Methods for providing chimeric antigen receptors (CARs) to personalized cells

Abstract

To provide methods for selecting cancer cell-specific antigens that are not expressed in healthy tissue, in order to design cells that kill / lyse cancer cells without attacking non-tumor cells.SOLUTION: Provided is a method for providing cells containing a chimeric antigen receptor (CAR) specific for one or more target antigens exposed to tumor cells, characterized by providing cell samples containing tumor cells and non-tumor cells and by repeating the following steps: contacting a cell tissue with a conjugate containing a fluorescent moiety and an antigen-recognizing moiety (hereinafter referred to as "conjugate"); removing the conjugate that is not bound from the cell tissue and detecting the cells bound to the conjugate by the fluorescent radiation emitted by the fluorescent moiety of the original conjugate; and extinguishing the fluorescent radiation emitted by the fluorescent moiety of the conjugate.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for selecting antigens exposed on cancer cells but not on healthy cells of an individual to provide the cells with an appropriate chimeric antigen receptor (CAR). [Background technology]

[0002] Cancer is a broad group of diseases involving unregulated cell proliferation. In cancer, cells divide and grow uncontrollably, forming malignant tumors that invade nearby parts of the body. Cancer can also spread to more distant parts of the body via the lymphatic system or bloodstream.

[0003] Chimeric antigen receptor (CAR) technology may provide a promising approach for adoptive cellular immunotherapy for cancer. Conventionally, CARs contain a single-chain fragment variable (scFv) of an antibody specific for a tumor-associated antigen (TAA) linked via a hinge and transmembrane region to the cytoplasmic domain of a T cell signaling molecule.

[0004] By introducing a chimeric antigen receptor (CRA) into T cells (hereafter referred to as CAR T cells), T cells are generated that are engineered to recognize tumor cells as "non-self" and thus help the immune system eradicate the tumor cells. For example, CAR T cells can contact a surface molecule on a target cell, causing activation of the CAR T cell and leading to lysis of the target cell.

[0005] However, developing effective CAR T cells faces challenges, among others: i) A single surface molecule may not be specific enough and may be expressed on non-tumor cells (safety issues) ii) The surface molecule is not expressed by all tumor cells, and surviving (escaped) tumor cells lead to recurrence iii) Tumor cells block expression of surface molecules to evade therapy iv) The expression level of the antigen or the affinity of a single binder is too low to allow efficient T cell activation. v) The strong heterogeneity of antigen expression within and between tumor samples requires individualized profiling (kinetics) of tumor marker expression; some markers appear in all or none of the tumor cells of tumors from the same type.

[0006] A potential way to address this problem is to use more than one surface molecule as a target, i.e. so-called "DUAL CAR T cells" that detect more than one target on a given cell.

[0007] The "DUAL CAR T-cell" approach has not been adequately established so far, especially due to the fact that more complex combinations of multiple targets are needed to adequately attack the tumor, making this particular combination less likely to suit more than one or several patients (requiring individualization of treatment). Moreover, if the tumor continues to grow after the first treatment, it is very likely that new combinations of CARs will need to be found to further eradicate the tumor.

[0008] Furthermore, to perform personalized phenotyping of tumor marker expression for individual patients, CAR and CAR T cell combinations need to be developed in an acceptable time frame as dictated by the patient and indication, with the optimal time frame being several weeks in clinical practice.

[0009] Therefore, the success of any CAR cell-based immunotherapy for cancer depends on the rapid and reliable selection of antigens specific for each tumor cell.

[0010] A specific group of cell surface antigens have been found to be expressed on human cancer cells, but not expressed or expressed at lower levels on non-malignant cells. These antigens (also called "markers") can be used to identify and / or mark and / or destroy and / or disable the escape mechanisms of such cancer cells via ligands that specifically bind to the markers.

[0011] Therefore, the object of the present invention was to provide a method for selecting antigens specific to cancer cells that are not expressed in healthy tissues in order to design cells that kill / lyse cancer cells without attacking non-tumor cells. Summary of the Invention [Means for solving the problem]

[0012] A first object of the present invention is to provide a cell sample containing tumor cells and non-tumor cells; - contacting the tissue with a conjugate comprising a fluorescent moiety and an antigen recognition moiety (hereinafter referred to as "conjugate"); - removing unbound conjugates from the tissue and detecting cells bound to the conjugates by fluorescent radiation emitted by the fluorescent moiety of the original conjugate. - quenching the fluorescence radiation emitted by the fluorescent moiety of the conjugate; a method for providing a cell comprising a chimeric antigen receptor (CAR) specific for one or more target antigens exposed on a tumor cell, the method being characterized by repeating In this case, the conjugate comprises a fluorescent moiety and an antigen recognition moiety that recognizes a different antigen, until at least two conjugates with antigen recognition moieties that recognize different antigens are identified that bind to tumor cells but essentially do not bind to non-tumor cells; At least two of the identified antigen recognition moieties, preferably two, three, four or five antigen recognition moieties, are provided to a cell or cell sample or cell population as a chimeric antigen receptor (CAR).

[0013] The cells comprising a chimeric antigen receptor (CAR) may be selected from the group consisting of T cells, NK cells, or cells genetically engineered to destroy (lyse) other cells when triggered by binding to other cells.

[0014] The identified conjugates do not have to bind to all tumor cells, as the tumor may be destroyed even if only a portion of the tumor cells are identified or attacked. Preferably, the method of the invention is carried out until at least two conjugates with antigen recognition moieties that recognize different antigens are identified and bind to at least 1%, more preferably at least 10% and most preferably at least 30% of the tumor cells.

[0015] When the conjugate is desired to bind to 0% of non-tumor cells, targeting a certain amount of non-tumor cells is acceptable for treatment. Preferably, the method of the present invention is carried out until at least two conjugates with antigen recognition moieties that recognize at least different antigens are identified and bind to tumor cells of several types of tissues or organs and to up to 10%, more preferably up to 5% and most preferably up to 1% of non-tumor cells. The term "several types of tissues or organs" refers, for example, to the pancreas or liver. Effect of the Invention

[0016] The method of the present invention allows for the rapid identification of one or more sets of two conjugates with antigen recognition moieties that recognize different antigens, and one or more different cells with at least two of the antigen recognition moieties subsequently identified as chimeric antigen receptors (CARs). Providing more than one set of conjugates and more than one CAR cell increases the chances of destroying tumor cells and / or targeting tumor cells to downregulate antigens and escape the target. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 shows the general domains of a chimeric antigen receptor (CAR). [Diagram 2] FIG. 2 shows the general structural concept of the chimeric antigen receptor. [Diagram 3] FIG. 3 shows a schematic diagram of the function of CAR-T cells providing AND logic. [Figure 4] FIG. 4 shows a schematic of the function of CAR-T cells providing NOT logic. [Diagram 5] FIG. 5 shows the staining of one and the same pancreatic cancer specimen with multiple conjugates containing fluorescent moieties and antigen recognition moieties to detect markers expressed in pancreatic cancer cells. [Figure 6] FIG. 6 shows comparative labeling of select markers in pancreatic cancer cells and healthy tissue to detect marker combinations that specifically label pancreatic cancer cells but not healthy tissue. [Figure 7] FIG. 7 shows labeling of high-grade serous ovarian cancer to identify potential target candidates. [Figure 8] FIG. 8 shows comparative expression analysis of select markers in high-grade serous ovarian cancer and healthy tissue to detect marker combinations that specifically label ovarian cancer cells but not healthy tissue.

[0018] definition The term "chimeric antigen receptor" or "CAR" refers to genetically engineered cells with new specificities derived from a different source.

[0019] The term "tumor" is medically known as a "neoplasm." Not all tumors are cancerous; benign tumors do not invade adjacent tissues and do not spread throughout the body. The methods of the present invention can be applied to both neoplasms and benign tumors.

[0020] The term "cancer" refers to a broad group of diseases involving unregulated cell growth. In cancer, cells (cancerous cells) divide and grow uncontrollably, forming malignant tumors and invading nearby sites in the body. Cancer can also spread to more distant sites in the body via the lymphatic system or bloodstream.

[0021] The terms "binder" or "specifically binds" or "specific for" in reference to an antigen-binding domain of a ligand such as an antibody, a fragment thereof, or of a CAR refer to an antigen-binding domain that recognizes and binds a particular antigen, but does not substantially recognize or bind other molecules in a sample. An antigen-binding domain that specifically binds to an antigen from one species may also bind to antigen from other species. This cross-species reaction does not violate the definition of a specific antigen-binding domain. An antigen-binding domain that specifically binds to an antigen may also bind to different allelic forms (allelic variants, splice variants, isoforms, etc.) of that antigen. This cross-reaction does not violate the definition of a specific antigen-binding domain.

[0022] As used herein, the terms "genetically engineered cells" and "genetically modified cells" can be used interchangeably. These terms refer to the inclusion and / or expression of foreign genes or nucleic acid sequences that in turn modify the genotype or phenotype of the cell or its progeny. In particular, these terms refer to cells, preferably T cells, that have been engineered by recombinant methods well known to those skilled in the art to stably or transiently express peptides or proteins that are not naturally expressed in these cells. For example, T cells are engineered to express artificial constructs, such as chimeric antigen receptors, on their cell surface. For example, a sequence encoding a CAR may be delivered into the cell using a retroviral or lentiviral vector.

[0023] The term "target" as used herein refers to an antigen or epitope associated with a cell that is to be specifically recognized by an antigen-binding domain, such as an antibody or the antigen-binding domain of a CAR. The antigen or epitope for antibody recognition can be bound to the cell surface, but may also be secreted, be part of the outer cell membrane, or be shed from the cell.

[0024] The term "antibody" as used herein refers to polyclonal or monoclonal antibodies and fragments thereof, which may be produced by methods well known to those skilled in the art. The antibody may be of any species, e.g., mouse, rat, sheep, human. For therapeutic purposes, if a non-human antigen-binding fragment is to be used, it may be humanized by any method known to those skilled in the art. The antibody may also be a modified antibody (e.g., an oligomer, a truncated antibody, an oxidized antibody, and a labeled antibody).

[0025] As used herein, the term "killer cell" refers to a cell that can kill / destroy / lyse other cells, such as cancer cells. Most frequently, T cells, NK cells, dendritic cells and macrophages can be used as killer cells.

[0026] The term "genetically engineered killer cells" as used herein refers to killer cells that have been genetically modified to enable specific killing of target cells, e.g., cells modified with a CAR against a target to kill tumor cells expressing the respective target.

[0027] Detailed Description of the Invention The method of the present invention allows for the rapid identification of a suitable set of conjugates, followed by rapid genetic engineering of suitable CAR cells to destroy tumor cells in a cell sample, the cell sample containing tumor cells and non-tumor cells preferably being derived from the same tissue or organ from the same patient.

[0028] To distinguish between tumor and non-tumor cells in a cell sample, a person skilled in the art, e.g., a pathologist, may determine whether the cell sample contains a tumor, and if so, which part of the cell sample, according to known criteria or experts in oncology. Such criteria may include, for example, guidelines published by the Arbeitsgemeinschaft der Wissenschaftlichen Medizinischen Fachgesellschaften (AWMF), and may include, for example, morphology of cells, tissue structure, ploidy, biomarker expression (e.g., Her2, p53, BRAC1, APC, EGFR, etc.).

[0029] The cell sample is then contacted with a plurality of binders to characterize the recognition pattern and select binders specific to tumor cells and binders specific to non-tumor cells, such that the identified binders can be used for subsequent treatment as later identified.

[0030] In a different method, at least two conjugates with antigen recognition moieties recognizing different antigens bind to at least 20% of the tumor cells and less than 5% of the non-tumor cells of the cell sample. Preferably, at least two conjugates with antigen recognition moieties recognizing different antigens bind to at least 50% of the tumor cells and less than 5% of the non-tumor cells of the cell sample. More preferably, at least two conjugates with antigen recognition moieties recognizing different antigens bind to at least 70% of the tumor cells and less than 5% of the non-tumor cells of the cell sample.

[0031] In another variation, the at least two conjugates comprising an antigen recognition moiety are characterized by destroying at least 10 times, preferably at least 50 times, and more preferably at least 100 times more tumor cells than non-tumor cells in a cell sample.

[0032] The methods of the invention involve identifying at least two conjugates with antigen recognition moieties that recognize different antigens that bind substantially to tumor cells but not to non-tumor cells.

[0033] When the CAR comprises at least two antigen recognition moieties, the CAR may be a trigger for implementing various logic gates. Logic gates are described in the literature as CAR cells of the "AND" type, "NOT" type, and "OR" type. As will be disclosed in more detail later, in the method of the present invention, the at least two antigen recognition moieties are provided as AND type, OR type, or NOT type, and / or at least two of the antigen recognition moieties are provided as ADAPTER type. In all these variants, the at least two antigen recognition moieties are provided as chimeric antigen receptors (CARs).

[0034] Chimeric antibody receptor (CAR) As generally shown in Figures 1 and 2, a chimeric antigen receptor (CAR) may comprise an extracellular domain comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain. The extracellular domain may be linked to the transmembrane domain by a linker. The extracellular domain may also be linked to a signal peptide.

[0035] The term "signal peptide" refers to a peptide sequence that directs the transport and localization of a protein within a cell, for example to a certain organelle (eg, the endoplasmic reticulum) and / or to the cell surface.

[0036] The term "antigen-binding domain" refers to the region of a CAR that specifically binds to an antigen (and thereby can target cells containing the antigen). A CAR obtained by the present invention may comprise one or more antigen-binding domains. Generally, the antigen-binding domain of a CAR is extracellular. The antigen-binding domain may comprise an antibody or a fragment thereof. The antigen-binding domain may comprise, for example, a full-length heavy chain, a Fab fragment, a single-chain Fv (scFv) fragment, a bivalent single-chain antibody or a diabody. Any molecule that specifically binds to a given antigen can be used as an antigen-binding domain, for example an affibody or a ligand-binding domain from a naturally occurring receptor. Often, the antigen-binding domain is an scFv. Usually, in an scFv, the variable regions of the heavy and light chains of an immunoglobulin are fused by a flexible linker to form the scFv. Such a linker may be, for example, a "(G4 / S1)3-linker".

[0037] The transmembrane domain of the CAR may, for example, comprise the sequence of the transmembrane domain of 4-1BB, CD8 and / or CD28. Additionally, an activating intracellular signaling domain may be present, including the sequence of the intracellular signaling domain of CD28, CD137 or CD3 zeta.

[0038] Instead of an activating intracellular signaling domain, a CAR may include an inhibiting intracellular signaling domain that has the sequence of an intracellular signaling domain such as PD1 or CTLA4.

[0039] In a preferred variant of this embodiment, the chimeric antigen receptor (CAR) comprises an antigen-binding domain specific for CD20 with or without additional antigen-binding domains, which is joined to a transmembrane domain and one or more signaling domains. This variant is shown by way of example in Figure 2A.

[0040] In some cases, it is beneficial for the antigen-binding domain to be derived from the same species as the species in which the CAR is to be used. For example, if the CAR is planned to be used therapeutically in humans, it may be beneficial for the antigen-binding domain of the CAR to comprise a human or humanized antibody or a fragment thereof. Human or humanized antibodies or fragments thereof can be produced by various methods well known to those skilled in the art.

[0041] The term "spacer" or "hinge" as used herein refers to a hydrophilic region between the antigen-binding domain and the transmembrane domain. The CAR identified by the present invention may include an extracellular spacer domain, but it is also possible to omit such a spacer. The spacer may include an Fc fragment of an antibody or a fragment thereof, a hinge region of an antibody or a fragment thereof, a CH2 or CH3 region of an antibody, an accessory protein, an artificial spacer sequence, or a combination thereof. A prominent example of a spacer is the CD8 alpha hinge.

[0042] The transmembrane domain of CAR can be derived from any desired natural or synthetic source for such a domain. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein.

[0043] The cytoplasmic domain or intracellular signaling domain of the CAR of the present invention is involved in the activation of at least one normal effector function of the immune cell in which the CAR is expressed. "Effector function" refers to a specialized function of a cell, for example, effector function in T cells can be cytolytic activity or helper activity, which includes secretion of cytokines. The intracellular signaling domain refers to the portion of a protein that transmits an effector function signal and instructs the cell expressing the CAR of the present invention to perform a specialized function. The intracellular signaling domain can include any complete or truncated portion of the intracellular signaling domain of a given protein that is sufficient to transmit an effector function signal.

[0044] Prominent examples of intracellular signaling domains for use in CARs include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement.

[0045] For CARs with at least two antigen-binding domains, the antigen-binding domains may have different functions. The method of the present invention shortens the development time for new CAR cells suitable for tumor therapy, which can be provided as "AND", "NOT" or "OR" CAR cells.

[0046] In one embodiment of the present invention, the CAR cell is only activated, i.e., the signal of the signal domain is induced only when both antigen-binding domains bind to their respective antigens. Such a CAR cell is hereafter referred to as "AND-CAR". The antigen-binding domain of an "AND-CAR" cell may be identified by the method of the present invention, for example, by identifying two antigens present in tumor cells, none or at most one of which is expressed in healthy cells. Figure 3 (published in Transl Cancer Res 2016;5(S1):S61-S65) shows how AND-CAR is inactivated by healthy cells (i) and (ii), but activated by tumor cells (iii).

[0047] In another embodiment of the present invention, the CAR cells are only activated, i.e., the signal of the signal domain is induced only when one antigen-binding domain binds to its respective antigen and not the other. Such CAR cells are hereafter referred to as "NOT-CAR". The antigen-binding domain of a "NOT-CAR" cell may be identified by the method of the present invention, for example, by identifying two antigens present in a healthy cell, only one of which is expressed in a tumor cell. Figure 4 (published in Transl Cancer Res 2016;5(S1):S61-S65) shows how a NOT-CAR is inactivated by a healthy cell (i) but activated by a tumor cell (ii).

[0048] In yet another embodiment of the present invention, the CAR cells are already activated, i.e., the signal of the signal domain is induced when only one of the two antigen-binding domains binds to its respective antigen. Such CAR cells are hereinafter referred to as "OR-CAR". The antigen-binding domain of the "OR-CAR" cell may be identified by the method of the present invention, for example, by identifying two antigens present on tumor cells but not on healthy cells.

[0049] Further reduction in processing time can be achieved by using the so-called "ADAPTER CAR" technology. "ADAPTER CAR" cells contain a signaling domain, a transmembrane domain and a linker domain that does not have antigen recognition properties. Then, one or more antigen recognition moieties are joined to this linker domain (linker + molecule = adapter). Thus, in the method according to the present invention, at least two with antigen recognition moieties as chimeric antigen receptors (CARs) may be provided as ADAPTER type.

[0050] In a variation of the invention, a cell is provided that contains biotin as an antigen recognition moiety of a chimeric antigen receptor (CAR), and a conjugate of at least two of the identified antigen recognition moieties with an anti-biotin moiety is provided to the cell, by conjugating the conjugate with the cell.

[0051] In another variation of the invention, a cell is provided that comprises biotin as an antigen recognition moiety of a first chimeric antigen receptor (CAR) and a second epitope as an antigen recognition moiety of a second chimeric antigen receptor (CAR), and a conjugate of the at least two identified antigen recognition moieties with an anti-biotin moiety and a second moiety, and conjugating the conjugate with the cell, thereby providing the at least two identified antigen recognition moieties as a chimeric antigen receptor (CAR).

[0052] Suitable linkers are molecules that are not present in the mammalian (human) body or that have low affinity for antibodies naturally occurring in the mammalian (human) body, for example biotin units or modified biotins that are identified by an appropriate adaptor, such as an anti-biotin antibody conjugated to the desired conjugated antigen recognition moiety.

[0053] The "ADAPTER CAR" technology is disclosed in International Application No. EP2017 / 077545 (PCT / EP2017 / 077545), which uses the term "Linker / Tag Epitope" (LLE) for the linker attached to the CAR cell.

[0054] Preferably, the LLE does not induce an immune response in a subject intended to be treated with cells expressing the CAR. This can be achieved by using the extracellular LLE-binding domain of the CAR, which is derived from a naturally occurring epitope-recognizing molecule, to bind the LLE with higher selectivity than endogenous targeting moieties that do not have a linker moiety, i.e., molecules that naturally occur in the subject (self-antigens).

[0055] For example, the extracellular LLE-binding domain of a CAR binds to said LLE with an affinity that is at least 2-fold, preferably at least 5-fold, and more preferably at least 10-fold higher than said naturally occurring molecule.

[0056] In the case of "ADAPTER" CAR cells, CAR cells with linker units and adapters are applied to patients in parallel. The advantage of this embodiment is that CAR cells with linkers can be produced independently of the identified antigen recognition moiety and do not need to be developed from scratch. Only the adapters need to be generated and tested. This approach also allows the intensity and kinetics of treatment to be controlled, similar to conventional chemotherapy.

[0057] Discovery Process To investigate multiple potential binders or conjugates, it is preferable to quench the fluorescence radiation emitted by the fluorescent moiety by enzymatic degradation of the conjugate. Such conjugates are disclosed, for example, in European Patent Publication No. 3037821 (EP3037821A1), European Patent Application No. 16203689.1 (EP16203689.1) or European Patent Application No. 16203607.3 (EP 16203607.3).

[0058] Instead, the fluorescent radiation emitted by the fluorescent moiety is quenched by the radiation.

[0059] Use for the treatment of cancer The cell populations obtainable by the methods of the present invention may be used as pharmaceutical compositions, preferably in methods for the treatment of human cancer, in particular human pancreatic cancer.

[0060] The pharmaceutical composition preferably comprises a genetically engineered cell population expressing a CAR obtained by the method of the invention. In a variant of the invention, the pharmaceutical composition is used in combination with chemotherapy, radiation, or immunomodulatory agents for the treatment of cancer.

[0061] The cancer to be treated may include hematopoietic tissue blood cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumor, minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological cancer, and solid tumor, or any combination thereof. In other embodiments, the cancer includes hematological cancer, such as leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), or chronic myeloid leukemia (CML)), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin's lymphoma, or Hodgkin's lymphoma), or multiple myeloma, or any combination thereof. Additionally, the cancer may include cancer of the oral cavity and pharynx (tongue, mouth, pharynx, head and neck), cancer of the digestive system (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct, gallbladder, pancreas), cancer of the respiratory system (larynx, lung and bronchi), cancer of the bones and joints, cancer of soft tissue, cancer of the skin (melanoma, basal cell carcinoma and squamous cell carcinoma), childhood tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), tumors of the central nervous system (brain, astrocytoma, glioblastoma, glioma), and adult carcinomas including cancer of the breast, reproductive system (cervix, uterus, ovary, vulva, vagina, prostate, testes, penis, endometrium), urinary system (bladder, kidney and renal pelvis, ureter), eye and orbit, endocrine system (thyroid), and brain, and other nervous system, or any combination thereof.

[0062] The treatment of cancer may include any method involving at least one of the disclosed antigens or any combination of the disclosed antigens as target molecules. Such a method may be, for example, treatment with an agent that binds to the antigen and affects the viability of, and preferably kills, cancerous cells expressing this antigen. Examples are oncolytic viruses, BiTEs®, ADCC and immunotoxins, which have already been disclosed.

[0063] For treatment, immune cells, such as T cells, of a subject may be isolated. The subject may be afflicted with said cancer or may be a healthy subject. These cells are genetically modified in vitro or in vivo to express one or more CARs of the invention. These engineered cells may be activated and expanded in vitro or in vivo. In cell therapy, these engineered cells may be injected into a recipient in need thereof. These cells may be a pharmaceutical composition (the cells + a pharma- tically acceptable carrier). The injected cells may kill (or at least stop the growth of) cancerous cells expressing one or more of the disclosed antigens in the recipient. The recipient may be the same subject from which the cells were obtained (autologous cell therapy) or from another subject of the same species (allogeneic cell therapy).

[0064] For example, cells carrying the following antigen recognition moieties as chimeric antigen receptors (CARs) may be used to treat certain types of cancer.

[0065] a) CLA, TSPAN8 as positive markers exclusively for tumors, e.g. pancreatic tumors.

[0066] b) CLA as a positive marker and CD45 as a NOT marker to distinguish tumor, e.g. pancreatic tumor, from healthy cells.

[0067] c) Epcam and CD90 as positive markers exclusively for tumors, e.g. ovarian cancer.

[0068] d) SSEA4 as a positive marker and CD34 and CD133 as negative markers to distinguish tumor, e.g., breast cancer, from healthy cells. EXAMPLES

[0069] The following examples are intended to illustrate the present invention in more detail, but the present invention is not limited to these examples.

[0070] Example 1: Identification of conjugate combinations comprising a fluorescent moiety and an antigen recognition moiety in combination that allows for the discrimination between pancreatic cancer (PaCa) and non-PaCa cells.

[0071] Pancreatic carcinomas (PaCa) from patients were analyzed by staining with different antibody fluorochrome conjugates, followed by imaging and destaining.

[0072] Frozen sections of PaCa samples were fixed and each specimen was stained with 101 fluorescently labeled antibodies and the nuclear labeling dye DAPI (Figure 5). Images so obtained were analyzed for antigen quantification and pattern recognition at the single pixel and divided cell levels.

[0073] To narrow the selection of potential targets, antibody conjugates were selected that showed staining patterns similar to those observed when the same specimens were stained with anti-EpCAM+(CD326) antibody fluorochrome conjugates. EpCAM is well known in the field for its expression on various human epithelial tissues and carcinomas. This was true, for example, for CLA, CD66c, CD318, and TSPAN8, as shown in FIG. 5.

[0074] Subsequently, 20 fluorochrome-conjugated antibodies of potential targets, such as CLA, CD66c, CD318 and TSPAN8, were assessed for their staining patterns on 16 healthy or near-normal (NAT) tissues in an identical staining / imaging / destaining process, the results of which are shown in Figure 6. This was performed to assess off tumor expression of antigens of interest and allow prediction of potential on-target off-tumor toxicity of potential targets. An example of this analysis is shown in Figure 6a-c for the markers CLA, CD66c, CD318 and TSPAN8.

[0075] The above examples show that these markers are expressed in PaCa samples, and we investigated whether these markers are also expressed in healthy tissues. For example, CD318 is also found in colon, lung, and liver tissues. Therefore, using CD318 as a single marker to direct CAR T cells will most likely result in on-target off-tumor toxicity in the indicated tissues.

[0076] Thus, the data from the above experiments were analyzed to find markers that show expression in the same pancreatic cancer specimens but not in the same healthy tissue specimens. As shown in Figures 6a-c, many combinations could be identified by visual comparison of images or bioinformatics analysis. Visual comparison indicates that if two images show substantially identical expression patterns for two different zygotes, each marker is expressed in substantially the same cells. On the other hand, if two images show different expression patterns for two different zygotes, each marker is not expressed in the same cells. For example, CD318 and CLA do not show combined expression in healthy tissue specimens.

[0077] Finally, the resulting target candidates were tested on a multi-patient sample array using 12 different PaCa and 1 ProCa samples to assess the breadth of applicability of these marker combinations, where the markers were shown to be co-expressed in the majority of pancreatic cancers.

[0078] Taken together, CLA, CD66c, CD318 and TSPAN8 have been identified as candidates for a CAR T cell approach to eradicate pancreatic cancer with minimal risk of affecting healthy tissue.

[0079] Example 2: Identification of conjugate combinations comprising a fluorescent moiety and an antigen recognition moiety in combination that allows discrimination between ovarian cancer (OvCa) and non-OvCa cells.

[0080] Fresh frozen high-grade serous ovarian carcinoma resections were sliced ​​and fixed with acetone. Screening was performed using a new high-content imaging platform that allows fully automated cyclic fluorescent imaging of individual biological samples. Sequential staining of two OvCa specimens with 20 antigen recognition moieties conjugated to fluorescent moieties showed expression of many antigens, including FOLR1, CD227, and CD340, in addition to EpCAM (CD326), a well-known marker of human epithelial tissue and carcinoma (Figure 7).

[0081] The target candidates CD326, FOLR1, CD227, and CD340 were validated using a staining / imaging / destaining process by comparing their expression levels in two independent high-grade serous ovarian cancer resection samples and healthy human tissue samples (kidney, lung, heart, skin, and colon) (Figure 8a), and in a third independent high-grade serous ovarian cancer resection sample and healthy human tissue samples (liver, pancreas, thyroid, and pituitary) (Figure 8b).

[0082] Data analysis and further co-staining revealed that all four markers presented were expressed in OvCa samples, but also in healthy tissues. However, we found that the expression in healthy tissues at least partially identifies the possibility of combining two or more markers to obtain selective labeling of OvCa cells. For example, CD227 and FOLR1 both showed expression in the majority of OvCa cells. CD227 showed more prominent expression in liver, lung and pancreas, while FOLR1 showed prominent expression in kidney, lung and skin. We found that when both markers were combined to examine the co-expression of cells, the healthy tissues did not show any co-expression of cells in a substantial proportion of cells. Therefore, the combination of CD227 and FOLR1 is an option to eradicate OvCa cells with CAR T (AND CAR T cells) that are activated only when both antigens CD227 and FOLR1 are not bound or are significantly reduced binding with on-target off-tumor toxicity. On the other hand, when performing similar analyses as outlined above, we found that, for example, the combination of FOLR1 and podoplanin offers the potential for NOT CAR T cell therapy in OvCa. FOLR1 is expressed not only in OvCa cells but also in kidney, lung and skin, whereas podoplanin shows no cellular co-expression with FOLR1 in OvCa cells and is strongly expressed in kidney, lung and skin.

[0083] Thus, using CAR T cells that are activated by the detection of FOLR1 but deactivated or inhibited by the detection of podoplanin results in efficient killing of OvCa cells, leaving healthy cells unaffected because they do not express FOLR1 or express podoplanin.

Claims

1. 1. A method for providing T cells comprising at least one chimeric antigen receptor (CAR) specific for one or more target antigens exposed on a tumor cell, comprising: - contacting a cell sample with a conjugate comprising a fluorescent moiety and an antigen recognition moiety, said cell sample comprising tumor cells and non-tumor cells; - removing unbound conjugate from said cell sample and detecting tumor and non-tumor cells bound to the conjugate by fluorescence radiation emitted by the fluorescent moiety of the conjugate; - quenching the fluorescence emitted by the fluorescent moiety of the conjugate; wherein the conjugate is at least two conjugates comprising a fluorescent moiety and an antigen recognition moiety that recognize different antigens; repeating the steps until at least two conjugates having antigen recognition moieties that recognize different antigens are identified in a combination that distinguishes between tumor cells and non-tumor cells; and providing at least two antigen recognition moieties that recognize the identified antigens different from each other to tumor cells and non-tumor cells as chimeric antigen receptors (CARs), wherein the at least two conjugates bind to at least 20% of tumor cells contained in the cell sample and to less than 5% of non-tumor cells contained in the cell sample.

2. 2. The method of claim 1, wherein at least two antigen recognition moieties are provided in AND or NOT form, The AND type is a type in which a CAR cell is activated and induces a signal from a signal domain only when both antigen-binding domains bind to their respective antigens; The method of claim 1, wherein the NOT type is a type in which the CAR cell is activated to induce a signal of the signal domain only when one antigen-binding domain binds to the respective antigen and the other does not bind to the antigen.

3. 3. The method of claim 1 or 2, characterized in that at least two of the conjugates bind tumor cells at least 10 times more than non-tumor cells of the cell sample.

4. 4. The method according to claim 1, wherein the fluorescence radiation emitted by the fluorescent moiety is quenched by enzymatic degradation of the conjugate.

5. 4. The method according to claim 1, wherein the fluorescence radiation emitted by the fluorescent moiety is quenched by radiation.

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