Kits for use in the treatment of hematological cancers

Abstract

The present invention relates to a kit for use in treating hematological cancer comprising T cells comprising a nucleotide sequence encoding a switchable chimeric antigen receptor (CAR) and a targeting module comprising at least one hematological cancer cell-binding domain and a tag-binding domain or tag, wherein at least one administration of a clinically effective amount of the T cells is administered to a subject having hematological cancer and a first administration of the targeting module is administered sequentially to the subject.

Description

[Technical Field]

[0001] The present invention relates to a kit for use in treating hematological cancer comprising T cells comprising a nucleotide sequence encoding a switchable chimeric antigen receptor (CAR) and a targeting module comprising at least one hematological cancer cell-binding domain and a tag-binding domain or tag, wherein at least one administration of a clinically effective amount of the T cells is administered to a subject having hematological cancer and a first administration of the targeting module is administered sequentially to the subject. [Background technology]

[0002] Chimeric antigen receptors (CARs) are artificial receptors that consist of a binding moiety that provides antigen specificity, such as a single-chain variable fragment (scFv) that recognizes surface antigens on tumor cells, a transmembrane domain, and one or several signaling chains derived from immune receptors, such as CD3ζ, that activate immune cells (Cartellieri et al., 2010) (Non-Patent Document 1).

[0003] Immune cells genetically engineered to express CARs can be used to bind cells or tissue structures expressing the appropriate target of the CAR-binding moiety. Crosslinking results in the induction of signaling pathways via the CAR signaling chain, which alters the biological properties of the CAR-grafted immune cells. Conversely, activation of CARs in genetically engineered regulatory T cells (Tregs) leads to the activation of Treg-specific immunomodulatory and immunosuppressive mechanisms, such as the secretion of interleukin (IL)-10 or transforming growth factor beta (TGF-β). Adoptive transfer of immune cells engineered with chimeric antigen receptors (CARs) is currently considered a highly promising therapeutic option for treating otherwise incurable malignant, infectious, or autoimmune diseases.

[0004] However, conventional CAR technology poses several significant challenges that need to be resolved before this therapeutic approach can be widely applied in clinical settings. First, several safety issues must be addressed. To date, immune responses from conventional CAR-engineered T cells are difficult to control after infusion into patients. The rate of serious adverse events is high (Titov et al., 2018) (Non-Patent Document 2). In particular, unexpected target gene expression in normal tissues can trigger rapid and severe immune responses of engineered T cells against normal cells, which can result in severe side effects (Morgan et al., 2010) (Non-Patent Document 3). Furthermore, because CAR-T cells are a new class of self-renewing cellular drugs, infused T cells can proliferate vigorously in the presence of large tumor burdens, resulting in tumor lysis syndrome, cytokine release syndrome, and macrophage activation syndrome (Brudno and Kochenderfer, 2016) (Non-Patent Document 4). Another drawback of conventional CAR technology is the limited retargeting of engineered T cells to a single antigen. Such a monotherapy approach carries the risk of tumor escape variants that lose their target antigen during treatment. The emergence of tumor escape variants after several months under conventional CAR T cell therapy has already been observed in clinical trials (Sotillo et al., 2015) (Non-Patent Document 5). Taken together, these obstacles limit the application of CAR T cells to very few indications. In fact, examples of clinical efficacy have so far been limited to CAR T cells targeting CD19 and BCMA.

[0005] The modular, switchable "universal" CAR T (UniCAR) approach overcomes these limitations by separating the antigen recognition and activation domains of the CAR into two separate operational units. T cells are engineered to express a CAR with a universal binding domain that recognizes a tag (Cartellieri et al., 2016). Antigen specificity is provided by a soluble adapter molecule composed of the antigen binding domain fused to a tag recognized by the universal CAR. Cartellieri et al. describe the in vitro and in vivo treatment of CD33-positive and / or CD123-positive acute myeloid leukemia cells.

[0006] Following the UniCAR approach (EP2990416A1) (Patent Document 1) for recognizing various antigens, a reversed universal CAR (RevCAR) approach is known (EP3581200A1) (Patent Document 2), which promotes the binding of immune cells engineered to express a tag-containing RevCAR to target cells via an adapter molecule containing a tag-binding domain and a target cell-binding domain.

[0007] Loff et al. describe the treatment of CD123-positive leukemia using a UniCAR platform containing a small peptide derived from the human nuclear La / SS-B protein as the extracellular domain and CD28 / CD3 zeta as the intracellular domain (Loff et al., 2020). CARs have been tested in mouse models of acute myeloid leukemia (AML).

[0008] Kittel-Boselli et al. describe the use of the RevCAR platform to target acute myeloid leukemia, specifically patient-derived AML cells expressing CD33 and CD123, where RevCAR consists of the extracellular peptide epitope E5B9 or E7B6 and the CD28(28) hinge domain (HiD), CD28 transmembrane domain (TMD), intracellular CD28 costimulatory domain (CSD), and CD28 zeta (3z) activating signaling domain (ASD) (Kittel-Boselli et al., 2021) (Non-Patent Document 8).

[0009] Wermke et al. have disclosed a CAR-T cell therapy for patients with relapsed / refractory acute myeloid leukemia using UniCAR-T cells and a targeting module (TM123) that recognizes peptide motifs and antigens present on cancer cells, particularly CD123 (Wermke et al., 2021) (Non-Patent Document 9). The dose administered to patient 1 was 1·10 8 Patient 2 received 2.5 10 8 Patient 3 received 2.5 10 UniCAR cells / day and 0.5 mg / day of TM123. 8 They received UniCAR cells / day and 1 mg / day of TM123. In three patients, UniCAR was administered on day 1 and TM123 was administered continuously from day 0 to day 24.

[0010] Furthermore, switchable CAR-T approaches such as UniCAR or RevCAR offer the possibility of resting CAR T cells between activation and stimulation cycles by interrupting the application of soluble adaptor molecules, which is expected to prevent the exhaustion seen with continuous stimulation of conventional CAR T cells and thereby improve their survival (Weber et al., 2021) (Non-Patent Document 10).

[0011] WO2018 / 160622A1 (Patent Document 3) discloses a method for treating a patient with cancer by administering to the patient a composition containing CAR T cells and administering to the patient a small molecule linked to a targeting moiety by a linker to reduce off-target toxicity and more precisely control CAR T cell activation. A small molecule ligand linked to the targeting moiety by a linker is used as a bridge between the cancer and CAR T cells, directing the CAR T cells to the cancer for cancer amelioration. WO2018 / 160622A1 describes methods for treating cancer, in which a small molecule is administered before the first and second doses of CAR T cells, particularly CAR T cells administered as a mixture of CAR T cells and non-transformed T cells; a method further comprising administering folic acid (salt); and a method for inhibiting or preventing cytokine release syndrome in a patient by sequentially administering a small molecule followed by CAR T cells, completing the sequential administration of the targeting module. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] EP2990416A1 [Patent Document 2] EP3581200A1 [Patent Document 3] WO2018 / 160622A1 [Non-patent literature]

[0013] [Non-Patent Document 1] Cartellieri et al., 2010 [Non-patent document 2] Titov et al., 2018 [Non-patent document 3] Morgan et al., 2010 [Non-patent document 4] Brudno and Kochenderfer, 2016 [Non-patent document 5] Sotillo et al., 2015 [Non-patent document 6] Cartellieri et al., 2016 [Non-Patent Document 7] Loff et al., 2020 [Non-patent document 8] Kittel-Boselli et al., 2021 [Non-Patent Document 9] Wermke et al., 2021 [Non-Patent Document 10] Weber et al., 2021 Summary of the Invention [Problem to be solved by the invention]

[0014] It is an object of the present invention to provide kits for use in the treatment of hematological cancers with reduced side effects and / or improved efficacy. [Means for solving the problem]

[0015] The above object has been achieved by a first aspect of the present invention, which relates to a kit for use in the treatment of blood cancer, comprising: a. A T cell comprising a nucleotide sequence encoding a switchable chimeric antigen receptor (CAR), wherein the switchable chimeric antigen receptor (CAR) tag-binding domains or tags, extracellular hinge and transmembrane domains, and an intracellular signaling domain comprising at least one signaling domain; T cells, including: b. a targeting module comprising: at least one hematological cancer cell binding domain, and tag-binding domains or tags, a targeting module comprising: Including, the tag-binding domain of the targeting module binds to the tag of the switchable CAR, or the tag of the targeting module binds to the tag-binding domain of the switchable CAR; a first dosage of the targeting module is administered continuously to the subject with hematological cancer from day 0 of treatment (start of treatment) at a dosage quantity ranging from 0.1 mg / day to 20 mg / day for a period of between 10 days and 25 days; At least one administration of a clinically effective amount of T cells comprising a nucleotide sequence encoding a switchable CAR is administered once within the range of days 0 to 5 of treatment, in a dose of 1 10 8 ~1·10 9 administered to a subject at a dosage within the range of cells; 7 to 21 days after administration of the first administration of the targeting module, at least one further administration of the targeting module is administered continuously to the subject for a period of between 4 and 25 days at a dosage in the range of 0.1 mg / day to 20 mg / day. The kit.

[0016] According to the present invention, a first administration of a targeting module is administered to a subject before, simultaneously with, and after administration of T cells comprising a nucleotide sequence encoding a switchable CAR. Advantageously, administration prior to administration of T cells comprising a nucleotide sequence encoding a switchable CAR allows for stimulation of the switchable CAR upon infusion, which is important for T cell engraftment and proliferation.

[0017] According to the present invention, the kit is used for combination therapy, and treatment begins on day 0 of combination therapy.

[0018] Advantageously, the switchable CAR of the present invention used in combination with a targeting module can actively target target cells, such as tumor cells, and induce a significant anti-tumor response, and the anti-tumor response of the switchable chimeric antigen receptor is only induced in the presence of the targeting module. This effect can be reversibly interrupted by ceasing the application of the targeting module. Further advantageously, the pharmacokinetic and pharmacodynamic half-life of the targeting module is short, providing a rapid and reversible switch-off mechanism for the immune response mediated by it. Advantageously, the use of the administration regimen according to the present invention can effectively reduce side effects such as fever (≧38°C).

[0019] According to the present invention, a first administration of a targeting module serves as an induction cycle for therapy, and at least one further administration of a targeting module serves as a consolidation cycle. Preferably, the first administration and / or the at least one further administration are administered sequentially to the subject to achieve a steady-state concentration of the targeting module in the subject.

[0020] As used herein, the term "continuously" refers to administration with little or no interruptions or gaps, preferably no interruptions or gaps. The term includes administration with interruptions of 1 minute to 72 hours for changing infusions or to accommodate side effects.

[0021] As used herein, the term "switchable chimeric antigen receptor" refers to an artificial chimeric fusion protein, particularly a receptor comprising a tag or tag-binding domain, an extracellular hinge and transmembrane domain, and an intracellular signaling domain. The domains can be derived from different sources, and thus the receptor is called chimeric. Advantageously, the receptor can be bound to the tag-binding domain or tag of a different targeting module via the tag or tag-binding domain, which in turn binds to an antigen on target cells, particularly on blood cancer cells. Thus, the tag or tag-binding domain serves as a target cell-binding domain. According to the present invention, T cells comprising a nucleotide sequence encoding a switchable CAR express a switchable CAR comprising a tag-binding domain or tag, an extracellular hinge and transmembrane domain, and an intracellular signaling domain comprising at least one signaling domain.

[0022] As used herein, the term "domain" refers to a portion of a protein sequence that can exist and function independently from the rest of the protein.

[0023] In several embodiments, the T cells comprising a nucleotide sequence encoding a targeting module and / or a switchable CAR are administered in the form of a pharmaceutical composition.

[0024] The pharmaceutical composition is preferably administered parenterally, particularly preferably intravenously.In some embodiments, the pharmaceutical composition is in a form suitable for intravenous administration.Preferably, the pharmaceutical composition is a solution, emulsion or suspension.

[0025] In some embodiments, the pharmaceutical composition is an injectable buffer solution comprising a targeting module at a concentration in the range of 1 ng / ml to 500 mg / ml, preferably a targeting module at a concentration in the range of 1 μg / ml to 5 mg / ml.

[0026] In some embodiments, the pharmaceutical composition contains 1·10 5 ~1·108 a buffered solution for injection comprising T cells comprising a nucleotide sequence encoding a switchable CAR at a concentration ranging from 1 ng / mL to 500 mg / mL, and optionally a targeting module at a concentration ranging from 1 ng / mL to 500 mg / mL.

[0027] In some embodiments, the pharmaceutical composition comprises a T cell comprising a nucleotide sequence encoding a targeting module and / or a switchable CAR, and a pharmaceutically acceptable thinner or carrier.

[0028] In some embodiments, the carrier is selected from water, an aqueous buffer solution, 0.9% saline, 5% glucose, 5% xylitol, 0.3% glycine solution, Ringer's solution, or an amino acid solution. In further embodiments, the aqueous buffer solution is selected from aqueous histidine, sodium succinate, sodium citrate, sodium phosphate, or potassium phosphate buffer solutions having a pH value ranging from pH 5.0 to pH 7.0. In some embodiments, the aqueous buffer solution has a buffer concentration ranging from 1 mmol / L (mM) to 500 mM, preferably from 5 mM to 20 mM, and particularly preferably from 5 mM to 10 mM.

[0029] In some embodiments, the carrier comprises sodium chloride, preferably at a concentration in the range of 1 mM to 300 mM, with 150 mM being especially preferred.

[0030] In some embodiments, the pharmaceutical composition further comprises a stabilizer, preferably at a concentration in the range of 1 mM to 900 mM, particularly preferably in the range of 50 mM to 600 mM. In some embodiments, the stabilizer is sucrose, trehalose, or L-methionine.

[0031] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. The term "pharmaceutically acceptable excipient" refers to a compound that provides approximately physiological conditions and / or increases stability, such as an agent for adjusting the pH value and a buffering agent, an agent for adjusting toxicity, etc. In several embodiments, the pharmaceutically acceptable excipient is selected from sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and polysorbate-80, preferably polysorbate-80, in the range of 0.0001% (w / v) to 1% (w / v), particularly preferably in the range of 0.001% (w / v) to 0.1% (w / v).

[0032] In a further embodiment, the pharmaceutical composition is sterile. The pharmaceutical composition is sterilized by conventional, well-known techniques, including, but not limited to, sterile filtration.

[0033] In some embodiments, the pharmaceutical composition is used for application to a subject.

[0034] In some embodiments, the pharmaceutical compositions are lyophilized prior to storage or stored as a solution at ambient temperature or below ambient temperature, including, but not limited to, frozen storage.

[0035] In some embodiments, the pharmaceutical composition is reconstituted and / or diluted in an infusion solution and a stabilizer solution before administration to a subject. The solution used for reconstitution or infusion / stabilization can contain any of the ingredients mentioned for the pharmaceutical composition or similar ingredients.

[0036] In some embodiments, the hematological cancer is leukemia. In some embodiments, the subject has acute myeloid leukemia (AML).

[0037] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human.

[0038] As used herein, the term "tag" refers to a marker, particularly a peptide sequence or organic molecule, that is attached to peptides or proteins to enable them to bind to specific atoms, ions or molecules, particularly tag-binding domains.

[0039] In some embodiments, the tag is selected from organic molecules, including fluorescent labels, such as FITC (fluorescein isothiocyanate), and biotin.

[0040] In some embodiments, the tag is a peptide epitope tag. In further embodiments, the tag comprises 10 to 20 amino acids.

[0041] In some embodiments, the peptide epitope tag is a myc tag, a His tag, preferably a short linear peptide sequence derived from the yeast transcription factor GCN4 according to SEQ ID NO: 18, SEQ ID NO: 19 or a mutant thereof; a leucine zipper sequence, preferably a sequence according to SYNZIP1 to SYNZIP48, BATF, FOS, ATF4, ATF3, BACH1, JUND, NFE2L3, HEPTAD (Reinke et al., 2010), SEQ ID NO: 20 or SEQ ID NO: 21 or a mutant thereof; or a short linear peptide sequence derived from a human protein, preferably derived from a human nuclear protein, more preferably derived from the human La protein, even more preferably according to SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or a mutant thereof.

[0042] As used herein, the term "mutation" intends the substitution, deletion or insertion of one or more amino acids in a polypeptide sequence.

[0043] As used herein, the term "nucleoprotein" refers to a protein found in the cell nucleus. Advantageously, the tag is a peptide sequence derived from a nuclear antigen, but cannot be accessed or bound by the corresponding tag-binding domain in nature or under physiological conditions, respectively. This means that the tag-binding domain cannot bind to antigens naturally present in the cell nucleus and therefore will not unintentionally bind in the absence of the applied tag or tag-containing molecule. Further advantageously, the tag is not immunogenic. This minimizes the risk of uncontrolled on-target off-site toxicity by CAR-expressing immune cells, such as the release of toxic levels of cytokines, variously referred to as cytokine storm or cytokine release syndrome (CRS).

[0044] In some embodiments, the human proteins are human alpha-fetoprotein and human nucleoprotein, more preferably derived from the human La protein.

[0045] In some embodiments, the His tag is an amino acid sequence consisting of histidine residues, preferably in the range of 6 to 14 histidine residues.

[0046] In some embodiments, the tag is a myc tag, a His tag, a short linear peptide sequence derived from the yeast transcription factor GCN4, a leucine zipper sequence, or a short linear peptide sequence derived from a human protein.

[0047] Preferably, said tag is the human La epitope E5B9 according to SEQ ID NO: 22 or E7B6 according to SEQ ID NO: 23 or SEQ ID NO: 24, most preferably the human La epitope E5B9 according to SEQ ID NO: 22 or E7B6 according to SEQ ID NO: 24.

[0048] In some embodiments, the tag is present at the amino terminus of the switchable CAR-containing polypeptide. Advantageously, locating the tag at the amino terminus avoids interfering with the access of the tag to the targeting module bound to the target cell.

[0049] In some embodiments, the tag binding domain is an antibody, antigen-binding fragment, protein, or peptide that binds to a myc tag, a His tag, a short linear peptide sequence derived from the yeast transcription factor GCN4, a leucine zipper sequence, or a short linear peptide sequence derived from a human protein, preferably derived from a human nuclear protein, most preferably derived from the human La protein.

[0050] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least three amino acids, and there is no limit to the maximum number of amino acids that may be included in a protein's or peptide's sequence.

[0051] Amino acid residues in polypeptide sequences are designated herein according to the single-letter code, eg, Q denotes a Gln or glutamine residue, R denotes an Arg or arginine residue, and D denotes an Asp or aspartic acid residue.

[0052] As used herein, the term "antibody" refers to a protein that binds an antigen through an antigen-binding fragment variable region (Fab). H ) and light chain (V L ) each of which is composed of one constant domain and one variable domain. As used herein, the term "antibody fragment or antigen-binding fragment" refers to at least the V L or V HIn some embodiments, the antibody fragment is selected from a single-chain variable fragment (scFv), a single-chain antibody, a F(ab')2 fragment, a Fab fragment, and fragments produced by a Fab expression library or a single-domain antibody (nanobody).

[0053] As used herein, the term "single-chain variable fragment (scFv)" refers to an artificial antibody fragment that contains the variable domain of the light chain and the variable domain of the heavy chain of an antibody that are covalently linked. In some embodiments, the V of an antibody L and V H are covalently linked by a short peptide of 10 to 25 amino acids. In a further embodiment, the short peptide is H N-terminus and V L or vice versa.

[0054] As used herein, the term "CDR (complementarity determining region)" refers to the portion of the variable chain of an antibody or antibody fragment (the antibody or antibody fragment binds to its specific antigen). Antibodies contain three CDRs (CDR1, CDR2, and CDR3) that are arranged non-contiguously in the amino acid sequence of each variable domain, and thus comprise two variable domains (V H and V L ) contains six CDRs, which can contact the antigen.

[0055] In some embodiments, V L and V H is the structure (G x S y ), where x and y are selected from 1 to 10, preferably 3 to 5. Most preferred is 1 to 10 repeats of the sequence G4S1 (SEQ ID NO: 1). Furthermore, linkers composed of peptide sequences that can increase the protease resistance of the antibody derivative are preferred.

[0056] In some embodiments of the invention, the linker of the tag-binding domain comprises 20 to 30 amino acids, preferably 25 amino acids.

[0057] In further embodiments, the linker of the tag binding domain comprises a linker according to SEQ ID NO:2 or SEQ ID NO:3.

[0058] In some embodiments, the antibody is obtained from an animal species, preferably a mammal, such as a human, monkey, mouse, rat, rabbit, guinea pig, horse, cow, sheep, goat, pig, dog, or cat. Preferably, the antibody or antibody fragment is a human antibody, a humanized antibody, or a deimmunized antibody. Humanized antibodies can be prepared by various methods, such as resurfacing and CDR grafting. In the case of resurfacing, a combination of molecular modeling, statistical analysis, and mutagenesis is used to modify all non-CDR regions on the surface of the antibody to resemble the surface of the antibody of the target organism. In CDR grafting, CDR regions according to the present invention, which are similar in sequence to the original CDR regions, are introduced into known human framework regions. Deimmunized antibodies can be obtained by specifically mutating residues to generate predicted immunogenic hotspots based on in silico peptide-MHC affinity prediction.

[0059] In some embodiments, the antibody or antibody fragment is a polyclonal antibody, a monoclonal antibody, or a chimeric antibody, in which the antigen-binding region of a non-human antibody has been transferred into the framework of a human antibody by recombinant DNA techniques, including in silico design.

[0060] In several embodiments, antibodies to a selected tag or antigen can be produced by immunizing various hosts, including but not limited to goats, rabbits, rats, mice, and humans, via injection of cells expressing the particular protein, DNA or RNA encoding the protein, the protein itself, or any portion, fragment, or oligopeptide that retains the immunogenic properties of the protein.

[0061] In a preferred embodiment, the tag binding domain is an antibody or antibody fragment.

[0062] In some embodiments, the tag-binding domain binds to a tag that is a short linear peptide sequence derived from a human La protein, preferably derived from the human La epitope E5B9 according to SEQ ID NO: 22 or E7B6 according to SEQ ID NO: 24.

[0063] In some embodiments, the tag binding domain preferably comprises: The following array: DIVMTQSPDSLAVSLGERATINCX 24 SSQSLLNSRTX 35 KNYLAWYQQKPGQPPKLLIYWASTR X 61 SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYNLX 101 TFGGGTKVElK (SEQ ID NO: 7)(X 24 , X 35 , X 61 and X 101 are independently selected from α-amino acid residues that constitute proteins), or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity to the sequence of SEQ ID NO: 8. V according to L and wherein the antibody or antigen-binding fragment comprises:

[0064] In some embodiments, the tag binding domain comprises an scFv of the anti-La epitope.

[0065] In some embodiments, X 24 , X 35 , X 61 and X 101 is chosen as follows: X 24is selected from polar and / or positively charged residues, such as serine, threonine, asparagine, glutamine, histidine, lysine and arginine, preferably lysine or arginine; X 35 is preferably selected from lysine and proline; X 61 is selected from polar and charged residues, such as asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine and arginine, preferably glutamic acid and lysine; X 101 is selected from hydrophobic residues such as isoleucine, leucine, valine, alanine, methionine, phenylalanine, proline and tryptophan, preferably leucine or proline.

[0066] Preferably, the tag binding domain is SEQ ID NO: 8 (V L ) and SEQ ID NO: 9 (V H ) each having at least 90% sequence identity, preferably at least 95% sequence identity to the sequences according to

[0067] Most preferably, the tag binding domain is SEQ ID NO: 8 (V L ) and SEQ ID NO: 9 (V H ) to construct the anti-La 5B9 scFv.

[0068] In some embodiments, the tag binding domain is V L -Linker V H an antibody or antigen-binding fragment that binds to the human La epitope E5B9 or E7B6, comprising the structure: V L The region comprises a sequence having at least 95% identity, preferably at least 99% identity, with a sequence according to SEQ ID NO: 8 or SEQ ID NO: 25, and / or a V H The region comprises a sequence having at least 95% identity, preferably at least 99% identity, with a sequence according to SEQ ID NO: 9 or SEQ ID NO: 26.

[0069] In some embodiments, at least one administration of a clinically effective amount of T cells comprising a nucleotide sequence encoding a switchable CAR is administered once within the range of days 0 to 5 of treatment, in a dose of 1:10 8 ~1·10 9 It is administered to a subject in a dosage that is within the range of cells.

[0070] In some embodiments, two administrations of a clinically effective amount of T cells comprising a nucleotide sequence encoding a switchable CAR are administered within a range of 1-10 days of treatment. 8 ~1·10 9 The total dose of both doses within the cell range is administered to the subject.

[0071] In some embodiments, the at least one administration of a clinically effective amount of T cells comprising a nucleotide sequence encoding a switchable CAR is 1-10 8 ~5·10 8 It is applied in a dosage within the range of cells.

[0072] In several embodiments, one administration of a clinically effective amount of T cells comprising a nucleotide sequence encoding a switchable CAR is administered to the subject.

[0073] In several embodiments, one administration of a clinically effective amount of T cells comprising a nucleotide sequence encoding a switchable CAR is administered once on day 1 of treatment.

[0074] In some embodiments, one administration of a clinically effective amount of T cells comprising a nucleotide sequence encoding a switchable CAR is administered 1 hour to 24 hours after the start of the sequential administration of the targeting module.

[0075] In some embodiments, at least one further administration of a clinically effective amount of T cells comprising a nucleotide sequence encoding a switchable CAR is administered in a dose of 1:10 8 ~1·10 9 It is administered to a subject in a dosage that is within the range of cells.

[0076] In some embodiments, one further administration of a clinically effective amount of T cells comprising a nucleotide sequence encoding a switchable CAR is administered in a dose of 1:10 8 ~1·10 9 It is administered to a subject in a dosage that is within the range of cells.

[0077] In some embodiments, two additional administrations of a clinically effective amount of T cells comprising a nucleotide sequence encoding a switchable CAR are administered in a dose of 1:10 8 ~1·10 8 It is administered to a subject in a dosage that is within the range of cells.

[0078] In some embodiments, three additional administrations of a clinically effective amount of T cells comprising a nucleotide sequence encoding a switchable CAR are administered in a dose of 1:10 8 ~1·10 9 It is administered to a subject in a dosage that is within the range of cells.

[0079] As used herein, the term "targeting module" refers to a molecule, preferably a polypeptide or protein, having at least two distinct domains, each specific for a target or a homogeneous group of targets, wherein at least one domain is specific for a target cell, particularly a blood cancer binding domain, such as a CD123 binding domain; and one domain is specific for a switchable chimeric antigen receptor, particularly a tag or tag-binding domain. In some embodiments, the targeting module is isolated. As used herein, the term "isolated" means altered or removed from a natural state.

[0080] Preferably, the targeting module according to the invention is expressed as a recombinant protein. In a further embodiment, the targeting module is chemically synthesized.

[0081] As used herein, the term "target cell binding domain" refers to a peptide, protein, or small organic ligand, particularly a hematological cancer binding domain, that specifically binds a protein or protein complex (antigen) on the surface of a target cell.

[0082] As used herein, the term "specific" refers to the ability of an antibody or antibody fragment, or a protein, peptide, or low molecular weight organic ligand, to recognize and bind to a binding partner (e.g., tumor antigen) protein present in a sample, but not to substantially recognize or bind to other molecules in the sample.

[0083] As used herein, the term "binding" or "bonding" refers to non-covalent bonds, particularly ionic bonds, hydrogen bonds, van der Waals forces and / or hydrophobic interactions.

[0084] The term "target cell binding domain" also includes soluble T cell receptors (TCRs) composed of the alpha and beta chains or the gamma and delta chains, or fragments or mutants thereof. Such TCR-derived binding moieties recognize and bind peptides 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 proteins such as the EGFR family, survivin, the sry-like high mobility group box (SOX) protein family, melanoma-associated antigens (e.g., autoimmunogenic cancer / testis antigen NY-ESO-1, members of melanoma antigen family A MAGEA, antigens preferentially expressed in melanoma PRAME), and leukemia-associated antigens (e.g., Wilms' tumor gene 1 WT1).

[0085] In further embodiments, the hematological cancer cell binding domain is a soluble T cell receptor consisting of the α and β or γ and δ chains of the T cell receptor (TCR).

[0086] In some embodiments, the targeting module is in a monomeric, dimeric or multimeric form, preferably in a monomeric form.

[0087] In further embodiments, the targeting module is monovalent, bivalent or multivalent.

[0088] In some embodiments, the hematological cancer cell binding domain is an antibody, antibody fragment, protein, peptide, or small organic ligand that binds to a surface antigen selected from the group including CD19, CD20, CD22, CD30, CD33, CD38, CD56, CD70, CD123, CD133, CD371, CXCR4, a member of the mucin protein family, and a ligand for the NKG2D receptor.

[0089] As used herein, the term "low molecular weight organic ligand" refers to an organic molecule having a molecular weight of up to 10 kilodaltons, preferably up to 3 kilodaltons, that specifically binds to a protein or protein complex (antigen) on the surface of a target cell, particularly a blood cancer cell.

[0090] In some embodiments, the hematological cancer cell binding domain is an antibody, antibody fragment, protein, peptide, or small organic ligand that binds to CD123.

[0091] In a preferred embodiment, the CD123 binding domain is an antibody or antigen-binding fragment.

[0092] In some embodiments, the CD123 binding domain comprises the sequences of SEQ ID NO:4 and SEQ ID NO:5.

[0093] In a preferred embodiment, the CD123 binding domain comprises the sequence according to SEQ ID NO:6.

[0094] In some embodiments, the hematological cancer cell binding domain comprises a humanized amino acid sequence.

[0095] In some embodiments, the different domains of the targeting module are linked to each other by a linker. The linker preferably comprises a short sequence of 10 to 20 amino acid residues. In some embodiments, the targeting module comprises a flexible peptide sequence selected such that the domains have a three-dimensional fold that allows them to exhibit specificity for effector cells and target cell binding. Preferred linkers have the structure (G x S y ) is a glycine-serine linker having the sequence G4S1 (SEQ ID NO: 1) repeated 1 to 10 times. Most preferred is a linker composed of a peptide sequence that can increase the protease resistance of the antibody derivative.

[0096] In some embodiments, the linker is SEQ ID NO:2 or SEQ ID NO:3.

[0097] In some embodiments, the targeting module has a length in the range of 20 to 1600 amino acids, preferably 200 to 800 amino acids.

[0098] In some embodiments, the targeting module comprises one of the sequences according to SEQ ID NO:10 to SEQ ID NO:17.

[0099] In some embodiments, the targeting module comprises a further domain selected from the group consisting of costimulatory ligands, radionuclides, cell death-inducing chemical compounds, and half-life-increasing domains, preferably IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, HSA, FcRn-binding peptides, or mutants thereof. As used herein, the term "mutant" refers to a protein having at least 90% sequence identity, preferably at least 95% sequence identity, with the half-life-increasing domain. Advantageously, the mutant may have one or more activities of the designated peptide or protein; in particular, the mutant increases half-life like the half-life-increasing domain.

[0100] In some embodiments, the first and / or at least one further administration of the targeting module is administered in the range of 0.5 mg / day to 12 mg / day, preferably in the range of 4 mg / day to 12 mg / day. In some embodiments, the first and / or at least one further administration of the targeting module is administered in the range of 0.5 mg / day to 8 mg / day, preferably in the range of 0.5 mg / day to 2 mg / day.

[0101] In some embodiments, the first administration of the targeting module is applied continuously for a period of between 20 and 24 days, preferably for a period of 20 or 21 days.

[0102] As used herein, the term "extracellular hinge and transmembrane domain" refers to a flexible peptide sequence connected to the tag, which anchors the switchable CAR within the plasma membrane of a cell and protrudes from the surface of the cell for optimal binding to its specific targeting module.

[0103] In some embodiments, the extracellular hinge and transmembrane domain are selected from the hinge and transmembrane domains of human CD8α, CD28, ICOS (CD278), NK cell receptors, preferably natural killer group NKG2D; or portions of antibody constant regions, and combinations thereof. As used herein, the term "combinations thereof" refers to combinations of different hinge and transmembrane domains.

[0104] In a preferred embodiment, the extracellular hinge and transmembrane domain comprises CD8α, CD28, ICOS (CD278) or mutants thereof and combinations thereof.

[0105] As used herein, the term "mutant" refers to a peptide or protein having at least 90% sequence identity, preferably at least 95% sequence identity, to a specified domain, antibody, antibody fragment, peptide, or protein. Advantageously, the mutant can retain one or more activities of the specified domain, antibody, antibody fragment, peptide, or protein.

[0106] In some embodiments, the mutation comprises a point mutation. As used herein, a "point mutation" is a mutation in which a single nucleotide base is changed, inserted, or deleted from the nucleotide sequence.

[0107] In some embodiments, the mutant is a truncated form of a peptide or protein. As used herein, the term "truncated form" refers to a shortened peptide or protein resulting from a mutation in the nucleotide sequence encoding the peptide or protein, which has at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 90% length and 100% sequence identity, most preferably at least 95% length and 100% sequence identity to the designated peptide or protein. Advantageously, the truncated form has at least 80%, preferably at least 90%, more preferably at least 95% of the activity of the designated peptide or protein.

[0108] Milone et al. and Zhao et al. describe the use of the hinge and transmembrane domains of the human CD8α molecule in CARs (Milone et al. 2009; Zhao et al. 2009).

[0109] Pinthus et al. and Cartellieri et al. described the use of the hinge and transmembrane domains of the human CD28 molecule in CARs (Pinthus et al. 2003; Cartellieri et al. 2016).

[0110] Zhang et al. described the use of the hinge and transmembrane domains of NKG2D in CARs (Zhang et al., 2005).

[0111] Frigault et al. and Wang et al. describe the use of a portion of the hinge and transmembrane domains of the constant region of immunoglobulin G1 (IgG) (Frigault et al. 2015; Wang et al. 2007). Frigault et al. describe the use of the hinge domain of the constant region of IgG4.

[0112] In some embodiments, the combination of extracellular hinge and transmembrane domain is a CD8α extracellular hinge and transmembrane domain, a CD28 extracellular hinge and transmembrane domain, a CD28 extracellular hinge domain combined with a CD8α or ICOS transmembrane domain, preferably a CD28 extracellular hinge and transmembrane domain, or a CD28 extracellular hinge domain combined with an ICOS transmembrane domain.

[0113] In a preferred embodiment, the extracellular hinge and transmembrane domain comprises the extracellular hinge domain of CD28, preferably a mutant of the extracellular hinge domain of CD28, more preferably a mutant of the extracellular hinge domain of CD28 comprising two point mutations in the B7 binding site. Advantageously, the mutation in the B7 binding site abolishes ligand binding. Preferably, the extracellular hinge and transmembrane domain comprises a mutant of the extracellular hinge domain of CD28 according to SEQ ID NO: 27.

[0114] In some embodiments, the extracellular hinge and transmembrane domain comprises SEQ ID NO:27 and SEQ ID NO:28 or SEQ ID NO:29.

[0115] As used herein, the term "signaling domain" refers to a peptide sequence that transmits a signal to a cell by cross-linking a cell expressing a switchable CAR (effector cell) to a human cell surface protein or protein complex (target cell). The cross-linking between the effector and target cell is mediated by a targeting module.

[0116] In some embodiments, the signaling domain is selected from CD3, CD28, CD137 (4-1BB), CD134 (OX40), the cytoplasmic region of CD278 (ICOS), DAP10, CD27, programmed cell death-1 (PD-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), the cytoplasmic region of the CD3 chain, DAP12, CD122 (interleukin-2 receptor beta), CD132 (interleukin-2 receptor gamma), CD127 (interleukin-7 receptor alpha), CD360 (interleukin-21 receptor), an activating Fc receptor, IL-2, IL-7, IL-15, or IL-21, and mutants thereof.

[0117] In preferred embodiments, the intracellular signaling domain comprises at least one signaling domain selected from the group consisting of the cytoplasmic region of CD3, CD28, 4-1BB (CD137), ICOS (CD278), IL-2, IL-7, IL-15 or IL-21, and mutants thereof.

[0118] In some embodiments, the intracellular signaling domain comprises two or three signaling domains selected from the group including CD28, 4-1BB, ICOS, CD3ζ, IL-7Rα, and mutants thereof.

[0119] In some embodiments, the intracellular signaling domain comprises two or three signaling domains selected from the group comprising the sequences of SEQ ID NO:30 to SEQ ID NO:38.

[0120] In some embodiments, the intracellular signaling domain comprises the cytoplasmic region of CD28 or a mutant thereof (preferably according to SEQ ID NO: 30 or SEQ ID NO: 31).

[0121] In some embodiments, the intracellular signaling domain comprises the cytoplasmic region of CD3ζ or a mutant thereof (preferably according to one of the sequences set forth in SEQ ID NO:32 to SEQ ID NO:34).

[0122] In some embodiments, the intracellular signaling domain comprises at least the cytoplasmic region of CD28 or a mutant thereof (preferably according to SEQ ID NO: 30 or SEQ ID NO: 31) and the cytoplasmic region of CD3ζ or a mutant thereof (preferably according to one of the sequences of SEQ ID NO: 32 to SEQ ID NO: 34).

[0123] In a further embodiment, the intracellular signaling domain comprises at least the cytoplasmic region of ICOS or a mutant thereof (preferably according to SEQ ID NO: 35).

[0124] In some embodiments, the intracellular signaling domain comprises at least the cytoplasmic region of ICOS or a mutant thereof (preferably according to SEQ ID NO: 35) and the cytoplasmic region of CD3ζ or a mutant thereof (preferably according to one of the sequences of SEQ ID NO: 32 to SEQ ID NO: 34).

[0125] Preferably, the switchable chimeric antigen receptor comprises a CD28 transmembrane domain and at least one CD28 cytoplasmic region or mutant thereof (preferably according to SEQ ID NO: 30 or SEQ ID NO: 31) as the intracellular signaling domain, or an ICOS transmembrane domain and at least one ICOS cytoplasmic region or mutant thereof (preferably according to SEQ ID NO: 35) as the intracellular signaling domain.

[0126] In some embodiments, the intracellular signaling domain further comprises the cytoplasmic region of 4-1BB (preferably according to SEQ ID NO: 36), IL-2Rβ, IL-7Rα and mutants thereof (preferably according to SEQ ID NO: 37 or 38), more preferably the cytoplasmic region of 4-1BB, IL-7Rα and mutants thereof (preferably according to SEQ ID NO: 36 or 37), and most preferably the cytoplasmic region of 4-1BB and mutants thereof.

[0127] In some embodiments, the switchable CAR is a universal chimeric antigen receptor comprising a tag-binding domain.

[0128] In some embodiments, the switchable CAR is a reversible chimeric antigen receptor that includes a tag. Advantageously, cells that include a reversible universal chimeric antigen cell surface receptor are less attenuated after stimulation, thereby improving persistence.

[0129] In a further embodiment, the switchable chimeric antigen receptor comprises an additional domain, which is a short peptide linker in the extracellular portion of the receptor that may serve to detect the chimeric antigen receptor on the cell surface or to stimulate chimeric antigen receptor T cells.

[0130] In preferred embodiments, the further domain forms a linear epitope for a monoclonal antibody (mab) that specifically binds to said further domain, hi some embodiments, the further domain comprises at least one linear epitope, preferably E7B6 according to SEQ ID NO:23 or SEQ ID NO:24.

[0131] In some embodiments, the additional domain is located between the tag-binding domain or tag and the extracellular hinge domain or an integral portion of the extracellular hinge domain.

[0132] Advantageously, switchable CAR-grafted cells bearing the additional domain can be specifically stimulated to preferentially proliferate and persist longer compared to non-grafted cells either in vitro or in vivo. Even more advantageously, the additional domain can also be used to purify switchable CAR-grafted cells from mixed cell populations or to suppress switchable CAR-grafted cell-mediated immune responses and eliminate switchable CAR-grafted cells in vivo.

[0133] In further embodiments, the switchable CAR comprises a signal peptide. Advantageously, the signal peptide allows expression on the cell surface of effector cells. In some embodiments, the signal peptide is located at the N-terminus of the switchable CAR nucleotide sequence before the tag-binding domain or tag. In some embodiments, the signal peptide is selected from the leader peptides derived from proteins such as CD28, CD8α, IL-2, lysozyme C, or the heavy or light chain of an antibody of human origin, to co-translationally or post-translationally target the protein to the secretory pathway and avoid immunogenic reactions.

[0134] In some embodiments, the tag-binding domain or tag is present at the amino terminus of the polypeptide comprising the switchable CAR. Advantageously, locating the tag-binding domain or tag at the amino terminus avoids interfering with the access of the tag-binding domain or tag to the targeting module bound to the target cell.

[0135] In some embodiments, the switchable CAR comprises the sequence of SEQ ID NO:39 to SEQ ID NO:56.

[0136] In some embodiments, the switchable CAR is a reversible CAR and comprises a sequence according to SEQ ID NO: 39 to SEQ ID NO: 47, preferably SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 44 or SEQ ID NO: 46, more preferably SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 46.

[0137] In a further embodiment, the switchable CAR is a UniCAR and comprises a sequence according to SEQ ID NO: 48 to SEQ ID NO: 56, preferably SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 53 or SEQ ID NO: 55, more preferably SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 55.

[0138] According to the present invention, after a period of between 5 and 21 days from the end of the administration period of the first administration of the targeting module, at least one further administration of the targeting module is continuously administered to the subject at a dosage in the range of 0.1 mg / day to 20 mg / day for a period of between 4 and 25 days. In some embodiments, after a period of between 7 and 21 days from the end of the administration period of the first administration of the targeting module, at least one further administration of the targeting module is continuously administered to the subject at a dosage in the range of 0.1 mg / day to 20 mg / day for a period of between 4 and 25 days.

[0139] In some embodiments, the administration of at least one additional dose of the targeting module is performed 5 to 14 days after the administration of the first dose of the targeting module, preferably 5 to 10 days after the administration of the first dose of the targeting module. In some embodiments, the administration of at least one additional dose of the targeting module is performed 7 to 14 days after the administration of the first dose of the targeting module, preferably 7 to 10 days after the administration of the first dose of the targeting module.

[0140] Preferably, 5 to 21 days after the first administration of the targeting module, at least one further administration of the targeting module is continuously administered to the subject in the range of 0.1 mg / day to 20 mg / day for a period of between 12 and 20 days. Preferably, 7 to 21 days after the first administration of the targeting module, at least one further administration of the targeting module is continuously administered to the subject in the range of 0.1 mg / day to 20 mg / day for a period of between 12 and 20 days.

[0141] In a preferred embodiment, administering at least one additional dose of the targeting module comprises administering two to four additional doses of the targeting module consecutively to the subject, each administered 5 to 21 days after the preceding dose, preferably 5 to 14 days after the preceding dose, more preferably 5 to 10 days after the preceding dose.

[0142] In a preferred embodiment, administering at least one additional dose of the targeting module comprises administering two to four additional doses of the targeting module consecutively to the subject, each administered 7 to 21 days after the preceding dose, preferably 7 to 14 days after the preceding dose, more preferably 7 to 10 days after the preceding dose.

[0143] In some embodiments, the administration of at least one additional dose of the targeting module comprises 3 to 4 consecutive administrations of the targeting module, each for 3 to 4 days, with a 24 to 72 hour break therebetween, preferably with a 24 to 48 hour break therebetween.

[0144] In a further embodiment, the kit for use in treating hematological cancer of the present invention comprises at least one additional targeting module, the at least one additional targeting module comprises at least one hematological cancer cell-binding domain and a tag-binding domain or tag; the at least one hematological cancer cell binding domain is an antibody, antibody fragment, protein, peptide, or small organic ligand that binds to a surface antigen selected from the group consisting of CD19, CD20, CD22, CD30, CD33, CD38, CD56, CD70, CD123, CD133, CD371, CXCR4, a member of the mucin protein family, and a ligand for the NKG2D receptor; The targeting module and the at least one additional targeting module comprise the same tag-binding domain or tag and different blood cancer cell-binding domains, and the additional targeting module is administered continuously to the subject at a dosage in the range of 0.1 mg / day to 20 mg / day for a period of between 4 days and 25 days, 7 days to 21 days after administration of the at least one additional dose of the targeting module.

[0145] A further aspect of the present invention is a method for treating hematological cancer in a subject, preferably a mammal, more preferably a human, by administering a kit according to the present invention, preferably to a subject in need thereof.

[0146] In some embodiments, the method for treating a hematological cancer in a subject comprises the following steps: a. administering a first administration of a targeting module continuously to a subject having a hematological cancer for a period of between 10 and 25 days, starting on day 0 of treatment (the start of treatment), at a dosage ranging from 0.1 mg / day to 20 mg / day; wherein the targeting module is at least one hematological cancer cell binding domain, and tag-binding domains or tags, Including, b. at least one administration of a clinically effective amount of T cells comprising a nucleotide sequence encoding a switchable CAR, administered once within days 0-5 of treatment in a dose of 1:10 8 ~1·10 9 administering to a subject a dose within the range of cells; wherein the switchable CAR is tag-binding domains or tags, extracellular hinge and transmembrane domains, and an intracellular signaling domain comprising at least one signaling domain; including; and c. 7 to 21 days after administration of the first administration of the targeting module, continuously administering to the subject at least one additional administration of the targeting module at a dosage in the range of 0.1 mg / day to 20 mg / day for a period of between 4 days and 25 days; Including, The tag-binding domain of the targeting module binds to the tag of the switchable CAR, or the tag of the targeting module binds to the tag-binding domain of the switchable CAR.

[0147] According to the present invention, the method is carried out in the order of steps a, b and c.

[0148] In several embodiments, the T cells comprising a nucleotide sequence encoding a targeting module and / or a switchable CAR are administered in the form of a pharmaceutical composition.

[0149] In some embodiments, the hematological cancer is leukemia. In some embodiments, the subject has acute myeloid leukemia (AML).

[0150] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human.

[0151] In some embodiments, the peptide epitope tag is a myc tag, a His tag, a short linear peptide sequence derived from the yeast transcription factor GCN4 (preferably according to SEQ ID NO: 18, SEQ ID NO: 19) or a mutant thereof; a leucine zipper sequence, preferably SYNZIP1 to SYNZIP48, BATF, FOS, ATF4, ATF3, BACH1, JUND, NFE2L3, HEPTAD (Reinke et al., 2010), a sequence according to SEQ ID NO: 20 or SEQ ID NO: 21 or a mutant thereof; or a short linear peptide sequence derived from a human protein, preferably derived from a human nuclear protein, more preferably derived from the human La protein, even more preferably SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or a mutant thereof.

[0152] In some embodiments, the tag binding domain is an antibody, antigen-binding fragment, protein, or peptide that binds to a myc tag, a His tag, a short linear peptide sequence derived from the yeast transcription factor GCN4, a leucine zipper sequence, or a short linear peptide sequence derived from a human protein, preferably derived from a human nuclear protein, most preferably derived from the human La protein.

[0153] In some embodiments, in step b., the clinically effective amount of T cells comprising a nucleotide sequence encoding a switchable CAR is 1·10 8 ~5·10 8 It is applied in a dosage within the range of cells.

[0154] In some embodiments, in step b., one administration of a clinically effective amount of T cells comprising a nucleotide sequence encoding a switchable CAR is administered to the subject.

[0155] In some embodiments, in step b., one administration of a clinically effective amount of T cells comprising a nucleotide sequence encoding a switchable CAR is administered once on day 1 of treatment.

[0156] In some embodiments, in step b., one administration of a clinically effective amount of T cells comprising a nucleotide sequence encoding a switchable CAR is administered 1 hour to 24 hours after the start of the sequential administration of the targeting module in step a.

[0157] In some embodiments, the hematological cancer cell binding domain is an antibody, antibody fragment, protein, peptide, or small organic ligand that binds to a surface antigen selected from the group including CD19, CD20, CD22, CD30, CD33, CD38, CD56, CD70, CD123, CD133, CD371, CXCR4, a member of the mucin protein family, and a ligand for the NKG2D receptor.

[0158] In some embodiments, the hematological cancer cell binding domain is an antibody, antibody fragment, protein, peptide, or small organic ligand that binds to CD123.

[0159] In a preferred embodiment, the CD123 binding domain is an antibody or antigen-binding fragment.

[0160] In some embodiments, the CD123 binding domain comprises the sequences of SEQ ID NO:4 and SEQ ID NO:5.

[0161] In a preferred embodiment, the CD123 binding domain comprises the sequence according to SEQ ID NO:6.

[0162] In some embodiments, the hematological cancer cell binding domain comprises a humanized amino acid sequence.

[0163] In some embodiments, the targeting module has a length in the range of 20 to 1600 amino acids, preferably 200 to 800 amino acids.

[0164] In some embodiments, the targeting module comprises one of the sequences according to SEQ ID NO:10 to SEQ ID NO:17.

[0165] In some embodiments, the first administration in step a. and / or at least one further administration of the targeting module in step c. is administered in the range of 0.5 mg / day to 12 mg / day, preferably in the range of 4 mg / day to 12 mg / day. In some embodiments, the first administration in step a. and / or at least one further administration of the targeting module in step c. is administered in the range of 0.5 mg / day to 8 mg / day, preferably in the range of 0.5 mg / day to 2 mg / day.

[0166] In some embodiments, the first administration of the targeting module in step a. is applied continuously for a period of between 20 and 24 days, preferably for a period of 20 or 21 days.

[0167] In some embodiments, the extracellular hinge and transmembrane domain comprises the transmembrane domain of CD8α, CD28, ICOS (CD278), or mutants thereof, and combinations thereof.

[0168] In some embodiments, the intracellular signaling domain comprises at least one signaling domain selected from the group consisting of CD3, CD28, 4-1BB (CD137), the cytoplasmic region of ICOS (CD278), IL-2, IL-7, IL-15, or IL-21, and mutants thereof.

[0169] In some embodiments, the method further comprises lymphodepletion prior to step a. As used herein, the term "lymphodepletion" refers to chemotherapy to reduce lymphocytes in the blood and bone marrow of a subject. Preferably, lymphodepletion is performed directly days prior to administering CAR-T cells in step b.

[0170] According to the method of the present invention, step c. is performed 5 to 21 days, preferably 5 to 14 days, and more preferably 5 to 10 days after step a. In some embodiments, step c. is performed 7 to 21 days, preferably 7 to 14 days, and more preferably 7 to 10 days after step a.

[0171] In some embodiments, step c. comprises sequential administration of at least one additional dose of the targeting module over a period of between 12 and 20 days.

[0172] In some embodiments, step c. comprises administering to the subject 2 to 4 additional doses of the targeting module sequentially, each 5 to 21 days after the previous dose, preferably 5 to 14 days after the previous dose, and more preferably 5 to 10 days after the previous dose. In some embodiments, step c. comprises administering to the subject 2 to 4 additional doses of the targeting module sequentially, each 7 to 21 days after the previous dose, preferably 7 to 14 days after the previous dose, and more preferably 7 to 10 days after the previous dose.

[0173] In some embodiments, step c. comprises administering multiple additional doses of the targeting module to the subject sequentially, each 5 to 21 days later, preferably 5 to 14 days later. In some embodiments, step c. comprises administering multiple additional doses of the targeting module to the subject sequentially, each 7 to 21 days later, preferably 7 to 14 days later. Suitably, the administration of multiple additional doses of the targeting module to the subject is carried out until at least one event selected from the group consisting of relapse, unacceptable toxicity, minimal residual disease (MRD) negativity, potentially curative treatment option, and a maximum treatment time of 2 years occurs.

[0174] As used herein, the term "unacceptable toxicity" refers to persistent side effects and / or adverse health conditions that substantially affect activities of daily living, are resistant to therapy (e.g., refractory seizures), have no treatment options (e.g., blindness), or require substantially invasive treatment (e.g., heart transplant).

[0175] As used herein, the term "minimal residual disease" (MRD) refers to the smallest amount of cancer cells that can be measured in the blood or bone marrow of a subject after cancer treatment, particularly as determined by qPCR.

[0176] In some embodiments, the potentially curative treatment option is allogeneic hematopoietic stem cell transplantation (alloHSCT) or donor lymphocyte infusion (DLI).

[0177] In some embodiments, step c. comprises three to four successive applications of the targeting module, each for three to four days, with a break of 24 to 72 hours therebetween, preferably a break of 24 to 48 hours therebetween.

[0178] In some embodiments, step c. comprises multiple sequential application of the targeting module, each for 3 to 4 days, each with a 24 to 72 hour break therebetween, preferably with a 24 to 48 hour break therebetween. Suitably, the multiple sequential application of the targeting module is administered for 3 to 4 days, each with a 24 to 72 hour break therebetween, until at least one event occurs selected from the group consisting of relapse, unacceptable toxicity, MRD negativity, potentially curative treatment option, and a maximum treatment time of 2 years.

[0179] In some embodiments, in step a. and / or step c., the targeting module is applied continuously until a stable plasma concentration of the targeting module is reached.

[0180] In some embodiments, the targeting modules in step a. and / or step c. are applied continuously until side effects occur.

[0181] In some embodiments, the methods of the present invention are practiced in combination with a therapy selected from the group including chemotherapy, radiation, immunosuppressants, antibodies and other immunoablative agents.

[0182] In a further embodiment, the method further comprises applying at least one additional targeting module, wherein the at least one additional targeting module comprises at least one hematological cancer cell binding domain and a tag-binding domain or tag; the at least one hematological cancer cell binding domain is an antibody, antibody fragment, protein, peptide, or small organic ligand that binds to a surface antigen selected from the group consisting of CD19, CD20, CD22, CD30, CD33, CD38, CD56, CD70, CD123, CD133, CD371, CXCR4, a member of the mucin protein family, and a ligand for the NKG2D receptor; the targeting module and the at least one further targeting module comprise the same tag-binding domain or tag and different hematological cancer cell-binding domains; The additional targeting module is administered continuously to the subject at a dosage in the range of 0.1 mg / day to 20 mg / day for a period of between 4 days and 25 days, 7 days to 21 days after administration of at least one additional dose of the targeting module.

[0183] Another aspect of the invention is a nucleic acid, vector, or cell comprising a nucleotide sequence encoding a switchable CAR for use in a method for treating a hematological cancer in a subject and / or for use in a kit for use in treating a hematological cancer according to the invention.

[0184] As used herein, "nucleic acid" or "nucleic acid molecule" refers to nucleotides and / or polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments produced by polymerase chain reaction (PCR), and fragments produced by ligation, cleavage, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally occurring nucleotides (e.g., DNA and RNA), or analogs of naturally occurring nucleotides (e.g., enantiomeric forms of naturally occurring nucleotides), or combinations of both. Nucleic acids can be either single-stranded or double-stranded. Nucleotides are designated as follows: one-letter designations are used to designate the base of a nucleoside: a is adenine, t is thymine, c is cytosine, and g is guanine.

[0185] In some embodiments, the nucleic acid, vector, and / or cell is isolated. In some embodiments, the nucleic acid is cDNA. As used herein, the term "cDNA" (complementary DNA) refers to double-stranded DNA synthesized from single-stranded RNA, such as mRNA, in a reaction catalyzed by the enzyme reverse transcriptase. In some embodiments, the cDNA is synthetically derived. In further embodiments, the cDNA is derived from mRNA and therefore, in contrast to genomic DNA, contains only exons and no introns.

[0186] In some embodiments, the vector is selected from the group comprising a DNA vector, an RNA vector, a plasmid, a lentiviral vector, a retroviral vector, an adenoviral vector, and an adeno-associated viral vector.

[0187] In some embodiments, the vector further comprises a promoter, wherein the promoter is selected from the group comprising an EF-1 promoter, a CMV IE gene promoter, an EF-1α promoter, a ubiquitin C promoter, or a phosphoglycerate kinase (PGK) promoter.

[0188] In several embodiments, the cells comprise an exogenous nucleotide sequence encoding a switchable CAR that is expressed on the surface of the cell.

[0189] In some embodiments, the cells are T cells, preferably cytotoxic T lymphocytes (CTLs) or regulatory T cells (T reg ); immune effector cells selected from the group including natural killer (NK) cells, and macrophages (MPs).

[0190] The present invention is not limited to the embodiments shown and described, but also includes all embodiments that have the same effect within the meaning of the present invention. Furthermore, the present invention is also not limited to the specifically described combinations of features, but may be defined by any other combination of all specific features of the individual features disclosed as a whole, unless the individual features are mutually exclusive or specific combinations of individual features are explicitly excluded.

[0191] Diagrams and Examples The invention will now be further illustrated by the following non-limiting figures and examples. [Brief explanation of the drawings]

[0192] FIG. 1 shows a schematic diagram of a Phase 1A trial design using a kit for use in the treatment of hematological cancer, including an initiation cycle (IC) followed by three consolidation cycles (CC) of targeting module administration.

[0193] FIG. 2 shows exemplary measurements of A) leukocytes (white blood cells, WBC) and B) neutrophils over the course of treatment.

[0194] Figure 3 shows UniCAR-T cell counts in peripheral blood (PB) and bone marrow (BM) over the course of treatment, including application of the targeting module (core cycle) and further application of the targeting module (additional cycle). Cell counts were calculated from vector copy numbers determined by digital droplet polymerase chain reaction, and the mean and standard deviation are shown.

[0195] Figure 4 shows a comparison of UniCAR-T growth and achieved responses in patients who received a further application (additional cycle) of one of the targeting modules after a shorter or longer interruption.

[0196] FIG. 5 shows the test for encephalopathy (CRES) using a writing test.

[0197] FIG. 6 shows transaminase (ASAT and ALAT) and ferritin levels during treatment and after stopping the targeted module infusion.

[0198] Figure 7 shows minimal residual disease (MRD) levels (NPM1 / ABL1 ratio) determined by qPCR in bone marrow and blood, reported as the ratio of cycle threshold (CT) values ​​of mutant NPM1 to ABL1 according to the European LeukemiaNet guideline for MRD in AML (Heuser et al. 2021). Treatment periods for the targeted module (TM123) are indicated by gray shading. CC: consolidation cycle.

[0199] Figure 8 shows VCN (copy number per μg of genomic DNA). UniCAR-T proliferation was determined by droplet digital PCR in both bone marrow and blood and reported as UniCAR copies per μg of genomic DNA. The treatment period of the targeting module (TM123) is indicated by gray shading. CC: consolidation cycle.

[0200] Figure 9 shows the responses obtained in relapsed / refractory AML (rrAML) patients treated with 100, 250, or 500 mM UniCAR-T cells (UniCAR02-T) and 0.5, 1, 2, or 4 mg / day of the targeted module (TM123): 8 patients (53%) received at least a partial remission (PR) and 4 patients (27%) received a complete remission / complete remission with incomplete hematologic recovery (CR / CRi). [Example]

[0201] Switchable CAR T cells Immune cells can be engineered to express switchable CARs. In genetic engineering to express UniCAR or RevCAR, a polynucleotide vector encoding UniCAR or RevCAR and all necessary elements to ensure its expression in the engineered immune cells are introduced into the immune cells. In particular, UniCAR and RevCAR contain IL-2LP (modified human IL-2 leader peptide), a tag-binding domain or tag, G4S1 (glycine-serine linker), ECD (extracellular domain), TMD (transmembrane domain), and at least two ICD (intracellular domain), as set forth in SEQ ID NO: 30 to SEQ ID NO: 38.

[0202] Transfer of the vector can be achieved by electroporation or transfection of the nucleic acid, or by utilizing viral vector systems such as adenovirus, adeno-associated virus, retrovirus, foamy virus or lentivirus gene transfer.

[0203] Lentiviral gene transfer is applied to the stable expression of switchable CARs in immune cells by first constructing a lentiviral vector encoding the selected switchable CAR. The lentiviral vector is pLVX-EF1alphaUniCAR 28 / ζ (Clontech, Takara Bio Group), in which the lentiviral portion of the vector is derived from human immunodeficiency virus (HIV), and the MSC / IRES / ZxGreenI portion is replaced by the switchable CAR construct.

[0204] Lentiviral particles are generated by transient transfection of human embryonic kidney (HEK) 293T (ACC 635) cells with a lentiviral vector plasmid encoding a switchable CAR and cotransfection with a plasmid (psPAX2) encoding a group-specific antigen (gag) and polymerase (pol) and a plasmid (pMD2.G) encoding an envelope. After transfection, the packaging plasmid expresses the HIV-1 Gag and Pol proteins. Plasmid MD2.G encodes the vesicular stomatitis virus glycoprotein (VSV-G). The VSV-G protein is used by lentiviral vectors to transduce a wide range of mammalian cells. Various envelopes from different viral species can be utilized for this purpose. Lentiviral vectors can be successfully pseudotyped with 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 viruses (PFVs), or chimeric envelope glycoprotein variants derived from gibbon ape leukemia virus (GaLV) and MLV.

[0205] Supernatants from transfected HEK293T cells are harvested 24 to 96 hours after transfection, and viral particles are concentrated from the supernatant by ultracentrifugation or other methods. For lentiviral transduction of immune cells, peripheral blood mononuclear cells (PBMCs) or isolated T cells are activated with mabs specific for the CD3 complex, such as clones OKT3 or UCHT1, in solution or coated on plastic cell culture dishes, magnetic beads, or biodegradable polymer matrices. Activation of PBMCs or isolated T cells can be further enhanced by stimulating costimulatory pathways with mabs or ligands specific for CD27, CD28, CD134, or CD137, alone or in combination, coated on plastic cell culture dishes, magnetic beads, or biodegradable polymer matrices, as well as by adding exogenous recombinant cytokines such as interleukin (IL)-2, IL-7, IL-12, IL-15, and IL-21. Concentrated or unconcentrated viral particles are added to PBMC or T cell cultures 24 to 96 hours after the initial administration of activating CD3-specific antibodies, and / or antibodies specific for the costimulatory receptors CD27, CD28, CD134, or CD137, and / or recombinant cytokines, either as a single dose or multiple doses. Electroporation, transduction, and expansion of T cells can be performed manually in open cell culture systems or in closed, partially, or fully automated systems.

[0206] Stable transduction of T cells can be determined by flow cytometry after staining with tag-containing molecules for surface expression of the switchable CAR or mab against the fourth domain of the switchable CAR 3 days or later after the final application of viral supernatant. Switchable CAR-transduced T cells can be expanded in vitro by culturing the T cells with recombinant cytokines and activating anti-CD3 mab.

[0207] In the case where the switchable CAR has an optional additional domain, which is a peptide sequence that forms a linear epitope for the mab, immune cells genetically modified to express the switchable CAR can be specifically propagated in vitro by coating the mab or its antibody fragment that binds to the additional domain on the surface of a culture dish, or on any type of bead or biodegradable polymer matrix that is added to cell culture at a predetermined ratio. When the surface-coated mab binds to the switchable CAR peptide domain, it induces cross-linking of the switchable CAR expressed on the cell surface and the formation of an immune synapse, which leads to the activation of a signal pathway specifically triggered by the signal domain of the switchable CAR. Depending on the induced signal pathway, this can lead to enhanced proliferation of immune cells bearing the switchable CAR and sustained resistance to activation-induced cell death, thereby enriching immune cells genetically modified with the switchable CAR in a mixed population.

[0208] The additional domain, which is a peptide sequence that forms a linear epitope for the mab, can be further used to enrich and purify immune cells expressing switchable CAR from a mixed population. Enrichment and purification are carried out by using a mab or its antibody fragment that binds to the additional switchable CAR domain to mark cells expressing switchable CAR for cell sorting, or to temporarily link immune cells expressing switchable CAR to small particles that can be used for cell isolation. In one embodiment, switchable CAR-grafted immune cells are incubated with a mab that recognizes the additional domain. Then, magnetic beads are added, which bind to antibodies or fragments thereof directed against the species-specific and isotype-specific heavy and light chains of the mab that binds to the additional domain. In this way, immune cells expressing switchable CAR and magnetic beads are linked, captured by a magnetic field, and separated from other immune cells.

[0209] Targeting Module The targeting module TM123 is a soluble recombinant fusion protein containing two antibody-derived binding domains. One selectively binds to the target antigen CD123, and the other recognizes the tag-binding domain or tag (epitope E5B9 from human La protein) displayed on switchable CAR-expressing cells. Therefore, TM123 functions as a bridging module between switchable CAR-T and CD123-expressing target cancer cells. The targeting module also contains an 8x-histidine tag at the C-terminus for detection and purification purposes.

[0210] In vitro characterization The successful preparation of switchable CAR can be demonstrated by flow cytometry and immunostaining of the CAR with EGFP expression.

[0211] Cytotoxicity assay The efficacy of the switchable CAR and CD123-binding TM to induce tumor cell elimination can be tested using a suspension cell-based co-culture assay with, for example, Nalm-6 or BxPC3 target cells in the presence of the targeting module. Target cells can be analyzed by flow cytometry or Incuyte (登録商標) Quantification can be performed using Zoom. For flow cytometry-based cytotoxicity, cell counts for each sample were normalized to a control sample seeded with tumor cells alone to calculate lysis. (登録商標) For cytotoxicity based on lysis, both live and dead target cells are quantified and lysis is assessed by calculating the ratio of dead to total target cells.

[0212] In vivo characterization Switchable CARs can be characterized in vivo in mice, where 1·10 5 MV4-11 luc cells were injected intravenously. After 3 days, 5 10 6UniCAR-T cells are injected intravenously to initiate targeting module administration, e.g., anti-CD123 targeting module is administered intraperitoneally (IP) twice daily (1 μg or 3 μg per mouse), weekdays for a total of 3 weeks. Tumor progression is monitored by IVIS. (登録商標) Disease progression can be monitored by fluorescent imaging using a SpectrumCT system. Mice should be monitored daily for signs of disease progression. When mice reach the endpoint criteria, they are sacrificed and organs are analyzed by flow cytometry.

[0213] In vivo characterization in humans – Phase 1A study design UniCAR-T cells were produced from autologous starting material (LEU), and patients could receive bridging therapy if necessary; lymphodepletion (LD) with standard doses of Flu / Cy was performed before the start of treatment; TM123 was administered as a continuous intravenous infusion starting on day 0 or day 1 for 20, 21, or 24 days, followed by UniCAR-T administration on day 1 (core cycle). Initially, patients were permitted to receive a second cycle (additional cycles) of TM123 based on initial safety and efficacy readouts in the core cycle. In the later phases of the study, patients were permitted to receive cycles 2, 3, and 4 (additional cycles) of TM123 based on initial safety and efficacy readouts in the core cycle.

[0214] Patients consisted of patients with rrAML and MRD. Treatment was generally well tolerated, regardless of age and prior therapy, with most side effects related to the underlying disease. Serious treatment-related adverse events included grade 3 CRS and grade 2 CRES. All cases resolved rapidly following completion of targeted module administration. Other events included cytopenias, but no treatment-induced, long-lasting cytopenias were observed.

[0215] Figure 2 shows the rapid recovery of white blood cells (WBC) and neutrophils after the end of targeting module application. Neutrophil recovery began 12 days after treatment initiation.

[0216] Figure 3 shows the number of UniCAR-T cells in peripheral blood (PB) and bone marrow (BM) over the course of treatment, including administration of the targeting module (core cycle) and further administration of the targeting module (additional cycle). Cell numbers were calculated from vector copy numbers determined by digital droplet polymerase chain reaction, and the mean and standard deviation are shown. Figure 3 shows the repopulation of UniCAR-T cells during additional TM123 cycles.

[0217] Figure 4 shows a comparison of UniCAR-T proliferation and achieved responses in patients who received an additional administration (additional cycle) of one of the targeting modules after a shorter (Patient 2: 21 days) or longer interruption (Patient 3: 81 days). Figure 4 shows the proliferation of UniCAR-T cells in peripheral blood and bone marrow during the core and additional cycles, despite a 3-month interruption of TM123 treatment.

[0218] Table 1 shows the results of UniCAR-T proliferation and achieved responses in patients who received a further administration (additional cycle) of one of the targeting modules after a shorter (patient 2: 21 days) or longer interruption (patient 3: 81 days).

[0219] [Table 1] This comparison shows improved results with respect to using a shorter break between the core and add cycles according to the present invention.

[0220] Figure 5 shows the treatment-emergent encephalopathy (CRES) test using a writing test. Figure 5 shows the improvement in writing ability after the completion of the targeted module cycle, thus demonstrating the effectiveness of the treatment switch-off mechanism. All reported CRES resolved within 24 hours of discontinuing the targeted module application.

[0221] Figure 6 shows transaminase (ASAT and ALAT) and ferritin levels during treatment and after cessation of targeted module infusion. Figure 6 demonstrates rapid resolution of acute side effects (grade IV hypertransaminasemia associated with CRS) in a patient with cessation of TM123 infusion during an additional TM123 cycle.

[0222] Anti-leukemic effects were observed: the overall response rate (ORR) was 53% in rrAML patients, with a CR / CRi rate of 27% (Figure 9), and three of four MRD patients showed a reduction in MRD load.

[0223] Figures 7 and 8 show results for a 64-year-old MRD+ patient who received four targeted module cycles: one 20-day core cycle and three consolidation cycles (each with 12-day continuous targeted module infusions and 7-14 days of treatment-free intervals between cycles). UniCAR-T cells were given as a one-time infusion on day 1 of cycle 1.

[0224] Long-term administration of the targeting module (20-day induction followed by three 12-day consolidation cycles at 1-2 week intervals) resulted in a durable, ongoing response lasting nearly 5 months. Figure 7 shows MRD levels (NPM1 / ABL1 ratio) determined by qPCR in bone marrow and blood, reported as the ratio of cycle threshold (CT) values ​​of mutant NPM1 to ABL1, according to the European LeukemiaNet guideline for MRD in AML (Heuser et al. 2021). A 64-year-old MRD+ patient with a history of four lines of therapy experienced a 3-log reduction in MRD levels and became transplant eligible. Figure 8 shows that re-administration of the targeting module resulted in significant repopulation of UniCAR T cells without further cell administration or lymphodepletion.

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Claims

1. 1. A kit for use in the treatment of hematological cancer, comprising: a. A T cell comprising a nucleotide sequence encoding a switchable chimeric antigen receptor (CAR), wherein the switchable CAR comprises: tag-binding domains or tags, extracellular hinge and transmembrane domains, and an intracellular signaling domain comprising at least one signaling domain; T cells comprising: b. a targeting module, at least one hematological cancer cell binding domain, and tag-binding domains or tags, a targeting module comprising: Including, the tag-binding domain of the targeting module binds to the tag of the switchable CAR, or the tag of the targeting module binds to the tag-binding domain of the switchable CAR; a first administration of the targeting module is administered continuously to a subject having a hematological cancer from day 0 of treatment at a dosage ranging from 0.1 mg / day to 20 mg / day for a period of between 10 days and 25 days; at least one administration of a clinically effective amount of T cells comprising a nucleotide sequence encoding a switchable CAR is administered within 1.10 days of treatment within the range of days 0 to 5 of treatment; ８ ~1.10 ９ administered to the subject at a dose within the range of cells; 7 to 21 days after administration of the first administration of the targeting module, at least one further administration of the targeting module is continuously administered to the subject for a period of between 4 and 25 days at a dosage in the range of 0.1 mg / day to 20 mg / day; The kit.

2. 2. The kit for use in treating hematological cancer of claim 1, wherein the T cells comprising a nucleotide sequence encoding the targeting module and / or switchable CAR are administered in the form of a pharmaceutical composition.

3. 3. The kit for use in treating blood cancer of claim 1 or 2, wherein the subject has acute myeloid leukemia (AML).

4. 4. The kit for use in treating blood cancer according to any one of claims 1 to 3, wherein the tag is a myc tag, a His tag, a short linear peptide sequence derived from the yeast transcription factor GCN4, a leucine zipper sequence, or a short linear peptide sequence derived from a human nuclear protein.

5. The kit for use in the treatment of blood cancer according to any one of claims 1 to 4, wherein the tag is a short linear peptide sequence derived from the human La protein.

6. 6. The kit for use in treating hematological cancer according to any one of claims 1 to 5, wherein the tag-binding domain is an antibody, antigen-binding fragment, protein or peptide that binds to a myc tag, a His tag, a short linear peptide sequence derived from yeast transcription factor GCN4, a leucine zipper sequence or a short linear peptide sequence derived from a human protein.

7. the at least one administration of a clinically effective amount of T cells comprising a nucleotide sequence encoding a switchable CAR is 1.10 ８ ~5.10 ８ The kit for use in the treatment of blood cancer according to any one of claims 1 to 6, wherein the kit is administered in a dosage within the cellular range.

8. 8. The kit for use in treating hematological cancer of any one of claims 1 to 7, wherein one administration of a clinically effective amount of T cells comprising a nucleotide sequence encoding a switchable CAR is administered to the subject.

9. 10. The kit for use in treating hematological cancer of claim 8, wherein one administration of a clinically effective amount of T cells comprising a nucleotide sequence encoding a switchable CAR is administered once on day 1 of treatment.

10. 10. The kit for use in treating hematological cancer of claim 8 or 9, wherein one administration of a clinically effective amount of T cells comprising a nucleotide sequence encoding a switchable CAR is administered 1 hour to 24 hours after the start of sequential administration of the targeting module.

11. 11. The kit for use in treating hematological cancer of any one of claims 1 to 10, wherein the hematological cancer cell binding domain is an antibody, antibody fragment, protein, peptide or small molecular weight organic ligand that binds to a surface antigen selected from the group consisting of CD19, CD20, CD22, CD30, CD33, CD38, CD56, CD70, CD123, CD133, CD371, CXCR4, a member of the mucin protein family and a ligand of the NKG2D receptor.

12. 12. The kit for use in treating hematological cancer according to any one of claims 1 to 11, wherein the hematological cancer cell binding domain is an antibody, antibody fragment, protein, peptide or low molecular weight organic ligand that binds to CD123.

13. The kit for use in treating hematological cancer of any one of claims 1 to 12, wherein the hematological cancer cell binding domain comprises a humanized amino acid sequence.

14. 14. The kit for use in treating hematological cancer of any one of claims 1 to 13, wherein the targeting module has a length in the range of 20 to 1600 amino acids.

15. 15. The kit for use in the treatment of hematological cancer of any one of claims 1 to 14, wherein the first administration and / or the at least one further administration of targeting module is administered at a dosage in the range of 4 mg / day to 12 mg / day.

16. 16. The kit for use in treating hematological cancer of any one of claims 1 to 15, wherein the first administration of targeting module is applied continuously for a period of between 20 and 24 days.

17. 17. The kit for use in treating hematological cancer according to any one of claims 1 to 16, wherein the extracellular hinge and transmembrane domain comprises the extracellular hinge and transmembrane domain of CD8α, CD28, ICOS (CD278), or mutants and combinations thereof.

18. The kit for use in treating hematological cancer according to any one of claims 1 to 17, wherein the intracellular signaling domain comprises at least one signaling domain selected from the group consisting of CD3, CD28, 4-1BB (CD137), the cytoplasmic region of ICOS (CD278), IL-2, IL-7, IL-15 or IL-21, and mutants thereof.

19. 19. The kit for use in treating hematological cancer of any one of claims 1 to 18, wherein administration of the at least one further administration of targeting module occurs 5 to 14 days after administration of the first administration of targeting module.

20. 20. The kit for use in treating hematological cancer of any one of claims 1-19, wherein administering the at least one additional dose of targeting module comprises sequentially administering two to four additional doses of targeting module to the subject, each dose being five to fourteen days after administration of a preceding dose.

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