Artificial signaling molecules

Abstract

The present invention provides a signaling molecule and an immune cell expressing the signaling molecule for use in the treatment of undesired immune activity, the signaling molecule being a fusion protein comprising a ligand domain, a spacer, a transmembrane domain and at least one intracellular signaling domain wherein the ligand domain comprises at least one epitope or all epitopes associated with an antigen, or a associated antigen, which is a target of undesired immunocompetence.

Description

[0001] This application is a divisional application of the Chinese patent application for invention with the application date of April 30, 2020, application number 202080032067.7, and invention title "Artificial Signaling Molecule". Technical Field

[0002] The present invention relates to artificial signaling molecules, which are fusion proteins for expression in immune cells and are specifically used for medical treatment. The artificial signaling molecules and immune cells expressing the artificial signaling molecules are suitable for treating undesired immune responses, such as for inhibiting undesired immune activities, specifically for treating immune rejection against grafts, such as treating HvG disease, for treating autoimmune diseases, or for treating allergic reactions.

[0003] The artificial signaling molecules are designed to direct the immune cells expressing them against immune cells that cause undesired immune responses, such as T cells, B cells, or NK cells. Background Art

[0004] For inhibiting undesired immune activities, it is generally known to inhibit the general activity of the immune system.

[0005] WO 2018 / 001874 describes CAR molecules expressed in Treg cells, which are used to treat HvG disease in graft recipients, where the CAR molecules produce inhibitory activity near the graft. Summary of the Invention

[0006] Object of the Invention

[0007] The object of the present invention is to provide an alternative for treating undesired immune activities, especially for treating undesired immune responses against specific associated antigens, such as for treating HvG disease, treating autoimmune diseases, or treating allergic reactions.

[0008] Description of the Invention

[0009] The present invention achieves this object by the features of the claims, in particular by providing a signal transduction molecule and an immune cell expressing the signal transduction molecule for treating undesired immune activity, wherein the signal transduction molecule is a fusion protein comprising a ligand domain, a spacer, a transmembrane domain and at least one intracellular signal transduction domain, or consisting of a ligand domain, a spacer, a transmembrane domain and at least one intracellular signal transduction domain, wherein the ligand domain comprises at least one epitope or all epitopes of an associated antigen or the associated antigen, and the associated antigen is a target of the undesired immune activity. Preferably, particularly in a heterodimeric ligand domain having, for example, two ligand domains of similar size, the signal transduction molecule comprises a dimerization domain arranged in its extracellular portion, such as arranged between the ligand domain and the transmembrane domain. Preferably, the domains of the signal transduction molecule are connected to each other from the N-terminus to the C-terminus, more preferably directly connected to each other from the N-terminus to the C-terminus, and the domains comprise or consist of: a ligand domain, a spacer, a transmembrane domain and at least one intracellular signal transduction domain, optionally a dimerization domain between the ligand domain and the transmembrane domain, preferably the extracellular portion of the HLA transmembrane domain located at the C-terminus of the ligand domain, preferably a linker of, for example, 8 to 12 amino acids between the extracellular portion of the HLA transmembrane domain and the dimerization domain or the spacer. The immune cell expressing the signal transduction molecule comprises a nucleic acid sequence encoding the signal transduction molecule, and the nucleic acid sequence is preferably integrated into the cell genome, for example, by transduction using a viral vector or by transfection of the nucleic acid sequence.

[0010] Thus, the ligand domain contains at least one epitope of the associated antigen, preferably all epitopes, or consists of at least one epitope of the associated antigen, preferably all epitopes. For example, the ligand domain may contain the associated antigen of a T cell receptor (TCR) and / or a B cell receptor (BCR), or consist of the associated antigen of a T cell receptor (TCR) and / or a B cell receptor (BCR). The undesired immune activity to be treated is directed against this associated antigen. Since the signaling molecule of the present invention contains at least one epitope of the associated antigen against which the undesired immune response is directed for its specificity, the binding of a T cell or B cell or NK cell to the ligand domain of the signaling molecule expressed by CD4+ T cells or CD8+ T cells results in the activation of T cell effector functions that are specific for the T cell or B cell or NK cell bound to the ligand domain of the signaling molecule. As a result of the binding of a CD4+ T cell or CD8+ T cell via its TCR or a B cell via its BCR or an NK cell via its receptor to the signaling molecule when expressed in CD4+ T cells or CD8+ T cells, the bound T cell or B cell or NK cell is inactivated or killed, depending on the functional characteristics of the signaling T cell. Since the binding of the TCR of a T cell or the BCR of a B cell or the KIR of an NK cell is mediated by the ligand domain of the signaling molecule, the inactivation or elimination of the bound T cell or bound B cell or bound NK cell is specific for the associated antigen targeted by the specific receptor of the T cell or B cell or NK cell. Thus, herein, the signaling molecule is also referred to as a chimeric ligand receptor (CLR).

[0011] Since the ligand domain provides the associated antigen for the TCR or BCR, the ligand domain does not have antibodies, such as no scFv and other antibody forms.

[0012] Thus, for treating HvG diseases, the ligand domain, also referred to as the associated antigen, can be the antigen against which the immune response to the graft is directed. For example, the ligand domain (i.e., the associated antigen) is heterologous, such as the HLA class I or class II molecules of a mismatched graft.

[0013] Optionally, for treating HvG diseases, immune cells expressing the signaling molecule can be used to treat a patient before or after transplantation, especially when the graft is HLA mismatched. In addition, immune cells expressing the signaling molecule can be used to treat patients who have or have not generated memory B cells against the mismatched HLA molecules of the graft after receiving the graft.

[0014] For treating an undesired immune response against a self-antigen, i.e., for treating an autoimmune disease, the ligand domain (i.e., the associated antigen) can be the self-antigen against which the autoimmune response is directed.

[0015] For treating allergy, the ligand domain (i.e., the associated antigen) can be the associated antigen of allergy. For example, a peanut antigen such as Ara h 1 (UniProtKB / Swiss-Prot: P43237.1).

[0016] For example, the associated antigen can be determined as the target of an antibody, a B cell receptor (BCR), a T cell receptor (TCR), or an NK cell receptor (such as KIR), which occurs during or is characteristic of an unwanted immune response. Preferably, the associated antigen is pre-determined directly or indirectly from a biopsy obtained from a patient, such as a blood sample. To indirectly pre-determine the associated antigen, the binding of antibodies or T cells in the biopsy can be analyzed. For treating HvG diseases, the associated antigen can be determined as a mismatched HLA molecule. The TCR of T cells and the BCR of B cells are usually natural components of patients with unwanted immune responses, respectively.

[0017] At least one intracellular signaling domain is a domain that activates T cell effector functions after the antigen domain in T cells. The intracellular signaling domain can be, for example, the hCD3ζ domain, preferably a combination of the h4-1BB domain and the hCD3ζ (zeta) domain, or a combination of the intracellular hCD28 signal transduction domain and the hCD3ζ domain.

[0018] The transmembrane domain can be, for example, the transmembrane domain of human CD28 (hCD28) or the transmembrane domain of CD4 (hCD4).

[0019] The immune cell expressing the signaling molecule is a T cell, which is preferably immunocompatible with the recipient, for example, autologous to the recipient. Alternatively, the immune cell expressing the signaling molecule is not immunocompatible with the recipient, for example, not HLA-matched to the recipient. Therefore, after administration of the incompatible immune cell expressing the signaling molecule, when it has not been genetically modified in a way that prevents allorecognition of this immune cell, such as by HLA knockdown or knockout, it may ultimately be eliminated by the host's immune system. Therefore, for T cells that are not immunocompatible with the recipient, such as not HLA-matched to the recipient, the T cells can be genetically modified to not express HLA molecules, for example, genetically modified to knockdown or knockout HLA molecule expression.

[0020] The immune cell expressing the signaling molecule is, for example, a primary CD4+ T cell or a primary CD8+ T cell, an NK cell, and / or a progenitor cell of one of these or a cytotoxic cell line, such as NK-92. Herein, the immune cell expressing the signaling molecule is also referred to as a T cell, which represents a primary CD4+ T cell or a primary CD8+ T cell, an NK cell, and a progenitor cell of one of these, or a cytotoxic cell line.

[0021] The use of signaling molecules and T cells expressing the signaling molecules in therapy is to eliminate the recipient's B cells, T cells, and NK cells that specifically bind to the antigenic domain of the signaling molecule without affecting other endogenous immune cells of the recipient. Thus, the use of signaling molecules and T cells expressing the signaling molecules in medical treatment results in selective inactivation in the case of regulatory T cells or immune cell elimination in the case of cytotoxic T cells, depending on their associated receptors that match the ligand domain of the signaling molecule, without general suppression of the immune system.

[0022] A dimerization domain, such as a leucine zipper domain, is preferably used for the signaling molecule that comprises or consists of two chains of an HLA class II molecule, such as an α chain and a β chain, as its ligand domain. For example, a first signaling molecule can comprise or consist of, preferably from the N-terminus to the C-terminus, an antigenic domain, a linker, a dimerization domain (also called a zipper), a spacer, a transmembrane domain, and at least one intracellular signaling domain, wherein the dimerization domain dimerizes with the dimerization domain of a second signaling molecule, and wherein the antigenic domains of the first and second signaling molecules can be different, such as the α chain and the β chain of an HLA class II molecule. Wherein, the second signaling molecule can comprise or consist of: a chain of an HLA class II molecule as its ligand domain, a linker, a dimerization domain, a spacer, a transmembrane domain, and at least one intracellular signaling domain. In another embodiment, the second signaling molecule can comprise or consist of: a chain of an HLA class II molecule as its ligand domain, a linker, and a dimerization domain, and the second signaling molecule binds to the transmembrane domain and at least one intracellular signaling domain of the first signaling molecule through the dimerized dimerization domain.

[0023] Preferably, the fusion protein comprises a dimerization domain in each of its component proteins, also called a signaling molecule, which does not form homodimers but only heterodimers. Thus, each signaling molecule (i.e., each protein covalently linked to or including one of the chains comprising a ligand domain of two chains) comprises different dimerization domains of a pair of dimerization domains that form a heterodimer. For example, preferred dimerization domains that form heterodimers are selected from the E and K peptides of the E / K coiled-coil domain, the N-terminal and complementary C-terminal portions of a split intein, such as the 36-residue N-terminal portion and the remaining C-terminal portion of a split intein, one knob and one hole of complementary knob-into-hole engineered human IgG CH3 domains, such as those containing mutations such as T366Y, F405A, T394W, and / or Y407T, or any other combination pair of complementary protein-protein interaction domains.

[0024] In a preferred embodiment, the fusion protein according to the invention consists of a first signaling molecule and a second signaling molecule, wherein the first signaling molecule consists of a ligand domain, a dimerization domain, a spacer, a transmembrane domain, and an intracellular signaling domain from the N-terminus to the C-terminus. In a preferred embodiment, the second signaling molecule comprises a dimerization domain, the dimerization domain of the first signaling molecule dimerizes with the dimerization domain of the second signaling molecule, and the ligand domain comprises at least one epitope of the associated antigen against which an unwanted immune response is directed.

[0025] Wherein, the second signaling molecule preferably consists of a ligand domain, a linker, and a dimerization domain. Alternatively, the second signaling molecule may consist of a signal transduction domain, a linker, a dimerization domain, a spacer, a transmembrane domain, and an intracellular signaling domain.

[0026] Herein, the domains of a signaling molecule are generally given from the N-terminus to the C-terminus of the signaling molecule.

[0027] Generally, for an HLA class II molecule as the ligand domain, the ligand domain may consist of the α1 domain and the β1 domain of the HLA class II heavy chain and optionally the α2 and β2 domains. Wherein, the ligand domain of the first signaling molecule may comprise or consist of the α1 and optionally the α2 domains, and the ligand domain of the second signaling molecule may comprise or consist of the β1 and optionally the β2 domains. Alternatively, the ligand domain of the first signaling molecule may comprise or consist of the β1 and optionally the β2 domains, and the ligand domain of the second signaling molecule may comprise or consist of the α1 and optionally the α2 domains.

[0028] It has been found that the extracellular portion of the HLA transmembrane domain (referred to as the extracellular portion of the transmembrane domain in the sequence example) arranged at the C-terminus of the α2 domain or the β2 domain results in a more stable molecule. The extracellular portion of the HLA transmembrane domain is preferably encoded by exon 4 of the HLA class II-encoding gene and exon 5 of the HLA class I-encoding gene.

[0029] Optionally, for a signaling molecule having an HLA class I heavy chain (α chain) as the ligand domain, β2-microglobulin can be bound through the dimerization domain, wherein for example β2-microglobulin has a dimerization domain at its N-terminus or C-terminus. The signaling molecule may comprise or consist of the following: preferably from its N-terminus to its C-terminus, a ligand domain that is the heavy chain of an HLA class I molecule, optionally the extracellular portion of the HLA transmembrane domain, a linker, a dimerization domain, a spacer, a transmembrane domain, and at least one intracellular signaling domain, wherein the dimerization domain dimerizes with the dimerization domain of a second molecule that comprises or consists of β2-microglobulin having a dimerization domain at its N-terminus or C-terminus.

[0030] In this text, the spacer is also referred to as the hinge domain.

[0031] Generally, according to European Patent Application No. 19166923.3 filed on April 2, 2019, the ligand domain can be an MHC I molecule comprising an α-chain and β2-microglobulin.

[0032] Generally, for HLA class I as the ligand domain, the ligand domain can consist of the α1 domain and α2 domain of the HLA class I molecule and optionally the α3 domain.

[0033] Optionally, T cells expressing CLR can encode two or more different CLR molecules, each molecule having a different ligand domain, such as from different HLA class I or different HLA class II molecules or different HLA class I and HLA class II molecules. For HLA class I as the ligand domain, CLR consists of at least one, two or three HLA class I heavy chain domains, such as consisting of the α1 domain, α2 domain and α3 domain. The light chain, i.e., β-2-microglobulin, can be the naturally expressed light chain or recombinant β-2-microglobulin, which can also optionally be covalently bound to one of the heavy chain domains. For HLA class II as the ligand domain, the ligand domain can consist of at least one or two α-chain domains, such as the α1 domain and α2 domain, and at least one or two β-chain domains, such as the β1 domain and β2 domain.

[0034] HLA class I or HLA class II antigens are known, for example from https: / / www.ebi.ac.uk / ipd / imgt / hla / download.html, version 0.3.35 or above, which can be downloaded from ftp: / / ftp.ebi.ac.uk / pub / databases / ipd / imgt / hla / .

[0035] Optionally, the signaling molecule as the ligand domain comprises the α-chain or heavy chain of the HLA class I molecule, and this ligand domain can contain at least one mutation at amino acid positions 74, 223, 224, 225, 226, 227, 229 and 245, where the numbering refers to the mature protein of the HLA class I molecule α-chain or heavy chain, i.e., without the signal peptide, such as at least one of the mutations 74L, 223A, 224F, 225D, 226A, 227K, 229A and 245V, in order to reduce or eliminate the binding of CD8 of CD8+ T cells and to reduce or eliminate the cytotoxic activity of CD8+ T cells against T cells expressing CLR comprising a portion of the HLA class I molecule as its ligand domain.

[0036] Optionally, the signaling molecule serving as the ligand domain comprises the α and / or β chain or heavy chain of an HLA class II molecule, and the ligand domain may contain at least one mutation at amino acid positions 88, 90, and 176, where the numbering refers to the mature protein of the HLA class II molecule α chain, i.e., without a signal peptide, resulting in a decrease or elimination of the interaction between the DR or DQ or DP α chain and CD4+ T cells, and / or a mutation at at least one amino acid position among positions 46, 54, 55, 56, 104, 114, 116, 134, 135, 136, 137, 138, 139, 141, 142, 143, 144, 145, 148, 158, 160, and 162, where the numbering refers to the mature protein of the HLA class II molecule β chain, i.e., without a signal peptide, so as to weaken or eliminate the interaction between the DR or DQ or DP β chain and CD4+ T cells.

[0037] Optionally, immune cells expressing at least one signaling molecule can be further genetically engineered to additionally express at least one of the complement inhibitors hDAF (CD55), CD46, or CD59, or a combination of at least two of these, particularly for the treatment of HvG diseases or for the treatment of adverse immune responses of HLA-mismatched grafts.

[0038] Optionally, the fusion protein may contain a ligand domain consisting of two chains associated with each other, for example, the chains of an MHC class I molecule or an MHC class II molecule that are not covalently bound to each other, where each chain of the ligand domain is covalently linked to a separate dimerization domain, and the dimerization domain is dimerized. Among them, the fusion protein contains two proteins dimerized at the dimerization domain or consists of two proteins dimerized at the dimerization domain, one dimerization domain of the fusion protein consisting of one chain of the ligand domain, (the first) dimerization domain, spacer, transmembrane domain, and intracellular signaling domain, and the other dimerization domain of the fusion protein consisting of the other chain of the ligand domain and (the second) dimerization domain, further optionally spacer, transmembrane domain, and intracellular signaling domain.

[0039] Thus, in one embodiment, the fusion protein containing a ligand domain consisting of two chains contains two proteins, one containing one chain of the ligand domain and the other containing the other chain of the ligand domain, and each of these chains of the ligand domain is covalently linked to an individual dimerization domain, and only one of the said proteins contains (e.g., covalently linked to) a spacer, transmembrane domain, and intracellular signaling domain. In this embodiment, the protein containing the other chain of the ligand domain consists of this chain of the ligand domain and a dimerization domain dimerized with the dimerization domain of another protein. Thus, in this embodiment, the fusion protein contains only one spacer covalently linked to only one transmembrane domain, and the transmembrane domain is covalently linked to the intracellular signaling domain.

[0040] In another embodiment, in a fusion protein comprising a ligand domain consisting of two chains, each of these chains is covalently linked to an individual dimerization domain, an individual spacer, an individual transmembrane domain, and an individual intracellular signaling domain. Among them, the various spacers, transmembrane domains, and intracellular signaling domains covalently linked to one of the two chains of the ligand domain can each have the same amino acid sequence or different amino acid sequences. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The present invention will now be described by way of example and with reference to the accompanying drawings shown below:

[0042] - Figure 1 is a schematic diagram of an embodiment of the signaling molecule of the present invention,

[0043] - Figure 2 is a schematic diagram of a further embodiment of the signaling molecule of the present invention,

[0044] - Figure 3 is a schematic diagram of a further embodiment of the signaling molecule of the present invention,

[0045] - Figure 4 is a schematic diagram of a further embodiment of the signaling molecule of the present invention,

[0046] - Figure 5 A), B), C), D), and E) of are schematic diagrams of embodiments of the domain arrangement of the signaling molecule,

[0047] - Figure 6 FACS results of CLR when expressed in immune cells detected by an antibody against the CLR antigen domain,

[0048] - Figure 7 A) of shows the FACS results of CLR when expressed in control cells (K562) detected by an antibody against the CLR antigen domain and Figure 7 B) of shows the FACS results of CLR when expressed in immune cells (T cells),

[0049] - Figure 8 shows LDH release as a measure of the cytotoxicity of T cells expressing CLR, and

[0050] - Figure 9A , Figure 9B and Figure 9C show the analysis results of the expression of activation markers in T cells expressing CLR. DETAILED DESCRIPTION

[0051] Figure 1Shows a schematic diagram of the signal transduction molecule CLR of the present invention, which is arranged in the membrane of the cell membrane and consists of: an extracellular ligand domain, such as a class I HLA molecule (which is represented in this example by a non-covalently bound light chain β-2-microglobulin and a class I HLA-A*02:01 heavy chain (HLA-A2), and the class I HLA-A*02:01 heavy chain consists of its α1, α2, and α3 domains), a spacer, a transmembrane domain spanning the cell membrane, and an intracellularly arranged signal transduction domain which is a combination of an intracellular 4-1BB domain and a CD3ζ (CD3z) domain. This exemplary ligand domain represents the associated antigen of the HvG disease.

[0052] Figure 2 Shows a schematic diagram of the signal transduction molecule CLR of the present invention, which comprises two heavy chains and in a preferred embodiment has an extracellular dimerization domain represented by two partners of a molecular zipper separated from each heavy chain by a linker, and wherein the two heavy chains are anchored in the membrane of the cell membrane via transmembrane domains. The signal transduction molecule consists of two proteins dimerized through its dimerization domain, and each protein contains an extracellular ligand domain, such as a class II HLA molecule (represented in this example by the HLA-DR1 antigen, which consists of the α1 and α2 domains of the HLA-DR1α chain HLA-DRA*01:01 and the β1 and β2 domains of the HLA-DR1β chain HLA-DRB1*01:01), and each heavy chain is connected to an intracellularly arranged signal transduction domain which is a combination of a spacer, a transmembrane domain spanning the cell membrane, and an intracellular 4-1BB domain and a CD3ζ (CD3z) domain. This exemplary ligand domain represents the associated antigen of the HvG disease. Generally, for class II HLA as the ligand domain, the ligand domain can consist of the α1 domain and β1 domain of the class II HLA heavy chain and optionally the α2 and β2 domains. Examples of the signal transduction molecule according to Figure 2 are given in SEQ ID NO:10, which has a short linker (also referred to as a spacer), wherein zipper a is embodied by a Jun-zipper. Examples of the signal transduction molecule with a short linker according to Figure 2 are given in SEQ ID NO:11, wherein zipper b is specifically embodied as zipper b by a Fos-zipper, which can dimerize with the Jun-zipper of SEQ ID NO:10.

[0053] According to Figure 2 further examples of the signal transduction molecule with a long linker are given in SEQ ID NO:12 which includes a Jun-zipper as the dimerization domain, and in SEQ ID NO:13 which has a Fos-zipper (for dimerizing with the Jun-zipper of SEQ ID NO:12).

[0054] Figure 3Shows a schematic diagram of an embodiment of the signal transduction molecule CLR of the present invention, wherein the transmembrane and signal transduction domains are attached only to one of the two heavy chains, here the α1 and α2 domains of the HLA-DR1α chain HLA-DRA*01:01. In this context, the first signal transduction molecule (CLR) consists of the HLA-DR1α chain HLA-DRA*01:01 as its ligand domain, a linker, a dimerization domain (zipper), a spacer, a transmembrane domain, and the intracellular 4-1BB domain and CD3ζ (CD3z) domain as signal transduction domains. The second CLR, consisting of a ligand domain represented by the β chain of HLA-DR1 (HLA-DRB1*01:01:01), a linker, and a dimerization domain (zipper), binds to the first CLR through the dimerized dimerization domain. According to Figure 3 Examples of signal transduction molecules are given in SEQ ID NO:14, which contains a short linker and in which zipper a is represented by a Jun-zipper for dimerization with the Fos-zipper of SEQ ID NO:15.

[0055] According to Figure 3 Further examples of signal transduction molecules with long linkers are given in SEQ ID NO:16, which includes a Jun-zipper as a dimerization domain, and SEQ ID NO:17 with a Fos-zipper (e.g., for dimerization with the Jun-zipper of SEQ ID NO:12).

[0056] Figure 4 Shows a schematic diagram of an embodiment of the signal transduction molecule CLR of the present invention, wherein the transmembrane and signal transduction domains are attached only to one of the two heavy chains, here the β1 and β2 domains of the HLA-DR1β chain. In this context, the first signal transduction molecule consists of HLA-DR1 (HLA-DRB1*01:01:01) as its ligand domain, a linker, a dimerization domain (zipper), a spacer, a transmembrane domain, and the intracellular 4-1BB domain and CD3ζ (CD3z) domain as signal transduction domains. The second CLR, consisting of a ligand domain represented by the β chain of HLA-DRA*01:01:01, a linker, and a dimerization domain (zipper), binds to the first CLR through the dimerized dimerization domain.

[0057] According to Figure 4 Examples of signal transduction molecules are given in SEQ ID NO:18, which contains a short linker and in which zipper a is represented by a Fos-zipper for dimerization with the Jun-zipper of SEQ ID NO:19, which also has a short linker.

[0058] According to Figure 4Further examples of signal transduction molecules with long linkers are given in SEQ ID NO:20, which includes a Fos-zipper as a dimerization domain, and SEQ ID NO:21, which has a Jun-zipper (e.g., for dimerization with the Fos-zipper of SEQ ID NO:20).

[0059] Generally, for HLA class II molecules as ligand domains, the ligand domain can consist of the α1 domain and the β1 domain, and optionally the α2 and β2 domains, such as the α1 and α2 domains or the β1 and β2 domains of the HLA class II heavy chain, preferably the extracellular portion of the HLA class II transmembrane domain at the C-terminus of the heavy chain.

[0060] Figure 5 The preferred signal transduction molecules of the present invention are shown, in Figure 5 A) of, the nucleic acid sequence encodes the following embodiments from 5' to 3': a signal transduction molecule and, as an optional component, a selection marker containing a secretory signal peptide (SP) and a selection marker and / or a suicide gene separated by a self-cleaving peptide element derived from Thosea asigna virus 2A (T2A), which is represented by a truncated epidermal growth factor (EGFRt). The encoded signal transduction molecule contains, from the N-terminus to the C-terminus, a ligand domain composed of a secretory signal peptide (SP, exon 1 of HLA-A*02:01:01) and exons 2-4 of HLA-A*02:01:01, a spacer (IgG4-Fc spacer), a transmembrane domain of human CD28 (hCD28TMD), and an intracellular signal transduction domain that is a combination of a co-stimulatory h4-1BB domain (h4-1BB) and an hCD3ζ domain (hDC3ζ).

[0061] The ligand domain acts as a ligand or binding domain for specific binding to the BCR of B cells and the TCR of T cells (BCR / TCR binding domain), respectively. Generally preferably, the antigen domain can be a partial or entire HLA class I heavy chain sequence. In this example, the antigen domain is a truncated HLA-A2 (A*02:01:01) heavy chain, which consists of the α1, α2, and α3 domains. The exemplary amino acid sequence is given in SEQ ID NO:1, which consists of an N-terminal signal peptide and an antigen domain composed of the α1, α2, and α3 domains of HLA-A*02:01.

[0062] For example, the spacer can contain 10 to 250 amino acids (AA) of a known spacer, such as those of the Ig hinge region, such as 12 AA to 229 AA of the Ig hinge region, such as the hinge region of IgG4-Fc. Here, a spacer of 12 AA (short) or 229 AA (long) of the IgG4-Fc hinge region is used.

[0063] In Figure 5There is no clear depiction of the preferred extracellular portion of the HLA transmembrane domain arranged at the C-terminus of the ligand domain between the ligand domain and the linker.

[0064] Figure 5 In B) and C) of, the ligand domains forming TCR and BCR ligands are composed of exon 1 of HLA-DRA*01:01:01 (exon 1 of HLA-DRA*01:01:01) and exons 2-3 of HLA-DRA*01:01:01, and exon 1 of HLA-DRB1*01:01:01 and exons 2-3 of HLA-DRB1*01:01:01, respectively. Embodiments comprising part or the whole of the HLA class II molecule as the ligand domain preferably comprise a dimerization domain to provide assembly with the HLA class II β-chain of the HLA class II molecule, also known as the zipping effect.

[0065] Figure 5 B) of shows an embodiment in which the dimerization domain is arranged between the ligand domain and the spacer, and preferably, the dimerization domain contains a linker at its N-terminus. The dimerization domain is shown as one partner of a molecule or leucine zipper (zipper a), which can be, for example, one of the dimerization partners of a jun / fos zipper, a basic / acidic zipper, or an EE1234L / RR1234L zipper. SEQ ID NO:2 gives the amino acid sequences of the signal peptide, the antigenic domain of the α1 and α2 domains of HLA-DRA*01:01, the extracellular portion of the transmembrane domain, the linker, and the Jun zipper (zipper a) as the dimerization domain.

[0066] Figure 5 C) of shows an embodiment in which the dimerization domain is the counterpart of zipper a (zipper b). SEQ ID NO:3 gives the amino acid sequences of the signal peptide, the ligand domain of the β1 and β2 domains of HLA-DRB1*01:01, the extracellular portion of the transmembrane domain, the linker, and the Fos zipper (zipper b) as the dimerization domain.

[0067] Alternative amino acid sequences of the linker domain and the dimerization domain are given in SEQ ID NO:4 and SEQ ID NO:5 (the zipper portions of which can dimerize) and SEQ ID NO:6 and SEQ ID NO:7 (the zipper portions of which can dimerize).

[0068] Examples

[0069] Example: Specific elimination of B cells

[0070] As an example of immune cells that elicit unwanted immune activity to be treated, mouse hybridoma B lymphocyte HB-82 cells were used, which express the HLA-A2-specific antibody BB7.2 as a BCR on their cell surface and secrete the HLA-A2-specific antibody BB7.2 into the supernatant in culture. Figure 6 The FACS results showing the detection of the BB7.2 antibody on the surface of HB-82 cells, which were stained with anti-mouse IgG(F(ab)2) and labeled with phycoerythrin, are shown. This shows that BB7.2 is not only secreted into the supernatant but also strongly expressed on the surface of HB-82 cells.

[0071] The antibody produced in the HB-82 cell supernatant was able to immunostain HLA-A2+ lymphocytes in FACS analysis using anti-mouse IgG(F(ab)2) labeled with phycoerythrin as a secondary antibody and kill HLA-A2+ lymphocytes in a complement-dependent cytotoxicity assay. This shows that the BB7.2 antibody binds to HLA-A2+ lymphocytes.

[0072] In separate cultures, CD8+ T cells were stimulated with anti-CD3 and anti-CD38 antibodies for controlled expansion. As a control, K562 cells were treated in the same way. The next day, the cells were transduced with a lentivirus encoding the nucleic acid sequence of a CLR containing exons 2 to 4 of HLA-A*02:01:01 as a ligand domain according to Figure 5 A). In parallel batches, the lentiviral vector encoded two CLR variants, namely a 12AA short spacer with an IgG4-Fc hinge region (SEQ ID NO:8) and a 229AA long spacer with an IgG4-Fc hinge region (SEQ ID NO:9). Nine days after stimulation, the transduced T cells and K562 control cells were sorted by flow cytometry (FACS) using co-expressed EGFRt as a selection marker.

[0073] The cells expressing the selection marker were cultured for another 5 days, and the expression of CLR and their activity against immune cells and against the ligand domain of CLR were analyzed. The transduced cells were contacted with the supernatant of HB-82 cells containing the BB7.2 antibody. Secondary staining was performed with anti-mouse IgG(F(ab)2) labeled with phycoerythrin. The FACS results are depicted in Figure 7 and show that the BB7.2 antibody effectively binds to immune cells expressing CLR and specifically binds to the ligand domain of CLR.

[0074] Figure 7Shows the FACS results of the following items, A) control cells, B) T cells, namely untransduced T cells (untransduced T cells) and untransduced control cells (untransduced K562), cells transduced with constructs containing short spacers T cells (HLAI CLR_short T cells) or control cells (HLA ICLR_short K562), and cells transduced with constructs containing long spacers T cells (HLA I CLR_long T cells) or control cells (HLA I CLR_long K562). The results show strong expression of CLR in both control cells (K562) and T cells, with the short spacer showing stronger CLR expression, especially in T cells.

[0075] The activity of T cells expressing CLR was analyzed by contacting and co-culturing them with freshly washed murine hybridoma B lymphocytes HB-82, which express the HLA-A2-specific BCR BB7.2 associated with the secretion of antibody BB7.2. T cells expressing CLR were used at a ratio of 0.5:1 or 1:1 or 5:1 with HB-82 cells. For all ratios, effective killing of HB-82 cells was observed, showing that CLR expression in T cells leads to effective elimination of B cells expressing BCR specific for the CLR ligand domain. For CLR with a short spacer, higher cytotoxic efficacy of T cells expressing it was determined. The results of the analysis of lactate dehydrogenase (LDH) released after 48 hours of co-culture are shown in Figure 8 and show effective cytotoxicity found in all ratios of T cells expressing CLR.

[0076] In addition, after co-culturing for 48 hours at effector-to-target ratios of 5:1 or 1:1 and 0.5:1, the expression of activation markers on T cells expressing CLR was analyzed. The results for CD8+ T cells for CD137 are shown in Figure 9A and for CD69 are shown in Figure 9B and for CD25 are shown in Figure 9C . These results show that CD8+ T cells transduced to express CLR clearly show the expression of activation markers CD137, CD69, and CD25, for all ratios and short and long spacer variants, with the short spacer variant having stronger activation.

[0077] These results of the B cell receptor example show that the signaling molecule CLR of the present invention guides T cells expressing the signaling molecule to kill immune cells, taking B cells as an example, which are specific for the associated antigen targeted by the immune cells, for example, specific for the B cell receptor and T cell receptor of the immune cells respectively.

[0078] The present invention provides the following embodiments:

[0079] 1. A fusion protein for use as a signal transduction molecule, comprising a ligand domain, a spacer, a transmembrane domain, and an intracellular signal transduction domain, wherein the ligand domain comprises at least one epitope of an associated antigen against which an undesired immune response is directed.

[0080] 2. The fusion protein according to item 1, comprising a dimerization domain arranged between the ligand domain and the transmembrane domain.

[0081] 3. The fusion protein according to one of the foregoing items, wherein the ligand domain comprises two chains, each chain of the ligand domain is covalently linked to the dimerization domain, and the dimerization domain is dimerized.

[0082] 4. The fusion protein according to one of the foregoing items, wherein the ligand domain comprises two chains, each of the chains is linked to the dimerization domain, and only one of the dimerization domains is covalently linked to the spacer, transmembrane domain, and intracellular signal transduction domain.

[0083] 5. The fusion protein according to one of items 1 to 2, wherein the ligand domain comprises two chains and each of these chains is covalently linked to the dimerization domain, spacer, transmembrane domain, and intracellular signal transduction domain, and the spacer, transmembrane domain, and intracellular signal transduction domain covalently linked to each chain may each have the same amino acid sequence or different amino acid sequences.

[0084] 6. The fusion protein according to one of the foregoing items, wherein the spacer has a length of 10 to 250 amino acids.

[0085] 7. The fusion protein according to one of the foregoing items, wherein the dimerization domain dimerizes only into a heterodimer.

[0086] 8. The fusion protein according to one of the foregoing items, wherein the intracellular signal transduction domain is a combination of the h4-1BB domain and the hCD3ζ domain, or a combination of the intracellular hCD28 signal transduction domain and the hCD3ζ domain.

[0087] 9. The fusion protein according to one of the foregoing items, wherein the ligand domain comprises at least part of HLA class I or at least part of HLA class II molecules.

[0088] 10. The fusion protein according to item 5, wherein the part of HLA class I comprises at least one mutation at amino acid positions 74, 223, 224, 225, 226, 227, 229, and 245, wherein the numbering refers to HLA class I without a signal peptide.

[0089] 11. The fusion protein according to item 5 or 6, wherein the portion of the HLA class II contains at least one mutation at amino acid positions 88, 90, and 176 of the α-chain, and / or at least one amino acid position among positions 46, 54, 55, 56, 104, 114, 116, 134, 135, 136, 137, 138, 139, 141, 142, 143, 144, 145, 148, 158, 160, and 162 of the β-chain, where the numbering refers to HLA class II without the signal peptide.

[0090] 12. The fusion protein according to one of the preceding items, which comprises a first signaling molecule, the first signaling molecule comprising a ligand domain, optionally an extracellular portion of the HLA transmembrane domain, a linker, a dimerization domain, a spacer, a transmembrane domain, and at least one intracellular signaling domain, wherein the dimerization domain dimerizes with the dimerization domain of a second signaling molecule, the second signaling molecule comprising a ligand domain, an extracellular portion of the HLA transmembrane domain, a linker, a dimerization domain, a spacer, a transmembrane domain, and at least one intracellular signaling domain, or the second signaling molecule consists of a ligand domain, a linker, and a dimerization domain.

[0091] 13. The fusion protein according to one of the preceding items, which comprises a first signaling molecule, the first signaling molecule comprising a ligand domain, optionally an extracellular portion of the HLA transmembrane domain, a spacer, a transmembrane domain, and at least one intracellular signaling domain, or consisting of a ligand domain, optionally an extracellular portion of the HLA transmembrane domain, a spacer, a transmembrane domain, and at least one intracellular signaling domain.

[0092] 14. The fusion protein according to one of the preceding items, which is used for treating immune rejection against a graft, for treating an autoimmune disease, or for treating an allergy.

[0093] 15. The fusion protein according to item 10, which is used for treating an autoimmune disease, wherein the ligand domain is the associated antigen targeted by the autoimmune disease.

[0094] 16. The fusion protein according to one of the preceding items, which consists of a first signaling molecule and a second signaling molecule, and comprises a ligand domain consisting of two chains, wherein the first signaling molecule consists of one chain of the ligand domain, a dimerization domain, a spacer, a transmembrane domain, and an intracellular signaling domain from the N-terminus to the C-terminus,

[0095] wherein the second signaling molecule comprises the other chain of the ligand domain and a dimerization domain,

[0096] wherein the dimerization domain of the first signaling molecule dimerizes with the dimerization domain of the second signaling molecule, and

[0097] Wherein the ligand domain comprises at least one epitope of an associated antigen against which an undesired immune response is directed.

[0098] 17. The fusion protein according to one of the preceding items, characterized in that the second signaling molecule consists, from the N-terminus to the C-terminus, of the other chain of the ligand domain, a linker, and a dimerization domain.

[0099] 18. The fusion protein according to one of the preceding items, characterized in that the second signaling molecule consists, from the N-terminus to the C-terminus, of the other chain of the signaling domain, a linker, a dimerization domain, a spacer, a transmembrane domain, and an intracellular signaling domain.

[0100] 19. The fusion protein according to one of the preceding items, characterized in that the ligand domain is an HLA class II molecule, wherein one chain of the ligand domain of the first signaling molecule consists of the α1 and optionally α2 domains of the HLA class II molecule, and the other chain of the ligand domain of the second signaling molecule consists of the β1 and optionally β2 domains of the HLA class II molecule.

[0101] 20. The fusion protein according to one of the preceding items, characterized in that the ligand domain is an HLA class II molecule, wherein one chain of the ligand domain of the first signaling molecule consists of the β1 and optionally β2 domains of the HLA class II molecule, and the other chain of the ligand domain of the second signaling molecule consists of the α1 and optionally α2 domains of the HLA class II molecule.

[0102] 21. The fusion protein according to one of the preceding items, characterized in that the ligand domain is an HLA class I molecule and one chain of the first signaling molecule is at least part of the heavy chain of the HLA class I molecule, and the other chain of the ligand domain of the second signaling molecule consists of β2-microglobulin, which has a dimerization domain at its N-terminus and / or at its C-terminus.

[0103] 22. An immune cell expressing the fusion protein according to one of the preceding items, for use in treating immune rejection against a graft, for treating an autoimmune disease, or for treating an allergy.

[0104] 23. The immune cell according to item 22, wherein the immune cell is a T cell, a primary T cell, an NK cell, or a progenitor cell or cell line of one of these.

[0105] 24. The immune cell according to one of items 22 to 23, wherein the immune cell is immunocompatible with the recipient.

[0106] 25. The immune cell according to one of items 22 to 24, wherein the immune cell is for administration to a patient before or after transplantation.

[0107] 26. A nucleic acid sequence encoding the fusion protein according to one of items 1 to 21.

Claims

1. Use of a fusion protein as a signal transduction molecule in the preparation of a medicament for treating immune rejection against a graft, wherein the fusion protein comprises a ligand domain, a spacer, a transmembrane domain, and an intracellular signal transduction domain, and the intracellular signal transduction domain activates T cell effector functions upon reaction of an antigen domain in T cells, wherein the ligand domain contains at least one epitope of an associated antigen against which an undesired immune response is directed; wherein the ligand domain consists of the α1 domain and the α2 domain of an HLA class I molecule and optionally the α3 domain; the ligand domain comprises at least one mutation that effectively reduces or eliminates the binding of CD8 of CD8+ T cells to T cells expressing the fusion protein.

2. The use according to claim 1, wherein The at least one mutation is at least one mutation at amino acid positions No. 74, 223, 224, 225, 226, 227, 229, and 245, where the numbering refers to the HLA class I molecule without a signal peptide.

3. Use of a fusion protein as a signal transduction molecule in the preparation of a medicament for treating immune rejection against a graft, wherein the fusion protein comprises a ligand domain, a spacer, a transmembrane domain, and an intracellular signal transduction domain, and the intracellular signal transduction domain activates T cell effector functions upon reaction of an antigen domain in T cells; wherein the ligand domain consists of the α1 domain and β1 of an HLA class II molecule and optionally the α2 domain and β2 domains; the ligand domain comprises at least one mutation that effectively weakens or eliminates the interaction with CD4+ T cells.

4. The use according to claim 3, characterized in that, The mutation is at least one mutation at amino acid positions No. 88, 90, and 176 in the α chain, and / or at least one mutation at amino acid positions No. 46, 54, 55, 56, 104, 114, 116, 134, 135, 136, 137, 138, 139, 141, 142, 143, 144, 145, 148, 158, 160, and 162 in the β chain, where the numbering refers to the HLA class II molecule without a signal peptide.

5. Use according to any one of the preceding claims, wherein the ligand domain comprises two chains, each chain of the ligand domain is covalently linked to a dimerization domain, and the dimerization domain is dimerized.

6. Use according to any one of the preceding claims, wherein the ligand domain comprises two chains, each chain is linked to a dimerization domain, and only one is covalently linked to the spacer, transmembrane domain, and intracellular signal transduction domain.

7. Use according to any one of claims 1 to 5, wherein the ligand domain comprises two chains, and each of these chains is covalently linked to a dimerization domain, a spacer, a transmembrane domain, and an intracellular signal transduction domain, and the spacer, transmembrane domain, and intracellular signal transduction domain covalently linked to each chain may each have the same amino acid sequence or different amino acid sequences.

8. Use according to any one of the preceding claims, wherein the spacer has a length of 10 to 250 amino acids.

9. Use according to any one of claims 6 to 8, wherein the dimerization domain dimerizes only to form a heterodimer.

10. The use according to any one of the preceding claims, wherein the intracellular signaling domain is a combination of the h4-1BB domain and the hCD3ζ domain, or a combination of the intracellular hCD28 signaling domain and the hCD3ζ domain.

11. Use according to any one of claims 6 to 10, characterized in that, The ligand domain of the first signaling molecule consists of the α1 domain and optionally the α2 domain of the HLA class II molecule, and the ligand domain of the second signaling molecule consists of the β1 domain and optionally the β2 domain of the HLA class II molecule.

12. Use according to any one of claims 6 to 10, characterized in that, One chain of the ligand domain of the first signaling molecule consists of the β1 domain and optionally the β2 domain of the HLA class II molecule, and the other chain of the ligand domain of the second signaling molecule consists of the α1 domain and optionally the α2 domain of the HLA class II molecule.

13. Use according to any one of claims 6 to 10, characterized in that, One chain of the signaling molecule is the heavy chain of the HLA class I molecule, and the other ligand domain of the second signaling molecule consists of β2-microglobulin, which has a dimerization domain at its N-terminus and / or C-terminus.

14. Use of immune cells in the preparation of a medicament for treating immune rejection against a graft, wherein the immune cells express the fusion protein according to any one of claims 1 to 13.

15. The use according to claim 14, wherein the immune cells are T cells, primary T cells, NK cells, progenitor cells or cell lines of one of these cells.

16. The use according to any one of claims 14 to 15, wherein the immune cells are immunocompatible with the recipient.

17. The use according to any one of claims 14 to 16, wherein the immune cells are for administration to a patient before or after transplantation.

18. A nucleic acid sequence encoding the fusion protein according to any one of claims 1 to 13.

Images