Targeting module for CD123 in a method for stimulating chimeric antigen receptor mediated immunoresponse

Abstract

The present invention relates to a targeting module comprising at least one CD123 binding domain and a tag binding domain that binds to human La epitope E5B9, a nucleic acid, vector or cell comprising a nucleotide sequence encoding the targeting module, a pharmaceutical composition and kit comprising a targeting module and a vector or cell comprising a nucleotide sequence encoding a reversible chimeric antigen receptor.

Description

Technical Field

[0001] The present invention relates to a targeting module comprising at least one CD123 binding domain and a tag binding domain that binds to the human La epitope E5B9, a nucleic acid, a vector or a cell comprising a nucleotide sequence encoding the targeting module, and a pharmaceutical composition and a kit comprising the targeting module and a vector or a cell comprising a nucleotide sequence encoding a reversible chimeric antigen receptor. Background Art

[0002] Chimeric antigen receptors (CARs) are artificial receptors composed of a binding moiety that provides antigen specificity and one or several signaling chains derived from immune receptors (Cartellieri et al., 2010). Immune cells that have been genetically modified to express chimeric antigen receptors (CARs) can be used to bind to cells or tissue structures corresponding to suitable targets carrying the CAR binding moiety. Cross-linking induces signaling pathways through the signaling chains of the chimeric antigen receptor (CAR), which will change the biological properties of the CAR-transplanted immune cells. Conversely, CAR activation in genetically modified regulatory T cells (Tregs) can induce Treg-specific immunomodulatory and inhibitory mechanisms, such as the secretion of interleukin (IL)-10 or tumor growth factor-β (TGF-β). Currently, adoptive transfer of immune cells engineered with chimeric antigen receptors (CARs) is considered a very promising therapeutic option for the treatment of otherwise incurable malignancies, infectious diseases, or autoimmune diseases.

[0003] However, conventional CAR technology is accompanied by a number of critical issues that need to be addressed before this form of treatment can be widely used in clinical treatment. First, several safety issues must be addressed. So far, the immune response of T cells engineered with conventional CARs after infusion into patients has been difficult to control, with a high rate of serious adverse events (Titov et al., 2018). In particular, unexpected target gene expression on normal tissues may trigger a rapid and strong immune response of engineered T cells against normal cells, which may lead to serious side effects (Morgan et al., 2010). In addition, because CAR-T cells are a new class of self-amplifying cell drugs, the infused T cells can undergo dramatic expansion in the presence of severe tumor burden, leading to tumor lysis syndrome, cytokine release syndrome, and macrophage activation syndrome (Brudno and Kochenderfer, 2016). Another disadvantage of conventional CAR technology is the limitation of engineered T cells to retarget a single antigen. This monotherapy approach means that there is a risk of generating tumor escape variants that have lost the target antigen during treatment. In clinical trials, tumor escape variants have been observed after several months of conventional CAR T cell therapy (Sotillo et al., 2015). Taken together, these obstacles limit the use of CAR T cells to very few indications. In fact, so far, examples of clinical effectiveness are limited to CAR T cells targeting CD19 and BCMA.

[0004] The modular switchable “universal” CAR T (UniCAR) approach can overcome these limitations by separating the antigen recognition domain and activation domain of the CAR into two independent 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 soluble adaptor proteins that consist of the antigen binding domain fused to a tag recognized by the universal CAR. Cartellieri et al. described in vitro and in vivo treatment of CD33 and / or CD123 positive acute myeloid leukemia cells.

[0005] In addition to the UniCAR method for recognizing various antigens (EP 2 990 416 A1), a reverse universal CAR (RevCAR) method is also known, which promotes the binding of immune cells engineered to express RevCAR containing tags to target cells via an adapter molecule containing a tag binding domain and a target cell binding domain (EP 3 581 200 A1). EP 3581 200A1 discloses an extracellular LA / SSB-derived tag and an adapter molecule containing a CD123 scFv and an scFv bound to a tag (5B9 or 7B6), which is added to bridge CAR and tumor cells, thereby generating antigen-specific cytotoxicity.

[0006] Darowski et al. described flexible chimeric antigen receptor adapter molecules (CAR adapters) for recruiting chimeric antigen receptor T cells with tags such as 5B9, GCN4, FITC, leucine zipper sequences, or biotinylated IgG, and targets such as CD33, CD123, CD19, CD20, CD22, HER2, EGFR, CCR4, G2D, MCSP, and ErbB2 (Darowski et al., 2019).

[0007] Feldmann et al. disclosed that RevCAR T cells effectively killed tumor cells and were flexibly redirected against multiple targets by replacing targeting modules, particularly targeting modules against PSMA and PSCA (Feldmann et al., 2020).

[0008] Kittel-Boselli et al. described the use of the RevCAR platform to target acute myeloid leukemia, specifically patient-derived AML cells expressing CD33 and CD123, wherein RevCAR consists of the extracellular peptide epitope E5B9 or E7B6 and the CD28 (28) hinge domain (HiD), CD28 transmembrane domain (TMD), intracellular CD28 co-stimulatory domain (CSD), and CDS zeta (3z) activation signaling domain (ASD) (Kittel-Boselli et al., 2021). The targeting module is composed of a variable heavy chain (VHD) derived from monoclonal antibodies (mAbs) CD33, CD123, 5B9, or 7B6. H ) and light chain (V L ) domains, connected by a glycine (G)-serine (S) linker. Summary of the invention

[0009] The object of the present invention is to provide an improved targeting module for the RevCAR system.

[0010] The present invention comprises at least one CD123 binding domain and a V L -Connector-V H The structure of the targeting module that binds the tag binding domain of the human La epitope E5B9 is realized.

[0011] The targeting module of the present invention comprises

[0012] i) at least one CD123 binding domain comprising a sequence having at least 95% identity, or at least 99% identity, to SEQ ID No. 22 and SEQ ID No. 23, and

[0013] ii) Contains V L -Connector-V H The structure of the tag binding domain that binds to the human La epitope E5B9, wherein the V L The region comprises a sequence having at least 95% identity, or at least 99% identity, to the sequence of SEQ ID No. 19, and / or a V of the tag binding domain. H The region comprises a sequence having at least 95% identity, or at least 99% identity to the sequence of SEQ ID No. 20.

[0014] In this specification, the term "targeting module" refers to a molecule having at least two different domains, preferably a polypeptide or protein, wherein each domain is specific to a target or a group of identical targets, wherein at least one domain is specific to a target cell, particularly a CD123 binding domain; and one domain is specific to a reversible chimeric antigen receptor, particularly a tag binding domain. In certain embodiments, the targeting module is separated. In certain embodiments, the targeting module of the present invention is expressed as a recombinant protein. In certain embodiments, the targeting module is chemically synthesized.

[0015] The term "autoimmune disease" refers to an abnormal immune response (autoimmunity) of the body to substances and tissues normally present in the body.

[0016] The term "domain" refers to a portion of a protein sequence that can exist and function independently of the rest of the protein.

[0017] The term "V L -Connector-V H Structure" refers to a structure in which V L The C-terminus of the region is connected to the linker, and the linker is connected to V H The N-terminus of the region is connected.

[0018] The term "linker" (also called spacer) refers to a molecule or a part of a molecule that separates at least two elements, the element being selected from a functional group, a tag, a binding domain or a binding domain subunit, such as V L and VH Structural domain.

[0019] The term "specificity" refers to the ability of an antibody or antibody fragment or protein, peptide or low molecular weight organic ligand to recognize and bind to a binding partner (eg, tumor antigen) protein present in a sample, but not substantially recognize or bind to other molecules in the sample.

[0020] The term "binding" refers to non-covalent binding, in particular ionic bonds, hydrogen bonds, Van der Waals forces and / or hydrophobic interactions.

[0021] The term "mutant" refers to a peptide or protein having at least 90% sequence identity, preferably at least 95% sequence identity with a specified antibody, antibody fragment, protein or peptide. The mutant can have one or more activities of a specified antibody, antibody fragment, peptide or protein.

[0022] In certain embodiments, the mutant is a truncated form of a peptide or protein. The term "truncated form" refers to a shortened peptide or protein having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 90% chain length and 100% sequence identity, and most preferably at least 95% chain length and 100% sequence identity with a specified peptide or protein. The activity of the truncated form is at least 80%, preferably at least 90%, and more preferably at least 95% of the activity of the specified peptide or protein.

[0023] The term "nucleoprotein" refers to proteins found in the nucleus of a cell.

[0024] The tag is a peptide sequence from a nuclear antigen that cannot be accessed and bound by the corresponding tag binding domain under physiological conditions in the context of the native protein. The tag is non-immunogenic, which minimizes the risk of uncontrolled off-site target toxicity in CAR-expressing immune cells, such as the release of toxic levels of cytokines, also known as cytokine storm or cytokine release syndrome (CRS).

[0025] The term "combined administration" refers to a therapeutic method in which a targeting module is administered before, simultaneously with and / or after administration of a vector or cell comprising a nucleotide sequence encoding a reversible chimeric antigen receptor (RevCAR).

[0026] The term "antibody" refers to a protein that binds to an antigen via its variable region (Fab). H ) and light chain (V L ) are composed of one constant domain and one variable domain, respectively. The term "antibody fragment or antigen-binding fragment" refers to a fragment that contains at least the V L or V HIn certain 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 a fragment produced by a Fab expression library or a single domain antibody (nanobody).

[0027] The term "single-chain variable fragment (scFv)" refers to an artificially synthesized antibody fragment comprising a covalently linked antibody light chain variable domain and a heavy chain variable domain. L and V H In certain embodiments, the short peptide is covalently linked to V H The N-terminal and V L or vice versa.

[0028] In certain embodiments, the CD123 binding domain comprises a CDR sequence as shown in SEQ ID No.33, SEQ ID No.34, SEQ ID No.35, SEQ ID No.36, the amino acid sequence WAS (tryptophan-alanine-serine) and SEQ ID No.37.

[0029] In certain embodiments, the tag binding domain that binds to the human La epitope E5B9 comprises the CDR sequences shown in SEQ ID No.38, SEQ ID No.39, SEQ ID No.40, SEQ ID No.41, the amino acid sequence WAS (tryptophan-alanine-serine) and SEQ ID No.42.

[0030] The term "CDR (complementarity determining region)" refers to the portion of the variable chain in an antibody or antibody fragment that binds to its specific antigen. Antibodies contain three non-contiguous CDRs (CDR1, CDR2, and CDR3) in the amino acid sequence of each variable domain, so that in two variable domains (V H and V L ) have six CDRs that can contact the antigen.

[0031] In certain embodiments, V L and V H The linker is connected via a glycine-serine linker having the structure (GxSy), wherein 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. 24). Preferably, the linker consists of a peptide sequence capable of increasing the protease resistance of the antibody derivative.

[0032] In certain embodiments, the linker of the tag binding domain comprises 20 to 30 amino acids, preferably 25 amino acids.

[0033] In certain embodiments, the sequence of the linker of the tag binding domain is shown as SEQ ID No.25 or SEQ ID No.26.

[0034] In certain embodiments, the antibody is derived from an animal, preferably from a mammal, such as a human, ape, mouse, rat, rabbit, guinea pig, horse, cattle, sheep, goat, pig, dog or cat. Preferably, the antibody or antibody fragment is a human, humanized or de-immunized antibody. Humanized antibodies can be prepared in various ways, for example, by surface reconstruction and CDR transplantation. In the case of surface reconstruction, all non-CDR regions on the antibody surface are modified using molecular modeling, statistical analysis and mutagenesis techniques in combination to make them similar to the antibody surface of the target organism. In CDR transplantation, the CDR region of the present invention is introduced into a known human framework region, and the sequence of the human framework region is similar to the original sequence. De-immunized antibodies can be obtained by specifically mutating the residues that will produce immunogenic hot spots determined based on computer peptide-MHC affinity prediction.

[0035] In certain embodiments, the antibody or antibody fragment is a polyclonal, monoclonal or chimeric antibody in which the antigen binding region of a non-human antibody is transferred into the framework of a human antibody by recombinant DNA technology (such as computer-designed recombinant DNA technology).

[0036] In certain embodiments, antibodies against a selected tag or antigen can be produced by immunizing various hosts including, but not limited to, goats, rabbits, rats, mice, and humans by injecting cells expressing a specific protein, DNA or RNA encoding the protein, the protein itself, or any portion, fragment, or oligopeptide that retains the immunogenicity of the protein.

[0037] In certain embodiments, the CD123 binding domain is an antibody or antigen binding fragment.

[0038] In certain embodiments, the CD123 binding domain comprises the sequences of SEQ ID No.22 and SEQ ID No.23.

[0039] In certain embodiments, the CD123 binding domain comprises the sequence shown in SEQ ID No.27.

[0040] The tag binding domain of the present invention binds to a tag from the human La epitope E5B9. In certain embodiments, the tag binding domain is an antibody or antigen binding fragment, preferably comprising a V having the following sequence: L :

[0041] DIVMTQSPDSLAVSLGERATINCX 24 SSQSLLNSRTX 35KNYLAWYQQKPGQPPKLLIYWASTRX 61 SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYNLX 101 TFGGGTKVE1K (SEQ ID No. 19), wherein X 24 , X 35 , X 61 and X 101 are independently selected from proteinogenic α-amino acid residues;

[0042] Or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity to the sequence SEQ ID No. 21.

[0043] In certain embodiments, the tag binding domain constitutes an anti-La epitope single chain variable fragment (scFv).

[0044] In certain embodiments, X 24 To X 101 The choices are as follows:

[0045] X 24 selected from polar and / or positively charged residues such as serine, threonine, asparagine, glutamine, histidine, lysine and arginine; preferably lysine or arginine;

[0046] X 35 Preferably selected from lysine and proline;

[0047] X 61 selected from polar and charged residues such as asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine and arginine, preferably glutamic acid and lysine;

[0048] X 101 Selected from hydrophobic residues such as isoleucine, leucine, valine, alanine, methionine, phenylalanine, proline and tryptophan; preferably leucine or proline.

[0049] Preferably, the tag binding domain comprises a sequence SEQ ID NO.20 (V H ) and SEQ ID NO.21 (V L ) have at least 90% sequence identity, preferably at least 95% sequence identity.

[0050] Most preferably, the tag binding domain is a protein having a sequence as shown in SEQ ID NO. 20 (V H ) and SEQ ID NO.21 (V L ) Anti-La 5B9 scFv with the sequence shown.

[0051] In certain embodiments, the targeting module is between 100 and 1600 amino acids in length, preferably between 500 and 800 amino acids in length.

[0052] In certain embodiments, the targeting module comprises one of the sequences shown in SEQ ID No. 3 to SEQ ID No. 10.

[0053] In another aspect, the present invention provides a nucleic acid, a vector or a cell comprising a nucleotide sequence encoding a targeting module of the present invention.

[0054] In certain embodiments, the nucleic acid, vector or cell comprises one of the sequences shown in SEQ ID No. 11 to 18. The sequences shown in SEQ ID No. 11 to 18 encode the targeting modules shown in SEQ ID No. 3 to 10.

[0055] In another aspect, the present invention provides a pharmaceutical composition comprising the targeting module of the present invention and a pharmaceutically acceptable diluent or carrier.

[0056] The pharmaceutical composition is preferably administered parenterally, particularly preferably intravenously. In certain embodiments, the pharmaceutical composition is in a form suitable for intravenous administration. Preferably, the pharmaceutical composition is a solution, emulsion or suspension.

[0057] In certain embodiments, the pharmaceutical composition is an injectable buffer solution comprising the targeting module, nucleic acid, vector and / or cell of the present invention at a concentration ranging from 1 ng / ml to 500 mg / ml, preferably ranging from 50 μg / ml to 5 mg / ml.

[0058] The pharmaceutical composition comprises a pharmaceutically acceptable diluent or carrier. In certain embodiments, the carrier is selected from water, aqueous buffer solution, 0.9% saline, 5% glucose solution, 5% xylitol solution, 0.3% glycine solution, Ringer solution or amino acid solution. In certain embodiments, the aqueous buffer solution is selected from aqueous histidine, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate buffer solution with a pH value in the range of 5.0 to 7.0. In certain embodiments, the buffer concentration of the aqueous buffer solution is in the range of 1mmol / L (mM) to 500mM, preferably in the range of 5mM to 20mM, and particularly preferably in the range of 5mM to 10mM.

[0059] In certain embodiments, the carrier comprises sodium chloride, preferably at a concentration ranging from 1 mM to 300 mM, particularly preferably 150 mM.

[0060] In certain embodiments, the pharmaceutical composition further comprises a stabilizer, preferably at a concentration ranging from 1 mM to 900 mM, particularly preferably ranging from 50 mM to 600 mM. In certain embodiments, the stabilizer is sucrose, trehalose or L-methionine.

[0061] In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. The term "pharmaceutically acceptable excipient" refers to a compound that provides approximate physiological conditions and / or increases stability, such as agents and buffers for adjusting pH, agents for adjusting toxicity, etc. In certain embodiments, the pharmaceutically acceptable excipient is selected from sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and polysorbate-80, preferably 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) Polysorbate-80.

[0062] In certain embodiments, the pharmaceutical composition comprises a targeting module in a dosage range of 25 μg / day to 100 mg / day, preferably 0.1 mg / day to 20 mg / day.

[0063] In certain embodiments, the pharmaceutical composition is sterile. The pharmaceutical composition is sterilized by conventional well-known techniques, including but not limited to sterile filtration.

[0064] In certain embodiments, the pharmaceutical compositions are for administration to a subject.

[0065] In certain embodiments, the pharmaceutical composition is lyophilized prior to storage, or stored in solution at ambient or subambient temperature, including but not limited to frozen storage.

[0066] In certain embodiments, the pharmaceutical composition is reconstituted and / or diluted in an infusion and stabilizer solution prior to administration to a subject. The solution for reconstitution or infusion / stabilization may contain any of the components mentioned above for the pharmaceutical composition or similar components.

[0067] In another aspect, the present invention provides a targeting module, a nucleic acid, a vector or a cell comprising a nucleotide sequence encoding the targeting module of the present invention, or a pharmaceutical composition comprising the targeting module of the present invention and a pharmaceutically acceptable diluent or carrier, which is used in a method for stimulating a chimeric antigen receptor-mediated immune response in a mammal, preferably for treating cancer, an infectious disease or an autoimmune disease.

[0068] In certain embodiments, the targeting module is administered in combination with a vector or cell comprising a nucleotide sequence encoding a RevCAR, wherein the RevCAR comprises

[0069] - a tag, wherein the tag is human La epitope E5B9 (SEQ ID No. 28),

[0070] - the extracellular hinge and transmembrane domain, and

[0071] - signal transduction domain,

[0072] The tag binding domain of the targeting module binds to the tag of the RevCAR.

[0073] In certain embodiments, the pharmaceutical composition for stimulating a chimeric antigen receptor-mediated immune response in a mammal further comprises a vector or cell comprising a nucleotide sequence encoding RevCAR, wherein the RevCAR comprises

[0074] - a tag, wherein the tag is human La epitope E5B9 (SEQ ID No. 28),

[0075] - the extracellular hinge and transmembrane domain, and

[0076] - signal transduction domain,

[0077] The tag binding domain of the targeting module binds to the tag of the RevCAR.

[0078] In another aspect, the present invention provides a kit comprising

[0079] a) a targeting module or a nucleic acid, a vector or a cell according to the invention, and

[0080] b) a vector or a cell comprising a nucleotide sequence encoding RevCAR,

[0081] The RevCAR comprises

[0082] - a tag, wherein the tag is human La epitope E5B9 (SEQ ID No. 28),

[0083] - the extracellular hinge and transmembrane domain, and

[0084] - signal transduction domain,

[0085] The tag binding domain of the targeting module binds to the tag of the RevCAR.

[0086] In certain embodiments, the extracellular hinge and transmembrane domain of the RevCAR are selected from one of the following: hinge and transmembrane domains of human CD28 molecules, CD8a chains, NK cell receptors, portions of antibody constant regions, and combinations of the above domains.

[0087] In certain embodiments, the signal transduction domain of RevCAR is selected from one of the following: CD28, CD137 (4-1BB), CD134 (OX40), CD278 (ICOS), DAP10 and the cytoplasmic region of CD27, programmed cell death protein-1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), the cytoplasmic region of the CD3 chain, DAP12, CD122 (interleukin-2 receptor β), CD132 (interleukin-2 receptor γ), CD127 (interleukin-7 receptor α), CD360 (interleukin-21 receptor), activated Fc receptor, and mutants of the above domains.

[0088] In certain embodiments, the kits of the invention comprise at least one other targeting module or at least one other nucleic acid, vector or cell encoding another targeting module, wherein the at least one other targeting module comprises at least one target cell binding domain and a tag binding domain.

[0089] The target cell binding domain is an antibody, antibody fragment, protein, peptide or low molecular weight organic ligand, or a mutant and analog thereof, and the target cell binding domain can bind to one or more of the following surface antigens: CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD15, CD19, CD20, CD22, CD23, CD25, CD30, CD33, CD38, CD44, CD44v6, CD52, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD90, CD99, CD123, CD133, CD135, CD150, CD181, CD182, CD184, CD223, CD229, CD269, CD273, CD274, CD276, CD279, CD319, CD366 and CD371, cytokine receptors, preferably interleukin receptors, particularly preferably IL-8Rα, IL-8Rβ, IL-11Rα, IL-11Rβ, IL13Rα1; CXCR4, c-Met, mesothelin, epidermal growth factor receptor family members and Mutants thereof, particularly preferably ErbB1, ErbB2, ErbB3, ErbB4 or their mutants; members of the tumor necrosis factor receptor superfamily, tight junction proteins, ephrins, ephrin receptors, particularly preferably EphA1-10, EphA5 or EphB1-6; fucosyltransferase, prostate specific antigen, preferably PSCA and PSMA; embryonic antigens, preferably CEA and fetal acetylcholine receptor; members of the vascular endothelial growth factor family, EpCAM, AFP, members of the intercellular adhesion molecule family, C-type lectins, integrins, mucin family family members, FSHR, HMW-MAA, FBP, folate receptor, somatostatin receptor, ligand of NKG2D receptor, epithelial cell glycoprotein family members, disialoganglioside, phosphatidylinositol glycan, G protein-coupled receptor, human papillomavirus protein, cancer-testis antigen, fibroblast activation protein, carbonic anhydrase family members, carbohydrate antigen family members, Notch ligand, MCSP, glycoprotein A33, guanylate cyclase 2C and tumor-specific glycans, wherein the targeting module and the at least one other targeting module comprise the same tag binding domain.

[0090] In certain embodiments, the kit of the present invention comprises a targeting module and / or a vector or cell comprising a nucleotide sequence encoding RevCAR in the form of a pharmaceutical composition.

[0091] In certain embodiments, the kits of the invention are used in a method of stimulating a chimeric antigen receptor-mediated immune response in a mammal, preferably for treating cancer, infectious disease, or autoimmune disease.

[0092] In certain embodiments, the targeting module is administered alone, preferably 1 hour to 2 days before administering a vector or cell comprising a nucleotide sequence encoding RevCAR, more preferably 4 to 24 hours before administering. The targeting module is administered before administering a vector or cell comprising a nucleotide sequence encoding RevCAR to stimulate RevCAR and increase the amplification of RevCAR carrying effector cells and their accumulation at the target site.

[0093] In certain embodiments, the targeting module is administered simultaneously with a vector or cell comprising a nucleotide sequence encoding RevCAR.

[0094] In certain embodiments, the targeting module is administered after the vector or cell comprising the nucleotide sequence encoding RevCAR, preferably within the range of 3 to 30 days. In addition, additional such doses of targeting modules can be administered after the intermission period to reactivate the effector cells carrying RevCAR.

[0095] The term "reversible chimeric antigen receptor" refers to an artificial chimeric fusion protein, in particular a receptor comprising a tag, an extracellular hinge, a transmembrane domain and a signal transduction domain. These domains can be derived from different sources, and therefore, the receptor is called a chimera. The receptor can be combined with different targeting modules through the tag.

[0096] A cell comprising a nucleotide sequence encoding RevCAR expresses RevCAR, which has binding specificity to the tag binding domain of the targeting module, which in turn binds to CD123 on the target cell.

[0097] In certain embodiments, the targeting moiety is in monomeric, dimeric or multimeric form, preferably in monomeric form.

[0098] In certain embodiments, the targeting moiety is monovalent, bivalent, or multivalent.

[0099] In certain embodiments, the targeting module is bivalent or multivalent and comprises at least one CD123 binding domain and a V L -Connector-V H Structure of the tag binding domain that binds to the human La epitope E5B9.

[0100] In certain embodiments, different domains of the targeting module of the present invention are connected to each other by a linker. The linker comprises a short sequence of preferably 10 to 20 amino acid residues. In certain embodiments, the targeting module comprises a flexible peptide sequence, and a suitable flexible peptide sequence is selected so that the domain has a three-dimensional folded structure so that it can show specificity for effector cells and target cells. A preferred linker is a glycine-serine linker with a structure (GxSy), wherein x and y are selected from 1 to 10, preferably 1 to 5. Most preferably, 1 to 10 repeats of the sequence G4S1 (SEQ ID No.24). In addition, the linker is preferably composed of a peptide sequence that can increase the protease resistance of the antibody derivative.

[0101] In certain embodiments, the sequence of the linker is shown as SEQ ID No.25 or SEQ ID No.26.

[0102] In certain embodiments, the targeting module of the present invention comprises another other domain selected from one of the following: a co-stimulatory ligand, a radionuclide, a cell death inducing compound, and a half-life extension domain, preferably IgG1Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, HSA, FcRn binding peptide or a mutant thereof. The term "mutant" refers to a protein having at least 90% sequence identity, preferably at least 95% sequence identity, to a half-life extension domain. The mutant can have one or more activities of a specified peptide or protein; in particular, the mutant, like a half-life extension domain, can extend the half-life.

[0103] In certain embodiments, the targeting module of the present invention is used in a method for stimulating a chimeric antigen receptor-mediated immune response in a mammal, wherein the targeting module is administered in combination with a vector or cell comprising a nucleotide sequence encoding a reversible chimeric antigen receptor and at least one other targeting module, wherein the at least one other targeting module comprises at least one target cell binding domain and a tag binding domain or tag, wherein the target cell binding domain is an antibody, antibody fragment, protein, peptide or low molecular weight organic ligand, or a mutant and analog thereof, and the target cell binding domain is capable of binding to one or more of the following surface antigens: CD2, CD3, CD4, CD8, CD10, CD19, CD20, CD22, CD23, CD25, CD30, CD33, CD38, CD44, CD44v6, CD52, CD90, CD99, CD133, CD150, CD181, CD182, CD184, CD223, CD229, CD269 (BCMA), CD273, CD274, CD276, CD279, CD319, CD366 and CD371, interleukin receptors, particularly preferably IL-8Rα (CXCR1), IL-8Rβ (CXCR2), IL-11Rα, IL-11Rβ, IL13Rα1; CXCR4, c-Met, interleukin receptors, particularly preferably IL-8Rα (CXCR1), IL-8Rβ (CXCR2), IL-11Rα, IL-11Rβ, IL13Rα1; ephrins, epidermal growth factor receptor family members and mutants thereof, particularly preferably ErbB1, ErbB2, ErbB3, ErbB4 or mutants thereof; tumor necrosis factor receptor superfamily members, ephrins, ephrin receptors, particularly preferably EphA1-10, EphA5 or EphB1-6; prostate specific antigens, preferably prostate stem cell antigen (PSCA) and prostate specific membrane antigen (PSMA); embryonic antigens, preferably carcinoembryonic antigen (CEA) and fetal acetylcholine receptor; vascular endothelial growth factor family members, epithelial cell adhesion molecule (EpCAM), alpha-fetoprotein (AFP), intercellular adhesion molecule family members, stick protein The targeting module of the present invention and at least one other targeting module comprise the same tag binding domain.

[0104] The term "target cell binding domain" refers to a peptide, protein or low molecular weight organic ligand that specifically binds to a protein or protein complex (antigen) on the surface of a target cell (preferably a cancer cell, T cell, infected cell, pathogen or parasite).

[0105] The terms "peptide", "polypeptide" and "protein" are used interchangeably to refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can be contained in a protein or peptide sequence.

[0106] The term "low molecular weight organic ligand" refers to an organic molecule with a molecular weight of up to 10 kilodaltons, preferably up to 3 kilodaltons, which specifically binds to a protein or protein complex (antigen) on the surface of a target cell (preferably a cancer cell, a T cell, an infected cell or a pathogen or parasite).

[0107] The term "target cell binding domain" also includes soluble T cell receptors, which are composed of α and β or γ and δ chains of T cell receptors (TCR), and also include fragments or mutants thereof. Such TCR-derived binding moieties recognize and bind peptides presented by human leukocyte antigen class I (HLA) and class II protein complexes. The TCR is specific for peptides derived from the following proteins: EGFR family, survivin, sex-determining region Y box (SOX) protein family, melanoma-associated antigens (e.g., autoimmunogenic cancer / testis antigen NY-ESO-1, melanoma antigen family member A MAGEA, antigens preferentially expressed in melanoma PRAME) and leukemia-associated antigens (e.g., Wilms tumor gene 1, WT1).

[0108] The term "analog" refers to a molecule that has a high degree of structural identity with a specified antibody, antibody fragment, protein, peptide or low molecular weight organic ligand, preferably at least one atom, atomic group, functional group or substructure is replaced by another atomic group such as hydroxyl. In certain embodiments, the analog of somatostatin (SRIF14) is octreotide or pasireotide. The analog binds to the same antigen as the specified antibody, antibody fragment, protein, peptide or low molecular weight organic ligand.

[0109] In certain embodiments, analogs of a given antibody, antibody fragment, protein or peptide are modified with the following functional groups: D amino acids, pseudopeptide bonds, amino alcohols, non-proteinogenic amino acids, unnatural amino acids, amino acids with modified side chains and / or cyclic proteins. These analogs exhibit increased stability.

[0110] In certain embodiments, the target cell binding domain is a soluble T cell receptor composed of the α and β or γ and δ chains of the T cell receptor (TCR).

[0111] In certain embodiments, at least one additional targeting module comprises at least one target cell binding domain and a tag binding domain or tag, wherein the target cell binding domain is an antibody or antibody fragment that is capable of binding to one or more of the following surface antigens: CD2, CD3, CD4, CD8, CD10, CD19, CD20, CD22, CD23, CD25, CD30, CD33, CD38, CD44, CD44v6, CD52, CD90, CD99, CD133, CD 150, CD181, CD182, CD184, CD223, CD229, CD269, CD273, CD274, CD276, CD279, CD319, CD366 and CD371, interleukin receptors, particularly preferably IL-8Rα, IL-8Rβ, IL-11Rα, IL-11Rβ, IL13Rα1; CXCR4, c-Met, mesothelin, epidermal growth factor receptor family members, particularly preferably ErbB1, ErbB 2, ErbB3 or ErbB4; members of the tumor necrosis factor receptor superfamily, ephrins, ephrin receptors, particularly preferably EphA1-10, EphA5 or EphB1-6; prostate specific antigens, preferably PSCA and PSMA; embryonic antigens, preferably CEA and fetal acetylcholine receptor; members of the vascular endothelial growth factor family, EpCAM, AFP, members of the intercellular adhesion molecule family, members of the mucin family, FSHR, HMW-MAA, folate binding protein FBP, folate receptors, somatostatin receptors, ligands of NKG2D receptors, cytokine receptors, members of the epithelial cell glycoprotein family, disialyl gangliosides, phosphatidylinositol glycans, G protein-coupled receptors, members of the carbonic anhydrase family, members of the carbohydrate antigen family, Notch ligands, MCSP, glycoprotein A33, guanylate cyclase 2C and tumor-specific glycans, wherein the targeting module of the present invention and at least one other targeting module comprise different target cell binding domains and the same tag binding domain or tag.

[0112] In certain embodiments, the nucleic acid, vector and / or cell is isolated. The term "isolated" means altered or removed from its natural state.

[0113] In certain embodiments, the nucleic acid is cDNA. The term "cDNA" (complementary DNA) refers to a double-stranded DNA synthesized from a single-stranded RNA, such as mRNA, by a reaction catalyzed by a reverse transcriptase. In certain embodiments, the cDNA is artificially synthesized. In certain embodiments, the cDNA is derived from mRNA and therefore, in contrast to genomic DNA, contains only exons and no introns.

[0114] The vector is preferably a plasmid, an artificial chromosome, linearized DNA or RNA, a viral particle or other vector which contains an expression cassette which is stably integrated into the genome of the host cell or host organism.

[0115] In certain embodiments, the cell is selected from immune cells, preferably immune cells with cytolysis, phagocytosis or immunosuppressive activity, such as T cells, natural killer (NK) cells and macrophages. In certain embodiments, the cell is selected from T cells, including α / β and γ / δ T cells or T cell subsets, such as stem cell memory T cells or central memory T cells, cytotoxic T cells or NK cells.

[0116] In certain embodiments, the nucleic acid, vector or cell further comprises an inducible expression system. In certain embodiments, the inducible expression system is based on a prokaryotic operon, including but not limited to a lactose operon, a transposon Tn10 or a tetracycline operon. In certain embodiments, the inducible expression system is based on a component of a eukaryotic signal transduction pathway, including but not limited to an expression system based on a steroid receptor, an estrogen receptor, a progesterone or a metallothionein.

[0117] In certain embodiments, the inducible expression system induces transcription of a nucleotide sequence encoding RevCAR and / or a nucleotide sequence encoding a targeting module of the present invention. Preferably, the inducible expression system induces transcription of a nucleotide sequence encoding a targeting module of the present invention.

[0118] In certain embodiments, a nucleic acid, vector or cell is administered in combination with a vector or cell comprising a nucleotide sequence encoding RevCAR and a nucleic acid, vector or cell comprising a nucleotide sequence encoding another targeting module, wherein at least one other targeting module comprises at least one target cell binding domain and a tag binding domain or tag, wherein the target cell binding domain is an antibody, antibody fragment, protein, peptide or low molecular weight organic ligand, or a mutant and analog thereof, and the target cell binding domain is capable of binding to one or more of the following surface antigens: CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD15, CD19, CD20, CD22, CD23, CD25, CD30, CD33, CD38, CD44, CD44v6, CD52, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD90, CD99, CD123, CD133, CD135, CD150, CD181, CD182, CD184, CD223, CD229, CD269, CD273, CD274, CD276, CD279, CD319, CD366 and CD371, cytokine receptors, preferably interleukin receptors, particularly preferably IL-8Rα, IL-8Rβ, I L-11Rα, IL-11Rβ, IL13Rα1; CXCR4, c-Met, mesothelin, epidermal growth factor receptor family members and mutants thereof, particularly preferably ErbB1, ErbB2, ErbB3, ErbB4 or their mutants; tumor necrosis factor receptor superfamily members, tight junction proteins, ephrins, ephrin receptors, particularly preferably EphA1-10, EphA5 or EphB1-6; fucosyltransferase, prostate specific antigen, preferably PSCA and PSMA; embryonic antigens, preferably CEA and fetal acetylcholine receptor; vascular endothelial growth factor family members, EpCAM, AFP, cell Intercellular adhesion molecule family members, C-type lectins, integrins, mucin family members, FSHR, HMW-MAA, FBP, folate receptors, somatostatin receptors, ligands of NKG2D receptors, epithelial cell glycoprotein family members, disialogangliosides, phosphatidylinositol glycans, G protein-coupled receptors, human papillomavirus proteins, cancer-testis antigens, fibroblast activation proteins, carbonic anhydrase family members, carbohydrate antigen family members, Notch ligands, MCSP, glycoprotein A33, guanylate cyclase 2C and tumor-specific glycans, wherein the targeting module of the present invention and at least one other targeting module comprise the same tag binding domain.

[0119] In certain embodiments, the pharmaceutical composition comprises at least one other targeting module or at least one other nucleic acid, vector or cell encoding the other targeting module, wherein at least one other targeting module comprises at least one target cell binding domain and a tag binding domain or tag, wherein the target cell binding domain is an antibody, antibody fragment, protein, peptide or low molecular weight organic ligand, or a mutant and analog thereof, and the target cell binding domain is capable of binding to one or more of the following surface antigens: CD2, CD3, CD4, CD8, CD10, CD19, CD20, CD22, C D23, CD25, CD30, CD33, CD38, CD44, CD44v6, CD52, CD90, CD99, CD133, CD150, CD181, CD182, CD184, CD223, CD229, CD269, CD273, CD274, CD276, CD279, CD319, CD366 and CD371, interleukin receptors, particularly preferably IL-8Rα, IL-8Rβ, IL-11Rα, IL-11Rβ, IL13Rα1 CXCR4, c-Met, mesothelin, epidermal growth factor receptor family members and mutants thereof, particularly preferably ErbB1, ErbB2, ErbB3, ErbB4 or mutants thereof; tumor necrosis factor receptor superfamily members, ephrins, ephrin receptors, particularly preferably EphA1-10, EphA5 or EphB1-6; prostate specific antigens, preferably PSCA and PSMA; embryonic antigens, preferably CEA and fetal acetylcholine receptor; vascular endothelial growth factor family members, EpCAM, AFP, intercellular Adhesion molecule family members, mucin family members, FSHR, HMW-MAA, FBP, folate receptor, somatostatin receptor, ligand of NKG2D receptor, cytokine receptor, epithelial cell glycoprotein family members, disialyl ganglioside, phosphatidylinositol glycan, G protein-coupled receptor, carbonic anhydrase family members, carbohydrate antigen family members, Notch ligand, MCSP, glycoprotein A33, guanylate cyclase 2C and tumor-specific glycans, wherein the targeting module and at least one other targeting module comprise the same tag binding domain.

[0120] In certain embodiments, the tag binding domain binds to a tag from human nuclear La protein. Preferably, the tag binding domain is an antibody or antigen binding fragment comprising V as shown in the following sequence: L :

[0121] DIVMTQSPDSLAVSLGERATINCX 24 SSQSLLNSRTX 35 KNYLAWYQQKPGQPPKLLIYWASTRX 61SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYNLX 101 TFGGGTKVE1K (SEQ ID No. 19), wherein X 24 , X 35 , X 61 and X 101 Independently of each other, they are selected from proteinogenic α-amino acid residues; or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity with SEQ ID No. 21.

[0122] In certain embodiments, the tag binding domain constitutes an anti-La epitope scFv.

[0123] In certain embodiments, X 24 To X 101 The choices are as follows:

[0124] X 24 selected from polar and / or positively charged residues such as serine, threonine, asparagine, glutamine, histidine, lysine and arginine; preferably lysine or arginine;

[0125] X 35 Preferably selected from lysine and proline;

[0126] X 61 selected from polar and charged residues such as asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine and arginine, preferably glutamic acid and lysine;

[0127] X 101 Selected from hydrophobic residues such as isoleucine, leucine, valine, alanine, methionine, phenylalanine, proline and tryptophan; preferably leucine or proline.

[0128] Preferably, the tag binding domain comprises SEQ ID No. 20 (V H ) and SEQ ID No.21 (V L ) sequences having at least 90% sequence identity, preferably at least 95% sequence identity.

[0129] Particularly preferably, the tag binding domain has a sequence as shown in SEQ ID No. 20 (V H ) and SEQ ID No.21 (V L ) Anti-La 5B9 scFv with the sequence shown.

[0130] The term "extracellular hinge and transmembrane domain" refers to the flexible peptide sequence connected to the tag, which anchors RevCAR into the cell membrane of the cell and protrudes from the cell surface for optimal binding to its specific targeting module.

[0131] In certain embodiments, the extracellular hinge and transmembrane domain are selected from the following hinges and transmembrane domains: human CD28 molecules, CD8a chains, NK cell receptors (preferably natural killer group NKG2D), or portions of antibody constant regions, and combinations thereof. The term "combination thereof" refers to a combination of different hinge and transmembrane domains.

[0132] Pinthus et al. and Cartellieri et al. described the use of the hinge and transmembrane domains of the human CD28 molecule in CAR (Pinthus et al., 2003, Cartellieri et al., 2016).

[0133] Milone et al. and Zhao et al. described the use of the hinge and transmembrane domains of the human CD8α molecule in CAR (Milone et al., 2009, Zhao et al., 2009).

[0134] Zhang et al. described the use of the hinge and transmembrane domains of NKG2D in CAR (Zhang et al., 2005).

[0135] Frigault et al. and Wang et al. describe the use of hinge and transmembrane domains of portions of the immunoglobulin G1 (IgG) constant region (Frigault et al., 2015, Wang et al., 2007). Frigault et al. describe the use of the hinge domain of the IgG4 constant region.

[0136] Examples of combinations of extracellular hinges and transmembrane domains include, but are not limited to, CD28 extracellular hinge and transmembrane domain, CD8α extracellular hinge and transmembrane domain, IgG1 or IgG4 constant regions in combination with CD28 or CD137 transmembrane domains.

[0137] The term "signal transduction domain" refers to a peptide sequence that transmits signals to cells by cross-linking cells expressing RevCAR (effector cells) with human cell surface proteins or protein complexes (target cells). The cross-linking between effector cells and target cells is mediated by the targeting module of the present invention.

[0138] In certain embodiments, the signal transduction domain is selected from the group consisting of: CD28, CD137 (4-1BB), CD134 (OX40), CD278 (ICOS), DAP10 and the cytoplasmic region of CD27, programmed cell death protein-1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), the cytoplasmic region of the CD3 chain, DAP12, CD122 (interleukin-2 receptor β), CD132 (interleukin-2 receptor γ), CD127 (interleukin-7 receptor α), CD360 (interleukin-21 receptor), activated Fc receptors and mutants thereof.

[0139] The term "mutant" refers to a protein having at least 90% sequence identity, preferably at least 95% sequence identity, to a signal transduction domain. The mutant transmits the signal to the cell in the same manner as the specified signal transduction domain by cross-linking cells expressing RevCAR (effector cells) with human cell surface proteins or protein complexes (target cells).

[0140] In certain embodiments, the mutant is a truncated form. The term "truncated form" refers to a shortened protein having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 90% chain length and 100% sequence identity, most preferably at least 95% chain length and 100% sequence identity with a signal transduction domain. The activity of the truncated form is at least 80%, preferably at least 90%, and more preferably at least 95% of that of a specified signal transduction domain.

[0141] Hombach et al. and Cartellieri et al. described the use of the cytoplasmic region of CD28 as a signaling domain in CAR (Hombach et al., 2001, Cartellieri et al., 2016). Guedan et al. described the use of mutants of the cytoplasmic region of CD28 as a signaling domain (Guedan et al., 2020).

[0142] Milone et al. and Finney et al. described the use of the cytoplasmic region of CD137 (4-1BB) as a signaling domain (Finney et al., 2004, Milone et al., 2009).

[0143] Finney et al. and Hombach and Abken described the use of the cytoplasmic region of CD134 (OX40) as a signaling domain in CARs (Finney et al., 2004, Hombach and Abken, 2011).

[0144] Guedan et al. described the use of the cytoplasmic region of CD278 (ICOS) as a signaling domain (Guedan et al., 2018).

[0145] Zhang et al. described the use of DAP10 as a signal transduction domain (Zhang et al., 2005).

[0146] Fedorov et al. described the use of programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte antigen 4 (CTLA-4) as signaling domains in CARs (Fedorov et al., 2013).

[0147] Gong et al. and Gade et al. described the use of the cytoplasmic region of the CD3 chain, in particular the CD3ζ chain, as a signaling domain in CAR (Gong et al., 1999, Gade et al., 2005).

[0148] et al. described the use of DAP12 as a signaling domain in CAR ( et al., 2015 ).

[0149] Kagoya et al. described the use of signaling chains or motifs derived from interleukin receptors as signaling domains in CARs (Kagoya et al., 2018).

[0150] Lamers et al. and Kershaw et al. describe the use of activating Fc receptors, in particular the Fcε receptor γ chain, as signaling domains (Lamers et al., 2004, Kershaw et al., 2006).

[0151] In certain embodiments, the signal transduction domain is selected from: CD28, CD137 (4-1BB), CD134 (OX40), CD278 (ICOS), DAP10 and the cytoplasmic region of CD27, programmed cell death protein-1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), the cytoplasmic region of the CD3 chain, DAP12, CD122 (interleukin-2 receptor β), CD132 (interleukin-2 receptor γ), CD127 (interleukin-7 receptor α) and CD360 (interleukin-21 receptor), activated Fc receptor.

[0152] In certain embodiments, RevCAR comprises a fourth domain, wherein the fourth domain is a short peptide linker to the extracellular portion of the receptor that can be used to detect chimeric antigen receptors on the cell surface or stimulate chimeric antigen receptor T cells.

[0153] In certain embodiments, the fourth domain is located between the tag binding domain or tag and the extracellular hinge domain or a portion of the extracellular hinge domain.

[0154] In vitro or in vivo, RevCAR-transplanted cells with the fourth domain can be specifically stimulated to preferentially proliferate and last longer than cells without RevCAR transplantation. The fourth domain can also be used to purify RevCAR-transplanted cells from a mixed cell population, or to inhibit RevCAR-transplanted cell-mediated immune responses and eliminate RevCAR-transplanted cells in vivo.

[0155] In certain embodiments, RevCAR includes a signal peptide. The signal peptide enables RevCAR to be expressed on the cell surface of effector cells. In certain embodiments, the signal peptide is located at the N-terminus of the RevCAR nucleotide sequence in front of the tag binding domain or the tag. In certain embodiments, the signal peptide targets the protein to the secretory pathway in the co-translational stage or the post-translational stage, and the signal peptide is selected from the leader peptide of the heavy chain or light chain of proteins such as CD28, CD8α, IL-2, lysozyme C or human antibodies to avoid immunogenic reactions.

[0156] In certain embodiments, the nucleic acid is cDNA.

[0157] In certain embodiments, the tag is present at the amino terminus of the polypeptide comprising RevCAR. Placing the tag at the amino terminus enables the tag to be combined with the targeting module bound to the target cell without hindrance.

[0158] In certain embodiments, the nucleic acid encodes a RevCAR having a sequence as shown in SEQ ID No. 29 or SEQ ID No. 30. Preferably, the sequence of the nucleic acid is shown in SEQ ID No. 31 or SEQ ID No. 32.

[0159] In certain embodiments, the cell comprising the nucleotide sequence encoding RevCAR is selected from immune cells, preferably immune cells with cell lysis, phagocytosis or immunosuppressive activity, such as T cells, natural killer (NK) cells and macrophages. In certain embodiments, the cell is selected from T cells, including α / β and γ / δ T cells or T cell subsets, such as stem cell memory T cells or central memory T cells, cytotoxic T cells or NK cells.

[0160] In certain embodiments, the kit further comprises at least one other targeting module or at least one other nucleic acid, vector or cell encoding the other targeting module, wherein at least one other targeting module comprises at least one target cell binding domain and a tag binding domain or tag, wherein the target cell binding domain is an antibody, antibody fragment, protein, peptide or low molecular weight organic ligand, or a mutant and analog thereof, and the target cell binding domain is capable of binding to one or more of the following surface antigens: CD2, CD3, CD4, CD8, CD10, CD19, CD20, CD22, C D23, CD25, CD30, CD33, CD38, CD44, CD44v6, CD52, CD90, CD99, CD133, CD150, CD181, CD182, CD184, CD223, CD229, CD269, CD273, CD274, CD276, CD279, CD319, CD366 and CD371, interleukin receptors, particularly preferably IL-8Rα, IL-8Rβ, IL-11Rα, IL-11Rβ, IL13Rα1 CXCR4, c-Met, mesothelin, epidermal growth factor receptor family members and mutants thereof, particularly preferably ErbB1, ErbB2, ErbB3, ErbB4 or mutants thereof; tumor necrosis factor receptor superfamily members, ephrins, ephrin receptors, particularly preferably EphA1-10, EphA5 or EphB1-6; prostate specific antigens, preferably PSCA and PSMA; embryonic antigens, preferably CEA and fetal acetylcholine receptor; vascular endothelial growth factor family members, EpCAM, AFP, intercellular Adhesion molecule family members, mucin family members, FSHR, HMW-MAA, FBP, folate receptor, somatostatin receptor, ligand of NKG2D receptor, cytokine receptor, epithelial cell glycoprotein family members, disialyl ganglioside, phosphatidylinositol glycan, G protein-coupled receptor, carbonic anhydrase family members, carbohydrate antigen family members, Notch ligand, MCSP, glycoprotein A33, guanylate cyclase 2C and tumor-specific glycans, wherein the targeting module and at least one other targeting module comprise the same tag binding domain. BRIEF DESCRIPTION OF THE DRAWINGS

[0161] The present invention will now be further explained by means of the following figures and examples.

[0162] Figure 1The schematic diagram of the mode of the combined action of the constructed T cells and antigen-specific targeting modules (TM) of the present invention is shown, and the two together constitute active drugs. Allogeneic T cells (Allo-RevCAR-T) expressing RevCAR carry RevCAR epitopes (RCE or tags) on their cell surfaces, which are preferably short, non-immunogenic peptide motifs derived from human nuclear La / SSB autoantigens. Since there is no ligand bound to RCE or tags in the human body, Allo-RevCAR-T remains in closed mode (left). The antigen specificity of Allo-RevCAR-T is provided by a soluble targeting module (TM) with a single specificity for the target antigen. The targeting module (TM) of Allo-RevCAR consists of a target binding domain and a tag binding domain specific to RCE or tags. RevCAR-T is cross-linked with tumor cells expressing target antigens by TM to activate RevCAR-T effector functions, thereby killing tumor cells (right).

[0163] Figure 2 The surface plasmon resonance (SPR) sensor diagram of the targeting module of the present invention and the reference targeting module is shown, wherein the targeting module of the present invention and the reference targeting module have the same CD123 binding domain, and the tag binding domain of the targeting module of the present invention comprises structure V L -Connector-V H , while the tag binding domain of the reference targeting module contains structure V H -Connector-V L In the figure: A) SEQ ID No.2 (reference), B) SEQ ID No.5, C) SEQ ID No.6, D) SEQ ID No.8. In order to perform data fitting and K D For calculations, a 1:1 binding model was used.

[0164] Figure 3 The surface plasmon resonance (SPR) sensor diagram of the targeting module of the present invention and the reference targeting module is shown, wherein the targeting module of the present invention and the reference targeting module have the same CD123 binding domain, and the tag binding domain of the reference targeting module comprises V H -Connector-V L The targeting module of the present invention binds to La epitope 5B9 (SEQ ID No. 28), and its tag binding domain comprises V L -Connector-V H Structure. In the figure: A) SEQ ID No.2 (reference), B) SEQ ID No.3, C) SEQ ID No.4, D) SEQ ID No.5 and E) SEQ ID No.6. In order to perform data fitting and K D For calculations, a 1:1 binding model was used.

[0165] Figure 4 The binding of the targeting module of the present invention to cells of CD123-positive AML cell lines is shown. In the figure: A) SEQ ID No.5 and Oci-AML3, B) SEQ ID No.9 and Oci-AML3, C) SEQ ID No.4 and Molm-13, and D) SEQ ID No.6 and Molm-13. The mean fluorescence intensity (MFI) was plotted to obtain the dose-response curve, and the half-maximal binding amount (K) was calculated after fitting with a four-parameter logistic regression. D ).

[0166] Figure 5 The thermodynamic stabilities of two targeting modules (SEQ ID No. 5 and SEQ ID No. 9) of the present invention evaluated by melting point analysis in the pH range of 4.0 to 9.0 are shown.

[0167] Figure 6 The results of cytotoxicity assays of RevCAR T cells and different targeting modules of the present invention against the AML cell line Molm-13 are shown, the assay being performed using a suspension cell-based co-culture assay in the presence of different concentrations of the targeting module. In the figure: A) SEQ ID No.2 (reference), B) SEQ ID No.4, C) SEQ ID No.5 and D) SEQ ID No.6. Target cell lysis was plotted to obtain dose-response curves and EC was calculated after fitting with a four-parameter logistic regression. 50 .

[0168] Figure 7 The results of cytotoxicity assays of RevCAR T cells and different targeting modules of the present invention against the AML cell line Oci-AML3 are shown, the assay being performed using a suspension cell-based co-culture assay in the presence of different concentrations of the targeting module. In the figure: A) SEQ ID No.2 (reference), B) SEQ ID No.3, C) SEQ ID No.4, D) SEQ ID No.5 and E) SEQ ID No.6. Target cell lysis was plotted to obtain dose-response curves and EC was calculated after fitting with a four-parameter logistic regression. 50 .

[0169] Figure 8 Shown is the surface expression of CD25 on Allo-RevCAR-T under R-TM123-mediated activation.

[0170] Fig. 9 The specific lysis of AML cell line MOLM-13 by R-TM123 redirected Allo-RevCAR-T is shown.

[0171] Fig.10 The release of cytokines and effector molecules by Allo-RevCAR-T redirected by R-TM123 against the AML cell line MOLM-13 is shown.

[0172] Fig.11 The cytotoxic response of Allo-RevCAR-T redirected by R-TM123 against primary leukemia cells from AML patients was shown.

[0173] Fig.12 The in vivo pharmacokinetics of R-TM123 in NSG mice are shown.

[0174] Fig.13 Leukemia elimination of extramedullary disease in AML by R-TM123-redirected Allo-RevCAR-T is shown. DETAILED DESCRIPTION

[0175] Design of the targeting module of the present invention

[0176] The targeting module R-TM123 is a soluble recombinant fusion protein that contains two antibody-derived binding domains. One of the binding domains can selectively bind to the target antigen CD123, and the other binding domain can recognize the RCE or tag (epitope E5B9 from human La protein) presented on cells expressing RevCAR. Therefore, R-TM123 can serve as a connection module between RevCAR-T and target cancer cells expressing CD123 ( Figure 1 ). The C-terminus of the targeting module also contains an 8x histidine tag for detection and purification.

[0177] Table 1 shows the design and characteristics of different targeting modules (SEQ ID No. 3 to SEQ ID No. 6, SEQ ID No. 8 and SEQ ID No. 9) of the present invention, wherein the tag binding domain comprises V L -Connector-V H The reference targeting module has the same CD123 binding domain as the targeting module of the present invention, but its tag binding domain comprises V H -Connector-V L The monomer content was determined by high performance liquid chromatography (HPLC), and the dissociation constant K was determined by surface plasmon resonance (SPR). D The 50% effective concentration (EC 50 )(mu = murine, hu = humanized).

[0178]

[0179] Surface plasmon resonance measurements

[0180] The functionality of the targeting module that binds CD123 can be confirmed in a binding assay with soluble recombinant CD123 using surface plasmon resonance measurements ( Figure 2 , 3 and Table 1).

[0181] The binding of different variants of the targeting module of the present invention was analyzed by SPR measurements using human CD123-Fc coupled to a CM5 sensor chip on a Biacore X100 device ( Figure 2 ). Except for the targeting module with the sequence shown in SEQ ID No. 8, the targeting modules were purified monomers. These targeting modules were concentrated to a concentration between 1.23 and 100 nM and technical replicates were performed. In order to perform data fitting and K D For calculations, a 1:1 binding model was used.

[0182] The equilibrium dissociation constant (K) generated by the interaction between the targeting module binding to CD123 and human CD123 D ) was in the range of 23 pM to 5.2 nM, while the K of the reference targeting module (SEQ ID No. 2) D It is 227pM.

[0183] In addition, the binding of different variants of the targeting module of the present invention was analyzed using La epitope 5B9 (SEQ ID No. 28) fused to a human Fc domain coupled to a CM5 sensor chip on a Biacore X100 device ( Figure 3 ). The targeting modules were purified monomers. The targeting modules were concentrated to concentrations between 2.47 and 200 nM and measured in technical replicates. In order to perform data fitting and K D For the calculations, a two-state reaction binding model was adopted.

[0184] The equilibrium dissociation constant (K) of the interaction between TM and 5B9 D ) was in the range of 2.0 pM to 3.4 nM, while the K of the reference targeting module (SEQ ID No. 2) D is 17nM.

[0185] Cell binding assay

[0186] Cell binding tests of targeting modules (TM) binding to CD123 were performed on CD123 positive target cell lines Oci-AML3 and Molm-13, where: A) SEQ ID No.5 and Oci-AML3, B) SEQ ID No.9 and Oci-AML3, C) SEQ ID No.4 and Molm-13, and D) SEQ ID No.6 and Molm-13. The affinity of the targeting modules was analyzed by flow cytometry. These targeting modules were titrated on AML cell lines and then detected with mouse anti-His-tag antibodies coupled to phycoerythrin.

[0187] Figure 4 The cell binding potential evaluated using CD123 positive target cell lines Oci-AML3 and Molm-13 is shown. Different concentrations of R-TM123 were incubated with cells, then washed and quantitatively analyzed. The geometric mean fluorescence (MFI) was plotted against the concentration of the targeting module (TM) to obtain a dose-response curve.

[0188] The cell binding data of the targeting module binding to CD123 on the CD123-positive target cell lines Oci-AML3 and Molm-13 were fitted using a four-parameter model suitable for sigmoidal curves with variable slope. The 50% effective concentration (EC 50 ) can be interpreted as a representative value of the affinity of the targeting module for cells overexpressing the target receptor (see Table 1).

[0189] Thermodynamic stability

[0190] The thermodynamic stability of the two targeting modules of the present invention was evaluated by melting point analysis ( Figure 5 ). The protein melting points of the targeting modules (SEQ ID No. 5 and SEQ ID No. 9) were determined using thermal shift assays in different buffers with pH values ​​ranging from 4.0 to 9.0. In general, the colloidal stability of a protein is related to the thermodynamic stability and enables comparative prediction of long-term stability.

[0191] RevCAR T cells

[0192] To express RevCAR by genetic engineering, the polynucleotide vector encoding RevCAR and ensuring that it is expressed in genetically engineered immune cells is transferred to immune cells. Specifically, RevCAR includes IL-2LP (modified human IL-2 leader peptide), RCE (RevCAR epitope, also a tag), G4S1 (glycine-serine linker), ECD (extracellular domain), TMD (transmembrane domain) and ICD (intracellular domain). The vector can be transferred by electroporation or nucleic acid transfection, or by means of a viral vector system such as adenovirus, adeno-associated virus, retrovirus, foamy virus or lentiviral gene.

[0193] RevCAR was stably expressed in immune cells by first constructing a lentiviral vector encoding the selected RevCAR and then using lentiviral gene transfer. The lentiviral vector was pLVX-EF1αUniCAR 28 / ζ (Clontech, Takara Bio Group), in which the lentiviral part of the vector was derived from human immunodeficiency virus (HIV), and the mesenchymal stem cell MSC / internal ribosome entry site IRES / ZxGreenI part was replaced with a RevCAR construct.

[0194] Lentiviral particles are prepared by transiently transfecting human embryonic kidney (HEK) 293T (ACC 635) cells with a lentiviral vector plasmid encoding RevCAR and co-transfecting with a plasmid (psPAX2) encoding a population-specific antigen (gag) and a polymerase (pol) and a plasmid (pMD2.G) encoding an envelope protein. After transfection, the packaging plasmid expresses the Gag and Pol proteins of HIV-1. Plasmid MD2.G encodes the glycoprotein of vesicular stomatitis virus (VSV-G). VSV-G protein (or various envelope proteins of different viral species) is used in lentiviral vectors to transduce a variety of mammalian cells. Lentiviral vectors can be successfully pseudotyped with the envelope glycoprotein (Env) of the dual-tropic murine leukemia virus (MLV) or the G protein (VSV-G) of the vesicular stomatitis virus, a modified envelope of the prototype foamy virus (PFV), or a chimeric envelope glycoprotein variant derived from the gibbon ape leukemia virus (GaLV) and MLV.

[0195] 24h to 96h after transfection, the supernatant of the transfected HEK293T cells is collected and the 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 by using monoclonal antibodies against the CD3 complex (e.g., clone OKT3 or UCHT1), which can be added after dissolution or coated on plastic cell culture dishes, magnetic beads, or biodegradable polymer matrices. The activation effect of peripheral blood mononuclear cells (PBMCs) or isolated T cells can be further enhanced by stimulating co-stimulatory pathways using monoclonal antibodies or ligands against CD27, CD28, CD134, or CD137 (used alone or in combination, coated on plastic cell culture dishes, magnetic beads, or biodegradable polymer matrices), and adding exogenous recombinant cytokines (such as interleukin (IL)-2, IL-7, IL-12, IL-15, and IL-21). Concentrated or non-concentrated viral particles are added to PBMC or T cell cultures 24h to 96h after the initial administration of activation CD3-specific antibodies and / or co-stimulatory receptor CD27, CD28, CD134 or CD137-specific antibodies and / or recombinant cytokines (single or multiple doses). T cell electroporation, transduction and expansion can be performed manually in an open cell culture system or in a closed partially or fully automated system.

[0196] From day 3 after the last administration of viral supernatant, stable transduction of T cells can be determined by flow cytometry by staining for surface expression of RevCAR using a molecule containing a tag or by staining with a monoclonal antibody against the fourth domain of RevCAR. RevCAR-transduced T cells can be propagated in vitro by culturing in the presence of recombinant cytokines and an activating anti-CD3 mAb.

[0197] If RevCAR contains an optional fourth domain, which is a peptide sequence that can form a linear epitope of a monoclonal antibody, then the immune cells genetically modified to express RevCAR can be specifically propagated in vitro by coating the monoclonal antibody or its antibody fragment that binds to the fourth RevCAR domain on the surface of a culture dish, various beads, or a biodegradable polymer matrix, and then adding them to the cell culture at a specific ratio. The binding of the surface-coated monoclonal antibody to the RevCAR peptide domain induces cross-linking of RevCAR expressed on the cell surface and forms an immune synapse, thereby activating the signaling pathway specifically triggered by the signaling domain of RevCAR. Depending on the induced signaling pathway, the proliferation capacity of immune cells carrying RevCAR may be enhanced, and persistent resistance to activation-induced cell death may be generated, thereby enriching immune cells genetically modified by RevCAR in a mixed population.

[0198] The optional fourth domain, i.e., the peptide sequence that forms the linear epitope of the monoclonal antibody, can be further used to enrich and purify immune cells expressing RevCAR from a mixed population. A monoclonal antibody or its antibody fragment that can bind to the fourth RevCAR domain is used for enrichment and purification to mark cells expressing RevCAR (for cell sorting), or immune cells expressing RevCAR are temporarily connected to small particles that can be used for cell separation. RevCAR-transplanted immune cells are incubated with monoclonal antibodies that recognize the fourth domain. Magnetic beads are then added, which are conjugated with the following antibodies or fragments thereof, which are directed against species-specific and isotype-specific heavy and light chains of monoclonal antibodies that bind to the optional fourth domain. Therefore, immune cells expressing RevCAR are connected to magnetic beads and captured in a magnetic field to be separated from other immune cells.

[0199] Cytotoxicity assay

[0200] The suspension cell co-culture assay was performed using the AML cell line Molm-13 ( Figure 6 ) and AML cell line Oci-AML3( Figure 7 ), the efficacy of the targeting module combined with CD123 in inducing RevCAR-T cells to eliminate tumor cells was tested at different concentrations of the targeting module. The convertible CAR-T cells were incubated with target cells at different concentrations of the targeting module at a ratio of effector cells (T cells) to target cells (E:T) of 2:1 for 48 hours. The human AML cell line Molm-13 ( Figure 6 ) or Oci-AML3( Figure 7) as CD123 positive target cells, the cell line was stained with efluor before the experiment. The target cells were quantitatively analyzed by flow cytometry, and the cell lysis rate was calculated by normalizing the cell count of each sample with the control sample in which only tumor cells were plated. The data were fitted with a four-parameter model suitable for S-shaped curves with variable slopes, and the EC 50 The calculated values ​​can be interpreted as a representative value of the efficacy of the targeting module against these tumor cells.

[0201] Dose-dependent activation of RevCAR-T by R-TM123

[0202] For the experiments described in the following sections, clinical-scale RevCAR-T was used. The high-affinity receptor for IL-2, CD25 (IL-2 receptor α, IL-2Rα), is expressed in human T cells and becomes detectable on the cell surface upon stimulation of the endogenous T cell receptor (TCR) complex (Kmieciak et al., 2009). IL-2Rα regulates T cell proliferative responses and is an indicator of the magnitude of TCR stimulation (Shatrova et al., 2016).

[0203] Stimulation of RevCAR by R-TM123 (SEQ ID No. 5) resembles activation of endogenous TCR, however in the artificial receptor the activation signal from the immunoreceptor tyrosine-based activation motif (ITAM) of the CD3ζ portion is accompanied by a simultaneous co-stimulatory signal from the CD28 signaling chain (Cartellieri et al., 2016), and this activation can be tracked by monitoring CD25 upregulation.

[0204] Figure 8 The surface expression of CD25 on RevCAR-T under R-TM123-mediated activation is shown. The RevCAR-T batches were co-cultured with CD123-expressing AML cell lines MOLM-13, MV4-11, and OCI-AML3 for 48 h in the presence of different concentrations of R-TM123. Cell samples were prepared and analyzed by flow cytometry for the surface expression of CD2, RevCAR, CD4, CD8, and CD25. All samples were pre-screened for CD2+ / RevCAR+ cells, and the frequencies of CD25+RevCAR-T cells were shown separately for CD4+ and CD8+ cells. Three technical replicates from co-culture were combined, and staining data for four clinical-scale batches generated from healthy donor material were shown.

[0205] Therefore, the surface expression of CD25 on RevCAR-T cells was measured in the presence of target cells expressing CD123 to clarify their response to R-TM123-mediated stimulation. The frequency of RevCAR-T cells expressing CD25 depends on the dose of R-TM123 ( Figure 8 After RevCAR-T cross-links with target cells via R-TM123, CD4+ and CD8+ T cells expressing RevCAR are activated ( Figure 8 The above T cell subsets (CD4+ and CD8+ T cells) showed similar R-TM123 dose-dependent responses after 48 h ( Figure 8 and Table 2 ).

[0206] Table 2. Half-maximal R-TM123 dose required for CD25 expression on RevCAR-T cells (EC 50 ).according to Figure 1 The dose-response curves shown were obtained by determining and listing the half-maximal R-TM123 dose (EC 50 ). GraphPad prism 9 software was used to fit the data points using four-parameter nonlinear regression.

[0207]

[0208] Dose-dependent cytotoxic response of R-TM123 to CD123 leukemia cell line

[0209] To evaluate dose-dependent target cell lysis, four clinical-scale batches of RevCAR-T were used in cytotoxicity assays. The cytotoxic responses of the four batches of RevCAR-T against three AML cell lines were analyzed by stepwise increasing concentrations of R-TM123. Fig. 9 Shown are the dose-response curves of R-TM123 against MOLM-13, OCI-AML3, and MV4-11.

[0210] Fig. 9 The specific lysis of the AML cell line MOLM-13 by RevCAR-T redirected by R-TM123 is shown. AML cell lines MOLM-13, MV4-11, and OCI-AML3 expressing CD123 were labeled with the cell dye eFluor670 and then incubated with 2×10 cells / T cells from four clinical-scale batches in a 1:1 ratio of effector cells (T cells) to target cells (E:T) in the absence or presence of R-TM123. 5RevCAR-T cells were co-cultured. After 48 h of co-culture, the number of live target cells was determined by cell counting, and specific lysis was determined. The figure shows the mean ± standard deviation of three technical replicates and the dose-response curves obtained for four clinical-scale RevCAR-T batches from independent donors. GraphPad9 software was used to fit the data points using four-parameter nonlinear regression, and the half-maximum dose of R-TM123 is listed in Table 3.

[0211] MOLM-13 cells were derived from the peripheral blood of a patient with relapsed acute monocytic leukemia (FAB M5a) that evolved from myelodysplastic syndrome (Matsuo et al., 1997). OCI-AML3 was obtained from a patient with AML (FAB M4) and carries an NPM1 mutation (type A) and an abnormal cytoplasmic translocation of nucleophosmin, an immunocytological hallmark of NPM1-mutated AML (Quentmeier et al., 2005). In addition, it also contains a R882C-type DNMT3A mutation (Tiacci et al., 2012). Therefore, these two cell lines represent the main AML subtypes and will be included in the upcoming clinical studies. The MV4-11 cell line was originally derived from a pediatric acute monocytic leukemia and has been described to also express CD123 (Mani et al., 2018).

[0212] RevCAR-T cells induced target cell lysis in all of the above cell lines, and lysis was strictly dependent on R-TM123. Half-maximal lysis (EC) in the single-digit picomolar range was observed for all three target cell lines. 50 )(Table 2). For all AML cell lines, all four clinical-scale batches of RevCAR-T showed similar half-maximal lysis and reached an upper limit (i.e., 100% target cell lysis) at approximately 1 nM.

[0213] Table 3. Half maximal dose (EC) of R-TM123 required for RevCAR-T-mediated lysis of AML target cell lines MOLM-13, MV4-11, and OCI-AML3 in vitro 50 ).

[0214]

[0215] Dose-dependent cytokine secretion of RevCAR-T cells directed against CD123-expressing leukemia cell lines Sub Release

[0216] Due to the engagement of TCR, T cells are activated and release a large number of cytokines. These cytokines can have effector, stimulatory, regulatory, chemoattractant and inflammatory functions. Similarly, CAR-engineered T cells release cytokines after stimulation via their artificial receptors (Rossi et al., 2018).

[0217] In order to characterize the cytokine release potential of RevCAR-T, co-culture assay was used.For this reason, the RevCAR-T from the same four clinical scale batches for specific target cell lysis and T cell activation studies described above were analyzed.The cells were thawed, co-cultured with MOLM-13AML cells for 48h in the presence of R-TM123, and the effector cytokines released in the cell culture supernatant were quantitatively analyzed using the MACSPlex cytotoxic T / NK cell kit (Miltenyi, Germany).

[0218] Qualitatively, very similar cytokine release profiles were observed for different RevCAR-T clinical-scale batches, which showed abundant release of effector cytokines (granzyme B and perforin) and elevated pro-inflammatory cytokines such as GM-CSF, IFN-γ, TNF-α, and IL-2 ( Fig.10 ). The anti-inflammatory and regulatory cytokine IL-4 was detected only in two RevCAR-T batches. In terms of quantity, the absolute content of individual cytokines was donor or product related. In addition, the half-maximal cytokine release (EC of R-TM123) was determined from the sigmoidal dose-response curve by nonlinear regression. 50 )(See Table 4). EC 50 Values ​​can be used as a direct measure of cytokine release kinetics and can be compared to other effector functions such as target cell lysis or T cell activation. In general, differences in half-maximal cytokine release between cytokines were more significant than differences between RevCAR-T cell products. In particular, the effector cytokines granzyme B and perforin were already secreted at lower R-TM123 concentrations and correlated with target cell lysis (EC values ​​for granzyme B / perforin were 2.3 and 1.3, respectively). 50 4-12pM, while the lysed EC 50 Proinflammatory cytokines showed half-maximal cytokine secretion at much higher R-TM123 doses, such as GM-CSF at about 35-40 pM, IFN-γ at about 20-25 pM, or TNF-α at about 50-80 pM.

[0219] In summary, the RevCAR-T clinical-scale products showed typical cytokine release from T cells. The amount of cytokine release varied quantitatively between different cytokines and between different T cell donors or products. Cytokine release correlated with other effector functions of the RevCAR-T tested (i.e., activation and target cell lysis), but was biased toward higher R-TM123 doses.

[0220] Fig.10 Cytokines and effector molecules released by R-TM123-redirected RevCAR-T against the AML cell line MOLM-13 are shown.

[0221] RevCAR-T cells from four healthy donors and prepared in a clinical-scale process were incubated with the AML cell line MOLM-13 expressing CD123 in the presence of a specified concentration of R-TM123 and a 1:1 ratio of effector cells (T cells) to target cells. After 48 h, T cell co-culture supernatants were collected, technical replicates were combined, and analyzed using a flow cytometry-based multiplex assay (MACSPlex cytotoxic T / NK cell kit; Miltenyi, Germany), and the respective dose-response curves were depicted. Four-parameter logistic regression was used in GraphPad Prism 9 to fit the S-shaped data points and determine the half-maximal cytokine release (EC 50 ) (summarized in Table 4).

[0222] Table 4. Half-maximal cytokine and effector molecule release of RevCAR-T redirected by R-TM123 against AML cell line MOLM-13. (nd=not detected).

[0223]

[0224] Allo-RevCAR-T cells lyse R-TM123 in a dose-dependent manner against primary AML cells

[0225] The ability of clinical-scale RevCAR-T batches to lyse primary patient AML materials was analyzed using a flow cytometry-based cytotoxicity detection method.Cytotoxicity was determined using a combination of primary AML cells (AML1, AML3, AML4, AML5) from four AML patients and three RevCAR-T batches (i.e., a total of 12 primary AML / RevCAR-T pairs).On the day of the assay start, patient-derived AML cells were thawed, washed, and characterized by flow cytometry for the expression of markers CD45, CD14, HLA-DR, CD33, and CD34, and the expression of target CD123 was confirmed (data not shown).RevCAR-T batches were co-cultured with primary AML cells in the presence of R-TM123 at an effector to target cell ratio of 1:2.After co-culture for 48h, the number of live AML cells was determined by flow cytometry staining ( Fig.11 ).

[0226] Fig.11 The cytotoxic response of RevCAR-T redirected by R-TM123 against primary leukemia cells from AML patients was shown. RevCAR-T from healthy donors and produced in a clinical-scale process were thawed and incubated with 1.25-1.5×10 4 Primary AML cells were co-cultured at a 1:2 ratio of effector cells (T cells) to target cells over the specified R-TM123 concentration range. IMDM supplemented with 5% FBS, 5 μM β-mercaptoethanol, 1% penicillin / streptomycin, 100 ng / mL stem cell factor (SCF), 10 ng / mL IL-3, 10 ng / mL thrombopoietin (TPO), and 10 ng / mL Fms-related tyrosine kinase 3 ligand (FLT-3L) was used as culture medium. After 48 h, viable AML cell counts were determined by flow cytometry. Dose-response curves were calculated using GraphPadprism 9 by performing a four-parameter nonlinear fit with a variable slope for log(agonist) versus response. The EC for target cell lysis was 1.0. 50 The calculated values ​​are shown in Table 5.

[0227] Table 5. EC of R-TM123 lysis of primary AML target cells in vitro 50 Calculated value summary.

[0228]

[0229] Single intravenous injection of R-TM123 in mice

[0230] In NOD.Cg-Prkdc scid Iq tm1WjlThe in vivo pharmacokinetic characteristics of R-TM123 were studied in the / SzJ immunocompromised mouse model (hereinafter referred to as NSG). NSG mice were injected intravenously (IV) with a single dose of 1000 or 3000 ng / g of R-TM123 drugs from a confirmed batch. Blood was collected by retroorbital puncture at 15, 30, 60, 120, 180, 210, 255, 270, 300 and 360 minutes after injection. After separating the plasma, the concentration of R-TM123 was measured using a specific sandwich ELISA assay, which used CD123-Fc as a capture agent and used an anti-poly His tag monoclonal antibody conjugated to horseradish peroxidase (HRP) to detect the bound R-TM123. The integrity of the anti-CD123 domain was captured with CD123-Fc, and the detection of the poly HIS tag indicated the presence of a second RCE-specific scFv because it was located at the N-terminus of the poly HIS tag. Quantification of the test samples was achieved by comparing the test samples to titrations of the R-TM123 standard.The detection limit of the sandwich ELISA assay was determined to be 0.6 ng / mL assay concentration.

[0231] To determine the plasma half-life, non-compartmental analysis (NCA) and two-compartmental analysis (2CA) were performed on the plasma data after intravenous bolus injection using PkSolver 2.0 software (Zhang et al., 2010). The 2CA data were converted to obs 2 Further weighting is performed to focus the fitted model on the terminal elimination phase T 1 / 2 -β. Fig.12 A graphical output of the experiments performed is provided and key pharmacokinetic data are summarized in Table 6 .

[0232] Fig.12 The in vivo pharmacokinetics of R-TM123 in NSG mice are presented. Graphical output results were obtained by non-compartmental analysis (NCA) and two-compartmental analysis (2CA) of R-TM123 concentrations in peripheral blood plasma obtained with R-TM123 drug product. Experimental mice (n=5) were injected intravenously with 1000 or 3000 ng of R-TM123 per gram of body weight. Peripheral blood samples were collected by retro-orbital puncture at 15, 30, 60, 120, 180, 210, 255, 270, 300 and 360 minutes after intravenous bolus injection. R-TM123 concentrations determined by ELISA were analyzed by NCA and 2CA using PkSolver 2.0 software (Zhang et al., 2010). Each data point shows the mean ± standard deviation of samples obtained from 5 individual mice.

[0233] Respectively, at a dose of 1000 ng / g body weight, the area under the curve value (AUC) was determined to be 255603.5 (NCA) and 220241.7 ng / mL*min (2CA), and at a dose of 3000 ng / g body weight, the area under the curve value (AUC) was determined to be 644179.4 (NCA) and 555559.9 ng / mL*min (2CA). At a dose of 1000 ng / g body weight, the elimination half-life (T 1 / 2 ) were calculated to be 36.5min (NCA) and 38.0min (2CA), while at a dose of 3000ng / g body weight, the elimination half-life (T 1 / 2 ) calculated values ​​were 47.8 min (NCA) and 45.3 min (2CA). The elimination half-life values ​​obtained are consistent with those reported in the literature for similar scFv constructs (Hutt et al., 2012), and as expected, the plasma concentration of R-TM123 increased 15 min after intravenous injection (T max ), that is, it reached the highest at the earliest time point measured. At 15 minutes after injection, the maximum plasma concentration (C max ) were 4682.6 and 11522.7 ng / mL, respectively. The observed short half-life suggests that R-TM123 should be administered by continuous infusion, as is done for bispecific T cell engagers with similarly short half-lives (Chichili et al., 2015; Hijazi et al., 2018).

[0234] Figure 6 .In vivo pharmacokinetics of R-TM123 in NSG mice The summary results of the above experiments are as follows.

[0235]

[0236] Results obtained at 255, 270, 300, and 360 min at 1000 ng / g bw or at 360 min at 3000 ng / g bw were not included in further analysis because these results were below the lower limit of quantification (LLoQ) of 0.67–1.42 ng / mL. For analysis, noncompartmental analysis (NCA) and two-compartment analysis (2CA) models were applied to the plasma data after the intravenous bolus using PkSolver 2.0 software (Zhang et al., 2010). The observed differences between the two doses are most likely due to technical differences in sampling and / or ELISA performance, especially at the lower R-TM123 concentrations. As plasma levels of R-TM123 are generally low, the LLoQ was reached significantly faster at the 1000 ng / g bw administered dose (210 min) than at the 3000 ng / g bw dose (300 min). The plasma half-life is likely to be underestimated due to the small number of valid measurement points in the resulting terminal elimination phase. Therefore, the estimated plasma half-life for the 3000 ng / g body weight dose, calculated from six measurement points above the lower limit of quantitation in the terminal phase, was 47.8 minutes for the noncompartmental analysis model (NCA) and 45.3 minutes for the two-compartment analysis model (2CA), reflecting the more reliable data set.

[0237] In vivo efficacy of RevCAR-T in different CDX AML models

[0238] In the AML-CDX model of extramedullary disease, the in vivo efficacy of R-TM123-redirected RevCAR-T was confirmed with a fluorescence-based readout. In this model, MOLM-13 cells expressing mCherry were injected subcutaneously into the flank of NSG mice alone or in combination with RevCAR-T cells on day 0. R-TM123 ( Fig.13 ), for a total of four cycles, each cycle lasting 5 days. Based on in vivo fluorescence imaging, a sustained anti-tumor response was observed against MOLM-13 AML cells.

[0239] Fig.13 The results show that R-TM123-redirected RevCAR-T targets extramedullary disease in AML and eliminates leukemia. NSG mice were injected subcutaneously with 1×10 6 MV4-11 cells expressing mCherry or these cells were injected subcutaneously with 5 × 10 5A combination of RevCAR-T cells from clinical-scale trials, and tumor growth was monitored by optical imaging. Subsequently, mice were injected with R-TM123 around the tumor every day at a specified dose per gram of body weight for four cycles, each cycle was five days, and each cycle was separated by a two-day drug withdrawal period. The percentage of tumor signal refers to the initial measurement value (geometric mean, t0) of the corresponding group. Statistical significance was assessed by two-way analysis of variance (ANOVA) with Dunn's multi-comparison test, and the P values ​​of all treatment groups were less than 0.05 compared with the group receiving RevCAR-T cells and tumor cells without R-TM123.

[0240] Cited Non-Patent Literature

[0241] Brudno JN,Kochenderfer JN(2016)Toxicities of chimeric antigenreceptor T cells:recognition and management. Blood 127,3321-3330.

[0242] Cartellieri M, Bachmann M, Feldmann A, Bippes C, Stamova S, Wehner R, TemmeA, Schmitz M (2010) Chimeric Antigen Receptor-Engineered T Cells for Immunotherapy of Cancer J.Biomed.Biotechnol.Article ID 956304, doi:10.1155 / 2010 / 956304.

[0243] Cartellieri M, Feldmann A, Koristka S, Arndt C, Loff S, Ehninger A, vonBonin M, Bejestani EP, Ehninger G, Bachmann MP (2016) Switching CAR T cells on and off: a novel modular platform for retargeting of T cells to AML blasts. Bloodcancer J.6(8),e458.

[0244] Chichili GR,Huang L,Li H,Burke S,He L,Tang Q,Jin L,Gorlatov S,Ciccarone V,Chen F,Koenig S,Shannon M,Alderson R,Moore PA,Johnson S,Bonvini E(2015)A CD3xCD123bispecific DART for redirecting host T cells to myelogenousleukemia:preclinical activity and safety in nonhuman primates.Sci Transl Med,7(289),289ra282.

[0245] Darowski D,Kobold S,Jost C,Klein C(2019)Combining the best of twoworlds:highly flexible chimeric antigen receptor adaptor molecules(CAR-adaptors)for the recruitment of chimeric antigen receptor T cells.MAbs.11(4),621-631.

[0246] Fedorov VD,Themeli M,Sadelain M(2013)PD-1-and CTLA-4-based inhibitorychimeric antigen receptors(iCARs)divert off-target immunotherapyresponses.Sci.Transl.Med.5(215),215ra172.

[0247] Feldmann A,Hoffmann A,Bergmann R,Koristka S,Berndt N,Arndt C,Rodrigues Loureiro L,Kittel-Boselli E,Mitwasi N,Kegler A,Lamprecht C,GonzálezSoto KE,Bachmann M(2020)Versatile chimeric antigen receptor platform forcontrollable and combinatorial T cell therapy.Oncoimmunology.9(1),1785608.

[0248] Finney HM,Akbar AN,Lawson AD(2004)Activation of resting human primaryT cells with chimeric receptors:costimulation from CD28,induciblecostimulator,CD134,and CD137 in series with signals from the TCR zetachain.J.Immunol.172(1),104-113.

[0249] Frigault MJ,Lee J,Basil MC,Carpenito C,Motohashi S,Scholler J,Kawalekar OU,Guedan S,McGettigan SE,Posey AD Jr,Ang S,Cooper LJ,Platt JM,Johnson FB,Paulos CM,Zhao Y,Kalos M,Milone MC,June CH(2015)Identification ofchimeric antigen receptors that mediate constitutive or inducibleproliferation of T cells.Cancer Immunol.Res.3(4),356-367.

[0250] Gade TP,Hassen W,Santos E,Gunset G,Saudemont A,Gong MC,Brentjens R,Zhong XS,Stephan M,Stefanski J,Lyddane C,Osborne JR,Buchanan IM,Hall SJ,Heston WD,Rivière I,Larson SM,Koutcher JA,Sadelain M(2005)Targetedelimination of prostate cancer by genetically directed human Tlymphocytes.Cancer Res.65(19),9080-9088.

[0251] Gong MC,Latouche JB,Krause A,Heston WDW,Bander NH,Sadelain M(1999)Cancer patient T cells genetically targeted to prostate-specific membraneantigen specifically lyse prostate cancer cells and release cytokines inresponse to prostate-specific membrane antigen.Neoplasia.1(2),123-127.

[0252] Guedan S,Posey AD,Shaw C,Wing A,Da T,Patel PR,McGettigan SE,Casado-Medrano V,Kawalekar OU,Uribe-Herranz M,Song D,Melenhorst JJ,Lacey SF,SchollerJ,Keith B,Young RM,June CH(2018)Enhancing CAR T cell persistence through ICOSand 4-1BB costimulation.JCI Insight.3(1),96976.

[0253] Guedan S,Madar A,Casado-Medrano V,Shaw CE,Wing A,Liu F,Young RM,JuneCH,Posey AD(2020)Single residue in CD28-costimulated CAR T cells limits long-term persistence and antitumor durability.J Clin Invest.133215.

[0254] Hijazi Y,Klinger M,Kratzer A,Wu B,Baeuerle PA,Kufer P,Wolf A,NagorsenD,Zhu M(2018)Pharmacokinetic and Pharmacodynamic Relationship of Blinatumomabin Patients with Non-Hodgkin Lymphoma.Curr Clin Pharmacol,13(1),55-64.

[0255] Hombach AA,Abken H(2011)Costimulation by chimeric antigen receptorsrevisited the T cell antitumor response benefits from combined CD28-OX40signalling.Int.J.Cancer.129(12),2935-2944.

[0256] Hombach A,Sent D,Schneider C,Heuser C,Koch D,Pohl C,Seliger B,Abken H(2001)T-cell activation by recombinant receptors:CD28 costimulation isrequired for interleukin 2 secretion and receptor-mediated T-cellproliferation but does not affect receptor-mediated target cell lysis.CancerRes.61(5),1976-1982.

[0257] Hutt M,Farber-Schwarz A,Unverdorben F,Richter F,and Kontermann RE(2012)Plasma Half-life Extension of Small Recombinant Antibodies by Fusion toImmunoglobulin-binding Domains.J Biol Chem.287(7):4462–4469.

[0258] Kagoya Y,Tanaka S,Guo T,Anczurowski M,Wang CH,Saso K,Butler MO,MindenMD,Hirano N(2018)A novel chimeric antigen receptor containing a JAK-STATsignaling domain mediates superior antitumor effects.Nat Med.24(3),352-359.

[0259] Kershaw MH,Westwood JA,Parker LL,Wang G,Eshhar Z,Mavroukakis SA,WhiteDE,Wunderlich JR,Canevari S,Rogers-Freezer L,Chen CC,Yang JC,Rosenberg SA,HwuP(2006)A phase I study on adoptive immunotherapy using gene-modified T cellsfor ovarian cancer.Clin.Cancer Res.12(20),6106-6115.

[0260] Kittel-Boselli E,Soto KEG,Loureiro LR,Hoffmann A,Bergmann R,Arndt C,Koristka S,Mitwasi N,Kegler A,Bartsch T,Berndt N,Altmann H,Fasslrinner F, M,Bachmann MP,Feldmann A(2021)Targeting Acute Myeloid LeukemiaUsing the RevCAR Platform:A Programmable,Switchable and CombinatorialStrategy.Cancers(Basel).13(19),4785.

[0261] Kmieciak M,Gowda M,Graham L,Godder K,Bear HD,Marincola FM,&Manjili MH(2009)Human T cells express CD25 and Foxp3 upon activation and exhibiteffector / memory phenotypes without any regulatory / suppressor function.JTransl Med,7,89.

[0262] Lamers CH,Sleijfer S,Willemsen RA,Debets R,Kruit WHJ,Gratama JW,Stoter G(2004)Adoptive immuno-gene therapy of cancer with single chainantibody[scFv(Ig)]gene modified T lymphocytes.J.Biol.Regul.Homeost.Agents.18(2),134-140.

[0263] Mani R,Goswami S,Gopalakrishnan B,Ramaswamy R,Wasmuth R,Tran M,Mo X,Gordon A,Bucci D,Lucas DM,Mims A,Brooks C,Dorrance A,Walker A,Blum W,Byrd JC,Lozanski G,Vasu S,Muthusamy N(2018)The interleukin-3 receptor CD123 targetedSL-401 mediates potent cytotoxic activity against CD34(+)CD123(+)cells fromacute myeloid leukemia / myelodysplastic syndrome patients and healthydonors.Haematologica,103(8),1288-1297.

[0264] Matsuo Y,MacLeod RA,Uphoff CC,Drexler HG,Nishizaki C,Katayama Y,Kimura G,Fujii N,Omoto E,Harada M,Orita K(1997)Two acute monocytic leukemia(AML-M5a)cell lines(MOLM-13 and MOLM-14)with interclonal phenotypicheterogeneity showing MLL-AF9 fusion resulting from an occult chromosomeinsertion,ins(11;9)(q23;p22p23).Leukemia,11(9),1469-1477.

[0265] Milone MC,Fish JD,Carpenito C,Carroll RG,Binder GK,Teachey D,SamantaM,Lakhal M,Gloss B,Danet-Desnoyers G,Campana D,Riley JL,Grupp SA,June CH(2009)Chimeric receptors containing CD137 signal transduction domains mediateenhanced survival of T cells and increased antileukemic efficacy invivo.Mol.Ther.17(8),1453-1464.

[0266] Morgan RA,Yang JC,Kitano M,Dudley ME,Laurencot CM,Rosenberg SA(2010)Case Report of a Serious Adverse Event Following the Administration of TCells Transduced With a Chimeric Antigen Receptor RecognizingERBB2.Mol.Ther.18,843-851.

[0267] Pinthus JH,Waks T,Kaufman-Francis K,Schindler DG,Harmelin A,Kanety H,Ramon J,Eshhar Z(2003)Immuno-gene therapy of established prostate tumorsusing chimeric receptor-redirected human lymphocytes.Cancer Res.63(10),2470-2476.

[0268] Quentmeier H,Martelli MP,Dirks WG,Bolli N,Liso A,Macleod RA,NicolettiI,Mannucci R,Pucciarini A,Bigerna B,Martelli MF,Mecucci C,Drexler HG,Falini B(2005)Exon-12 NPM gene mutation-A and cytoplasmic expression of cell line OCI / AML3bears nucleophosmin.Leukemia,19(10),1760-1767.

[0269] Rossi J,Paczkowski P,Shen YW,Morse K,Flynn B,Kaiser A,Ng C,GallatinK,Cain T,Fan R,Mackay S,Heath JR,Rosenberg SA,Kochenderfer JN,Zhou J,Bot,A outcomes in NHL.Blood, 132(8), 804-814.

[0270] Shatrova AN,Mityushova EV,Vassilieva IO,Aksenov ND,Zenin VV,NikolskyNN,Marakhova,II(2016)Time-Dependent Regulation of IL-2Rα-Chain(CD25)Expression by TCR Signal Strength and IL-2-Induced STAT5 Signaling inActivated Human Blood T Lymphocytes.PLoS One,11(12),e0167215.

[0271] Sotillo E,Barrett DM,Black K.,Bagashev A,Oldridge D,Wu G,Sussman R,Lanauze C,Ruella M,Gazzara MR,Martinez NM,Harrington CT,Chung EY,PerazzelliJ,Hofmann TJ,Maude SL,Raman P,Barrera A,Gill S,Lacey SF,Melenhorst JJ,AllmanD,Jacoby E,Fry T,Mackall C,Barash Y,Lynch KW,Maris JM,Grupp SA,Thomas-Tikhonenko A(2015)Convergence of Acquired Mutations and Alternative Splicingof CD19 Enables Resistance to CART-19 Immunotherapy.Cancer Discov.5,1282-1295.

[0272] Tiacci E,Spanhol-Rosseto A,Martelli MP,Pasqualucci L,Quentmeier H,Grossmann V,Drexler HG,Falini B(2012)The NPM1 wild-type OCI-AML2 and theNPM1-mutated OCI-AML3 cell lines carry DNMT3A mutations.Leukemia,26(3),554-557.

[0273] Titov A,Petukhov A,Staliarova A,Motorin D,Bulatov E,Shuvalov O,SoondSM,Piacentini M,Melino G,Zaritskey A,Barlev NA(2018)The biological basis andclinical symptoms of CAR-T therapy-associated toxicites.Cell Death Dis 9,897.

[0274] K,Cartellieri M,Michen S,Wiedemuth R,Müller N,Lindemann D,Bachmann M,Füssel M,Schackert G,Temme A(2015)DAP12-based activating chimericantigen receptor for NK cell tumor immunotherapy.J.Immunol.194(7),3201-3212.

[0275] Wang J,Jensen M,Lin Y,Sui X,Chen E,Lindgren CG,Till B,Raubitschek A,Forman SJ,Qian X,James S,Greenberg P,Riddell S,Press OW(2007)Optimizingadoptive polyclonal T cell immunotherapy of lymphomas,using a chimeric T cellreceptor possessing CD28 and CD137costimulatory domains.Hum.Gene Ther.18(8),712-725.

[0276] Zhang T,Lemoi BA,Sentman CL(2005)Chimeric NK-receptor-bearing T cellsmediate antitumor immunotherapy.Blood.106(5),1544-1551.

[0277] Zhang Y,Huo M,Zhou J,Xie S(2010)PKSolver:An add-in program forpharmacokinetic and pharmacodynamic data analysis in Microsoft Excel.ComputMethods Programs Biomed.99(3),306-14.

[0278] Zhao Y,Wang QJ,Yang S,Kochenderfer JN,Zheng Z,Zhong X,Sadelain M,Eshhar Z,Rosenberg SA,Morgan RA(2009)A herceptin-based chimeric antigenreceptor with modified signaling domains leads to enhanced survival oftransduced T lymphocytes and antitumor activity.J.Immunol.183(9),5563-5574.

Claims

1. A targeting module comprising i) at least one CD123 binding domain comprising a CDR sequence as shown in SEQ ID No.33, SEQ ID No.34, SEQ ID No.35, SEQ ID No.36, the amino acid sequence WAS (tryptophan-alanine-serine) and SEQ ID No.37, and ii) a tag binding domain that binds to the human La epitope E5B9, comprising the CDR sequences shown in SEQ ID No.38, SEQ ID No.39, SEQ ID No.40, SEQ ID No.41, the amino acid sequence WAS (tryptophan-alanine-serine) and SEQ ID No.42, The tag binding domain comprises V L -Connector-V H structure.

2. The targeting module according to claim 1, wherein i) the at least one CD123 binding domain comprises a sequence having at least 95% identity, or at least 99% identity, to the sequences SEQ ID No. 22 and SEQ ID No. 23, and ii) the tag binding domain that binds to human La epitope E5B9 comprises V L -Connector-V H structure, wherein the tag binding domain V L The region comprises a sequence having at least 95% identity, or at least 99% identity, to the sequence shown in SEQ ID No. 19, and / or the V of the tag binding domain H The region comprises a sequence having at least 95% identity, or at least 99% identity to the sequence shown as SEQ ID No.

20.

3. The targeting module according to claim 1 or 2, wherein the linker of the tag binding domain comprises 20 to 30 amino acids, preferably 25 amino acids.

4. The targeting module according to any one of claims 1 to 3, wherein the linker of the tag binding domain comprises a linker of one of the sequences shown as SEQ ID No. 25 or SEQ ID No.

26.

5. The targeting module according to any one of claims 1 to 4, wherein the CD123 binding domain is an antibody or an antigen binding fragment.

6. The targeting module according to any one of claims 1 to 5, wherein the CD123 binding domain comprises the sequence shown in SEQ ID No.

27.

7. The targeting module according to any one of claims 1 to 6, wherein the targeting module has a length ranging from 500 to 800 amino acids.

8. The targeting module according to any one of claims 1 to 7, comprising one of the sequences shown in SEQ ID No. 3 to SEQ ID No.

10.

9. A nucleic acid, vector or cell comprising a nucleotide sequence encoding the targeting module according to any one of claims 1 to 8.

10. The nucleic acid, vector or cell according to claim 9, comprising one of the sequences shown in SEQ ID No. 11 to SEQ ID No.

18.

11. A pharmaceutical composition comprising the targeting module according to any one of claims 1 to 8 and a pharmaceutically acceptable diluent or carrier.

12. A targeting module according to any one of claims 1 to 8, a nucleic acid, a vector or a cell comprising a nucleotide sequence encoding the targeting module according to any one of claims 1 to 8, or a pharmaceutical composition comprising the targeting module according to any one of claims 1 to 8 and a pharmaceutically acceptable diluent or carrier, for use in a method of stimulating a chimeric antigen receptor-mediated immune response in a mammal, or for treating cancer, an infectious disease or an autoimmune disease.

13. The targeting module in the method for stimulating a chimeric antigen receptor-mediated immune response in a mammal according to claim 12, wherein the targeting module is administered in combination with a vector or cell comprising a nucleotide sequence encoding a reversible chimeric antigen receptor, wherein the reversible chimeric antigen receptor comprises Tag, wherein the tag is human La epitope E5B9, The extracellular hinge and transmembrane domain, and signal transduction domain, The tag binding domain of the targeting module binds to the tag of the reversible chimeric antigen receptor.

14. The pharmaceutical composition for stimulating a chimeric antigen receptor-mediated immune response in a mammal according to claim 12, further comprising a vector or cell comprising a nucleotide sequence encoding a reversible chimeric antigen receptor, wherein the reversible chimeric antigen receptor comprises Tag, wherein the tag is human La epitope E5B9, The extracellular hinge and transmembrane domain, and signal transduction domain, The tag binding domain of the targeting module binds to the tag of the reversible chimeric antigen receptor.

15. A kit comprising a) a targeting module according to any one of claims 1 to 8 or a nucleic acid, a vector or a cell according to claim 9 or 10, and b) a vector or a cell comprising a nucleotide sequence encoding a reversible chimeric antigen receptor, wherein the reversible chimeric antigen receptor comprises Tag, wherein the tag is human La epitope E5B9, The extracellular hinge and transmembrane domain, and signal transduction domain, The tag binding domain of the targeting module binds to the tag of the reversible chimeric antigen receptor.

16. The kit according to claim 15, wherein the extracellular hinge and transmembrane domain are selected from one of the following: hinge and transmembrane domain of human CD28 molecule, CD8a chain, NK cell receptor, part of antibody constant region, and combinations thereof.

17. A kit according to claim 15 or 16, wherein the signal transduction domain is selected from one of the following: CD28, CD137 (4-1BB), CD134 (OX40), CD278 (ICOS), DAP10 and the cytoplasmic region of CD27, programmed cell death protein-1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), the cytoplasmic region of the CD3 chain, DAP12, CD122 (interleukin-2 receptor β), CD132 (interleukin-2 receptor γ), CD127 (interleukin-7 receptor α), CD360 (interleukin-21 receptor), activated Fc receptor, and mutants of the above domains.

18. The kit according to any one of claims 15 to 17, further comprising at least one other targeting module or a nucleic acid, vector or cell encoding the other targeting module, wherein the at least one other targeting module comprises at least one target cell binding domain and a tag binding domain, wherein the at least one other target cell binding domain is an antibody, antibody fragment, protein, peptide or low molecular weight organic ligand capable of binding one or more of the following surface antigens: CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD15, CD19, CD20, CD22, CD23, CD25, CD30, CD33, CD38, CD44, CD44v6, CD52, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD90, CD99, CD133, CD135, CD150, CD181, CD182, CD184, CD223, CD229, CD269, CD273, CD274, CD276, CD279, CD319, CD366 and CD371, cytokine receptors, CXCR4, c-Met, mesothelin, epidermal growth factor receptor, leukocyte antigen, leukocyte antigen, cytokine receptor, leukocyte antigen, cytokine receptor, leukocyte antigen, cytokine receptor, leukocyte antigen, cytokine receptor, cytokine receptor, leukocyte antigen, cytokine receptor, cytokine receptor, cytokine receptor, cytokine receptor, cytokine receptor, cytokine receptor, Long factor receptor family members and mutants thereof, tumor necrosis factor receptor superfamily members, tight junction proteins, ephrins, ephrin receptors, fucosyltransferases, prostate specific antigens, embryonic antigens, vascular endothelial growth factor family members, EpCAM, AFP, intercellular adhesion molecule family members, C-type lectins, integrins, mucin family members, FSHR, HMW-MAA, FBP, folate receptors, somatostatin receptors, ligands of NKG2D receptors, epithelial cell glycoprotein family members, disialyl gangliosides, phosphatidylinositol glycans, G protein-coupled receptors, human papillomavirus proteins, cancer-testis antigens, fibroblast activation proteins, carbonic anhydrase family members, carbohydrate antigen family members, Notch ligands, MCSP, glycoprotein A33, guanylate cyclase 2C and tumor-specific glycans, wherein the targeting module and the at least one other targeting module comprise the same tag binding domain.

19. The kit according to any one of claims 15 to 18, wherein the targeting module and / or the vector or cell comprising a nucleotide sequence encoding a reversible chimeric antigen receptor is in the form of a pharmaceutical composition.

20. The kit according to any one of claims 15 to 19, for use in a method of stimulating a chimeric antigen receptor mediated immune response in a mammal, preferably for the treatment of cancer, infectious diseases or autoimmune diseases.

Images