cell

Abstract

To provide a cell.SOLUTION: There is provided an effector immune cell which expresses a cell surface receptor or receptor complex which specifically binds an antigen recognition receptor of a target immune cell. The effector immune cell is engineered such that, when a synapse is formed between the effector immune cell and the target immune cell, the capacity of the effector immune cell to kill the target immune cell is greater than the capacity of the target immune cell to kill the effector immune cell. There is also provided the use of such a cell in methods for treating cancer and preventing allograft rejection and GVHD.SELECTED DRAWING: None

Description

Technical Field

[0001] Field of the Invention The present invention relates to effector immune cells that specifically bind to antigen recognition receptors of target immune cells, and particularly to an approach for controlling the killing of such effector immune cells by target cells.

Background Art

[0002] Background of the Invention Prevention of Rejection In solid organ transplantation or hematopoietic stem cell transplantation (HSCT), if the HLA is not compatible between the recipient and the donor, organ rejection or graft-versus-host disease (GVHD) can occur, respectively. Although immunosuppressive drugs can alleviate these outcomes, since they have a wide-ranging inhibitory effect on immune cells, they increase the risk of opportunistic infections.

[0003] Alloreactive T cells that recognize HLA incompatibility via the T cell receptor (TCR) are the main mediators of rejection and GVHD. The specificity of CD8+ T cells is determined by the clonotype of the TCR that recognizes short antigenic peptides presented on MHC class I molecules. MHC class I molecules are non-covalently bound heterodimers composed of a transmembrane highly polymorphic α-chain and a non-membrane-bound non-polymorphic β2-microglobulin (β2m).

[0004] Margalit et al ((2002) International Immunology 15:1379-1387) described an approach for converting TCR ligands into T cell activating receptors. This paper describes T cells expressing a β2 microglobulin polypeptide containing an antigen peptide attached to the N-terminus via a transmembrane domain and a CD3ζ-derived endodomain and linker attached to the C-terminus. Such cells were found to express high levels of surface peptide-class I complexes and respond in a peptide-specific manner to antibodies and target T cells. Expression of such peptide-linker-β2m-TM-CD3ζ polypeptides in T cells makes it possible to specifically target pathogenic CD8+ T cells that recognize specific antigen peptides.

[0005] CAR-T cells Traditionally, antigen-specific T cells have been generated by the selective expansion of peripheral blood T cells that are naturally specific for the target antigen. However, it is difficult and almost impossible to select and expand a large number of T cells specific for most cancer antigens. Since transgenic expression of chimeric antigen receptors (CARs) can generate a large number of T cells specific for any surface antigen by ex vivo viral vector transduction of peripheral blood T cell populations, gene therapy using integrating vectors is a solution to this problem.

[0006] A chimeric antigen receptor is a protein that links the specificity of a monoclonal antibody (mAb) to the effector function of a T cell. Its normal form is that of a type I transmembrane domain protein with an antigen recognition amino terminus, a spacer, a transmembrane domain all connected to a compound endodomain that transmits survival and activation signals for the T cell.

[0007] The most common form of these molecules is a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody that recognizes a target antigen, via a spacer and a transmembrane domain, to a signaling endodomain. Such molecules activate T cells in response to recognition by their target scFv. When T cells express such a CAR, the T cells recognize and kill target cells that express the target antigen. Several CARs have been developed against tumor-associated antigens, and adoptive transfer approaches using such CAR-expressing T cells are currently in clinical trials for various cancer treatments.

[0008] After injection, CAR T cells engraft within the recipient and expand after encountering target-bearing cells. The CAR T cells then persist, and their population slowly shrinks over time. The persistence of CAR T cells can be determined in clinical trials by real-time PCR for the transgene in blood samples or by flow cytometry for the CAR in blood samples, and clinical investigators have found a correlation between persistence and duration of response. This correlation is particularly prominent in the treatment of CD19 CAR in B acute lymphoblastic leukemia (ALL). In this context, engraftment failure of CAR T cells often occurs prior to leukemia relapse.

[0009] CAR T cells can activate a cellular immune response that can induce rejection of the CAR T cells. This is due to the immunogenicity of engineered components in the cell by either a non-self protein or a non-self sequence formed at the junction between the receptor and self proteins used to create other engineered components.

[0010] CAR is an artificial protein typically composed of a targeting domain, a spacer domain, a transmembrane domain, and a signaling domain. The targeting domain typically derives from an scFv that can be murine. This scFv can be a human scFv or a humanized scFv, and while the other components individually derive from self-proteins, the junctions between them can, as before, exhibit immunogenicity. For example, within the scFv, there are junctions between the heavy chain and the linker and between the linker and the light chain. Then, there is a junction between the scFv and the spacer domain. If the transmembrane domain is not continuous with the spacer, there is an additional junction in that portion. Similarly, if the transmembrane domain is not continuous with the amino-terminal portion of the endodomain, there is an additional junction in that portion. Finally, most endodomains have at least two components, and occasionally, subsequently, there are additional junctions between each component.

[0011] Furthermore, CAR T cells are often engineered using additional components. These components include suicide genes (e.g., the HSV-TK enzyme). This enzyme has been found to be highly immunogenic and to cause cellular immune depletion of CAR T cells in situations distinct from the marked immunosuppression in haplotype-matched hematopoietic stem cell transplantation. Since almost all engineered components, including fusions between two proteins or the use of heterologous proteins, can exhibit immunogenicity, other low-immunogenic suicide genes can still exhibit some immunogenicity.

[0012] In many situations, CAR T cells are generated from autologous T cells. In this situation, an allogeneic response does not occur. Depending on the situation, T cells from an allogeneic donor are used. This can occur, for example, if the patient has undergone an allogeneic hematopoietic stem cell transplantation. In this case, the harvested T cells will be allogeneic. Otherwise, the patient may have insufficient T cells for generating the CAR T cell product due to chemotherapy-induced lymphopenia.

[0013] Allogeneic cell rejection can be due to minor or major incompatibility. Minor incompatibility occurs when allogeneic T cells match the recipient's human leukocyte antigen (HLA). In this case, there is no HLA difference between individuals, and thus rejection occurs due to minor histocompatibility antigens that present non-self (donor) epitopes / immunogenic peptides on HLA. When the donor and recipient are incompatible or only partially compatible, the T cell receptor (TCR) on the recipient's endogenous T cells can interact with the incompatible HLA in a non-specific way and consequently cause rejection. Both minor and major forms of allogeneic rejection are caused by HLA interacting with TCR.

[0014] WO2019 / 073248 and UK Patent Application Publication No. 1904971.7 describe an approach that includes coupling MHC class I or II on CAR-expressing cells to TCR on T cells to directly or indirectly induce signal transduction in CAR-expressing cells. When CAR-expressing cells are administered to a subject, MHC class I or II on these cells interacts with any endogenous reactive T cells present in the subject by recognition of the peptide / MHC complex. Any such reactive T cells in the subject are depleted by activation of cytotoxicity-mediated cell death by the CAR-expressing cells.

[0015] CAR-mediated approach for treating T cell malignancies Lymphoid malignancies can be broadly divided into those derived from either T cells or B cells. T cell malignancies are a group of clinically and biologically heterogeneous disorders that together account for 10 - 20% of non-Hodgkin lymphomas and 20% of acute leukemias. The most commonly identified histological subtypes are peripheral T cell lymphoma, not otherwise specified (PTCL-NOS); angioimmunoblastic T cell lymphoma (AITL), and anaplastic large cell lymphoma (ALCL). A subset of 20% of all acute lymphoblastic leukemias (ALL) have a T cell phenotype.

[0016] These conditions typically behave aggressively, for example, compared to B-cell malignancies, and the estimated 5-year survival rate is only 30%. In the case of T-cell lymphoma, there is a high proportion of patients with disseminated disease, an unfavorable International Prognostic Index (IPI) score, and extranodal disease. Chemotherapy alone is usually not effective, and less than 30% of patients are cured with current treatments. WO2015 / 132598 describes a method capable of depleting malignant T cells in a subject without affecting a significant proportion of healthy T cells. In particular, WO2015 / 132598 describes a chimeric antigen receptor (CAR) that specifically binds to the T-cell receptor beta constant region 1 (TRBC1) or TRBC2. All of the above approaches involve specific binding of the T-cell receptor on the target T cells. In this context, the targeted T cells "fight back" due to the ligation of their TCR, thereby being able to deplete the transplanted / desirable T cells.

Prior Art Documents

Patent Documents

[0017]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0018]

Non-Patent Document 1

Brief Description of the Drawings

[0019]

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[0020] Summary of Aspects of the Invention The inventors have developed an approach for manipulating the balance between effector immune cells (cell A), which target autoreactive or pathogenic immune cells (cell B), such that the engineered immune cells have a selective advantage and kill cell B, and cell B, which kills cell A, in favor of cell A that kills cell B.

[0021] Accordingly, in a first aspect, the present invention provides an effector immune cell that expresses a cell surface receptor or receptor complex that specifically binds to an antigen recognition receptor of a target immune cell, wherein when a synapse is formed between the effector immune cell and the target immune cell, the effector immune cell is engineered such that its ability to kill the target immune cell is higher than the ability of the target immune cell to kill the effector immune cell.

[0022] In a first embodiment of the first aspect of the present invention, the effector immune cell is engineered to be resistant to an immunosuppressive agent.

[0023] For example, the effector immune cell can be engineered to be resistant to one or more calcineurin inhibitors.

[0024] In this regard, the effector immune cell can express: Calcineurin A containing mutations T351E and L354A with reference to the sequence shown as SEQ ID NO: 65; Calcineurin A containing mutations V314R and Y341F with reference to the sequence shown as SEQ ID NO: 65; or Calcineurin B containing mutations L124T and K-125-LA-Ins with reference to the sequence shown as SEQ ID NO: 66.

[0025] The effector immune cell can be engineered to be resistant to rapamycin.

[0026] Effector immune cells may express a dominant negative C-terminal Src kinase (dnCSK) that confers resistance to multiple immunosuppressive agents.

[0027] In a second embodiment of the first aspect of the present invention, effector immune cells are engineered to express or overexpress a fusion protein comprising an immunosuppressive molecule or an extracellular domain of an immunosuppressive molecule.

[0028] The immunosuppressive molecule may bind to: PD-1, LAG3, TIM-3, TIGIT, BTLA, VISTA, CEACAM1-R, KIR2DL4, B7-H3, or B7-H4.

[0029] The immunosuppressive molecule may be selected from: PD-L1, PD-L2, HVEM, CD155, VSIG-3, galectin-9, HLA-G, CEACAM-1, LSECTin, FGL1, B7-H3, and B7-H4.

[0030] Effector immune cells may be engineered to express a fusion protein comprising an extracellular domain and a membrane localization domain of an immunosuppressive molecule.

[0031] Effector immune cells may be engineered to express a fusion protein comprising an extracellular domain of an immunosuppressive molecule and a costimulatory endodomain (such as those selected from CD28, ICOS, CTLA4, 41BB, CD27, CD30, OX-40, TACI, CD2, CD27, and GITR).

[0032] The antigen recognition receptor of the target immune cell may be, for example, a T cell receptor (TCR) or an activating killer cell immunoglobulin-like receptor (KAR).

[0033] The cell surface receptor of the effector immune cell may be, for example, a chimeric antigen receptor (CAR), and the antigen recognition receptor may be a T cell receptor (TCR).

[0034] When an effector immune cell expresses a TCR-specific CAR, the CAR can bind to TCR beta constant region 1 (TRBC1) or TRBC2.

[0035] Alternatively, the cell surface receptor complex of an effector immune cell can be an engineered MHC class I complex or an engineered MHC class II complex.

[0036] For example, the cell surface receptor complex can include an MHC class I polypeptide linked to an intracellular signaling domain; an MHC class II polypeptide; or beta-2 microglobulin.

[0037] The cell surface receptor complex has the following structure: peptide-L-B2M-endo (wherein "peptide" is a peptide that binds to the peptide binding groove of the MHC class I alpha chain; "L" is a linker, "B2M" is beta-2 microglobulin; "endo" is an intracellular signaling domain) and can be an engineered MHC class I complex that includes a molecule having the above structure.

[0038] The effector immune cell can include an MHC class I polypeptide: an MHC class II polypeptide; or beta-2 microglobulin linked to a component of the TCR / CD3 complex.

[0039] The effector immune cell can include an MHC class I polypeptide: an MHC class I polypeptide; an MHC class II polypeptide; or beta-2 microglobulin linked to CD3-zeta, CD3-epsilon, CD3-gamma, or CD3-delta via a linker peptide.

[0040] Effector immune cells can express a bispecific polypeptide comprising: (i) a first binding domain that binds to an MHC class I polypeptide, an MHC class II polypeptide, or β-2 microglobulin; and (ii) a second binding domain that binds to a component of the TCR / CD3 complex.

[0041] Effector immune cells can express an engineered polypeptide comprising a CD79α chain and / or a CD79β chain linked to an intracellular signaling domain.

[0042] Effector immune cells can express an engineered polypeptide comprising a binding domain that binds to an MHC class I polypeptide or an MHC class II polypeptide linked to an intracellular signaling domain. The binding domain can be an antibody-like binding domain.

[0043] Effector immune cells can express an engineered polypeptide comprising an MHC class II binding domain of CD4 or an MHC class I binding domain of CD8 linked to an intracellular signaling domain.

[0044] Effector immune cells of a first aspect of the invention are engineered to express a cell surface receptor (such as a CAR) or a receptor complex (such as an engineered MHC class I complex or an engineered MHC class II complex), and then further engineered such that when a synapse is formed between the effector immune cell and a target immune cell, the ability of the effector immune cell to kill the target immune cell is higher than the ability of the target immune cell to kill the effector immune cell.

[0045] Further engineering of effector immune cells can be, as described above, the following: (i) engineering the cells to be resistant to immunosuppressive agents, or (ii) engineering the cells to express or overexpress a fusion protein comprising an immune inhibitory molecule or an extracellular domain of an immune inhibitory molecule and may include.

[0046] The synapse formed between an effector immune cell and a target immune cell is formed when a cell surface receptor or receptor complex of the effector immune cell specifically binds to an antigen recognition receptor of the target immune cell.

[0047] In a second aspect, (i) a first nucleic acid sequence encoding a part of a cell surface receptor or cell surface receptor complex as defined herein; and (ii) a second nucleic acid sequence that, when expressed in a cell, confers resistance to an immunosuppressive agent on the aforementioned cell; and / or (iii) a third nucleic acid sequence encoding a fusion protein comprising an immune inhibitory molecule or an extracellular domain of an immune inhibitory molecule A nucleic acid construct comprising is provided.

[0048] In a third aspect, a vector comprising the nucleic acid construct of the second aspect of the invention is provided.

[0049] In a fourth aspect, a kit of vectors, (i) a first vector comprising a nucleic acid sequence encoding a part of a cell surface receptor or cell surface receptor complex as defined herein; and (ii) a second vector comprising a nucleic acid sequence that, when expressed in a cell, confers resistance to an immunosuppressive agent on the aforementioned cell; and / or (iii) a third vector comprising a nucleic acid sequence encoding a fusion protein comprising an immune inhibitory molecule or an extracellular domain of an immune inhibitory molecule A kit of vectors comprising is provided.

[0050] In a fifth aspect, a pharmaceutical composition comprising a plurality of effector immune cells of the first aspect of the invention is provided.

[0051] In a sixth aspect, a pharmaceutical composition of the fifth aspect of the invention for use in the treatment of a disease is provided.

[0052] In a seventh aspect, there is provided a method of treating a disease, comprising the step of administering to a subject the pharmaceutical composition of the fifth aspect of the present invention.

[0053] The method may include the following steps: (i) a step of administering a pharmaceutical composition to a subject, the pharmaceutical composition comprising a plurality of effector immune cells of the first aspect of the present invention engineered to be resistant to an immunosuppressive agent; and (ii) a step of administering the aforementioned immunosuppressive agent to the aforementioned subject.

[0054] In an eighth aspect, there is provided the use of a plurality of effector immune cells of the first aspect of the present invention in the manufacture of a medicament for the treatment of a disease.

[0055] The disease can be cancer.

[0056] In a ninth aspect, there is provided a method of producing effector immune cells of the first aspect of the present invention, the method comprising the step of introducing ex vivo into the aforementioned cells a kit of the nucleic acid construct of the second aspect of the present invention, the vector of the third aspect of the present invention, or the vector of the fourth aspect of the present invention.

[0057] In a tenth aspect, there is provided a method of depleting alloreactive immune cells from a population of immune cells, the method comprising the step of contacting the aforementioned population of immune cells with a plurality of effector immune cells of the first aspect of the present invention, wherein the aforementioned plurality of effector immune cells express an engineered MHC class I complex or MHC class II complex as defined herein.

[0058] In an eleventh aspect, there is provided a method of treating or preventing rejection of a graft after allotransplantation, the method comprising the step of administering to a recipient subject a plurality of effector immune cells derived from a donor subject for the aforementioned allotransplantation, wherein the aforementioned plurality of effector immune cells express an engineered MHC class I complex or MHC class II complex as defined herein.

[0059] In a twelfth aspect, a method of treating or preventing graft-versus-host disease (GVHD) associated with allogeneic transplantation, the method comprising contacting the allograft described above with a plurality of effector immune cells of the first aspect of the present invention, wherein the plurality of effector immune cells express an engineered MHC class I complex or MHC class II complex as defined herein.

[0060] Allogeneic transplantation may include adoptive transfer of allogeneic or autologous immune cells.

[0061] In a thirteenth aspect, an allograft depleted of alloreactive immune cells by the method of the twelfth aspect of the present invention is provided.

Mode for Carrying Out the Invention

[0062] Detailed Description Some clinical applications relate to the generation of effector immune cells that recognize and deplete a normal subset of immune cells by recognizing their antigen recognition receptors.

[0063] In this context, the targeted normal immune cells can "fight back" and deplete the effector immune cells. The present invention relates to the engineering of effector immune cells such that the effector immune cells are immunologically "advantageous" over the target immune cells, such that when a synapse is formed between the effector immune cells and the targeted immune cells, the effector immune cells are dominant.

[0064] There are various situations in which the effector cells can "fight back", including the following: (i) A situation in which the effector immune cells express a CAR that specifically binds to the T cell receptor of T cells; (ii) A situation in which the effector immune cells express an MHC I or II complex engineered to deplete alloreactive or autoreactive T cells.

[0065] These situations will be described in more detail below.

[0066] Chimeric antigen receptor against the TCR complex The effector immune cells of the present invention can express a chimeric antigen receptor (CAR). In particular, the aforementioned cells can express a CAR that specifically binds to a component of the T cell receptor (TCR) or TCR:CD3 complex.

[0067] Classical chimeric antigen receptors (CARs) are chimeric type I transmembrane proteins in which an extracellular antigen recognition domain (binder) is connected to an intracellular signaling domain (endodomain) (see Figure 3). The binder is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but can be based on other formats containing antibody-like antigen-binding sites. The spacer domain can be used to isolate the binder from the membrane and orient it appropriately. A common spacer domain used is the Fc of IgG1. Smaller spacers, for example, depending on the antigen, a stalk from CD8α, or even just the IgG1 hinge alone may be sufficient. The transmembrane domain anchors the protein within the cell membrane and connects the spacer to the endodomain.

[0068] Initial CAR designs had an endodomain derived from the intracellular portion of either the γ-chain of FcεR1 or CD3ζ. As a result, these first-generation receptors transmitted immunological signal 1 and were sufficient to induce T cell killing of cognate target cells but were unable to fully activate T cells to proliferate and survive. To overcome this limitation, composite endodomains were constructed such that fusing the intracellular portion of a T cell costimulatory molecule with the intracellular portion of CD3ζ results in a second-generation receptor capable of simultaneously transmitting activation and costimulatory signals upon antigen recognition. The most commonly used costimulatory domain is the costimulatory domain of CD28. This provides the most potent costimulatory signal (i.e., immunological signal 2 that induces T cell proliferation). Some receptors have also been described that include TNF receptor family endodomains (such as closely related OX40 and 41BB that transmit survival signals). Even more potent third-generation CARs having endodomains capable of transmitting activation, proliferation, and survival signals are described herein.

[0069] When the CAR binds to the target antigen, this binding transmits an activation signal to the T cell in which the CAR is expressed. Thus, the CAR directs the specificity and cytotoxicity of the T cell towards tumor cells expressing the targeted antigen.

[0070] Thus, a CAR typically includes, or is associated with, an intracellular domain that includes: (i) an antigen-binding domain; (ii) a spacer; (iii) a transmembrane domain; and (iii) a signaling domain.

[0071] A CAR can have the following general structure: Antigen-binding domain - spacer domain - transmembrane domain - intracellular signaling domain (endodomain).

[0072] Antigen-binding domain The antigen-binding domain is part of the CAR that recognizes the antigen. In classical CARs, the antigen-binding domain contains a single-chain variable fragment (scFv) derived from a monoclonal antibody. CARs with domain antibodies (dAbs) or VHH or Fab-based antigen-binding domains have also been produced.

[0073] Alternatively, the CAR may contain a ligand for the target antigen. For example, a B cell maturation antigen (BCMA)-binding CAR with an antigen-binding domain based on a ligand (a proliferation-inducing ligand (APRIL)) has been described.

[0074] Spacer Classical CARs contain a spacer sequence that connects the antigen-binding domain to the transmembrane domain and spatially separates the antigen-binding domain from the endodomain. A flexible spacer allows the antigen-binding domain to be oriented in different directions to facilitate binding.

[0075] Various sequences are commonly used as the spacer of the CAR (e.g., the IgG1 Fc region, the IgG1 hinge, or the human CD8 stalk).

[0076] WO2016 / 151315 describes a spacer that forms a coiled-coil domain and forms a multimeric CAR. For example, this describes a spacer based on cartilage oligomeric matrix protein (COMP) that forms a pentamer. The COMP spacer may include the sequence shown in SEQ ID NO: 1, or a shortened version thereof that forms a coiled-coil and thereby retains the ability to form a multimer. SEQ ID NO: 1 (COMP spacer) DLGPQMLRELQETNAALQDVRELLRQQVREITFLKNTVMECDACG

[0077] Transmembrane domain The transmembrane domain is part of the CAR that crosses the membrane. The transmembrane domain can be any protein structure that is thermodynamically stable within the membrane. This is typically an α-helix composed of several hydrophobic residues. The transmembrane domain of any transmembrane protein can be used to provide the transmembrane portion of the CAR. Those skilled in the art can determine the sequence and full length of the transmembrane domain of a protein using the TMHMM algorithm (http: / / www.cbs.dtu.dk / services / TMHMM-2.0 / ). Alternatively, an artificially designed TM domain may be used.

[0078] End domain The end domain is the signaling part of the CAR. The end domain can be part of the intracellular domain of the CAR or can associate with the aforementioned intracellular domain. After antigen recognition, the receptors cluster, native CD45 and CD148 are excluded from the synapse, and signals are transmitted to the cell. The most commonly used end domain component is the end domain component of CD3-zeta containing three ITAMs. This transmits an activation signal to T cells after binding to the antigen. CD3-zeta may not provide a fully competent activation signal, and additional co-stimulatory signaling may be required. Co-stimulatory signals promote the proliferation and survival of T cells. There are two main types of co-stimulatory signals: those belonging to the Ig family (CD28, ICOS) and those belonging to the TNF family (OX40, 41BB, CD27, GITR, etc.). For example, chimeric CD28 and OX40 can be used together with CD3-zeta to transmit proliferation / survival signals, or all three can be used together.

[0079] The end domain can include: (i) An ITAM-containing end domain (such as the end domain derived from CD3 zeta); and / or (ii) A co-stimulatory domain (such as the end domain derived from CD28 or ICOS); and / or (iii) A domain that transmits a survival signal (e.g., a TNF receptor family end domain such as OX-40, 4-1BB, CD27, or GITR).

[0080] Several systems have been described in which the antigen recognition moiety is present on a separate molecule derived from the signaling moiety (such as those described in WO015 / 150771; WO2016 / 124930, and WO2016 / 030691). Thus, the CAR of the present invention may include an antigen-binding component that includes an antigen-binding domain and a transmembrane domain, and this component may be capable of interacting with a separate intracellular signaling component that includes a signaling domain. The vector of the present invention may express a CAR signaling system that includes such an antigen-binding component and an intracellular signaling component.

[0081] Since the CAR may include a signal peptide, when the signal peptide is expressed inside the cell, the nascent protein goes to the endoplasmic reticulum and then towards the cell surface where it is expressed. The signal peptide may be present at the amino terminus of the molecule.

[0082] Target antigen A "target antigen" is an entity that is specifically recognized and bound by the antigen-binding domain of the CAR.

[0083] The target antigen may be an antigen present on cancer cells (e.g., a tumor-associated antigen).

[0084] As shown in Table 1 below, various tumor-associated antigens (TAAs) are known. The CAR may be capable of binding to such TAAs.

Table 1

[0085] The effector immune cells of the present invention may bind to the T cell receptor (TCR) complex on the target T cell. In particular, the effector immune cells of the present invention may bind to the TCRβ constant region (TRBC) of the TCR complex on the target T cell.

[0086] The T cell receptor (TCR) is expressed on the surface of T lymphocytes and is responsible for the recognition of antigens bound to major histocompatibility complex (MHC) molecules. When the TCR associates with an antigen peptide and MHC (peptide / MHC), T lymphocytes are activated through a series of biochemical events mediated by associated enzymes, co-receptors, specialized adapter molecules, and activated or released transcription factors.

[0087] The TCR is a disulfide-bonded membrane-anchored heterodimer, usually consisting of highly variable alpha (α) and beta (β) chains that are expressed as part of a complex with invariant CD3 chain molecules. T cells expressing this receptor are called α:β (or αβ) T cells (about 95% of total T cells). A small number of T cells express another receptor formed by variable gamma (γ) and delta (δ) chains, and this receptor is called γδ T cells (about 5% of total T cells).

[0088] Each of the α and β chains is composed of two extracellular domains: a variable (V) region and a constant (C) region (both are immunoglobulin superfamily (IgSF) domains that form an antiparallel β-sheet). The constant region is proximal to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail, while the variable region binds to the peptide / MHC complex. The constant region of the TCR consists of a short connecting sequence, and in this sequence, cysteine residues form disulfide bonds to link the two chains.

[0089] The variable domains of both the α and β chains of the TCR have three hypervariable regions or complementarity-determining regions (CDRs). Also, the variable region of the β chain has an additional hypervariable region (HV4), however, this usually does not contact the antigen and is therefore not considered a CDR.

[0090] In addition, the TCR includes up to five invariant chains γ, δ, ε (collectively referred to as CD3) and ζ. The CD3 and ζ subunits mediate TCR signaling via specific cytoplasmic domains, and the aforementioned domains interact with second messengers and adapter molecules after αβ or γδ recognition. Prior to cell surface expression of the TCR complex, pairs of the transmembrane and extracellular domains of the α and β chains of the TCR and the subunits that both CD3γ and CD3δ play roles in are assembled.

[0091] Thus, the TCR generally consists of the CD3 complex as well as the α and β chains of the TCR, and these chains are composed of variable and constant regions.

[0092] The locus (Chr7:q34) that supplies the TCRβ constant region (TRBC) produces two almost identical and functionally equivalent genes: TRBC1 and TRBC2 by duplication during evolution, and these genes differ by four amino acids in the mature protein. Each TCR contains either TRBC1 or TRBC2 in a mutually exclusive manner, and as such, each αβ T cell will express either TRBC1 or TRBC2 in a mutually exclusive manner.

[0093] Effector immune cells may be able to selectively bind to either TRBC1 or TRBC2 in a mutually exclusive manner.

[0094] As described above, each αβ T cell expresses a TCR that contains either TRBC1 or TRBC2. In clonal T cell disorders (such as T cell lymphoma or T cell leukemia), malignant T cells derived from the same clone will express either TRBC1 or TRBC2.

[0095] When administering TRBC1- or TRBC2-specific CAR-T cells to a patient with T-cell lymphoma or T-cell leukemia, malignant T cells are selectively depleted along with normal T cells expressing the same TRBC as the malignant T cells, but such treatment does not significantly deplete normal T cells expressing other TRBCs derived from malignant T cells.

[0096] Since TRBC-selective CAR-T cells do not significantly deplete normal T cells expressing other TRBCs derived from malignant T cells, they do not deplete the entire T-cell compartment. Maintaining the ratio of the subject's T-cell compartment (i.e., T cells that do not express the same TRBC as the malignant T cells) reduces toxicity and cellular and humoral immunodeficiency, thereby reducing the risk of infection.

[0097] TRBC1-binding CAR-T cells CAR-T cells specific for TRBC1 and TRBC2 are described in International Application No. WO2015 / 132598.

[0098] A CAR that selectively binds to TRBC1 can have a variable heavy chain (VH) and a variable light chain (VL) that include the following complementarity-determining regions (CDRs): VH CDR1: GYTFTGY (SEQ ID NO: 2); VH CDR2: NPYNDD (SEQ ID NO: 3); VH CDR3: GAGYNFDGAYRFFDF (SEQ ID NO: 4); VL CDR1: RSSQRLVHSNGNTYLH (SEQ ID NO: 5); VL CDR2: RVSNRFP (SEQ ID NO: 6); and VL CDR3: SQSTHVPYT (SEQ ID NO: 7).

[0099] Provided that the resulting antibody retains the ability to selectively bind to TRBC1, one or more CDRs may or may not each independently contain one or more amino acid mutations (e.g., substitutions) compared to the sequences shown in SEQ ID NOs: 8-13.

[0100] The antigen-binding domain of the TRBC1-selective CAR may comprise a variable heavy chain (VH) having the amino acid sequence shown as SEQ ID NO: 8 and a variable light chain (VL) having the amino acid sequence shown as SEQ ID NO: 9.

Chem.

Chem.

[0101] The CAR may comprise a ScFv having the amino acid sequence shown as SEQ ID NO: 10.

Chem.

[0102] TRBC1-binding CAR-T cells CAR-T cells specific for TRBC2 are described in International Application No. PCT / GB2019 / 053100.

[0103] The TRBC2-specific CAR may have an antigen-binding domain that contains at least one mutation in the VH domain compared to a reference antibody having a VH domain with the sequence shown in SEQ ID NO: 7 and a VL domain with the sequence shown in SEQ ID NO: 8, where at least one mutation in the VH domain is selected from T28K, Y32K, and A100N. Such an antigen-binding domain should have an increased affinity for TRBC2 compared to the TRBC-1-binding reference antibody (JOVI-1).

[0104] The variant antigen-binding domain may contain at least two mutations selected from T28K, Y32K, and A100N in the VH domain. For example, this may include the mutations Y32K and A100N. The variant antigen-binding domain may further contain the mutation T28R in the VH domain, or the mutation G31K in the VH domain.

[0105] The variant antigen-binding domain may contain the T28K, Y32K, and A100N mutations.

[0106] The variant antigen-binding domain may further comprise at least one mutation at a position selected from the group consisting of V2, Y27, G31, R98, Y102, N103, and A107 in the VH domain, N35 in the VL domain, and R55 in the VL domain. The at least one additional mutation may be selected from: a) In the VH domain: - V2K, V2R, - Y27F, Y27M, Y27N, Y27W, - G31K, G31R, G31S, - R98K, - Y102F, Y102L, - N103A, N103E, N103F, N103H, N103L, N103M, N103Q, N103S, N103W, N103Y, - A107S, and b) In the VL domain: - N35M, N35F, N35Y, N35K, N35R, and - R55K.

[0107] The variant antigen-binding domain may be selected from variant antigen-binding domains comprising the following combinations of mutations: - T28K, Y32F, A100N in the VH domain and N35K in the VL domain, - T28K, Y32F, A100N in the VH domain, - T28K, Y32F, A100N, Y27N in the VH domain - T28K, Y32F, A100N, G31K in the VH domain - T28K, Y32F, A100N, Y27M in the VH domain - T28K, Y32F, A100N, Y27W in the VH domain - T28K, Y32F, A100N in the VH domain and R55K in the VL domain, - T28K, Y32F, A100N, N103H in the VH domain - T28K, Y32F, A100N, N103A in the VH domain - T28K, Y32F, A100N, N103Y in the -VH domain - T28K, Y32F, A100N in the -VH domain and N35R in the VL domain, - T28K, Y32F, A100N, N103S in the -VH domain and N35M in the VL domain, - T28K, Y32F, A100N, N103M in the -VH domain - T28K, Y32F, A100N, N103W in the -VH domain and N35R in the VL domain - T28K, Y32F, A100N in the -VH domain and N35F in the VL domain, - T28K, Y32F, A100N, N103S in the -VH domain and N35K in the VL domain, - T28K, Y32F, A100N, R98K in the -VH domain - T28K, Y32F, A100N, N103S in the -VH domain and N35R in the VL domain - T28K, Y32F, A100N, N103L in the -VH domain - T28K, Y32F, A100N, N103S in the -VH domain and N35F in the VL domain - T28K, Y32F, A100N, N103S in the -VH domain and N35Y in the VL domain - T28K, Y32F, A100N, N103L in the -VH domain and N35M in the VL domain - T28K, Y32F, A100N, N103L in the -VH domain and N35R in the VL domain - T28K, Y32F, A100N, N103W in the -VH domain and N35K in the VL domain - T28K, Y32F, A100N, N103L in the -VH domain and N35Y in the VL domain - T28K, Y32F, A100N, N103F in the -VH domain - T28K, Y32F, A100N, N103W in the -VH domain - T28K, Y32F, A100N, N103L in the -VH domain and N35K in the VL domain - T28K, Y32F, A100N in the -VH domain, N35F in the VL domain, - T28K, Y32F, A100N, N103W in the -VH domain, N35M in the VL domain, - T28K, Y32F, A100N, N103F in the -VH domain, N35Y in the VL domain, - T28K, Y32F, A100N, Y27F in the -VH domain, - T28K, Y32F, A100N, N103Q in the -VH domain, - T28K, Y32F, A100N, N103S in the -VH domain, - T28K, Y32F, A100N, N103M in the -VH domain, N35F in the VL domain, - T28K, Y32F, A100N, N103F in the -VH domain, N35M in the VL domain, - T28K, Y32F, A100N, N103F in the -VH domain, N35F in the VL domain, - T28K, Y32F, A100N, G31R in the -VH domain, - T28K, Y32F, A100N, N103W in the -VH domain, N35F in the VL domain, - T28K, Y32F, A100N, V2R in the -VH domain, - T28K, Y32F, A100N, G31S in the -VH domain, - T28K, Y32F, A100N, A107S in the -VH domain, - T28K, Y32F, A100N, N103E in the -VH domain, N35M in the VL domain, - T28K, Y32F, A100N, V2K in the -VH domain, - T28K, Y32F, A100N, N103E - T28K, Y32F, A100N, Y102F, N103M in the -VH domain, N35K in the VL domain, - T28K, Y32F, A100N, Y102F, N103M in the -VH domain, N35F in the VL domain, -T28K, Y32F, A100N in the VH domain, Y102F, N103M, and N35R in the VL domain, -T28K, Y32F, A100N, Y102F in the VH domain, and N35R in the VL domain, -T28K, Y32F, A100N, N103M in the VH domain, and N35M in the VL domain, -T28K, Y32F, A100N, N103M in the VH domain, and N35Y in the VL domain, -T28K, Y32F, A100N, N103M in the VH domain, and N35R in the VL domain, -T28K, Y32F, A100N, N103F in the VH domain, and N35K in the VL domain, -T28K, Y32F, A100N, Y102L, N103W in the VH domain, and N35R in the VL domain, -T28K, Y32F, A100N, Y102L, N103W in the VH domain, and N35K in the VL domain, -T28K, Y32F, A100N, Y102F, and -T28K, Y32F, A100N, Y102L, N103M in the VH domain, and N35R in the VL domain.

[0108] The variant antigen-binding domain may contain the T28K, Y32F, A100N mutations in the VH domain and the N35K mutation in the VL domain.

[0109] The variant antigen-binding domain may contain the T28K, Y32F, and A100N mutations in the VH domain.

[0110] Engineered MHC I or II complex The major histocompatibility complex (MHC) is a large genetic locus on the DNA of vertebrates that contains a series of closely related polymorphic genes encoding cell surface proteins essential for the adaptive immune system. The MHC is a tissue antigen that enables the immune system (more specifically, T cells) to bind to, recognize, and tolerate the MHC itself (self-recognition). Additionally, the MHC is a chaperone for intracellular peptides that complex with the MHC and are presented as potential foreign antigens to the T cell receptor (TCR). The MHC interacts with the TCR and its coreceptor to optimize the binding conditions of TCR-antigen interactions regarding antigen binding affinity and specificity, as well as signal transduction efficacy.

[0111] Essentially, the MHC-peptide complex is a self-antigen / allogeneic antigen complex. T cells, when bound, are in principle tolerant to self-antigens but should be activated when exposed to allogeneic antigens.

[0112] MHC molecules bind to both the T cell receptor and the CD4 / CD8 coreceptor on T lymphocytes. The antigen epitope held in the peptide-binding groove of the MHC molecule interacts with the variable Ig-like domain of the TCR to induce T cell activation.

[0113] MHC class I molecules are expressed in all nucleated cells and also in platelets (essentially all cells other than erythrocytes). MHC class I presents peptide epitopes to cytotoxic T lymphocytes (CTLs). CTLs express the CD8 receptor in addition to the TCR. When the CD8 receptor of the CTL docks with the MHC class I molecule, if the TCR of the CTL fits the epitope within the MHC class I molecule, the CTL induces programmed cell death by apoptosis in the cell. Thus, MHC class I aids in mediating cellular immunity (the main means of combating intracellular pathogens such as viruses and some bacteria). In humans, MHC class I includes HLA-A, HLA-B, and HLA-C molecules.

[0114] MHC-I molecules are heterodimers, having a polymorphic heavy chain α-subunit (whose gene occurs within the MHC locus) and a small invariant β2-microglobulin subunit (whose gene is usually outside the MHC locus). The polymorphic heavy chain of the MHC-I molecule contains an N-terminal extracellular region composed of three domains α1, α2, and α3, a transmembrane helix for retaining the MHC-I molecule on the cell surface, and a short cytoplasmic-side terminus. The two domains α1 and α2 form a deep peptide-binding groove between two long α-helices, and the bottom of the groove is formed by eight β-strands. The immunoglobulin-like domain α3 is involved in the interaction with the CD8 coreceptor. β2-microglobulin confers stability to the complex and is involved in the recognition of the peptide-MHC class I complex by the CD8 coreceptor. The peptide is non-covalently bound to MHC-I and is retained by several pockets on the bottom of the peptide-binding groove. The most polymorphic amino acid side chains in the human alleles fill the center and the widest part of the accommodating groove, while the conserved side chains form clusters at the narrower ends of the groove.

[0115] Although all cell types can conditionally express MHC class II, it is usually expressed only on "professional" antigen-presenting cells (APCs): macrophages, B cells, and especially dendritic cells (DCs). APCs take up antigen proteins, process the antigens, return molecular fragments of the proteins (antigen epitopes), and present the aforementioned fragments on the surface of the APCs coupled within MHC class II molecules (antigen presentation). On the cell surface, the epitope can be recognized by an immunogenic-structure-like T cell receptor (TCR).

[0116] On the surface of helper T cells, not only TCRs but also CD4 receptors are present. When the CD4 molecule of a naive helper T cell docks to the MHC class II molecule of an APC, its TCR can encounter and bind to the epitope coupled within the MHC class II. This event primes the naive T cell.

[0117] Class II MHC molecules are also heterodimers, with genes for both the α and β subunits being polymorphic and located within the small region of MHC class II. The peptide-binding groove of the MHC-II molecule is formed by the N-terminal domains of both subunits α1 and β1 of the heterodimer; this is different from MHC-I molecules, in which two domains of the same chain are involved. Furthermore, both subunits of MHC-II contain an immunoglobulin domain α2 or β2 that can be recognized by the transmembrane helix and the CD4 coreceptor. In this way, because different lymphocytes express different T cell receptor (TCR) coreceptors, MHC molecules mediate via the type of lymphocyte that can bind a given antigen with high affinity.

[0118] The effector immune cells of the present invention may comprise an MHC class I polypeptide linked to an intracellular signaling domain; an MHC class II polypeptide; or beta-2 microglobulin.

[0119] Peptide-specific approach CD8+ T cells are important mediators of graft rejection and graft-versus-host disease and contribute to the development of autoimmune diseases. As described above, the TCR ligand is converted into a T cell activating receptor by the expression of a beta2 microglobulin polypeptide containing an intracellular signaling domain attached to one end via a linker and an antigen peptide attached to the other end. Cells engineered to express such molecules were found to express high levels of surface peptide-class I complexes presenting antigen peptides and to respond in a peptide-specific manner to antibodies and target T cells. Expression of such peptide-linker-signaling domain polypeptides in effector immune cells such as T cells makes it possible to specifically target pathogenic CD8-T cells that recognize specific antigen peptides.

[0120] Accordingly, the effector immune cells of the present invention may comprise an engineered MHC class I complex comprising a molecule having the following structure: Peptide-L-B2M-endo (In the formula, "peptide" is a peptide that binds to the peptide-binding groove of the MHC class I α-chain; "L" is a linker, "B2M" is β-2 microglobulin; "endo" is an intracellular signaling domain).

[0121] The peptide can be an alloantigen or an autoantigen.

[0122] Autoimmune disorders are characterized by the immune system reacting against endogenous antigens and, as a result, tissue being damaged. More than 80 chronic autoimmune diseases have been characterized, and these affect virtually all organ systems in the body. The most common autoimmune diseases are insulin-dependent diabetes mellitus (IDDM), multiple sclerosis (MS), systemic lupus erythematosus (SLE), rheumatoid arthritis, several forms of anemia (pernicious anemia, aplastic anemia, hemolytic anemia), thyroiditis, and uveitis.

[0123] Allograft rejection typically results from an excessive adaptive immune response against foreign organs or tissues. This is the main risk factor in organ transplantation and a cause of post-transplant complications. The main complication associated with bone marrow (BM) transplantation, known as graft-versus-host (GVH) reaction or graft-versus-host disease (GVHD), occurs in at least half of patients when donor lymphocytes are transplanted into an allogeneic recipient with a reduced immune system function, and the donor lymphocytes begin to attack the host tissue, and the immune-suppressed host has its immune response against the graft blocked.

[0124] The linker connects the peptide to β-2 microglobulin and provides mobility such that the peptide can bind to the peptide-binding groove of the associated MHC molecule. The linker can contain, for example, 5 to 20 amino acids, or 10 to 15 amino acids.

[0125] Alternatively, the molecule may include a peptide bridge that crosslinks β-2 microglobulin to the cell membrane. The peptide bridge may include 13 membrane-proximal amino acids of the extracellular portion of HLA-A2 (having the sequence LRWEPSSNPTIPI (SEQ ID NO: 11)).

[0126] The molecule may include a membrane targeting domain such as a transmembrane domain. As examples, the transmembrane domains of CD8 alpha and CD28 are shown as SEQ ID NO: 12 and SEQ ID NO: 13, respectively.

Chemical formula

Chemical formula

[0127] The amino acid sequence of human β-2 microglobulin is available from Uniprot accession number P61769 and is shown below as SEQ ID NO: 14.

Chemical formula

[0128] The engineered MHC class I complex may include a variant of the β-2 microglobulin sequence shown as SEQ ID NO: 14 (e.g., a variant having at least 80%, 90%, 95%, or 99% amino acid identity to the sequence shown as SEQ ID NO: 14), provided that the resulting peptide-L-B2M-endo molecule retains the ability to associate with the MHC class I alpha chain.

[0129] The endodomain derived from human CD3 zeta has the sequence shown in SEQ ID NO: 15.

Chemical formula

[0130] The engineered MHC class I complex can include a variant having at least 80%, 90%, 95%, or 99% amino acid identity to the sequence set forth in SEQ ID NO: 15, provided that the engineered MHC class I complex has an intracellular signaling domain having the sequence set forth in SEQ ID NO: 15 or that the resulting peptide-L-B2M-endo molecule retains the ability to induce activation of effector immune cells upon TCR recognition.

[0131] Additional intracellular signaling domains and co-stimulatory domains are described below.

[0132] Peptide cross - approach To directly or indirectly induce signaling in effector cells, it is possible to couple MHC class I or II on effector immune cells to the TCR on target immune cells. In these approaches, the MHC signaling system can present the same range of peptides as the corresponding endogenous MHC class I and II molecules. As such, any peptide that is naturally presented by an MHC class I or II molecule can be presented by the engineered MHC complex. Such peptides can include, for example, peptides derived from any heterologous sequence or junctional sequence that can exhibit immunogenicity derivable from a chimeric antigen receptor expressed by a cell. In an allogeneic context, such peptides can also include minor histocompatibility antigens. Thus, such engineered MHC class complexes will interact with any endogenous reactive T cells present in the recipient of the engineered cells upon recognition of the peptide / MHC complex. Thus, reactive T cells can be depleted by activation of cytotoxic - mediated cell death by the cells of the present invention. Therefore, the cellular immune response against the cells of the present invention can be reduced.

[0133] In this regard, effector immune cells can include an MHC class I polypeptide; an MHC class II polypeptide; or a polypeptide capable of co - existing with β - 2 microglobulin having an intracellular signaling domain.

[0134] An effector immune cell may include: (i) An ectodomain derived from an MHC class I polypeptide or an ectodomain derived from an MHC class II polypeptide linked to an intracellular signaling domain; or an engineered polypeptide comprising β-2 microglobulin linked to an intracellular signaling domain (see Figures 2a, 4, 5, 6c, 8a, and 10a); (ii) An engineered polypeptide comprising an MHC class I polypeptide, an MHC class II polypeptide, or β-2 microglobulin linked to a component of the CD3 / TCR complex (such as CD3-zeta, CD3-epsilon, CD3-gamma, or CD3-delta) (see Figures 2c, 8c, and 10c); (iii) An engineered polypeptide comprising a binding domain (such as an antibody-like binding domain) that binds to an MHC class I polypeptide, an MHC class II polypeptide, or β-2 microglobulin linked to an intracellular signaling domain (see Figures 8b and 10b); (iv) An engineered polypeptide comprising CD79α or CD79β linked to an intracellular signaling domain (see Figure 6b); (v) An engineered polypeptide comprising the MHC class II binding domain of CD4 linked to an intracellular signaling domain; or the MHC class I binding domain of CD8 linked to an intracellular signaling domain (see Figure 11).

[0135] Alternatively, an effector immune cell can be engineered to express a bispecific polypeptide comprising (i) a first binding domain that binds to an MHC class I polypeptide; an MHC class II polypeptide; β-2 microglobulin; and (ii) a second binding domain that binds to a component of the TCR / CD3 complex (see Figures 2b, 8d, and 10d).

[0136] HLA class I MHC class I molecules are heterodimers consisting of two polypeptide chains (an α polypeptide and β2-microglobulin (b2m)). The two chains are non-covalently linked via the interaction of the b2m domain and the α3 domain. The α chain is polymorphic and, in humans, is encoded by the human leukocyte antigen gene complex (HLA). The b2m subunit is not polymorphic and is encoded by the beta-epsilon macroglobulin gene. HLA gene. The HLAs corresponding to MHC class I are HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G.

[0137] HLA-A, HLA-B, and HLA-C are typically highly polymorphic, while HLA-E, HLA-F, HLA-G are less polymorphic.

[0138] The engineered polypeptide of the effector cells of the present invention may comprise the extracellular domain of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G.

[0139] The engineered polypeptide or bispecific polypeptide expressed by the effector cells of the present invention may bind to HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G.

[0140] The most common haplotypes vary between populations. Thus, the effector immune cells of the present invention can be designed for a particular population having a specific common haplotype. Exemplary class I haplotypes are summarized in the following table:

Table 2

[0141] The amino acid sequence of HLA class I - HLA-A is HLA-A01 shown as SEQ ID NO: 16:

Figure

[0142] Amino acid sequence of HLA Class I - HLA-A is HLA-A02 shown as SEQ ID NO: 17: [Chemical formula]

[0143] Amino acid sequence of HLA Class I - HLA-A is HLA-A-A03 shown as SEQ ID NO: 18: [Chemical formula]

[0144] Amino acid sequence of HLA Class I - HLA-B is HLA-B07 shown as SEQ ID NO: 19: [Chemical formula]

[0145] Amino acid sequence of HLA Class I - HLA-B is HLA-B08 shown as SEQ ID NO: 20: [Chemical formula]

[0146] Exemplary amino acid sequence of HLA Class I - HLA-B is HLA-B44 shown as SEQ ID NO: 21: [Chemical formula]

[0147] Amino acid sequence of HLA Class I - HLA-C is HLA-C01 shown as SEQ ID NO: 22: [Chemical formula]

[0148] The engineered polypeptide of the effector cell of the present invention includes an extracellular domain of any one of SEQ ID NOs: 16-22, or a variant thereof having at least 80, 85, 90, 95, 98, or 99% identity, provided that the variant maintains the ability to assemble with the β2-microglobulin chain and facilitate productive peptide presentation by the MHC class I complex.

[0149] Also, the engineered polypeptide may include a transmembrane domain.

[0150] The transmembrane domain can be any peptide domain capable of inserting into and traversing the cell membrane. The transmembrane domain can be any protein structure that is thermodynamically stable within the membrane. This is typically an α-helix composed of several hydrophobic residues. The transmembrane domain of any transmembrane protein can be used to provide the transmembrane portion of the present invention. One of ordinary skill in the art can determine the sequence and full length of the transmembrane domain of a protein using the TMHMM algorithm (http: / / www.cbs.dtu.dk / services / TMHMM-2.0 / ). Additionally, considering that the transmembrane domain of a protein has a relatively simple structure (i.e., a polypeptide sequence predicted to form a hydrophobic α-helix long enough to span the membrane), artificially designed TM domains can also be used (U.S. Patent No. 7,052,906 B1 describes synthetic transmembrane components). For example, the transmembrane domain can include a hydrophobic alpha helix. The transmembrane domain can be derived from, for example, CD8 alpha or CD28.

[0151] HLA class II In humans, the MHC class II protein complex is encoded by the human leukocyte antigen gene complex (HLA). The HLAs corresponding to MHC class II are HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR.

[0152] Activated human T cells express MHC class II molecules of all isotypes (HLA-DR, HLA-DQ, and HLA-DP) on their surface. The expression of MHC class II molecules is found approximately 3 to 5 days after T cell activation, and this expression is a late event compared to the induction of various other effector molecules after T cell receptor (TCR) triggering and co-stimulation. Since adoptively transferred immune effectors are expected to be activated at several time points after injection, allogeneic rejection can occur when HLA class II is expressed.

[0153] HLA class II molecules are formed as the following two polypeptide chains: alpha and beta. Although some haplotypes are much more common in certain populations than in others, these molecules are typically highly polymorphic on an individual basis.

[0154] Polypeptides of any haplotype or any combination of haplotypes may be used in the present invention (including any of the haplotypes listed in the following table:

Table 3

[0155] HLA-DR is very low in polymorphism and is particularly suitable for use in the present invention. In one embodiment, the engineered polypeptide comprises an ectodomain and an intracellular signaling domain derived from HLA-DR. The ectodomain may be derived from HLA-DRα or HLA-DRβ.

[0156] The amino acid sequence of the HLA class II histocompatibility antigen DRα chain (having UniProtKB accession number P01903) is shown in SEQ ID NO: 23:

Chemical formula

[0157] The engineered polypeptide may include a variant having at least 80, 85, 90, 95, 98, or 99% sequence identity to the HLA-DRα-derived ectodomain set forth in SEQ ID NO: 23 (such as from about amino acid 26 to about amino acid 216 of SEQ ID NO: 23), provided that the variant assembles with the β chain and retains the ability to facilitate productive peptide presentation by the MHC class II complex.

[0158] The amino acid sequence of the HLA class II histocompatibility antigen DRβ chain (having UniProtKB accession number Q04826) is shown in SEQ ID NO: 24:

Chemical formula

[0159] The engineered polypeptide may include a variant having at least 80, 85, 90, 95, 98, or 99% sequence identity to the HLA-DRβ-derived ectodomain set forth in SEQ ID NO: 24 (such as from about amino acid 25 to about amino acid 308 of SEQ ID NO: 24), provided that the variant assembles with the α chain and retains the ability to facilitate productive peptide presentation by the MHC class II complex.

[0160] HLA-DP and HLA-DQ have polymorphic α and β chains. Thus, allogeneic production can be restricted to recipients having a common HLA-DP or HLA-DQ α and β chain and having that haplotype. Preferably, the recipient may be homozygous for that haplotype. If the recipient is not homozygous for the haplotype, two HLA-DP and two HLA-DQ (in combination with HLA-DR as needed, such as HLA-DRα) may be used.

[0161] The amino acid sequence of HLA class II histocompatibility antigen DP (having UniProtKB accession number Q30058) is shown in SEQ ID NO: 25:

Chemical formula

[0162] The engineered polypeptide may include a variant having at least 80, 85, 90, 95, 98, or 99% sequence identity thereto, provided that the variant assembles and maintains the ability to facilitate productive peptide presentation by the MHC class II complex, such as the ectodomain derived from HLA-DP set forth in SEQ ID NO: 25 (about amino acids 29 to about amino acids 224 of SEQ ID NO: 25).

[0163] The amino acid sequence of HLA class II histocompatibility antigen DQ (having UniProtKB accession number O19764) is shown in SEQ ID NO: 26:

Chemical formula

[0164] The engineered polypeptide may include a variant having at least 80, 85, 90, 95, 98, or 99% sequence identity thereto, provided that the variant assembles and maintains the ability to facilitate productive peptide presentation by the MHC class II complex, such as the ectodomain derived from HLA-DQ set forth in SEQ ID NO: 26 (about amino acids 32 to about amino acids 228 of SEQ ID NO: 26).

[0165] The engineered polypeptide may include an extracellular domain derived from any of SEQ ID NOs: 23-26. The engineered polypeptide may also include the transmembrane domain described above.

[0166] The sequences of MHC polypeptides are provided in the ImMunoGeneTics (IMGT) database (Lefranc, M.-P. et al., Nucleic Acids Res., 27:209-212 (1999); doi:10.1093 / nar / 27.1.209).

[0167] The identity rate between two polypeptide sequences can be readily determined by programs such as BLAST that are freely available at http: / / blast.ncbi.nlm.nih.gov. Appropriately, the identity rate is determined by the completeness with respect to the reference sequence and / or query sequence.

[0168] As used herein, "able to coexist an MHC class I polypeptide or an MHC class II polypeptide with an intracellular signaling domain within a cell" means that when a target T cell binds to a peptide / MHC complex on the effector immune cell of the present invention, the polypeptide enables the intracellular signaling domain to transmit an activation signal into the effector immune cell of the present invention so as to transmit an activation signal in the effector immune cell of the present invention.

[0169] CD79 CD79 is composed of two chains, CD79α and CD79β, which form a heterodimer on the B cell surface. CD79α a / β assembles with membrane-bound immunoglobulin to form a complex with the B cell receptor (BCR). CD79α and CD79β are members of the immunoglobulin superfamily and contain an ITAM signaling motif capable of B cell signaling in response to cognate antigen recognition by the BCR.

[0170] Furthermore, CD79α and CD79β associate with HLA class II, thereby enabling CD79-mediated signaling of HLA class II in a manner similar to membrane-bound immunoglobulins (Lang, P. et al.. Science 291, 1537-1540 (2001) and Jin, L. et al. Immunol. Lett. 116, 184-194 (2008).

[0171] In one aspect, the present invention provides a cell comprising: (i) a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR); and (ii) at least one polypeptide capable of co-existing with an MHC class I polypeptide or an MHC class II polypeptide in an intracellular signaling domain within the cell; wherein the at least one polypeptide capable of co-existing with the MHC class I polypeptide or the MHC class II polypeptide in the intracellular signaling domain is CD79 or a variant thereof.

[0172] The cell may comprise an engineered polypeptide comprising CD79α or CD79β linked to an intracellular signaling domain. The cell may comprise two engineered polypeptides: one comprising CD79α linked to an intracellular signaling domain; and one comprising CD79β linked to an intracellular signaling domain.

[0173] The amino acid sequence of human CD79α (having UniProtKB accession number P11912) is shown in SEQ ID NO: 27:

Chemical formula

[0174] The CD79α sequence for use in the present invention may include the sequence shown in SEQ ID NO: 27 or a variant thereof having at least 80, 85, 90, 95, 98, or 99% sequence identity, provided that the variant maintains the ability to assemble with HLA class I and / or HLA class II and facilitate signal transduction.

[0175] The engineered polypeptide may include the extracellular domain, transmembrane domain, and intracellular signaling domain of CD79α. The engineered polypeptide may include the extracellular domain (ecotdomain) of CD79α corresponding to about amino acids 33 to about amino acids 143 of SEQ ID NO: 27.

[0176] The engineered polypeptide may include the transmembrane domain of CD79α corresponding to about amino acids 144 to about amino acids 165 of SEQ ID NO: 27.

[0177] The engineered polypeptide may include the intracellular signaling domain of CD79α corresponding to about amino acids 166 to about amino acids 226 of SEQ ID NO: 27.

[0178] The amino acid sequence of human CD79β (having UniProtKB accession number P40259) is shown in SEQ ID NO: 28:

Chemical formula

[0179] The CD79β sequence for use in the present invention may include the sequence shown in SEQ ID NO: 8 or a variant thereof having at least 80, 85, 90, 95, 98, or 99% sequence identity, provided that the variant maintains the ability to assemble with HLA class I and / or HLA class II and facilitate signal transduction.

[0180] The engineered polypeptide may include the extracellular domain, transmembrane domain, and intracellular signaling domain of CD79β. The engineered polypeptide may include the extracellular domain of CD79β corresponding to about amino acid 29 to about amino acid 159 of SEQ ID NO: 28.

[0181] The engineered polypeptide may include the transmembrane domain of CD79β corresponding to about amino acid 160 to about amino acid 180 of SEQ ID NO: 28.

[0182] The engineered polypeptide may include the intracellular signaling domain of CD79β corresponding to about amino acid 181 to about amino acid 229 of SEQ ID NO: 28.

[0183] Effector immune cells may express the following two engineered polypeptides: those containing the extracellular domain of CD79α and those containing the extracellular domain of CD79β.

[0184] CD3-linked polypeptide Effector immune cells may include: (i) a chimeric antigen receptor (CAR) or transgenic T cell receptor (TCR); and (ii) an engineered polypeptide comprising an MHC class I polypeptide or MHC class II polypeptide linked to a component of the CD3 / TCR complex.

[0185] CD3 is a T cell co-receptor involved in the activation of both cytotoxic T cells and T helper cells. It is formed from a protein complex composed of four individual chains. As used herein, the term "CD3 complex" also includes the CD3ζ chain. In mammals, this complex includes the CD3γ chain, CD3δ chain, and two CD3ε chains. These chains assemble with the TCR to generate a TCR complex, which can generate activation signals in T lymphocytes.

[0186] The CD3ζ chain, CD3γ chain, CD3δ chain, and CD3ε chain are highly related cell surface proteins of the immunoglobulin superfamily that contain a single extracellular immunoglobulin domain. The transmembrane region of the CD3 chains contains several negatively charged aspartic acid residues, which are characterized by their ability to associate with the positively charged TCR chains. The intracellular tail of the CD3 molecule contains a single conserved motif known as the immunoreceptor activation tyrosine motif (ITAM) that is involved in TCR signaling.

[0187] Polypeptides linked to components of the TCR complex can assemble to facilitate productive peptide presentation by MHC class I complexes or MHC class II complexes on the cell surface. Furthermore, the TCR / CD3 components can assemble into the TCR / CD3 complex. Therefore, binding of the TCR to a peptide / MHC complex containing a polypeptide linked to a component of the TCR complex will induce signaling by the CD3 / TCR complex.

[0188] The polypeptide can be linked to a component of the TCR or CD3 complex. The polypeptide can be linked to an engineered TCR polypeptide lacking a variable domain.

[0189] The engineered polypeptide can be linked to a component of the CD3 complex (e.g., selected from CD3-zeta, CD3-epsilon, CD3-gamma, and CD3-delta).

[0190] Examples of the human CD3ζ, CD3γ, CD3δ, and CD3ε amino acid sequences are shown in SEQ ID NOs: 29-32, respectively.

Chemical formula

Chemical formula

[0191] The MHC class I / MHC class II or B2M polypeptide can be linked to the CD3 component by any suitable means. For example, the polypeptide can be fused to the components of the CD3 complex by a linker peptide.

[0192] Suitable linker peptides are known in the art. For example, a range of suitable linker peptides are described by Chen et al. (Adv Drug Deliv Rev. 2013 October 15;65(10):1357-1369 - see especially Table 3).

[0193] A suitable linker is (SGGGG)n (including one or more copies of SEQ ID NO: 33). For example, a suitable linker peptide is shown as SEQ ID NO: 34.

Chemical formula

[0194] The polypeptide can be linked to the extracellular domain of the components of the CD3 complex. The polypeptide can be linked to the N-terminus of the components of the CD3 complex.

[0195] An exemplary polypeptide for use in the present invention is shown as SEQ ID NO: 35.

Chemical formula

[0196] This polypeptide sequence contains an extracellular domain, a transmembrane domain, and an intracellular CD3-ζ domain derived from HLA-DRα.

[0197] An exemplary polypeptide for use in the present invention is shown as SEQ ID NO: 36.

Chemical formula

[0198] This polypeptide sequence includes an ectodomain, a transmembrane domain, a 41BB endodomain, and an intracellular CD3-ζ endodomain derived from HLA-DRα.

[0199] An exemplary polypeptide for use in the present invention is shown as SEQ ID NO: 37.

Chemical Structure

[0200] This polypeptide sequence includes an ectodomain, a transmembrane domain, a CD28 endodomain, and an intracellular CD3-ζ endodomain derived from HLA-DRα.

[0201] An exemplary polypeptide for use in the present invention is shown as SEQ ID NO: 38.

Chemical Structure

[0202] This polypeptide sequence includes an ectodomain derived from CD79α, a 41BB domain, and an endodomain derived from CD79.

[0203] An exemplary polypeptide for use in the present invention is shown as SEQ ID NO: 39.

Chemical Structure

[0204] This polypeptide sequence includes an ectodomain derived from CD79β, a CD28 domain, and an endodomain derived from CD79.

[0205] An exemplary polypeptide for use in the present invention is shown as SEQ ID NO: 40.

Chemical Structure

[0206] This polypeptide sequence includes an ectodomain derived from CD79α, a CD28 domain, and an endodomain derived from CD79.

[0207] An exemplary polypeptide for use in the present invention is shown as SEQ ID NO: 41.

Chemical formula

[0208] This polypeptide sequence includes an ectodomain derived from CD79β, a 41BB domain, and an endodomain derived from CD79.

[0209] An exemplary polypeptide for use in the present invention is shown as SEQ ID NO: 42.

Chemical formula

[0210] This polypeptide sequence includes an ectodomain derived from CD79α, a 41BB domain, and a CD3-zeta domain.

[0211] An exemplary polypeptide for use in the present invention is shown as SEQ ID NO: 43.

Chemical formula

[0212] This polypeptide sequence includes an ectodomain derived from CD79β, a 41BB domain, and a CD3-zeta domain.

[0213] The polypeptide sequences for use in the present invention are the sequences shown as SEQ ID NOs: 35 to 43, or variants that assemble and facilitate productive peptide presentation by MHC class II complexes at the cell surface, and maintain the ability to transmit activation signals after binding of the TCR to the peptide / MHC complex containing the aforementioned polypeptide, provided that they have at least 80, 85, 90, 95, 98, or 99% sequence identity, and may include variants.

[0214] Intracellular signaling domain The present invention provides at least one polypeptide capable of coexisting an MHC class I polypeptide or an MHC class II polypeptide with an intracellular signaling domain within a cell.

[0215] The engineered polypeptide of the present invention may include an intracellular signaling domain.

[0216] As used herein, the intracellular signaling domain refers to the signaling portion of the endomain.

[0217] The intracellular signaling domain may be or include a T cell signaling domain.

[0218] The intracellular signaling domain may include one or more immunoreceptor tyrosine-based activation motifs (ITAMs). An ITAM is a conserved sequence of four amino acids repeated twice in the cytoplasmic tail of certain cell surface proteins of the immune system. This motif contains tyrosine separated from leucine or isoleucine by any two other amino acids, giving the signature YxxL / I. Two of these signatures are typically separated by amino acids between 6 and 8 amino acids in the tail of the molecule (YxxL / I x(6-8) YxxL / I).

[0219] ITAM is important for signal transduction in immune cells. Therefore, ITAM is found in the tails of important cell signaling molecules such as CD3 and ζ chains of the T cell receptor complex, CD79 alpha and beta chains of the B cell receptor complex, and certain Fc receptors. The tyrosine residues within these motifs become phosphorylated after the interaction of the receptor molecule with its ligand, forming docking sites for other proteins involved in the cell's signal transduction pathways.

[0220] Preferably, the intracellular signaling domain component comprises, consists essentially of, or consists of a CD3-ζ end domain containing three ITAMs. Classically, the CD3-ζ end domain transmits activation signals to T cells after antigen binding. However, in the context of the present invention, the CD3-ζ end domain transmits activation signals to effector cells after its MHC complex interacts with the TCR on an adjacent T cell.

[0221] The intracellular signaling domain may include additional co-stimulatory signaling. For example, 4-1BB (also known as CD137) can be used together with CD3-ζ, or CD28 and OX40 can be used together with CD3-ζ to transmit proliferation / survival signals.

[0222] Thus, the intracellular signaling domain can include the CD3-ζ end domain alone, the CD3-ζ end domain in combination with one or more co-stimulatory domains selected from the 4-1BB end domain, the CD28 end domain, or the OX40 end domain, and / or combinations of some or all of 4-1BB, CD28, or OX40.

[0223] The end domain can include one or more of the following: ICOS end domain, CD2 end domain, CD27 end domain, or CD40 end domain.

[0224] The endodomain may include the sequence shown as SEQ ID NOs: 44-47, or a variant thereof having at least 80, 85, 90, 95, 98, or 99% sequence identity, provided that the variant sequence retains the ability to transmit an activation signal to the cell. [Chem.] [Chem.]

[0225] Antigen-binding domain linked to a signal transduction domain The engineered polypeptide of the present invention may include an MHC class I polypeptide linked to an intracellular signal transduction domain; an MHC class II polypeptide, or a binding domain that binds to β2 microglobulin.

[0226] The binding domain may be or include an antibody or antibody-like molecule.

[0227] "Antibody," as used herein, refers to a polypeptide having an antigen-binding site that includes at least one complementarity determining region, i.e., a CDR. An antibody may include three CDRs and may have an antigen-binding site equivalent to a single domain antibody (dAb), a heavy chain antibody (VHH), or a nanobody. An antibody may include six CDRs and may have an antigen-binding site equivalent to the antigen-binding site of a classical antibody molecule. The remainder of the polypeptide may be any sequence that provides a scaffold appropriate for the antigen-binding site and presents the antigen-binding site in a manner appropriate for antigen binding to the antigen-binding site.

[0228] Full-length antibodies or immunoglobulins typically consist of the following four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each heavy chain contains one N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen-binding site of the antibody. The variable regions are characterized by an identical general structure in which relatively conserved regions called frameworks (FR) are joined to three hypervariable regions called complementarity-determining regions (CDR). The term "complementarity-determining region" or "CDR" as used herein refers to a region within an antibody that complements the shape of an antigen. Thus, the CDRs determine the affinity and specificity of the protein for a particular antigen. The CDRs of each pair of two chains are aligned by the framework regions, thereby acquiring the function of specifically binding to an epitope. Thus, in the case of the VH and VL domains, both the heavy and light chains are characterized by three CDRs each, CDRH1, CDRH2, CDRH3, and CDRL1, CDRL2, CDRL3, respectively.

[0229] The engineered polypeptide of the present invention may comprise a full-length antibody or an antigen-binding fragment thereof.

[0230] Full-length antibodies can be, for example, IgG, IgM, IgA, IgD, or IgE.

[0231] "Antibody fragment" refers to one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen. Antibody fragments can include, for example, one or more CDRs, variable regions (or portions thereof), constant regions (or portions thereof), or combinations thereof. Examples of antibody fragments include, but are not limited to, Fab fragments, F(ab’)2 fragments, Fv fragments, single-chain Fv (scFv), domain antibodies (dAb or VH), single-domain antibodies (sdAb), VHH, nanobodies, diabodies, triabodies, trimerbodies, and monobodies.

[0232] The engineered polypeptide of the present invention may comprise an antigen-binding domain based on a non-immunoglobulin scaffold. These antibody-binding domains are also referred to as antibody mimetics. Non-limiting examples of non-immunoglobulin antigen-binding domains include affibodies, fibronectin artificial antibody scaffolds, anticalins, affilins, DARPins, VNARs, iBodies, affimers, finomerans, abdualins / nanobodies, centyrins, alphabodies, nanofitins, and D-domains.

[0233] Some antibodies that specifically bind to MHC class I or MHC class II have been described.

[0234] For example, WO05 / 023299 (incorporated by reference) describes antibodies that bind to MHC class II antigens, particularly antibodies against the HLA-DR alpha chain. Table 1 of this document includes the sequence characteristics of clones MS-GPC-1 (scFv-17), MS-GPC-6 (scFv-8A), MS-GPC-8 (scFv-B8), and MS-GPC-10 (scFv-E6), and FIG. 15 shows the VH and VL sequences of MS-GPC-1; MS-GPC-6; MS-GPC-8; MS-GPC-10; MS-GPC-8-6; MS-GPC-8-10; MS-GPC-8-17; MS-GPC-8-27; MS-GPC-8-6-13; MS-GPC-8-10-57; MS-GPC-8-27-41; MS-GPC-8-1; MS-GPC-8-9; MS-GPC-8-18; MS-GPC-8-6-2; MS-GPC-8-6-19; MS-GPC-8-6-27; MS-GPC-8-6-45; MS-GPC-8-6-47; MS-GPC-8-27-7; and MS-GPC-8-27-10.

[0235] The engineered polypeptide may comprise an MHC class II binding domain that includes one of these pairs of VH and VL sequences. In particular, the engineered polypeptide may comprise an MHC class II binding domain based on the binder MS-GPC-8.

[0236] Andris et al (1995 Mol Immunol 32:14-15) describe six antibodies specific for human HLA class I and class II antigens, including an antibody against the HLA-DQ beta chain with the antibody clone name anti-HLAII / DQB1-MP1.

[0237] Watkins et al (2000 Tissue Antigens 55:219-28) describe the isolation and characterization of human monoclonal HLA-A2 antibodies. Antibody clones include: anti-HLA-A2 / A28-3PF12, anti-HLA-A2 / A28-3PC4, and anti-HLA-A2 / A28-3PB2.

[0238] The engineered polypeptide of the present invention may comprise a binding domain of MHC class I or MHC class II derived from any of these antibodies.

[0239] The engineered polypeptide may comprise a short flexible linker for introducing a chain break. The chain break separates two individual domains but allows them to be oriented at different angles. Such sequences include sequence SDP and sequence SGGGSDP (SEQ ID NO: 48).

[0240] The linker may comprise a serine-glycine linker (such as SGGGGS (SEQ ID NO: 49)).

[0241] The engineered polypeptide may comprise a transmembrane domain as defined above. The engineered polypeptide may comprise, for example, the transmembrane domain of CD8-alpha or CD28.

[0242] The engineered polypeptide comprises an intracellular signaling domain as defined above. The engineered polypeptide may comprise, for example, the CD3ζ end domain.

[0243] The engineered polypeptide may have the following general structure: MHC class I or II binding domain - transmembrane domain - intracellular signaling domain, or MHC class I or II binding domain - linker - transmembrane domain - intracellular signaling domain

[0244] CD4 / CD8 fusion protein The engineered polypeptide of the present invention may comprise the MHC class II binding domain of CD4 linked to an intracellular signaling domain or the MHC class I binding domain of CD8 linked to an intracellular signaling domain.

[0245] CD4 and CD8 are co - receptors of the T - cell receptor (TCR) and assist T cells in their communication with antigen - presenting cells.

[0246] CD4 (cluster of differentiation 4) is a glycoprotein found on the surface of immune cells such as T - helper cells, monocytes, macrophages, and dendritic cells. CD4 is a member of the immunoglobulin superfamily having the following four immunoglobulin domains (D1 - D4) exposed on the extracellular surface of the cell: D1, similar to the immunoglobulin variable (IgV) domain; and D2, D3, and D4, similar to the immunoglobulin constant (IgC) domain.

[0247] The immunoglobulin variable (IgV) domain of D1 adopts an immunoglobulin - like β - sandwich fold with seven β - strands in two β - sheets. CD4 interacts with the β2 - domain of MHC class II molecules via its D1 domain. Thus, T cells displaying CD4 molecules on their surface are specific for antigens presented by MHCII (i.e., this T cell is MHC class II - restricted).

[0248] The short cytoplasmic tail / intracellular tail (C) of CD4 contains an amino acid sequence that enables it to recruit and interact with the tyrosine kinase Lck. When the extracellular D1 domain of CD4 binds to the β2 region of MHC class II, the TCR complex and CD4 come very close to each other, thereby allowing the tyrosine kinase Lck to bind to the cytoplasmic end of CD4 and phosphorylate the tyrosine residues of the immunoreceptor tyrosine activation motif (ITAM) on the cytoplasmic domain of CD3, amplifying the signal generated by the TCR. The phosphorylated ITAM on CD3 recruits and activates SH2 domain-containing protein tyrosine kinases (PTKs) such as ZAP70, further mediating downstream signaling via tyrosine phosphorylation. These signals activate transcription factors (including NF-κB, NFAT, and AP-1), promoting T cell activation.

[0249] The amino acid sequence of human CD4 is available from UniProt accession number P01730. The engineered polypeptide of the present invention may include the D1 domain of CD4 having the sequence shown in SEQ ID NO: 50. The positions of Gln40 and Thr45 are shown in underlined bold.

Chemical formula

[0250] The engineered polypeptide may include a variant D1 domain of CD4 that contains one or more amino acid mutations that increase its binding affinity for the β2 region of MHC class II compared to the wild-type D1 domain.

[0251] For example, Wang et al (2011, PNAS 108: 15960-15965) described the affinity maturation of human CD4 by yeast surface display to increase its affinity for HLA-DR1. A CD4 variant carrying the substitution mutations Gln40Tyr and Thr45Trp was found to bind to HLA-DR1 with a KD = 8.8 μM, compared to KD > 400 μM for wild-type CD4.

[0252] The engineered polypeptide may comprise a variant D1 domain of CD4 that contains an amino acid mutation(s) at position Gln40 and / or Thr45 with reference to the sequence shown as SEQ ID NO: 50.

[0253] The engineered polypeptide may comprise a variant D1 domain of CD4 that contains an amino acid substitution(s) Gln40Tyr and / or Thr45Trp with reference to the sequence shown as SEQ ID NO: 50.

[0254] The CD8 (cluster of differentiation 8) co-receptor is mainly expressed on the surface of cytotoxic T cells, but can also be found on natural killer cells, cortical thymocytes, and dendritic cells. There are two isoforms of CD8 (alpha and beta), each encoded by a different gene.

[0255] To function, CD8 forms a dimer consisting of a pair of CD8 chains. The most common form of CD8 is composed of a CD8-alpha chain and a CD8-beta chain, although homodimers of the CD8-alpha chain are also expressed on some cells. Both CD8-alpha and CD8-beta are members of the immunoglobulin superfamily with an immunoglobulin variable (IgV)-like extracellular domain connected to the membrane by a thin stalk, and an intracellular tail.

[0256] The extracellular IgV-like domain of CD8-alpha interacts with the alpha3 portion of class I MHC molecules. The major recognition site is a mobile loop in the alpha3 domain of the MHC molecule that exists between residues 223 and 229. When CD8-alpha binds to MHC class I, the close association between the T cell receptor of the cytotoxic T cell and the target cell during antigen-specific activation is maintained. The cytoplasmic side terminus of the CD8 co-receptor interacts with Lck (lymphocyte-specific protein tyrosine kinase). When the T cell receptor binds to its specific antigen, Lck phosphorylates the cytoplasmic CD3 and zeta chains of the TCR complex, thereby initiating a phosphorylation cascade that ultimately activates transcription factors such as NFAT, NF-κB, and AP-1.

[0257] The engineered polypeptide of the present invention may include an IgV-like domain derived from CD8-α.

[0258] The amino acid sequence of human CD8α is available from UniProt accession number P01732. The engineered polypeptide of the present invention may include the amino acid residues 22-135 of this sequence and may include the Ig-like V-type domain of CD8 having the sequence shown in SEQ ID NO: 51.

Chemical formula

[0259] The engineered polypeptide may include a variant CD8α Ig-like V-type domain that contains one or more amino acid mutations that increase its binding affinity for the α3 portion of class I MHC molecules as compared to the wild-type CD8α domain.

[0260] For example, high-affinity variants of CD8α can be generated and characterized using the in vitro evolution method described by Wang et al (2011, PNAS 108:15960-15965).

[0261] The engineered polypeptide may include a dimer of CD8. Devine et al (1999, J. Immunol. 162:846-851) described a molecule containing two CD8α Ig domains linked via one carboxyl terminus to the other amino terminus by a peptide spacer. Using a 20-amino acid peptide spacer of a four-repeat unit of GGGGS (SEQ ID NO: 52), it was possible to accurately identify the two IG-like domains.

[0262] The engineered polypeptide may include the CD8αα homodimer described by Devine et al 1999. The CD8αα homodimer may have the sequence shown in SEQ ID NO: 53.

Chemical formula

[0263] The engineered polypeptide may comprise a CD8αβ heterodimer. For example, the engineered polypeptide may comprise a CD8α Ig-like V domain having the sequence shown in SEQ ID NO: 51 joined to the CD8β Ig-like V domain by a peptide spacer. The peptide spacer may be between 10 and 20 amino acids in length, such as between 15 and 25 amino acids in length. The peptide spacer may be approximately 20 amino acids in length. With respect to the CD8αα homodimer described in Devine et al 1999, the peptide spacer may comprise four repeat units of GGGGS (SEQ ID NO: 52).

[0264] The amino acid sequence of the CD8β Ig-like V domain is shown below as SEQ ID NO: 54.

Chemical Structure

[0265] The engineered polypeptide may comprise a CD8αβ heterodimer in which the CD8α domain and the CD8β domain are present in either order in the construct (i.e., CD8αβ or CD8βα).

[0266] The engineered polypeptide may comprise a short flexible linker for introducing a chain break between the CD8α monomer, CD8αα homodimer, or CD8αβ heterodimer and the stalk and / or transmembrane domain. The chain break separates the two individual domains but allows them to be oriented at different angles. Such sequences include SEQ SDP and SEQ SGGGSDP (SEQ ID NO: 48).

[0267] The linker may comprise a serine-glycine linker (such as SGGGGS (SEQ ID NO: 49)).

[0268] The engineered polypeptide may include a transmembrane domain as defined above. For example, the engineered polypeptide may include the transmembrane domain of CD8-alpha or CD28.

[0269] The engineered polypeptide includes an intracellular signaling domain as defined above. The engineered polypeptide may include, for example, the CD3ζ end domain.

[0270] The engineered polypeptide may have the following general structure: CD4 D1 domain - linker - transmembrane domain - intracellular signaling domain; CD8α Ig-like V domain - linker - transmembrane domain - intracellular signaling domain; CD8αα homodimer - linker - transmembrane domain - intracellular signaling domain; or CD8αβ homodimer - linker - transmembrane domain - intracellular signaling domain

[0271] Bispecific polypeptide In a further embodiment of the invention, a polypeptide capable of co-existing an MHC class I polypeptide or an MHC class II polypeptide with an intracellular signaling domain may be a bispecific polypeptide comprising: (a) A first binding domain that binds to an MHC class I polypeptide or an MHC class II polypeptide (b) A second binding domain capable of binding to a polypeptide comprising an intracellular signaling domain or a component of the CD3 complex.

[0272] The bispecific polypeptide may be membrane-tethered.

[0273] When expressed by a cell or expressed on the cell surface, the bispecific molecule of the invention co-exists MHC class I or II with the TCR, facilitating TCR signaling in the cells of the invention after binding of the TCR on different T cells to the peptide / MHC complex bound by the bispecific molecule.

[0274] Several different formats of bispecific molecules have been developed. One of the most common formats is a fusion consisting of two single-chain variable fragments (scFvs) of different antibodies.

[0275] The first and / or second binding domains of the bispecific molecule can be antibody or immunoglobulin-based binding domains.

[0276] As used herein, "antibody" means a polypeptide having an antigen-binding site that includes at least one complementarity determining region CDR. An antibody can include three CDRs and have an antigen-binding site equivalent to that of a domain antibody (dAb). An antibody can include six CDRs and have an antigen-binding site equivalent to that of a classical antibody molecule. The remainder of the polypeptide can be any sequence that provides a scaffold appropriate for the antigen-binding site and presents the antigen-binding site in a manner appropriate for antigen to bind to the antigen-binding site. An antibody can be an entire immunoglobulin molecule or a portion thereof (such as Fab, F(ab)'2, Fv, single-chain Fv (ScFv) fragment, nanobody, or single-chain variable domain (which can be a VH or VL chain having three CDRs), etc.). An antibody can be a bifunctional antibody. An antibody can be a non-human antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0277] Alternatively, the first and / or second binding domains of the bispecific molecule of the present invention can include domains not derived from and not based on immunoglobulins. To utilize the binding ability of non-antibody polypeptides, several "antibody mimetic" designed repeat proteins (DRPs) have been developed. Such molecules include ankyrin or leucine-rich repeat proteins (such as DARPin (designed ankyrin repeat protein), Anticalin, Avimer, and Versabody).

[0278] The first binding domain of the bispecific molecule of the present invention can bind to a polypeptide of MHC class I or MHC class II.

[0279] As described above, several antibodies that specifically bind to MHC class I or MHC class II have been described.

[0280] For example, WO05 / 023299 (incorporated by reference) describes antibodies that bind to MHC class II antigens, in particular, antibodies against the HLA-DR alpha chain. Table 1 of this document includes the sequence characteristics of clones MS-GPC-1 (scFv-17), MS-GPC-6 (scFv-8A), MS-GPC-8 (scFv-B8), and MS-GPC-10 (scFv-E6), and FIG. 15 shows the VH and VL sequences of MS-GPC-1; MS-GPC-6; MS-GPC-8; MS-GPC-10; MS-GPC-8-6; MS-GPC-8-10; MS-GPC-8-17; MS-GPC-8-27; MS-GPC-8-6-13; MS-GPC-8-10-57; MS-GPC-8-27-41; MS-GPC-8-1; MS-GPC-8-9; MS-GPC-8-18; MS-GPC-8-6-2; MS-GPC-8-6-19; MS-GPC-8-6-27; MS-GPC-8-6-45; MS-GPC-8-6-47; MS-GPC-8-27-7; and MS-GPC-8-27-10.

[0281] The bispecific polypeptide may include an MHC class II binding domain that includes one of these pairs of VH and VL sequences. In particular, the bispecific polypeptide may include an MHC class II binding domain based on binder MS-GPC-8.

[0282] Andris et al (1995 Mol Immunol 32:14-15) describe six antibodies specific for human HLA class I and class II antigens, including an antibody against the HLA-DQ beta chain with the antibody clone name anti-HLAII / DQB1-MP1.

[0283] Watkins et al (2000 Tissue Antigens 55:219-28) described the isolation and characterization of human monoclonal HLA-A2 antibodies. Antibody clones include: anti-HLA-A2 / A28-3PF12, anti-HLA-A2 / A28-3PC4, and anti-HLA-A2 / A28-3PB2.

[0284] The bispecific polypeptide of the present invention may comprise a binding domain of MHC class I or MHC class II derived from any of these antibodies.

[0285] The second domain of the bispecific molecule of the present invention can bind to a polypeptide comprising an intracellular signaling domain or a component of the CD3 complex. In particular, the second domain may be capable of binding to CD3 on the surface of T cells. In this regard, the second domain may comprise CD3 or a TCR-specific antibody or a portion thereof.

[0286] The second domain may comprise complementarity-determining regions (CDRs) derived from the scFv sequence shown as SEQ ID NO: 55.

[0287] The second domain may comprise an scFv sequence (such as that shown as SEQ ID NO: 55). The second domain may comprise a variant of such a sequence having at least 80% sequence identity and binding to CD3.

[0288] The second domain may comprise an antibody or a portion thereof (such as OKT3, WT32, anti-leu-4, UCHT-1, SPV-3TA, TR66, SPV-T3B, or an affinity-modified variant thereof) that specifically binds to CD3.

[0289] The second domain of the bispecific molecule of the present invention may comprise all or a portion of the monoclonal antibody OKT3, which was the first monoclonal antibody approved by the FDA. OKT3 is available from ATCC CRL 8001. The sequence of the antibody is disclosed in U.S. Patent No. 7,381,803.

[0290] The second domain may include one or more CDRs from OKT3. The second binding domain may include CDR3 from the heavy chain of OKT3 and / or CDR3 from the light chain of OKT3. The second binding domain may include all six CDRs from OKT3 as shown below.

Chemical formula

[0291] The second binding domain may include a scFv that includes a CDR sequence from OKT3. The second binding domain may include the scFv sequence shown below as SEQ ID NO: 55 or 62 or a variant thereof having at least 80% sequence identity and retaining the ability to bind to CD3.

Chemical formula

[0292] SEQ ID NO: 55 and 62 provide another structure of scFV suitable for use in the present invention. SEQ ID NO: 55 is provided as a VL-VH arrangement. SEQ ID NO: 55 is provided as a VH-VL arrangement.

[0293] Variant sequences derived from SEQ ID NO: 55 or 62 may have at least 80, 85, 90, 95, 98, or 99% sequence identity and may have equivalent or improved CD3 binding ability compared to the sequences shown as SEQ ID NO: 55 or 62.

[0294] The bispecific molecule of the present invention may include a spacer sequence that connects the first domain to the second domain and spatially separates the two domains.

[0295] For example, the first and second binding domains may be connected via a short 5-residue peptide linker (GGGGS).

[0296] The spacer array can include, for example, an IgG1 hinge or a CD8 stalk. The linker can alternatively include an alternative linker array having a length and / or domain spacing characteristic similar to that of the IgG1 hinge or the CD8 stalk.

[0297] The spacer can be a short spacer, for example, a spacer comprising less than 100, less than 80, less than 60, or less than 45 amino acids. The spacer can be or can include an IgG1 hinge or a CD8 stalk or a modified version thereof.

[0298] Examples of amino acid sequences for these linkers are shown below:

Chemical formula

Chemical formula

[0299] The CD8 stalk has a sequence that can induce homodimer formation. If this is not desired, one or more cysteine residues can be substituted or removed from the CD8 stalk sequence. The bispecific molecule of the present invention is a molecule having the sequence shown in SEQ ID NO: 64 or a variant sequence that substantially equivalently spaces the first domain and the second domain and / or a variant sequence that homodimerizes the bispecific molecule, and can include a spacer comprising or consisting of a variant having at least 80, 85, 90, 95, 98, or 99% sequence identity thereto.

[0300] The bispecific molecule of the present invention can have the following general formula: First domain - Spacer - Second domain.

[0301] In addition, the spacer may include one or more linker motifs for introducing a chain break. The chain break separates two individual domains but allows them to be oriented at different angles. Such sequences include sequence SDP and sequence SGGGSDP (SEQ ID NO: 48).

[0302] The linker may include a serine-glycine linker (such as SGGGGS (SEQ ID NO: 49)).

[0303] The spacer can cause the bispecific molecule to form a homodimer, for example, due to the presence of one or more cysteine residues in the spacer, and this spacer can form a disulfide bond with another molecule containing the same spacer.

[0304] The bispecific molecule can be tethered to the membrane. In other words, the bispecific molecule may include a transmembrane domain such that the molecule is localized to the cell membrane after expression in the cells of the present invention.

[0305] By way of example, the transmembrane domain can be the transmembrane domain described herein. For example, the transmembrane domain may include a hydrophobic alpha helix. The transmembrane domain can be derived from CD8 alpha or CD28.

[0306] The bispecific molecule of the present invention may have the following general formula: First domain - Spacer - Second domain - Transmembrane domain; or Transmembrane domain - First domain - Spacer - Second domain.

[0307] Transgenic T cell receptor The engineered immune cells of the present invention may express a transgenic T cell receptor (TCR).

[0308] The T cell receptor (TCR) is a molecule found on the surface of T cells that is responsible for recognizing a fragment of an antigen as a peptide bound to a major histocompatibility complex (MHC) molecule.

[0309] The TCR is a heterodimer composed of two different protein chains. In humans, in 95% of T cells, the TCR consists of an alpha (α) chain and a beta (β) chain (encoded by TRA and TRB, respectively), while in 5% of T cells, the TCR consists of gamma and delta (γ / δ) chains (encoded by TRG and TRD, respectively).

[0310] When the TCR associates with an antigen peptide and MHC (peptide / MHC), T lymphocytes are activated via signal transduction.

[0311] In contrast to conventional antibody-directed target antigens, the antigens recognized by TCRs can include the entire array of potential intracellular proteins that are processed and delivered to the cell surface as peptide / MHC complexes.

[0312] It is possible to engineer cells to express heterologous (i.e., non-natural) TCR molecules by artificially introducing the TRA gene and the TRB gene; or the TRG gene and the TRD gene into the cells using vectors. For example, the gene of the engineered TCR can be reintroduced into autologous T cells and returned to the patient for adoptive T cell therapy. Such "heterologous" TCRs can also be referred to herein as "transgenic TCRs".

[0313] Effector immune cells and cell surface receptor / receptor complexes The effector immune cells of the present invention can be cytolytic immune cells (such as T cells, natural killer (NK) cells, or cytokine-induced killer cells).

[0314] The T cells can be alpha-beta T cells or gamma-delta T cells.

[0315] The cells can be derived from the patient's own peripheral blood (first party), or donor peripheral blood (second party) in the context of hematopoietic stem cell transplantation, or peripheral blood from an unrelated donor (third party). Prior to the introduction of the nucleic acid molecule(s) encoding the polypeptide of the present invention, T cells or NK cells can be activated and / or expanded, for example, by treatment with anti-CD3 monoclonal antibody.

[0316] Alternatively, the cells can be derived from the ex vivo differentiation of induced or embryonic progenitor cells into T cells. Alternatively, immortalized T cell lines retaining lytic function can be used.

[0317] The cells can be hematopoietic stem cells (HSCs). HSCs can be obtained for transplantation from the bone marrow of a suitably matched donor by leukapheresis of peripheral blood after mobilization by administration of pharmacological doses of cytokines such as G-CSF (peripheral blood stem cells (PBSCs)), or from umbilical cord blood (UCB) recovered from the placenta after childbirth. Bone marrow, PBSCs, or UCB can be transplanted untreated, or HSCs can be enriched by immunoselection with monoclonal antibodies against the CD34 surface antigen.

[0318] The cell surface receptor or receptor complex binds to the antigen recognition receptor of the target immune cell, and the aforementioned receptor or receptor complex can be an MHC class I receptor or complex; an MHC class II receptor or complex; or a TCR or TCR / CD3 complex.

[0319] Target immune cells and antigen recognition receptors The target immune cells of the present invention can be cytolytic immune cells (such as T cells, natural killer (NK) cells, or cytokine-induced killer cells).

[0320] The target immune cells can be present in vitro, ex vivo, or in vivo within a population of immune cells. The target immune cells can be present, for example, in the patient or in the graft prior to administration to the patient.

[0321] The target immune cells can specifically recognize self-antigens or allogeneic antigens.

[0322] The antigen recognition receptor of the target immune cells can be the T cell receptor (such as αβ-TCR or γδ-TCR) described in more detail above. Alternatively, the antigen recognition receptor can be an NK cell activation receptor. There are two different types of surface receptors responsible for inducing NK-mediated natural cytotoxicity: NK KAR (killer activation receptor) and NK KIR (killer inhibitory receptor), which generate opposite signals. There is a balance between these competing signals that determines whether the cytotoxic activity of NK cells should be induced.

[0323] KAR typically has a non-covalently linked subunit containing an immunoreceptor activation tyrosine motif (ITAM) in the cytoplasmic side terminus (such as CD3ζ), the γc chain, or one of two adapter proteins, DAP10 and DAP12. In a manner similar to the TCR on T cells, the ITAM associated with KAR is involved in promoting signal transduction in NK cells. When the activating ligand binds to the KAR complex, the tyrosine residues in the ITAM in the associated chain are phosphorylated by kinases, and signals promoting natural cytotoxicity are transmitted into the NK cell.

[0324] Operations for withstanding the "counterattack" of target immune cells The effector immune cells of the present invention are engineered such that when the cell surface receptor or receptor complex of the effector immune cells specifically binds to the antigen recognition receptor of the target immune cells, the target immune cells are killed by the effector immune cells rather than the effector immune cells being killed by the target immune cells, so that the effector immune cells win the battle between the two immune cells.

[0325] There are various ways to engineer the effector immune cells to have a selective superiority over the target immune cells at the time and place where the two cells meet.

[0326] For example: 1) Effector immune cells can be engineered to be resistant to one or more immunosuppressive drugs. 2) Effector immune cells can be engineered to be able to transmit one or more inhibitory immune signals.

[0327] Resistance to immunosuppression Effector immune cells can be engineered to be resistant to one or more immunosuppressive drugs. This means that in the presence of immunosuppressive drugs, the target immune cells are suppressed, and the effector cells are resistant to suppression, giving the effector immune cells a selective advantage.

[0328] The immunosuppressive drug can be administered to the population of immune cells in vivo or in vitro. For example, the immunosuppressive drug can be administered to a patient before or simultaneously with the administration of a composition comprising effector immune cells. Alternatively, the immunosuppressive drug can be administered to a graft before or simultaneously with the administration of a composition comprising effector immune cells to the graft and before introducing the graft into the patient.

[0329] Immunosuppressive drugs, also known as immunosuppressive substances, immunosuppressants, and antirejection drugs, are drugs that inhibit or prevent the activity of the immune system. Immunosuppressive drugs are generally used in immunosuppressive therapy for, for example, the following purposes: (i) To prevent rejection of transplanted organs and tissues (e.g., bone marrow, heart, kidney, liver) and cells (e.g., during hematopoietic stem cell transplantation and allogeneic immunotherapy approaches); (ii) To treat autoimmune diseases or diseases most likely of autoimmune origin (e.g., rheumatoid arthritis, multiple sclerosis, myasthenia gravis, psoriasis, vitiligo, granulomatosis with polyangiitis, systemic lupus erythematosus, systemic sclerosis / scleroderma, sarcoidosis, focal segmental glomerulosclerosis, Crohn's disease, Behçet's disease, pemphigus, and ulcerative colitis); and (iii) for treating several other non-autoimmune inflammatory diseases (e.g., suppression of chronic allergic asthma), ankylosing spondylitis.

[0330] A number of immunosuppressive drugs are known and are routinely used in transplantation and immunotherapy approaches. Immunosuppressive drugs can be, for example, small molecules or antibodies or other biologics. Immunosuppressive drugs can be glucocorticoids, cytostatic agents, polyclonal or monoclonal antibodies, or drugs that act on immunophilins. These are described in more detail below.

[0331] Glucocorticoids Glucocorticoids are a class of corticosteroids within the steroid hormone class. Glucocorticoids are corticosteroids that bind to glucocorticoid receptors. Examples include: cortisol (hydrocortisone), cortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, fludrocortisone acetate, and deoxycorticosterone acetate.

[0332] At pharmacological (i.e., supra-physiological) doses, glucocorticoids are used to suppress various allergic, inflammatory, and autoimmune disorders. Glucocorticoids are also administered as post-transplant immunosuppressive agents to prevent acute graft rejection and graft-versus-host disease.

[0333] Glucocorticoids suppress cellular immunity. Glucocorticoids act by inhibiting the genes encoding the cytokines interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, and TNF-alpha, of which the most important is IL-2. When the production level of cytokines is reduced, T cell proliferation decreases. Glucocorticoids also suppress humoral immunity and reduce the expression levels of IL-2 and the IL-2 receptor by B cells. This reduces both the expansion and proliferation of B cell clones and antibody synthesis.

[0334] Glucocorticoids affect all types of inflammatory events, regardless of their cause. Glucocorticoids induce the synthesis of lipocortin-1 (annexin-1), which then binds to the cell membrane and prevents phospholipase A2 from contacting arachidonic acid, its substrate. This reduces eicosanoid production. The expression of cyclooxygenase (both COX-1 and COX-2) is also suppressed, enhancing the effect.

[0335] In addition, glucocorticoids stimulate the escape of lipocortin-1 into the extracellular space, where lipocortin-1 binds to leukocyte membrane receptors and inhibits the following various inflammatory events: epithelial adhesion, migration, chemotaxis, phagocytosis, respiratory burst, and the release of various inflammatory mediators (lysosomal enzymes, cytokines, tissue plasminogen activator, chemokines, etc.) from neutrophils, macrophages, and mast cells.

[0336] Cell growth inhibitors Cell growth inhibitors inhibit cell division. In immunotherapy, cell growth inhibitors are used at lower doses than for the treatment of malignant diseases. Cell growth inhibitors affect the growth of both T cells and B cells. Purine analogs are the most frequently administered because of their highest effectiveness. Examples of cell growth inhibitors include alkylating agents, antimetabolites, methotrexate, azathioprine and mercaptopurine, and cytotoxic antibiotics.

[0337] Alkylating agents used in immunotherapy include nitrogen mustard (cyclophosphamide), nitrosoureas, and platinum compounds. Cyclophosphamide (Baxter's Cytoxan) is perhaps the most powerful immunosuppressive compound. At low doses, cyclophosphamide is very effective in the treatment of systemic lupus erythematosus, autoimmune hemolytic anemia, granulomatosis with polyangiitis, and other immune diseases. At high doses, it causes pancytopenia and hemorrhagic cystitis.

[0338] Metabolic antagonists interfere with nucleic acid synthesis. These include: folic acid analogs (such as methotrexate); purine analogs (such as azathioprine and mercaptopurine); pyrimidine analogs (such as fluorouracil); and protein synthesis inhibitors.

[0339] Methotrexate is a folic acid analog. Methotrexate binds to dihydrofolate reductase and prevents the synthesis of tetrahydrofolate. Methotrexate is used in the treatment of autoimmune diseases (such as rheumatoid arthritis or Behcet's disease) and in transplantation.

[0340] Azathioprine (Imuran by Prometheus) is a major immunosuppressive cytotoxic substance. Azathioprine is widely used to control graft rejection. Azathioprine is non-enzymatically cleaved to mercaptopurine and acts as a purine analog and a DNA synthesis inhibitor. Also, mercaptopurine itself can be administered directly.

[0341] It affects both cellular and humoral immunity by preventing the clonal expansion of lymphocytes during the induction phase of the immune response. Also, this prevention is effective in the treatment of autoimmune diseases.

[0342] Among the cytotoxic antibiotics, dactinomycin is the most important. Dactinomycin is used in kidney transplantation. Other cytotoxic antibiotics are anthracyclines, mitomycin C, bleomycin, mitramycin.

[0343] Antibody Antibodies are sometimes used as rapid and potent immunosuppressive therapy to prevent acute rejection reactions and as targeted treatment for lymphocyte proliferative disorders or autoimmune disorders (such as anti-CD20 monoclonal). Antibodies can be polyclonal or monoclonal antibodies.

[0344] Heterologous polyclonal antibodies are obtained from the sera of animals (e.g., rabbits, horses) and injected together with the patient's thymocytes or lymphocytes. Anti-lymphocyte globulin (ALG) and anti-thymocyte globulin (ATG) are used. These are part of the treatment for steroid-resistant acute rejection and severe aplastic anemia. However, they are initially added to reduce the dosage and toxicity of other immunosuppressive drugs. Also, they can be transitioned to cyclosporine treatment.

[0345] Polyclonal antibodies inhibit T lymphocytes, cause their lysis (both complement-mediated cell lysis and cell-mediated opsonization), and then reticuloendothelial cells remove them from the circulation into the spleen and liver. In this way, polyclonal antibodies inhibit the cellular immune response (including transplant rejection, delayed-type hypersensitivity (i.e., tuberculin skin reaction), and graft-versus-host disease (GVHD)), but affect thymus-dependent antibody production.

[0346] Two commercially available preparations are: Atgam obtained from horse serum and Thymoglobulin obtained from rabbit serum. Polyclonal antibodies affect all lymphocytes, cause systemic immunosuppression, and may lead to post-transplant lymphoproliferative disorder (PTLD) or severe infections (especially by cytomegalovirus). To reduce these risks, treatment is carried out in a hospital where isolation from infection is sufficient.

[0347] Monoclonal antibodies have fewer side effects. Antibodies directed against the IL-2 receptor (CD25) and CD3 are particularly important. Monoclonal antibodies are used to prevent rejection of transplanted organs, but also to track changes in lymphocyte subsets. This is reasonable for anticipating similar new drugs in the future.

[0348] Muromonab-CD3 is an IgG2a type mouse anti-CD3 monoclonal antibody that prevents T cell activation and proliferation by binding to the T cell receptor complex present on all differentiated T cells. As such, Muromonab-CD3 is one of the most potent immunosuppressive substances and is administered for the control of acute rejection episodes that are resistant to steroids and / or polyclonal antibodies. Acting more specifically than polyclonal antibodies, Muromonab-CD3 is also used prophylactically in transplantation.

[0349] Interleukin-2 is an important immune system regulatory factor required for the clonal expansion proliferation and survival of activated T lymphocytes. Its effects are mediated by the trimeric cell surface receptor IL-2a, which consists of an alpha chain, a beta chain, and a gamma chain. IL-2a (CD25, T cell activation antigen, TAC) is expressed only by already activated T lymphocytes. Therefore, this is particularly important for selective immunosuppressive treatment, and research for developing effective and safe anti-IL-2 antibodies has been intensively conducted. Basiliximab (Simulect) and daclizumab (Zenapax) are chimeric mouse / human anti-Tac antibodies. These drugs act by binding to the alpha chain of the IL-2a receptor, preventing IL-2-induced clonal proliferation of activated lymphocytes, and shortening their survival period. These drugs are used, for example, in the prevention of acute organ rejection after bilateral kidney transplantation.

[0350] Calcineurin inhibitors and other drugs Tacrolimus and cyclosporine are calcineurin inhibitors (CNIs). Calcineurin has been used since 1983 and is one of the most widely used immunosuppressive drugs. Calcineurin is a fungal cyclic peptide composed of 11 amino acids.

[0351] Cyclosporine is thought to bind to the cytoplasmic protein cyclophilin (immunophilin) of immune lymphocytes, particularly T-lymphocytes. The complex of this cyclosporine and cyclophilin inhibits calcineurin, a phosphatase that induces the transcription of interleukin-2 under normal conditions. In addition, this drug inhibits lymphokine production and interleukin release and reduces the function of effector T cells.

[0352] Tacrolimus is a product of the bacterium Streptomyces tsukubaensis. Tacrolimus is a macrolide lactone and acts by inhibiting calcineurin.

[0353] This drug is mainly used in liver and kidney transplants, but in some clinics it is also used in heart, lung, and heart-lung transplants. This drug binds to immunophilin FKBP1A, and then the complex binds to calcineurin, inhibiting its phosphatase activity. In this way, this drug prevents cells from transitioning from the G0 phase to the G1 phase of the cell cycle. Tacrolimus is more potent than cyclosporine and its side effects are not as prominent.

[0354] Sirolimus (rapamycin) is a macrolide lactone produced by the actinomycete Streptomyces hygroscopicus. It is used to prevent rejection. Sirolimus is similar in structure to tacrolimus but has somewhat different actions and different side effects.

[0355] In contrast to cyclosporine and tacrolimus, which affect the first stage of T lymphocyte activation, sirolimus affects the second stage, namely signal transduction and clonal expansion of lymphocytes. Sirolimus binds to FKBP1A like tacrolimus, however, the complex inhibits another protein mTOR instead of calcineurin. Therefore, sirolimus acts synergistically with cyclosporine and has few side effects when combined with other immunosuppressive agents. Also, sirolimus indirectly inhibits some T lymphocyte-specific kinases and phosphatases, and thus prevents the transition from the G1 phase to the S phase of the cell cycle. In a similar manner, sirolimus prevents the differentiation of B cells into plasma cells and reduces the production of IgM, IgG, and IgA antibodies. Also, sirolimus is active against PI3K / AKT / mTOR-dependent tumors.

[0356] Everolimus is an analog of sirolimus and is also an mTOR inhibitor.

[0357] Other immunosuppressive drugs include interferon, opoid, TNF-binding protein, mycophenolate, and biologic agents.

[0358] IFN-β inhibits the production of Th1 cytokines and the activation of monocytes. IFN-β is used to delay the progression of multiple sclerosis. IFN-γ can induce lymphocytic apoptosis.

[0359] Opioid is a substance that acts on opioid receptors to obtain a morphine-like effect. Long-term use of opioid can cause immunosuppression of both innate and adaptive immunity. A decrease in proliferation and a decline in immune function have been observed not only in lymphocytes but also in macrophages. These effects are thought to be mediated by opioid receptors expressed on the surface of these immune cells.

[0360] TNF-α (Tumor necrosis factor-alpha) binding proteins are monoclonal antibodies or circulating receptors (such as infliximab (Remicade), etanercept (Enbrel), or adalimumab (Humira)), which bind to TNF-α and prevent TNF-α from inducing the synthesis of IL-1 and IL-6 and the adhesion of lymphocyte activation molecules. These are used in the treatment of rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, and psoriasis.

[0361] Also, the effects of TNF or TNF are inhibited by various natural compounds, including curcumin (a component in turmeric) and catechins (components in green tea).

[0362] Mycophenolic acid acts as a non-competitive, selective, irreversible inhibitor of inosine-5'-monophosphate dehydrogenase (IMPDH), an important enzyme in de novo guanosine nucleotide synthesis. In contrast to other human cell types, lymphocytes B and T are highly dependent on this process. Mycophenolate mofetil is used in transplant patients in combination with cyclosporine or tacrolimus.

[0363] As a small biologic agent, fingolimod, a synthetic immunosuppressant, can be mentioned. This increases the expression or changes the function of a specific adhesion molecule (α4 / β7 integrin) in lymphocytes, thereby causing lymphocytes to accumulate in lymphoid tissue (lymph nodes) and reducing their number in circulation. In this regard, small biologic agents are different from all other known immunosuppressants.

[0364] Milocillin is an unusual amino acid and an antibiotic derived from a specific thermophilic fungus. Milocillin has been shown to inhibit the proliferation of cytotoxic T cells.

[0365] Resistance by mutation The effector cells of the present invention may contain one or more mutations that increase resistance to one or more immunosuppressive drugs. For example, the effector cells may contain one or more mutations that make the cells resistant to tacrolimus and / or cyclosporine.

[0366] The effector cells may contain a nucleic acid sequence encoding a calcineurin (CN) having one or more mutations. Calcineurin (CaN) is a calcium- and calmodulin-dependent serine / threonine protein phosphatase that activates T cells of the immune system. Calcineurin activates the nuclear factor of activated T cell cytoplasm (NFATc) (a transcription factor) by dephosphorylating it. The activated NFATc then translocates into the nucleus, where it controls the expression of interleukin 2 (IL-2) and stimulates the T cell response. Calcineurin is the target of a class of drugs called calcineurin inhibitors (including cyclosporine, voclosporin, pimecrolimus, and tacrolimus). Brewin et al (2009; Blood 114:4792-4803) describe various calcineurin mutants that make cytotoxic T lymphocytes resistant to tacrolimus and / or cyclosporine.

[0367] Calcineurin is a heterodimer of calcineurin A, a 61 kD calmodulin-binding catalytic subunit, and calcineurin B, a 19 kD Ca 2+ binding regulatory subunit. There are three isozymes in the catalytic subunit, each encoded by a separate gene (PPP3CA, PPP3CB, and PPP3CC), and two isoforms in the regulatory subunit, also encoded by separate genes (PPP3R1, PPP3R2). The amino acid sequences of all polypeptides encoded by these genes are available from Uniprot under the following accession numbers: PPP3CA: Q08209; PPP3CB: P16298; PPP3CC: P48454; PPP3R1: P63098; and PPP3R2: Q96LZ3.

[0368] The amino acid sequence of the alpha isoform of calcineurin A is shown below as SEQ ID NO: 65.

Chemical formula

[0369] Mutant calcineurin A may contain mutations at one or more of the following positions with reference to SEQ ID NO: 65: V314; Y341; M347; T351; W352; S353;: L354; F356; and K360.

[0370] Mutant calcineurin A may contain one or more of the following substitution mutations with reference to SEQ ID NO: 65: V314K, V314R, or V314F; Y341F; M347W, M347R, or M347E; T351E; W352A, W352C, or W352E; S353H or S353N; L354A; F356A; and K360A or K360F.

[0371] Mutant calcineurin A may contain one or more of the following combinations of mutations with reference to SEQ ID NO: 65: L354A and K360A; L354A and K360F; T351E and K360F; W352A and S353H; T351E and L354A; W352C and K360F; W352C; L354A and K360F; V314K and Y341F; and V314R and Y341F.

[0372] The amino acid sequence of calcineurin B type 1 is shown below as SEQ ID NO: 66.

Chemical formula

[0373] Mutant calcineurin B may contain a mutation at one or more of the following positions with reference to SEQ ID NO: 66: Q51; L116; M119; V120; G121; N122; N123; L124; K125; and K165.

[0374] Mutant calcineurin B may contain one or more of the following mutations by substitution and, if necessary, insertion with reference to SEQ ID NO: 66: Q51S; L116R or L116Y; M119A, M119W, or M119-F-Ins; V120L, V120S, V120D, or V120F; G121-LF-Ins; N122A, N122H, N122F, or N122S; N123H, N123R, N123F, N123K, or N123W; L124T; K125A, K125E, K125W, K125-LA-Ins, K125-VQ-Ins, or K125-IE-Ins; and K165Q.

[0375] Mutant calcineurin B may contain one or more of the following combinations of mutations with reference to SEQ ID NO: 66: V120S and L124T; V120D and L124T; N123W and K125-LA-Ins; L124T and K125-LA-Ins; V120D and K125-LA-Ins; and M119-F-Ins and G121-LF-Ins.

[0376] In particular, the mutant calcineurin B may include the following combinations of mutations with reference to SEQ ID NO: 66: L124T and K125-LA-Ins. This is a module known as "CnB30" described in the Examples section. CnB30 has the amino acids shown as SEQ ID NO: 131.

Chemical formula

[0377] In the study described in Brewin et al 2009 (supra), the following CNa mutants showed resistance to FK506: L354A and K360F; W352A; W352C; T351E and L354A; M347W; and M347E.

[0378] The following CNa mutants showed resistance to cyclosporin A: V314K; V314R; Y341F; V314K and Y341F; and V314R and Y341F.

[0379] The following CNa mutants showed resistance to FK506: N123W; K125-VQ-Ins; K125-IE-Ins; K-125-LA-Ins; and L124T and K-125-LA-Ins.

[0380] The following CNa mutants showed resistance to cyclosporin A: K125-VQ-Ins; K125-IE-Ins; K-125-LA-Ins; V120S and L124T; and L124T and K-125-LA-Ins.

[0381] In particular, in Brewin et al 2009 (supra), the following have been reported: A combination of the mutations T351E and L354A in CNa confers resistance to CsA but not to FK506; A combination of the mutations V314R and Y341F in CNa confers resistance to FK506 but not to CsA; and A combination of the mutations L124T and K-125-LA-Ins in CNB confers resistance to both calcineurin inhibitors on CTLs.

[0382] The effector immune cells of the present invention can express variant calcineurin A containing one or more mutations in the CNa amino acid sequence and / or variant calcineurin B containing one or more mutations in the CNB amino acid sequence, in which the resistance of the effector immune cells to one or more calcineurin inhibitors is increased.

[0383] In particular, the effector immune cells can express the variant calcineurin A and / or variant calcineurin B listed above to confer resistance to cyclosporin A and / or tacrolimus (FK506).

[0384] Dominant negative CSK Effector immune cells can be engineered to express dominant negative C-terminal Src kinase (dnCSK). It has previously been shown that co-expression of dnCSK can enhance the function of CAR-expressing cells (such as CAR-T cells) (UK Patent Application Publication No. 1919017.2). Expression of dominant negative CSK in CAR-T cells appears to increase the sensitivity of CAR-T cells and improve cytotoxicity and cytokine release, particularly in response to low-density target antigens.

[0385] The present invention has here found that the expression of dnCSK also confers on cells an overall resistance to immunosuppression. The expression of dnCSK confers an “inclusive” resistance to immunosuppression, thereby generally reducing the sensitivity of cells to immunosuppressive drugs.

[0386] C-terminal Src kinase (CSK), also known as tyrosine-protein kinase, is an enzyme that phosphorylates tyrosine residues present at the C-terminus of Src-family kinases (SFK) (including SRC, HCK, FYN, LCK, LYN, and YES1), thereby suppressing their activity.

[0387] Src-family kinases (SFK) (such as Lck) are composed of an N-terminal myristoyl group, which enables membrane localization and is attached to the SH4 domain, SH3 domain, SH2 domain, and protein tyrosine kinase domain (SH1 domain).

[0388] Tyrosine residues are conserved in the activation loop and C-terminal tail. Phosphorylation of the activation loop tyrosine by trans-autophosphorylation increases SFK activity, whereas phosphorylation of the C-terminal tyrosine by C-terminal Src kinase (CSK) inhibits SFK activity.

[0389] Csk phosphorylates the C-terminal tyrosine residue Y505 of negative regulatory Lck to maintain the inactive state of Lck. In resting T cells, Csk is targeted to lipid rafts by the association of its SH2 domain with the phosphotyrosine residue pY317 of PAG. PAG is expressed as a tyrosine-phosphorylated protein in unstimulated T cells. This interaction between Csk and PAG causes activation of Csk and inhibition of Lck.

[0390] Upon TCR activation, CD45 is excluded from the membrane microdomain and dephosphorylates PAG, thereby causing Csk to dissociate from the plasma membrane.

[0391] The amino acid sequence of human CSK is available from Uniprot accession number 41240, and this sequence is shown below as SEQ ID NO: 67. In this sequence, residues 9 - 70 correspond to the SH3 domain, residues 82 - 171 correspond to the SH2 domain; and residues 195 - 449 correspond to the protein kinase domain.

Chemical

[0392] The cells of the present invention can express dominant negative C-terminal Src kinase (dnCSK).

[0393] Dominant negative CSK can lack a functional protein kinase domain. DnCSK may not contain a kinase domain, or may contain a partially or completely inactive kinase domain. The kinase domain can be inactivated, for example, by truncation or mutation of one or more amino acids.

[0394] DnCSK can be, for example, the following: i) Truncated CSK that is mobilized to the cell membrane but lacks a functional kinase domain; ii) Mutant CSK that lacks the ability to phosphorylate Y505 of Lck; or iii) Mutant CSK whose catalytic activity is inhibited by a drug (see Figure 14).

[0395] Effector immune cells can express dnCSK that completely lacks a kinase domain. For example, dnCSK can contain an SH2 domain and, optionally, an SH3 domain, but can be truncated to remove the kinase domain.

[0396] Alternatively, the effector immune cell may express a dnCSK comprising a partial truncated kinase domain comprising a part of a phosphatase (e.g., a portion of the sequence derived from residues 195 to 449 of SEQ ID NO: 67), provided that the ability of the truncated kinase to phosphorylate the C-terminal tyrosine residue Y505 of Lck is reduced compared to wild-type CSK. The truncated kinase may in fact have no remaining kinase activity.

[0397] dnCSK may be a truncated CSK that retains the ability to bind to transmembrane adapter proteins (such as PAG, Lime, and / or Dok1 / 2, etc.) that recruit wild-type CSK to the cell membrane, but lacks a functional kinase domain.

[0398] dnCSK may have the sequence shown in SEQ ID NO: 68 corresponding to the wild-type CSK sequence (SEQ ID NO: 67) with the kinase domain subtracted.

Chemical formula

[0399] Alternatively, dnCSK may have the sequence shown in SEQ ID NO: 69 corresponding to the wild-type CSK sequence (SEQ ID NO: 67) with the kinase and SH3 domains subtracted.

Chemical formula

[0400] The effector immune cells of the present invention may express a dnCSK comprising an inactivated kinase domain with reduced or no ability to phosphorylate proteins such as Lck.

[0401] The kinase domain may contain one or more amino acid mutations such that the kinase activity is reduced compared to the wild-type sequence.

[0402] The mutations may be, for example, additions, deletions, or substitutions.

[0403] The mutation may include deletion or substitution of one or more lysine residues.

[0404] The variant kinase sequence may have a mutation at lysine 222 with reference to the sequence shown as SEQ ID NO: 67.

[0405] The dnCSK of the present invention may have the sequence shown as SEQ ID NO: 70 corresponding to the full-length CSK sequence having the K222R substitution. This mutation is shown in SEQ ID NO: 70 in underlined bold. Alternatively, the dnCSK of the present invention may have a sequence equivalent to SEQ ID NO: 70 lacking the SH3 domain.

Chemical formula

[0406] The dnCSK may include a mutant CSK whose catalytic activity is inhibited by a drug. For example, the dnCSK may have the sequence shown as SEQ ID NO: 71, also known as "CSKas", which includes the mutant T266G compared to the wild-type sequence shown as SEQ ID NO: 67. The substitution is shown in SEQ ID NO: 71 in underlined bold. Alternatively, the dnCSK of the present invention may have a sequence equivalent to SEQ ID NO: 71 lacking the SH3 domain domain.

Chemical formula

[0407] The catalytic activity of CSKas is inhibited by 3-iodo-benzyl-PP1. Thus, in the presence of this molecule, CSKas acts as a dominant negative version of CSK that competes with the wild-type enzyme for binding to membrane proteins (such as PAG, Lime, and / or Dok1 / 2) that recruit wild-type CSK to the cell membrane.

[0408] Inhibiting the transmission of immune signals The effector immune cells of the present invention may express or overexpress an immunosuppressive molecule or a fusion protein containing the extracellular domain of an immunosuppressive molecule.

[0409] In vivo, membrane-bound immunosuppressive receptors (such as PD-1, LAG-3, 2B4, or BTLA1) inhibit T cell activation. During T cell activation (schematically shown in Figure 15a), when antigen is recognized by the T cell receptor (TCR), the immunoreceptor tyrosine-based activation motif (ITAM) on CD3ζ is phosphorylated. The phosphorylated ITAM is recognized by the ZAP70 SH2 domain and the T cell is activated. As schematically shown in Figure 15b, inhibitory immunoreceptors such as PD1 effectively reverse this process. PD1 has an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its endodomain that is recognized by the SH2 domain of PTPN6 (SHP-1). When PD1 binds to its ligand (PD-L1) or tumor cells, PTPN6 is recruited to the membrane-proximal region and its phosphatase domain then dephosphorylates the ITAM domain, inhibiting immune activation.

[0410] Target immune cells will naturally express immunosuppressive receptors (such as PD-1, LAG3, TIM-3, TIGIT, BTLA, VISTA, CEACAM1-R, KIR2DL4, B7-H3, and B7-H4) that contain various such ITIMs.

[0411] By engineering effector immune cells of the invention to express the ligand of one or more immunosuppressive receptors or the extracellular domain of such a ligand, when a synapse is formed between the two cells, the effector immune cell will inhibit T cell activation in the target immune cell. This "unidirectional" inhibition favors the effector immune cell over the target immune cell with respect to activation, which means that the effector immune cell will predominate and kill the target immune cell.

[0412] Effector immune cells can express or overexpress the ligand of an immunosuppressive receptor on the target immune cell. The immunosuppressive receptor expressed by the target cell can be selected, for example, from: PD-1, LAG3, TIM-3, TIGIT, BTLA, VISTA, CEACAM1-R, KIR2DL4, B7-H3, and B7-H4.

[0413] The immunosuppressive molecule expressed by effector immune cells or its extracellular domain may be selected, for example, from the following: PD-L1, PD-L2, HVEM, CD155, VSIG-3, galectin-9, HLA-G, CEACAM-1, LSECTin, FGL1, B7-H3, and B7-H4.

[0414] PD-L1 Programmed cell death ligand 1 (PD-L1), also known as surface antigen classification 274 (CD274) or B7 homolog 1 (B7-H1), is a 40 kDa type I transmembrane protein expressed by cancer cells that aids in evasion from anti-tumor immunity. When PD-L1 associates with its receptor PD-1 on T cells, signals are delivered that inhibit TCR-mediated activation of IL-2 production and T cell proliferation.

[0415] The amino acid sequence of human PD-L1 is available from Uniprot accession number Q9NZQ7 and is shown below as SEQ ID NO: 72.

Chemical formula

[0416] The signal peptide, extracellular domain, and transmembrane domain of PD-L1 are shown below as SEQ ID NOs: 73, 74, and 75, respectively.

Chemical formula

Chemical formula

[0417] The effector immune cells of the present invention may contain the PD-L1 extracellular domain and, optionally, the PD-L1 signal peptide and / or the PD-L1 transmembrane domain.

[0418] PD-L2 Programmed cell death 1 ligand 2 (PD-L2, also known as B7-DC) is an immune checkpoint receptor ligand that plays a role in the negative regulation of the adaptive immune response. PD-L2 is one of the two known ligands of programmed cell death protein 1 (PD-1), the other being PD-L1.

[0419] PD-L2 is expressed mainly on professional antigen-presenting cells (including dendritic cells (DCs) and macrophages). When PD-L2 binds to PD-1, it can activate a pathway that inhibits TCR / BCR-mediated immune cell activation, and the expression of PD-L2, PD-L1, and PD-1 is important in the immune response to certain cancers.

[0420] The amino acid sequence of human PD-L2 is available from Uniprot accession number Q9BQ51, and this sequence is shown below as SEQ ID NO: 76.

Chemical formula

[0421] The signal peptide, extracellular domain, and transmembrane domain of PD-L2 are shown below as SEQ ID NOs: 77, 78, and 79, respectively.

Chemical formula

Chemical formula

[0422] The effector immune cells of the present invention may comprise the PD-L2 extracellular domain and, optionally, the PD-L2 signal peptide and / or the PD-L2 transmembrane domain.

[0423] HVEM Herpesvirus entry mediator (HVEM), also known as tumor necrosis factor receptor superfamily member 14 (TNFRSF14), is a human cell surface receptor of the TNF-receptor superfamily. The cytoplasmic region of this receptor binds to several TNF receptor-associated factor (TRAF) family members that mediate signaling pathways activating the immune response. TNFRSF14 has been shown to interact with TRAF2, TNFSF14, and TRAF5.

[0424] The amino acid sequence of HVEM is available from Uniprot accession number Q92956 and is shown below as SEQ ID NO: 80.

Chem.

[0425] The signal peptide, extracellular domain, and transmembrane domain of HVEM are shown below as SEQ ID NOs: 81, 82, and 83, respectively.

Chem.

Chem.

[0426] The effector immune cells of the present invention may comprise the HVEM extracellular domain and, optionally, the HVEM signal peptide and / or the HVEM transmembrane domain.

[0427] CD155 CD155 (surface antigen classification 155), also known as the poliovirus receptor, is a type I transmembrane glycoprotein in the immunoglobulin superfamily. CD155 is involved in the intestinal humoral immune response and the positive selection of selected MHC-independent T cells in the thymus.

[0428] The amino acid sequence of CD155 is available from Uniprot accession number P15151 and is shown below as SEQ ID NO: 84. [Chemical formula]

[0429] The signal peptide, extracellular domain, and transmembrane domain of CD155 are shown below as SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87, respectively. [Chemical formula] [Chemical formula]

[0430] The effector immune cells of the present invention may contain the CD155 extracellular domain and, if necessary, the CD155 signal peptide and / or the CD155 transmembrane domain.

[0431] VSIG-3 VSIG-3, also known as IGSF11, is a ligand of the B7 family member VISTA. VSIG-3 inhibits human T cell proliferation in the presence of T cell receptor signaling and significantly reduces the production of cytokines and chemokines (including IFN-γ, IL-2, IL-17, CCL5 / Rantes, CCL3 / MIP-1α, and CXCL11 / I-TAC) by human T cells.

[0432] The amino acid sequence of VSIG-3 is available from the Uniprot accession number Q5DX21 and is shown below as SEQ ID NO: 88. [Chemical formula]

[0433] The signal peptide, extracellular domain, and transmembrane domain of VSIG-3 are shown below as SEQ ID NO: 89, SEQ ID NO: 90, and SEQ ID NO: 91, respectively. [Chemical formula]

[0434] The effector immune cells of the present invention may include the VSIG-3 extracellular domain and, optionally, the VSIG-3 signal peptide and / or the VSIG-3 transmembrane domain.

[0435] Galectin-9 Galectin-9 is a ligand of HAVCR2 (TIM-3) and is expressed on various tumor cells. When an interaction occurs between galectin-9 and HANCR2, it weakens the expansion and proliferation and effector functions of T cells in the tumor microenvironment. Binding to HAVCR2 induces the death of type 1 T helper lymphocytes (Th1). Galectin-9 has N-terminal and C-terminal carbohydrate-binding domains connected by a linking peptide.

[0436] The amino acid sequence of galectin-9 is available from the Uniprot accession number O00182, and this sequence is shown below as SEQ ID NO: 92.

Chemical formula

[0437] The signal peptide, galectin 1 domain, and galectin 2 domain of galectin-9 are shown below as SEQ ID NOs: 93, 94, and 95, respectively.

Chemical formula

[0438] The effector immune cells of the present invention may include the full-length galectin-9 sequence regardless of the presence or absence of the signal peptide. Alternatively, the effector immune cells may include only the galectin 1 domain or the galectin 2 domain or the HAVCR2-binding domain derived from galectin-9, -1, or -2.

[0439] The effector immune cells of the present invention may comprise galectin-9 or a membrane-anchored version of a part thereof. Galectin-9 may be anchored to the membrane using a transmembrane domain and, optionally, a spacer sequence and / or an endodomain. For example, galectin-9 or a part thereof may be anchored to the membrane using the CD8 stalk spacer, transmembrane domain, and shortened endodomain previously described in WO2013 / 153391 for the selection-suicide gene RQR8.

[0440] HLA-G The HLA-G histocompatibility antigen class I, also known as human leukocyte antigen G (HLA-G), belongs to the HLA non-classical class I heavy chain paralog. This class I molecule is a heterodimer consisting of a heavy chain and a light chain (beta-2 microglobulin). HLA-G is a ligand for the NK cell inhibitory receptor KIR2DL4, and when this HLA is expressed by the trophoblast during pregnancy, pregnancy is protected from NK cell-mediated death.

[0441] The amino acid sequence of HLA-G is available from Uniprot accession number P17693, and this sequence is shown below as SEQ ID NO: 96.

Chemical formula

[0442] The signal peptide, extracellular domain, and transmembrane domain of HLA-G are shown below as SEQ ID NOs: 97, 98, and 99, respectively.

Chemical formula

[0443] The effector immune cells of the present invention may comprise the HLA-G extracellular domain and, optionally, the HLA-G signal peptide and / or the HLA-G transmembrane domain.

[0444] CEACAM-1 Carcinoembryonic antigen-related cell adhesion molecule 1 (biliary glycoprotein) (CEACAM1), also known as CD66a (surface antigen classification 66a), is a human glycoprotein and a member of the carcinoembryonic antigen (CEA) gene family.

[0445] CEACAM-1 functions as a co-inhibitory receptor in the immune responses of T cells, natural killer (NK), and neutrophils. When the TCR / CD3 complex is stimulated, CEACAM-1 mediates homophilic binding to adjacent cells, recruits PTPN6 through interaction with LCK and phosphorylation by LCK as well as interaction with the TCR / CD3 complex, and as a result, granule exocytosis is blocked by dephosphorylation of CD247 and ZAP70, inhibiting TCR-mediated cytotoxicity. In addition, CEACAM-1 inhibits T cell proliferation and cytokine production by inhibiting the JNK cascade and plays a crucial role in the regulation of autoimmune and antitumor immunity by inhibiting T cells through interaction with HAVCR2 of T cells. During natural killer (NK) cell activation, CEACAM-1 inhibits KLRK1-mediated cell lysis of CEACAM1-bearing tumor cells by trans homophilic interaction with CEACAM1 on target cells, leading to cis interaction between CEACAM1 and KLRK1, thereby recruiting PTPN6, and then VAV1 dephosphorylation occurs.

[0446] The amino acid sequence of CEACAM-1 is available from Uniprot accession number P13688 and is shown below as SEQ ID NO: 100.

Chem.

[0447] The signal peptide, extracellular domain, and transmembrane domain of CEACAM-1 are shown below as SEQ ID NOs: 101, 102, and 103, respectively.

Chem.

[0448] The effector immune cells of the present invention may include the CEACAM-1 extracellular domain and, optionally, the CEACAM-1 signal peptide and / or the CEACAM-1 transmembrane domain.

[0449] LSECTin LSECTin, that is, liver sinusoidal endothelial cell lectin, is a ligand of LAG-3 and a negative regulator of T cell proliferation and T cell-mediated immunity.

[0450] The amino acid sequence of LSECTin is available from the Uniprot accession number Q6UXB4, and this sequence is shown below as SEQ ID NO: 104. [Chemical formula]

[0451] The cytoplasmic domain, transmembrane domain, and extracellular domain of LSECTin are shown below as SEQ ID NOs: 105, 106, and 107, respectively. [Chemical formula] [Chemical formula]

[0452] The effector immune cells of the present invention may include the LSECTin extracellular domain and, optionally, the LSECTin signal peptide and / or the LSECTin transmembrane domain.

[0453] FGL1 Fibrinogen-like protein 1 (FGL-1) is a protein that is structurally related to fibrinogen. It is an immunosuppressive molecule that inhibits antigen-specific T cell activation by acting as a major ligand for LAG3. FGL-1 is responsible for the T cell inhibitory function of LAG3 and binds to LAG3 independently of major histocompatibility complex class II (MHC-II).

[0454] The amino acid sequence of FGL1 is available from Uniprot accession number Q08830 and is shown below as SEQ ID NO: 108.

Chemical formula

[0455] The signal peptide of FGL1 is shown below as SEQ ID NO: 109. SEQ ID NO: 109 (FGL1 signal peptide) MAKVFSFILVTTALTMGREISA

[0456] The effector immune cells of the present invention may comprise FGL1 or an LAG-3 binding domain derived from FGL1 and, optionally, an FGL1 signal peptide.

[0457] The effector immune cells of the present invention may comprise FGL1 or a membrane-tethered version of a part thereof. FGL1 can be tethered to the membrane using a transmembrane domain and, optionally, a spacer sequence and / or an endodomain. For example, FGL1 or a part thereof can be tethered to the membrane using the CD8 stalk spacer, transmembrane domain, and truncated endodomain previously described for the selection-suicide gene RQR8 in WO2013 / 153391.

[0458] B7-H3 B7-H3, also known as CD276, is an immune checkpoint molecule that is expressed by some solid tumors and is involved in the control of T-cell mediated immune responses.

[0459] The amino acid sequence of B7-H3 is available from the Uniprot accession number Q5ZPR3, and this sequence is shown below as SEQ ID NO: 110.

Chem.

[0460] The signal peptide, extracellular domain, and transmembrane domain of B7-H3 are shown below as SEQ ID NOs: 111, 112, and 113, respectively.

Chem.

Chem.

[0461] The effector immune cells of the present invention may contain the B7-H3 extracellular domain and, optionally, the B7-H3 signal peptide and / or the B7-H3 transmembrane domain.

[0462] B7-H4 B7-H4, also known as V-set domain-containing T cell activation inhibitor 1, is another member of the B7 family of costimulatory proteins that act as immune checkpoint molecules. B7-H4 negatively regulates T-cell-mediated immune responses by inhibiting T cell activation, proliferation, cytokine production, and the development of cytotoxicity. B7-H4 plays an important role in the suppression of tumor-associated antigen-specific T cell immunity, together with regulatory T cells (Tregs), when expressed on the cell surface of tumor macrophages.

[0463] The amino acid sequence of B7-H4 is available from the Uniprot accession number Q7Z7D3, and this sequence is shown below as SEQ ID NO: 114.

Chem.

[0464] The signal peptide, extracellular domain, and transmembrane domain of B7-H4 are shown below as SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117, respectively. [Chemical formula]

[0465] The effector immune cells of the present invention may include the B7-H4 extracellular domain and, optionally, the B7-H4 signal peptide and / or the B7-H4 transmembrane domain.

[0466] Effector immune cells may express a protein comprising the extracellular domain of PD-L1, PD-L2, HVEM, CD155, VSIG-3, galectin-9, HLA-G, CEACAM-1, LSECTin, FGL1, B7-H3, B7-H4 having the sequences shown above, or variants thereof (e.g., variants having at least 80%, 90%, 95%, or 99% amino acid identity, provided that the resulting protein molecule retains the ability to bind to an inhibitory immune receptor on the target immune cell and inhibit the activation of the target immune cell).

[0467] Membrane localization domain Effector immunity may express a fusion protein comprising the extracellular domain and the membrane localization domain of an immune inhibitory molecule.

[0468] The membrane localization domain can be any sequence that attaches or retains the fusion protein in a position proximal to the plasma membrane.

[0469] The membrane localization domain can be or can include a sequence that initially attaches the nascent polypeptide to the ER membrane. Since the membrane material "flows" from the ER to the Golgi and ultimately to the plasma membrane, the protein remains associated with the membrane at the end of the synthesis / trafficking process.

[0470] The membrane localization domain can include, for example, a transmembrane sequence, a membrane permeation stop sequence, a GPI anchor, or a myristoylation / prenylation / palmitoylation site.

[0471] Myristoylation is a lipid modification in which a myristoyl group derived from myristic acid is covalently attached to the alpha-amino group of the N-terminal glycine residue by an amide bond. Myristic acid is a 14-carbon saturated fatty acid also known as n-tetradecanoic acid. The modification can be added either simultaneously with or after translation. N-myristoyltransferase (NMT) catalyzes the myristic acid addition reaction in the cytoplasm. Since the hydrophobic myristoyl group interacts with phospholipids in the cell membrane, myristoylation targets the protein to the membrane and the membrane adheres to the protein.

[0472] A fusion protein can contain a sequence that can be myristoylated by an NMT enzyme. The fusion protein can contain a myristoyl group when expressed in a cell.

[0473] A fusion protein can contain a consensus sequence such as: NH2-G1-X2-X3-X4-S5-X6-X7-X8, which is recognized by an NMT enzyme.

[0474] Palmitoylation is the covalent attachment of a fatty acid (such as palmitic acid) to cysteine residues of a protein, and to a lesser extent, serine and threonine residues. Palmitoylation can be used to enhance the hydrophobicity of a protein and induce membrane association. In contrast to prenylation and myristoylation, palmitoylation is usually irreversible (because the bond between palmitic acid and the protein is often a thioester bond). The reverse reaction is catalyzed by palmitoyl protein thioesterase.

[0475] In G-protein-mediated signal transduction, palmitoylation of the alpha subunit, prenylation and myristoylation of the gamma subunit are involved in tethering the G protein to the inner surface of the plasma membrane, as a result of which the G protein can interact with its receptor.

[0476] The fusion protein may contain a sequence that can be palmitoylated. The fusion protein may contain additional fatty acids that cause membrane localization when expressed intracellularly.

[0477] Prenylation (also known as isoprenylation or lipidation) is the addition of a hydrophobic molecule to a protein or chemical. The prenyl group (3-methyl-but-2-en-1-yl) facilitates attachment to the cell membrane, similar to a lipid anchor such as a GPI anchor.

[0478] Protein prenylation involves the transfer of either a farnesyl moiety or a geranylgeranyl moiety to the C-terminal cysteine(s) of the target protein. There are three enzymes that prenylate intracellularly (farnesyl transferase, Caax protease, and geranylgeranyl transferase I).

[0479] The fusion protein may contain a sequence that can be prenylated. The fusion protein may contain one or more prenyl groups that cause membrane localization when expressed intracellularly.

[0480] Cytoplasmic domain The fusion protein may contain a cytoplasmic domain derived from a protein other than the immunosuppressive molecule from which the extracellular domain is derived.

[0481] The cytoplasmic domain may stabilize the fusion protein. The cytoplasmic domain may be derived from, for example, CD19. The complete cytoplasmic domain of CD19 is shown below as SEQ ID NO: 118. The fusion protein may contain all or part of this sequence. For example, the fusion protein may contain the first 10, 15, 20, or 25 amino acids of the cytoplasmic portion of CD19. The fusion protein may contain the first 19 amino acids of the cytoplasmic portion of CD19 and have the sequence shown in SEQ ID NO: 119.

Chemical formula

[0482] Costimulatory end domain Effector immune cells can express a fusion protein comprising an extracellular domain of an immunosuppressive molecule and a costimulatory end domain.

[0483] The costimulatory end domain can be, or can include, an end domain selected from one of the following proteins: CD28, ICOS, CTLA4, 41BB, CD27, CD30, OX-40, TACI, GITR, CD2, and CD40. The amino acid sequences of these end domains are shown below as SEQ ID NOs: 120-130, respectively.

Chemical formula

Chemical formula

[0484] The fusion protein can include a combination of end domains (such as CD28 and OX-40 or CD28 and 4-1BB, etc.).

[0485] The fusion protein can include a variant of one of the sequences shown as SEQ ID NOs: 120-130 (for example, a variant having at least 80%, 90%, 95%, or 99% amino acid identity, provided that the resulting sequence retains the ability to provide a growth and / or survival signal to effector immune cells).

[0486] Nucleic acid sequence Furthermore, the present invention provides a nucleic acid sequence encoding a fusion protein comprising an extracellular domain of an immunosuppressive molecule together with: (a) a heterologous transmembrane domain (i.e., not derived from an immunosuppressive molecule); and / or (b) a heterologous end domain (i.e., not derived from an immunosuppressive molecule). The end domain can include one or more of the costimulatory domains defined above.

[0487] As used herein, the terms "polynucleotide", "nucleotide", and "nucleic acid" are intended to be synonymous with each other.

[0488] One of ordinary skill in the art will understand that a number of different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. Further, one of ordinary skill in the art will understand that using routine techniques, nucleotides can be substituted in the polynucleotides described herein that do not affect the polypeptide sequence encoded by the polynucleotides so as to reflect the codon usage frequency of any particular host organism in which the polypeptide is to be expressed.

[0489] The nucleic acids of the present invention can include DNA or RNA. The nucleic acids of the present invention can be single-stranded or double-stranded. The nucleic acids of the present invention can also be polynucleotides that include nucleotides synthesized or modified within the nucleic acids of the present invention. Several different types of modifications to oligonucleotides are known in the art. These modifications include methylphosphonate and phosphorothioate backbones, and the addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. It should be understood that the polynucleotides can be modified by any method available in the art for the uses described herein. Such modifications can be carried out to enhance the in vivo activity and extend the life span of the polynucleotide of interest.

[0490] The terms "variant", "homolog", or "derivative" with respect to a nucleotide sequence include any substitution, variation, modification, replacement, deletion, or addition of one (or more than one) nucleic acid from, or to, the foregoing sequence.

[0491] Nucleic acid construct Also, the present invention relates to (i) a first nucleic acid sequence encoding a cell surface receptor or a portion of a cell surface receptor complex as defined above; and (ii) a second nucleic acid sequence that, when expressed in a cell, confers resistance to an immunosuppressive agent on the cell; and / or (iii) a third nucleic acid sequence encoding a fusion protein comprising an immunosuppressive molecule or an extracellular domain of an immunosuppressive molecule to provide a nucleic acid construct comprising.

[0492] The first nucleic acid sequence may encode: (a) a chimeric antigen receptor (CAR); and / or (b) an engineered polypeptide comprising an ectodomain derived from an MHC class I polypeptide or an MHC class II polypeptide linked to an intracellular signaling domain; or β-2 microglobulin linked to an intracellular signaling domain; (c) an engineered polypeptide comprising an MHC class I polypeptide or an MHC class II polypeptide or β-2 microglobulin linked to a component of the CD3 / TCR complex (such as CD3-zeta, CD3-epsilon, CD3-gamma, or CD3-delta); (d) an engineered polypeptide comprising a binding domain (such as an antibody-like binding domain) that binds to an MHC class I polypeptide, an MHC class II polypeptide, or β-2 microglobulin linked to an intracellular signaling domain; (f) an engineered polypeptide comprising CD79α or CD79β linked to an intracellular signaling domain; (g) an MHC class II binding domain of CD4 linked to an intracellular signaling domain; or an engineered polypeptide comprising an MHC class I binding domain of CD8 linked to an intracellular signaling domain; or (e) (i) an MHC class I polypeptide; an MHC class II polypeptide; a first binding domain that binds to β-2 microglobulin; and (ii) a bispecific polypeptide comprising a second binding domain that binds to a component of the TCR / CD3 complex.

[0493] The second nucleic acid sequence may encode: (e) A variant calcineurin with increased resistance to one or more calcineurin inhibitors compared to wild-type calcineurin; and / or (f) Dominant negative CSK.

[0494] The third nucleic acid sequence may encode: (g) An immunosuppressive molecule or a fusion protein comprising an extracellular domain of an immunosuppressive molecule.

[0495] In a first embodiment, the present invention provides a nucleic acid construct comprising: (i) A first nucleic acid sequence encoding a CAR that specifically binds to TRBC1 or TRBC2; and (ii) A second nucleic acid sequence encoding a variant calcineurin with increased resistance to one or more calcineurin inhibitors compared to wild-type calcineurin, and / or dominant negative CSK; and / or (iii) A third nucleic acid sequence encoding an immunosuppressive molecule or a fusion protein comprising an extracellular domain of an immunosuppressive molecule.

[0496] In a second embodiment, the present invention provides a nucleic acid construct comprising: (i) A first nucleic acid sequence encoding β-2 microglobulin linked to an intracellular signaling domain; and (ii) A second nucleic acid sequence encoding a variant calcineurin with increased resistance to one or more calcineurin inhibitors compared to wild-type calcineurin, and / or dominant negative CSK; and / or (iii) A third nucleic acid sequence encoding an immunosuppressive molecule or a fusion protein comprising an extracellular domain of an immunosuppressive molecule.

[0497] Also, the nucleic acid construct of the second embodiment may comprise a nucleic acid sequence encoding a CAR.

[0498] The nucleic acids may be in any order within the nucleic acid. Nucleic acids encoding separate polypeptides may be separated by co-expression sites capable of co-expressing the two polypeptides as separate entities. A co-cleavage site may be a sequence encoding a cleavage site such that the nucleic acid construct produces both polypeptides linked by the cleavage site(s). The cleavage site may be self-cleaving such that when the polypeptide is produced, it is immediately cleaved into separate peptides without the need for any external cleavage activity.

[0499] The cleavage site can be any sequence that allows the two polypeptides to become separated.

[0500] The term "cleavage" is used herein for convenience, but the cleavage site can separate the peptides into individual entities by mechanisms other than classical cleavage. For example, for the foot-and-mouth disease virus (FMDV) 2A self-cleaving peptide (see below), various models have been proposed to explain the following "cleavage" activity: proteolytic activity by host cell proteases, autoproteolytic activity, or translational effects (Donnelly et al. (2001) J. Gen. Virol. 82:1027-1041). The exact mechanism of such "cleavage" is not important for the purposes of the present invention as long as the cleavage site expresses the proteins as separate entities when placed between nucleic acid sequences encoding the proteins.

[0501] The cleavage site can be, for example, a furin cleavage site, a tobacco etch virus (TEV) cleavage site, or can encode a self-cleaving peptide.

[0502] A "self-cleaving peptide" refers to a peptide that functions such that when a polypeptide containing the protein and the self-cleaving peptide is produced, it is immediately "cleaved" or separated into individual and separate first and second polypeptides without the need for any external cleavage activity.

[0503] The self-cleaving peptide can be a 2A self-cleaving peptide derived from aphthovirus or cardiovirus. The primary 2A / 2B cleavage of aphthovirus and cardiovirus is mediated by the "cleavage" of 2A at its own C-terminus. In aphthoviruses (such as foot-and-mouth disease virus (FMDV) and equine rhinitis A virus), the 2A region is a short segment of about 18 amino acids and, together with the N-terminal residue (conserved proline residue) of protein 2B, exhibits an autonomous element that can mediate "cleavage" at its own C-terminus (Donelly et al. (2001) supra).

[0504] "2A-like" sequences have been found in picornaviruses other than aphthovirus or cardiovirus, "picornavirus-like" insect viruses, type C rotavirus, as well as repetitive sequences within Trypanosoma spp and bacterial sequences (Donnelly et al. (2001) supra).

[0505] The cleavage site may include a 2A-like sequence (RAEGRGSLLTCGDVEENPGP) shown as SEQ ID NO: 132.

[0506] Vector Furthermore, the present invention provides a vector or a kit of vectors comprising one or more nucleic acid sequences and / or nucleic acid constructs of the present invention. Such vectors can be used to introduce nucleic acid sequences into host cells to express a cell surface receptor or receptor complex together with one or more proteins that confer selective superiority to the host cells over target immune cells (i.e., effector immune cells).

[0507] The kit of vectors may include: (i) a first vector comprising a nucleic acid sequence encoding a part of a cell surface receptor or cell surface receptor complex; and (ii) a second vector comprising a nucleic acid sequence that confers resistance to an immunosuppressive agent to the aforementioned cells when expressed in the cells; and / or (iii) A third vector comprising a nucleic acid sequence encoding a fusion protein comprising an immunosuppressive molecule or an extracellular domain of an immunosuppressive molecule.

[0508] In a first embodiment, the present invention provides a kit of vectors comprising: (i) A first vector comprising a nucleic acid sequence encoding a CAR that specifically binds to TRBC1 or TRBC2; and (ii) A second vector comprising a nucleic acid sequence encoding a variant calcineurin with increased resistance to one or more calcineurin inhibitors compared to wild-type calcineurin, and / or a dominant negative CSK; and / or (iii) A third vector comprising a nucleic acid sequence encoding a fusion protein comprising an immunosuppressive molecule or an extracellular domain of an immunosuppressive molecule.

[0509] In a second embodiment, the present invention provides a kit of vectors comprising: (i) A first vector comprising a nucleic acid sequence encoding β-2 microglobulin linked to an intracellular signaling domain; and (ii) A second vector comprising a nucleic acid sequence encoding a variant calcineurin with increased resistance to one or more calcineurin inhibitors compared to wild-type calcineurin, and / or a dominant negative CSK; and / or (iii) A third vector comprising a nucleic acid sequence encoding a fusion protein comprising an immunosuppressive molecule or an extracellular domain of an immunosuppressive molecule.

[0510] Also, the kit of vectors of the second embodiment may comprise a vector comprising a nucleic acid sequence encoding a CAR.

[0511] The vector can be, for example, a plasmid or a viral vector (such as a retroviral vector or a lentiviral vector), or a transposon-based vector or synthetic mRNA.

[0512] The vector may be capable of being transfected or transduced into cells such as T cells or NK cells.

[0513] Cell The present invention provides effector immune cells.

[0514] The cell may contain the nucleic acid sequence, nucleic acid construct, or vector of the present invention.

[0515] The cell may be a cytolytic immune cell (such as a T cell or NK cell).

[0516] T cells or T lymphocytes are one type of lymphocyte that play a central role in cellular immunity. These can be distinguished from other lymphocytes (such as B cells and natural killer cells (NK cells)) by the presence of T cell receptors (TCRs) on the cell surface. As summarized below, there are various types of T cells.

[0517] Helper T helper cells (TH cells) assist other white blood cells in immunological processes (including the maturation of B cells into plasma cells and memory B cells and the activation of cytotoxic T cells and macrophages). TH cells express CD4 on their surface. TH cells become activated when peptide antigens are presented by MHC class II molecules on the surface of antigen-presenting cells (APCs). These cells can differentiate into one of several subtypes (TH1, TH2, TH3, TH17, Th9, or TFH) that secrete different cytokines to facilitate different types of immune responses.

[0518] Cytolytic T cells (TC cells, or CTLs) destroy virus-infected cells and tumor cells and are also involved in graft rejection. CTLs express CD8 on their surface. These cells recognize their targets by binding to antigens associated with MHC class I, which are present on the surface of all nucleated cells. CD8+ cells can be inactivated in an anergic state via IL-10, adenosine, and other molecules secreted by regulatory T cells, thereby preventing autoimmune diseases such as experimental autoimmune encephalomyelitis.

[0519] Memory T cells are a subset of antigen-specific T cells that are maintained for long periods after recovery from an infection. Memory T cells "remember" past infections for the immune system because they rapidly become numerous effector T cells upon re-exposure to their cognate antigen. Memory T cells include the following three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells can be either CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.

[0520] Regulatory T cells (Treg cells), formerly known as suppressor T cells, are essential for maintaining immune tolerance. Their main roles are to halt T cell-mediated immunity towards the end of an immune response and to suppress autoreactive T cells that have avoided the negative selection process in the thymus.

[0521] Two main classes of CD4+ Treg cells (intrinsic Treg cells and adaptive Treg cells) have been described.

[0522] Intrinsic Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) are generated in the thymus and are involved in the interaction of developing T cells with both myeloid (CD11c+) dendritic cells and plasmacytoid (CD123+) dendritic cells activated by TSLP. Intrinsic Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutating the FOXP3 gene blocks the development of regulatory T cells and can lead to the development of the lethal autoimmune disease IPEX.

[0523] Adaptive Treg cells (also known as Tr1 cells or Th3 cells) can be generated during a normal immune response.

[0524] The cells can be natural killer cells (or NK cells). NK cells form part of the innate immune system. NK cells respond rapidly in an MHC-dependent manner to innate signals from virus-infected cells.

[0525] NK cells (which belong to the innate lymphocyte cell group) are defined as large granular lymphocytes (LGL) and constitute a third cell type that differentiates from a common lymphoid progenitor cell that gives rise to B lymphocytes and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus and then enter the circulation.

[0526] The cells of the present invention can be any of the above cell types.

[0527] The cells of the present invention can be generated ex vivo from any of the patient's own peripheral blood (first party), or donor peripheral blood in the context of a hematopoietic stem cell transplant (second party), or peripheral blood from an unrelated donor (third party).

[0528] Alternatively, the cells can be derived from the ex vivo differentiation of induced progenitor cells or embryonic progenitor cells, for example, into T cells or NK cells. Alternatively, an immortalized T cell line that retains lytic function and can act as a therapeutic agent can be used.

[0529] In all of these embodiments, the chimeric polypeptide-expressing cells are generated by introducing DNA or RNA encoding the chimeric polypeptide by one of a number of means, including transduction using viral vectors, transfection using DNA or RNA.

[0530] The cells of the invention can be ex vivo cells derived from a subject. The cells can be derived from a peripheral blood mononuclear cell (PBMC) sample. The cells can be activated and / or expanded, for example, by treatment with an anti-CD3 monoclonal antibody, prior to being transfected with a nucleic acid encoding a molecule from which the chimeric polypeptide of the first aspect of the invention is obtained.

[0531] The cells of the invention can be made by: (i) isolation of a cell-containing sample from the subject or other source listed above; and (ii) transduction or transfection of the cells with one or more nucleic acid sequences, nucleic acid constructs, or vectors of the invention.

[0532] The cells can then be purified (e.g., selected) based on the expression of, for example, one or more heterologous nucleic acid sequences.

[0533] Effector immune cells can recognize and kill target immune cells. The target immune cells can be cytolytic immune cells, such as T cells or NK cells as defined above.

[0534] Pharmaceutical composition The invention also relates to a pharmaceutical composition comprising a plurality of the cells of the invention.

[0535] The pharmaceutical composition can further comprise a pharmaceutically acceptable carrier, diluent, or excipient. The pharmaceutical composition can optionally comprise one or more additional pharmaceutically active polypeptides and / or compounds. Such formulations can be, for example, in a form suitable for intravenous infusion.

[0536] Treatment method The present invention provides a method for treating a disease, which includes a step of administering the cells of the present invention to a subject (for example, in the above-mentioned pharmaceutical composition).

[0537] The method for treating a disease relates to the therapeutic use of the cells of the present invention. In this specification, cells can be administered to a subject already having a disease or symptom in order to alleviate, reduce, or improve at least one symptom related to the disease and / or delay, reduce, or block the progression of the disease.

[0538] The method for preventing a disease relates to the prophylactic use of the cells of the present invention. In this specification, such cells can be administered to a subject who has not yet suffered from and / or does not show any symptoms of the disease in order to prevent or impair the cause of the disease and / or reduce or prevent the occurrence of at least one symptom related to the disease. The subject may have a predisposition to the disease or may be considered to be at risk of developing the disease.

[0539] The method may include the following steps: (i) A step of isolating a cell-containing sample; (ii) A step of transducing or transfecting such cells with the nucleic acid sequence or vector provided by the present invention; (iii) A step of administering the cells derived from (ii) to a subject.

[0540] The sample containing cells can be isolated from the above-mentioned subject or other sources.

[0541] Furthermore, the present invention provides a method for treating a disease in a subject, which includes the following steps: (i) A step of administering a pharmaceutical composition to a subject, wherein the pharmaceutical composition contains a plurality of effector immune cells that are engineered to be resistant to an immunosuppressive agent; and (ii) A step of administering the aforementioned immunosuppressive agent to the aforementioned subject.

[0542] Effector immune cells can express, for example, a variant calcineurin that has been engineered to be resistant to one or more of the following calcineurin inhibitors: Calcineurin A containing the mutations T351E and L354A with reference to the sequence shown as SEQ ID NO: 65; Calcineurin A containing the mutations V314R and Y341F with reference to the sequence shown as SEQ ID NO: 65; or Calcineurin B containing the mutations L124T and K-125-LA-Ins with reference to the sequence shown as SEQ ID NO: 66.

[0543] Step (ii) can include administering cyclosporine and / or tacrolimus to the cell or patient.

[0544] Effector cells can express dnCSK, and step (ii) can include administering any immunosuppressant (e.g., rapamycin) to the subject.

[0545] The present invention provides the cells of the present invention for use in treating and / or preventing a disease.

[0546] The present invention also relates to the use of the cells of the present invention in the manufacture of a medicament for treating a disease.

[0547] Diseases to be treated by the methods of the present invention can be cancer diseases (such as bladder cancer, breast cancer, colon cancer, endometrial cancer, kidney cancer (renal cell carcinoma), leukemia, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, and thyroid cancer, etc.).

[0548] The disease can be multiple myeloma (MM), B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), neuroblastoma, T-cell acute lymphoblastic leukemia (T-ALL), or diffuse large B-cell lymphoma (DLBCL).

[0549] The disease can be a plasma cell disorder, such as plasmacytoma, plasma cell leukemia, multiple myeloma, macroglobulinemia, amyloidosis, Waldenström macroglobulinemia, solitary bone plasmacytoma, extramedullary plasmacytoma, osteosclerotic myeloma, heavy chain disease, monoclonal gammopathy of undetermined significance, or smoldering multiple myeloma.

[0550] The effector immune cells of the present invention can kill target immune cells, which can be cancer cells or normal immune cells that are reactive to the effector immune cells.

[0551] The present invention also provides a method for depleting alloreactive immune cells from a population of immune cells, the method comprising contacting the population of immune cells with a plurality of effector immune cells having the engineered MHC class I complex or MHC class II complex as defined above.

[0552] The present invention also provides a method for treating or preventing graft rejection after allotransplantation, the method comprising administering to a recipient subject a plurality of effector immune cells derived from a donor subject for the allogeneic transplantation as described above, wherein the plurality of effector immune cells express the engineered MHC class I complex or MHC class II complex as defined above.

[0553] The effector immune cells can be administered to a patient before, after, or simultaneously with transplantation. For example, for organ transplantation, effector T cells derived from an organ donor expressing the engineered MHC class I complex or MHC class II complex as defined above can be injected into the recipient before transplantation to eliminate alloreactive T cells that can mediate graft rejection. Alternatively, in the case of HSCT, recipient T cells expressing the engineered MHC class I complex or MHC class II complex as defined above can be cultured with the stem cell graft before injection to eliminate donor alloreactive T cells that can attack host tissue.

[0554] Also provided is a method for treating or preventing graft-versus-host disease (GVHD) associated with allotransplantation, the method comprising contacting an allograft with a plurality of effector immune cells having an engineered MHC class I complex or MHC class II complex as defined above.

[0555] Allotransplantation may include adoptive transfer of allogeneic immune cells.

[0556] Also provided are allografts in which alloreactive immune cells are depleted by the method of the invention. Also provided are allografts comprising the effector immune cells of the invention.

[0557] Also provided are the effector immune cells of the invention for use in the following: Depletion of alloreactive immune cells from a population of immune cells; Treatment or prevention of graft rejection after allotransplantation; or Treatment or prevention of graft-versus-host disease (GVHD) associated with allotransplantation.

[0558] Also provided is the use of the effector immune cells of the invention in the manufacture of a pharmaceutical composition for the following: Depletion of alloreactive immune cells from a population of immune cells; Treatment or prevention of graft rejection after allotransplantation; or Treatment or prevention of graft-versus-host disease (GVHD) associated with allotransplantation.

[0559] The present invention will now be further illustrated by way of examples, which are intended to assist those skilled in the art in practicing the invention and are in no way intended to limit the scope of the invention.

Examples

[0560] Example 1 - Generation of a model system demonstrating "reverse" killing of TRBC1-binding CAR-T cells by target T cells WO2015 / 132598 describes a CAR that specifically binds to TCR beta constant region 1 (TRBC1) comprising VH and VL domains shown as SEQ ID NOs: 7 and 8, respectively.

[0561] A shortened version of this CAR lacking the signaling domain called dJOVI was created; dJOVI binds to TRBC1 on target cells but cannot induce T cell activation and death. PBMCs were transduced with a vector expressing dJOVI or full-length CAR (JOVI) together with the selection-suicide gene RQR8 described in WO2013 / 153391. JOVI- or dJOVI-transduced PBMCs were co-cultured with TRBC1+ target PBMCs at an effector:target ratio of 1:2, and viable transduced (RQR8+) T cells were counted 24 hours after co-culture. The results are shown in Figure 12. Killing of effector cells by TRBC1+ target cells was more in dJOVI-transduced PBMCs, indicating that binding of JOVI to TRBC1 on the target was sufficient to induce target T cell activation and reverse-kill effector cells.

[0562] Example 2 - Expression of PDL1 or PDL2 by TRBC1-binding CAR-T cells reduces reverse-killing of effector cells To investigate the effect of engineering CAR-T cells to transmit inhibitory immune signals on reverse-killing by target T cells, PBMCs were transduced to express JOVI- or dJOVI- together with truncated versions of PD-L1 or PDL2 lacking the cytoplasmic domain (dPDL1 and dPDL2). For this assay, TRBC1+ target PBMCs were transduced to express full-length PD1.

[0563] Co-cultures using PBMCs transduced with JOVI or dJOVI expressing dPDL1 or dPDL2 together with RQR8; and TRBC1+ target PBMCs expressing PD1 were set up at an effector:target ratio of 1:1. Viable transduced (RQR8+) T cells were counted 72 hours after co-culture and each condition was normalized to its respective JOVI (or dJOVI) co-culture. The results are shown in Figure 13. The number of recovered transduced cells increased when dPDL1 or dPDL2 was expressed on the CAR compared to CAR alone.

[0564] Example 3 - Expression of calcineurin mutants by TRBC1-binding CAR-T cells reduces effector cell anergy in the presence of calcineurin inhibitors Co-culture of JOVI-RQR8 transduced PBMCs expressing calcineurin mutants with TRBC1+ target PBMCs is set up at effector:target ratios of 1:1 and 1:4. Different concentrations of calcineurin inhibitors are added to the co-cultures. Live transduced (RQR8+) T cells are counted by flow cytometry 72 hours after co-culture and each condition is normalized to the co-culture without added inhibitor.

[0565] Example 4 - Expression of dnCSK by TRBC1-binding CAR-T cells reduces effector cell anergy in the presence of immunosuppressants Co-culture of JOVI-RQR8 transduced PBMCs expressing dnCSK with TRBC1+ target PBMCs is set up at effector:target ratios of 1:1 and 1:4. Different concentrations of immunosuppressants are added to the co-cultures. Live transduced (RQR8+) T cells are counted by flow cytometry 72 hours after co-culture and each condition is normalized to the co-culture without added immunosuppressant.

[0566] Example 5 - Expression of PDL1 or PDL2 by effector T cells expressing β2m-CD3ζ reduces anergy by target cells To investigate the effect of engineering CAR-T cells to transmit inhibitory immune signals to target T cells undergoing necroptotic cell death with a necroptosis-associated response; PBMCs are transduced to express JOVI-, dJOVI-, or irrelevant CARs together with a fusion protein consisting of a truncated version of PD-L1 or PDL2 lacking the cytoplasmic domain (dPDL1 and dPDL2) and B2M tethered to CD3 zeta (β2m-CD3ζ). For this assay, TRBC1+ target PBMCs are transduced to express full-length PD1 in the presence or absence of a superantigen (SAg) that ligates enhanced MHC to the TCR. The superantigen is not processed intracellularly. Instead, the superantigen binds to class II MHC molecules as intact molecules and binds outside of the peptide antigen-binding groove. SAg is a molecule that indiscriminately stimulates up to 20% of total T cells (a normal response to an antigen stimulates only 0.01% of T cells).

[0567] Co-cultures of PBMCs transduced with JOVI- or dJOVI- or irrelevant CARs expressing dPDL1 or dPDL2 together with β2m-CD3ζ; and TRBC1+ target PBMCs expressing PD1+SAg are set up at a 1:1 effector:target ratio. Live transduced T cells are counted 72 hours after co-culture and each condition is normalized to its respective JOVI (or dJOVI) co-culture.

[0568] Example 6 - Expression of calcineurin mutants by T cells expressing β2m-CD3ζ reduces effector cell necroptosis in the presence of calcineurin inhibitors The co - culture of PBMCs transduced with vectors encoding CAR (JOVI, dJOVI, or an unrelated CAR), β2m - CD3ζ, and a calcineurin variant with TRBC1+ target PBMCs is set at effector:target ratios of 1:1 and 1:4. Different concentrations of calcineurin inhibitors and SAg are added to the co - cultures. Live transduced T cells are counted by flow cytometry 72 hours after co - culture, and each condition is normalized to the co - culture without the inhibitor or SAg added.

[0569] Example 7 - Expression of dnCSK by T cells expressing β2m - CD3ζ reduces the anergy of effector cells in the presence of immunosuppressive agents The co - culture of PBMCs transduced with vectors encoding CAR (JOVI, dJOVI, or an unrelated CAR), β2m - CD3ζ, and dnCSK with TRBC1+ target PBMCs is set at effector:target ratios of 1:1 and 1:4. Different concentrations of immunosuppressive agents and SAg are added to the co - cultures. Live transduced T cells are counted by flow cytometry 72 hours after co - culture, and each condition is normalized to the co - culture without the immunosuppressive agent or SAg added.

[0570] Example 8 - Expression of a calcineurin variant by TRBC2 - binding CAR - T cells confers resistance to growth inhibition by calcineurin inhibitors PBMCs were transduced with a vector expressing CAR together with the selection - suicide gene RQR8 described in WO2013 / 153391. The CARs tested are summarized below: CD19 CAR: A second - generation CAR having an antigen - binding domain derived from Fmc63, a hinge spacer, and a 41BB / CD3z endodomain TRBC1 CAR: A second - generation CAR having an antigen - binding domain, a hinge spacer, and a 41BB / CD3z endodomain described in WO2018 / 224844 TRBC2 CAR: A second-generation CAR having the antigen-binding domain, CD8 stalk spacer, and CD28 / CD3z endodomain described in WO2020 / 089644

[0571] One cell population was transduced with a tricistronic vector expressing RQR8, TRBC2 CAR, and the CnB30 calcineurin mutant module having SEQ ID NO: 131 above.

[0572] The transduced cells were co-cultured with one of the following target cell types: Jurkat TRBC1: Wild-type Jurkat cells expressing TRBC1 Jurkat KO: Jurkat cells engineered to lack TRBC1 expression Jurkat TRBC2: Jurkat cells in which the TRBC1 gene has been replaced with the TRBC2 gene such that the expression of TRBC2 is identical to the expression of TRBC1 on wild-type cells using CRISPR-Cas9 technology.

[0573] Cells were co-cultured for 96 hours at an E:T ratio of 1:4 in the presence or absence of 20 ng / ml tacrolimus. Transduced effector cells were identified based on RQR8 expression and proliferation of the aforementioned cells analyzed using cell-trace violet (CTV) dilution. The results are shown in Figures 16 and 17. As expected, in the absence of tacrolimus, cells expressing the TRBC1 CAR had an increased percentage and number of proliferating cells after co-culture with target cells expressing TRBC1; cells expressing the TRBC2 CAR had an increased percentage and number of proliferating cells after co-culture with target cells expressing TRBC2 (Figure 16). As seen by comparison of "TRBC2 CAR" in Figure 16B (without tacrolimus) and Figure 17B (with tacrolimus), in the presence of tacrolimus, CAR-T cell proliferation is inhibited. Only cells co-expressing the TRBC2 CAR and the CnB30 calcineurin mutant had an increased absolute number of transduced effector cells after co-culture with targets expressing TRBC2 (Figure 17B). Also, this population had the highest percentage of transduced proliferating cells (Figure 17A).

[0574] Also, proliferation analysis was performed using the FlowJo proliferation tool with CD19 CAR as a negative control for single / live / CellTrace Violet positive cells. The number of cells at each division was plotted for each of the above CAR+ target combinations, and the results are shown in Figure 18 (without tacrolimus) and Figure 19 (with tacrolimus). Also, the results for two individual donors are shown in the histogram plot of Figure 20. Also, in the presence of tacrolimus, only cells co-expressing the TRBC2 CAR and the CnB30 calcineurin mutant had increased effector cell proliferation after co-culture with targets expressing TRBC2 (Figure 19, lower graph; and Figure 20).

[0575] In a similar study, cells transduced to express either only the TRBC2 CAR or the TRBC2 CAR in combination with the calcineurin variant (CnB30) were co-cultured with the TRBC2+ positive target in the presence or absence of 20 ng / ml of tacrolimus for 4 days. The number of CAR-expressing cells after 4 days of co-culture is shown in FIG. 21. The only population with a large number of TRBC2 CAR-expressing cells after co-culture in the presence of tacrolimus was the cells co-expressing the TRBC2 CAR with the calcineurin variant (TRBC2 CAR + CnB30).

[0576] FIG. 22 shows the percentage of RQR8-expressing cells. The percentage of CD19-expressing cells was constant, while the percentage of cells expressing the TRBC2 CAR increased after co-culture with the TRBC2+ target in the absence of tacrolimus. This was true for cells expressing only the TRBC2 CAR or cells co-expressing the TRBC2 CAR in combination with the calcineurin variant. In the presence of tacrolimus, the percentage of RQR8+ cells expressing only the TRBC2 CAR decreased, indicating that tacrolimus inhibits the increase of these cells. In contrast, the percentage of RQR8+ cells co-expressing TRBC2 CAR / CnB30 was the same as that in the co-culture without tacrolimus, indicating that these cells are resistant to calcineurin inhibition.

[0577] Example 9 - Investigation of the effect of calcineurin variant expression by anti-TRBC2-expressing cells on reverse killing by target cells expressing TRBC2 PBMCs from healthy donors were magnetically sorted into the TRBC1+ fraction and the TRBC2+ fraction. Two days after activation, the TRBC1+ fraction was transduced with a retroviral vector expressing either of the above RQR8 and CD19 or the TRBC2 CAR. One cell population was transduced with a tricistronic vector expressing RQR8, the TRBC2 CAR, and the CnB30 calcineurin variant module having the above SEQ ID NO: 131.

[0578] Cells were left untreated or treated with 20 ng / ml tacrolimus 3 days after transduction and allowed to expand for an additional 4 days under these conditions. Seven days after transduction, killing assays were set up at effector:target ratios of 1:1 and 1:4, and the non-transduced TRBC2+ fraction was labeled with Cell Trace Violet and used as a self-target.

[0579] Killing was assayed by flow cytometry 72 hours later, and supernatants were harvested from the co-cultures and analyzed for IFNγ and IL-2 production. Transduced effector cells were identified based on their RQR8 expression. The results are shown in Figures 23 - 26.

[0580] Target cell killing was improved in the effector cell population co-expressing the TRBC2 CAR and the CnB30 calcineurin mutant compared to the effector cell population expressing only the TRBC2 CAR after expansion and co-culture in the presence of tacrolimus (Figure 23). This effect was particularly prominent when the cells were co-cultured at an E:T ratio of 1:4.

[0581] After 72 hours of co-culture with TRBC2-expressing PBMCs at a 1:1 ratio in the absence of tacrolimus, some anti-TRBC2 CAR-expressing cells were detected (Figure 24, first graph). However, when CAR-expressing cells were co-cultured with TRBC2-expressing PBMCs at a 1:4 ratio, the CAR T cell count was near zero (Figure 24, second graph), which was probably due to the reverse killing of CAR-expressing cells by the target cells.

[0582] However, after expansion and co-culture in the presence of tacrolimus, the effector cell population co-expressing the TRBC2 CAR and the CnB30 calcineurin mutant had somewhat similar survival / growth after co-culture at a 1:4 ratio. At a 1:1 co-culture ratio, the effector cell population co-expressing the TRBC2 CAR and the CnB30 calcineurin mutant showed much higher survival / growth than the effector cell population expressing only the TRBC2 CAR (Figure 24, third and fourth graphs).

[0583] In addition, in a cell population co-expressing TRBC2 CAR and the CnB30 calcineurin mutant, T cell activation regarding expansion and proliferation in the presence of tacrolimus and cytokine release after co-culture was increased compared to a cell population expressing only TRBC2 CAR. This applies to both IFNγ (Figure 25) and IL-2 (Figure 26).

[0584] In summary, these data indicate that the expression of the calcineurin mutant by CAR-expressing cells favors effector cells over target T cells and prevents reverse killing by target cells.

[0585] All publications described in the above specification are hereby incorporated by reference herein. Various modifications and variations of the described methods and systems of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in conjunction with specific preferred embodiments, it should be understood that the invention claimed in the claims should not be unduly limited to the specific embodiments described. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art of molecular biology or related fields are intended to be within the scope of the following claims. In certain embodiments, for example, the following items are provided. (Item 1) An effector immune cell expressing a cell surface receptor or receptor complex that specifically binds to an antigen recognition receptor of a target immune cell, wherein the effector immune cell is engineered such that when a synapse is formed between the effector immune cell and the target immune cell, the ability of the effector immune cell to kill the target immune cell is higher than the ability of the target immune cell to kill the effector immune cell. (Item 2) The effector immune cell according to Item 1, which is engineered to be resistant to an immunosuppressive agent. (Item 3) The effector immune cell according to item 2, which is engineered to be resistant to one or more calcineurin inhibitors. (Item 4) The effector immune cell according to item 3, which expresses: Calcineurin A containing the mutations T351E and L354A with reference to the sequence shown as SEQ ID NO: 65; Calcineurin A containing the mutations V314R and Y341F with reference to the sequence shown as SEQ ID NO: 65; or Calcineurin B containing the mutations L124T and K-125-LA-Ins with reference to the sequence shown as SEQ ID NO: 66. (Item 5) The effector immune cell according to item 2, which is engineered to be resistant to rapamycin. (Item 6) The effector immune cell according to item 2, which expresses a dominant negative C-terminal Src kinase (dnCSK). (Item 7) The effector immune cell according to item 1, which is engineered to express or overexpress an immunosuppressive molecule or a fusion protein comprising the extracellular domain of an immunosuppressive molecule. (Item 8) The effector immune cell according to item 7, wherein the immunosuppressive molecule binds to PD-1, LAG3, TIM-3, TIGIT, BTLA, VISTA, CEACAM1-R, KIR2DL4, B7-H3, or B7-H4. (Item 9) The effector immune cell according to item 7, wherein the immunosuppressive molecule is selected from PD-L1, PD-L2, HVEM, CD155, VSIG-3, galectin-9, HLA-G, CEACAM-1, LSECTin, FGL1, B7-H3, B7-H4. (Item 10) The effector immune cell according to any one of items 7 to 9, which is engineered to express a fusion protein comprising the extracellular domain and the membrane localization domain of the immunosuppressive molecule. (Item 11) The effector immune cell according to any one of items 7 to 9, which is engineered to express a fusion protein comprising an extracellular domain and a costimulatory endodomain of the immunosuppressive molecule. (Item 12) The costimulatory endodomain is selected from CD28, ICOS, CTLA4, 41BB, CD27, CD30, OX-40, TACI, CD2, CD27, and GITR The effector immune cell according to item 11, comprising one or more than one endodomain. (Item 13) The effector immune cell according to any one of the preceding items, wherein the antigen recognition receptor is a T cell receptor (TCR) or an activating killer cell immunoglobulin-like receptor (KAR). (Item 14) The effector immune cell according to any one of the preceding items, wherein the cell surface receptor is a chimeric antigen receptor (CAR), and the antigen recognition receptor is a T cell receptor (TCR). (Item 15) The effector immune cell according to item 14, wherein the CAR binds to TCR beta constant region 1 (TRBC1) or TRBC2. (Item 16) The effector immune cell according to any one of items 1 to 12, wherein the cell surface receptor complex is an engineered MHC class I complex or MHC class II complex. (Item 17) The effector immune cell according to item 16, wherein the cell surface receptor complex comprises an MHC class I polypeptide linked to an intracellular signaling domain; an MHC class II polypeptide; or beta-2 microglobulin. (Item 18) The cell surface receptor complex has the following structure: Peptide-L-B2M-endo (wherein, "Peptide" is a peptide that binds to the peptide-binding groove of the MHC class I alpha chain; "L" is a linker, 「B2M」 is β-2 microglobulin; 「endo」 is an intracellular signaling domain) The effector immune cell according to item 17, which is an engineered MHC class I complex comprising a molecule having (Item 19) The effector immune cell according to item 16, comprising an MHC class I polypeptide, an MHC class II polypeptide linked to a component of the TCR / CD3 complex; or β-2 microglobulin. (Item 20) The effector immune cell according to item 19, comprising an MHC class I polypeptide, an MHC class II polypeptide; or β-2 microglobulin linked to CD3-zeta, CD3-epsilon, CD3-gamma, or CD3-delta via a linker peptide. (Item 21) The effector immune cell according to item 16, wherein the effector immune cell is engineered to express a bispecific polypeptide comprising (i) a first binding domain that binds to an MHC class I polypeptide, an MHC class II polypeptide; or β-2 microglobulin; and (ii) a second binding domain that binds to a component of the TCR / CD3 complex. (Item 22) The effector immune cell according to item 16, comprising a CD79α chain and / or a CD79β chain linked to an intracellular signaling domain. (Item 23) The effector immune cell according to item 16, comprising an engineered polypeptide comprising a binding domain that binds to an MHC class I polypeptide or an MHC class II polypeptide linked to an intracellular signaling domain. (Item 24) The effector immune cell according to item 16, comprising an engineered polypeptide comprising an MHC class II binding domain of CD4 or an MHC class I binding domain of CD8 linked to an intracellular signaling domain. (Item 25) A nucleic acid construct, (i) A first nucleic acid sequence encoding a cell surface receptor or a part of a cell surface receptor complex as defined in any of the preceding items; and (ii) A second nucleic acid sequence which, when expressed in a cell, confers resistance to an immunosuppressive agent on the cell; and / or (iii) A third nucleic acid sequence encoding an immunosuppressive molecule or a fusion protein comprising an extracellular domain of an immunosuppressive molecule A nucleic acid construct comprising. (Item 26) A vector comprising the nucleic acid construct according to item 25. (Item 27) A kit of vectors, (i) A first vector comprising a nucleic acid sequence encoding a cell surface receptor or a part of a cell surface receptor complex as defined in any of items 1 to 24; and (ii) A second vector comprising a nucleic acid sequence which, when expressed in a cell, confers resistance to an immunosuppressive agent on the cell; and / or (iii) A third vector comprising a nucleic acid sequence encoding an immunosuppressive molecule or a fusion protein comprising an extracellular domain of an immunosuppressive molecule A kit of vectors comprising. (Item 28) A pharmaceutical composition comprising a plurality of effector immune cells as defined in any of items 1 to 24. (Item 29) The pharmaceutical composition according to item 28 for use in the treatment of a disease. (Item 30) A method of treating a disease, the method comprising administering the pharmaceutical composition according to item 29 to a subject. (Item 31) The following steps: (i) A step of administering a pharmaceutical composition to a subject, the pharmaceutical composition comprising a plurality of effector immune cells as defined in item 1 which have been engineered to be resistant to an immunosuppressive agent; and (ii) A step of administering the immunosuppressive agent to the subject The method according to item 30 comprising. (Item 32) Use of a plurality of effector immune cells as described in any of items 1 to 24 in the manufacture of a medicament for the treatment of a disease. (Item 33) The use according to item 29, the method according to item 30 or 31, or a pharmaceutical composition for the use according to item 32, wherein the disease is cancer. (Item 34) A method for producing effector immune cells as described in any of items 1 to 24, comprising the step of introducing ex vivo into the cells a nucleic acid construct as described in item 25, a vector as described in item 26, or a kit of vectors as described in item 27. (Item 35) A method for depleting alloreactive immune cells from a population of immune cells, comprising the step of contacting the population of immune cells with a plurality of effector immune cells as described in any of items 16 to 24. Including, method. (Item 36) A method for treating or preventing rejection of a graft after allotransplantation, comprising the step of administering to a recipient subject a plurality of effector immune cells derived from a donor subject for the allotransplantation, wherein the plurality of effector immune cells express an engineered MHC class I complex or MHC class II complex as defined in any of items 16 to 24. (Item 37) A method for treating or preventing graft-versus-host disease (GVHD) associated with allotransplantation, comprising the step of contacting the allograft with a plurality of effector immune cells as described in any of items 16 to 24. (Item 38) The method according to item 36 or 37, wherein the allotransplantation comprises adoptive transfer of allogeneic immune cells. (Item 39) An allograft depleted of alloreactive immune cells by the method according to item 35.

Claims

【Claim 1】 The invention described in the specification.

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