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-recognizing receptors on target immune cells, and in particular to approaches for controlling the killing of such effector immune cells by target cells. [Background technology]

[0002] Background of the Invention Preventing rejection In solid organ transplants or hematopoietic stem cell transplants (HSCT), HLA mismatch between the recipient and donor can lead to organ rejection or graft-versus-host disease (GVHD), respectively. Immunosuppressants can mitigate these outcomes, but their broad inhibitory effects on immune cells increase the risk of opportunistic infections.

[0003] Alloreactive T cells, which recognize HLA mismatches via the T cell receptor (TCR), are major mediators of rejection and GVHD. CD8+ T cell specificity is determined by the TCR clonotype, which recognizes short antigenic peptides presented on MHC class I molecules. MHC class I molecules are noncovalent heterodimers composed of an integral membrane-resident, highly polymorphic α chain and a nonmembrane-bound, nonpolymorphic β2-microglobulin (β2m).

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

[0005] CAR-T cells Traditionally, antigen-specific T cells have been generated by selective expansion of peripheral blood T cells that are innately specific for the target antigen. However, it is difficult, if not impossible, to select and expand large numbers of T cells specific for most cancer antigens. Gene therapy using integrating vectors offers a solution to this problem, as transgenic expression of chimeric antigen receptors (CARs) can generate large numbers of T cells specific for any surface antigen by ex vivo viral vector transduction of peripheral blood T cell populations.

[0006] Chimeric antigen receptors are proteins that combine the specificity of monoclonal antibodies (mAbs) with the effector functions of T cells. Their usual form is a type I transmembrane domain protein with an antigen-recognizing amino terminus, a spacer, a compound endodomain that transmits T cell survival and activation signals, all connected by a transmembrane domain.

[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, fused to a signaling endodomain via a spacer and a transmembrane domain. Such molecules activate T cells in response to the recognition of the target by the 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 the treatment of various cancers.

[0008] After infusion, CAR T cells are transplanted into the recipient and proliferate after encountering target-bearing cells. CAR T cells then persist, and their population slowly shrinks over time. In clinical trials, CAR T cell persistence can be determined by real-time PCR for the transgene in blood samples or flow cytometry for the CAR in blood samples, and clinical researchers have found a correlation between persistence and sustained response. This correlation is particularly evident in CD19 CAR therapy for B-acute lymphoblastic leukemia (ALL). In this setting, CAR T cell engraftment failure often occurs before leukemia relapse.

[0009] CAR T cells can activate a cellular immune response that can induce rejection of the CAR T cells due to the immunogenicity of the engineered components in the cell, either due to non-self proteins or non-self sequences formed from the junctions between the receptor and self proteins used to create the other engineered components.

[0010] CAR is an artificial protein typically composed of a targeting domain, a spacer domain, a transmembrane domain, and a signal transduction domain. The targeting domain is typically derived from an scFv, which may be mouse. This scFv can be a human scFv or a humanized scFv, and while other components are derived from individual self-proteins, the junction between them may still exhibit immunogenicity. For example, within an scFv, there is a junction 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 at that location. Similarly, if the transmembrane domain is not continuous with the amino-terminal portion of the endodomain, there is an additional junction at that location. Finally, most endodomains have at least two components, and sometimes there is an additional junction between each component.

[0011] In addition, CAR T cells are often engineered with additional components. These components include suicide genes (such as HSV-TK enzyme). This enzyme is highly immunogenic and has been found to cause cellular immune depletion of CAR T cells in a situation different from the significant immunosuppression in haploidentical hematopoietic stem cell transplantation. Almost all engineered components, including fusion between two proteins or the use of heterologous proteins, can exhibit immunogenicity, so 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. In some situations, T cells from an allogeneic donor are used. This can occur, for example, if the patient has undergone an allogeneic hematopoietic stem cell transplant. In this case, the harvested T cells will be allogeneic. Otherwise, the patient may have insufficient T cells to generate the CAR T cell product due to chemotherapy-induced lymphopenia.

[0013] Rejection of allogeneic cells can be due to minor or major mismatches. Minor mismatches occur when allogeneic T cells match the recipient's human leukocyte antigens (HLA). In this case, HLA does not differ between individuals, resulting in rejection due to minor histocompatibility antigens presenting non-self (donor) epitopes / immunogenic peptides on HLA. When the donor and recipient are mismatched or only partially matched, the T cell receptor (TCR) on the recipient's endogenous T cells can interact with the mismatched HLA in a nonspecific manner, resulting in rejection. Both minor and major forms of allorejection are due to HLA interacting with the TCR.

[0014] WO2019 / 073248 and UK Patent Application Publication No. 1904971.7 describe an approach involving coupling MHC class I or II on CAR-expressing cells to TCR on T cells to directly or indirectly induce signal transduction in the CAR-expressing cells. When CAR-expressing cells are administered to a subject, MHC class I or II on the cells interact with any endogenous reactive T cells present in the subject through recognition of peptide / MHC complexes. 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 Approaches to Treat T-Cell Malignancies Lymphoid malignancies can be broadly divided into those derived from either T cells or B cells. T-cell malignancies are a clinically and biologically heterogeneous group of disorders, collectively accounting for 10-20% of non-Hodgkin lymphomas and 20% of acute leukemias. The most commonly identified histologic subtypes are peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS); angioimmunoblastic T-cell lymphoma (AITL); and anaplastic large cell lymphoma (ALCL). Approximately 20% of all acute lymphoblastic leukemias (ALL) are of the T-cell phenotype.

[0016] These conditions typically behave aggressively compared to, for example, B-cell malignancies, with an estimated 5-year survival rate of only 30%. In the case of T-cell lymphoma, a high proportion of patients present with disseminated disease, unfavorable International Prognostic Index (IPI) scores, and extranodal disease. Chemotherapy alone is usually ineffective, with fewer than 30% of patients being cured with current treatments. WO2015 / 132598 describes a method that can deplete malignant T cells in a subject without affecting a significant proportion of healthy T cells. In particular, WO2015 / 132598 describes a CAR that specifically binds to TCR beta constant region 1 (TRBC1) or TRBC2. All of the above approaches involve specific binding of T cell receptors on target T cells, in which the targeted T cells can "fight back" due to ligation of their TCRs, thereby depleting the transplanted / desired T cells. [Prior art documents] [Patent documents]

[0017] [Patent Document 1] International Publication No. 2019 / 073248 [Patent Document 2] UK Patent Application Publication No. 1904971.7 [Patent Document 3] International Publication No. 2015 / 132598 [Non-patent literature]

[0018] [Non-Patent Document 1] Margalit et al((2002)International Immunology 15:1379-1387 [Brief explanation of the drawings]

[0019] [Figure 1]Figure 1 - (a) MHC class I molecule complex composed of MHC and B2M; (b) TCR complex composed of TCR alpha / beta chains surrounded by CD3 elements. [Figure 2] Figure 2 - (a) B2M-Z construct: B2M construct is fused in frame to a transmembrane domain and CD3-zeta endodomain; (b) B2M-TCR bispecific construct: scFv recognizing B2M is fused using a linker to a second scFv recognizing the CD3 / TCR complex, which is then tethered to the membrane via the transmembrane domain; (c) Fusion between B2M and CD3 / TCR: As an example, a fusion between B2M and CD3 epsilon via a flexible linker is shown. [Figure 3] Figure 3-a) Schematic diagram showing classical CARs. (b)-(d) Different generations and orders of CAR endodomains: (b) Early designs in which only ITAM signals are transduced via the FcεR1-γ or CD3ζ endodomains, whereas later designs in which an additional (c) one or (d) two costimulatory signals are transduced within the endodomains of the same compound. [Figure 4] Figure 4 - Schematic diagram showing MHC class I CAR. Major histocompatibility complex (MHC) class I CAR is a heterodimer composed of two non-covalently linked polypeptide chains, α and β2-microglobulin (β2m). The α1 and α2 subunits, both loaded with peptides, bind to the T cell receptor (TCR) expressed on the surface of T cells. The β2-microglobulin is connected to a transmembrane domain, which anchors the molecule in the cell membrane and is further linked to an endodomain, which acts to transmit intracellular signals to the cell. The endodomain may be composed of one or more signaling domains. [Figure 5]Figure 5 - Schematic diagram showing three possible β2m-based CAR designs. In the first CAR (A), β2-microglobulin is linked via a bridge to the CD3ζ transmembrane domain, which is then linked to the CD3ζ endodomain. Two other CAR designs (B and C) add the costimulatory domains 41BB or CD28, respectively. [Figure 6] Figure 6 - (a) A naturally occurring MHC class II molecule complex composed of an α chain and a β chain (e.g., HLA-DRα and HLA-DRβ) and presenting a peptide; (b) An MHC class II molecule comprising an α chain and a β chain associated with CD79, including CD79α and CD79β, both of which may contain signaling domains; (c) An engineered MHC class II molecule comprising an α chain and a β chain, where the α chain contains the signaling domain. [Figure 7] Figure 7 - MHC class I and TCR. (a) MHC class I molecules are heterodimers consisting of two polypeptide chains (α and β2-microglobulin (B2M)); (b) TCR complexes consisting of TCR alpha / beta chains surrounded by CD3 elements [Figure 8-1] Figure 8 - Different MHC1α / TCR fusion constructs. (a) MHC1α-CD3z construct: the MHC class I alpha chain is fused in frame to the TM domain and CD3-zeta endodomain; (b) Ab-CD3z construct: an antibody or antibody-like binder specific for the MHC class I alpha chain is fused to the TM domain and CD3-zeta endodomain; (c) Fusion between MHC1α and CD3 / TCR: As an example, the fusion of the MHC class I alpha chain to CD3 epsilon via a flexible linker is shown; (d) MHC1α-TCR BiTE construct: an scFv recognizing the MHC class I alpha chain is fused using a linker to a second scFv recognizing the CD3 / TCR complex, which is then tethered to the membrane via the transmembrane domain. [Figure 8-2] Same as above. [Figure 9]Figure 9 - MHC class II and TCR. (a) MHC class II molecules are heterodimers consisting of α and β chains; (b) TCR complexes composed of TCR alpha / beta chains surrounded by CD3 elements. [Figure 10] Figure 10 - Different MHCII / TCR fusion constructs. (a) MHCII-CD3z construct: The α or β chain of MHC class II is fused to the TM domain and the CD3-zeta endodomain; (b) Ab-CD3z construct: An antibody or antibody-like binder specific for the α or β chain of MHC class II is fused to the TM domain and the CD3-zeta endodomain; (c) Fusion between MHCII and CD3 / TCR: The α or β chain of MHC class I is fused to a component of the TCR / CD3 complex via a flexible linker. For example, CD3 epsilon is shown; (d) MHCII-TCR BiTE construct: An scFv recognizing the α or β chain of MHC class II is fused to a second scFv recognizing the CD3 / TCR complex using a linker. This is then tethered to the membrane via a transmembrane domain. [Figure 11] Figure 11 - CD4 / CD8 fusion molecules. CD4 and CD8 are TCR co-receptors. The extracellular domain of CD4 binds to the β2 region of MHC class II; whereas, the extracellular domain of CD8 binds to the α3 portion of class I MHC molecules. (a) CD4-CD3z construct: The MHC class II binding domain of CD4 is fused to the TM domain and CD3-zeta endodomain; (b) CD8-CD3z construct: The MHC class I binding domain of CD8 is fused to the TM domain and CD3-zeta endodomain. [Figure 12] Figure 12 - Data showing killing of cells expressing a truncated version of the TRBC1-specific CAR lacking the signaling domain (reverse killing) by TRBC1+ target T cells. [Figure 13] Figure 13 - Data showing persistence of JOVI (or dJOVI) CAR T cells with or without dPDL1 (or dPDL2). [Figure 14]Figure 14: Schematic diagram showing CSK and various dnCSK constructs. A - Wild-type CSK with SH3, SH2, and protein tyrosine kinase domains. B - dnCSK lacking the kinase domain. C - dnCSK lacking the kinase and SH3 domains. 6 - dnCSK with the mutation K222R. [Figure 15] FIG. 15: Schematic diagram showing the mechanism of (a) T cell activation; and (b) inhibition of T cell activation by inhibitory immunoreceptors. [Figure 16] Figure 16: Graphs showing (A) the percentage and (B) number of CAR-expressing (RQR8-positive) cell proliferation after 96 hours of co-culture with Jurkat KO, Jurkat TRBC1, and Jurkat TRBC2 target cells in the absence of tacrolimus. [Figure 17] Figure 17: Graphs showing (A) percentage and (B) number of CAR-expressing (RQR8-positive) cell proliferation after 96 hours of co-culture with Jurkat KO, Jurkat TRBC1, and Jurkat TRBC2 target cells in the presence of 20 ng / ml tacrolimus. [Figure 18-1] Figure 18: Graph showing the number of CAR-expressing (RQR8-positive) cells at each division after co-culture with Jurkat KO, Jurkat TRBC1, and Jurkat TRBC2 target cells in the absence of tacrolimus. Proliferation analysis was performed on single / viable / CellTrace Violet-positive cells using the FlowJo™ proliferation tool and a CD19 CAR was used as a negative control for all conditions. The number of cells at each division is plotted for each CAR+target combination. [Figure 18-2] Same as above. [Figure 19-1]Figure 19: Graph showing the number of CAR-expressing (RQR8-positive) cells at each division after co-culture with Jurkat KO, Jurkat TRBC1, and Jurkat TRBC2 target cells in the presence of 20 ng / ml tacrolimus. Proliferation analysis was performed on single / viable / CellTrace Violet-positive cells using the FlowJo™ proliferation tool and a CD19 CAR was used as a negative control for all conditions. The number of cells at each division is plotted for each CAR+target combination. [Figure 19-2] Same as above. [Figure 20-1] Figure 20: Histogram plot showing proliferation of CAR-expressing (RQR8-positive) cells after co-culture with Jurkat KO, Jurkat TRBC1, and Jurkat TRBC2 target cells with or without 20 ng / ml tacrolimus. Proliferation analysis was performed on single / viable / CellTrace Violet-positive cells using the FlowJo™ proliferation tool and a CD19 CAR was used as a negative control for all conditions. Results are shown using cells from two separate donors. [Figure 20-2] Same as above. [Figure 21] Figure 21: Graph showing cell counts of non-transduced (NT) and TRBC2 CAR-expressing (RQR8-positive) cells before (day 0) and after (day 4) co-culture with TRBC2 targets with or without the addition of 20 ng / ml tacrolimus. [Figure 22] Figure 22: Graph showing the percentage of TRBC2 CAR-expressing (RQR8-positive) cells before (day 0) and after (day 4) co-culture with TRBC2 targets with or without the addition of 20 ng / ml tacrolimus. [Figure 23]Figure 23: Graph showing the killing of TRBC2-expressing PBMCs after co-culture with PBMCs transduced to express a CD19 CAR, a TRBC2 CAR, or co-expressing a TRBC2 CAR and a calcineurin mutant module (TRBC2+CnB30). Co-cultures were set up at an E:T ratio of 1:1 or 1:4 in the presence or absence of 20 ng / ml tacrolimus. [Figure 24] Figure 24: Graph showing survival / proliferation of PBMCs transduced to express CD19 CAR, TRBC2 CAR, or co-express TRBC2 CAR and calcineurin mutant module (TRBC2+CnB30) after co-culture with TRBC2-expressing PBMCs. Co-culture was set up at an E:T ratio of 1:1 or 1:4 in the presence or absence of 20 ng / ml tacrolimus. [Figure 25] Figure 25: Graph showing IFNγ secretion after co-culture of TRBC2-expressing PBMCs with PBMCs transduced to express a CD19 CAR, a TRBC2 CAR, or co-expressing a TRBC2 CAR and a calcineurin mutant module (TRBC2+CnB30). Co-cultures were set up at an E:T ratio of 1:1 or 1:4 in the presence or absence of 20 ng / ml tacrolimus. [Figure 26] Figure 26: Graph showing IL-2 secretion after co-culture of TRBC2-expressing PBMCs with PBMCs transduced to express a CD19 CAR, a TRBC2 CAR, or co-expressing a TRBC2 CAR and a calcineurin mutant module (TRBC2+CnB30). Co-cultures were set up at an E:T ratio of 1:1 or 1:4 in the presence or absence of 20 ng / ml tacrolimus. Summary of the Invention [Means for solving the problem]

[0020] Summary of the Invention The inventors have developed an approach to engineer effector immune cells (cell A) such that when targeting autoreactive or pathogenic immune cells (cell B), the engineered immune cells have a selective advantage, and the balance between cell A killing cell B and cell B killing cell A favors cell A killing 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-recognizing receptor on a target immune cell; wherein the effector immune cell has been engineered such that, upon formation of a synapse between said effector immune cell and said target immune cell, said effector immune cell has a greater ability to kill said target immune cell than said target immune cell.

[0022] In a first embodiment of the first aspect of the present invention, the effector immune cells are engineered to be resistant to immunosuppressants.

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

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

[0025] Effector immune cells can be engineered to be resistant to rapamycin.

[0026] Effector immune cells can express dominant-negative C-terminal Src kinases (dnCSKs), which confer resistance to multiple immunosuppressive drugs.

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

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

[0029] The immunoinhibitory molecule may be selected from the following: 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 can be engineered to express a fusion protein comprising the extracellular and membrane-localizing domains of an immunoinhibitory molecule.

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

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

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

[0034] When effector immune cells express 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 the 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 (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 beta-2 microglobulin; "endo" is the intracellular signaling domain) The compound may be an engineered MHC class I complex comprising a molecule having the formula:

[0038] Effector immune cells can comprise an MHC class I polypeptide linked to a component of the TCR / CD3 complex; an MHC class II polypeptide; or beta-2 microglobulin.

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

[0040] The effector immune cell 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 beta-2 microglobulin; and (ii) a second binding domain that binds to a component of the TCR / CD3 complex.

[0041] Effector immune cells can express engineered polypeptides comprising the CD79 α chain and / or the CD79 β chain linked to an intracellular signaling domain.

[0042] The effector immune cells can express an engineered polypeptide that includes a binding domain that binds to an MHC class I or 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 engineered polypeptides comprising the MHC class II binding domain of CD4 or the MHC class I binding domain of CD8 linked to an intracellular signaling domain.

[0044] The effector immune cells of the first aspect of the present invention may be engineered to express a cell surface receptor (such as a CAR) or receptor complex (such as an engineered MHC class I complex or an engineered MHC class II complex), and then further engineered such that upon formation of a synapse between the effector immune cell and the target immune cell, the effector immune cell has a greater ability to kill the target immune cell than the target immune cell has a greater ability to kill the effector immune cell.

[0045] Further manipulation of effector immune cells, as described above, can be performed as follows: (i) engineering cells to be resistant to immunosuppressants, or (ii) engineering cells to express or overexpress an immunoinhibitory molecule or a fusion protein comprising the extracellular domain of an immunoinhibitory molecule; may include:

[0046] A synapse formed between an effector immune cell and a target immune cell is formed when a cell surface receptor or receptor complex on the effector immune cell specifically binds to an antigen-recognizing receptor on the target immune cell.

[0047] In a second aspect, (i) a first nucleic acid sequence encoding a portion of a cell surface receptor or cell surface receptor complex as defined herein; and (ii) a second nucleic acid sequence that, when expressed in the cell, confers resistance to the immunosuppressant to said cell; and / or (iii) a third nucleic acid sequence encoding an immunoinhibitory molecule or a fusion protein comprising the extracellular domain of an immunoinhibitory molecule; The present invention provides a nucleic acid construct comprising:

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

[0049] In a fourth aspect, there is provided a kit of vectors, comprising: (i) a first vector comprising a nucleic acid sequence encoding a portion 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 the cell, confers resistance to an immunosuppressant to said cell; and / or (iii) a third vector comprising a nucleic acid sequence encoding an immunoinhibitory molecule or a fusion protein comprising the extracellular domain of an immunoinhibitory molecule. A vector kit is provided, comprising:

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

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

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

[0053] The method may include the steps of: (i) administering to a subject a pharmaceutical composition, wherein the pharmaceutical composition comprises a plurality of effector immune cells of the first aspect of the invention that have been engineered to be resistant to an immunosuppressant; and (ii) administering said immunosuppressant to said subject.

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

[0055] The disease may be cancer.

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

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

[0058] In an eleventh aspect, there is provided a method of treating or preventing graft rejection following allogeneic transplantation, comprising administering to a recipient subject, for said allogeneic transplant, a plurality of effector immune cells derived from a donor subject, wherein said 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, there is provided a method of treating or preventing graft-versus-host disease (GVHD) associated with an allogeneic transplant, comprising contacting said allogeneic transplant with a plurality of effector immune cells of the first aspect of the invention, wherein said plurality of effector immune cells express an engineered MHC class I complex or MHC class II complex as defined herein.

[0060] Allogeneic transplantation can involve the adoptive transfer of allogeneic or autologous immune cells.

[0061] In a thirteenth aspect, there is provided an allograft which has been depleted of alloreactive immune cells by the method of the twelfth aspect of the invention. DETAILED DESCRIPTION OF THE INVENTION

[0062] Detailed Description Several clinical applications involve the generation of effector immune cells that recognize and deplete subsets of conventional immune cells by recognizing their antigen-recognizing receptors.

[0063] In this situation, the targeted conventional immune cells can "fight back" and deplete the effector immune cells. The present invention relates to the manipulation of effector immune cells such that they have an immunological "advantage" over the targeted immune cells, thereby prevailing when a synapse is formed between the effector immune cells and the targeted immune cells.

[0064] There are a variety of situations in which these effector cells can "fight back," including: (i) A situation in which an effector immune cell expresses a CAR that specifically binds to the T cell receptor on a T cell; (ii) situations in which effector immune cells express engineered MHCI or II complexes to deplete alloreactive or autoreactive T cells;

[0065] These situations are described in more detail below.

[0066] Chimeric antigen receptor for the TCR complex The effector immune cells of the present invention may express a chimeric antigen receptor (CAR). In particular, the cells may express a CAR that specifically binds to a T cell receptor (TCR) or a component of the 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. A spacer domain can be used to separate the binder from the membrane and properly orient it. A common spacer domain used is the Fc of IgG1. Depending on the antigen, smaller spacers, such as a stalk derived from CD8α or even just an IgG1 hinge, may be sufficient. The transmembrane domain anchors the protein within the cell membrane and connects the spacer to the endodomain.

[0068] Early CAR designs had endodomains derived from the intracellular portion of either the γ chain of FcεR1 or CD3ζ. Consequently, these first-generation receptors were sufficient to transduce immunological signals 1 and trigger T cell killing of cognate target cells, but were unable to sufficiently activate T cells to proliferate and survive. To overcome this limitation, composite endodomains were constructed: fusion of the intracellular portion of a T cell costimulatory molecule with the intracellular portion of CD3ζ results in second-generation receptors that can simultaneously transduce activation and costimulatory signals after antigen recognition. The most commonly used costimulatory domain is that of CD28, which provides the most potent costimulatory signal (i.e., immunological signal 2 that induces T cell proliferation). Several receptors have also been described, including those containing TNF receptor family endodomains (such as the closely related OX40 and 41BB, which transduce survival signals). Here, we describe even more potent third-generation CARs with endodomains capable of transducing activation, proliferation, and survival signals.

[0069] When a CAR binds to a 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 to tumor cells expressing the targeted antigen.

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

[0071] A CAR may 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 the part of CAR that recognizes the antigen. In classical CAR, the antigen-binding domain comprises the single-chain variable fragment (scFv) of monoclonal origin. CARs with domain antibody (dAb) or VHH or Fab-based antigen-binding domains have also been produced.

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

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

[0075] Various sequences are commonly used as spacers in CARs (e.g., IgG1 Fc region, IgG1 hinge, or human CD8 stalk).

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

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

[0078] End Domain The endodomain is the signaling portion of the CAR. It can be part of the intracellular domain of the CAR or can be associated with the intracellular domain. After antigen recognition, receptors cluster, native CD45 and CD148 are excluded from the synapse, and a signal is transmitted to the cell. The most commonly used endodomain component is that of CD3-zeta, which contains three ITAMs. This transmits an activation signal to T cells after binding to an antigen. CD3-zeta may not provide a sufficient activation signal, and additional costimulatory signaling may be required. Costimulatory signals promote T cell proliferation and survival. There are two main types of costimulatory 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 with CD3-zeta to transmit proliferation / survival signals, or all three can be used together.

[0079] The endodomain may include: (i) an ITAM-containing endodomain (such as the endodomain from CD3 zeta); and / or (ii) a costimulatory domain (such as the endodomain from CD28 or ICOS); and / or (iii) domains that transmit survival signals (e.g., TNF receptor family endodomains 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 from the signal transduction moiety (such as those described in WO015 / 150771; WO2016 / 124930, and WO2016 / 030691). Thus, the CAR of the present invention may comprise an antigen-binding component comprising an antigen-binding domain and a transmembrane domain, which can interact with a separate intracellular signal transduction component comprising a signal transduction domain. The vector of the present invention can express a CAR signal transduction system comprising such an antigen-binding component and an intracellular signal transduction component.

[0081] The CAR may contain a signal peptide so that when the signal peptide is expressed inside the cell, the nascent protein is directed to the endoplasmic reticulum and then to the cell surface, where it is expressed. The signal peptide may be 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 a CAR.

[0083] The target antigen can be an antigen present on a cancer cell (eg, a tumor-associated antigen).

[0084] A variety of tumor-associated antigens (TAA) are known, as shown in Table 1 below. The CAR may be capable of binding to such a TAA. [Table 1]

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

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

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

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

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

[0090] The TCR also contains up to five invariant chains: γ, δ, ε (collectively referred to as CD3), and ζ. The CD3 and ζ subunits mediate TCR signaling through specific cytoplasmic domains that interact with second messengers and adapter molecules after recognition of αβ or γδ. Prior to cell surface expression of the TCR complex, the α and β transmembrane and extracellular domains of the TCR, as well as both CD3γ and CD3δ, play a role in paired assembly of the subunits.

[0091] Thus, a TCR generally consists of a CD3 complex and the α and β chains of the TCR, which in turn consist of variable and constant regions.

[0092] The locus (Chr7:q34) supplying the TCRβ constant region (TRBC) has duplicated during evolution to produce two nearly identical and functionally equivalent genes: TRBC1 and TRBC2, which differ by four amino acids in the mature proteins. 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 capable of selectively binding to either TRBC1 or TRBC2 in a mutually exclusive manner.

[0094] As explained above, each αβ T cell expresses a TCR containing 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 TRBC1 or TRBC2-specific CAR-T cells are administered to patients with T-cell lymphoma or T-cell leukemia, the malignant T cells are selectively depleted along with normal T cells that express the same TRBCs as the malignant T cells, but such treatment does not significantly deplete normal T cells that express other TRBCs derived from the malignant T cells.

[0096] Because TRBC-selective CAR-T cells do not significantly deplete normal T cells that express other TRBCs derived from the malignant T cells, they do not deplete the entire T cell compartment. Preserving the proportion of the subject's T cell compartment (i.e., T cells that do not express the same TRBCs as the malignant T cells) reduces toxicity and reduces cellular and humoral immune deficiencies, 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 may have a variable heavy chain (VH) and a variable light chain (VL) that comprise 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] One or more CDRs may or may not each independently contain one or more amino acid mutations (e.g., substitutions) compared to the sequences set forth in SEQ ID NOs: 8 to 13, provided that the resulting antibody retains the ability to selectively bind to TRBC1.

[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. [ka] [ka]

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

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

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

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

[0105] The variant antigen-binding domain may comprise 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 further mutation may be selected from the following: 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, N103L in the VH domain and N35F in the VL domain, - T28K, Y32F, A100N, N103W in the VH domain and N35M in the VL domain, - T28K, Y32F, A100N, N103F in the VH domain and 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 and N35F in the VL domain, - T28K, Y32F, A100N, N103F in the VH domain and N35M in the VL domain, - T28K, Y32F, A100N, N103F in the VH domain and N35F in the VL domain, -T28K, Y32F, A100N, G31R in the VH domain, - T28K, Y32F, A100N, N103W in the VH domain and 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 and N35M in the VL domain, -T28K, Y32F, A100N, V2K in the VH domain, -T28K, Y32F, A100N, N103E in the VH domain - T28K, Y32F, A100N, Y102F, N103M in the VH domain and N35K in the VL domain, - T28K, Y32F, A100N, Y102F, N103M in the VH domain and N35F in the VL domain, - T28K, Y32F, A100N, Y102F, N103M in the VH domain 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 in the VH domain, and - T28K, Y32F, A100N, Y102L, N103M in the VH domain and N35R in the VL domain.

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

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

[0110] Engineered MHCI or II complex The major histocompatibility complex (MHC) is a large genetic locus on vertebrate DNA 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 allows the immune system (more specifically, T cells) to bind, recognize, and tolerate the MHC itself (self-recognition). The MHC also chaperones intracellular peptides that are complexed with the MHC and presented to the T cell receptor (TCR) as potential foreign antigens. The MHC interacts with the TCR and its co-receptors to optimize the binding conditions of the TCR-antigen interaction for antigen binding affinity and specificity, as well as signaling efficacy.

[0111] Essentially, the MHC-peptide complex is a self-antigen / alloantigen complex: upon binding, T cells essentially tolerate the self-antigen but must become activated when exposed to an alloantigen.

[0112] MHC molecules bind to both the T cell receptor and the CD4 / CD8 co-receptor on T lymphocytes, and antigen epitopes held in the peptide-binding groove of the MHC molecule interact with the variable Ig-like domain of the TCR to trigger T cell activation.

[0113] MHC class I molecules are expressed in all nucleated cells and in platelets (essentially all cells except red blood cells). MHC class I presents peptide epitopes to cytotoxic T lymphocytes (CTLs). CTLs express CD8 receptors in addition to TCRs. When the CD8 receptor of a CTL docks with an MHC class I molecule, if the TCR of the CTL matches an epitope within the MHC class I molecule, the CTL induces programmed cell death by apoptosis in the cell. Thus, MHC class I helps mediate cell-mediated immunity, the primary 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 containing a polymorphic heavy chain α-subunit (the gene for which occurs within the MHC locus) and a small, invariant β2-microglobulin subunit (the gene for which is usually present outside the MHC locus). The polymorphic heavy chain of MHC-I molecules 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 tail. The two domains, α1 and α2, form a deep peptide-binding groove between two long α-helices, the bottom of which is formed by eight β-strands. The immunoglobulin-like domain, α3, is involved in interaction with the CD8 coreceptor. β2-microglobulin confers stability to the complex and is involved in recognition of peptide-MHC class I complexes by the CD8 coreceptor. Peptides are noncovalently bound to MHC-I and are retained by several pockets at the bottom of the peptide-binding groove. The most polymorphic amino acid side chains among human alleles fill the central and widest part of the accommodation groove, while the conserved side chains are clustered at the narrower end of the groove.

[0115] Although MHC class II can be conditionally expressed by all cell types, 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 antigen, return molecular fragments of the protein (antigen epitopes), and present these fragments on their surface coupled within MHC class II molecules (antigen presentation). On the cell surface, the epitopes can be recognized by T cell receptors (TCRs) as immunogenic structures.

[0116] Helper T cells have not only TCRs but also CD4 receptors on their surface. When the CD4 molecules of naive helper T cells dock with the MHC class II molecules of APCs, the TCR can encounter and bind to epitopes coupled within the MHC class II. This event primes the naive T cells.

[0117] Class II MHC molecules are also heterodimers, with polymorphic genes for both the α and β subunits located within the MHC class II subregion. The peptide-binding groove of MHC-II molecules is formed by the N-terminal domains of both α1 and β1 subunits of the heterodimer; this differs from MHC-I molecules, which involve two domains from the same chain. Furthermore, both MHC-II subunits contain transmembrane helices and immunoglobulin domains α2 and β2, which can be recognized by the CD4 co-receptor. In this way, MHC molecules identify the type of lymphocyte that can bind a given antigen with high affinity, since different lymphocytes express different T cell receptor (TCR) co-receptors.

[0118] Effector immune cells of the present invention can 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 pathogenesis of autoimmune diseases. As explained above, TCR ligands are converted into T cell activating receptors by expression of a β2-microglobulin polypeptide containing an intracellular signaling domain attached to one end via a linker and an antigenic peptide attached to the other end. Cells engineered to express such molecules have been found to express high levels of surface peptide-class I complexes that present antigenic peptides and respond to antibodies and target T cells in a peptide-specific manner. Expression of such peptide-linker-signaling domain polypeptides in effector immune cells such as T cells allows for the specific targeting of pathogenic CD8- T cells that recognize specific antigenic peptides.

[0120] Thus, 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 beta-2 microglobulin; "endo" is the intracellular signaling domain).

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

[0122] Autoimmune disorders are characterized by immune system responses to endogenous antigens resulting in tissue damage. Over 80 chronic autoimmune diseases have been characterized, affecting virtually every organ system in the body. The most common autoimmune diseases are insulin-dependent diabetes mellitus (IDDM), multiple sclerosis (MS), systemic lupus erythematosus (SLE), rheumatoid arthritis, some forms of anemia (pernicious anemia, aplastic anemia, hemolytic anemia), thyroiditis, and uveitis.

[0123] Allograft rejection typically results from an excessive adaptive immune response against a foreign organ or tissue. It is a major risk factor in organ transplantation and a cause of post-transplant complications. A major complication of bone marrow (BM) transplantation, known as the 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 compromised immune system. The donor lymphocytes begin to attack host tissue, preventing the immunocompromised host from mounting an immune response against the graft.

[0124] The linker connects the peptide to the beta-2 microglobulin and provides flexibility that allows the peptide to 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] The molecule may also include a peptide bridge that bridges beta-2 microglobulin to the cell membrane. The peptide bridge may include the 13 membrane-proximal amino acids of the extracellular portion of HLA-A2 (having the sequence LRWEPSSNPTIPI (SEQ ID NO: 11)).

[0126] The molecule may comprise a membrane targeting domain, such as a transmembrane domain. By way of example, the transmembrane domains of CD8 alpha and CD28 are shown as SEQ ID NO: 12 and SEQ ID NO: 13, respectively. [ka] [ka]

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

[0128] The engineered MHC class I complex may include a variant of the beta-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 α chain.

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

[0130] The engineered MHC class I complex may comprise an intracellular signaling domain having the sequence set forth in SEQ ID NO: 15 or 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 resulting Peptide-L-B2M-endo molecule retains the ability to induce activation of effector immune cells upon TCR recognition.

[0131] Additional intracellular signaling and costimulatory domains are described below.

[0132] Cross-peptide approach It is possible to couple MHC class I or II on effector immune cells to TCRs on target immune cells to directly or indirectly induce signal transduction in effector cells. In these approaches, the MHC signaling system can present peptides from the same range as the corresponding endogenous MHC class I and II molecules. As such, any peptide naturally presented by MHC class I or II molecules can be presented by engineered MHC complexes. This includes peptides derived from any heterologous or junctional sequences that may exhibit immunogenicity, for example, from chimeric antigen receptors expressed by cells. In allogeneic situations, this peptide may 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 through peptide / MHC complex recognition. Thus, reactive T cells can be depleted by activating cytotoxic-mediated cell killing 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, the effector immune cell can comprise an MHC class I polypeptide; an MHC class II polypeptide; or a polypeptide capable of co-localizing with beta-2 microglobulin having an intracellular signaling domain.

[0134] Effector immune cells may include: (i) an ectodomain from an MHC class I polypeptide or an ectodomain from an MHC class II polypeptide linked to an intracellular signaling domain; or an engineered polypeptide comprising beta-2 microglobulin linked to an intracellular signaling domain (see Figures 2a, 4, 5, 6c, 8a, and 10a); (ii) engineered polypeptides including MHC class I or MHC class II polypeptides or β-2 microglobulin linked to components 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 beta-2 microglobulin, linked to an intracellular signaling domain (see Figures 8b and 10b); (iv) engineered polypeptides containing 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, effector immune cells 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; beta-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 (alpha polypeptide and beta2-microglobulin (b2m)). The two chains are non-covalently linked via interactions between the b2m and alpha3 domains. The alpha 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-e macroglobulin gene. HLA genes. The HLAs corresponding to MHC class I are HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G.

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

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

[0139] The engineered or bispecific polypeptides expressed by the effector cells of the 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, effector immune cells of the present invention can be designed for a particular population with a specific common haplotype. Exemplary Class I haplotypes are summarized in the table below: [Table 2]

[0141] The amino acid sequence of HLA class I - HLA-A is HLA-A01 shown as SEQ ID NO: 16: [ka] In the sequences shown as SEQ ID NOs: 16 to 22: Ectodomain = no character modification Bold / underlined = transmembrane Italics = endodomain

[0142] The amino acid sequence of HLA class I - HLA-A is HLA-A02 shown as SEQ ID NO: 17: [ka]

[0143] The amino acid sequence of HLA class I - HLA-A is HLA-A-A03 shown as SEQ ID NO: 18: [ka]

[0144] The amino acid sequence of HLA class I - HLA-B is HLA-B07 shown as SEQ ID NO: 19: [ka]

[0145] The amino acid sequence of HLA class I - HLA-B is HLA-B08 shown as SEQ ID NO: 20: [ka]

[0146] An exemplary HLA class I amino acid sequence - HLA-B is HLA-B44, shown as SEQ ID NO: 21: [ka]

[0147] The amino acid sequence of HLA class I - HLA-C is HLA-C01 shown as SEQ ID NO: 22: [ka]

[0148] The engineered polypeptide of the effector cells of the invention may comprise the extracellular domain of any 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] The engineered polypeptide may also include a transmembrane domain.

[0150] The transmembrane domain can be any peptide domain that can insert into and cross a cell membrane. The transmembrane domain can be any protein structure that is thermodynamically stable within the membrane. It 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. The sequence and total length of the transmembrane domain of a protein can be determined by those skilled in the art using the TMHMM algorithm (http: / / www.cbs.dtu.dk / services / TMHMM-2.0 / ). Furthermore, given 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), an artificially designed TM domain can also be used (U.S. Patent No. 7,052,906 B1 describes a synthetic transmembrane component). 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. Expression of MHC class II molecules is observed approximately 3–5 days after T cell activation, a slow event compared to the induction of various other effector molecules after T cell receptor (TCR) triggering and costimulation. Because adoptively transferred immune effectors are expected to be activated at some time point after infusion, the expression of HLA class II may lead to allorejection.

[0153] HLA class II molecules are formed as two polypeptide chains: alpha and beta. These molecules are typically highly polymorphic from one individual to another, although some haplotypes are much more common in certain populations than others.

[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 table below: [Table 3]

[0155] HLA-DR has very low 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 can 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: [ka] Bold underlined = the ectodomain of this HLADRα sequence corresponds to amino acids 26-216 of the sequence.

[0157] The engineered polypeptide can comprise the ectodomain from HLA-DRα set forth in SEQ ID NO:23 (e.g., from about amino acid 26 to about amino acid 216 of SEQ ID NO:23), 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 the β chain and facilitate productive peptide presentation by the MHC class II complex.

[0158] The amino acid sequence of the HLA class II histocompatibility antigen DR β chain (having UniProtKB accession number Q04826) is shown in SEQ ID NO: 24: [ka] Bold underlined = the ectodomain of this HLA-DRβ sequence, corresponding to amino acids 25-308 of the sequence.

[0159] The engineered polypeptide can comprise the ectodomain from HLA-DRβ set forth in SEQ ID NO:24 (e.g., from about amino acid 25 to about amino acid 308 of SEQ ID NO:24), 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 the α chain and facilitate productive peptide presentation by the MHC class II complex.

[0160] HLA-DP and HLA-DQ have polymorphic α and β chains. Therefore, the α and β chains of common HLA-DP or HLA-DQ can be selected to limit allogeneic production to recipients with that haplotype. Preferably, the recipient can be homozygous for that haplotype. If the recipient is not homozygous for the haplotype, two HLA-DP and two HLA-DQ (optionally in combination with HLA-DR, e.g., HLA-DRα) can 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: [ka] Italics indicate the transmembrane domain, corresponding to amino acids 225-244. Bold underlined = ectodomain of this HLA-DP sequence, corresponding to amino acids 29-224 of the sequence

[0162] The engineered polypeptide may comprise an ectodomain from HLA-DP set forth in SEQ ID NO:25 (such as from about amino acid 29 to about amino acid 224 of SEQ ID NO:25) 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 and facilitate productive peptide presentation by MHC class II complexes.

[0163] The amino acid sequence of HLA class II histocompatibility antigen DQ (having UniProtKB accession number O19764) is shown in SEQ ID NO: 26: [ka] Italics = transmembrane domain, corresponding to amino acids 229-249 Bold underlined = the ectodomain of this HLA-DQ sequence, corresponding to amino acids 32-228 of the sequence.

[0164] The engineered polypeptide can comprise the ectodomain from HLA-DQ set forth in SEQ ID NO:26 (e.g., from about amino acid 32 to about amino acid 228 of SEQ ID NO:26) 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 and facilitate productive peptide presentation by MHC class II complexes.

[0165] The engineered polypeptide may comprise an extracellular domain derived from any of SEQ ID NOs: 23 to 26. The engineered polypeptide may also comprise a transmembrane domain as 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 percent identity between two polypeptide sequences can be readily determined using programs such as BLAST, which is available free of charge at http: / / blast.ncbi.nlm.nih.gov. Suitably, percent identity is determined by completeness to a reference and / or query sequence.

[0168] As used herein, "the MHC class I polypeptide or MHC class II polypeptide can co-localize with an intracellular signaling domain in a cell" means that the polypeptide causes the MHC class I polypeptide or MHC class II polypeptide to co-localize with the intracellular signaling domain such that when a target T cell binds to a peptide / MHC complex on the effector immune cell of the invention, the intracellular signaling domain transmits an activation signal into the effector immune cell of the invention.

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

[0170] CD79α and CD79β also associate with HLA class II, thereby enabling HLA class II to signal through CD79 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) chimeric antigen receptor (CAR) or transgenic T cell receptor (TCR); and (ii) at least one polypeptide that enables an MHC class I polypeptide or an MHC class II polypeptide to co-localize with an intracellular signaling domain in a cell; wherein the at least one polypeptide that enables an MHC class I polypeptide or an MHC class II polypeptide to co-localize with an intracellular signaling domain is CD79 or a variant thereof.

[0172] The cells can contain an engineered polypeptide comprising CD79α or CD79β linked to an intracellular signaling domain. The cells can contain 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: [ka] Underlined = signal peptide (amino acids 1-32) Bold = extracellular (amino acids 33-143) No text modification = transmembrane domain (amino acids 144-165) Italics = cytoplasmic domain (amino acids 166-226)

[0174] CD79α sequences for use in the present invention may include the sequence set forth 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 and facilitate signaling to HLA class I and / or HLA class II.

[0175] The engineered polypeptide may comprise the ectodomain, transmembrane domain, and intracellular signaling domain of CD79α. The engineered polypeptide may comprise the ectodomain of CD79α corresponding to about amino acid 33 to about amino acid 143 of SEQ ID NO:27.

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

[0177] The engineered polypeptide can include the intracellular signaling domain of CD79α, corresponding to about amino acid 166 to about amino acid 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: [ka] Underlined = signal peptide (amino acids 1-28) Bold = extracellular (amino acids 29-159) No text modification = transmembrane domain (amino acids 160-180) Italics = cytoplasm (amino acids 181-229)

[0179] A CD79β sequence for use in the present invention may comprise the sequence set forth 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 and facilitate signaling to HLA class I and / or HLA class II.

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

[0181] The engineered polypeptide can 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 can 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 can express two engineered polypeptides: one comprising the extracellular domain of CD79α and one comprising the extracellular domain of CD79β.

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

[0185] CD3 is a T cell coreceptor 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, the CD3δ chain, and two CD3ε chains. These chains assemble with the TCR to form a TCR complex, which can generate an activation signal in T lymphocytes.

[0186] The CD3ζ, CD3γ, CD3δ, and CD3ε chains are closely related cell surface proteins of the immunoglobulin superfamily that contain a single extracellular immunoglobulin domain. The transmembrane region of the CD3 chains is characterized by several negatively charged aspartic acid residues that can associate with the positively charged TCR chains. The intracellular tail of the CD3 molecule contains a single conserved motif known as an immunoreceptor tyrosine-based activation motif (ITAM), which 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 or MHC class II complexes on the cell surface. Furthermore, TCR / CD3 components can assemble into TCR / CD3 complexes. Therefore, binding of a TCR to a peptide / MHC complex containing a polypeptide linked to a component of the TCR complex will trigger signaling by the CD3 / TCR complex.

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

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

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

[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 a component 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(SEQ ID NO: 33) (comprising one or more copies of SEQ ID NO: 33). For example, a suitable linker peptide is shown as SEQ ID NO: 34. [ka]

[0194] The polypeptide may be linked to the ectodomain of a component of the CD3 complex. The polypeptide may be linked to the N-terminus of a component of the CD3 complex.

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

[0196] This polypeptide sequence contains the ectodomain derived from HLA-DRα, the transmembrane domain, and the intracellular CD3-ζ endodomain.

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

[0198] This polypeptide sequence contains the HLA-DRα-derived ectodomain, the transmembrane domain, the 41BB endodomain, and the intracellular CD3-ζ endodomain.

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

[0200] This polypeptide sequence contains the ectodomain from HLA-DRα, the transmembrane domain, the CD28 endodomain, and the intracellular CD3-ζ endodomain.

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

[0202] This polypeptide sequence contains the ectodomain from CD79α, the 41BB domain, and the endodomain from CD79.

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

[0204] This polypeptide sequence contains the ectodomain from CD79β, the CD28 domain, and the endodomain from CD79.

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

[0206] This polypeptide sequence contains the ectodomain from CD79α, the CD28 domain, and the endodomain from CD79.

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

[0208] This polypeptide sequence contains the ectodomain from CD79β, the 41BB domain, and the endodomain from CD79.

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

[0210] This polypeptide sequence contains the ectodomain from CD79α, the 41BB domain, and the CD3-zeta domain.

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

[0212] This polypeptide sequence contains the ectodomain from CD79β, the 41BB domain, and the CD3-zeta domain.

[0213] Polypeptide sequences for use in the present invention may include the sequences set forth as SEQ ID NOs: 35-43 or variants having at least 80, 85, 90, 95, 98, or 99% sequence identity, provided that the variants retain the ability to assemble and facilitate productive peptide presentation by MHC class II complexes at the cell surface and transmit an activation signal following binding of a TCR to a peptide / MHC complex containing said polypeptide.

[0214] Intracellular signaling domains The present invention provides at least one polypeptide that can colocalize an MHC class I or MHC class II polypeptide with an intracellular signaling domain within a cell.

[0215] The engineered polypeptides of the present invention may comprise an intracellular signaling domain.

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

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

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

[0219] ITAMs are important for signal transduction in immune cells. Therefore, ITAMs are found in the tails of important cell signaling molecules, such as the CD3 and zeta chains of the T cell receptor complex, the CD79 alpha and beta chains of the B cell receptor complex, and certain Fc receptors. Tyrosine residues within these motifs become phosphorylated after interaction of the receptor molecule with its ligand, forming docking sites for other proteins involved in cell signaling pathways.

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

[0221] The intracellular signaling domain may include additional costimulatory signaling, for example, 4-1BB (also known as CD137) can be used in conjunction with CD3-ζ, or CD28 and OX40 can be used in conjunction with CD3-ζ to deliver growth / survival signals.

[0222] Thus, the intracellular signaling domain may comprise the CD3-ζ endodomain alone, the CD3-ζ endodomain in combination with one or more costimulatory domains selected from the 4-1BB endodomain, the CD28 endodomain, or the OX40 endodomain, and / or a combination of some or all of 4-1BB, CD28, or OX40.

[0223] The endodomain may comprise one or more of the following: an ICOS endodomain, a CD2 endodomain, a CD27 endodomain, or a CD40 endodomain.

[0224] The endomain may comprise the sequences set forth as SEQ ID NOs: 44-47 or variants 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. [ka] [ka]

[0225] Antigen-binding domain linked to a signaling domain The engineered polypeptides of the invention can comprise an MHC class I polypeptide linked to an intracellular signaling domain; an MHC class II polypeptide, or a binding domain that binds to β2 microglobulin.

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

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

[0228] A full-length antibody or immunoglobulin typically consists of 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 an N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain contains an 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 the same general structure: relatively conserved regions called framework regions (FR) are joined to three hypervariable regions called complementarity-determining regions (CDRs). The term "complementarity-determining region" or "CDR" as used herein refers to the region in an antibody that complements the shape of the antigen. Thus, the CDRs determine the protein's affinity and specificity for a specific antigen. The CDRs of the two chains of each pair are aligned by the framework regions, thereby acquiring the function of specifically binding an epitope. Thus, for the VH and VL domains, both the heavy and light chains are characterized by three CDRs: CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3, respectively.

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

[0230] The full-length antibody can be, for example, an 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. An antibody fragment can include, for example, one or more CDRs, a variable region (or a portion thereof), a constant region (or a portion thereof), or a combination thereof. Examples of antibody fragments include, but are not limited to, Fab fragments, F(ab')2 fragments, Fv fragments, single-chain Fvs (scFvs), domain antibodies (dAbs or VHs), single-domain antibodies (sdAbs), VHHs, nanobodies, diabodies, triabodies, trimeric bodies, and monobodies.

[0232] The engineered polypeptides of the present invention can include antigen-binding domains based on non-immunoglobulin scaffolds. These antibody-binding domains are also called antibody mimics. Non-limiting examples of non-immunoglobulin antigen-binding domains include affibodies, fibronectin artificial antibody scaffolds, anticalins, affines, DARPins, VNARs, iBody, affimers, finomelans, abdulins / nanoantibodies, centilins, alphabodies, nanophytins, and D domains.

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

[0234] 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 contains 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 Figure 15 shows the 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- The VH and VL sequences of 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 are shown.

[0235] The engineered polypeptide may comprise an MHC class II binding domain comprising 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. The antibody clones include: anti-HLA-A2 / A28-3PF12, anti-HLA-A2 / A28-3PC4, and anti-HLA-A2 / A28-3PB2.

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

[0239] The engineered polypeptide may contain a short flexible linker to introduce a chain break, which separates the two individual domains but allows them to be oriented at different angles. Such sequences include the sequence SDP and the 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, for example, comprise the transmembrane domain of CD8-alpha or CD28.

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

[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 An engineered polypeptide of the invention can 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 for the T cell receptor (TCR) and assist T cells in communicating 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 with four immunoglobulin domains (D1-D4) exposed on the extracellular surface of the cell: D1, which resembles the immunoglobulin variable (IgV) domain; and D2, D3, and D4, which resemble immunoglobulin constant (IgC) domains.

[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 through its D1 domain. Therefore, T cells that display CD4 molecules on their surface are specific for antigens presented by MHC class II (i.e., the T cells are MHC class II restricted).

[0248] The short cytoplasmic / intracellular tail (C) of CD4 contains an amino acid sequence that allows 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 are brought into close proximity, allowing the tyrosine kinase Lck to bind to the cytoplasmic tail of CD4 and phosphorylate tyrosine residues in immunoreceptor tyrosine activation motifs (ITAMs) on the cytoplasmic domain of CD3, amplifying the signal generated by the TCR. The phosphorylated ITAMs on CD3 recruit and activate SH2 domain-containing protein tyrosine kinases (PTKs) (e.g., ZAP70) to further mediate 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. Engineered polypeptides of the invention may comprise the D1 domain of CD4 having the sequence shown in SEQ ID NO: 50. The positions of Gln40 and Thr45 are shown in bold and underlined. [ka]

[0250] The engineered polypeptide may comprise a variant D1 domain of CD4 that contains one or more amino acid mutations that increase its binding affinity to 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 affinity maturation of human CD4 by yeast surface display to increase the affinity of CD4 for HLA-DR1. A CD4 variant carrying the substitution mutations Gln40Tyr and Thr45Trp was found to bind to HLA-DR1 with a KD of 8.8 μM, compared to a KD of over 400 μM for wild-type CD4.

[0252] The engineered polypeptide may comprise a variant D1 domain of CD4 comprising amino acid mutation(s) at positions 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 comprising the 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) coreceptor is expressed primarily on the surface of cytotoxic T cells, but can also be found on natural killer cells, cortical thymocytes, and dendritic cells. Two isoforms of CD8 (alpha and beta) exist, 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 consists of the CD8-α and CD8-β chains, but homodimers of the CD8-α chain are also expressed on some cells. Both CD8-α and CD8-β 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-α interacts with the α3 portion of class I MHC molecules. The primary recognition site is a flexible loop in the α3 domain of the MHC molecule located between residues 223 and 229. CD8-α binding to MHC class I maintains tight association between the T cell receptor of cytotoxic T cells and target cells during antigen-specific activation. The cytoplasmic tail of the CD8 coreceptor interacts with Lck (lymphocyte-specific protein tyrosine kinase). Upon binding of the T cell receptor to its specific antigen, Lck phosphorylates the cytoplasmic CD3 and ζ chains of the TCR complex, initiating a phosphorylation cascade that ultimately activates transcription factors such as NFAT, NF-κB, and AP-1.

[0257] The engineered polypeptides of the invention may comprise an IgV-like domain derived from CD8-α.

[0258] The amino acid sequence of human CD8α is available from UniProt accession number P01732. Engineered polypeptides of the invention include amino acid residues 22 to 135 of this sequence and may comprise the Ig-like V-type domain of CD8 having the sequence set forth in SEQ ID NO:51. [ka]

[0259] The engineered polypeptide may comprise a variant CD8αIg-like V-type domain that contains one or more amino acid mutations that increase its binding affinity for the α3 portion of a class I MHC molecule 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 in Wang et al. (2011, PNAS 108:15960-15965).

[0261] The engineered polypeptide may contain a dimer of CD8. Devine et al. (1999, J. Immunol. 162:846-851) described a molecule containing two CD8αIg domains linked via the carboxyl terminus of one to the amino terminus of the other by a peptide spacer. Using a 20-amino acid peptide spacer of four repeating units of GGGGS (SEQ ID NO: 52), it was possible to precisely identify the two IG-like domains.

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

[0263] The engineered polypeptide can include a CD8αβ heterodimer. For example, the engineered polypeptide can include a CD8αIg-like V-domain having the sequence set forth in SEQ ID NO: 51 joined to a CD8βIg-like V-domain by a peptide spacer. The peptide spacer can be 10 to 20 amino acids in length, e.g., between 15 and 25 amino acids in length. The peptide spacer can be approximately 20 amino acids in length. Devine et al. For the CD8αα homodimer described in [SEQ ID NO: 52], the peptide spacer may comprise four repeating units of GGGGS (SEQ ID NO: 52).

[0264] The amino acid sequence of the CD8βIg-like V-type domain is shown below as SEQ ID NO:54. [ka]

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

[0266] The engineered polypeptides may contain a short, flexible linker between the CD8α monomer, CD8αα homodimer, or CD8αβ heterodimer and the stalk and / or transmembrane domain to introduce a chain break. The chain break separates the two individual domains but allows them to be oriented at different angles. Such sequences include the sequence SDP and the sequence 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 comprise a transmembrane domain as defined above, for example, the engineered polypeptide may comprise the transmembrane domain of CD8-alpha or CD28.

[0269] The engineered polypeptide comprises an intracellular signaling domain as defined above. The engineered polypeptide may, for example, comprise a CD3ζ endodomain.

[0270] The engineered polypeptide may have the following general structure: CD4 D1 domain-linker-transmembrane domain-intracellular signaling domain; CD8αIg-like V-type 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 Polypeptides In a further embodiment of the invention, the polypeptide that allows an MHC class I or MHC class II polypeptide to co-localize with an intracellular signaling domain can 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-anchored.

[0273] When expressed by or on the cell surface, the bispecific molecules of the invention colocalize the TCR with MHC class I or II and facilitate TCR signaling in the cells of the invention following binding of the TCR on a different T cell 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" refers to a polypeptide having an antigen-binding site comprising at least one complementarity-determining region (CDR). An antibody may comprise three CDRs and have an antigen-binding site equivalent to that of a domain antibody (dAb). An antibody may comprise six CDRs and have an antigen-binding site equivalent to that of a classical antibody molecule. The remainder of the polypeptide may be any sequence that provides a suitable scaffold for the antigen-binding site and presents the antigen-binding site in a manner suitable for antigen binding to the antigen-binding site. An antibody may be a whole immunoglobulin molecule or a portion thereof, such as a Fab, F(ab)'2, Fv, single-chain Fv (ScFv) fragment, nanobody, or single-chain variable domain (which may be a VH or VL chain having three CDRs). An antibody may be a bifunctional antibody. An antibody may 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 molecules of the invention may comprise domains not derived from or based on immunoglobulins. To exploit the binding capacity of non-antibody polypeptides, several "antibody-mimetic" designed repeat proteins (DRPs) have been developed. Such molecules include ankyrin or leucine-rich repeat proteins (e.g., DARPins (designed ankyrin repeat proteins), anticalins, avimers, and versabodies).

[0278] The first binding domain of the bispecific molecule of the invention is capable of binding to an MHC class I or MHC class II polypeptide.

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

[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 contains 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 Figure 15 shows the 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- The VH and VL sequences of 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 are shown.

[0281] The bispecific polypeptide may comprise an MHC class II binding domain comprising one of these VH and VL sequence pairs. In particular, the bispecific polypeptide may comprise an MHC class II binding domain based on the 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) describe the isolation and characterization of human monoclonal HLA-A2 antibodies. The antibody clones include: anti-HLA-A2 / A28-3PF12, anti-HLA-A2 / A28-3PC4, and anti-HLA-A2 / A28-3PB2.

[0284] The bispecific polypeptides of the invention may comprise an MHC class I or MHC class II binding domain 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 can be capable of binding to CD3 on the surface of T cells. In this regard, the second domain can comprise a CD3- or TCR-specific antibody or a portion thereof.

[0286] The second domain may comprise the complementarity determining regions (CDRs) 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 that has at least 80% sequence identity and that binds to CD3.

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

[0289] The second domain of the bispecific molecules of the 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 published in U.S. Patent No. 7,381,803.

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

[0291] The second binding domain may comprise an scFv comprising CDR sequences from OKT3. The second binding domain may comprise 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. [ka]

[0292] SEQ ID NOs: 55 and 62 provide alternative structures for scFVs suitable for use in the present invention. SEQ ID NO: 55 is provided in a VL-VH configuration. SEQ ID NO: 55 is provided in a VH-VL configuration.

[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 sequence set forth as SEQ ID NO: 55 or 62.

[0294] The bispecific molecules of the invention may comprise a spacer sequence connecting the first domain with the second domain and spatially separating the two domains.

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

[0296] The spacer sequence may comprise, for example, an IgG1 hinge or a CD8 stalk. The linker may alternatively comprise an alternative linker sequence having similar length and / or domain spacing characteristics as an IgG1 hinge or a CD8 stalk.

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

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

[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. Bispecific molecules of the invention can comprise a spacer comprising or consisting of the sequence set forth in SEQ ID NO: 64 or a variant thereof having at least 80, 85, 90, 95, 98, or 99% sequence identity, provided that the variant sequence provides approximately equivalent spacing between the first and second domains and / or allows the bispecific molecule to homodimerize.

[0300] Bispecific molecules of the invention may have the following general formula: First domain - spacer - second domain.

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

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

[0303] The spacer can allow the bispecific molecule to form homodimers, for example, due to the presence of one or more cysteine residues in the spacer, which can form disulfide bonds with another molecule containing an identical spacer.

[0304] The bispecific molecule may be membrane-anchored, in other words, the bispecific molecule may comprise a transmembrane domain such that the molecule is localized to the cell membrane after expression in a cell of the invention.

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

[0306] Bispecific molecules of the 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 receptors The engineered immune cells of the present invention may express a transgenic T cell receptor (TCR).

[0308] T cell receptors (TCRs) are molecules found on the surface of T cells that are responsible for recognizing fragments of antigens as peptides bound to major histocompatibility complex (MHC) molecules.

[0309] The TCR is a heterodimer composed of two different protein chains: in humans, in 95% of T cells, the TCR consists of alpha (α) and beta (β) chains (encoded by TRA and TRB, respectively), whereas 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 engages with an antigen peptide and MHC (peptide / MHC), the T lymphocyte is activated via signal transduction.

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

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

[0313] Effector immune cells and cell surface receptors / receptor complexes 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 from donor peripheral blood in the context of hematopoietic stem cell transplantation (second party), or from peripheral blood from an unrelated donor (third party). Prior to introduction of the nucleic acid molecule(s) encoding the polypeptide of the invention, the T cells or NK cells can be, for example, activated and / or expanded, for example, by treatment with an anti-CD3 monoclonal antibody.

[0316] Alternatively, the cells can be derived from ex vivo differentiation of inducible or embryonic precursor cells into T cells. Alternatively, immortalized T cell lines that retain 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 suitable matched donor by leukapheresis of peripheral blood after mobilization with the administration of cytokines such as G-CSF (peripheral blood stem cells (PBSCs)) at pharmacological doses, or from umbilical cord blood (UCB) collected from the placenta after delivery. Bone marrow, PBSCs, or UCB can be transplanted untreated, or HSCs can be enriched by immunoselection with a monoclonal antibody against the CD34 surface antigen.

[0318] The cell surface receptor or receptor complex binds to an antigen-recognizing receptor on the target immune cell, and the 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 a population of immune cells in vitro, ex vivo, or in vivo. The target immune cells can be present in the patient or in a graft, for example, prior to administration to the patient.

[0321] Target immune cells may specifically recognize self- or alloantigens.

[0322] The antigen-recognizing receptor of the target immune cell can be a T cell receptor (such as an αβ-TCR or γδ-TCR), as described in more detail above. Alternatively, the antigen-recognizing receptor can be an NK cell-activating receptor. There are two different types of surface receptors responsible for triggering NK-mediated innate cytotoxicity: NK KARs (killer-activating receptors) and NK KIRs (killer-inhibitory receptors), which generate opposing signals. A balance exists between these competing signals that determines whether or not NK cell cytotoxic activity should be triggered.

[0323] KARs typically have noncovalently linked subunits containing an immunoreceptor tyrosine-based activation motif (ITAM) in their cytoplasmic tail (e.g., CD3ζ), the γc chain, or one of two adaptor proteins, DAP10 and DAP12. Similar to TCRs on T cells, ITAMs associated with KARs are involved in promoting signal transduction in NK cells. When an activating ligand binds to the KAR complex, tyrosine residues in the ITAMs in the associated chains are phosphorylated by kinases, transducing signals that promote innate cytotoxicity into the NK cell.

[0324] Manipulation to withstand "counterattack" by targeted immune cells The effector immune cells of the present invention are engineered such that when a cell surface receptor or receptor complex of the effector immune cell specifically binds to an antigen-recognizing receptor of a target immune cell, the effector immune cell wins the battle between the two immune cells, rather than the target immune cell being killed by the effector immune cell.

[0325] There are a variety of ways in which effector immune cells can be engineered to have a selective advantage over target immune cells when and 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 capable of transmitting one or more inhibitory immune signals.

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

[0328] The immunosuppressant can be administered to the population of immune cells in vivo or in vitro.For example, the immunosuppressant can be administered to the patient before or at the same time as the composition comprising effector immune cells is administered.Alternatively, the immunosuppressant can be administered to the graft before or at the same time as the composition comprising effector immune cells is administered to the graft and before the graft is introduced into the patient.

[0329] Immunosuppressants, also known as immunosuppressants, immunosuppressants, and anti-rejection drugs, are drugs that inhibit or prevent the activity of the immune system. Immunosuppressants are commonly used in immunosuppressive therapy, for example, to: (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 to be autoimmune in 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, Behcet's disease, pemphigus, and ulcerative colitis); and (iii) To treat several other non-autoimmune inflammatory diseases (e.g., long-term suppression of allergic asthma), ankylosing spondylitis.

[0330] Numerous immunosuppressants are known and routinely used in transplantation and immunotherapy approaches. Immunosuppressants can be, for example, small molecules or antibodies or other biologics. Immunosuppressants can be glucocorticoids, cytostatics, 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 the glucocorticoid receptor. Examples include: cortisol (hydrocortisone), cortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, fludrocortisone acetate, and deoxycorticosterone acetate.

[0332] At pharmacological (i.e., supraphysiological) doses, glucocorticoids are used to suppress a variety of allergic, inflammatory, and autoimmune disorders. Glucocorticoids are also administered as posttransplant immunosuppressants to prevent acute graft rejection and graft-versus-host disease.

[0333] Glucocorticoids suppress cell-mediated immunity 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, the most important of which is IL-2. Lower cytokine production levels result in decreased T-cell proliferation. Glucocorticoids also suppress humoral immunity, reducing the expression of IL-2 and IL-2 receptors by B cells. This reduces both the expansion of B-cell clones and antibody synthesis.

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

[0335] Glucocorticoids also stimulate the escape of lipocortin-1 into the extracellular space, where it binds to leukocyte membrane receptors and inhibits various inflammatory events, including epithelial adhesion, transmigration, chemotaxis, phagocytosis, respiratory burst, and the release of various inflammatory mediators (such as lysosomal enzymes, cytokines, tissue plasminogen activator, and chemokines) from neutrophils, macrophages, and mast cells.

[0336] Cytostatics Cytostatics inhibit cell division. In immunotherapy, cytostatics are used in lower doses than in the treatment of malignant diseases. Cytostatics affect the proliferation of both T cells and B cells. Purine analogs are the most frequently administered because they are the most effective. Cytostatics include alkylating agents, antimetabolites, methotrexate, azathioprine, and mercaptopurine, as well as cytotoxic antibiotics.

[0337] Alkylating agents used in immunotherapy include nitrogen mustards (cyclophosphamide), nitrosoureas, and platinum compounds. Cyclophosphamide (Baxter's Cytoxan) is perhaps the most potent immunosuppressant compound. At low doses, cyclophosphamide is highly effective in treating systemic lupus erythematosus, autoimmune hemolytic anemia, granulomatosis with polyangiitis, and other immune disorders. At high doses, it can cause pancytopenia and hemorrhagic cystitis.

[0338] Antimetabolites 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. It binds to dihydrofolate reductase and prevents the synthesis of tetrahydrofolate. Methotrexate is used in the treatment of autoimmune diseases (e.g., rheumatoid arthritis or Behcet's disease) and in transplantation.

[0340] Azathioprine (Imuran, Prometheus) is a major immunosuppressive cytotoxic agent. It is widely used to control transplant rejection. Azathioprine is non-enzymatically cleaved to mercaptopurine, which acts as a purine analog and DNA synthesis inhibitor. Mercaptopurine itself can also be administered directly.

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

[0342] Among cytotoxic antibiotics, dactinomycin is the most important. It is used in kidney transplants. Other cytotoxic antibiotics are anthracyclines, mitomycin C, bleomycin, and mithramycin.

[0343] antibody Antibodies are sometimes used as rapid and potent immunosuppressive therapy to prevent acute rejection and as targeted treatment for lymphoproliferative or autoimmune disorders (e.g., anti-CD20 monoclonals). Antibodies can be polyclonal or monoclonal.

[0344] Heterologous polyclonal antibodies are obtained from the serum of animals (e.g., rabbits, horses) and injected with the patient's thymocytes or lymphocytes. Antilymphocyte antigen (ALG) and antithymocyte antigen (ATG) have been used. These are part of the treatment for steroid-resistant acute rejection and severe aplastic anemia. However, they are added first to reduce the dosage and toxicity of other immunosuppressants. They can also be used to transition to cyclosporine therapy.

[0345] Polyclonal antibodies inhibit T lymphocytes, causing their lysis (both complement-mediated cytolysis and cell-mediated opsonization), followed by removal of reticuloendothelial cells from the circulation into the spleen and liver. In this way, polyclonal antibodies inhibit cell-mediated immune responses, including graft 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, derived from horse serum, and thymoglobulin, derived from rabbit serum. Polyclonal antibodies affect all lymphocytes, causing generalized immunosuppression and possibly leading to post-transplant lymphoproliferative disease (PTLD) or severe infections, particularly with cytomegalovirus. To mitigate these risks, treatment is typically given in hospitals with adequate isolation from infection.

[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 the rejection of transplanted organs, but they are also used to track changes in lymphocyte subpopulations. This makes it reasonable to anticipate similar new drugs in the future.

[0348] Muromonab-CD3 is an IgG2a-type murine 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 immunosuppressants and is administered to control acute rejection episodes refractory to steroids and / or polyclonal antibodies. Because it acts more specifically than polyclonal antibodies, muromonab-CD3 is also used prophylactically in transplantation.

[0349] Interleukin-2 is a key immune system regulator required for the clonal expansion and survival of activated T lymphocytes. Its effects are mediated by the trimeric cell surface receptor IL-2a, consisting of α, β, and γ chains. IL-2a (CD25, T-cell activation antigen, TAC) is expressed only by activated T lymphocytes. Therefore, it is particularly important for selective immunosuppressive treatment, and research to develop effective and safe anti-IL-2 antibodies has been intensively pursued. Basiliximab (Simulect) and daclizumab (Zenapax) are chimeric mouse / human anti-TAC antibodies. These drugs act by binding to the α chain of the IL-2a receptor, preventing IL-2-induced clonal expansion of activated lymphocytes and shortening their survival. 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 consisting of 11 amino acids.

[0351] Cyclosporine is thought to bind to the cytoplasmic protein cyclophilin (immunophilin) of immunocompetent lymphocytes, particularly T-lymphocytes. This cyclosporine-cyclophilin complex inhibits calcineurin, a phosphatase that under normal conditions induces the transcription of interleukin-2. This drug also inhibits lymphokine production and interleukin release, impairing effector T-cell function.

[0352] Tacrolimus is a product of the bacterium Streptomyces tsukubaensi. It is a macrolide lactone that acts by inhibiting calcineurin.

[0353] This drug is primarily used in liver and kidney transplants, but in some clinics it is also used in heart, lung, and cardiopulmonary transplants. It binds to the immunophilin FKBP1A, which then binds to calcineurin, inhibiting its phosphatase activity. In this way, it prevents cells from moving from the G0 to G1 phase of the cell cycle. Tacrolimus is more potent than cyclosporine and has less pronounced side effects.

[0354] Sirolimus (rapamycin) is a macrolide lactone produced by the actinomycete Streptomyces hygroscopicus. It is used to prevent rejection. Although sirolimus is structurally similar to tacrolimus, it has somewhat different mechanisms of action and different side effects.

[0355] In contrast to cyclosporine and tacrolimus, which affect the first step of T lymphocyte activation, sirolimus affects the second step, i.e., signal transduction and lymphocyte clonal proliferation. Like tacrolimus, sirolimus binds to FKBP1A; however, the complex inhibits another protein, mTOR, rather than calcineurin. Therefore, sirolimus acts synergistically with cyclosporine and, when combined with other immunosuppressants, has minimal side effects. Sirolimus also indirectly inhibits several T lymphocyte-specific kinases and phosphatases, thus preventing the transition from the G1 to S phase of the cell cycle. In a similar manner, sirolimus prevents B cell differentiation into plasma cells and reduces the production of IgM, IgG, and IgA antibodies. Sirolimus is also active against PI3K / AKT / mTOR-dependent tumors.

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

[0357] Other immunosuppressants include interferons, opioids, TNF binding proteins, mycophenolates, and small biologic agents.

[0358] IFN-β suppresses Th1 cytokine production and monocyte activation. IFN-β is used to slow the progression of multiple sclerosis. IFN-γ can induce lymphocytic apoptosis.

[0359] Opioids are substances that act on opioid receptors to produce morphine-like effects. Chronic opioid use can cause immunosuppression of both innate and adaptive immunity. Decreased proliferation and impaired 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)) that bind to TNF-α and prevent it from inducing the synthesis of IL-1 and IL-6 and the adhesion of lymphocyte activation molecules. They are used in the treatment of rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, and psoriasis.

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

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

[0363] Small-cell immunosuppressants include the synthetic immunosuppressant fingolimod, which increases the expression or alters the function of certain adhesion molecules (α4 / β7 integrin) in lymphocytes, leading to their accumulation in lymphoid tissues (lymph nodes) and a decrease in their number in circulation. In this respect, small-cell immunosuppressants differ from all other known immunosuppressants.

[0364] Myriocin is an atypical amino acid and an antibiotic derived from certain thermophilic fungi. It has been shown to inhibit the proliferation of cytotoxic T cells.

[0365] Mutational resistance Effector cells of the invention may contain one or more mutations that increase resistance to one or more immunosuppressive drugs, for example, effector cells may contain one or more mutations that render the cells resistant to tacrolimus and / or cyclosporine.

[0366] Effector cells may contain a nucleic acid sequence encoding calcineurin (CN) with 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 cytoplasmic nuclear factor of activated T cells (NFATc), a transcription factor, by dephosphorylating it. Activated NFATc is then translocated into the nucleus, where it upregulates the expression of interleukin-2 (IL-2), stimulating T cell responses. 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) described various calcineurin mutants that render cytotoxic T lymphocytes resistant to tacrolimus and / or cyclosporine.

[0367] Calcineurin is composed of a 61 kD calmodulin-binding catalytic subunit, calcineurin A, and a 19 kD Ca 2+ PPP3R1 is a heterodimer of the catalytic subunit and the binding regulatory subunit, calcineurin B. There are three isozymes of the catalytic subunit, each encoded by a separate gene (PPP3CA, PPP3CB, and PPP3CC), and two isoforms of the regulatory subunit, each encoded by a separate gene (PPP3R1 and 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. [ka]

[0369] The 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] The mutant calcineurin A may include 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] The mutant calcineurin A may comprise 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. [ka]

[0373] The mutant calcineurin B may contain mutations 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] The mutant calcineurin B may include one or more of the following substitution and optional insertion mutations 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] The mutant calcineurin B may comprise 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 comprise the following combination of mutations with reference to SEQ ID NO: 66: L124T and K125-LA-Ins. This is the module known as "CnB30" described in the Examples section. CnB30 has the amino acid sequence shown as SEQ ID NO: 131. [ka]

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

[0378] The following CNa mutants were resistant to cyclosporine A: V314K; V314R; Y341F; V314K and Y341F; and V314R and Y341F.

[0379] The following CNb mutants were resistant to FK506: N123W; K125-VQ-Ins; K125-IE-Ins; K-125-LA-Ins; and L124T and K-125-LA-Ins.

[0380] The following CNb mutants were resistant to cyclosporine A: K125-VQ-Ins; K125-IE-Ins; K-125-LA-Ins; V120S and L124T; and L124T and K-125-LA-Ins.

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

[0382] The effector immune cells of the present invention may 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 that increase the resistance of the effector immune cells to one or more calcineurin inhibitors.

[0383] In particular, effector immune cells may express the above-listed variant calcineurin A and / or variant calcineurin B that 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, improving cytotoxicity and cytokine release, particularly in response to low-density target antigens.

[0385] The present inventors have now discovered that expression of dnCSK also confers global resistance to immunosuppression to cells. Expression of dnCSK confers "global" resistance to immunosuppression, thereby generally reducing the sensitivity of cells to immunosuppressive drugs.

[0386] C-terminal Src kinases (CSKs), also known as tyrosine-protein kinases, are enzymes that phosphorylate tyrosine residues at the C-terminus of Src-family kinases (SFKs) (including SRC, HCK, FYN, LCK, LYN, and YES1), thereby inhibiting their activity.

[0387] Src family kinases (SFKs), such as Lck, consist of an N-terminal myristoyl group that allows membrane localization and is attached to SH4, SH3, SH2, and protein tyrosine kinase domains (SH1 domains).

[0388] Tyrosine residues are conserved in the activation loop and C-terminal tail, and 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 negative regulatory Lck on its C-terminal tyrosine residue Y505, maintaining its inactive state. In resting T cells, Csk is targeted to lipid rafts by association of its SH2 domain with the phosphotyrosine residue pY317 of PAG, which is expressed as a tyrosine-phosphorylated protein in unstimulated T cells. This interaction between Csk and PAG results in the activation of Csk and the inhibition of Lck.

[0390] Upon TCR activation, CD45 is excluded from membrane microdomains and dephosphorylates PAG, which leads to the shedding of Csk from the plasma membrane.

[0391] The amino acid sequence of human CSK is available from Uniprot accession number 41240, and 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; residues 195-449 correspond to the protein kinase domain. [ka]

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

[0393] Dominant negative CSKs may lack a functional protein kinase domain. dnCSKs may not contain a kinase domain, or may contain a partially or completely inactive kinase domain. The kinase domain may be inactivated, for example, by truncating or mutating one or more amino acids.

[0394] The dnCSK can be, for example: i) truncated CSKs that are recruited to the plasma membrane but lack a functional kinase domain; ii) a 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 lacks the kinase domain entirely, for example, dnCSK can include the SH2 and optional SH3 domains, but can be truncated to remove the kinase domain.

[0396] Alternatively, effector immune cells can express dnCSK containing a partially truncated kinase domain that includes a portion of the phosphatase (e.g., the portion of the sequence from residues 195-449 of SEQ ID NO: 67), provided that the truncated kinase has a reduced ability to phosphorylate the C-terminal tyrosine residue Y505 of Lck compared to wild-type CSK. The truncated kinase may have virtually no residual kinase activity.

[0397] dnCSKs may be truncated CSKs that retain the ability to bind to transmembrane adaptor proteins (such as PAG, Lime, and / or Dok1 / 2) that recruit wild-type CSKs to the plasma membrane but lack a functional kinase domain.

[0398] The dnCSK may have the sequence shown in SEQ ID NO: 68, which corresponds to the wild-type CSK sequence (SEQ ID NO: 67) minus the kinase domain. [ka]

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

[0400] Effector immune cells of the present invention may express dnCSKs that contain an inactivated kinase domain that has reduced or no ability to phosphorylate proteins such as Lck.

[0401] The kinase domain can contain, for example, one or more amino acid mutations that result in reduced kinase activity compared to the wild-type sequence.

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

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

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

[0405] A dnCSK of the invention may have the sequence shown in SEQ ID NO: 70, which corresponds to the full-length CSK sequence with a K222R substitution. This mutation is shown in bold and underlined in SEQ ID NO: 70. Alternatively, a dnCSK of the invention may have a sequence equivalent to SEQ ID NO: 70 in which the SH3 domain is deleted. [ka]

[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 in SEQ ID NO: 71, also known as "CSKas," which contains the mutation T266G compared to the wild-type sequence shown in SEQ ID NO: 67. The substitution is shown in bold and underlined in SEQ ID NO: 71. Alternatively, the dnCSK of the present invention may have a sequence equivalent to SEQ ID NO: 71, in which the SH3 domain is deleted. [ka]

[0407] The catalytic activity of CSKas is inhibited by 3-iodobenzyl-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 plasma membrane.

[0408] Inhibitory immune signal transduction The effector immune cells of the present invention may express or overexpress an immunoinhibitory molecule or a fusion protein comprising the extracellular domain of an immunoinhibitory molecule.

[0409] In vivo, membrane-bound immunoinhibitory receptors (such as PD-1, LAG-3, 2B4, or BTLA1) inhibit T cell activation. During T cell activation (schematically shown in Figure 15a), antigen recognition by the T cell receptor (TCR) results in the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) on CD3ζ. The phosphorylated ITAMs are recognized by the ZAP70 SH2 domain, resulting in T cell activation. Inhibitory immunoreceptors such as PD1 effectively reverse this process, as shown schematically in Figure 15b. PD1 contains an ITIM in its endodomain that is recognized by the SH2 domain (SHP-1) of PTPN6. When PD1 binds to its ligand (PD-L1) or tumor cells, PTPN6 is recruited to the juxtamembrane region, where its phosphatase domain subsequently dephosphorylates the ITAM domain, inhibiting immune activation.

[0410] Target immune cells will naturally express a variety of such ITIM-containing immunoinhibitory receptors (such as PD-1, LAG3, TIM-3, TIGIT, BTLA, VISTA, CEACAM1-R, KIR2DL4, B7-H3, and B7-H4).

[0411] By engineering the effector immune cells of the present invention to express ligands of one or more immunoinhibitory receptors or the extracellular domains of such ligands, the effector immune cells will inhibit T cell activation in target immune cells when a synapse is formed between the two cells. This "one-way" inhibition gives the effector immune cells an activation advantage over the target immune cells, meaning that the effector immune cells predominate and kill the target immune cells.

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

[0413] The immunoinhibitory molecule or its extracellular domain expressed by the effector immune cells can be selected from, for example, 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 cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a 40 kDa type 1 transmembrane protein expressed by cancer cells and aids in evasion from antitumor immunity. Engagement of PD-L1 with its receptor PD-1 on T cells delivers a signal that inhibits 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. [ka]

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

[0417] Effector immune cells of the invention may comprise 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 adaptive immune responses. PD-L2 is one of two known ligands for programmed cell death protein 1 (PD-1), the other being PD-L1.

[0419] PD-L2 is primarily expressed on professional antigen-presenting cells, including dendritic cells (DCs) and macrophages. When PD-L2 binds to PD-1, it can activate pathways that inhibit 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 is shown below as SEQ ID NO: 76. [ka]

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

[0422] Effector immune cells of the 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 domain of this receptor binds to several TNF receptor-associated factor (TRAF) family members, which mediate signal transduction pathways that activate immune responses. 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. [ka]

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

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

[0427] CD155 CD155 (cluster of differentiation 155), also known as the poliovirus receptor, is a type I transmembrane glycoprotein in the immunoglobulin superfamily. CD155 is involved in the humoral immune response in the gut and in 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. [ka]

[0429] The signal peptide, extracellular domain, and transmembrane domain of CD155 are shown below as SEQ ID NOs: 85, 86, and 87, respectively. [ka] [ka]

[0430] Effector immune cells of the present invention may comprise the CD155 extracellular domain and optionally the CD155 signal peptide and / or the CD155 transmembrane domain.

[0431] VSIG-3 VSIG-3, also known as IGSF11, is a ligand for 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 by human T cells, including IFN-γ, IL-2, IL-17, CCL5 / Rantes, CCL3 / MIP-1α, and CXCL11 / I-TAC.

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

[0433] The signal peptide, extracellular domain, and transmembrane domain of VSIG-3 are shown below as SEQ ID NOs: 89, 90, and 91, respectively. [ka]

[0434] Effector immune cells of the present invention may comprise a VSIG-3 extracellular domain and optionally a VSIG-3 signal peptide and / or a VSIG-3 transmembrane domain.

[0435] Galectin-9 Galectin-9 is a ligand for HAVCR2 (TIM-3) and is expressed on various tumor cells. Interaction between galectin-9 and HAVCR2 attenuates T cell proliferation and effector function in the tumor microenvironment. Binding to HAVCR2 induces the death of T helper type 1 (Th1) lymphocytes. Galectin-9 has N- and C-terminal carbohydrate-binding domains connected by a linking peptide.

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

[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. [ka]

[0438] The effector immune cells of the present invention may comprise the full-length galectin-9 sequence with or without the signal peptide, or may comprise only the galectin-1 domain or the galectin-2 domain or the HAVCR2-binding domain from galectin-9, -1, or -2.

[0439] The effector immune cells of the present invention may comprise a membrane-tethered version of galectin-9 or a portion thereof. Galectin-9 may be membrane-tethered using a transmembrane domain and, optionally, a spacer sequence and / or an endodomain. For example, galectin-9 or a portion thereof may be membrane-tethered using the CD8 stalk spacer, transmembrane domain, and truncated endodomain previously described in WO 2013 / 153391 for the sorting-suicide gene RQR8.

[0440] HLA-G HLA-G histocompatibility antigen class I, also known as human leukocyte antigen G (HLA-G), belongs to the HLA nonclassical class I heavy chain paralogs. 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 its expression by trophoblasts during pregnancy protects the pregnancy from NK cell-mediated death.

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

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

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

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

[0445] CEACAM-1 serves as a corepressor receptor in the immune response of T cells, natural killer (NK), and neutrophils. Upon stimulation of the TCR / CD3 complex, CEACAM-1 mediates homophilic binding to neighboring cells. Its interaction with and phosphorylation by LCK and its interaction with the TCR / CD3 complex recruits PTPN6, which subsequently dephosphorylates CD247 and ZAP70, blocking granule exocytosis and inhibiting TCR-mediated cytotoxicity. CEACAM-1 also plays a crucial role in regulating autoimmunity and antitumor immunity by inhibiting T cell proliferation and cytokine production through inhibition of the JNK cascade and by inhibiting T cells through its interaction with HAVCR2 on T cells. During natural killer (NK) cell activation, CEACAM-1 inhibits KLRK1-mediated cytolysis of CEACAM1-bearing tumor cells through homophilic interaction in trans with CEACAM1 on target cells, leading to interaction of CEACAM1 with KLRK1 in cis, which recruits PTPN6 and subsequently leads to VAV1 dephosphorylation.

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

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

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

[0449] LSECTin LSECTin, liver sinusoidal endothelial cell lectin, is a ligand for 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 Uniprot accession number Q6UXB4 and is shown below as SEQ ID NO:104. [ka]

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

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

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

[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 FGL1-derived LAG-3 binding domain and optionally an FGL1 signal peptide.

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

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

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

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

[0461] The effector immune cells of the present invention may comprise 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 acts as an immune checkpoint molecule. B7-H4 negatively regulates T-cell-mediated immune responses by inhibiting T-cell activation, proliferation, cytokine production, and the development of cytotoxicity. When expressed on the cell surface of tumor macrophages, B7-H4, together with regulatory T cells (Tregs), plays an important role in suppressing tumor-associated antigen-specific T-cell immunity.

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

[0464] The signal peptide, extracellular domain, and transmembrane domain of B7-H4 are shown below as SEQ ID NOs: 115, 116, and 117, respectively. [ka]

[0465] The effector immune cells of the present invention may comprise 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 proteins comprising the extracellular domains of PD-L1, PD-L2, HVEM, CD155, VSIG-3, Galectin-9, HLA-G, CEACAM-1, LSECTin, FGL1, B7-H3, B7-H4 having the sequences set forth above, or variants thereof (e.g., variants with at least 80%, 90%, 95%, or 99% amino acid identity, provided that the resulting protein molecule retains the ability to bind to an inhibitory immunoreceptor on the target immune cell and inhibit activation of the target immune cell).

[0467] Membrane localization domain Effector antibodies can express fusion proteins containing the extracellular and membrane-localizing domains of immunoinhibitory molecules.

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

[0469] The membrane localization domain may be or contain a sequence that initially attaches the nascent polypeptide to the ER membrane, where the protein remains membrane-associated at the end of the synthesis / translocation process as membrane material "flows" from the ER to the Golgi and ultimately to the plasma membrane.

[0470] The membrane localization domain can include, for example, a transmembrane sequence, a membrane-stopping 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 an N-terminal glycine residue via an amide bond. Myristic acid, also known as n-tetradecanoic acid, is a 14-carbon saturated fatty acid. The modification can be added either co-translationally or post-translationally. N-myristoyltransferase (NMT) catalyzes the myristate addition reaction in the cytoplasm. Myristoylation targets and attaches proteins to membranes because the hydrophobic myristoyl group interacts with phospholipids in the cell membrane.

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

[0473] The fusion protein may comprise 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, and less commonly, serine and threonine residues, of proteins. Palmitoylation increases the hydrophobicity of proteins and can be used to 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 signaling, palmitoylation of the α subunit, prenylation of the γ subunit, and myristoylation are involved in anchoring the G protein to the inner surface of the plasma membrane so that 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 an additional fatty acid that directs it to membrane localization when expressed intracellularly.

[0477] Prenylation (also known as isoprenylation or lipidation) is the addition of hydrophobic molecules to proteins or chemicals. The prenyl group (3-methyl-but-2-en-1-yl) facilitates attachment to cell membranes, similar to lipid anchors such as GPI anchors.

[0478] Protein prenylation involves the transfer of either a farnesyl or geranyl-geranyl moiety to the C-terminal cysteine(s) of a target protein. Three enzymes exist that perform prenylation in cells: farnesyltransferase, Caax protease, and geranylgeranyltransferase I.

[0479] The fusion protein may contain a sequence that can be prenylated. The fusion protein may contain one or more prenyl groups that allow it to be membrane-localized when expressed in a cell.

[0480] Cytoplasmic domain The fusion protein may include a cytoplasmic domain derived from a protein other than the immunoinhibitory 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. [ka]

[0482] Costimulatory endodomain Effector immune cells can express fusion proteins comprising the extracellular domain and a costimulatory endodomain of an immunoinhibitory molecule.

[0483] The costimulatory endodomain may be or may include an endodomain 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 which are set forth below as SEQ ID NOs: 120-130, respectively. [ka] [ka]

[0484] Fusion proteins may include combinations of endodomains, such as CD28 and OX-40 or CD28 and 4-1BB.

[0485] The fusion protein may comprise a variant of one of the sequences set forth as SEQ ID NOs: 120-130 (e.g., a variant having at least 80%, 90%, 95%, or 99% amino acid identity, provided that the resulting sequence retains the ability to provide proliferation and / or survival signals to effector immune cells).

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

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

[0488] Those skilled in the art will understand that many different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. Furthermore, those skilled in the art should understand that, using routine techniques, nucleotides that do not affect the polypeptide sequence encoded by the polynucleotides described herein can be substituted to reflect the codon usage of any particular host organism in which the polypeptide is to be expressed.

[0489] The nucleic acids of the present invention may comprise DNA or RNA. The nucleic acids of the present invention may be single-stranded or double-stranded. The nucleic acids of the present invention may also be polynucleotides that contain synthetic or modified nucleotides 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 polynucleotides can be modified for the uses described herein by any method available in the art. Such modifications may be performed to enhance the in vivo activity and longevity of the polynucleotide of interest.

[0490] The terms "variant," "homologue," or "derivative" in reference to a nucleotide sequence include any substitution, variation, modification, replacement, deletion, or addition of one (or more) nucleic acid from or to the aforementioned sequence.

[0491] nucleic acid construct The present invention also provides (i) a first nucleic acid sequence encoding a portion of a cell surface receptor or cell surface receptor complex as defined above; and (ii) a second nucleic acid sequence that, when expressed in the cell, confers resistance to the immunosuppressant to said cell; and / or (iii) a third nucleic acid sequence encoding an immunoinhibitory molecule or a fusion protein comprising the extracellular domain of an immunoinhibitory molecule; The present invention provides 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 from an MHC class I polypeptide or an ectodomain from an MHC class II polypeptide linked to an intracellular signaling domain; or beta-2 microglobulin linked to an intracellular signaling domain; (c) an engineered polypeptide comprising an MHC class I or MHC class II polypeptide or beta-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 beta-2 microglobulin, linked to an intracellular signaling domain; (f) an engineered polypeptide comprising CD79α or CD79β linked to an intracellular signaling domain; (g) 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; or (e) A bispecific polypeptide comprising: (i) an MHC class I polypeptide; an MHC class II polypeptide; a first binding domain that binds to beta-2 microglobulin; and (ii) 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 that has 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 can encode: (g) A fusion protein comprising an immunoinhibitory molecule or the extracellular domain of an immunoinhibitory 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 that has increased resistance to one or more calcineurin inhibitors compared to wild-type calcineurin, and / or a dominant-negative CSK; and / or (iii) a third nucleic acid sequence encoding an immunoinhibitory molecule or a fusion protein comprising the extracellular domain of an immunoinhibitory molecule.

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

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

[0498] The nucleic acids may be in any order in the nucleic acid. Nucleic acids encoding separate polypeptides may be separated by a co-expression site, which allows the two polypeptides to be co-expressed as separate entities. The 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, so that when the polypeptides are produced, they are immediately cleaved into individual 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, although cleavage sites may separate 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 "cleavage" activity: proteolytic activity by host cell proteinases, 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 purposes of the present invention, as long as the cleavage site, when placed between the nucleic acid sequences encoding the proteins, results in expression of the proteins as individual entities.

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

[0502] A "self-cleaving peptide" refers to a peptide that functions such that when a protein and polypeptide comprising 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 from an aphthovirus or cardiovirus. The primary 2A / 2B cleavage of aphthoviruses and cardioviruses is mediated by 2A "cleavage" at its own C-terminus. In apthoviruses (such as foot-and-mouth disease virus (FMDV) and equine rhinitis A virus), the 2A region is a short stretch of approximately 18 amino acids that, together with the N-terminal residue of protein 2B (a conserved proline residue), represents 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 apthoviruses or cardioviruses, "picornavirus-like" insect viruses, type C rotaviruses, and repeat sequences within Trypanosoma spp. and bacterial sequences (Donnelly et al. (2001) supra).

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

[0506] vector The present invention also provides vectors or kits of vectors comprising one or more nucleic acid sequence(s) or nucleic acid construct(s) of the invention. Such vectors can be used to introduce the nucleic acid sequence(s) into host cells so as to express a cell surface receptor or receptor complex together with one or more proteins that confer a selective advantage on the host cells (i.e., effector immune cells) over target immune cells.

[0507] The vector kit may include: (i) a first vector comprising a nucleic acid sequence encoding a cell surface receptor or a portion of a cell surface receptor complex; and (ii) a second vector comprising a nucleic acid sequence that, when expressed in the cell, confers resistance to an immunosuppressant to said cell; and / or (iii) a third vector comprising a nucleic acid sequence encoding an immunoinhibitory molecule or a fusion protein comprising the extracellular domain of an immunoinhibitory 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 that has 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 an immunoinhibitory molecule or a fusion protein comprising the extracellular domain of an immunoinhibitory 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 beta-2 microglobulin linked to an intracellular signaling domain; and (ii) a second vector comprising a nucleic acid sequence encoding a variant calcineurin that has 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 an immunoinhibitory molecule or a fusion protein comprising the extracellular domain of an immunoinhibitory molecule.

[0510] The second embodiment vector kit may also include a vector comprising a nucleic acid sequence encoding a CAR.

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

[0512] The vector may be capable of transfecting or transducing cells such as T cells or NK cells.

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

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

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

[0516] T cells or T lymphocytes are a type of lymphocyte that play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes (such as B cells and natural killer cells (NK cells)) by the presence of a T cell receptor (TCR) on the cell surface. There are various types of T cells, as summarized below.

[0517] 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 THF) that secrete different cytokines to facilitate different types of immune responses.

[0518] Cytolytic T cells (TC cells, or CTLs) destroy virus-infected 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 is present on the surface of all nucleated cells. Through IL-10, adenosine, and other molecules secreted by regulatory T cells, CD8+ cells can be inactivated to an anergic state, thereby preventing autoimmune diseases such as experimental autoimmune myelitis.

[0519] Memory T cells are a subset of antigen-specific T cells that persist for extended periods after recovery from infection. Memory T cells rapidly become effector T cells in large numbers upon re-exposure to their cognate antigen, providing the immune system with a "memory" of past infection. Memory T cells include 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 primary role is to shut down T cell-mediated immunity toward the end of an immune response and to suppress autoreactive T cells that have escaped the negative selection process in the thymus.

[0521] Two main classes of CD4+ Treg cells have been described: endogenous and adaptive Treg cells.

[0522] Endogenous Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) arise in the thymus and are involved in the interaction of developing T cells with both myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells activated by TSLP. Endogenous Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutation of the FOXP3 gene blocks the development of regulatory T cells and can lead to the fatal 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 are part of the innate immune system. NK cells respond rapidly to innate signals from virus-infected cells in an MHC-dependent manner.

[0525] NK cells (belonging to the innate lymphoid cell group) are defined as large granular lymphocytes (LGLs) and constitute a third cell type differentiated from a common lymphoid progenitor cell that generates B 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 cell types described above.

[0527] The cells of the invention can be produced ex vivo from either 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 may be derived from ex vivo differentiation of inducible or embryonic progenitor cells, e.g., into T cells or NK cells. Alternatively, immortalized T cell lines may be used that retain lytic function and can act as therapeutic agents.

[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 with a viral vector, transfection with DNA or RNA.

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

[0531] The cells of the invention can be produced by: (i) isolation of a cell-containing sample from a subject or other source as listed above; and (ii) Transduction or transfection of cells with one or more nucleic acid sequence(s), nucleic acid construct(s), or vector(s) of the invention.

[0532] Cells can then be purified (eg, selected), for example, based on expression of one or more heterologous nucleic acid sequences.

[0533] Effector immune cells are capable of recognizing and killing target immune cells, which may be cytolytic immune cells (such as T cells or NK cells, as defined above).

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

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

[0536] Treatment method The invention provides methods of treating disease comprising administering to a subject cells of the invention (eg, in a pharmaceutical composition described above).

[0537] Methods of treating disease relate to therapeutic uses of the cells of the invention, wherein the cells can be administered to a subject already suffering from a disease or condition to alleviate, relieve, or ameliorate at least one symptom associated with the disease and / or to slow, reduce, or block the progression of the disease.

[0538] Methods for preventing disease relate to the prophylactic use of the cells of the present invention. Herein, such cells can be administered to a subject who is not yet afflicted and / or does not show any symptoms of the disease to prevent or impair the cause of the disease, or to reduce or prevent the occurrence of at least one symptom associated with the disease. The subject may have a predisposition to the disease or may be considered at risk of developing the disease.

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

[0540] The sample containing the cells may be isolated from a subject as described above or from another source.

[0541] The present invention also provides a method of treating a disease in a subject, comprising the steps of: (i) administering to a subject a pharmaceutical composition, wherein the pharmaceutical composition comprises a plurality of effector immune cells that have been engineered to be resistant to an immunosuppressant; and (ii) administering said immunosuppressant to said subject.

[0542] Effector immune cells may, for example, express a variant calcineurin 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 set forth as SEQ ID NO: 65; Calcineurin A comprising the mutations V314R and Y341F with reference to the sequence set forth as SEQ ID NO: 65; or Calcineurin B comprising the mutations L124T and K-125-LA-Ins with reference to the sequence set forth as SEQ ID NO: 66.

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

[0544] The effector cells may express dnCSK, and step (ii) may comprise administering to the subject an optional immunosuppressant (eg, rapamycin).

[0545] The present invention provides the cells of the present invention for use in the treatment and / or prevention of disease.

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

[0547] The disease to be treated by the method of the present invention may be a cancer disease (such as bladder cancer, breast cancer, colon cancer, endometrial cancer, kidney cancer (renal cell carcinoma), leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, and thyroid cancer).

[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, Waldenstrom's macroglobulinemia, isolated bone plasmacytoma, extramedullary plasmacytoma, osteosclerosing myeloma, heavy chain disease, monoclonal gammopathy of undetermined significance, or smoldering multiple myeloma.

[0550] The effector immune cells of the present invention are capable of killing target immune cells, which may be cancer cells or normal immune cells that are reactive against the effector immune cells.

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

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

[0553] Effector immune cells can be administered to patients before transplantation, after transplantation, or simultaneously with transplantation.For example, for organ transplantation, the effector T cells from organ donors that express the above-defined engineered MHC class I complex or MHC class II complex can be infused into recipients before transplantation to eliminate the alloreactive T cells that can mediate the rejection of the transplant.Alternatively, for HSCT, the recipient T cells that express the above-defined engineered MHC class I complex or MHC class II complex can be cultured with stem cell grafts before infusion to eliminate the 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 an allogeneic transplant, comprising contacting the allogeneic transplant with a plurality of effector immune cells having an engineered MHC class I complex or MHC class II complex as defined above.

[0555] Allogeneic transplantation can involve the adoptive transfer of allogeneic immune cells.

[0556] Also provided are allografts that have been depleted of alloreactive immune cells by the methods of the invention.Also provided are allografts that comprise effector immune cells of the invention.

[0557] Also provided are effector immune cells of the invention for use in: depletion of alloreactive immune cells from the immune cell population; To treat or prevent graft rejection after allogeneic transplantation; or Treatment or prevention of graft-versus-host disease (GVHD) associated with allogeneic transplantation.

[0558] Also provided is the use of an effector immune cell of the invention in the manufacture of a pharmaceutical composition for: depletion of alloreactive immune cells from the immune cell population; To treat or prevent graft rejection after allogeneic transplantation; or Treatment or prevention of graft-versus-host disease (GVHD) associated with allogeneic transplantation.

[0559] The present invention will now be further illustrated by examples which are intended to aid those skilled in the art in the practice of the invention and are not intended to limit the scope of the invention in any way. [Example]

[0560] Example 1 - Creation 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 the VH and VL domains shown as SEQ ID NOs: 7 and 8, respectively.

[0561] We created a truncated version of this CAR lacking the signaling domain, called dJOVI; dJOVI binds to TRBC1 on target cells but is unable to induce T cell activation and killing. PBMCs were transduced with vectors expressing dJOVI or full-length CAR (JOVI) along with the selection-suicide gene RQR8 described in WO 2013 / 153391. JOVI- or dJOVI-transduced PBMCs were cocultured with TRBC1+ target PBMCs at an effector:target ratio of 1:2, and viable transduced (RQR8+) T cells were counted 24 hours after coculture. The results are shown in Figure 12. Effector cell killing by TRBC1+ target cells was greater than that by dJOVI-transduced PBMCs, indicating that JOVI binding to TRBC1 on targets was sufficient to induce target T cell activation and reverse effector cell killing.

[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 deliver inhibitory immune signals on target T cell killing, PBMCs were transduced to express JOVI- or dJOVI- along 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] Cocultures were established using JOVI- or dJOVI-transduced PBMCs expressing dPDL1 or dPDL2 together with RQR8 and TRBC1+ target PBMCs expressing PD1 at an effector:target ratio of 1:1. Viable transduced (RQR8+) T cells were counted 72 hours after coculture, and each condition was normalized to its respective JOVI (or dJOVI) coculture. The results are shown in Figure 13. Compared to CAR alone, the recovery of transduced cells increased when dPDL1 or dPDL2 was expressed on the CAR.

[0564] Example 3 - Expression of calcineurin mutants by TRBC1-binding CAR-T cells reduces reverse killing of effector cells in the presence of calcineurin inhibitors JOVI-RQR8-transduced PBMCs expressing calcineurin mutants were cocultured with TRBC1+ target PBMCs at effector:target ratios of 1:1 and 1:4. Different concentrations of calcineurin inhibitors were added to the cocultures. Viable transduced (RQR8+) T cells were counted by flow cytometry 72 hours after coculture, and each condition was normalized to cocultures without the addition of inhibitors.

[0565] Example 4 - Expression of dnCSK by TRBC1-binding CAR-T cells reduces reverse killing of effector cells in the presence of immunosuppressants JOVI-RQR8-transduced PBMCs expressing dnCSK were cocultured with TRBC1+ target PBMCs at effector:target ratios of 1:1 and 1:4. Different concentrations of immunosuppressants were added to the cocultures. Viable transduced (RQR8+) T cells were counted by flow cytometry 72 hours after coculture, and each condition was normalized to cocultures without immunosuppressants.

[0566] Example 5 - Expression of PDL1 or PDL2 by effector T cells expressing β2m-CD3ζ reduces reverse killing by target cells To investigate the effect of engineering CAR-T cells to deliver inhibitory immune signals on the reverse killing of target T cells associated with anti-rejection killing responses, PBMCs were transduced to express JOVI-, dJOVI-, or an unrelated CAR along with a fusion protein consisting of truncated versions 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 were transduced to express full-length PD1 in the presence or absence of a superantigen (SAg) to ligate the enriched MHC to the TCR. Superantigens are not processed intracellularly. Instead, they bind to class II MHC molecules as intact molecules and outside the peptide antigen-accommodating groove. SAg molecules indiscriminately stimulate up to 20% of all T cells (compared to a typical response to antigen, which stimulates only 0.01% of T cells).

[0567] Cocultures of PBMCs transduced with JOVI-, dJOVI-, or an irrelevant CAR expressing dPDL1 or dPDL2 with β2m-CD3ζ and TRBC1+ target PBMCs expressing PD1+ SAg were set up at an effector:target ratio of 1:1. Viable transduced T cells were counted 72 hours after coculture, and each condition was normalized to its respective JOVI (or dJOVI) coculture.

[0568] Example 6 - Expression of calcineurin mutants by β2m-CD3ζ-expressing T cells reduces reverse killing of effector cells in the presence of calcineurin inhibitors PBMCs transduced with vectors encoding CARs (JOVI, dJOVI, or an unrelated CAR), β2m-CD3ζ, and calcineurin mutants were cocultured with TRBC1+ target PBMCs at effector:target ratios of 1:1 and 1:4. Different concentrations of calcineurin inhibitors and SAg were added to the cocultures. Viable transduced T cells were counted by flow cytometry 72 hours after coculture, and each condition was normalized to cocultures without inhibitors or SAg.

[0569] Example 7 - Expression of dnCSK by β2m-CD3ζ-expressing T cells reduces reverse killing of effector cells in the presence of immunosuppressants PBMCs transduced with vectors encoding CAR (JOVI, dJOVI, or an unrelated CAR), β2m-CD3ζ, and dnCSK were cocultured with TRBC1+ target PBMCs at effector:target ratios of 1:1 and 1:4. Different concentrations of immunosuppressants and SAg were added to the cocultures. Viable transduced T cells were counted by flow cytometry 72 hours after coculture, and each condition was normalized to cocultures without immunosuppressants or SAg.

[0570] Example 8 - Expression of calcineurin mutants by TRBC2-binding CAR-T cells confers resistance to growth inhibition by calcineurin inhibitors PBMCs were transduced with vectors expressing CARs together with the selection-suicide gene RQR8 described in WO2013 / 153391. The CARs tested are summarized below: CD19 CAR: A second-generation CAR containing an antigen-binding domain, hinge spacer, and 41BB / CD3z endodomain derived from Fmc63 TRBC1 CAR: A second-generation CAR having an antigen-binding domain, hinge spacer, and 41BB / CD3z endodomain as described in WO2018 / 224844 TRBC2 CAR: A second-generation CAR having an antigen-binding domain, a CD8 stalk spacer, and a CD28 / CD3z endodomain as 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 described 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 was replaced with the TRBC2 gene using CRISPR-Cas9 technology such that expression of TRBC2 is identical to expression of TRBC1 on wild-type cells.

[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 showed an increased percentage and number of proliferating cells after co-culture with TRBC1-expressing target cells; cells expressing the TRBC2 CAR showed an increased percentage and number of proliferating cells after co-culture with TRBC2-expressing target cells (Figure 16). In the presence of tacrolimus, CAR-T cell proliferation is inhibited, as seen by comparison of "TRBC2 CAR" in Figure 16B (without tacrolimus) and Figure 17B (with tacrolimus). Only cells co-expressing the TRBC2 CAR and the CnB30 calcineurin mutant increased the absolute number of transduced effector cells after co-culture with TRBC2-expressing targets (Fig. 17B), and this population also had the highest percentage of transduced proliferating cells (Fig. 17A).

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

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

[0576] Figure 22 shows the percentage of RQR8-expressing cells. The percentage of CD19-expressing cells remained constant, while the percentage of cells expressing TRBC2 CAR increased after co-culture with TRBC2+ targets in the absence of tacrolimus. This was true for cells expressing TRBC2 CAR alone or cells co-expressing TRBC2 CAR in combination with a calcineurin mutant. In the presence of tacrolimus, the percentage of RQR8+ cells expressing TRBC2 CAR alone decreased, indicating that tacrolimus inhibited the expansion of these cells. In contrast, the percentage of RQR8+ cells co-expressing TRBC2 CAR / CnB30 was identical to that in co-cultures without tacrolimus, indicating that these cells were resistant to calcineurin inhibition.

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

[0578] Three days after transduction, cells were either left untreated or treated with 20 ng / ml tacrolimus and expanded under these conditions for an additional four days. Seven days after transduction, killing assays were performed at effector:target ratios of 1:1 and 1:4, and the untransduced TRBC2+ fraction was labeled with Cell Trace Violet and used as autologous targets.

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

[0580] After expansion and co-culture in the presence of tacrolimus, target cell killing was improved in effector cell populations that co-expressed the TRBC2 CAR with the CnB30 calcineurin mutant compared to effector cell populations expressing the TRBC2 CAR alone (Figure 23). This effect was particularly pronounced when 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 could be detected (Figure 24, first graph). However, when CAR-expressing cells were co-cultured with TRBC2-expressing PBMCs at a 1:4 ratio, CAR T cell counts were close to zero (Figure 24, second graph), likely due to reverse killing of CAR-expressing cells by target cells.

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

[0583] Furthermore, cell populations co-expressing the TRBC2 CAR and the CnB30 calcineurin mutant exhibited increased T cell activation in terms of cytokine release after expansion and co-culture in the presence of tacrolimus compared to cell populations expressing the TRBC2 CAR alone, for both IFNγ (Figure 25) and IL-2 (Figure 26).

[0584] Together, these data indicate that expression of calcineurin mutants by CAR-expressing cells gives effector cells an advantage over target T cells and prevents reverse killing by the target cells.

[0585] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in 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 that expresses a cell surface receptor or receptor complex that specifically binds to an antigen-recognizing receptor on a target immune cell; wherein the effector immune cell has been engineered such that, upon formation of a synapse between the effector immune cell and the target immune cell, the effector immune cell has a greater ability to kill the target immune cell than the target immune cell. (Item 2) 2. The effector immune cell of item 1, which is engineered to be resistant to immunosuppressants. (Item 3) 3. The effector immune cell of item 2, which is engineered to be resistant to one or more calcineurin inhibitors. (Item 4) The effector immune cell of item 3, which expresses: Calcineurin A containing the mutations T351E and L354A with reference to the sequence set forth as SEQ ID NO: 65; Calcineurin A comprising the mutations V314R and Y341F with reference to the sequence set forth as SEQ ID NO: 65; or Calcineurin B comprising the mutations L124T and K-125-LA-Ins with reference to the sequence set forth as SEQ ID NO: 66. (Item 5) 3. The effector immune cell of item 2, which is engineered to be resistant to rapamycin. (Item 6) 3. The effector immune cell according to item 2, which expresses a dominant-negative C-terminal Src kinase (dnCSK). (Item 7) 2. The effector immune cell of item 1, which is engineered to express or overexpress an immunoinhibitory molecule or a fusion protein comprising the extracellular domain of an immunoinhibitory molecule. (Item 8) 8. The effector immune cell of item 7, wherein the immunoinhibitory molecule binds to PD-1, LAG3, TIM-3, TIGIT, BTLA, VISTA, CEACAM1-R, KIR2DL4, B7-H3, or B7-H4. (Item 9) 8. The effector immune cell according to item 7, wherein the immunoinhibitory molecule is selected from PD-L1, PD-L2, HVEM, CD155, VSIG-3, galectin-9, HLA-G, CEACAM-1, LSECTin, FGL1, B7-H3, and B7-H4. (Item 10) 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 membrane-localized domain of the immunoinhibitory molecule. (Item 11) 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 costimulatory endodomain of the immunoinhibitory molecule. (Item 12) Item 13. The effector immune cell according to Item 11, wherein the costimulatory endodomain comprises one or more endodomains selected from CD28, ICOS, CTLA4, 41BB, CD27, CD30, OX-40, TACI, CD2, CD27, and GITR. Item 14. The effector immune cell according to any one of the preceding items, wherein the antigen-recognizing receptor is a T cell receptor (TCR) or an activated killer cell immunoglobulin-like receptor (KAR). 10. The effector immune cell of any 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 of item 14, wherein the CAR binds to TCR beta constant region 1 (TRBC1) or TRBC2. (Item 16) 13. 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) 17. The effector immune cell of 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 (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 beta-2 microglobulin; "endo" is the intracellular signaling domain) 18. The effector immune cell of item 17, which is an engineered MHC class I complex comprising a molecule having the formula: (Item 19) 17. The effector immune cell of item 16, comprising an MHC class I polypeptide, an MHC class II polypeptide, or beta-2 microglobulin linked to a component of the TCR / CD3 complex. (Item 20) 20. The effector immune cell of item 19, comprising an MHC class I polypeptide linked to CD3-zeta, CD3-epsilon, CD3-gamma, or CD3-delta via a linker peptide; an MHC class II polypeptide; or beta-2 microglobulin. (Item 21) 17. The effector immune cell of 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 beta-2 microglobulin; and (ii) a second binding domain that binds to a component of the TCR / CD3 complex. (Item 22) 17. The effector immune cell of item 16, comprising a CD79 α chain and / or a CD79 β chain linked to an intracellular signaling domain. (Item 23) 17. The effector immune cell of 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) 17. The effector immune cell of item 16, comprising an engineered polypeptide comprising the MHC class II binding domain of CD4 or the MHC class I binding domain of CD8 linked to an intracellular signaling domain. (Item 25) A nucleic acid construct comprising: (i) a first nucleic acid sequence encoding a portion of a cell surface receptor or cell surface receptor complex as defined in any preceding item; and (ii) a second nucleic acid sequence that, when expressed in the cell, confers resistance to the immunosuppressant to the cell; and / or (iii) a third nucleic acid sequence encoding an immunoinhibitory molecule or a fusion protein comprising the extracellular domain of an immunoinhibitory molecule; A nucleic acid construct comprising: (Item 26) 26. A vector comprising the nucleic acid construct of item 25. (Item 27) A vector kit comprising: (i) a first vector comprising a nucleic acid sequence encoding a cell surface receptor or a portion 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 that, when expressed in a cell, confers resistance to the cell to an immunosuppressant; and / or (iii) a third vector comprising a nucleic acid sequence encoding an immunoinhibitory molecule or a fusion protein comprising the extracellular domain of an immunoinhibitory molecule. A vector kit comprising: (Item 28) 25. A pharmaceutical composition comprising a plurality of effector immune cells according to any one of items 1 to 24. (Item 29) 29. A pharmaceutical composition according to item 28 for use in the treatment of a disease. (Item 30) 30. A method for treating a disease, comprising administering to a subject the pharmaceutical composition of item 29. (Item 31) The following steps: (i) administering to a subject a pharmaceutical composition comprising a plurality of effector immune cells according to item 1 that have been engineered to be resistant to immunosuppressants; and (ii) administering the immunosuppressant to the subject. Item 31. The method according to Item 30, comprising: (Item 32) 25. Use of a plurality of effector immune cells according to 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 the pharmaceutical composition for the use according to item 32, wherein the disease is cancer. (Item 34) A method for producing the effector immune cell according to any one of Items 1 to 24, comprising the step of introducing the nucleic acid construct according to Item 25, the vector according to Item 26, or the vector kit according to Item 27 into the cell ex vivo. (Item 35) 25. 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 according to any one of items 16 to 24. (Item 36) 25. A method for treating or preventing graft rejection after allogeneic transplantation, comprising administering to a recipient subject for the allogeneic transplant a plurality of effector immune cells derived from a donor subject, 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) 25. A method for treating or preventing graft-versus-host disease (GVHD) associated with an allogeneic transplant, the method comprising contacting the allogeneic transplant with a plurality of effector immune cells according to any one of items 16 to 24. (Item 38) Item 39. The method of item 36 or 37, wherein the allogeneic transplantation comprises adoptive transfer of allogeneic immune cells. 36. An allograft in which alloreactive immune cells have been depleted by the method described in item 35.

Claims

[Claim 1] The invention described in the specification.

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