Targeting modules for common chimeric antigen receptor-expressing immune cells and uses in the treatment of cancer, infection and autoimmune disorders

Abstract

The present invention relates to targeting modules comprising chemically synthesized peptide-binding moieties specific for human cell surface proteins or protein complexes, kits comprising the targeting modules, and vectors or cells comprising nucleic acids encoding common chimeric antigen receptors, and uses for the treatment of cancer, infectious diseases and autoimmune disorders.

Description

[Technical Field]

[0001] The present invention relates to targeting modules comprising chemically synthesized peptide-binding moieties specific for human cell surface proteins or protein complexes, kits comprising the targeting modules, and vectors or cells comprising nucleic acids encoding common chimeric antigen receptors, and uses for the treatment of cancer, infectious diseases and autoimmune disorders.

[0002] Chimeric antigen receptors (CARs) are artificial receptors consisting of a binding moiety that provides antigen specificity and one or several signaling chains derived from immune receptors (Cartellieri et al. 2010). These two main CAR domains are connected by a connecting peptide chain containing a transmembrane domain that anchors the CAR within the cell's plasma membrane. Immune cells, particularly T lymphocytes and NK lymphocytes, can be genetically engineered to express CARs inserted into their plasma membrane. When such CAR-modified immune cells encounter other cells or tissue structures that express or are modified with the appropriate target of the CAR binding moiety, they cross-link to the target upon binding of the CAR binding moiety to the target antigen. Cross-linking results in the induction of signaling pathways via the CAR signaling chain, which alters the biological properties of the CAR-engrafted immune cells. Adoptive transfer of CAR-engineered immune cells is currently considered a highly promising therapeutic option for the treatment of otherwise incurable malignant, infectious, or autoimmune diseases. Initial clinical trials have demonstrated both the safety and feasibility of this treatment strategy (Lamers et al. 2006, Kershaw et al. 2006). However, conventional CAR technology poses several critical safety issues. The immune response of T cells engineered with conventional CARs is difficult to control after infusion into patients, and unexpected target gene expression in healthy tissues, in particular, can trigger immune responses of the engineered T cells against healthy cells, potentially resulting in serious side effects (Lamers et al. 2006, Morgan et al. 2010). Another drawback of conventional CAR technology is the limitation of engineered T cells to retarget a single antigen. This single-treatment approach highlights the risk of the development of tumor escape variants that lose their target antigen during treatment. The emergence of tumor escape variants after several months of conventional CAR T cell therapy has already been observed during clinical trials (Grupp et al. 2013).

[0003] International Publication No. 2012082841 discloses common anti-tag chimeric antigen receptor-expressing T cells and methods for treating cell-related disorders, such as cancer. Furthermore, International Publication No. 2013044225 discloses a common immune receptor expressed by T cells for targeting a wide variety of antigens. Both methods describe the use of engineered T cells expressing a common anti-tag immune receptor. These T cells can be redirected to disease-related cell surface antigens by further applying modules that bind the surface antigens of these T cells and carry individual tags. A drawback is the redirection of genetically engineered T cells using foreign tags, which are likely immunogenic and therefore put patients at risk and negatively affect the efficacy of treatment.

[0004] Alternatively, EP2990416 discloses a therapy based on genetically modified immune cells, particularly T cells and NK cells, that enables redirection of various disorders in a safe and efficient manner using endogenous tags based on nucleoproteins. In particular, EP2990416 discloses a nucleic acid encoding a universal chimeric antigen receptor (UniCAR) and a targeting module composed of a binding moiety specific for a specific human cell surface protein or protein complex and a tag derived from a human nucleoprotein, as well as the use of the nucleic acid and the targeting module to stimulate an immune response in mammals. In contrast to conventional CARs, the scFv in the universal CAR does not recognize cell surface antigens but rather recognizes a short, non-immunogenic peptide motif derived from a human nucleoprotein. Therefore, T cells engineered to express UniCAR remain inactive after reinfusion because the UniCAR target is not available on the surface of intact cells under physiological conditions (Figure 1). Targeting modules are based on recombinant proteins including antibody fragments (i.e., scFv or Fab), ligands, or soluble receptors.

[0005] The drawback of the disclosed method is the highly complex production and purification procedures that must be established and implemented to obtain clinical-grade targeting modules. These procedures include recombinant expression in prokaryotic or mammalian cell cultures, complex multi-step chromatography procedures for purification, and a sophisticated analytical panel for quality control. Moreover, the stability of targeting modules is often limited, and long-term storage conditions are not favorable.

[0006] In a first aspect, the present invention provides specific targeting modules that are easily, rapidly and economically manufactured at clinical grade levels.

[0007] Furthermore, the present invention provides specific targeting modules with improved pharmaceutical properties and improved stability.

[0008] An embodiment of the present invention relates to a targeting module comprising a chemically synthesized peptide binding moiety specific for a human cell surface protein or protein complex and a tag that is a peptide derived from any protein, wherein the targeting module is a peptide comprising 10 to 120 amino acids.

[0009] In an embodiment, the linear peptide epitope is derived from a human protein to minimize the risk of immunogenicity.

[0010] In a further embodiment, the linear peptide epitope is derived from a human nuclear protein to minimize the risk of immunogenicity and to prevent off-target activity of UniCAR-expressing immune cells. The peptide of the nuclear human protein will not be presented on the cell surface, and therefore, UniCAR-grafted immune cells cannot be activated in the absence of the targeting module (Figure 1).

[0011] Advantageously, the targeting modules according to the invention are easily synthetic. Even more advantageously, the targeting modules according to the invention are capable of binding to the universal chimeric antigen receptor (UniCAR) and to proteins or protein complexes on the surface of human cells.

[0012] A targeting module according to the present invention is a molecule that enables the genetically modified immune cell to reach its target, in particular a cell surface protein or an extracellular structure.

[0013] As used herein, the term "chemically synthesized" refers to a method of producing a peptide by chemical synthesis, particularly by coupling the carboxyl group of a first amino acid with the amino group of a second amino acid. In embodiments, the chemical synthesis is selected from liquid phase synthesis or solid phase synthesis.

[0014] As used herein, the term "specific" refers to the ability of a peptide or protein to bind exclusively to a target or to a group of targets.

[0015] As used herein, the term "cell surface protein or protein complex" refers to a cell surface protein or an extracellular structure. As used herein, the term "protein complex" refers to a group of two or more associated protein chains.

[0016] Brady et al. disclosed peptides specific for human cell surface proteins or protein complexes and corresponding receptor types or subtypes overexpressed in human tumor cells (Brady et al. 2014).

[0017] As used herein, the term "tag" refers to a peptide sequence attached to a peptide or protein so that the peptide or protein can be bound to a specific atom, ion, or molecule.

[0018] A human protein according to the present invention is a protein found in the human body.

[0019] A nuclear protein according to the present invention is a protein found within the cell nucleus.

[0020] In a further embodiment, the targeting module is chemically synthesized.

[0021] According to the present invention, the chemically synthesized peptide binding moiety is selected from somatostatin and somatostatin analogs, somatostatin antagonists, bombesin and bombesin analogs, gastrin-releasing peptide (GRP) and GRP analogs, neuromedin B and neuromedin B analogs, the vasoactive secretin family, melanocyte-stimulating hormone (MSH) and MSH analogs, cholecystokinin (CCK), gastrin, neurotensin and neurotensin analogs, the gonadotropin-releasing hormone family, neurokines, exendin or exenatide, Arg-Gly-Asp (RGD) peptides, Asn-Gly-Arg (NGR) peptides, neuregulins, or the chemically synthesized peptide binding moiety has binding specificity for a membrane receptor.

[0022] As used herein, the term "analog" refers to a compound exhibiting an amino acid sequence that is at least 75% identical to the peptide, preferably at least 80% identical to the peptide, and particularly preferably at least 85% identical to the peptide.

[0023] As used herein, the term "antagonist" refers to a compound that binds a peptide receptor without triggering a signal transduction cascade.

[0024] Merlo et al., Raderer et al., and Kaltsas et al. describe somatostatin analogs, particularly octreotide, octreotate, and lanreotide, and their somatostatin receptor binding (Merlo et al. 1999, Raderer et al. 2000, Kaltsas et al. 2005). In an embodiment, the somatostatin analog is octreotide, octreotate, or lanreotide.

[0025] Wild et al. 2011 reported that somatostatin antagonists, especially pNO2-Phe-c( D Cys-Tyr- D Trp-Lys-Thr-Cys) D TyrNH2 (BASS) and its somatostatin receptor binding (Wild et al. 2011).

[0026] Gonzalez et al. described bombesin, gastrin-releasing peptide (GRP), neuromedin B, and their analogs (Gonzalez et al. 2008). Furthermore, Ohki-Hamazaki et al. described bombesin, gastrin-releasing peptide (GRP), neuromedin B, and their analogs, particularly the bombesin analog allitesin (Ohki-Hamazaki et al. 2005).

[0027] Hessenius et al. and Raderer et al. 2000 described their binding to vasoactive intestinal peptide (VIP) and VIP receptors (Hessenius et al. 2000, Raderer et al. 2000).

[0028] Chen et al. and Yang et al. describe α-melanocyte-stimulating hormone (α-MSH), α-MSH analogs, and their receptor binding (Chen et al. 2002, Yang et al. 2009). In an embodiment, the MSH analog is selected from cyclic α-MSH, melanotan I, or melanotan II.

[0029] Froberg et al. and Kolenc-Peitl et al. described DOTA-binding cholecystokinin (CCK)2 and gastrin and their receptor binding (Froberg et al. 2009, Kolenc-Peitl et al. 2011).

[0030] de Visser et al. and Buchegger et al. have described neurotensin analogs, particularly DOTA- and DTPA-conjugated neurotensin analogs, as well as carboxy-terminal hexapeptide analogs of neurotensin (de Visser et al. 2003, Buchegger et al. 2003).

[0031] Reubi et al. describe the effects of neuropeptide Y (NPY) and neuropeptide Y analogs and their receptor binding in cancer (Reubi et al. 2001).

[0032] Nagy and Schally and Popovics et al. describe luteinizing hormone-releasing hormone (LHRH) and cytotoxic LHRH complexes and their receptors as specific targets for cancer therapy (Nagy and Schally 2005, Popovics et al. 2014).

[0033] Beaujouan et al. describe the tachykinins or neurokinins, respectively, in particular substance P, neurokinins A and B (NKA and NKB) and neuropeptides Y and K (NPY and NPK) and their receptors (Beaujouan et al. 2004).

[0034] Wild et al. and Brom et al. described exendin 3, exendin 4, their complexes, and their binding to the glucagon-like peptide 1 (GLP-1) receptor (Wild et al. 2006, Wild et al. 2010, Brom et al. 2010).

[0035] Haubner et al., Decristoforo et al., and Dumont et al. have described Arg-Gly-Asp (RGD) peptide conjugates for binding of avβ3 integrin (Haubner et al. 2005, Decristoforo et al. 2008, Dumont et al. 2011).

[0036] Arap et al. described Arg-Gly-Asp (RGD) and Asn-Gly-Arg (NGR) peptides, complexes with these peptides, and the binding of these complexes to cancer cells (Arap et al. 1998).

[0037] In embodiments, the peptides having binding specificity for a membrane receptor have binding specificity for a membrane receptor selected from cluster of differentiation (CD) molecules, cytokine and chemokine receptors, tyrosine kinase receptor family members, members of the epidermal growth factor receptor family, members of the ephrin receptor family, so-called prostate-specific antigens, embryonic antigens and carcinoembryonic antigens, members of the vascular endothelial growth factor receptor family, members of the mucin protein family, folate-binding proteins and receptors, ligands for the NKG2D receptor, members of the epithelial glycoprotein (EGP) family, disialogangliosides, members of the carbonic anhydrase family, and members of the carbohydrate antigen family, lectins, lectin-like molecules, members of the tumor necrosis factor receptor family, members of the keratin family, and variants of membrane receptors.

[0038] As used herein, the term "variant" refers to a membrane receptor that has at least 75%, preferably at least 90%, identity in the extracellular region.

[0039] In a preferred embodiment, the peptide having binding specificity for a membrane receptor is a membrane receptor selected from CD2, CD3, CD4, CD8, CD10, CD13, CD19, CD20, CD22, CD23, CD30, CD25, CD33, CD38, CD44, CD52, CD90, CD99, CD123, CD181, CD182, CD184, CD223, CD269, CD274, CD276, CD279 and CD366, interleukin receptors being particularly preferred. IL-8Rα (CXCR1), IL-8Rβ (CXCR2), IL-11Rα, IL-11Rβ, IL-13Rα1 and 2, CXCR4; c-Met, transforming growth factor β receptor, ErbB1, ErbB2, ErbB3, ErbB4 and variants thereof, ephrin receptors, particularly preferably EphA1-10 or EphB1-6; prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), embryonic antigens (e.g., carcinoembryonic antigen (CEA)), fetal acetylcholine receptor), carcinoembryonic antigen (onco-fetal antigens), tumor-specific glycans (e.g., serine- or threonine-linked N-acetylgalactosamine (Tn) or derivatives such as sialyl Tn); VEGFR1, VEGFR2, or VEGFR3, neuropilin 1, epidermal cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), alpha-fetoprotein (AFP), mucins, particularly preferred MUC1, MUC16, or MUC18; follicle-stimulating hormone receptor (FSHR), human high molecular weight melanoma-associated antigen (HMW-MAA), folate-binding protein Proteins (FBP), folate receptors, NKG2D, major histocompatibility complex (MHC) class I molecules, particularly preferred are MHC class I chain-related gene A (MICA) or MHC class I chain-related gene B (MICB), UL16 binding protein (ULPB) 1, ULPB2, ULPB3, ribonucleic acid transporter 1 (Rae-1) family members or histocompatibility 60 (H-60); chaperones and heat shock proteins, particularly preferred are heat shock protein (HSP) 90 or 78 kDa glucose-regulated protein (GRP78);They have binding specificity for EGP-2 or EGP-4, diasialoganglioside 2 (GD2) or GD3, carbonic anhydrase 9 (CAIX), Lewis Y (LeY), C-type lectin-like molecule 1 (CLL-1), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor, apoptosis antigen 1 (APO-1, Fas, CD95), Notch ligands (e.g., Delta-like 1 and 4), members of the keratin family or integrins, with avβ3 or avβ5 being particularly preferred, aminopeptidase A, aminopeptidase N, or neural / glial antigen 2 (NG2);

[0040] Clark-Lewis et al. describe interleukin 8 (IL-8) and IL-8 analogs and their ability to bind to specific membrane receptors on neutrophils (Clark-Lewis et al. 1991).

[0041] Cardo-Vila et al. describe interleukin 11 (IL-11) and IL-11 analogues and their ability to bind to IL-11 receptors, for example, on the surface of tumor cells (Cardo-Vila et al. 2008).

[0042] Sai et al. described a peptide that specifically binds to interleukin-13 receptor subunit alpha 2 (Sai ​​et al. 2017).

[0043] Hanaoka et al. described the design of a 14-residue peptide as an inhibitor of the chemokine receptor CXCR4, the synthesis of a DTPA conjugate of the peptide, and its receptor binding (Hanaoka et al. 2006). Furthermore, Jacobson et al. described the development of a highly selective CXCR4 antagonist based on a short peptide (Jacobson et al. 2010). Laverman et al. described receptor-binding peptides, particularly targeting receptors overexpressed on tumor cells. Laverman et al. disclosed CKK peptides, GLP-1 peptides, CXCR4-binding peptides, and gastrin-releasing peptide receptor-targeting peptides (BN, GRP, and BN analogs) (Laverman et al. 2012).

[0044] Broda et al. describe the c-Met binding peptide cMBP2 and how to select appropriate binding peptides to the receptor, especially on tumor cells (Broda et al. 2015).

[0045] Wang et al. described a complex of erbB2-binding peptide (LTVSPWY) (Wang et al. 2007a).

[0046] Kolonin et al. and Staquicini et al. described peptide ligands to the ephrin receptor EphA5 for targeting human cancer cells (Kolonin et al. 2006, Staquicini et al. 2015).

[0047] Barrett et al., Vallabhajosula et al., and Afshar-Oromieh et al. have described peptides that bind prostate-specific membrane antigen (PSMA) (Barrett et al. 2013, Vallabhajosula et al. 2014, Afshar-Oromieh et al. 2015). Furthermore, WO 2010 / 108125 and WO 2015 / 055318 disclose PSMA-binding peptides.

[0048] De Rosa et al. describe the design and synthesis of a peptide that exhibits VEGF-like receptor binding to its receptor VEGFR2 (De Rosa et al. 2016). Michaloski et al. describe a peptide that binds to all VEGFRs (Michaloski et al. 2016).

[0049] Karjalainen et al. described a peptide that binds to neuropilin 1 (Karjalainen et al. 2011).

[0050] Iwasaki et al. 2015 described a 14-mer macrocyclic peptide that binds to the extracellular domain of epithelial cell adhesion molecule (EpCAM) (Iwasaki et al. 2015).

[0051] Cardo-Vila et al. 2010 described a peptide that binds to EGFR (Cardo-Vila et al. 2010).

[0052] Staquicini et al. 2008 describe MUC18-derived 9-mer and 10-mer peptides that bind to MUC18, mimicking the homophilic interactions of MUC18 (Staquicini et al. 2008).

[0053] Arap et al. described a stress response chaperone GRP78-binding peptide motif and a synthetic chimeric peptide containing a GRP78-binding motif for targeting tumor cells (Arap et al. 2004), and Vidal et al. described a peptide that binds to HSP90 (Vidal et al. 2004).

[0054] Kajiwara et al. describe methods for the design of synthetic peptides that bind to receptors, in particular the design and synthesis of synthetic peptide-binding members of the tumor necrosis factor receptor family TRAIL (tumor necrosis factor-related apoptosis-inducing ligand receptor), TNFR1 or Fas (apoptosis antigen 1, APO-1, CD95) (Kajiwara et al. 2004).

[0055] Soudy et al. described two peptides, a 12-mer and a 10-mer, and a complex of this peptide that binds keratin 1, a member of the keratin family (Soudy et al. 2017).

[0056] Cardo-Vila et al. described an αvβ5 binding peptide (Cardo-Vila et al. 2003).

[0057] Marchio et al. described peptides that target aminopeptidase A (Marchio et al., 2004). Pasqualini et al. described targeting of drugs to aminopeptidase N (CD13) via an NGR peptide tag (Pasqualini et al., 2000).

[0058] Burg et al. described peptides that target NG2 in the context of angiogenesis (Burg et al. 1999).

[0059] Preferably, the peptides having binding specificity for membrane receptors are selected from the group disclosed by Clark-Lewis et al., Cardo-Vila et al. (2003, 2008, 2010), Sai et al., Hanaoka et al., Jacobson et al., Laverman et al., Broda et al., Wang et al., Kolonin et al., Staquicini et al. (2008, 2015), Barrett et al., Vallabhajosula et al. and Afshar-Oromieh et al., De Rosa et al., Michaloski et al., Karjalainen et al., Iwasaki et al., Arap et al., Kajiwara et al., Soudy et al., Marchio et al., Pasqualini et al. or Burg et al.

[0060] In a preferred embodiment, the chemically synthesized peptide binding moiety is selected from somatostatin, bombesin, gastrin-releasing peptide (GRP), vasoactive intestinal peptide (VIP), α-melanocyte-stimulating hormone (α-MSH), melanotan 2 (α-M2), cholecystokinin (CCK) or gastrin, neurotensin, neuropeptide Y, luteinizing hormone-releasing hormone (LHRH), substance P, exendin, Arg-Gly-Asp (RGD) peptide, or Asn-Gly-Arg (NGR) peptide.

[0061] In an embodiment, the targeting module is a peptide comprising 13 to 85 amino acids, particularly preferably 20 to 60 amino acids.

[0062] In an embodiment, the chemically synthesized peptide binding moiety is a peptide having 3 to 75 amino acids, preferably 10 to 50 amino acids.

[0063] In further embodiments, the chemically synthesized peptide binding moiety comprises one peptide (monospecific), two, three or more peptides (bispecific and multispecific).

[0064] In further embodiments, the chemically synthesized peptide-binding moiety comprises at least two peptides selected from somatostatin and somatostatin analogs, somatostatin antagonists, bombesin and bombesin analogs, gastrin-releasing peptide (GRP) and GRP analogs, neuromedin B and neuromedin B analogs, the vasoactive secretin family, melanocyte-stimulating hormone (MSH) and MSH analogs, cholecystokinin (CCK), gastrin, neurotensin and neurotensin analogs, the gonadotropin-releasing hormone family, neurokines, exendin or exenatide, Arg-Gly-Asp (RGD) and Asn-Gly-Arg (NGR) peptides, neuregulins, or peptides with binding specificity for membrane receptors.

[0065] Examples of chemically synthesized peptide-binding moieties having at least two peptides include, but are not limited to, a combination of two different peptides that bind to the same cell surface protein (e.g., IL-11R, IL-13RA2, ErbB2, PSCA, PSMA, VEGFR, or GD2), a combination of a PSMA-binding peptide and a peptide that binds to VEGFR-2, PSCA, IL-11R, or MUC1, a combination of peptides that bind to GD2 and CD90, or a combination of melanotan I and II, analogs thereof, or cyclic peptides.

[0066] In further embodiments, the chemically synthesized peptide binding moiety and / or tag comprises D-amino acids, pseudopeptide bonds, amino alcohols, non-proteinogenic amino acids, amino acids with modified side chains, and / or the chemically synthesized peptide binding moiety and / or tag is a cyclic peptide.

[0067] According to the present invention, a tag is a peptide derived from any protein for which an antibody or other binding domain is available.

[0068] In a further embodiment, the tag is a peptide derived from any human protein for which an antibody or other binding domain is available.

[0069] In a further embodiment, the tag is a peptide derived from a human nucleoprotein for which an antibody or other binding domain is available.

[0070] The present invention further comprises the use of a targeting module according to the invention for preparing a medicament for therapeutic and / or diagnostic use in the case of cancer or autoimmune diseases.

[0071] The present invention also encompasses methods for the treatment of humans suffering from cancer, infectious diseases, inflammatory diseases or autoimmune diseases by administration of a targeting module according to the present invention.

[0072] For therapeutic applications, a sterile pharmaceutical composition containing a pharmacologically effective amount of a targeting module according to the present invention is administered to a patient to treat the above-mentioned diseases.

[0073] The invention will be explained in more detail with the help of the following figures and embodiments, without limiting the invention to said figures and embodiments.

[0074] The invention will now be further illustrated by the following non-limiting figures and examples. [Brief explanation of the drawings]

[0075] [Figure 1] Figure 1 shows a scheme of the modular composition of the UniCAR platform and its mode of action by binding a target-specific peptide targeting module (pTM) to a target structure on the surface of a target cell, e.g., a tumor cell as shown in the scheme. [Figure 2] Figure 2 shows a scheme of a universal chimeric antigen receptor (UniCAR) with three domains, in which the first domain is a tag-binding domain (e.g., scFv anti-tag), the second domain is an extracellular hinge (ECD) and transmembrane domain (TMD), the third domain is a signaling domain (ICD), and the optional fourth domain is a short peptide linker in the extracellular portion of the receptor (not shown). [Figure 3] Figure 3 shows the vector pLVX-EF1alphaUniCAR28 / ζ (Clontech, Takara Bio Group), which contains a 5' long terminal repeat (5'LTR), primer binding site (PBS), packaging signal (Ψ), reverse response element (RRE), central polypurine tract / central terminal sequence (cPPT / CTS), human elongation factor 1 alpha promoter (PEF1α), multiple cloning site (MCS), internal ribosome entry site (IRES), optimized human codons (ZsGreen1), woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), 3' long terminal repeat (3'LTR), origin of replication (pUC), ampicillin resistance gene (Amp), and β-lactamase. [Figure 4] Figure 4 shows the plasmid psPAX2 encoding the group-specific antigen (gag) and polymerase (pol) with the CMV enhancer and promoter (CMVenh), splicing donor (SD), splicing acceptor (SA), group-specific antigen (Gag), precursor protein (Pro) encoding the protease protein, protein (PoI) encoding reverse transcriptase and integrase, reverse response element (RRE), and ampicillin (Amp). [Figure 5] FIG. 5 shows the plasmid pMD2.G, which encodes for the envelope with the CMV enhancer and promoter (CMV), beta-globin intron, and beta-globin polyadenosine tail (beta-globin pA). [Figure 6]Figure 6 shows the peptide targeting module dose-dependent cytotoxic activity of human naive T cells engineered to express UniCAR toward prostate-specific membrane antigen (PSMA)-expressing target cells (OCI-AML3 engineered to express PSMA) in the presence of a PSMA-specific peptide targeting module (TM-pPSMA). Lysis is shown compared to control samples using target cells alone after 24 and 48 hours of incubation. For alloreactivity control, UniCAR-T was incubated with target cells in the absence of TM-pPSMA (cells only). [Figure 7] Figure 7 shows peptide targeting module dose-dependent secretion of T cell-specific cytokines (granulocyte-macrophage colony-stimulating factor GM-CSF, interferon gamma IFN-γ, interleukin 2 IL-2, and tumor necrosis factor alpha TNF□) by human naive T cells engineered to express UniCAR upon establishment of an immune synapse on target cells by the peptide targeting module. [Figure 8] Figure 8 shows peptide targeting module dose-dependent activation of human naive T cells engineered to express UniCAR as determined by activation marker CD25 surface expression upon establishment of an immune synapse to the target cell by the peptide targeting module. [Figure 9] FIG. 9 illustrates a targeting module according to the invention binding to PSMA that combines a glutamic acid-urea-lysine motif followed by Z, which is a single chelator (i.e., an N,N-bis(2-hydroxybenzyl)ethylenediamine-N,N-diacetic acid (HBED) chelator) or an aromatic amino acid or complex thereof, and O, which is a linker (i.e., a PEG linker) of two or more repeats connected to the La5B9 epitope. [Figure 10]FIG. 10 illustrates a targeting module according to the invention that binds to PSMA, which comprises a glutamic acid-urea-lysine motif followed by Z, which is a single chelator (i.e., an N,N-bis(2-hydroxybenzyl)ethylenediamine-N,N-diacetic acid (HBED) chelator) or Z, which is an aromatic amino acid or complex thereof, and O, which is a linker (i.e., a PEG linker) of two or more repeats connected to the La7B6 epitope.

[0076] In a preferred embodiment, the tag is a peptide derived from the human nuclear La protein. In an embodiment, the peptide derived from the human nuclear La protein is selected from the short linear epitopes recognized by the monoclonal anti-La antibodies 5B9 or 7B6. Preferably, the tag is a short linear epitope derived from the human nuclear La protein (E5B9) according to SEQ ID NO: 25 or from E7B6 according to SEQ ID NO: 27.

[0077] In a further embodiment, the targeting module according to the present invention further comprises at least one additional ligand. The additional ligand is not involved in target antigen binding. In an embodiment, the at least one additional ligand is selected from a costimulatory ligand or cytokine fused to the N-terminus or C-terminus of the targeting module, preferably CD28, CD137 (41BB), CD134 (OX40), CD27, or the extracellular domains of IL-2, IL-7, IL-12, IL-15, IL-17, and IL-21. In a further embodiment, the at least one additional ligand is selected from compounds that induce cell death in the target cell and neighboring cells.

[0078] In a further embodiment, the targeting module according to the present invention further comprises a chelator. As used herein, the term "chelator" refers to a compound that forms two or more separate coordinate bonds with a metal ion. In an embodiment, the chelator is selected from diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), mercaptoacetyltriglycine (MAG3), 6-hydrazinopridine-3-carboxylic acid (Hynic), hydroxybenzylethylenediamine (HBED), N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid (HBED-CC), or 2-(3-(1-carboxy-5-[(6-fluoro-pyridine-3-carbonyl)-amino]-pentyl)-ureido)-pentanedioic acid (DCFPyL).

[0079] In a preferred embodiment, a targeting module according to the present invention comprises a chelator at the C-terminus.

[0080] In an embodiment, a targeting module according to the present invention comprising a chelator further comprises a metal or metal ion, preferably a radionuclide. The term "radionuclide" refers to an atom that has excess nuclear energy that makes it unstable. In an embodiment, the radionuclide is 51 Cr, 89 Sr, 90 Y, 99 mTc, 111 In, 133 Xe, 153 Sm, 169 Er, 186 Re, 201 Tl or 224 Ra.

[0081] In an embodiment, a targeting module according to the present invention comprising a chelator is used for the preparation of a radiolabeled compound.

[0082] In an embodiment, a targeting module according to the invention binds to PSMA and has a structure according to formula (I), which comprises PSMA conjugated to a glutamic acid-urea-lysine motif followed by a short linker and an N,N-bis(2-hydroxybenzyl)ethylenediamine-N,N-diacetic acid (HBED) chelator. A PEG linker is attached to the chelator followed by the La5B9 epitope.

[0083] [ka]

[0084] In an embodiment, a targeting module according to the invention binds to PSMA and has a structure according to formula (II), which comprises PSMA conjugated to a glutamic acid-urea-lysine motif followed by a short linker and an N,N-bis(2-hydroxybenzyl)ethylenediamine-N,N-diacetic acid (HBED) chelator.

[0085] [ka]

[0086] In an embodiment, a targeting module according to the invention binds to IL13RA2 and has a structure according to SEQ ID NO: 26, comprising an IL13RA2 binding motif followed by a linker and the La5B9 epitope.

[0087] Generate targeting module The peptide targeting module (pTM) contains two domains: a binding moiety specific for a specific human cell surface protein or protein complex, and a tag recognized by the binding moiety of UniCAR. pTM can be produced by techniques known to those skilled in the art. These techniques include, but are not limited to, artificial synthesis of polypeptide chains or solid-phase and liquid-phase chemical synthesis.

[0088] In one embodiment, pTM may be synthesized in a single, two, or multiple solution phase chemical synthesis procedure. As a first step, 1-(9-fluorenylmethyloxycarbonyl-amino)-4,7,10-trioxa-13-tridecanamine hydrochloride (Fmoc-TOTA * The Fmoc-TOTA (HCl) linker molecule was dissolved in dichloromethane (DCM) containing N,N-diisopropylethylamine (DIPEA) and loaded onto 2-chlorotrityl polystyrene resin (2-chlorotrityl PS). * The HCl is covalently attached to the resin for further processing. A capping solution consisting of methanol and DIPEA in DCM can be used to block the two unreacted chlorotrityl chloride groups on the 2-chlorotrityl chloride resin. The covalently attached structure 4,7,10-trioxa-13-tridecanamine (TOTA), which later serves as a linker in the final molecule, is then deprotected from its fluorenylmethyloxycarbonyl (Fmoc) protecting group using 20% ​​piperidine in dimethylformamide (DMF). Amino acids can then be sequentially added to the immobilized TOTA using a fluorenylmethyloxycarbonyl / tert-butyl (Fmoc / tBu) strategy. Each amino acid is protected with its corresponding protecting group. Coupling was carried out using N,N'-diisopropylcarbodiimide (DIC) and ethyl 2-cyano-2-hydroxyimino)acetate (Oxyma Pure) in DMC / N-methyl-2-pyrrolidone (NMP) or with Boc-O-tert-butyl-L-serine (dicyclohexylammonium) salt (Boc-Ser(tBu)-OH *This can be done using DCHA and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) as coupling reagents. In this way, amino acid chains of varying lengths from 2 to 100 can be generated. The constructed peptides with the TOTA linker are cleaved from the resin using hexafluoroisopropanol (HFIP) in dimethyl carbonate (DCM). The side-chain protecting groups and the N-terminal tert-butyloxycarbonyl (Boc) are unaffected. DCM is used as the solvent.

[0089] HPLC processing is used to replace the trifluoroacetic acid (TFA) counterion with an acetate counterion, followed by final lyophilization.

[0090] The final product can be purified using high performance liquid chromatography (HPLC). In particular, reverse-phase HPLC can be useful. A common purification buffer is TFA. Alternatively or additionally, ion exchange chromatography can be applied to purify the final peptide product.

[0091] In a further embodiment, a targeting module according to the present invention is used in the treatment of cancer, infectious diseases, inflammatory disorders and autoimmune disorders.

[0092] In a further aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) at least one targeting module according to the present invention; b) a vector or cell containing a nucleic acid encoding a common chimeric antigen receptor; wherein the common chimeric antigen receptor comprises three domains, wherein: the first domain is a tag-binding domain; the second domain is an extracellular hinge and transmembrane domain, and The third domain is a signaling domain, In this, the tag binding domain binds to the tag of the targeting module according to the present invention.

[0093] As used herein, the term "universal chimeric antigen receptor" refers to an artificial chimeric fusion protein, particularly a receptor comprising a tag-binding domain, an extracellular hinge and transmembrane domain, and a signaling domain (Figure 2). The domains can be derived from different sources, and therefore the receptor is called chimeric. Advantageously, the receptor can use the tag-binding domain to bind to different targeting modules, and therefore is universal.

[0094] Advantageously, cells containing a nucleic acid encoding a universal chimeric antigen receptor (UniCAR) express the UniCAR, which has binding specificity for the tag of the targeting module, which in turn binds to a cell surface protein or extracellular structure.

[0095] 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.

[0096] In an embodiment, the kit comprises at least two targeting modules according to the invention, wherein the at least two targeting modules comprise different chemically synthesized peptide binding moieties specific for a human cell surface protein or protein complex and the same tag, wherein the tag is a peptide derived from a human nuclear protein.

[0097] In an embodiment, the kit comprises 1 to 5 targeting modules according to the present invention, preferably 1 to 3 targeting modules.

[0098] In embodiments, the tag-binding domain is present at the amino terminus of the polypeptide comprising UniCAR. Advantageously, placing the tag-binding domain at the amino terminus allows the tag-binding domain access to the tagged targeting module bound to the target cell.

[0099] In a further embodiment, the tag-binding domain is an antibody or antigen-binding fragment. As used herein, the term "antibody" refers to a protein that binds an antigen via the variable region of the Fab. A fragment antigen-binding (Fab) fragment is the region on an antibody that binds to an antigen. A Fab fragment is composed of one constant domain and one variable domain from each of the heavy and light chains. As used herein, the term "antigen-binding fragment" refers to a protein that contains at least the variable domain of an antibody light or heavy chain. In an embodiment, the antigen-binding fragment is selected from a single-chain variable fragment (scFv), a single-chain antibody, a F(ab')2 fragment, a Fab fragment, and a fragment produced by a Fab expression library.

[0100] In embodiments, the tag binding domain is obtained from an animal species, preferably a mammal such as human, monkey, mouse, rat, rabbit, guinea pig, horse, cow, sheep, goat, pig, dog, or cat. Preferably, the tag binding domain is a human or humanized antibody.

[0101] In embodiments, the tag binding domain is a polyclonal antibody, a monoclonal antibody, or a chimeric antibody, in which the antigen-binding region of a non-human antibody is transferred into the framework of a human antibody by recombinant DNA techniques.

[0102] In embodiments, antibodies to a selected tag can be produced by immunization of various hosts, including but not limited to goats, rabbits, rats, mice, and humans, via injection with the particular protein or any portion, fragment, or oligopeptide that retains the immunogenic properties of that protein.

[0103] In an embodiment, the tag binding domain binds to a tag derived from human nuclear La protein, preferably the tag binding domain is an antibody or antigen-binding fragment, wherein the tag binding domain comprises an anti-La epitope scFv, more preferably an anti-La epitope scFv according to SEQ ID NOs: 21 and 22 or 23 and 24.

[0104] Advantageously, the tag is a peptide sequence derived from a nuclear antigen that cannot be accessed and bound by the corresponding tag-binding domain in the context of the native protein under physiological conditions. Even more advantageously, the tag is not immunogenic. This minimizes the risk of uncontrolled on-target off-site toxicity by UniCAR-expressing immune cells, such as the release of toxic levels of cytokines, variously referred to as cytokine storm or cytokine release syndrome (CRS).

[0105] As used herein, the term "single-chain variable fragment (scFv)" refers to an artificial antigen-binding fragment comprising covalently linked antibody light and heavy chain variable domains. In embodiments, the antibody light chain variable domain (VL) and heavy chain variable domain (VH) are covalently linked by a short peptide of 10 to 25 amino acids. In further embodiments, the short peptide links the N-terminus of the VH to the C-terminus of the VL, or vice versa.

[0106] As used herein, the term "extracellular hinge" refers to a flexible peptide sequence connecting the tag-binding domain and the transmembrane domain that allows the UniCAR to protrude from the surface of the cell for optimal binding to its specific tag.

[0107] As used herein, the term "transmembrane domain" refers to a peptide sequence that is thermodynamically stable within the membrane and therefore anchors UniCAR into the plasma membrane of a cell.

[0108] In further embodiments, the extracellular hinge and transmembrane domain are selected from the hinge and transmembrane domain of the human CD28 molecule, the CD8a chain, a portion of the constant region of an NK cell receptor, preferably the natural killer group NKG2D, or an antibody, and combinations thereof. As used herein, the term "combinations thereof" refers to combinations of different hinge and transmembrane domains.

[0109] Pinthus et al. 2003, Pinthus et al. 2004, Cartellieri et al. 2014, and Cartellieri et al. 2016 describe the use of the hinge and transmembrane domains of the human CD28 molecule in CARs (Pinthus et al. 2003, Pinthus et al. 2004, Cartellieri et al. 2014, Cartellieri et al. 2016).

[0110] Carpentino et al., Milone et al., and Zhao et al. have described the use of the hinge and transmembrane domains of the human CD8α molecule in CARs (Carpentino et al. 2009, Milone et al. 2009, Zhao et al. 2009).

[0111] Zhang et al. 2005 and Zhang et al. 2006 describe the use of the hinge and transmembrane domains of NKG2D in CARs (Zhang et al. 2005, Zhang et al. 2006).

[0112] Hombach et al., Frigault et al., and Wang et al. described the use of the hinge and transmembrane domains of portions of the constant region of immunoglobulin G1 (IgG) (Hombach et al. 2007, Frigault et al. 2015, Wang et al. 2007b). Frigault et al. described the use of the hinge domain of the constant region of IgG4.

[0113] Examples of combinations of extracellular hinge and transmembrane domains include, but are not limited to, CD28 extracellular hinge and transmembrane domain, CD8 alpha extracellular hinge and transmembrane domain, IgG1 or IgG4 constant region combined with CD28, or CD137 transmembrane domain.

[0114] As used herein, the term "signaling domain" refers to an amino acid sequence that transmits a signal into a cell by crosslinking a cell expressing UniCAR (effector cell) to a protein or protein complex on the surface of a human cell (target cell). The crosslinking between the effector and target cell is mediated by a targeting module according to the present invention.

[0115] In further embodiments, the signaling domain is selected from CD28, CD137 (4-1BB), CD134 (OX40), DAP10 and the cytoplasmic region of CD27, programmed cell death 1 (PD-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), the cytoplasmic region of the CD3 chain, DAP12 and an activating Fc receptor.

[0116] Hombach et al., Maher 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, Maher et al. 2002, Cartellieri et al. 2014, Cartellieri et al. 2016).

[0117] Milone et al. and Finney et al. described the use of the cytoplasmic region of CD137 (4-1BB) as a signaling domain (Finney et al. 2004, Milone et al. 2009).

[0118] Finney et al., Hombach and Abken 2011, and Hombach and Abken 2013 describe the use of the cytoplasmic region of CD134 (OX40) as a signaling domain in CAR (Finney et al. 2004, Hombach and Abken 2011, Hombach and Abken 2013).

[0119] Zhang et al. describe the use of DAP10 as a signaling domain (Zhang et al. 2005).

[0120] Fedorov et al. describe 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).

[0121] Gong et al. and Gade et al. have described the use of the cytoplasmic region of the CD3 chain, specifically the CD3Cζ chain, as a signaling domain in CARs (Gong et al. 1999, Gade et al. 2005).

[0122] Toepfer et al. describe the use of DAP12 as a signaling domain in CAR (Toepfer et al. 2015).

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

[0124] In an embodiment, the common chimeric antigen receptor comprises at least one signaling domain, preferably two, three, four or more signaling domains, particularly preferably selected from CD28, CD137 (4-1BB), CD134 (OX40), DAP10 and the cytoplasmic region of CD27, programmed cell death 1 (PD-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), the cytoplasmic region of the CD3 chain, DAP12 and an activating Fc receptor.

[0125] In a further embodiment, a nucleic acid encoding a common chimeric antigen receptor designated UniCAR01 according to SEQ ID NO: 1 is provided. This nucleic acid sequence encodes a human IL-2m leader peptide according to SEQ ID NO: 2, a humanized heavy chain of anti-La5B9scFv according to SEQ ID NO: 3, a humanized light chain of anti-La5B9scFv according to SEQ ID NO: 4, a human CD28 portion according to SEQ ID NOs: 5-7 comprising a human CD28 extracellular portion with a mutated binding motif according to SEQ ID NO: 5, a CD28 transmembrane domain according to SEQ ID NO: 6, and a human CD28 intracellular portion comprising a mutated internalization motif according to SEQ ID NO: 7, and a human CD3 zeta intracellular domain according to SEQ ID NO: 8.

[0126] The product of protein expression of the nucleic acid according to SEQ ID NO: 1 can be obtained in SEQ ID NO: 17.

[0127] The nucleic acid sequence of the humanized anti-La5B9 variable region heavy chain according to SEQ ID NO:3 encodes the protein according to SEQ ID NO:21, whereas the humanized anti-La5B9 variable region light chain according to SEQ ID NO:4 encodes for the protein according to SEQ ID NO:22.

[0128] In a further embodiment, a nucleic acid sequence is provided that encodes a common chimeric antigen receptor designated UniCAR02 according to SEQ ID NO: 9. This nucleic acid sequence encodes a human IL-2m leader peptide according to SEQ ID NO: 2, a humanized heavy chain of anti-La5B9scFv according to SEQ ID NO: 3, a humanized light chain of anti-La5B9scFv according to SEQ ID NO: 4, the extracellular hinge and transmembrane regions of the human CD8 alpha chain according to SEQ ID NOs: 10 and 11, the human CD137 intracellular signaling domain according to SEQ ID NO: 12, and the human CD3 zeta intracellular domain according to SEQ ID NO: 8.

[0129] The product of protein expression of the isolated nucleic acid sequence according to SEQ ID NO: 9 can be obtained in SEQ ID NO: 18.

[0130] A nucleic acid encoding a common chimeric antigen receptor designated UniCAR03 according to SEQ ID NO: 13 is provided. This nucleic acid sequence encodes a human IL-2m leader peptide according to SEQ ID NO: 2, a humanized heavy chain of anti-La7B6scFv according to SEQ ID NO: 14, a humanized light chain of anti-La7B6scFv according to SEQ ID NO: 15, a human CD28 extracellular portion with a mutated binding motif according to SEQ ID NO: 5, a CD28 transmembrane domain according to SEQ ID NO: 6, a human CD28 intracellular portion comprising a mutated internalization motif according to SEQ ID NO: 7, and a human CD3 zeta intracellular domain according to SEQ ID NO: 8.

[0131] The product of protein expression of the nucleic acid according to SEQ ID NO: 13 can be obtained in SEQ ID NO: 19.

[0132] The nucleic acid sequence of the humanized anti-7B6 variable region heavy chain according to SEQ ID NO: 14 codes for a protein according to SEQ ID NO: 23, whereas the humanized anti-7B6 variable region light chain according to SEQ ID NO: 15 codes for a protein according to SEQ ID NO: 24.

[0133] In a further embodiment, a nucleic acid is provided that encodes a reverse common chimeric antigen receptor designated UniCAR04 according to SEQ ID NO: 16. This nucleic acid sequence encodes a human IL-2m leader peptide according to SEQ ID NO: 2, a humanized heavy chain of anti-La7B6scFv according to SEQ ID NO: 14, a humanized light chain of anti-La7B6scFv according to SEQ ID NO: 15, the extracellular hinge and transmembrane regions of the human CD8 alpha chain according to SEQ ID NOs: 10 and 11, the human CD137 intracellular signaling domain according to SEQ ID NO: 12, and the human CD3 zeta intracellular domain according to SEQ ID NO: 8.

[0134] The product of protein expression of the isolated nucleic acid sequence according to SEQ ID NO: 16 can be obtained in SEQ ID NO: 20.

[0135] In a further embodiment, the common chimeric antigen receptor comprises a fourth domain, wherein the fourth domain is a short peptide linker in the extracellular portion of the receptor.

[0136] In a further embodiment, the fourth domain forms a linear epitope for a monoclonal antibody (mab) that specifically binds to the fourth domain. In an embodiment, the fourth domain comprises at least one linear epitope.

[0137] In a further embodiment, the fourth domain is located within the tag-binding domain, between the tag-binding domain and the extracellular hinge domain or an integral portion of the extracellular hinge domain.

[0138] Advantageously, UniCAR-grafted immune cells with the fourth domain can be specifically stimulated to preferentially proliferate and persist longer than non-grafted immune cells either in vitro or in vivo. Advantageously, the fourth domain can also be used to purify UniCAR-grafted immune cells from mixed cell populations, or to attenuate UniCAR-grafted immune cell-mediated immune responses and eliminate UniCAR-grafted immune cells in vivo.

[0139] In a further embodiment, the common chimeric antigen receptor comprises a signal peptide. Advantageously, this signal peptide enables expression on the cell surface of effector cells. In an embodiment, the signal peptide is located at the N-terminus of the UniCAR nucleic acid sequence in front of the tag-binding domain. In an embodiment, the signal peptide is selected from leader peptides derived from proteins such as CD28, CD8 alpha, IL-2, or the heavy or light chains of antibodies of human origin to target the protein to the secretory pathway either co- or post-translationally and avoid immunogenic responses.

[0140] In a further embodiment, the nucleic acid is cDNA. 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.

[0141] In embodiments, the cells are selected from autologous, syngeneic, or allogeneic cells, depending on the disease to be treated and the means available for treating the disease. In embodiments, the cells are selected from immune cells with cytolytic, phagocytic, or immunosuppressive activity, such as T cells, including regulatory T cells, natural killer (NK) cells, and macrophages. In preferred embodiments, the cells are selected from subpopulations of T cells, such as alpha / beta T cells and gamma / delta T cells, or stem cell memory or central memory T cells, cytotoxic T cells, T cells, including regulatory T cells, or NK cells. In one embodiment, the effector cells are derived from a specific HLA background and are utilized in an autologous or allogeneic system. Effector cells can be isolated from any source, including tumor explants or intratumoral cells of the subject being treated. In embodiments, effector cells may be generated by in vitro differentiation from pluripotent or multipotent stem or progenitor cells, before or after genetic engineering to express UniCAR in individual cells. In the following, the term "effector cell" refers to any type of immune cell as described above that has been genetically modified to express UniCAR on its cell surface.

[0142] Generation of UniCAR cells Immune cells can be genetically engineered to express UniCAR by various methods.The polynucleotide vector encoding UniCAR and all the necessary elements that ensure the expression of UniCAR in genetically engineered immune cells are transferred into immune cells.Transfer of vector can be carried out by electroporation, or nucleic acid transfection, or with the help of viral vector systems such as adenovirus, adeno-associated virus, retrovirus, foamy virus or lentivirus viral gene transfer.

[0143] Lentiviral gene transfer is applied for stable expression of UniCAR in immune cells by first constructing a lentiviral vector encoding the selected UniCAR. The lentiviral vector is pLVX-EF1alphaUniCAR28 / ζ (Clontech, Takara Bio Group), in which the lentiviral portion of the vector is derived from human immunodeficiency virus (HIV), and the MSC / IRES / ZxGreenI portion is replaced by the UniCAR construct, as shown in Figure 3.

[0144] Lentiviral particles are generated by transient transfection of human embryonic kidney (HEK) 293T (ACC635) cells with a lentiviral vector plasmid encoding UniCAR and cotransfection with a plasmid (psPAX2) encoding a group-specific antigen (gag) and polymerase (pol), as illustrated in Figure 4, and a plasmid (pMD2.G) encoding an envelope, as illustrated in Figure 5. The MD2.G plasmid encodes the glycoprotein of vesicular stomatitis virus (VSV-G). The VSV-G protein is used with lentiviral vectors to transduce a wide range of mammalian cells. Various envelopes from different viral species can be utilized for this purpose. Lentiviral vectors can be successfully pseudotyped with the envelope glycoprotein (Env) of the amphotropic murine leukemia virus (MLV) or the G protein of vesicular stomatitis virus (VSV-G), modified envelopes of prototype foamy virus (PFV), or chimeric envelope glycoprotein variants derived from gibbon ape leukemia virus (GaLV) and MLV.

[0145] Supernatants from transfected HEK293T cells are collected 24–96 h after transfection, and viral particles are concentrated from the supernatant by ultrafiltration or other methods. For lentiviral transduction of immune cells, peripheral blood mononuclear cells (PBMCs) or isolated T cells are activated with mabs specific for the CD3 complex, such as clones OKT3 or UCHT1, either in solution or coated onto plastic cell culture dishes or magnetic beads. Activation of PBMCs or isolated T cells is further enhanced by stimulating costimulatory pathways with mabs or ligands specific for CD27, CD28, CD134, or CD137, either alone or in combination with exogenous recombinant cytokines such as interleukin (IL)-2, IL-7, IL-12, IL-15, and IL-21. Concentrated or non-concentrated viral particles are added to PBMC or T cell cultures 24-96 hours after the first dose of activating CD3 antibodies and / or recombinant cytokines as a single or multiple doses.

[0146] Stable transduction of T cells may be measured by flow cytometry after staining with a tag-containing targeting module for surface expression of UniCAR or a mab directed against the fourth domain of UniCAR starting 3 days after the final administration of viral supernatant. UniCAR-transduced T cells can be expanded in vitro by culturing them in the presence of recombinant cytokines and an activating anti-CD3 mab.

[0147] If UniCAR has an optional fourth domain, i.e., a peptide sequence that forms a linear epitope for a mab, immune cells genetically engineered to express UniCAR can be specifically propagated in vitro by coating the mab or its antibody fragment binding to the fourth UniCAR domain onto the surface of any type of culture dish or onto beads that are added to cell culture at a defined ratio of 1 bead:1–4 UniCAR-grafted effector cells. Binding of the surface-coated mab to the UniCAR peptide domain induces cross-linking of the cell surface-expressed UniCAR and the formation of an immune synapse, which results in the activation of a signaling pathway specifically triggered by the UniCAR signaling domain. Depending on the induced signaling pathway, this may enhance the proliferation of UniCAR-bearing immune cells and sustain resistance to activation-induced cell death, thus leading to an expansion of UniCAR-engineered immune cells in a mixed population.

[0148] The optional fourth domain, which is a peptide sequence that forms a linear epitope for the mab, can be further used to enrich and purify UniCAR-expressing immune cells from a mixed population. Enrichment and purification are carried out with the aid of a mab or its antibody fragment that binds to the fourth UniCAR domain, either to label UniCAR-expressing cells for cell sorting or to transiently link UniCAR-expressing immune cells to small particles that can be used for cell isolation. In one embodiment, UniCAR-transplanted immune cells are incubated with a mab that recognizes the fourth domain. Then, magnetic beads that are bound to an antibody or its fragment that is directed against the species-specific and isotype-specific heavy and light chains of the mab that binds to any fourth domain are added. Thus, the UniCAR-expressing immune cells and the magnetic beads are linked, captured in a magnetic field, and separated from other immune cells.

[0149] The present invention further includes pharmaceutical compositions comprising kits according to the present invention.

[0150] The pharmaceutical composition is preferably administered parenterally, particularly preferably intravenously. In an embodiment, the pharmaceutical composition is in a form suitable for intravenous administration. Preferably, the pharmaceutical composition is a solution, emulsion, or suspension.

[0151] In an embodiment, the pharmaceutical composition is an injectable buffer solution containing 0.1 μg / ml to 50 mg / ml of the targeting module, preferably 0.5 μg / ml to 5 mg / ml of the targeting module.

[0152] In an embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable diluent or carrier. In an embodiment, the carrier is water, buffered water, 0.4% saline, 0.3% glycine, or a similar solvent. In an embodiment, the buffered water is selected from histidine buffered water at a pH value of 5.0 to 7.0, with histidine buffered water at a pH of 6.0 being particularly preferred; or sodium succinate, sodium citrate, sodium phosphate, or potassium phosphate buffered water. In an embodiment, the buffer has a concentration of 1 mmol / L (mM) to 500 mM, preferably 1 mM to 50 mM, and particularly preferably 5 mM to 10 mM. In an embodiment, the carrier comprises sodium chloride, preferably at a concentration of 0 mM to 300 mM, and particularly preferably 150 mM. In an embodiment, the pharmaceutical composition further comprises a stabilizer, preferably at a concentration of 1 mM to 50 mM, and particularly preferably 5 mM to 10 mM. In an embodiment, the stabilizer is L-methionine.

[0153] In an embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. The term "pharmaceutically acceptable excipient" refers to a compound that provides suitable physiological conditions and / or a compound that enhances stability, such as an agent for adjusting pH and buffering, an agent for adjusting toxicity, and the like. In an embodiment, the pharmaceutically acceptable excipient is selected from sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate.

[0154] In a preferred embodiment, the pharmaceutical composition comprises the targeting module at a dosage of 0.1 μg / kg to 1 mg / kg, preferably 1 μg / kg to 100 μg / kg body weight per administration.

[0155] In a further embodiment, the pharmaceutical composition is sterile. The pharmaceutical composition is sterilized by conventional, well-known techniques.

[0156] In an embodiment, the pharmaceutical composition comprising the kit according to the present invention is used for administration to a subject.

[0157] The present invention further includes a kit according to the present invention or a pharmaceutical composition according to the present invention for use in the treatment of cancer, infectious diseases and autoimmune disorders. The term "autoimmune disorder" refers to an abnormal immune response of the body against substances and tissues normally present in the body (autoimmunity).

[0158] In an embodiment, the kit according to the invention or the pharmaceutical composition according to the invention is used for preparing a medicament for therapeutic and / or diagnostic use in the case of cancer or autoimmune diseases.

[0159] The present invention also encompasses a method for treating a human suffering from cancer or an autoimmune or inflammatory disease by administering a kit according to the present invention or a pharmaceutical composition according to the present invention. For therapeutic applications, a sterile kit according to the present invention or a pharmaceutical composition according to the present invention, comprising a pharmacologically effective amount of a targeting module according to the present invention and a vector or cell comprising a nucleic acid encoding a common chimeric antigen receptor, is administered to a patient to treat the above-mentioned disease.

[0160] In an embodiment, the kit according to the invention or the pharmaceutical composition according to the invention is used to stimulate an immune response mediated by a chimeric common antigen receptor in a mammal.

[0161] 1. A method for stimulating an immune response mediated by a chimeric common antigen receptor in a mammal, comprising: administering to a mammal an effective amount of a vector or cells comprising a nucleic acid encoding a common chimeric antigen receptor, the common chimeric antigen receptor comprising three domains, a first domain being a tag-binding domain, a second domain being an extracellular hinge and transmembrane domain, and a third domain being a signaling domain, the tag-binding domain binding to a tag derived from a human nuclear protein; administering a targeting module according to the present invention; A method comprising: The targeting module is administered to the subject prior to, concurrently with, or following administration of the effector cells expressing the common chimeric antigen receptor.

[0162] In a preferred embodiment, the kit according to the invention, in particular the targeting module and vector or cell according to the invention, or the pharmaceutical composition is administered to a human.

[0163] In further embodiments, the recently described embodiments can be combined.

[0164] Cited Non-Patent Literature [ka]

[0165] [ka]

[0166] [ka]

[0167] [ka]

[0168] [ka]

[0169] [ka]

[0170] [ka]

[0171] [ka]

[0172] Reference sign 1. The first domain is the tag-binding domain 2. The second domain is the extracellular hinge and transmembrane domain 3. The third domain is the signaling domain 4. An optional fourth domain that is a short peptide linker

Claims

1. A targeting module comprising a chemically synthesized peptide binding moiety specific for a human cell surface protein or protein complex and a tag, Formula (I): 【Chemical 1】 Or formula (II): 【Chemistry 2】 A targeting module having a structure according to the formula: (wherein [Z] is a single chelator or aromatic amino acid or a complex thereof, and o is two or more repeating linkers).

2. A targeting module as described in claim 1, wherein Z is a single chelator.

3. 3. The targeting module of claim 1 or 2 for use in the treatment of cancer, an infectious disease, an inflammatory disorder or an autoimmune disorder.

4. a) a targeting module according to claim 1 or 2; b) a vector or cell comprising a nucleic acid encoding a common chimeric antigen receptor; and wherein the common chimeric antigen receptor comprises three domains; the first domain is a tag-binding domain; the second domain is an extracellular hinge and transmembrane domain; and the third domain is a signaling domain; A kit, wherein the tag-binding domain binds to the tag of the targeting module of claim 1 or 2.

5. 5. The kit of claim 4, wherein the tag binding domain binds to a tag derived from human nuclear La protein, and the tag binding domain is an antibody or antigen-binding fragment, and the tag binding domain constitutes an anti-La epitope scFv.

6. 6. The kit of claim 4 or 5, wherein the tag binding domain constitutes an anti-La epitope scFv according to SEQ ID NOs: 21 and 22 or SEQ ID NOs: 23 and 24.

7. 7. The kit of claim 4, wherein the extracellular hinge and transmembrane domain are selected from the hinge and transmembrane domains of the human CD28 molecule, the CD8a chain NK cell receptor, portions of the constant region of an antibody, and combinations thereof.

8. 8. The kit of claim 7, wherein the CD8a chain NK cell receptor is a natural killer group NKG2D receptor.

9. 9. The kit of any one of claims 4 to 8, wherein the signaling domain is selected from CD28, CD137 (4-1BB), CD134 (OX40), DAP10 and the cytoplasmic region of CD27, programmed cell death 1 (PD-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), the cytoplasmic region of the CD3 chain, DAP12 and an activating Fc receptor.

10. 10. The kit according to any one of claims 4 to 9, wherein the nucleic acid is represented by SEQ ID NO: 1, 9, 13 or 16, which encodes a common chimeric antigen receptor having an amino acid sequence according to SEQ ID NO: 17, 18, 19 or 20.

11. A pharmaceutical composition comprising the kit of any one of claims 4 to 10.

12. 12. A kit according to any one of claims 4 to 10 or a pharmaceutical composition according to claim 11 for use in the treatment of cancer, an infectious disease, an inflammatory disorder or an autoimmune disorder.

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