding site for the anti-CD123 antigen is provided in SEQ ID NO: 23. The isolated nucleic acid sequence encodes the leader peptide IgG kappa according to SEQ ID NO: 11, the humanized heavy chain of anti-CD123 scFv according to SEQ ID NO: 24, the humanized light chain of anti-CD123 scFv according to SEQ ID NO: 25, the La 5B9 epitope according to SEQ ID NO: 14, a myc tag according to SEQ ID NO: 15 and a his tag according to SEQ ID NO: 16.

[0091] The product of protein expression of the nucleic acid set forth in SEQ ID NO: 23 can be derived from SEQ ID NO: 31. The nucleic acid sequence of the humanized heavy chain of the anti-CD123 scFv set forth in SEQ ID NO: 24 encodes the protein domain set forth in SEQ ID NO: 42, whereas the humanized light chain of the anti-CD123 scFv set forth in SEQ ID NO: 25 encodes the protein domain set forth in SEQ ID NO: 43. The La 5B9 epitope set forth in SEQ ID NO: 14 encodes the protein set forth in SEQ ID NO: 44.

[0092] In a further embodiment of the invention, an isolated nucleic acid sequence encoding a targeting module having a binding site for anti-CD123-anti-CD33 antigen is provided in SEQ ID NO: 26. The isolated nucleic acid sequence encodes the leader peptide IgG kappa according to SEQ ID NO: 11, the humanized heavy chain of anti-CD123 scFv according to SEQ ID NO: 24, the humanized light chain of anti-CD123 scFv according to SEQ ID NO: 25, the La 5B9 epitope according to SEQ ID NO: 14, the humanized heavy chain of anti-CD33 scFv according to SEQ ID NO: 22, the humanized light chain of anti-CD33 scFv according to SEQ ID NO: 21, a myc tag according to SEQ ID NO: 15 and a his tag according to SEQ ID NO: 16.

[0093] The product of protein expression of the nucleic acid set forth in SEQ ID NO: 26 can be obtained from SEQ ID NO: 32. The nucleic acid sequence of the humanized heavy chain of the anti-CD123 scFv set forth in SEQ ID NO: 24 encodes the protein domain set forth in SEQ ID NO: 42, whereas the humanized light chain of the anti-CD123 scFv set forth in SEQ ID NO: 25 encodes the protein domain set forth in SEQ ID NO: 43. The La 5B9 epitope set forth in SEQ ID NO: 14 encodes the protein set forth in SEQ ID NO: 44. The nucleic acid sequence of the humanized heavy chain of the anti-CD33 scFv set forth in SEQ ID NO: 22 encodes the protein domain set forth in SEQ ID NO: 41, whereas the humanized light chain of the anti-CD33 scFv set forth in SEQ ID NO: 21 encodes the protein domain set forth in SEQ ID NO: 40.

Claims

1. A nucleic acid encoding a universal chimeric antigen receptor, the receptor comprising three domains: - the first domain is a tag-binding domain, - the second domain is the extracellular hinge and transmembrane domain, - The third domain is the signal transduction domain, wherein the tag binding domain is an antibody or antigen-binding fragment thereof and binds to a tag derived from any human nuclear protein, and said tag is a short linear epitope (E5B9) from human nuclear La protein encoded by the nucleic acid sequence set forth in SEQ ID NO:

14.

2. 2. The nucleic acid according to claim 1, wherein the hinge and transmembrane regions are selected from the hinge and transmembrane regions of the human CD28 molecule, the CD8a chain, the NK cell receptor, the natural killer group NKG2D, or portions of the constant regions of antibodies and combinations of their various hinge and transmembrane domains.

3. 3. The nucleic acid of claim 1 or 2, wherein the signaling domain is selected from CD28, CD137 (4-1BB), CD134 (OX40), DAP10 and the cytoplasmic domain of CD27, programmed cell death-1 (PD-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4) and the cytoplasmic domains of the CD3 chain, DAP12 and T-cell activation-inducing Fc receptor.

4. 4. The nucleic acid of claim 1, wherein the receptor comprises a fourth domain that is a short peptide linker in the extracellular portion of the receptor.

5. A cell or vector comprising a nucleic acid according to any one of claims 1 to 4.

6. The cell of claim 5, wherein the cell is selected from the group of immune cells including T cells, natural killer cells, cytotoxic T lymphocytes and regulatory T cells.

7. a vector according to claim 5, a targeting module consisting of a binding moiety specific for a particular human cell surface protein or protein complex and a tag and / or a vector comprising a nucleic acid encoding said targeting module, A kit comprising: The tag is a short linear epitope (E5B9) derived from human nuclear La protein encoded by the nucleic acid sequence set forth in SEQ ID NO: 14; and The binding moiety comprises an antibody or antigen-binding fragment that binds to an antigen selected from the group consisting of surface antigen CD33, surface antigen CD123, prostate stem cell antigen PSCA, and prostate specific membrane antigen PSMA. The kit.

8. A preparation for administration to a subject, comprising the UniCAR-expressing immune cell according to claim 5 or 6 and a targeting module composed of a binding moiety specific to a certain human cell surface protein or protein complex and a tag, The tag is a short linear epitope (E5B9) derived from human nuclear La protein encoded by the nucleic acid sequence set forth in SEQ ID NO: 14; and The binding moiety comprises an antibody or antigen-binding fragment that binds to an antigen selected from the group consisting of surface antigen CD33, surface antigen CD123, prostate stem cell antigen PSCA, and prostate specific membrane antigen PSMA. The formulation.

9. A pharmaceutical composition for use in the treatment of cancer comprising a cell according to claim 5 or 6 and a targeting module comprising a binding moiety specific for a particular human cell surface protein or protein complex and a tag, The tag is a short linear epitope (E5B9) derived from human nuclear La protein encoded by the nucleic acid sequence set forth in SEQ ID NO: 14; and The binding moiety comprises an antibody or antigen-binding fragment that binds to an antigen selected from the group consisting of surface antigen CD33, surface antigen CD123, prostate stem cell antigen PSCA, and prostate specific membrane antigen PSMA. The pharmaceutical composition.

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