Targeting module for CD123 for use in a method for stimulating a chimeric antigen receptor-mediated immune response in a mammal

Abstract

The present invention relates to a pharmaceutical composition and a kit comprising 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, vector or cell comprising a nucleotide sequence encoding the targeting module, a vector or cell comprising the targeting module and 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, vector or cell comprising a nucleotide sequence encoding the targeting module, and a pharmaceutical composition and kit comprising the targeting module and a vector or cell comprising a nucleotide sequence encoding a reversible chimeric antigen receptor.

Background Art

[0002] A chimeric antigen receptor (CAR) is an artificial receptor composed of a binding moiety that confers antigen specificity and one or several signaling chains derived from immunoreceptors (Cartellieri et al., 2010). Immune cells genetically modified to express a CAR can be used to bind to cells or tissue structures expressing an appropriate target of the CAR binding moiety. By cross-linking, a signaling pathway via the CAR signaling chain is induced, which changes the biological properties of the CAR-grafted immune cells. Conversely, activation of CAR in genetically modified regulatory T cells (Tregs) results in activation of Treg-specific immunomodulatory and immunosuppressive mechanisms such as the secretion of interleukin (IL)-10 or tumor growth factor beta (TGF-β). Adoptive transfer of immune cells engineered with chimeric antigen receptors (CARs) is currently considered a very promising therapeutic option for treating malignant, infectious or autoimmune diseases that cannot be cured by other means.

[0003] However, conventional CAR technology is associated with several important problems that need to be solved before this treatment method can be widely applied to clinical treatment. First, several safety issues need to be addressed. So far, the immune response of T cells engineered with conventional CARs has been difficult to control after injection into patients. The severe adverse event rate is high (Titov et al., 2018). In particular, unexpected target gene expression in normal tissues can trigger a rapid and severe immune reaction of engineered T cells against normal cells, which can result in severe side effects (Morgan et al., 2010). Furthermore, since CAR-T cells are a new class of self-proliferating cell drugs, the injected T cells can proliferate actively in the presence of a large tumor mass, causing tumor lysis syndrome, cytokine release syndrome, and macrophage activation syndrome (Brudno and Kochenderfer, 2016). Another drawback of conventional CAR technology is the limited retargeting of engineered T cells to a single antigen. Such monotherapy approaches carry the risk of generating tumor escape variants that lack the target antigen during treatment. The emergence of tumor escape variants several months after conventional CAR T cell therapy has already been observed in clinical trials (Sotillo et al., 2015). Collectively, these obstacles severely limit the application of CAR T cells to a very narrow indication. In fact, examples of clinical efficacy have so far been limited to CD19-targeted CAR T cells and BCMA-targeted CAR T cells.

[0004] The modular, switchable "universal" chimeric antigen receptor T cell (UniCAR) approach can overcome these limitations by separating the antigen recognition and activation domains of the CAR into two distinct operating units. T cells are engineered to express a CAR with a universal binding domain that recognizes a tag (Cartellieri et al., 2016). Antigen specificity is provided by a soluble adapter protein composed of an antigen-binding domain fused to a tag recognized by the universal CAR. Cartellieri et al. have described the in vitro and in vivo treatment of CD33-positive and / or CD123-positive acute myeloid leukemia cells.

[0005] Subsequent to the UniCAR approach (EP2990416A1 (Patent Document 1)) for recognizing various antigens, the reversed universal CAR (RevCAR) approach is known, which promotes the binding of immune cells engineered to express a RevCAR containing a tag to target cells via an adapter molecule containing a tag-binding domain and a target cell-binding domain (EP3581200A1 (Patent Document 2)). EP3581200A1 (Patent Document 2) discloses an extracellular LA / SSB-derived tag and an adapter molecule containing a CD123 scFv and an scFv (5B9 or 7B6) that binds to the tag, which is added to crosslink the CAR and tumor cells to produce antigen-specific cytotoxicity.

[0006] Darowski et al. have described flexible chimeric antigen receptor adapter molecules (CAR-adapters) for the recruitment of 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, ErbB2, etc. (Darowski et al., 2019).

[0007] Feldmann et al. have disclosed that RevCART cells, which are flexibly redirected to multiple targets by exchanging targeting modules, particularly targeting modules against PSMA and PSCA, can efficiently kill tumor cells (Feldmann et al., 2020).

[0008] Kittel-Boselli et al. have described the targeting of acute myeloid leukemia, particularly patient-derived AML cells expressing CD33 and CD123, using a RevCAR platform, where the RevCAR is composed of an 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 constructed from the variable heavy chain (V H ) domain and the variable light chain (V L ) domain derived from monoclonal antibodies (mAb) CD33, CD123, 5B9, or 7B6, which are linked via a glycine (G)-serine (S) linker.

Prior Art Documents

Patent Documents

[0009]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0010] An object of the present invention is to provide an improved targeting module for use in the RevCAR system.

[0011] This object is achieved by at least one CD123 binding domain according to the present invention and V L-Linker-V H It was solved by a targeting module that includes a tag-binding domain that binds to the human La epitope E5B9 containing the structure.

Means for Solving the Problem

[0012] Advantageously, the targeting module according to the present invention i) at least one CD123-binding domain comprising a sequence having at least 95% identity, preferably at least 99% identity, with the sequences according to SEQ ID NO: 22 and SEQ ID NO: 23, and ii) V L -Linker-V H A tag-binding domain that binds to the human La epitope E5B9, which includes the structure, wherein the V L region of the tag-binding domain includes a sequence having at least 95% identity, preferably at least 99% identity, with the sequence according to SEQ ID NO: 19, and / or the V H region of the tag-binding domain includes a sequence having at least 95% identity, preferably at least 99% identity, with the sequence according to SEQ ID NO: 20, and includes a tag-binding domain.

[0013] As used herein, the term "targeting module" refers to a molecule, preferably a polypeptide or protein, having at least two different domains, each domain being specific for a target or a homogeneous group of targets, at least one domain being specific for target cells, particularly a CD123-binding domain, and one domain being specific for a reversible chimeric antigen receptor, particularly a tag-binding domain. In a plurality of embodiments, the targeting module is isolated. Preferably, the targeting module according to the present invention is expressed as a recombinant protein.

[0014] In a further embodiment, the targeting module is chemically synthesized.

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

[0016] As used herein, the term "domain" refers to a portion of a protein sequence that can exist and function independently from the rest of the protein.

[0017] As used herein, "V L -linker-V H structure" refers to a structure in which the C-terminus of the V L region is linked to a linker, and the linker is linked to the N-terminus of the V H region.

[0018] As used herein, the term "linker" (also referred to as a spacer) refers to a molecule or molecular moiety that separates at least two elements under consideration, particularly selected from functional groups, tags, binding domains or binding domain subunits, such as the V L domain and the V H domain.

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

[0020] As used herein, the terms "bind" or "binding" refer to non-covalent bonds, particularly ionic bonds, hydrogen bonds, van der Waals forces and / or hydrophobic interactions.

[0021] As used herein, the term "mutant" refers to a peptide or protein having at least 90% sequence identity, preferably at least 95% sequence identity, to a specified antibody, antibody fragment, protein or peptide. Advantageously, the mutant may have one or more activities of the specified antibody, antibody fragment, peptide or protein.

[0022] In multiple embodiments, the mutant is a truncated peptide or protein. As used herein, the term "truncated versions" 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, most preferably at least 95% chain length and 100% sequence identity, to a specified peptide or protein. Advantageously, the truncated version has at least 80%, preferably at least 90%, more preferably at least 95% of the activity of the specified peptide or protein.

[0023] As used herein, the term "nuclear protein" refers to a protein found in the cell nucleus.

[0024] Advantageously, the tag is a peptide sequence derived from a nuclear antigen, but under the circumstances of the native protein under physiological conditions, it cannot be accessed or bound by the corresponding tag-binding domain. Even more advantageously, the tag is not immunogenic. This minimizes the risk of uncontrolled on-target off-site toxicity by immune cells expressing CAR, such as the release of toxic levels of cytokines variously called cytokine storm or cytokine release syndrome (CRS).

[0025] As used herein, the term "administered in combination" refers to a treatment in which the targeting module is administered before, at the same time as, and / or after the administration of a vector or cell comprising a nucleotide sequence encoding a reversible chimeric antigen receptor (RevCAR).

[0026] As used herein, the term "antibody" refers to a protein that binds an antigen via the variable region of the antigen-binding fragment (Fab). This consists of one constant domain and one variable domain each of the heavy chain (V H ) and the light chain (V L ). As used herein, the term "antibody fragment or antigen-binding fragment" refers to a protein comprising at least V L or V H of an antibody. In a plurality of embodiments, the antibody fragment is selected from a single-chain variable fragment (scFv), a single-chain antibody, an F(ab’)2 fragment, a Fab fragment, and a fragment generated by a Fab expression library or a single-domain antibody (nanobody).

[0027] As used herein, the term "single-chain variable fragment (scFv)" refers to an artificial antibody fragment comprising the variable domains of the light and heavy chains of an antibody that are covalently linked. In a plurality of embodiments, V L and V H of the antibody are covalently linked by a short-chain peptide of 10 to 25 amino acids. In a further embodiment, the short-chain peptide links the N-terminus of V H to the C-terminus of V L , or vice versa.

[0028] In a plurality of embodiments, at least one CD123-binding domain comprises CDR sequences according to SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, the amino acid sequence WAS (Trp-Ala-Ser), and SEQ ID NO: 37.

[0029] In multiple embodiments, the tag-binding domain that binds to the human La epitope E5B9 includes CDR sequences according to SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, the amino acid sequence WAS (Trp-Ala-Ser), and SEQ ID NO: 42.

[0030] As used herein, the term "CDR (Complementary Determining Region)" refers to the portions of the variable chains of antibodies or antibody fragments that bind to their specific antigens. An antibody contains three CDRs (CDR1, CDR2, and CDR3) that are non-contiguously arranged in the amino acid sequence of each variable domain, and thus, two variable domains (V H and V L ) contain six CDRs, which can contact the antigen.

[0031] In multiple embodiments, V L and V H are linked via a glycine-serine linker having the structure (G x S y ), where x and y are selected from 1 to 10, preferably 3 to 5. Most preferred are repeats from 1 to 10 of the sequence G4S1 (SEQ ID NO: 24). Further, a linker composed of a peptide sequence that can increase the protease resistance of the antibody derivative is preferred.

[0032] In multiple embodiments of the present invention, the linker of the tag-binding domain includes 20 to 30 amino acids, preferably 25 amino acids.

[0033] In a further embodiment, the linker of the tag-binding domain includes a linker according to SEQ ID NO: 25 or SEQ ID NO: 26.

[0034] In multiple embodiments, the antibody is obtained from an animal species, preferably a mammal such as a human, monkey, mouse, rat, rabbit, guinea pig, horse, cow, sheep, goat, pig, dog, or cat. Preferably, the antibody or antibody fragment is a human antibody, humanized antibody, or deimmunized antibody. Humanized antibodies can be prepared in various ways, such as by resurfacing and CDR grafting. In the case of resurfacing, a combination of molecular modeling, statistical analysis, and mutagenesis is used to modify all non-CDR regions on the surface of the antibody to resemble the surface of the antibody of the target organism. In CDR grafting, CDR regions according to the present invention that have sequences similar to the original CDR regions are introduced into known human framework regions. Deimmunized antibodies can be obtained by specifically mutating residues to generate immunogenic hotspots predicted based on in silico peptide-MHC affinity prediction.

[0035] In multiple embodiments, the antibody or antibody fragment is a polyclonal antibody, monoclonal antibody, or chimeric antibody, and in the chimeric antibody, the antigen-binding region of a non-human antibody has been transferred into the framework of a human antibody by recombinant DNA technology including in silico design.

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

[0037] In a preferred embodiment, the CD123 binding domain is an antibody or antigen-binding fragment.

[0038] In multiple embodiments, the CD123 binding domain comprises the sequences of SEQ ID NO: 22 and SEQ ID NO: 23.

[0039] In a preferred embodiment, the CD123 binding domain comprises a sequence according to SEQ ID NO: 27.

[0040] According to the present invention, the tag binding domain binds to a tag derived from the human La epitope E5B9. In a plurality of embodiments, the tag binding domain preferably has the following sequence: DIVMTQSPDSLAVSLGERATINCX 24 SSQSLLNSRTX 35 KNYLAWYQQKPGQPPKLLIYWASTRX 61 SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYNLX 101 TFGGGTKVElK (SEQ ID NO: 19) (X 24 , X 35 , X 61 and X 101 are each independently selected from the alpha amino acid residues that make up the protein), or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity, to the sequence of SEQ ID NO: 21 and is an antibody or antigen-binding fragment comprising V L .

[0041] In a plurality of embodiments, the tag binding domain comprises an scFv against the La epitope.

[0042] In some embodiments, X 24 ~X 101 is selected as follows: X 24 is selected from polar and / or positively charged residues such as serine, threonine, asparagine, glutamine, histidine, lysine and arginine, preferably lysine or arginine, X 35 is preferably selected from lysine and proline, X 61is selected from polar and charged residues such as asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine and arginine, preferably glutamic acid and lysine, X 101 is a hydrophobic residue, for example, isoleucine, leucine, valine, alanine, methionine, phenylalanine, proline and tryptophan, preferably selected from leucine or proline.

[0043] Preferably, the tag-binding domain has at least 90% sequence identity, preferably at least 95% sequence identity, respectively, to the sequences according to SEQ ID NO: 20 (V H ) and SEQ ID NO: 21 (V L ).

[0044] Most preferably, the tag-binding domain constitutes an anti-La 5B9 scFv according to SEQ ID NO: 20 (V H ) and SEQ ID NO: 21 (V L ).

[0045] In a plurality of embodiments, the length of the target module ranges from 100 to 1600 amino acids, preferably from 500 to 800 amino acids.

[0046] In a plurality of embodiments, the targeting module comprises one of the sequences according to SEQ ID NOs: 3 to 10.

[0047] In a further aspect, the invention provides a nucleic acid, vector or cell comprising a nucleotide sequence encoding a targeting module according to the invention.

[0048] In a plurality of embodiments, the nucleic acid, vector or cell comprises one of the sequences according to SEQ ID NOs: 11 to 18. The sequences according to SEQ ID NOs: 11 to 18 encode a targeting module according to SEQ ID NOs: 3 to 10.

[0049] In a further aspect, the present invention provides a pharmaceutical composition comprising a targeting module according to the present invention and a pharmaceutically acceptable diluent or carrier.

[0050] The pharmaceutical composition is preferably administered parenterally, particularly preferably intravenously. In a plurality of embodiments, the pharmaceutical composition is in a form suitable for intravenous administration. Preferably, the pharmaceutical composition is a solution, emulsion or suspension.

[0051] In a plurality of embodiments, the pharmaceutical composition is an injectable buffered solution comprising a targeting module, nucleic acid, vector and / or cell according to the present invention at a concentration in the range of 1 ng / ml to 500 mg / ml, preferably in the range of 50 μg / ml to 5 mg / ml.

[0052] The pharmaceutical composition comprises a pharmaceutically acceptable diluent (thinner or dilution agent) or carrier. In a plurality of embodiments, the carrier is selected from water, aqueous buffer solution, 0.9% saline, 5% glucose, 5% xylitol, 0.3% glycine solution, Ringer's solution or amino acid solution. In a further embodiment, the aqueous buffer solution is selected from aqueous buffer solutions of histidine, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate having a pH value in the range of pH 5.0 to pH 7.0. In a plurality of embodiments, the aqueous buffer solution has a buffer concentration in the range of 1 mmol / l (mM) to 500 mM, preferably in the range of 5 mM to 20 mM, particularly preferably in the range of 5 mM to 10 mM.

[0053] In a plurality of embodiments, the carrier preferably contains sodium chloride at a concentration in the range of 1 mM to 300 mM, particularly preferably 150 mM.

[0054] In a plurality of embodiments, the pharmaceutical composition further comprises a stabilizer at a concentration preferably in the range of 1 mM to 900 mM, particularly preferably in the range of 50 mM to 600 mM. In a plurality of embodiments, the stabilizer is sucrose, trehalose or L-methionine.

[0055] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. The term "pharmaceutically acceptable excipient" refers to compounds that provide approximately physiological conditions and / or increase stability, such as agents and buffers for adjusting the pH value, agents for adjusting toxicity, and the like. In a plurality of embodiments, the pharmaceutically acceptable excipient is selected from sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and polysorbate-80 in the range of 0.0001% (w / v) to 1% (w / v), particularly preferably in the range of 0.001% (w / v) to 0.1% (w / v), and preferably is polysorbate-80.

[0056] In a preferred embodiment, the pharmaceutical composition comprises a targeting module with a dosage in the range of 25 μg / day to 100 mg / day, preferably in the range of 0.1 mg / day to 20 mg / day.

[0057] In a further embodiment, the pharmaceutical composition is sterile. The pharmaceutical composition is sterilized by conventional well-known techniques including, but not limited to, sterile filtration.

[0058] In a plurality of embodiments, the pharmaceutical composition is used for administration to a subject.

[0059] In a plurality of embodiments, the pharmaceutical composition is lyophilized before storage or stored as a solution at a temperature below ambient temperature, including, but not limited to, frozen storage.

[0060] In a plurality of embodiments, the pharmaceutical composition is reconstituted and / or diluted in an injection and stabilization solution before administration to a subject. The solution used for reconstitution or injection / stabilization may contain any of the components described for the pharmaceutical composition or similar components.

[0061] In a further aspect, the present invention provides a targeting module according to the present invention, a nucleic acid, vector or cell comprising a nucleotide sequence encoding a targeting module according to the present invention, or a pharmaceutical composition comprising a targeting module according to the present invention and a pharmaceutically acceptable diluent or carrier, for use in a method for stimulating a chimeric antigen receptor-mediated immune response in a mammal, preferably for use in the treatment of cancer, infectious diseases or autoimmune diseases.

[0062] In a plurality of embodiments, the targeting module is administered in combination with a vector or cell comprising a nucleotide sequence encoding a RevCAR, wherein the RevCAR - a tag that is the human La epitope E5B9 (SEQ ID NO: 28), - an extracellular hinge and transmembrane domain and - a signaling domain and the tag-binding domain of the targeting module binds to the tag of the RevCAR.

[0063] In a plurality of embodiments, a pharmaceutical composition for use in a method for stimulating a chimeric antigen receptor-mediated immune response in a mammal according to the present invention further comprises a vector or cell comprising a nucleotide sequence encoding a RevCAR, wherein the RevCAR - a tag that is the human La epitope E5B9 (SEQ ID NO: 28), - an extracellular hinge and transmembrane domain and - a signaling domain and the tag-binding domain of the targeting module binds to the tag of the RevCAR.

[0064] In a further aspect, the present invention provides a) a targeting module or nucleic acid, vector or cell according to the present invention, and b) a vector or cell comprising a nucleotide sequence encoding a RevCAR A kit comprising: wherein the RevCAR comprises: - A tag that is the human La epitope E5B9 (SEQ ID NO: 28), - An extracellular hinge and transmembrane domain and - A signaling domain and wherein: Provided is a kit in which the tag-binding domain of the targeting module binds to the tag of the RevCAR.

[0065] In a plurality of embodiments, the extracellular hinge and transmembrane domain of the RevCAR is selected from the group consisting of the human CD28 molecule, the CD8a chain, the hinge and transmembrane domain of an NK cell receptor, a portion of the constant region of an antibody, and combinations thereof.

[0066] In a further embodiment, the signaling domain of the RevCAR is selected from the group consisting of the cytoplasmic regions of CD28, CD137 (4-1BB), CD134 (OX40), CD278 (ICOS), DAP10 and CD27, programmed cell death-1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), the cytoplasmic region of the CD3 chain, DAP12, CD122 (interleukin-2 receptor β), CD132 (interleukin-2 receptor γ), CD127 (interleukin-7 receptor α), CD360 (interleukin-21 receptor), activating Fc receptors, and mutants thereof.

[0067] In a further embodiment, the kit according to the invention comprises at least one further distinct targeting module or at least one further distinct nucleic acid, vector or cell encoding a further distinct targeting module, wherein the at least one further distinct targeting module comprises at least one target cell-binding domain and a tag-binding domain, At least one target cell binding domain is 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, members of the epidermal growth factor receptor family and mutants thereof, particularly preferably ErbB1, ErbB2, ErbB3, ErbB4 or mutants thereof; members of the tumor necrosis factor receptor superfamily, claudin, ephrin, ephrin receptor, particularly preferably EphA1-10, EphA5 or EphB1-6; fucosyltransferase, prostate specific antigen, preferably PSCA and PSMA; fetal antigens, preferably CEA and fetal acetylcholine receptor;An antibody, antibody fragment, protein, peptide or low molecular weight organic ligand that binds to a surface antigen selected from the group consisting of members of the vascular endothelial growth factor family, EpCAM, AFP, members of the intercellular adhesion molecule family, C-type lectins, integrins, members of the mucin protein family, FSHR, HMW-MAA, FBP, folate receptor, somatostatin receptor, ligands of the NKG2D receptor, members of the epithelial glycoprotein family, diasialogangliosides, glypicans, G protein-coupled receptors, human papillomavirus proteins, cancer testicular antigens, fibroblast activation proteins, members of the carbonic anhydrase family, members of the carbohydrate antigen family, Notch ligands, MCSP, glycoprotein A33, guanylate cyclase 2C and tumor-specific glycans, including mutants and analogs of the specified antibody, antibody fragment, protein, peptide or low molecular weight organic ligand; The targeting module and at least one further distinct targeting module comprise the same tag-binding domain.

[0068] In a further embodiment, the kit according to the invention comprises a vector or cell comprising a nucleotide sequence encoding a targeting module and / or a RevCAR in the form of a pharmaceutical composition.

[0069] In a further embodiment, the kit according to the invention is used in a method for stimulating a chimeric antigen receptor-mediated immune response in a mammal, preferably for use in the treatment of cancer, infectious disease or autoimmune disease.

[0070] In multiple embodiments, the targeting module is administered alone, preferably 1 hour to 2 days, more preferably 4 to 24 hours, prior to administration of a vector or cell comprising a nucleotide sequence encoding a RevCAR. Advantageously, administration of the targeting module prior to administration of a vector or cell comprising a nucleotide sequence encoding a RevCAR stimulates the RevCAR and increases the proliferation and accumulation of effector cells having the RevCAR at the target site.

[0071] In a further embodiment, the targeting module is administered simultaneously with a vector or cell comprising a nucleotide sequence encoding a RevCAR.

[0072] In a further embodiment, the targeting module is administered up to after administration of a vector or cell comprising a nucleotide sequence encoding a RevCAR, preferably in the range of 3 days to 30 days. Further, such additional doses of the targeting module can be administered after a rest period to reactivate effector cells having the RevCAR.

[0073] As used herein, the term "reversible chimeric antigen receptor" refers to an artificial chimeric fusion protein, particularly a receptor comprising a tag, an extracellular hinge and transmembrane domain, and a signaling domain. The domains can be derived from different sources and thus the receptor is called chimeric. Advantageously, the receptor can be tagged to bind to different targeting modules.

[0074] Advantageously, cells comprising a nucleotide sequence encoding a RevCAR express the RevCAR, which has binding specificity for the tag-binding domain of the targeting module, and the targeting module in turn binds to CD123 on the target cells.

[0075] In multiple embodiments, the targeting module is in monomeric, dimeric or polymeric form, preferably in monomeric form.

[0076] In further embodiments, the targeting module is monovalent, bivalent or multivalent.

[0077] In some embodiments, the targeting module according to the present invention is bivalent or multivalent and comprises at least one CD123 binding domain and a tag binding domain that binds to the human La epitope E5B9 comprising a V L -linker-V H structure.

[0078] In a plurality of embodiments, the different domains of the targeting module according to the present invention are linked to each other by a linker. The linker preferably comprises a short chain sequence of 10 to 20 amino acid residues. In a plurality of embodiments, the targeting module comprises a flexible peptide sequence selected such that the domains have a three-dimensional folding that can exhibit specificity for effector cells and target cell binding. Preferred linkers have a structure (G x S y ) where x and y are selected from 1 to 10, preferably 1 to 5, and are glycine-serine linkers. Most preferred are 1 to 10 repeats of the sequence G4S1 (SEQ ID NO: 24). Additionally, linkers composed of peptide sequences that can increase the protease resistance of the antibody derivative are preferred.

[0079] In a plurality of embodiments, the linker is SEQ ID NO: 25 or SEQ ID NO: 26.

[0080] In several embodiments, the targeting module according to the invention comprises yet another domain selected from the group consisting of a co-stimulatory ligand, a radionuclide, a cell death-inducing chemical compound and a half-life increasing domain, preferably IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, HSA, an FcRn binding peptide or a mutant thereof. As used herein, the term "mutant" refers to a protein having at least 90% sequence identity, preferably at least 95% sequence identity, to the half-life increasing domain. Advantageously, the mutant may have one or more activities of the designated peptide or protein, and in particular, the mutant increases the half-life, such as a half-life increasing domain.

[0081] In multiple embodiments, the targeting module according to the present invention is used in a method for stimulating a chimeric antigen receptor-mediated immune response in a mammal, 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 further distinct targeting module, the at least one further distinct targeting module comprising at least one target cell binding domain and a tag binding domain or tag, the at least one target cell binding domain being 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, mesothelin, members of the epidermal growth factor receptor family and mutants thereof, particularly preferably ErbB1, ErbB2, ErbB3, ErbB4 or mutants thereof, members of the tumor necrosis factor receptor superfamily, ephrins, ephrin receptors, particularly preferably EphA1-10, EphA5 or EphB1-6; prostate specific antigen, preferably prostate stem cell antigen (PSCA) and prostate specific membrane antigen (PSMA);An antibody, antibody fragment, protein, peptide or low molecular weight organic ligand that binds to a surface antigen selected from the group consisting of fetal antigens, preferably carcinoembryonic antigen (CEA) and fetal acetylcholine receptor, members of the vascular endothelial growth factor family, epithelial cell adhesion molecule (EpCAM), alpha-fetoprotein (AFP), members of the intercellular adhesion molecule family, members of the mucin protein family, follicle stimulating hormone receptor (FSHR), human high molecular weight melanoma-associated antigen (HMW-MAA), folic acid binding protein FBP, folic acid receptor, somatostatin receptor, ligands of the NKG2D receptor, cytokine receptors, members of the epithelial glycoprotein family, disialogangliosides, glypicans, G protein-coupled receptors, members of the carbonic anhydrase family, members of the carbohydrate antigen family, Notch ligands, melanoma-associated chondroitin sulfate proteoglycan (MCSP), glycoprotein A33, guanylate cyclase 2C and tumor-specific glycans, including mutants and analogs of the specified antibody, antibody fragment, protein, peptide or low molecular weight organic ligand, and the targeting module according to the invention and at least one further distinct targeting module comprise the same tag-binding domain.;

[0082] As used herein, 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.

[0083] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and 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 included in the sequence of the protein or peptide.

[0084] As used herein, the term "low molecular weight organic ligand" refers to an organic molecule having a molecular weight of up to 10 kilodaltons, preferably up to 3 kilodaltons, that specifically binds to a protein or protein complex (antigen) on the surface of a target cell, preferably a cancer cell, T cell, infected cell or pathogen or parasite.

[0085] The term "target cell binding domain" also includes soluble T cell receptors composed of the alpha and beta chains or gamma and delta chains of the T cell receptor (TCR), fragments or mutants thereof. Such binding moieties derived from TCR recognize and bind to peptides presented by human leukocyte antigen class (HLA) I and II protein complexes. Examples include, but are not limited to, TCRs specific for peptides derived from proteins such as the EGFR family, survivin, sry-like high mobility group box (SOX) protein family, melanoma-associated antigens (e.g., the autoimmunogenic cancer / testis antigen NY-ESO-1, members of the melanoma antigen family A MAGEA, antigens preferentially expressed in melanoma PRAME), and leukemia-associated antigens (e.g., Wilms tumor gene 1 WT1).

[0086] As used herein, the term "analogue" refers to a molecule having a high degree of structural identity to a specified antibody, antibody fragment, protein, peptide or low molecular weight organic ligand, preferably with at least one atom, atomic group, functional group or substructure replaced by another atomic group, such as a hydroxy group. In a plurality of embodiments, analogues of somatostatin (SRIF14) are octreotide or pasireotide. Advantageously, the analogue binds to the same antigen as the specified antibody, antibody fragment, protein, peptide or low molecular weight organic ligand.

[0087] In some embodiments, analogs of the specified antibody, antibody fragment, protein or peptide include modifications selected from the group consisting of D-amino acids, peptidomimetic bonds, amino alcohols, amino acids not constituting proteins, unnatural amino acids, amino acids with modified side chains and / or cyclic proteins. Advantageously, these analogs exhibit increased stability.

[0088] In a further embodiment, the target cell binding domain is a soluble T cell receptor consisting of the alpha and beta chains or the gamma and delta chains of a T cell receptor (TCR).

[0089] In a preferred embodiment, at least one further targeting module comprises at least one target cell binding domain and a tag binding domain or tag, wherein the at least one target cell binding domain is selected from the group consisting of 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, 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, members of the epidermal growth factor receptor family, particularly preferably ErbB1, ErbB2, ErbB3 or ErbB4; members of the tumor necrosis factor receptor superfamily, ephrins, ephrin receptors, particularly preferably EphA1-10, EphA5 or EphB1-6, prostate specific antigen, preferably PSCA and PSMA; fetal 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 protein family, FSHR, HMW-MAA, folate binding protein FBP, folate receptor, somatostatin receptor, ligands of the NKG2D receptor, cytokine receptors, members of the epithelial glycoprotein family, disialogangliosides, glypicans, 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, and is an antibody or antibody fragment that binds to a surface antigen selected from the group, and the targeting module according to the invention and at least one further targeting module comprise different target cell binding domains, and the same tag binding domain or tag.

[0090] According to the present invention, the nucleic acid, vector and / or cell are isolated. As used herein, the term "isolated" means that it has been changed from its natural state or removed.

[0091] In a plurality of embodiments, the nucleic acid is cDNA. As used herein, the term "cDNA" (complementary DNA) refers to double-stranded DNA synthesized from single-stranded RNA, such as mRNA, in a reaction catalyzed by the enzyme reverse transcriptase. In a plurality of embodiments, the cDNA is of synthetic origin. In a further embodiment, the cDNA is derived from mRNA and thus contains only exons and no introns, in contrast to genomic DNA.

[0092] The vector is preferably a plasmid, artificial chromosome, linear DNA or RNA, viral particle, or another vector containing an expression cassette stably integrated into the genome of a host cell or host organism.

[0093] In a plurality of embodiments, the cell is selected from immune cells that preferably have cytolytic activity, phagocytic activity or immunosuppressive activity, such as T cells, natural killer (NK) cells and macrophages. In a preferred embodiment, the cell is selected from T cells, including alpha / beta and gamma / delta T cells, or subpopulations of T cells such as stem cell memory T cells or central memory T cells, cytotoxic T cells or NK cells.

[0094] In a plurality of embodiments, the nucleic acid, vector or cell further comprises an inducible expression system. In some embodiments, the inducible expression system is based on a prokaryotic operon, including but not limited to the lac operon, transposon Tn 10 or the tetracycline operon. In other embodiments, the inducible expression system is based on components of a eukaryotic signaling pathway, including but not limited to expression systems based on steroid receptors, estrogen receptors, progesterone or metallothionein.

[0095] In multiple embodiments, the inducible expression system induces the transcription of the nucleotide sequence encoding the RevCAR according to the present invention and / or the nucleotide sequence encoding the targeting module, and preferably, the inducible expression system induces the transcription of the nucleotide sequence encoding the targeting module according to the present invention.

[0096] In multiple embodiments, the nucleic acid, vector or cell is administered in combination with a vector or cell comprising a nucleotide sequence encoding a RevCAR, and a nucleic acid, vector or cell comprising a nucleotide sequence encoding at least one further targeting module, wherein the at least one further targeting module comprises at least one target cell binding domain and a tag binding domain or tag, and the at least one target cell binding domain is 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, a cytokine receptor, preferably an interleukin receptor, particularly preferably IL-8Rα, IL-8Rβ, IL-11Rα, IL-11Rβ, IL13Rα1; CXCR4, c-Met, mesothelin, a member of the epidermal growth factor receptor family and mutants thereof, particularly preferably ErbB1, ErbB2, ErbB3, ErbB4 or mutants thereof; a member of the tumor necrosis factor receptor superfamily, claudin, ephrin, ephrin receptor, particularly preferably EphA1-10, EphA5 or EphB1-6; fucosyltransferase, prostate specific antigen, preferably PSCA and PSMA; fetal antigen, preferably CEA and fetal acetylcholine receptor;An antibody, antibody fragment, protein, peptide or low molecular weight organic ligand that binds to a surface antigen selected from the group consisting of members of the vascular endothelial growth factor family, EpCAM, AFP, members of the intercellular adhesion molecule family, C-type lectins, integrins, members of the mucin protein family, FSHR, HMW-MAA, FBP, folate receptor, somatostatin receptor, ligands of the NKG2D receptor, members of the epithelial glycoprotein family, disialogangliosides, glypicans, G protein-coupled receptors, human papillomavirus proteins, cancer testicular antigens, fibroblast activation proteins, members of the carbonic anhydrase family, members of the carbohydrate antigen family, Notch ligands, MCSP, glycoprotein A33, guanylate cyclase 2C and tumor-specific glycans, including mutants and analogs of the specified antibody, antibody fragment, protein, peptide or low molecular weight organic ligand, and the targeting module according to the invention and at least one further distinct targeting module comprise the same tag-binding domain.;

[0097] In a plurality of embodiments, the pharmaceutical composition comprises at least one further distinct targeting module, or at least one further distinct nucleic acid, vector or cell encoding a further distinct targeting module, the at least one further distinct targeting module comprising at least one target cell binding domain and a tag binding domain or tag, the at least one target cell binding domain being 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, 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, members of the epidermal growth factor receptor family and mutants thereof, particularly preferably ErbB1, ErbB2, ErbB3, ErbB4 or mutants thereof, members of the tumor necrosis factor receptor superfamily, ephrins, ephrin receptors, particularly preferably EphA1-10, EphA5 or EphB1-6; prostate specific antigen, preferably PSCA and PSMA; fetal antigens, preferably CEA and fetal acetylcholine receptor;an antibody, antibody fragment, protein, peptide, or small organic ligand that binds to a surface antigen selected from the group consisting of a member of the vascular endothelial growth factor family, EpCAM, AFP, a member of the intercellular adhesion molecule family, a member of the mucin protein family, FSHR, HMW-MAA, FBP, a folate receptor, a somatostatin receptor, a ligand for the NKG2D receptor, a cytokine receptor, a member of the epithelial glycoprotein family, a disialoganglioside, a glypican, a G protein-coupled receptor, a member of the carbonic anhydrase family, a member of the carbohydrate antigen family, a Notch ligand, MCSP, glycoprotein A33, guanylate cyclase 2C, and a tumor-specific glycan, including mutants and analogs of the specified antibody, antibody fragment, protein, peptide, or small organic ligand, wherein the targeting module and at least one further targeting module comprise identical tag-binding domains;

[0098] According to the present invention, the tag-binding domain binds to a tag derived from the human nuclear La protein. Preferably, the tag-binding domain is The following array: DIVMTQSPDSLAVSLGERATINCX 24 SSQSLLNSRTX 35 KNYLAWYQQKPGQPPKLLIYWASTRX 61 SGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYNLX 101 TFGGGTKVElK (SEQ ID NO: 19) (X 24 , X 35 , X 61 and X 101 are independently selected from proteinogenic alpha amino acid residues), or a sequence having at least 90% sequence identity, preferably at least 95% sequence identity to SEQ ID NO: 21; V according to L and wherein the antibody or antigen-binding fragment comprises:

[0099] In multiple embodiments, the tag-binding domain comprises an scFv of an anti-La epitope.

[0100] In some embodiments, X 24 ~X 101 is selected as follows: X 24 is selected from polar and / or positively charged residues such as serine, threonine, asparagine, glutamine, histidine, lysine and arginine, preferably lysine or arginine, X 35 is preferably selected from lysine and proline, X 61 is selected from polar and charged residues such as asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine and arginine, preferably glutamic acid and lysine, X 101 is selected from hydrophobic residues such as isoleucine, leucine, valine, alanine, methionine, phenylalanine, proline and tryptophan, preferably leucine or proline.

[0101] Preferably, the tag-binding domain has at least 90% sequence identity, preferably at least 95% sequence identity, respectively, to the sequences according to SEQ ID NO: 20 (V H ) and SEQ ID NO: 21 (V L ).

[0102] Particularly preferably, the tag-binding domain comprises an anti-La 5B9 scFv according to SEQ ID NO: 20 (V H ) and SEQ ID NO: 21 (V L ).

[0103] As used herein, the term "extracellular hinge and transmembrane domain" refers to a flexible peptide sequence linked to the tag, which anchors the RevCAR within the cell membrane and protrudes from the surface of the cell to optimally bind to its specific targeting module.

[0104] In multiple embodiments, the extracellular hinge and transmembrane domain are selected from the hinge and transmembrane domain of the human CD28 molecule, CD8a chain, NK cell receptor, preferably the natural killer group NKG2D; or a portion of the constant region of an antibody and combinations thereof. As used herein, the term "combinations thereof" refers to combinations of different hinge and transmembrane domains.

[0105] Pinthus et al. and Cartellieri et al. have described the use of the hinge and transmembrane domain of the human CD28 molecule in CARs (Pinthus et al., 2003; Cartellieri et al., 2016).

[0106] Milone et al. and Zhao et al. have described the use of the hinge and transmembrane domain of the human CD8α molecule in CARs (Milone et al., 2009; Zhao et al., 2009).

[0107] Zhang et al. have described the use of the hinge and transmembrane domain of NKG2D in CARs (Zhang et al., 2005).

[0108] Frigault et al. and Wang et al. have described the use of the hinge and transmembrane domain of a portion of the constant region of immunoglobulin G1 (IgG) (Frigault et al., 2015; Wang et al., 2007). Frigault et al. have described the use of the hinge domain of the constant region of IgG4.

[0109] Examples of combinations of extracellular hinge and transmembrane domains include, but are not limited to, the extracellular hinge and transmembrane domain of CD28, the extracellular hinge and transmembrane domain of CD8 alpha, the constant region of IgG1 or IgG4 combined with the transmembrane domain of CD28 or CD137.

[0110] As used herein, the term "signaling domain" refers to a peptide sequence that transmits a signal to a cell by crosslinking a cell expressing a RevCAR (effector cell) to a human cell surface protein or protein complex (target cell). The crosslinking between the effector and target cells is mediated by a targeting module according to the present invention.

[0111] In several embodiments, the signaling domain is selected from the cytoplasmic regions of CD28, CD137 (4-1BB), CD134 (OX40), CD278 (ICOS), DAP10 and CD27, programmed cell death-1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), the cytoplasmic region of the CD3 chain, DAP12, CD122 (interleukin-2 receptor β), CD132 (interleukin-2 receptor γ), CD127 (interleukin-7 receptor α), CD360 (interleukin-21 receptor), activating Fc receptors, and mutants thereof.

[0112] As used herein, the term "mutant" refers to a protein having at least 90% sequence identity, preferably at least 95% sequence identity, to a signaling domain. Advantageously, the mutant transmits a signal to a cell by crosslinking a cell expressing a RevCAR (effector cell) to a human cell surface protein or protein complex (target cell) in the same manner as the designated signaling domain.

[0113] In several embodiments, the mutant is a truncated form. As used herein, 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 to the signaling domain. Advantageously, the truncated form has at least 80%, preferably at least 90%, more preferably at least 95% activity of the designated signaling domain.

[0114] Hombach et al. and Cartellieri et al. have described the use of the cytoplasmic region of CD28 as a signaling domain in CARs (Hombach et al., 2001; Cartellieri et al., 2016). Guedan et al. have described the use of a mutant of the cytoplasmic region of CD28 as a signaling domain (Guedan et al., 2020).

[0115] Milone et al. and Finney et al. have described the use of the cytoplasmic region of CD137 (4-1BB) as a signaling domain (Finney et al., 2004; Milone et al., 2009).

[0116] Finney et al. as well as Hombach and Abken have described the use of the cytoplasmic region of CD134 (OX40) as a signaling domain in CARs (Finney et al., 2004; Hombach and Abken, 2011).

[0117] Guedan et al. have described the use of the cytoplasmic region of CD278 (ICOS) as a signaling domain (Guedan et al., 2018).

[0118] Zhang et al. have described the use of DAP10 as a signaling domain (Zhang et al., 2005).

[0119] Fedorov et al. described the use of programmed cell death 1 (PD-1) and cytotoxic T lymphocyte antigen 4 (CTLA-4) as signaling domains in CARs (Fedorov et al., 2013).

[0120] Gong et al. and Gade et al. described the use of the cytoplasmic region of the CD3 chain, particularly the CD3ζ chain, as a signaling domain in CARs (Gong et al., 1999; Gade et al., 2005).

[0121] Toepfer et al. described the use of DAP12 as a signaling domain in CARs (Toepfer et al., 2015).

[0122] Kagoya et al. described the use of a signaling chain or motif derived from an interleukin receptor as a signaling domain in CARs (Kagoya et al., 2018).

[0123] Lamers et al. and Kershaw et al. described the use of activating Fc receptors, particularly the Fc epsilon receptor gamma chain, as signaling domains (Lamers et al., 2004; Kershaw et al., 2006).

[0124] In a preferred embodiment, the signaling domain is selected from the cytoplasmic regions of CD28, CD137 (4-1BB), CD134 (OX40), CD278 (ICOS), DAP10 and CD27, programmed cell death-1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), the cytoplasmic region of the CD3 chain, DAP12, CD122 (interleukin-2 receptor beta), CD132 (interleukin-2 receptor gamma), CD127 (interleukin-7 receptor alpha) and CD360 (interleukin-21 receptor), and activating Fc receptors.

[0125] In a further embodiment, the RevCAR comprises a fourth domain, which is a short-chain peptide linker in the extracellular portion of the receptor that can serve to detect the chimeric antigen receptor on the cell surface or stimulate chimeric antigen receptor T cells.

[0126] In some embodiments, the fourth domain is located between the tag-binding domain or tag and the extracellular hinge domain or an essential part of the extracellular hinge domain.

[0127] Advantageously, the RevCAR-grafted cells having the fourth domain can be specifically stimulated to preferentially proliferate and survive longer than non-grafted cells, either in vitro or in vivo. Even more advantageously, the fourth domain can also be used to purify RevCAR-grafted cells from a mixed cell population or, in vivo, to suppress the immune response mediated by RevCAR-grafted cells and to eliminate RevCAR-grafted cells.

[0128] In a further embodiment, the RevCAR comprises a signal peptide. Advantageously, the signal peptide enables expression on the cell surface of effector cells. In a plurality of embodiments, the signal peptide is located at the N-terminus of the RevCAR nucleotide sequence before the tag-binding domain or tag. In some embodiments, the signal peptide targets the protein to the secretory pathway either during or after translation and is selected from leader peptides of proteins such as CD28, CD8 alpha, IL-2, lysozyme C or the heavy or light chain of an antibody derived from humans to avoid an immunogenic reaction.

[0129] In a plurality of embodiments, the nucleic acid is cDNA.

[0130] In a plurality of embodiments, the tag is present at the amino terminus of the polypeptide comprising the RevCAR. Advantageously, the location of the tag at the amino terminus allows access of the tag to the targeting module bound to the target cell without being hindered.

[0131] In multiple embodiments, the nucleic acid encodes a RevCAR according to SEQ ID NO: 29 or SEQ ID NO: 30. Preferably, the nucleic acid is SEQ ID NO: 31 or SEQ ID NO: 32.

[0132] In some embodiments, the cell comprising the nucleotide sequence encoding RevCAR is selected from immune cells, such as T cells, natural killer (NK) cells, and macrophages, preferably having cytolytic activity, phagocytic activity, or immunosuppressive activity. In preferred embodiments, the cell is selected from T cells, including alpha / beta and gamma / delta T cells, or subpopulations of T cells such as stem cell memory T cells or central memory T cells, cytotoxic T cells, or NK cells.

[0133] In multiple embodiments, the kit further comprises at least one further distinct targeting module, or at least one further distinct nucleic acid, vector or cell encoding a further distinct targeting module, the at least one further distinct targeting module comprising at least one target cell binding domain and a tag binding domain or tag, the at least one target cell binding domain being 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, 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, members of the epidermal growth factor receptor family and mutants thereof, particularly preferably ErbB1, ErbB2, ErbB3, ErbB4 or mutants thereof, members of the tumor necrosis factor receptor superfamily, ephrins, ephrin receptors, particularly preferably EphA1-10, EphA5 or EphB1-6, prostate specific antigen, preferably PSCA and PSMA; fetal antigens, preferably CEA and fetal acetylcholine receptor;An antibody, antibody fragment, protein, peptide or low molecular weight organic ligand that binds to a surface antigen selected from the group consisting of members of the vascular endothelial growth factor family, EpCAM, AFP, members of the intercellular adhesion molecule family, members of the mucin protein family, FSHR, HMW-MAA, FBP, folate receptor, somatostatin receptor, ligands of the NKG2D receptor, cytokine receptor, members of the epithelial glycoprotein family, disialoganglioside, glypican, G protein-coupled receptor, members of the carbonic anhydrase family, members of the carbohydrate antigen family, Notch ligand, MCSP, glycoprotein A33, guanylate cyclase 2C and tumor-specific glycans, including mutants and analogs of the specified antibody, antibody fragment, protein, peptide or low molecular weight organic ligand, and the targeting module and at least one further distinct targeting module comprise the same tag-binding domain.;

[0134] The present invention is further illustrated herein by the following non-limiting drawings and examples.

Brief Description of the Drawings

[0135]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10-1

Figure 10-2

Figure 11

Figure 12

Figure 13

Mode for Carrying Out the Invention

[0136] Design of the targeting module according to the present invention The targeting module R-TM123 is a soluble recombinant fusion protein having two antibody-derived binding domains. One selectively binds to the target antigen CD123, and the other recognizes the RCE or tag presented on RevCAR-expressing cells (epitope E5B9 of human La protein). Therefore, R-TM123 functions as a bridging module between RevCAR-T and target cancer cells expressing CD123 (Figure 1). The targeting module further includes an 8x-histidine tag at the C-terminus for detection and purification purposes.

[0137]

Table 1

[0138] Surface plasmon resonance measurement The functionality of the CD123-binding TM can be confirmed in a binding assay to soluble recombinant CD123 using surface plasmon resonance measurements (Figures 2 and 3 and Table 1).

[0139] Binding of various variants of the targeting module according to the invention was analyzed by SPR measurements using human CD123-Fc conjugated to a CM5 sensor chip on a Biacore X100 instrument (Figure 2). Except for the targeting module according to SEQ ID NO: 8, the targeting modules were purified monomers. The targeting modules were concentrated between 1.23 and 100 nM and measured in technical duplicates. For data fitting and calculation of K D a 1:1 binding model was applied.

[0140] The interaction of the CD123-binding TM with human CD123 had an equilibrium dissociation constant (K D ) in the range between 23 pM and 5.2 nM, while the reference TM according to SEQ ID NO: 2 had a K D of 227 pM.

[0141] Furthermore, binding of various mutants of the targeting module according to the invention was analyzed using La epitope 5B9 (SEQ ID NO: 28) fused to the human Fc domain conjugated to a CM5 sensor chip on a Biacore X100 instrument (Figure 3). The targeting modules were purified monomers. The targeting modules were concentrated between 2.47 and 200 nM and measured in technical duplicates. For data fitting and calculation of KD, a two-state reaction binding model was applied.

[0142] The interaction of the TM with 5B9 had an equilibrium dissociation constant (K D ) in the range between 2.0 nM and 3.4 nM, while the reference TM according to SEQ ID NO: 2 had a K D of 17 nM.

[0143] Cell binding assay The cell binding of the CD123-binding targeting module (TM) was tested in the CD123-positive target cell lines Oci-AML3 and Molm-13: A) SEQ ID NO: 5 against Oci-AML3, B) SEQ ID NO: 9 against Oci-AML3, C) SEQ ID NO: 4 against Molm-13, and D) SEQ ID NO: 6 against Molm-13. The affinity of the TM was analyzed by flow cytometry. The TM was titrated in the AML cell lines and then detected with a mouse anti-His tag antibody conjugated to phycoerythrin.

[0144] Figure 4 shows the cell binding ability evaluated using the CD123-positive target cell lines Oci-AML3 and Molm-13. Different concentrations of R-TM123 were incubated with the cells, washed, and quantified. The geometric mean fluorescence (MFI) was plotted against the TM concentration to obtain a dose-response curve.

[0145] The cell binding data of the CD123-binding TM in the CD123-positive target cell lines Oci-AML3 and Molm-13 were fitted to a four-parameter model with a variable slope of a sigmoid curve. The 50% effective concentration (EC 50 ) obtained from this model can be interpreted as a representative value of the affinity of the TM for cells overexpressing the target receptor (see Table 1).

[0146] Thermodynamic stability The thermodynamic stability of the two targeting modules according to the present invention was evaluated by melting point analysis (Figure 5). The protein melting points of the targeting modules according to SEQ ID NO: 5 and SEQ ID NO: 9 were determined using a thermal shift assay in various buffers in the pH range of pH 4.0 to 9.0. Typically, the colloidal stability of a protein correlates with its thermodynamic stability, providing a predictive possibility for comparing long-term stabilities.

[0147] RevCAR T cells In genetic manipulation to express RevCAR, a polynucleotide vector encoding RevCAR and all necessary elements to ensure vector expression in the genetically engineered immune cells are transferred into the immune cells. In particular, 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). Vector transfer can be performed by nucleic acid electroporation or transfection, or by using viral vector systems such as gene transfer of adenovirus, adeno-associated virus, retrovirus, foamy virus, or lentivirus.

[0148] Lentiviral gene transfer is applied to stable expression of RevCAR in immune cells by first constructing a lentiviral vector encoding the selected RevCAR. The lentiviral vector is pLVX-EF1 alpha UniCAR 28 / ζ (Clontech, Takara Bio Group), where the lentiviral part of the vector is derived from human immunodeficiency virus (HIV) and the MSC / IRES / ZxGreenI part is replaced by the RevCAR construct.

[0149] Lentiviral particles are produced by transient transfection of human embryonic kidney (HEK) 293T (ACC 635) cells with a RevCAR encoding a lentiviral vector plasmid, and co-transfection with a plasmid (psPAX2) encoding group-specific antigen (gag) and polymerase (pol), and a plasmid (pMD2.G) encoding an envelope. 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). The VSV-G protein is used in lentiviral vectors to introduce a wide range of mammalian cells. A variety of envelopes derived from different viral species can be utilized for this purpose. Lentiviral vectors can be successfully pseudotyped with the envelope glycoprotein (Env) of bidirectional murine leukemia virus (MLV) or the G protein of vesicular stomatitis virus (VSV-G), a modified envelope of prototype foamy virus (PFV), or a chimeric envelope glycoprotein variant derived from gibbon ape leukemia virus (GaLV) and MLV.

[0150] The supernatant of transfected HEK293T cells is collected 24 to 96 hours after transfection, and viral particles are concentrated from the supernatant by ultracentrifugation or other methods. For lentiviral transduction of immune cells, peripheral blood mononuclear cells (PBMCs) or isolated T cells are activated with a mab specific for the CD3 complex, such as clone OKT3 or UCHT1, which is in solution or coated on plastic cell culture dishes or magnetic beads or biodegradable polymer matrices. Activation of PBMCs or isolated T cells is further enhanced by stimulating the co-stimulatory pathway with mab or ligand specific for CD27, CD28, CD134 or CD137, alone or in combination, coated on plastic cell culture dishes or magnetic beads or biodegradable polymer matrices, and by 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 the culture of PBMCs or T cells 24 to 96 hours after the first administration as a single dose or multiple doses of an activating CD3-specific antibody and / or an antibody specific for the co-stimulatory receptors CD27, CD28, CD134 or CD137 and / or a recombinant cytokine. Electroporation, transduction and proliferation of T cells can be performed manually in an open cell culture system or in a closed, partially or fully automated system.

[0151] Stable transduction of T cells can be determined by flow cytometry after staining with a tag-containing molecule for surface expression of RevCAR or a mab against the fourth domain of RevCAR 3 days or more after the final administration of the viral supernatant. T cells transduced with RevCAR can be expanded in vitro by culturing the T cells in the presence of recombinant cytokines and activating anti-CD3 mab.

[0152] In cases where the RevCAR has an optional fourth domain that is a peptide sequence forming a linear epitope for the mab, genetically modified immune cells expressing the RevCAR can be specifically expanded in vitro by coating the surface of the culture dish, or any type of beads or biodegradable polymer matrix added to the cell culture at a predetermined ratio, with a mab or an antibody fragment thereof that binds to the fourth RevCAR domain. When the mab coated on the surface binds to the RevCAR peptide domain, cross-linking of the RevCAR expressed on the cell surface and formation of an immune synapse are induced, which results in activation of the signaling pathway specifically triggered by the signal domain of the RevCAR. Depending on the signaling pathway induced, this can lead to enhanced proliferation of immune cells having the RevCAR and persistence of resistance to activation-induced cell death, and thus enrichment of the immune cells genetically modified with the RevCAR in the mixed population.

[0153] The optional fourth domain that is a peptide sequence forming a linear epitope for the mab can be further utilized to enrich and purify immune cells expressing the RevCAR from a mixed population. Enrichment and purification are performed by using a mab or an antibody fragment thereof that binds to the fourth RevCAR domain to mark cells expressing the RevCAR for cell sorting, or to temporarily link immune cells expressing the RevCAR to small particles that can be used for cell isolation. In one aspect, RevCAR-grafted immune cells are incubated with a mab that recognizes the fourth domain. Next, magnetic beads are added, which conjugate with an antibody or a fragment thereof against the species-specific and isotype-specific heavy and light chains of the mab that binds to the optional fourth domain. In this way, the immune cells expressing the RevCAR and the magnetic beads are linked, captured in a magnetic field, and separated from other immune cells.

[0154] Cytotoxicity assay The efficacy of CD123-binding TM in inducing tumor cell elimination by RevCAR-T cells was tested using a suspension cell-based co-culture assay with AML cell line Molm-13 (Figure 6) and AML cell line Oci-AML3 (Figure 7) in the presence of various concentrations of the targeting module. Switchable CAR-T cells were incubated with target cells at an E:T ratio of 2:1 for 48 hours in the presence of various TM concentrations. As CD123-positive target cells, human AML cell line Molm-13 (Figure 6) or Oci-AML3 (Figure 7) stained with efluor before the setting was used respectively. Target cells were quantified by flow cytometry, and the cell numbers of each sample were normalized to the control sample seeded with only tumor cells to calculate lysis. The data was fitted to a four-parameter model with a variable slope of a sigmoid curve. The calculated EC 50 values can be interpreted as representative values of the efficacy of TM against these tumor cells.

[0155] Dose-dependent activation of RevCAR-T by R-TM123 In the experiments described in the following sections, clinical-scale RevCAR-T was used. CD25 (IL-2 receptor α, IL-2Rα), a high-affinity receptor for IL-2, is expressed in human T cells and becomes detectable on the cell surface upon stimulation of the endogenous TCR complex (Kmieciak et al., 2009). IL-2Rα regulates the proliferative response of T cells and is an indicator of the degree of TCR stimulation (Shatrova et al., 2016).

[0156] Stimulation of RevCAR via R-TM123 according to SEQ ID NO: 5 is similar to activation by the endogenous TCR, except that the artificial receptor activation signal from the immunoreceptor tyrosine-based activation motif (ITAM) of the CD3ζ subunit is accompanied by a co-stimulation signal from the CD28 signaling chain (Cartellieri et al., 2016), and upregulation of CD25 can be monitored after this activation.

[0157] Figure 8 shows the surface expression of CD25 in RevCAR-T during R-TM123-mediated activation. RevCAR-T batches were co-cultured with AML cell lines MOLM-13, MV4-11, and OCI-AML3 expressing CD123 for 48 hours in the presence of various concentrations of R-TM123. Cell samples were prepared and the surface expressions of CD2, RevCAR, CD4, CD8, and CD25 were analyzed by flow cytometry. All samples were pre-gated for CD2+ / RevCAR+ cells. The frequencies of CD25+ RevCAR-T cells are shown separately for CD4+ cells and CD8+ cells. Technical triplicates obtained from the co-cultures were pooled and staining data from four clinical-scale batches made from healthy donor material are shown.

[0158] Thus, the surface expression of CD25 in RevCAR-T in response to R-TM123-mediated stimulation in the presence of target cells expressing CD123 was determined. The frequency of RevCAR-T expressing CD25 is dependent on the dose of R-TM123 (Figure 8). Both CD4+ RevCAR-expressing T cells and CD8+ RevCAR-expressing T cells are activated when RevCAR-T crosslinks to target cells via R-TM123 (Figure 8). The degree of response dependent on the dose of R-TM123 after 48 hours is similar in both subpopulations (Figure 8 and Table 2).

[0159]

Table 2

[0160] R-TM123 dose-dependent cytotoxic response against CD123 leukemia cell lines To evaluate dose-dependent target cell lysis, four clinical-scale batches of RevCAR-T were used in a cytotoxicity assay. Increasing concentrations of R-TM123 were used to analyze the cytotoxic response against three AML cell lines for all four RevCAR-T batches. The R-TM123 dose-response curves for MOLM-13, OCI-AML3, and MV4-11 are shown in Figure 9.

[0161] Figure 9 shows specific lysis of the AML cell line MOLM-13 by RevCAR-T redirected by R-TM123. The AML cell lines MOLM-13, MV4-11, and OCI-AML3 expressing CD123 were labeled with the cell tracer eFluor670 and then co-cultured with 2 × 10 5 cells of four clinical-scale batches of RevCAR-T at an E:T ratio of 1:1. After 48 hours of co-culture, the number of viable target cells was determined by cytometry to determine specific lysis. The mean ± SD values of technical triplicates and the induced dose-response curves for four clinical-scale batches of RevCAR-T from independent donors are shown. Data points were fit with four-parameter nonlinear regression in GraphPad 9, and the half-maximal doses of R-TM123 are shown in Table 3.

[0162] The MOLM-13 cells are derived from the peripheral blood of a patient (FAB M5a) with relapsed acute monocytic leukemia transformed from myelodysplastic syndrome (Matsuo et al., 1997). OCI-AML3 was established from a patient with AML (FAB M4) and has abnormal cytoplasmic translocation of nucleophosmin, an immunocytological feature of AML with NPM1 mutation (type A) and NPM1-mutated AML (Quentmeier et al., 2005). Furthermore, it also has a DNMT3A mutation of the R882C type (Tiacci et al., 2012). Thus, both cell lines are major AML subtypes that will be included in future clinical studies. The MV4-11 cell line is originally derived from pediatric acute monocytic leukemia and has also been described as expressing CD123 (Mani et al., 2018).

[0163] RevCAR-T cells induced lysis of target cells in all of these cell lines, and lysis occurred in a strictly R-TM123-dependent manner. Half-maximal lysis (EC 50) was observed in all three target cell lines (Table 2). All four clinical-scale batches of RevCAR-T showed similar maximum half-maximal lysis and reached the upper plateau region (i.e., 100% target cell lysis) at approximately 1 nM in all AML cell lines.

[0164]

Table 3

[0165] R-TM123 dose-dependent cytokine release by RevCAR-T redirected against leukemia cell lines expressing CD123 Through the involvement of the TCR, T cells are activated and release large amounts of cytokines. Cytokines can have effector, stimulatory, regulatory, chemotactic, and inflammatory functions. Similarly, CAR-engineered T cells release cytokines when stimulated via their artificial receptors (Rossi et al., 2018).

[0166] To characterize the cytokine release capacity of RevCAR-T, a co-culture assay was used. For this purpose, RevCAR-T derived from the same four clinical-scale batches was used for specific target cell lysis and the T cell activation studies described in the previous section were analyzed. Cells were thawed and co-cultured with MOLM-13 AML cells in the presence of R-TM123 for 48 h, and the effector cytokines released into the cell culture supernatant were quantified using the MACSPlex Cytotoxic T / NK Cell kit (Miltenyi, Germany).

[0167] Qualitatively, very similar cytokine release profiles, showing elevated levels of effector cytokines (granzyme B and perforin) as well as inflammatory cytokines such as GM-CSF, IFN-γ, TNF-α, and IL-2, were seen across different RevCAR-T clinical scale batches (Figure 10). The anti-inflammatory and regulatory cytokine IL-4 was detected in only two RevCAR-T batches. Quantitatively, it became clear that the absolute amounts of individual cytokines were donor- or product-dependent. Furthermore, the half-maximal cytokine release (EC 50 of R-TM123) was determined from sigmoid dose-response curves via non-linear regression (see Table 4). The EC 50 values can be used as a direct indicator of cytokine release dynamics and compared to other effector functions such as target cell lysis or T cell activation. Typically, the half-maximal cytokine release varied significantly more between cytokines than between RevCAR-T cell products. The effector cytokines granzyme B and perforin were already secreted at low R-TM123 concentrations and correlated with target cell lysis (2 - 6 pM for lysis vs. 4 - 12 pM for the EC 50 of granzyme B / perforin), which is noteworthy. The inflammatory cytokines showed half-maximal cytokine secretion at much higher R-TM123 doses; for example, GM-CSF was approximately 35 - 40 pM, IFN-γ was approximately 20 - 25 pM, or TNF-α was approximately 50 - 80 pM.

[0168] In conclusion, RevCAR-T clinical scale products show cytokine release typical of T cells. The release varies in amount between cytokines and between T cell donors or products. Cytokine release correlates with other effector functions (i.e., activation and target cell lysis) of the RevCAR-T tested but shifts to higher R-TM123 doses.

[0169] Figure 10 shows the release of cytokines and effector molecules by RevCAR-T redirected by R-TM123 against the AML cell line MOLM-13.

[0170] RevCAR-T, derived from four healthy donors and manufactured by a clinical-scale process, was incubated with the AML cell line MOLM-13 expressing CD123 in the presence of the indicated concentrations of R-TM123 and at an effector-to-target cell ratio of 1:1. After 48 hours, T cell co-culture supernatants were harvested, technical replicates were pooled, and analyzed using a flow cytometry-based multiplex assay (MACSPlex Cytotoxic T / NK Cell kit; Miltenyi, Germany). The respective dose-response curves are shown. Sigmodial data points were fit using four-parameter logistic regression in GraphPad Prism 9 to determine the half-maximal cytokine release (EC 50 ), as summarized in Table 4.

[0171] [Table 4]

[0172] R-TM123 dose-dependent lysis of primary AML cells by Allo-RevCAR-T The ability of clinical-scale RevCAR-T batches to lyse primary patient AML material was analyzed in a flow-based cytotoxicity assay. The cytotoxicity assay was performed on primary AML cells (AML1, AML3, AML4, AML5) from four AML patients, each combined with three RevCAR-T batches (i.e., a total of 12 primary AML / RevCAR-T pairs). On the day the assay was set up, patient-derived AML cells were thawed, washed, and characterized via flow cytometry for the expression of markers CD45, CD14, HLA-DR, CD33, CD34, and to confirm the expression of the target CD123 (data not shown). RevCAR-T batches were co-cultured with primary AML cells at an effector-to-target ratio of 1:2 in the presence of R-TM123. After 48 hours of co-culture, the number of viable AML cells was determined via flow cytometry staining (Figure 11).

[0173] Figure 11 shows the cytotoxic response of RevCAR-T redirected against primary leukemia cells from AML patients by R-TM123. RevCAR-T, derived from healthy donors and manufactured in a clinical-scale process, was thawed and co-cultured with 1.25 - 1.5×10 4 primary AML cells at an effector-to-target ratio of 1:2 in the indicated 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 the culture medium. After 48 hours, the number of viable AML cells was determined by flow cytometry. A four-parameter non-linear fit of log(agonist) vs. response with variable slope was applied to calculate the dose-response curve using GraphPad prism 9. The calculated EC50 values for target cell lysis are summarized in Table 5.

[0174]

Table 5

[0175] Intravenous administration of a single dose of R-TM123 in mice The pharmacokinetic profile of R-TM123 was investigated in vivo in NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ immunodeficient mouse model (hereinafter referred to as NSG). NSG mice were injected intravenously (IV) with a single dose of 1000 or 3000 ng / g of the R-TM123 pharmaceutical from the confirmation experiment. Blood was collected by retro-orbital puncture at 15, 30, 60, 120, 180, 210, 255, 270, 300 and 360 minutes after injection. After plasma was isolated, the concentration of R-TM123 was measured using a specific sandwich ELISA assay with CD123-Fc as the capture agent and a horseradish peroxidase (HRP)-conjugated anti-poly His tag monoclonal antibody for detecting the bound R-TM123. Capture with CD123-Fc indicates the integrity of the anti-CD123 domain, while detection of the poly His tag indicates the presence of the second scFv specific for RCE, since this is located at the N-terminus of the poly His tag. Quantification of the test samples was performed by comparing them with a titration of the R-TM123 standard. The detection limit of the sandwich ELISA assay was determined to be 0.6 ng / mL of assay concentration.

[0176] To determine the plasma half-life, non-compartmental analysis (NCA) and two-compartment analysis (2CA) of plasma data after intravenous bolus injection were performed using PkSolver 2.0 (Zhang et al., 2010). The data of 2CA were further weighted via W = 1 / C obs 2 to focus the fitting model on the final elimination phase T 1 / 2 -β. The graphic output for the experiments performed is shown in Figure 12, and an overview of the important pharmacokinetic data is shown in Table 6.

[0177] Figure 12 shows the in vivo pharmacokinetics of R-TM123 in NSG mice. Graphic output of non-compartmental analysis (NCA) and two-compartment analysis (2CA) of R-TM123 concentration in peripheral blood plasma obtained with the R-TM123 pharmaceutical. Experimental mice (n = 5) were injected IV 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 the IV bolus injection. The R-TM123 concentration determined by ELISA was analyzed using PkSolver 2.0 (Zhang et al., 2010) NCA and 2CA. The mean ± SD values of samples obtained from 5 individual mice are shown per data point.

[0178] The area under the curve values (AUC) were determined to be 255603.5 (NCA) and 220241.7 ng / mL×min (2CA) for the dose of 1000 ng per gram of body weight and 644179.4 (NCA) and 555559.9 ng / mL×min (2CA) for the dose of 3000 ng per gram of body weight, respectively. The calculated elimination half-life (T 1 / 2 ) was 36.5 minutes (NCA) and 38.0 minutes (2CA) for the dose of 1000 ng per gram of body weight and 47.8 minutes (NCA) and 45.3 minutes (2CA) for the dose of 3000 ng per gram of body weight, respectively. The obtained T 1 / 2 is consistent with values reported in the literature for similar scFv constructs (Hutt et al., 2012), and as expected, the plasma concentration of R-TM123 was highest at 15 minutes after intravenous injection (T max ), i.e., the earliest time point measured. The maximum plasma concentration (C max) was detected 15 minutes after injection and determined to be doses of 4682.6 and 11522.7 ng / mL at 1000 ng per gram of body weight or 3000 ng per gram of body weight, respectively. The short half-life observed, as was done with bispecific T cell engagers with comparable short half-lives (Chichili et al., 2015; Hijazi et al., 2018), supports the delivery of R-TM123 by continuous infusion.

[0179]

Table 6

[0180] The results obtained at 255, 270, 300 and 360 minutes at 1000 ng per gram of body weight or at 360 minutes at 3000 ng per gram of body weight were below the lower limit of quantification (LLoQ) of 0.67 - 1.42 ng / mL and were thus excluded from further analysis. For the analysis, the NCA model and the 2CA model of plasma data after intravenous bolus injection were applied using PkSolver 2.0 (Zhang et al., 2010). The observed differences between the two doses are most likely due to technical variations in sample collection and / or ELISA performance, especially at lower R-TM123 concentrations. Due to the overall lower plasma levels of R-TM123, the LLoQ was reached significantly earlier at a dose of 1000 ng per gram of body weight (210 minutes) than at a dose of 3000 ng per gram of body weight (300 minutes). The plasma half-life is likely underestimated due to the smaller number of valid measurement points obtained in the final elimination phase. Therefore, the estimated plasma half-lives of the 3000 ng per gram of body weight dose, 47.8 minutes (NCA model) and 45.3 minutes (2CA model), calculated at 6 measurement points above the LLoQ in the final phase, reflect a more robust dataset.

[0181] In vivo efficacy of RevCAR-T in different CDX AML models The in vivo efficacy of RevCAR-T redirected by R-TM123 was confirmed in an extramedullary AML-CDX model using a fluorescence-based readout. In this model, MOLM-13 cells expressing mCherry were subcutaneously injected into the flanks of NSG mice on day 0, either alone or in combination with RevCAR-T cells. R-TM123 was administered once daily for 5 days in 4 cycles at the indicated dose per gram of body weight around the tumor (Figure 13). A sustained antitumor response against MOLM-13 AML cells was observed based on fluorescence in vivo imaging.

[0182] Figure 13 shows the elimination of leukemia by RevCAR-T redirected against extramedullary lesions of AML by R-TM123. NSG mice were subcutaneously injected with 1×10 6 MV4-11 cells expressing mCherry, either alone or in combination with 5×10 5 RevCAR-T cells from a clinical-scale experiment, and tumor growth was monitored by optical imaging. Subsequently, the mice were injected with R-TM123 (at the indicated dose per gram of body weight, daily, around the tumor) for 5 days in 4 cycles with a 2-day off period. The percentage of tumor signal was referenced to the first measurement (geometric mean, t0) of each group. Statistical significance was evaluated by two-way ANOVA using Dunnett's multiple comparison test, and as a result, the P value was less than 0.05 in all treatment groups compared to the group administered RevCAR-T cells and tumor cells without R-TM123.

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Claims

1. i) At least one CD123 binding domain comprising a CDR sequence according to SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, amino acid sequence WAS, and SEQ ID NO: 37, and ii) A tag-binding domain for binding human La epitope E5B9, comprising a CDR sequence according to SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, amino acid sequence WAS, and SEQ ID NO: 42, V Ｌ -Linker-V Ｈ Tag-binding domain including structure, A targeted module, including the following.

2. i) The at least one CD123 binding domain comprises a sequence having at least 95% identity, preferably at least 99% identity, with the sequence according to Sequence ID No. 22 and Sequence ID No. 23, ii) The tag-binding domain that binds the human La epitope E5B9 is V Ｌ -Linker-V Ｈ The structure includes the V of the tag binding domain. Ｌ The region includes a sequence having at least 95% identity, preferably at least 99% identity, with the sequence according to sequence number 19, and / or the V of the tag-binding domain. Ｈ The region includes a sequence that has at least 95% identity, preferably at least 99% identity, with the sequence according to sequence number 20. The targeting module according to claim 1.

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 targeted module according to claim 1 or 2, wherein the linker of the tag-binding domain includes a linker according to one of the sequences of sequence number 25 or sequence number 26.

5. The targeting module according to claim 1 or 2, wherein the CD123-binding domain is an antibody or antigen-binding fragment.

6. The targeting module according to claim 1 or 2, wherein the CD123 binding domain comprises a sequence according to sequence number 27.

7. A targeting module according to claim 1 or 2, wherein the length is in the range of 500 to 800 amino acids.

8. A targeting module according to claim 1 or 2, comprising one of the sequences of sequence numbers 3 to 10.

9. A nucleic acid, vector, or cell comprising a nucleotide sequence encoding the targeting module described in claim 1.

10. The nucleic acid, vector, or cell according to claim 9, comprising a nucleotide sequence encoding a targeting module according to one of the sequences of SEQ ID NOs: 3 to 10.

11. The nucleic acid, vector, or cell according to claim 9 or 10, comprising one of the sequences of sequence numbers 11 to 18.

12. A pharmaceutical composition comprising a targeting module according to claim 1 or 2 and a pharmaceutically acceptable diluent or carrier.

13. The pharmaceutical composition according to claim 12, wherein the targeting module includes one of the sequences of Sequence IDs 3 to 10.

14. A targeting module according to claim 1 or 2 for use in a method for stimulating a chimeric antigen receptor-mediated immune response in mammals, preferably for use in the treatment of cancer, infectious diseases or autoimmune diseases; a nucleic acid, vector or cell comprising a nucleotide sequence encoding the targeting module according to claim 1 or 2; or a pharmaceutical composition comprising the targeting module according to claim 1 or 2 and a pharmaceutically acceptable diluent or carrier.

15. A pharmaceutical composition comprising the targeting module according to claim 8, for use in a method for stimulating a chimeric antigen receptor-mediated immune response in mammals, preferably for use in the treatment of cancer, infectious disease or autoimmune disease, a nucleic acid, vector or cell comprising a nucleotide sequence encoding the targeting module according to claim 8, or the targeting module according to claim 8 and a pharmaceutically acceptable diluent or carrier.

16. A targeted module for use in a method for stimulating a chimeric antigen receptor-mediated immune response in a mammal, according to claim 14, The targeting module is administered in combination with a vector or cells containing a nucleotide sequence encoding a reversible chimeric antigen receptor. The aforementioned reversible chimeric antigen receptor - The tag is human La epitope E5B9. - Extracellular hinge and transmembrane domain, and - Signal transduction domain, Includes, The tag-binding domain of the targeting module binds to the tag of the reversible chimeric antigen receptor. The aforementioned targeting module.

17. A pharmaceutical composition for use in a method for stimulating a chimeric antigen receptor-mediated immune response in a mammal according to claim 14, further comprising a vector or cells comprising a nucleotide sequence encoding a reversible chimeric antigen receptor, wherein the reversible chimeric antigen receptor is - The tag is human La epitope E5B9. - Extracellular hinge and transmembrane domain, and - Signal transduction domain, Includes, The tag-binding domain of the targeting module binds to the tag of the reversible chimeric antigen receptor. Pharmaceutical composition.

18. a) The targeting module according to claim 1 or 2 or the nucleic acid, vector or cell according to claim 9 or 10, and b) A vector or cell containing a nucleotide sequence encoding a reversible chimeric antigen receptor, A kit that includes, The aforementioned reversible chimeric antigen receptor - The tag is human La epitope E5B9. - Extracellular hinge and transmembrane domain, and - Signal transduction domain, Includes, The tag-binding domain of the targeting module binds to the tag of the reversible chimeric antigen receptor. kit.

19. The kit according to claim 18, wherein the targeting module includes one of the sequences of sequence numbers 3 to 10.

20. The kit according to claim 18, wherein the extracellular hinge and transmembrane domain are selected from the group comprising human CD28 molecules, CD8a chain NK cell receptor hinges and transmembrane domains, portions of the constant region of an antibody, and combinations thereof.

21. The kit according to claim 8, wherein the signal transduction domain is selected from the group comprising the cytoplasmic regions of CD28, CD137 (4-1BB), CD134 (OX40), CD278 (ICOS), DAP10 and CD27, programmed cell death-1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), the cytoplasmic region of the CD3 chain, DAP12, CD122 (interleukin-2 receptor β), CD132 (interleukin-2 receptor γ), CD127 (interleukin-7 receptor α), CD360 (interleukin-21 receptor), activated Fc receptor, and mutants thereof.

22. Further comprising at least one yet another targeting module, or at least one yet another nucleic acid, vector, or cell encoding yet another targeting module, The at least one yet another targeting module comprises at least one target cell binding domain and a tag binding domain, The aforementioned at least one yet another target cell binding domain is 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, members of the epidermal growth factor receptor family and their mutants, members of the tumor necrosis factor receptor superfamily, claudin, ephrin, ephrin receptor, fucosyltransferase, prostate-specific antigen, fetal antigen, members of the vascular endothelial growth factor family, EpCAM, AFP, members of the intercellular adhesion molecule family, type C lectins, integrins, mucin proteins Antibodies, antibody fragments, proteins, peptides, or low molecular weight organic ligands that bind to surface antigens selected from the group including family members, ligands for FSHR, HMW-MAA, FBP, folate receptor, somatostatin receptor, NKG2D receptor, members of the epithelial glycoprotein family, disiaroganglioside, glypican, G protein-coupled receptors, human papillomavirus protein, cancer testicular antigen, fibroblast-activating protein, members of the carbonic anhydrase family, members of the glycosylation antigen family, Notch ligand, MCSP, glycoprotein A33, guanylate cyclase 2C, and tumor-specific glycans. The targeting module and the at least one further targeting module include the same tag binding domain, The kit according to claim 8.

23. The kit according to claim 8, wherein the vector or cells comprising the targeting module and / or the nucleotide sequence encoding the reversible chimeric antigen receptor are in the form of a pharmaceutical composition.

24. A kit according to claim 8, for use in a method for stimulating a chimeric antigen receptor-mediated immune response in mammals, preferably for use in the treatment of cancer, infectious diseases or autoimmune diseases.

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