Length-matched chimeric antigen receptors and length-matched coreceptors for improved CAR T cell activation

Abstract

The present invention relates to optimizing the effector function of immune effector cells expressing a chimeric antigen receptor (CAR) co-localized with a coreceptor by ensuring that the intermembrane distance spanned by the CAR-antigen complex is similar to the intermembrane distance spanned by a coreceptor-ligand complex.

Description

[Technical Field]

[0001] The present invention relates to chimeric antigen receptors (CARs) and accessory receptors, and methods for optimizing effector function when the CARs and / or accessory receptors are expressed in immune effector cells. [Background technology]

[0002] T cells patrol the body in search of antigens derived from infectious organisms or cancer cells. They use their T cell antigen receptor (TCR) to recognize peptide antigens on the major histocompatibility complex (pMHC). T cells have exquisite antigen sensitivity and can be activated by recognizing only one pMHC. This high sensitivity is important because infectious organisms or cancers deploy evasive mechanisms to reduce the amount of antigen presented to T cells.

[0003] An exciting new treatment for cancer involves reprogramming a patient's T cells to target the cancer. This is done by using genetic engineering to express chimeric antigen receptors (CARs) on T cells. These CARs enable the patient's T cells to recognize and kill cancer cells. This therapy has been approved for treating B-cell cancers. However, many patients relapse with B cells expressing low levels of the target antigen. It is now clear that CARs have a significant defect in antigen sensitivity. CARs require 100 to 1000 times more antigen than TCRs to activate T cells. The mechanism responsible for this defect in CAR antigen sensitivity was unknown.

[0004] Many attempts have been made to improve CARs, focusing on the intracellular signaling domain of CARs and / or targeting multiple different antigens. We previously discovered that the defective sensitivity of CARs may be primarily due to their inability to utilize accessory receptors (1).

[0005] There is an urgent need to improve the sensitivity of CARs to prevent cancer recurrence, and more sensitive CARs should also enable the use of CAR T cells in the treatment of a wider range of cancers.

[0006] It is therefore an object of the present invention to provide further improved CARs and / or co-receptors, thereby improving CAR T-cell therapy. Summary of the Invention

[0007] The present inventors have discovered that optimal antigen recognition by CAR requires optimal alignment between the membrane of immune effector cells and the membrane of antigen-presenting cells (APCs). Membrane alignment is affected by the dimensions of receptor-ligand complexes, such as CARs and co-receptors, co-localized on immune effector cells, their respective antigens, and the ligands on APCs. Interestingly, the present inventors have discovered that antigen recognition by CARs is optimized when the intermembrane distance spanned by the complex of CAR and target antigen (CAR-antigen complex) is comparable to the intermembrane distance spanned by the complex of CAR and co-localized co-receptor and its ligand (co-ligand complex). Therefore, the present invention provides CARs and / or co-receptors of appropriate sizes for optimizing membrane alignment, and these CARs and co-receptors of the present invention are also referred to herein as variable-size CARs (vsCARs) and variable-size co-receptors, respectively.

[0008] Specifically, the inventors have demonstrated that adjusting the size of the extracellular portion of a CAR (and thus the height of the extracellular antigen-binding domain of the CAR) results in changes in antigen sensitivity, as evidenced by modulation of T cell effector function. Example 1 shows that CARs prepared according to conventional designs in the art, using a CD8a or CD28 hinge between the extracellular antigen-binding domain and the transmembrane domain, exhibited low antigen sensitivity. On the other hand, CARs with smaller hinges and therefore shorter extracellular antigen-binding domains from the membrane of immune effector cells compared to CARs prepared according to conventional designs, exhibited good antigen sensitivity comparable to T cell receptors (TCRs) and synthetic T cell receptors and antigen receptors (STARs), but higher than conventional CARs and epsilon T cell receptor fusion constructs (εTRuCs). This indicates that extracellular dimensions, rather than intracellular signaling, optimize antigen recognition. Furthermore, smaller CARs do not associate with the TCR-CD3 complex, which is an advantageous feature for clinical use.

[0009] The inventors have also shown that modulating the size of the extracellular portion of the coreceptor (and thus the height of the extracellular ligand-binding domain of the coreceptor (e.g., CD2) from the membrane of the immune effector cell) results in altered antigen sensitivity of the CAR, as demonstrated by modulation of T cell effector function. Example 2 shows that the antigen sensitivity of a CAR prepared according to conventional designs in the art was improved by increasing the height of the extracellular ligand-binding domain of the coreceptor (e.g., CD2). Thus, engineering the coreceptor-ligand complex by varying the dimensions of the extracellular portion maximizes the ability to potentiate the CAR-antigen complex.

[0010] As shown in Figure 1A, the coreceptor-ligand complex spans an intermembrane distance corresponding to the TCR-pMHC complex (e.g., 14 nm for the CD2-CD58 complex). However, this intermembrane distance is not sufficient for CARs prepared according to conventional designs in the art (CARs). CON, i.e., CARs comprising a CD28 hinge or a CD8a hinge) and are not optimized for the target antigen. The utility of the vsCARs and variable-sized coreceptors of the present invention is described below.

[0011] For example, as shown in Figure 1B, CAR CON If the intermembrane distance spanned by the complex between CAR and the target antigen is large compared to the intermembrane distance spanned by the coreceptor-ligand complex, CAR signaling will be weakened (Figure 1B, left). CON A smaller vsCAR can be used compared to the corresponding endogenous co-receptor, resulting in a vsCAR-antigen complex spanning the intermembrane distance (e.g., 14 nm for the CD2-CD58 complex (2,3)). Therefore, optimal membrane alignment is achieved (Figure 1B, right). Alternatively, optimal membrane alignment can be achieved by using a co-receptor of variable size (e.g., elongated CD2) that is larger than the corresponding endogenous co-receptor, as shown in Figure 1C, thereby improving antigen recognition. Therefore, co-receptor-ligand complexes of variable size can be used to enhance the CAR CON -Spans the intermembrane distance corresponding to the antigen complex.

[0012] As a further example, as shown in Figure 1D, in situations where optimizing membrane alignment using endogenous coreceptor-ligand (e.g., CD2-CD58) complexes is difficult because the target antigen is large and even the small size of the vsCAR may span too long a membrane distance to form a complex with the target antigen, the vsCAR may be combined with coreceptors of variable sizes to optimize membrane alignment, thereby improving antigen recognition and CAR signaling.

[0013] Coreceptors of variable size can be prepared by modifying the size of the stalk between the extracellular ligand-binding domain and the transmembrane domain, thereby determining the height of the extracellular ligand-binding domain of the coreceptor from the membrane of the immune effector cell. For example, as shown in Figure 1E, various sequences (also referred to herein as inflexible spacers) that physically increase the height of the ligand-binding domain from the membrane of the immune effector cell can be used. Thus, the present invention provides coreceptors comprising a stalk of variable size that controls the height of the extracellular ligand-binding domain of the coreceptor to optimize the effector function of the immune effector cell induced by the CAR, wherein the coreceptor and CAR are expressed on the immune effector cell.

[0014] The vsCAR can be prepared by modifying the size of the hinge between the antigen-binding domain and the transmembrane domain, thereby determining the height of the extracellular antigen-binding domain of the CAR from the membrane of the immune effector cell. For example, as shown in Figure 1F, various sequences (also referred to herein as inflexible spacers) that physically increase the height of the ligand-binding domain from the membrane of the immune effector cell can be used. Thus, the present invention provides CARs that contain variable-sized hinges to control the height of the extracellular antigen-binding domain of the CAR in order to optimize the effector function of the immune effector cell in which the CAR is expressed.

[0015] Furthermore, the present inventors have overcome a significant problem in the field of CAR technology. Specifically, CARs that have been introduced into the clinic and / or have been successful in clinical trials typically target membrane-proximal epitopes expressed on APCs compared to the antigenic site of the peptide-MHC complex. This allows such CARs to utilize endogenous co-receptors, such as the CD2-CD58 complex, to enhance T cell activation. The present inventors have now provided a mechanism by which CARs can successfully target membrane-distal antigens expressed on APCs (e.g., the peptide-MHC complex itself or larger antigens) while maintaining the effect achieved by co-receptors to enhance T cell activation.

[0016] Accordingly, the present invention provides an immune effector cell comprising: - a chimeric antigen receptor (CAR) capable of binding to an antigen on an antigen-presenting cell (APC), the CAR comprising a fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain and the transmembrane domain are linked by a hinge; and - a coreceptor capable of binding to a ligand on the APC, the coreceptor comprising an extracellular ligand-binding domain and a transmembrane domain, the extracellular ligand-binding domain and the transmembrane domain being connected by a stalk. Including, (a) the coreceptor is an exogenous coreceptor and the stalk comprises a sequence that physically increases the height of the extracellular ligand-binding domain from the membrane of the immune effector cell; (b) the hinge of the CAR comprises or consists of a sequence that physically increases the height of the antigen-binding domain from the membrane of the immune effector cell, the sequence comprising a mucin-like sequence, one or more folded polypeptide domains, or a fragment of the CD28 hinge set forth in SEQ ID NO: 10 or the CD8a hinge set forth in SEQ ID NO: 43; and / or (c) the CAR and the co-receptor are each sized such that the intermembrane distance spanned by the CAR-antigen complex is comparable to the intermembrane distance spanned by the co-receptor-ligand complex; Immune effector cells are provided.

[0017] The present invention also provides a chimeric antigen receptor (CAR) capable of binding to an antigen on an antigen-presenting cell (APC), the CAR comprising a fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain and the transmembrane domain are linked by a hinge, the hinge comprising or consisting of a sequence that physically increases the height of the extracellular antigen-binding domain from the membrane of the immune effector cell, the sequence comprising a mucin-like sequence, one or more folded polypeptide domains, or a fragment of the CD28 hinge set forth in SEQ ID NO: 10 or the CD8a hinge set forth in SEQ ID NO: 43.

[0018] The present invention also provides a coreceptor capable of binding to a ligand on an APC, comprising an extracellular ligand-binding domain and a transmembrane domain, said extracellular ligand-binding domain and said transmembrane domain being linked by a stalk, said stalk comprising or consisting of a sequence that physically increases the height of said ligand-binding domain from the membrane of said immune effector cell, and optionally said stalk comprising a mucin-like sequence.

[0019] The present invention also provides immune effector cells comprising or encoding a CAR and / or co-receptor of the present invention.

[0020] The present invention also provides a method of preparing immune effector cells, the method comprising introducing a nucleic acid encoding a CAR and / or a coreceptor of the present invention into an immune effector cell.

[0021] The present invention also provides a method for optimizing effector function of an immune effector cell, the immune effector cell comprising: - a chimeric antigen receptor (CAR) capable of binding to an antigen on an antigen-presenting cell (APC), the CAR comprising a fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain and the transmembrane domain are linked by a hinge; and - a coreceptor capable of binding to a ligand on the APC, the coreceptor comprising an extracellular ligand-binding domain and a transmembrane domain, the extracellular ligand-binding domain and the transmembrane domain being connected by a stalk. Including, The method comprises altering the height of the extracellular antigen binding domain of the CAR and / or the height of the extracellular ligand binding domain of the co-receptor from the membrane of the immune effector cell to optimize the effector function of the immune effector cell upon contact with the APC. A method is also provided.

[0022] The present invention also provides a method for identifying an improved immune effector cell, the immune effector cell comprising: - a chimeric antigen receptor (CAR) capable of binding to an antigen on an antigen-presenting cell (APC), the CAR comprising a fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain and the transmembrane domain are linked by a hinge; and - a coreceptor capable of binding to a ligand on the APC, the coreceptor comprising an extracellular ligand-binding domain and a transmembrane domain, the extracellular ligand-binding domain and the transmembrane domain being connected by a stalk. wherein the method comprises: (a) altering the height of the extracellular antigen-binding domain of the CAR and / or the height of the extracellular ligand-binding domain of the coreceptor from the membrane of the immune effector cell; and (b) determining whether immune effector cells expressing the modified CAR and / or modified coreceptor have improved effector function when compared to immune effector cells expressing the unmodified CAR and / or unmodified coreceptor; Also provided is a method, including:

[0023] The present invention also provides a method for identifying an improved immune effector cell, comprising determining whether an immune effector cell of the invention has improved effector function when compared to an immune effector cell expressing an unmodified CAR and / or an unmodified coreceptor.

[0024] The present invention also provides immune effector cells obtained or obtainable by the methods of the present invention.

[0025] The present invention also provides methods, such as ex vivo methods, for preparing populations of immune effector cells for adoptive cell therapy, comprising culturing immune effector cells of the invention to produce the population of immune effector cells.

[0026] The present invention also provides a population of immune effector cells produced by the method of the present invention.

[0027] The present invention also provides a method of treating cancer in a subject, comprising administering to said subject an effective amount of an immune effector cell or population of immune effector cells of the present invention.

[0028] The present invention also provides an immune effector cell or population of immune effector cells of the invention for use in a method of treating cancer in a subject.

[0029] The invention also provides the use of an immune effector cell or population of immune effector cells of the invention in the manufacture of a medicament for the treatment of cancer.

[0030] The present invention also provides the use of an immune effector cell or population of immune effector cells of the present invention to treat cancer.

[0031] The present invention also provides the use of an immune effector cell or population of immune effector cells of the present invention for adoptive cell therapy. [Brief explanation of the drawings]

[0032] [Figure 1-1]Schematic diagram of the mechanism of optimizing antigen sensitivity through membrane alignment. (A) A coreceptor-ligand complex (e.g., CD2-CD58) spans the intermembrane distance (e.g., approximately 14 nm) corresponding to TCR / pMHC binding. (B) In situations where the size of a standard CAR does not correspond to the size of the coreceptor-ligand complex (e.g., CD2-CD58) (left), an appropriately sized hinge can be selected and inserted into the CAR to match this distance (right). For example, a variable-sized CAR (vsCAR) can be used, such that the vsCAR-antigen complex spans the intermembrane distance corresponding to the coreceptor-ligand complex, thereby achieving optimal membrane alignment and improving antigen recognition. (C) Alternatively, a variable-sized coreceptor (e.g., extended CD2) can be used, such that the variable-sized coreceptor-ligand complex spans the intermembrane distance corresponding to a conventional CAR-antigen complex, thereby achieving optimal alignment of the conventional CAR with the target antigen and improving antigen recognition. (D) In ​​situations where optimizing membrane alignment using naturally occurring coreceptor-ligand (e.g., CD2-CD58) complexes is difficult because the target antigen is large and may span a membrane distance too long for even the vsCAR wild-type complex (left), vsCARs can be combined with coreceptors of variable sizes to optimize membrane alignment (right). (E) Design of coreceptors of variable sizes. These coreceptors can be varied in size using inflexible spacers consisting of various numbers of amino acid residues (e.g., 4, 8, 20, 40, or 234 amino acid residues) derived from CD43 (e.g., any of SEQ ID NOS: 50-62). See, for example, the extended CD2 variants described in SEQ ID NOS: 27-36. (F) Design of vsCARs. The shortest CARs contain "mini," "micro," "nano," or truncated hinges from therapeutic CD28 CARs (see, for example, SEQ ID NOS: 37, 41, and 42). The CAR can be varied in size using inflexible spacers consisting of various numbers of amino acid residues (e.g., 4, 8, 20, 40, or 234 amino acid residues) derived from CD43 (e.g., any of SEQ ID NOs: 50-62).See, for example, SEQ ID NOs: 38-40, which may also be applicable to hinges other than the mini-CD28 hinge. [Figure 1-2] Same as description for Figure 1-1. [Figure 1-3] Same as description for Figure 1-1. [Figure 1-4] Same as description for Figure 1-1. [Figure 2] Schematic diagram of the 1G4 T cell receptor ("TCR"), D52N CAR ("CAR"), D52N Fab CAR ("Fab CAR"), and IgG1 antibody. The 1G4 TCR comprises a variable alpha domain and a constant alpha domain, a P2A self-cleaving peptide, followed by a variable beta domain and a constant beta domain. The 1G4 TCR uses endogenous CD3 components. The D52N CAR is a CAR prepared according to a conventional design using a D52N single-chain variable fragment (scFv). D52N is an antigen-binding domain that recognizes the same peptide antigen recognized by the 1G4 TCR. Conventional designs include, for example, the D52N scFv, followed by a hinge and transmembrane domain (e.g., CD28 or CD8a), and an intracellular signaling domain (e.g., CD28 cytoplasmic domain and zeta chain (CD247) cytoplasmic domain). The D52N Fab CAR is a CAR of the present invention. It comprises the variable heavy domain of D52N scFv conjugated to IgG1-CH1 and the variable light domain of D52N scFv conjugated to IgG1-CL domain, each of which is conjugated to a CD28 transmembrane domain, a CD28 cytoplasmic domain, and a zeta chain (CD247) signaling tail. [Figure 3]Schematic diagram of antigen receptors: TCR(1G4), STAR(D52N), ε-TRuC(D52N), Fab CAR(D52N Fab-28z), and CAR(D52N-CD28-28z). TCR(1G4), Fab CAR(D52N Fab-28z), and CAR(D52N-CD28-28z) are as illustrated in Figure 2. All of these antigen receptors recognize the same peptide antigen (SLLMWITQV; SEQ ID NO: 66) presented on HLA-A*02:01. In the case of the TCR, the variable domain of the 1G4 TCR is used, while in the case of the other receptors tested, the D52N variable domain from the 3M4E5 antibody is used (4, 5). [Figure 4] Surface CD3ε and surface antigen receptor expression profiles on TCRα-β-Jurkat cells transduced with 1G4 TCR, D52N-CD28-28z CAR, and D52N-Fab CAR-28z receptor. Staining was performed on E6.1 TCRα-β-Jurkat cells lentivirally transduced with the indicated constructs. pMHC antigen tetramers detect the surface levels of each antigen receptor (y-axis), and UCHT antibodies detect surface CD3ε levels. CAR and Fab CAR constructs were detected on the cell surface without upregulation of CD3ε, indicating that these receptors are CD3-independent, which is not the case for 1G4 TCR. [Figure 5] Expression profiles of TCR(1G4), STAR(D52N), ε-TRuC(D52N), D52N-Fab CAR, and D52N-CD28-28z CAR. Staining was performed on primary human CD8+ T cells subjected to antibiotic selection after lentiviral transduction of the corresponding receptors. [Figure 6-1]Activation of primary CD8+ T cells expressing various antigen receptors was assessed by flow cytometry. T cells were incubated with T2 target cell lines loaded with various concentrations of NY-ESO-1 9V peptide antigen for 20 hours, after which surface activation markers (A) 4-1BB and (B) CD25 were assessed. (C) EC50 values ​​from three independent donors. EC50 is the antigen concentration required to elicit 50% of the maximal response. Statistical analysis was performed on log-transformed EC50 values ​​for 4-1BB and CD25 readings using Dunnett's multiple comparison test (ns = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001). [Figure 6-2] Same as description for Figure 6-1. [Figure 6-3] Same as description for Figure 6-1. [Figure 7-1] Primary CD8+ T cell activation expressing various antigen receptors was assessed by cytokine production. T cells were incubated with T2 target cell lines loaded with various concentrations of NY-ESO-1 9V peptide antigen for 20 hours, after which cytokines IFN-γ (top row) and IL-2 (bottom row) were assessed by ELISA. Data are shown from three independent donors. [Figure 7-2] Same as description for Figure 7-1. [Figure 7-3] Same as description for Figure 7-1. [Figure 8-1] Varying the hinge size can improve the antigen sensitivity of CARs. (A) Schematic of a conventional CAR with a CD28 hinge (SEQ ID NO: 10) and a panel of CARs of varying sizes with the indicated hinges. The CD28 minihinge contains a smaller hinge compared to the conventional CAR (SEQ ID NO: 37). Larger hinges are generated by including spacers of the indicated sizes from CD43 (see SEQ ID NOs: 38-40). (B) Primary human CD8+ T cells expressing the indicated antigen receptors are co-cultured with Nalm6 target cells pulsed with the indicated concentrations of peptide antigens. Surface expression of the 4-1BB activation marker is measured 5 hours later. (C) EC50s matched to the indicated antigen receptors. [Figure 8-2] Same as description for Figure 8-1. [Figure 9-1] Schematic diagram of the mechanism for optimizing antigen sensitivity through membrane alignment using variable-sized coreceptors. (A) Design of variable-sized coreceptors. In this study, the ligand-binding domain of the coreceptor is the extracellular domain of CD2, extended by a fragment of the mucin-like sequence of the extracellular portion of CD43. The transmembrane and intracellular domains are obtained from wild-type CD2. (B) Schematic diagram of the experimental system. Jurkat TCRα-β- T cells, which do not express CD8, were subjected to CRISPR to remove endogenous CD2. Jurkat TCRα-β- CD2- cells were transduced with antigen receptors, and then transduced with CD2 WT or various variable-sized coreceptors using the design in (A). [Figure 9-2] Same as description for Figure 9-1. [Figure 10-1] Wild-type CD2 optimizes antigen sensitivity of the T cell receptor. (A) Schematic of the antigen receptor. (B, C) Surface CD69 on T cells co-cultured with U87 target cell line pulsed with the indicated concentrations of peptide antigen. Representative dose response (B) and EC50 (C) from multiple experiments. Arrows indicate CD2 that optimizes antigen sensitivity. [Figure 10-2] Same as description for Figure 10-1. [Figure 11-1] Wild-type CD2 optimizes antigen sensitivity of STAR. (A) Schematic of the antigen receptor. (B, C) Surface CD69 on T cells co-cultured with U87 target cell line pulsed with the indicated concentrations of peptide antigen. Representative dose response (B) and EC50 (C) from multiple experiments. Arrows indicate CD2 that optimizes antigen sensitivity. [Figure 11-2] Same as description for Figure 11-1. [Figure 12-1]CD2-CD43(40) optimizes antigen sensitivity of D52N-CD8a-z CAR. (A) Schematic of the antigen receptor. (B, C) Surface CD69 on T cells co-cultured with U87 target cell line pulsed with the indicated concentrations of peptide antigen. Representative dose response (B) and EC50 (C) from multiple experiments. Arrow indicates CD2, which optimizes antigen sensitivity. [Figure 12-2] Same as description for Figure 12-1. [Figure 13-1] CD2-CD43 (20) optimizes antigen sensitivity of the D52N-CD28-28z CAR. (A) Schematic of the antigen receptor. (B, C) Surface CD69 on T cells co-cultured with U87 target cell line pulsed with the indicated concentrations of peptide antigen. Representative dose response (B) and EC50 (C) from multiple experiments. Arrow indicates CD2, which optimizes antigen sensitivity. [Figure 13-2] Same as description for Figure 13-1. [Figure 14-1] CD2-CD43 (20) optimizes antigen sensitivity of ε-TRuC(D52N). (A) Schematic of the antigen receptor. (B, C) Surface CD69 on T cells cocultured with U87 target cell line pulsed with the indicated concentrations of peptide antigen. Representative dose response (B) and EC50 (C) from multiple experiments. Arrow indicates CD2, which optimizes antigen sensitivity. [Figure 14-2] Same as description for Figure 14-1. [Figure 15] Stretching the CD2-CD58 complex reduces antigen recognition by the TCR (top) but improves antigen recognition by the CAR (bottom) based on suboptimal and optimal membrane alignment, respectively. [Figure 16-1]A. Jurkat T cells were transduced with the FM63-CD8a-41BBz (Kymriah) chimeric antigen receptor. Surface levels were detected with GFP-Spycatcher fused to Spytag-CD19. B. Kymriah-expressing Jurkat T cells were transduced with CD2 molecules of variable size and sorted for matched expression using anti-CD2 PE. C. Nalm6 CombiCells expressing Spycatcher were conjugated with various concentrations of purified Spytag-CD19 and detected by flow cytometry. D. Representative dose-response of Kymriah CAR Jurkat T cells containing CD2 molecules of the indicated variable sizes. Summary of antigen sensitivity (EC50) measurements from EN=3 independent experiments. A t-test was used to determine the p-value for the null hypothesis that EC50 is identical between CD2 WT and other CD2 conditions, with a Sidak-Holm correction for multiple comparisons. Abbreviations: * = p-value ≤ 0.05, ** = p-value ≤ 0.01, *** = p-value ≤ 0.01. [Figure 16-2] Same as description for Figure 16-1. [Figure 16-3] Same as description for Figure 16-1. [Figure 16-4] Same as description for Figure 16-1. [Figure 16-5] Same as description for Figure 16-1. DETAILED DESCRIPTION OF THE INVENTION

[0033] Detailed Description of the Invention Chimeric antigen receptor (CAR) The immune effector cells of the present invention comprise a chimeric antigen receptor (CAR), a non-naturally occurring protein that comprises an extracellular portion that comprises an antigen-specific antigen-binding domain linked via a hinge and transmembrane domain to an intracellular portion that comprises one or more signaling moieties.

[0034] The immune effector cells of the invention can comprise a CAR of the invention. In this embodiment, the immune effector cells can also comprise a coreceptor of the invention.

[0035] The immune effector cells of the present invention can be prepared according to conventional designs, such as those described in Reference 6 (herein referred to as conventional CARs or CARs). CON (also referred to as a CAR) wherein the CAR comprises a CD28 hinge set forth in SEQ ID NO: 10 or a CD8a hinge set forth in SEQ ID NO: 43. In this embodiment, the immune effector cell also comprises a coreceptor of the invention.

[0036] The CAR of the present invention has improved antigen sensitivity compared to conventional CARs. The CAR of the present invention is sized so that the intermembrane distance spanned by the CAR-antigen complex is comparable to the intermembrane distance spanned by the co-receptor-ligand complex. The size (e.g., height) of the extracellular portion of the CAR, specifically the height of the extracellular antigen-binding domain of the CAR from the membrane of the immune effector cell, is important in determining antigen sensitivity. The optimal size (e.g., height) of the extracellular portion of the CAR, specifically the optimal height of the extracellular antigen-binding domain of the CAR from the membrane of the immune effector cell, depends on the size (e.g., height) of the target antigen, co-receptor, and its ligand, as further described below.

[0037] A CAR of the invention may comprise an extracellular antigen-binding domain that has a height that is similar in size to (e.g., within 5% or 10%, or identical to) the height of the extracellular antigen-binding domain of a T cell receptor from the membrane of an immune effector cell.

[0038] The extracellular antigen-binding domain of the CAR of the invention can be approximately 7 nm in height from the membrane of the immune effector cell.

[0039] The CAR of the invention can comprise an extracellular antigen-binding domain that has a height that is higher than the extracellular antigen-binding domain of the T cell receptor from the membrane of the immune effector cell, for example, no more than 15%, no more than 20%, no more than 25%, or no more than 30% higher.

[0040] The extracellular antigen-binding domain of the CAR of the invention can be greater than about 7 nm but less than about 47 nm in height from the membrane of the immune effector cell. The extracellular antigen-binding domain of the CAR of the invention can be greater than 7 nm, 10 nm or more, 15 nm or more, 20 nm or more, 25 nm or more, 30 nm or more, 35 nm or more, 40 nm or more, or 45 nm or more in height from the membrane of the immune effector cell. The extracellular antigen-binding domain of the CAR of the invention can be 45 nm or less, 40 nm or less, 35 nm or less, 30 nm or less, 25 nm or less, 20 nm or less, 15 nm or less, or 10 nm or less in height from the membrane of the immune effector cell.

[0041] The CAR of the invention can comprise an extracellular antigen-binding domain that has a height that is lower than the extracellular antigen-binding domain of the T cell receptor from the membrane of the immune effector cell, for example, no more than 15%, no more than 20%, no more than 25%, or no more than 30% lower.

[0042] The extracellular antigen-binding domain of the CAR of the present invention may be greater than about 3 nm but less than about 7 nm in height from the membrane of the immune effector cell. The extracellular antigen-binding domain of the CAR of the present invention may be greater than 3 nm, greater than 4 nm, greater than 5 nm, or greater than 6 nm in height from the membrane of the immune effector cell. The extracellular antigen-binding domain of the CAR of the present invention may be less than 7 nm, less than 6 nm, less than 5 nm, or less than 4 nm in height from the membrane of the immune effector cell.

[0043] The height of the extracellular antigen-binding domain of a CAR of the invention from the membrane of an immune effector cell can be determined according to routine methods in the art, as described herein.

[0044] The hinge between the antigen-binding domain and the transmembrane domain can determine the size (e.g., height) of the extracellular portion of the CAR of the present invention, specifically the height of the extracellular antigen-binding domain. The present inventors have discovered that conventional CARs have low antigen sensitivity. This can be improved by adjusting (e.g., increasing or decreasing) the size of the hinge of the CAR, allowing for optimal membrane alignment.

[0045] Thus, the CARs of the invention comprise hinges of different sizes compared to conventional CARs, and as a result, the CARs of the invention may have shorter or longer extracellular portions compared to conventional CARs, and as a result, may have lower or higher extracellular antigen-binding domains compared to conventional CARs.

[0046] The CAR of the present invention does not comprise a hinge consisting of (a) the CD8a hinge set forth in SEQ ID NO:43, or (b) the CD28 hinge set forth in SEQ ID NO:10.

[0047] The hinge of the CAR of the invention comprises or consists of a sequence that physically increases the height of the antigen-binding domain from the membrane of the immune effector cell.

[0048] The sequence can be inflexible, e.g., each block of amino acid residues in the sequence physically increases the height of the antigen-binding domain from the membrane of the immune effector cell because the sequence does not compress or fold back on itself. The block can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid residues.

[0049] The sequence may be a bulky sequence that physically increases the height of the antigen-binding domain from the membrane of the immune effector cell, e.g., by steric hindrance, thereby preventing the antigen-binding domain from contacting the membrane.

[0050] The sequence may comprise or consist of a mucin-like sequence, one or more folded domains, or a fragment of the CD28 or CD8a hinge set forth in SEQ ID NOs: 10 and 43, respectively. The mucin-like peptide may be a fragment of the extracellular domain of CD43, as further described below. The one or more folded domains may be one or more domains having an immunoglobulin fold, such as an immunoglobulin constant domain or FNIII, as further described below.

[0051] The sequence that physically increases the height of the antigen-binding domain from the membrane of the immune effector cell can be of any suitable length. The sequence can be at least one amino acid long, e.g., at least 5, at least 10, or at least 20 amino acids long. The sequence can be 250 or fewer amino acids long, 200 or fewer amino acids long, 150 or fewer amino acids long, or 100 or fewer amino acids long, e.g., 80 or fewer, 60 or fewer, 40 or fewer, 30 or fewer, or 20 or fewer amino acids long. The sequence can be 1 to 40 amino acids long, e.g., 2 to 30, 3 to 25, 4 to 20, or 5 to 15 amino acids long.

[0052] The sequence that physically increases the height of the antigen-binding domain from the membrane of the immune effector cell can be an immunoglobulin domain, e.g., an immunoglobulin constant domain such as the CH2 and CH3 regions of IgG1, IgG2, IgG3, IgG4, CD8 (e.g., CD8α hinge), or CD28. CON In comparison, the CAR of the present invention may have the CD28 or CD8a hinge replaced with an immunoglobulin constant domain that is smaller in size compared to the hinge.

[0053] In some cases, the sequence that physically increases the height of the antigen-binding domain from the membrane of the immune effector cell is not an immunoglobulin domain.

[0054] The sequence that physically increases the height of the antigen-binding domain from the membrane of an immune effector cell can be a fragment of the CD28 hinge set forth in SEQ ID NO: 10. The fragment preferably retains the cysteine ​​residue at position 29 of SEQ ID NO: 10. Alternatively, the fragment can include a modification that removes the cysteine ​​residue at position 29 of SEQ ID NO: 10, e.g., by substitution. For example, the fragment can consist of 20 or fewer contiguous amino acids from the C-terminus of SEQ ID NO: 10, or 30 or fewer contiguous amino acids from the N-terminus of SEQ ID NO: 10. The fragment can consist of 5 or fewer, 10 or fewer, 15 or fewer, 20 or fewer, 25 or fewer, or 30 or fewer contiguous amino acids from SEQ ID NO: 10, and includes a cysteine ​​residue at position 29 of SEQ ID NO: 10. The fragment can consist of a sequence set forth in any one of SEQ ID NOs: 37, 41, or 42. For example, a CAR comprising the full length of SEQ ID NO: 10 can be prepared. CON In comparison, the CAR of the present invention may have a shorter CD28 hinge.

[0055] The sequence that physically increases the height of the antigen-binding domain from the membrane of an immune effector cell can be a fragment of the CD8a hinge set forth in SEQ ID NO:43. The fragment preferably retains the cysteine ​​residue at position 27 of SEQ ID NO:43. Alternatively, the fragment can include a modification that removes the cysteine ​​residue at position 27 of SEQ ID NO:43, e.g., by substitution. For example, the fragment can consist of no more than 30 or no more than 25 consecutive amino acids from the C-terminus of SEQ ID NO:43, or no more than 30 consecutive amino acids from the N-terminus of SEQ ID NO:43. The fragment can consist of no more than 5, no more than 10, no more than 15, no more than 20, no more than 25, or no more than 30 consecutive amino acids from SEQ ID NO:43, and includes a cysteine ​​residue at position 27 of SEQ ID NO:43. The fragment can consist of any one of the sequences set forth in SEQ ID NOs:46, 63, and 64. For example, compared to a conventional CAR comprising the full length of SEQ ID NO:43, the CAR of the present invention can have a shorter CD8a hinge.

[0056] A CAR of the invention can comprise one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten) immunoglobulin domains (e.g., immunoglobulin constant domains). A CAR of the invention can comprise ten or fewer immunoglobulin constant domains. Multiple immunoglobulin domains can be coupled in series or in parallel, e.g., when a CAR of the invention comprises multiple polypeptides, each polypeptide can comprise one or more immunoglobulin domains, e.g., two or more, three or more, four or more, or five or more immunoglobulin domains.

[0057] The sequence that physically increases the height of the antigen-binding domain from the membrane of immune effector cells can be a mucin-like sequence, or a fragment or derivative thereof. Mucin-like sequences are characterized by being rich in serine and threonine residues in proteins, and these residues are highly O-glycosylated. The mucin-like sequence can be the mucin-like sequence of the extracellular portion of CD43 (i.e., the mucin-like sequence set forth in SEQ ID NO: 62). Mucin-like sequences from other proteins, such as MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC13, MUC15, MUC16, MUC17, MUC18, MUC20, MUC21, and PSGL-1, can also be used. The stalks of mucin-like surface proteins, such as CD8a and CD28, can also be used.

[0058] The mucin-like sequence can be a fragment of the extracellular domain of CD43, as set forth in SEQ ID NO: 62. For example, the fragment can be 4 to 234 amino acids (e.g., as shown in Figure 1F). The fragment can consist of 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 50 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or more, 180 or more, 190 or more, 200 or more, 210 or more, 220 or more, or 230 or more consecutive amino acids from the N-terminus or C-terminus of SEQ ID NO: 62. The fragment can consist of 234 amino acids or less. The fragment can be 4 to 120 amino acids, 8 to 80 amino acids, or 20 to 40 amino acids. The fragment may be any of SEQ ID NOs: 53-62.

[0059] The CAR of the present invention can be a dimer, such as a heterodimer. For example, the CAR can comprise two fusion proteins that form a dimer, and for each fusion protein, an antigen-binding domain can be conjugated to a folded polypeptide domain (e.g., an immunoglobulin constant domain), followed by a transmembrane domain (e.g., a CD28 transmembrane domain) and an intracellular signaling domain (e.g., a CD28 zeta chain signaling domain). For example, the first fusion protein can comprise a light chain variable domain as the antigen-binding domain and a light chain constant domain as the folded polypeptide domain, and the second fusion protein can comprise a heavy chain variable domain as the antigen-binding domain and a heavy chain constant domain as the folded polypeptide domain. Such a CAR can essentially resemble an antibody Fab conjugated to a transmembrane domain (e.g., a CD28 transmembrane domain) and an intracellular signaling domain (e.g., a CD28 zeta chain signaling domain). Thus, the extracellular portion of such a CAR contains two immunoglobulin constant domains (one from each chain) and two immunoglobulin light chains (one from each chain), comparable in size (e.g., height) to a T cell receptor. For example, the CAR may be a Fab CAR, as shown in Figure 2. This allows immune effector cells containing such a CAR to strongly interact with antigen-presenting APCs, resulting in a CAR-antigen complex that spans a membrane distance of approximately 14 nm.

[0060] The antigen-binding domain of the CAR of the present invention can be an scFv, a monoclonal antibody (comprising two heavy chains and two light chains), a polyclonal antibody, Fab, Fab', F(ab')2 fragment, a heavy chain variable domain (VH), or a nanobody (VHH). The ScFv domain comprises the heavy chain variable domain (VH) and the light chain variable domain (VL) of an immunoglobulin, connected by a short linker peptide.

[0061] The CAR of the present invention can comprise multiple extracellular antigen-binding domains, for example, two or three extracellular antigen-binding domains. The two or more extracellular antigen-binding domains can bind to different antigens, i.e., the CAR can be bispecific or multispecific.

[0062] The antigen-binding domain of the CAR of the invention can be specific for any antigen, such as those listed in Table 2. The antigen can be a peptide-MHC complex, CD19, mesothelin, BCMA, CD22, EGFR, or EGFRvIII. For example, the peptide of the peptide-MHC complex can be a NY-ESO-1 peptide, i.e., a peptide derived from proteolytic cleavage of NY-ESO-1.

[0063] The transmembrane domain of the CAR of the present invention can be derived from a naturally occurring transmembrane protein, such as a type I transmembrane protein. This transmembrane domain spans the cell membrane, for example, the cell membrane of a eukaryotic cell. This transmembrane domain plays a role in transmitting an activation signal to the cytoplasmic signaling domain after ligand binding of the extracellular antigen-binding domain (e.g., scFv). The transmembrane domain of the CAR of the present invention is typically the transmembrane domain of CD28. This transmembrane domain can be the transmembrane domain of the α, β, δ, or γ subunit of the T cell receptor, CD3ε, CD3ζ, CD4, CD6, CD8α, CD28, CD86, OX-40, 4-1BB, or CD40L (CD154). This transmembrane domain can be the transmembrane domain of CD8, for example, when the immune effector cell is an NK cell.

[0064] The intracellular signaling domain of the CAR of the present invention can include any activation domain known in the art. This activation domain plays a role in activating immune effector cells after binding of the extracellular domain (e.g., scFv). The intracellular signaling domain of the CAR of the present invention can include one or more of the following: CD3ζ (zeta) activation domain, 4-1BB (CD137) activation domain, CD3ε (epsilon) activation domain, OX40 (CD134) activation domain, CD28 activation domain, and / or CD27 activation domain.

[0065] This intracellular signaling domain may comprise a CD3ζ (zeta) activation domain, as in the first generation of CARs in the art.

[0066] This intracellular signaling domain may comprise a CD3ζ (zeta) activation domain and a CD28 activation domain (which may also be referred to as a CD28z domain), as is the case for second generation CARs in the art.

[0067] This intracellular signaling domain may include a 4-1BB activation domain and a CD3ζ (zeta) activation domain (which may also be referred to as a 4-1BBz domain), for example, as in the case of third generation CARs in the art.

[0068] The intracellular signaling domain may include the 4-1BBz and CD28z domains, which include the CD3 zeta, CD28, and 4-1BB activation domains.

[0069] The intracellular signaling domain may comprise a CD3ζ (zeta) activation domain, alone or in combination with a CD28, CD27, OX-40 (CD134), and / or 4-1BB (CD137) domain.

[0070] Typically, this intracellular signaling domain does not include a CD2 domain, such as a CD2 intracellular signaling domain.

[0071] Other activation domains include IL-15Rα, CD2, CDS, ICAM-1, LTA-1, and ICOS, which may be used in combination with the above activation domains.

[0072] When the immune effector cell in which the CAR is expressed is a phagocyte, the intracellular signaling domain can include the intracellular domain of Megf10 or FcRv.

[0073] The CARs of the present invention can be expressed on the surface of immune effector cells independently of the endogenous TCR-CD3 complex. The CARs of the present invention may not require endogenous TCR and / or CD3 for expression on the surface of immune effector cells.

[0074] Coreceptors The immune effector cells of the present invention contain a co-receptor, which co-localizes with the CAR on the cell membrane at the immunological synapse with the antigen-presenting cell (APC). The co-receptor is capable of binding to a ligand on the antigen-presenting cell and contains an extracellular ligand-binding domain, a transmembrane domain, and an intracellular signaling domain.

[0075] The immune effector cells of the present invention can comprise a coreceptor of the present invention that is an engineered coreceptor. In this embodiment, the immune effector cells can also comprise a CAR of the present invention.

[0076] The coreceptors of the present invention provide improved antigen sensitivity of CARs expressed on the same immune effector cells compared to the corresponding endogenous coreceptors. The coreceptors of the present invention are sized so that the intermembrane distance spanned by the coreceptor-ligand complex is comparable to the intermembrane distance spanned by the CAR-antigen complex. Therefore, the size (e.g., height) of the extracellular portion of the coreceptor, specifically the height of the extracellular ligand-binding domain of the coreceptor from the membrane of the immune effector cell, is important in determining the antigen sensitivity of CARs expressed on the same immune effector cells. The optimal size (e.g., height) of the extracellular portion of the coreceptor, specifically the optimal height of the extracellular ligand-binding domain of the coreceptor from the membrane of the immune effector cell, depends on the size (e.g., height) of its ligand, CAR, and target antigen, as further described below.

[0077] A coreceptor of the present invention can comprise an extracellular ligand-binding domain that has a height that is greater than that of the extracellular ligand-binding domain of the corresponding endogenous coreceptor from the membrane of the immune effector cell, e.g., no greater than 15%, no greater than 20%, no greater than 25%, or no greater than 30% greater.

[0078] The extracellular ligand-binding domain of a coreceptor of the invention can be greater than about 7 nm but less than about 47 nm above the membrane of an immune effector cell. The extracellular ligand-binding domain of a coreceptor of the invention can be greater than 7 nm, 10 nm or more, 15 nm or more, 20 nm or more, 25 nm or more, 30 nm or more, 35 nm or more, 40 nm or more, or 45 nm or more above the membrane of an immune effector cell. The extracellular ligand-binding domain of a coreceptor of the invention can be 45 nm or less, 40 nm or less, 35 nm or less, 30 nm or less, 25 nm or less, 20 nm or less, 15 nm or less, or 10 nm or less above the membrane of an immune effector cell.

[0079] The coreceptors of the present invention may comprise an extracellular ligand-binding domain that is shorter than the extracellular ligand-binding domain of the corresponding endogenous coreceptor from the membrane of the immune effector cell, e.g., no more than 15%, no more than 20%, no more than 25%, or no more than 30% shorter.

[0080] The extracellular ligand-binding domain of a coreceptor of the invention can be greater than about 3 nm but less than about 7 nm in height from the membrane of an immune effector cell. The extracellular ligand-binding domain of a coreceptor of the invention can be 3 nm or more, 4 nm or more, 5 nm or more, or 6 nm or more in height from the membrane of an immune effector cell. The extracellular ligand-binding domain of a coreceptor of the invention can be less than 7 nm, 6 nm or less, 5 nm or less, or 4 nm or less in height from the membrane of an immune effector cell.

[0081] The height of the extracellular ligand-binding domain of a coreceptor of the invention from the membrane of an immune effector cell can be determined according to routine methods in the art, as described herein.

[0082] The stalk between the ligand-binding domain and the transmembrane domain can determine the size (e.g., height) of the extracellular portion of the coreceptor of the present invention, specifically the height of the extracellular ligand-binding domain. The present inventors have discovered that conventional CARs have low antigen sensitivity. This can be improved by adjusting (e.g., increasing or decreasing) the size of the stalk of the coreceptor (e.g., CD2), allowing for optimal membrane alignment of the conventional CAR.

[0083] Thus, the coreceptors of the present invention comprise a stalk that differs in size compared to the corresponding endogenous coreceptor, such that the CARs of the present invention may have a shorter or longer extracellular portion compared to the corresponding endogenous coreceptor, and consequently, a shorter or longer extracellular ligand-binding domain compared to the corresponding endogenous coreceptor.

[0084] The stalk of the coreceptor of the present invention comprises or consists of a sequence that physically increases the height of the ligand binding domain from the membrane of the immune effector cell.

[0085] The sequence can be inflexible, e.g., each block of residues in the sequence physically increases the height of the ligand-binding domain from the membrane of the immune effector cell because the sequence does not compress or fold back on itself. The block can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more residues.

[0086] The sequence can be a bulky sequence that physically increases the height of the ligand-binding domain from the membrane of the immune effector cell, e.g., by steric hindrance, thereby preventing the ligand-binding domain from contacting the membrane. The sequence that physically increases the height of the ligand-binding domain from the membrane of the immune effector cell can be as described above with respect to the hinge of the CAR of the invention.

[0087] The sequence that physically increases the height of the ligand-binding domain from the membrane of immune effector cells can be a mucin-like sequence, or a fragment or derivative thereof. Mucin-like sequences are characterized by being rich in serine and threonine residues in proteins, and the serine and threonine residues are highly O-glycosylated. The mucin-like sequence can be the mucin-like sequence of the extracellular portion of CD43 set forth in SEQ ID NO: 62. Mucin-like sequences from other proteins, such as MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC13, MUC15, MUC16, MUC17, MUC18, MUC20, MUC21, and PSGL-1, can also be used. The stalks of mucin-like surface proteins, such as CD8a and CD28, can also be used.

[0088] The mucin-like sequence can be a fragment of the extracellular domain of CD43, as set forth in SEQ ID NO: 62. For example, the fragment can be 4 to 234 amino acids (e.g., as shown in Figure 1E). The fragment can consist of 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or more, 180 or more, 190 or more, 200 or more, 210 or more, 220 or more, or 230 or more consecutive amino acids from SEQ ID NO: 62 (e.g., from the N-terminus or C-terminus of SEQ ID NO: 62). The fragment can consist of 234 amino acids or less. The fragment may be 4 to 120 amino acids, 8 to 80 amino acids, or 20 to 40 amino acids. The fragment may be any of SEQ ID NOs: 53 to 62.

[0089] In some cases, the sequence that physically increases the height of the ligand binding domain from the membrane of the immune effector cell is not an immunoglobulin domain.

[0090] The co-receptor can be any of the receptors listed in Table 1. Thus, the ligand can be CD2, L-selectin, alpha 4 integrin, LFA-1, CD28, PD-1, 4-1BB, or CD6. The co-receptor can be an adhesion receptor. For example, the co-receptor can be CD2.

[0091] [Table 1]

[0092] Thus, the ligand can be CD58, GLYCAM1, VCAM-1, ICAM-1, ICAM-2, ICAM-3, CD80, CD86, PD-L1, PD-L2, 4-1BBL, or CD166. The ligand can be CD58. Typically, the ligand on the APC is different from the antigen on the APC.

[0093] The co-receptor may comprise the extracellular ligand-binding domain of CD2 and the ligand may be CD58. The co-receptor may be CD2 modified according to the present invention and the ligand may be CD58.

[0094] Membrane alignment and intermembrane distance Membrane alignment is influenced by the dimensions of the receptor-ligand complex, such as the CAR and co-receptor, co-localized on the immune effector cell, and their respective antigens, as well as the ligand on the APC. The membrane alignment for antigen recognition by the CAR is optimized when the intermembrane distance spanned by the complex between the CAR and the target antigen is comparable to the intermembrane distance spanned by the complex between a particular co-receptor and its ligand.

[0095] The intermembrane distance spanned by the complex of the CAR and the target antigen is similar to (e.g., within 5%, 10%, 15%, 20%, 25%, or 30% of, or the same as) the intermembrane distance spanned by the complex of the co-receptor and the ligand.

[0096] The intermembrane distance spanned by the complex of a CAR of the present invention and a target antigen can be similar to (e.g., within 5%, 10%, 15%, 20%, 25%, or 30% of, or the same as) the intermembrane distance spanned by the complex of a T cell receptor and its peptide-MHC antigen.

[0097] The intermembrane distance spanned by the complex of the CAR of the present invention and its antigen can be approximately 6 nm to 14 nm. The intermembrane distance spanned by the complex of the CAR of the present invention and its antigen can be 6 nm or more, 7 nm or more, 8 nm or more, 9 nm or more, 10 nm or more, 11 nm or more, 12 nm or more, or 13 nm or more. The intermembrane distance spanned by the complex of the CAR of the present invention and its antigen can be 14 nm or less, 13 nm or less, 12 nm or less, 11 nm or less, 10 nm or less, 9 nm or less, 8 nm or less, or 7 nm or less.

[0098] The intermembrane distance spanned by the complex of the CAR of the present invention and its antigen can be approximately 14 nm to 54 nm. The intermembrane distance spanned by the complex of the CAR of the present invention and its antigen can be 15 nm or more, 20 nm or more, 25 nm or more, 30 nm or more, 35 nm or more, 40 nm or more, 45 nm or more, or 50 nm or more. The intermembrane distance spanned by the complex of the CAR of the present invention and its antigen can be 50 nm or less, 45 nm or less, 40 nm or less, 35 nm or less, 30 nm or less, 25 nm or less, 20 nm or less, or 15 nm or less.

[0099] Similarly, the intermembrane distance spanned by a complex of a coreceptor and its ligand of the present invention may be approximately 14 nm to 54 nm. The intermembrane distance spanned by a complex of a coreceptor and its ligand of the present invention may be 15 nm or more, 20 nm or more, 25 nm or more, 30 nm or more, 35 nm or more, 40 nm or more, 45 nm or more, or 50 nm or more. The intermembrane distance spanned by a complex of a coreceptor and its ligand of the present invention may be 50 nm or less, 45 nm or less, 40 nm or less, 35 nm or less, 30 nm or less, 25 nm or less, 20 nm or less, or 15 nm or less.

[0100] In one embodiment, the immune effector cells of the present invention comprise a CAR of the present invention (e.g., FIG. 1B), and the intermembrane distance spanned by the complex of the CAR of the present invention and the target antigen is comparable to (e.g., within 5%, 10%, 15%, 20%, 25%, or 30% of, or the same as) the intermembrane distance spanned by the complex of an endogenous co-receptor and its ligand. CAR CARs of the invention having an extracellular antigen-binding domain of 100 nm (100 nm) are coupled to a target cell of interest (CAR) using a CAR-specific antibody against a target cell of interest (CAR-C). The CAR-specific antibody is coupled to ... ant ) can be determined based on h CAR is, h CAR =xh ant may be comparable to (e.g., within 5%, 10%, 15%, 20%, 25%, or 30% of, or identical to) the value determined by CAR a, xh ant Examples of CARs of the invention having an extracellular antigen-binding domain at an appropriate height from the membrane of the immune effector cell are provided herein.

[0101] The immune effector cells of the present invention are CAR CON (i.e., a CAR comprising a CD28 hinge or a CD8a hinge) and a co-receptor of the invention (e.g., FIG. 1C), a CAR CON The intermembrane distance spanned by the complex of the co-receptor and the target antigen is comparable to (e.g., within 5%, 10%, 15%, 20%, 25%, or 30% of, or the same as) the intermembrane distance spanned by the complex of the co-receptor and its ligand of the present invention. acc ) is a co-receptor of the present invention having an extracellular ligand-binding domain of CAR CON The intermembrane distance (y) spanned by the complex of the ligand and the target antigen, and the height of the epitope of the ligand (h lig ) can be determined by h acc is, h acc =yhlig may be comparable to (e.g., within 5%, 10%, 15%, 20%, 25%, or 30% of, or identical to) the value determined by acc , h acc =yh lig Examples of coreceptors of the invention having an extracellular ligand-binding domain at an appropriate height from the membrane of the immune effector cell are provided herein.

[0102] In embodiments in which the immune effector cell comprises a CAR of the invention and a coreceptor of the invention (e.g., FIG. 1D), the intermembrane distance spanned by the complex of the CAR of the invention and the target antigen is comparable (e.g., within 5%, 10%, 15%, 20%, 25%, or 30% of, or the same) to the intermembrane distance spanned by the complex of the coreceptor of the invention and its ligand. CAR CARs of the invention having an extracellular antigen-binding domain of about 100 nm (h), and an appropriate height (h) from the membrane of immune effector cells. acc The coreceptor of the present invention having an extracellular ligand-binding domain of about 100 nm in height (h ant ) and the height of the ligand epitope (h lig ) can be determined by taking into account, for example, h CAR +h ant =h acc +h lig The appropriate height (h CAR ) and examples of co-receptors of the invention having an extracellular ligand-binding domain at an appropriate height from the membrane of the immune effector cell are provided herein.

[0103] Thus, the present invention also relates to methods for determining the appropriate size (e.g., height) of the extracellular antigen-binding domain of a CAR of the present invention and / or the appropriate size (e.g., height) of the extracellular ligand-binding domain of a coreceptor of the present invention.

[0104] The intermembrane distance, which is the height of the extracellular antigen-binding domain or extracellular ligand-binding domain from the membrane of an immune effector cell of any of the membrane proteins described herein, can be determined according to conventional methods in the art. For example, (1) using theoretical methods such as those used herein; (2) predicting the overall size using existing structural information, such as from protein data banks; (3) using bioinformatics prediction tools based on the amino acid sequence of the protein (such as Alphafold, which converts the sequence into a structure and can estimate the distance from this structure); or (4) using microscopy methods such as electron microscopy (7), immunofluorescence, or quantum dot labeling. For example, the intermembrane distance spanned by the CD2-CD58 complex was determined in References 3 and 6. Theoretical methods, as used herein, involve adding the size to the known height of the extracellular portion of the protein, for example, adding 3.7 nm for each immunoglobulin domain and / or 0.2 nm for each additional inflexible amino acid (based on experimental data from Reference 8). Electron microscopy can be used to measure the intermembrane distance directly. For example, T cells are incubated with APC that presents high concentrations of antigen to TCR or CAR.Direct intermembrane distance is measured at several points at the contact interface (see references 7 and 9).Indirect methods can also be used, such as comparing the activity of CAR of the present invention with the activity of TCR (see, for example, the following examples).

[0105] Immune effector cells The immune effector cells of the invention comprise a CAR and / or a coreceptor of the invention. The immune cells express the CAR and / or coreceptor of the invention.

[0106] Immune effector cells of the invention can comprise a CAR of the invention. The CAR of the invention can co-localize with an endogenous coreceptor (e.g., endogenous CD2) on the surface of the immune effector cell at the immunological synapse (e.g., Figure 1B).

[0107] The immune effector cells of the invention can comprise a coreceptor of the invention. The coreceptor of the invention binds to a CAR on the surface of the immune effector cell at the immunological synapse. CON (i.e., having the CD28 hinge set forth in SEQ ID NO: 10 or the CD8a hinge set forth in SEQ ID NO: 43) (e.g., FIG. 1C).

[0108] Immune effector cells of the invention can comprise a CAR of the invention and a coreceptor of the invention. The CAR of the invention can co-localize with the coreceptor of the invention on the surface of the immune effector cell at the immunological synapse (e.g., Figure ID).

[0109] The immune effector cells of the present invention can be T cells, γδ T cells, natural killer (NK) cells, NKT cells, induced pluripotent stem cell (iPSC)-derived NK cells (iPSC-NK), phagocytes, or macrophages.

[0110] The immune effector cells can be T cells. The T cells can be CD8+ T cells or cytotoxic T cells. The T cells can be CD4-CD8+ T cells.

[0111] The T cells can be CD4+ T cells or helper T cells (TH cells), such as TH1, TH2, TH3, TH17, TH9, or T FH cells. The T cells can be regulatory T cells (Tregs). The T cells can be naive, effector, memory, effector memory, central memory, or memory stem T cells. The T cells can be peripheral lymphocytes.

[0112] The T cells may be expanded from PBMCs. The T cells may be autologous to the subject to be administered. The T cells may be allogeneic to the subject to be administered. The T cells may be partially HLA-mismatched to the subject to be administered.

[0113] The NK cells may be cells of the NK92 cell line. The NK cells may be isolated from plasma mononuclear cells (PBMCs) of the subject to be treated or a healthy donor. The NK cells may be isolated from umbilical cord blood. The NK cells may be isolated from CD34 + They can be differentiated from hematopoietic progenitor cells (HPCs).

[0114] Macrophages can be differentiated into an "M1" phenotype. M1 macrophages express proinflammatory cytokines and have potent anti-tumor activity. Undifferentiated macrophages expressing a CAR described herein can be induced to differentiate into an M1 phenotype by culturing in the presence of an antigen.

[0115] Sources of cells for use in accordance with the present invention are known to those of skill in the art and include, for example, peripheral blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, the cells are derived from whole blood.

[0116] The immune effector cells of the present invention can be derived from autologous cells. The immune effector cells can be derived from allogeneic cells. The term "autologous" refers to any material derived from an individual that is later reintroduced into the same individual. The term "allogeneic" refers to any material derived from another individual of the same species as the individual to whom it is introduced. Two or more individuals are said to be allogeneic to one another if their genes at one or more loci are not identical. In some embodiments, allogeneic material from individuals of the same species can be sufficiently genetically distinct to interact antigenically.

[0117] The immune effector cells may comprise a nucleic acid described herein. The immune effector cells may comprise a vector described herein. The immune effector cells may comprise an RNA or RNA vector described herein.

[0118] The immune effector cells can express CARs specific for one or more antigens.

[0119] The immune effector cells may persistently or transiently express the CAR and / or accessory proteins of the present invention.

[0120] The immune effector cells can include genomic modifications to reduce or eliminate the expression of endogenous co-receptors. The endogenous co-receptors can be any of the co-receptors described herein (e.g., Table 1). The endogenous co-receptor can be an adhesion receptor such as CD2. Genetic modification techniques for such modifications are known in the art.

[0121] The present invention also provides immune effector cells obtained or obtainable by any of the methods described herein.

[0122] The present invention also relates to methods for preparing immune effector cells of the invention or populations of immune effector cells of the invention, comprising introducing nucleic acid encoding a CAR and / or coreceptor of the invention into immune effector cells, e.g., by transformation (e.g., transfection or transduction).

[0123] The term "transduction" may be used to describe virally mediated nucleic acid transfer. Viral vectors may be used to introduce one or more constructs into cells. Conventional viral-based expression systems may include retroviral, alpharetroviral, lentiviral, adenoviral, adeno-associated (AAV), and herpes simplex virus (HSV) vectors for gene transfer. Non-viral transduction vectors include transposon-based systems, including PiggyBac and Sleeping Beauty systems. Methods for producing and purifying such vectors are known in the art. The vector is preferably a vector described herein. Immune effector cells may be transduced using any method known in the art. Transduction may be in vitro or ex vivo.

[0124] The term "transfection" can be used to describe non-viral-mediated nucleic acid transfer. Immune effector cells can be transfected using any method known in the art. Transfection can be in vitro or ex vivo. Any vector capable of transfecting immune effector cells can be used, for example, conventional plasmid DNA or RNA transfection, preferably mRNA transfection. Human artificial chromosomes and / or naked RNA can be used to transfect cells with nucleic acid sequences or nucleic acid constructs. Human artificial chromosomes are described, for example, in Kazuki et al., Mol. Ther. 19(9): 1591-1601 (2011) and Kouprina et al., Expert Opinion on Drug Delivery 11(4): 517-535 (2014). Alternative non-viral delivery systems include DNA plasmids, naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes. Non-viral methods for delivery of nucleic acids include lipofection, microinjection, biolistic bombardment, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, naked RNA, artificial virions, and drug-enhanced DNA uptake.

[0125] The immune effector cells may be transfected with nucleic acid sequences using nanoparticle delivery systems, including, but not limited to, lipid-based systems, liposomes, micelles, microvesicles, and exosomes. For nanoparticles capable of delivering RNA, see, e.g., Alabi et al., Proc Natl Acad Sci US A. 2013 Aug 6;110(32):12881-6; Zhang et al., Adv Mater. 2013 Sep 6;25(33):4641-5; Jiang et al., Nano Lett. 2013 Mar 13;13(3):1059-64; Karagiannis et al., ACS Nano. 2012 Oct 23;6(10):8484-7; Whitehead et al., ACS Nano. 2012 Aug 28;6(8):6922-9; and Lee et al., Nat Nanotechnol. 2012 Jun 3;7(6):389-93. Lipid nanoparticles, spherical nucleic acid (SNA™) constructs, nanoplexes, and other nanoparticles (particularly gold nanoparticles) are also contemplated as delivery vehicles for the nucleic acids or vectors of the invention.

[0126] The immune effector cells can be transfected by electroporation, which is mRNA electroporation, which has the advantage of allowing transient expression of the CAR and / or co-receptor.

[0127] The present invention also relates to methods (e.g., ex vivo methods) for preparing a population of immune effector cells (e.g., for adoptive cell therapy), comprising culturing immune effector cells of the invention to produce a population of immune effector cells. The present invention also provides populations of immune effector cells obtained or obtainable by any of the methods described herein. The present invention also provides populations of immune effector cells expressing a CAR and / or co-receptor of the invention.

[0128] The immune effector cells induce effector functions in the antigen-presenting cells they contact. The effector functions can be immune effector cell activation (e.g., T cell activation), immune effector cell proliferation (e.g., T cell proliferation), cytolytic activity (e.g., APC apoptosis or cell death), arrest or reduction of cell proliferation (e.g., of APCs), and / or modulation of cytokine release (e.g., release of proinflammatory cytokines). Methods for measuring effector functions are known in the art. Immune effector cell activation can be measured by increased surface expression of 4-1BB and / or CD25 and / or increased expression of CD69. Cytolytic activity can be measured by target cell death and / or release of perforin and / or granzymes. Modulation of cytokine release can be increased release of proinflammatory cytokines. The proinflammatory cytokines can be selected from GM-CSF, TNF-α, IL-2, IL-6, IL-1β, and / or IFN-γ.

[0129] Surface expression of 4-1-BB and / or CD25 can be measured as a normalized percentage of maximum surface expression, as shown in FIGS.

[0130] The release of pro-inflammatory cytokines may be measured as shown in Figure 7. Preferably, the cytokines IL-2 and / or IFN-γ are measured.

[0131] CD69 expression can be measured as the percentage of cells positive for CD69, as shown in Figures 10-14.

[0132] Immune effector cells expressing the CAR of the present invention and / or the coreceptor of the present invention may have improved or optimized effector function when compared with immune effector cells expressing a conventional CAR containing the same antigen-binding domain. Improved effector function is observed when immune effector cell activation is increased, immune effector cell proliferation is increased, cytolytic activity is increased, APC proliferation is slowed or stopped, pro-inflammatory cytokine release is increased, and / or anti-inflammatory cytokine release is reduced. Effector function is measured using EC for comparison, as shown in the figure. 50 The effector function can be measured as an increase of 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, or 200% or more. The effector function is optimal when altering the size of the CAR and / or coreceptor does not further improve the effector function. The effector function can be considered optimized when it is within ±25% (e.g., ±20%, ±15%, ±10%, or ±5%) of the optimal effector function.

[0133] antigen presenting cells Any antigen-presenting cell (APC) is useful in the present invention. The APC can be a professional APC, such as a dendritic cell, macrophage, B cell, or epithelial cell. The APC can be a non-professional APC, such as a fibroblast, thymic epithelial cell, thyroid epithelial cell, glial cell, pancreatic beta cell, or vascular endothelial cell. The APC can be a tumor cell, such as a tumor cell of a tumor listed in Table 2.

[0134] The APC comprises on its cell surface an antigen capable of binding to the antigen-binding domain of a CAR, such as a CAR of the invention. The antigen can be any antigen, such as a tumor antigen (e.g., a tumor-associated antigen, a development tumor antigen, and / or a neoantigen) listed in Table 2. The antigen can be a peptide-MHC complex, CD19, mesothelin, BCMA, CD22, EGFR, EGFRvIII, or NY-ESO-1.

[0135] [Table 2-1]

[0136] [Table 2-2]

[0137] Nucleic acids, vectors, and host cells Also provided are one or more isolated nucleic acids encoding the CAR and / or coreceptor of the invention, which in some cases are present in multiple nucleic acids that, when combined, are capable of collectively encoding the CAR and / or coreceptor of the invention.

[0138] Nucleic acids encoding the CAR and / or coreceptor of the invention can be obtained by methods known to those skilled in the art. For example, DNA sequences encoding part or all of the heavy and light chains of an antibody can be synthesized from the corresponding amino acid sequences, as desired.

[0139] The nucleic acid may be a DNA sequence. The nucleic acid may be an RNA sequence, such as an mRNA. A vector may comprise the nucleic acid.

[0140] This vector can be a viral vector. Conventional viral expression systems may include retrovirus, alpharetrovirus, lentivirus, adenovirus, adeno-associated (AAV) and herpes simplex virus (HSV) vectors for gene transfer. Non-viral transduction vectors include transposon-based systems, including PiggyBac and Sleeping Beauty systems. Methods for producing and purifying such vectors are known in the art.

[0141] The vector may be a cloning vector or an expression vector. A suitable vector may be any vector that is capable of carrying a sufficient amount of genetic information and allowing the expression of the polypeptide of the present invention.

[0142] This vector is preferably an RNA vector.Suitable RNA vectors include those described in Schutsky, Keith, et al., Oncotarget 6.30 (2015): 28911 and Beatty, Gregory L., et al., Gastroenterology 155.1 (2018): 29-32.

[0143] General methods for constructing vectors, transfection methods, and culture methods are known to those skilled in the art, see, in this regard, "Current Protocols in Molecular Biology", 1999, FM Ausubel (ed), Wiley Interscience, New York, and the Maniatis Manual produced by Cold Spring Harbor Publishing.

[0144] The nucleic acid may be provided in the form of an expression cassette comprising a control sequence operably linked to the inserted sequence, thereby allowing for the expression of the CAR and / or coreceptor of the present invention in vivo. Accordingly, one or more expression cassettes encoding one or more nucleic acids encoding the CAR and / or coreceptor of the present invention are also provided. The expression cassette is further typically provided within a vector (e.g., a plasmid or a recombinant viral vector). Accordingly, vectors encoding the CAR and / or coreceptor of the present invention are also provided. Further provided are multiple vectors encoding the CAR and / or coreceptor of the present invention collectively.

[0145] This vector can be a human artificial chromosome, as described, for example, in Kazuki et al., Mol. Ther. 19(9): 1591-1601 (2011) and Kouprina et al., Expert Opinion on Drug Delivery 11(4): 517-535 (2014).

[0146] The vector can be a DNA plasmid, naked nucleic acid (eg, naked RNA), and non-viral delivery systems, such as nucleic acid complexed with a delivery vehicle such as a liposome.

[0147] The nucleic acids, expression cassettes, or vectors described herein can be introduced into host cells, for example, by transfection. Accordingly, host cells containing one or more nucleic acids, expression cassettes, or vectors of the present invention are also provided. The nucleic acids, expression cassettes, or vectors described herein can be transiently or permanently introduced into host cells, thereby allowing expression of antibodies from one or more nucleic acids, expression cassettes, or vectors. Such host cells include transient or preferably stable higher eukaryotic cell lines (e.g., mammalian cells or insect cells), lower eukaryotic cells (e.g., yeast), or prokaryotic cells (e.g., bacterial cells). Specific examples of cells include mammalian HEK293, e.g., HEK293F, HEK293T, HEK293S, or HEK Expi293F, CHO, HeLa, NS0, and COS cells, or any other cell line used herein.

[0148] Typically, the host cell is an immune effector cell of the invention. The nucleic acids, expression cassettes, or vectors described herein can be transiently introduced into the host cell.

[0149] The polynucleotide or vector of the present invention can be mRNA for administration to patients (for example, mRNA vaccination).The patient's T cells can then express the CAR of the present invention and / or the co-receptor of the present invention in vivo.Such mRNA molecules and related methods are described in reference 10.

[0150] Also provided are kits suitable for transforming and / or transfecting immune effector cells or populations of immune effector cells to generate the immune effector cells or populations of immune effector cells of the present invention. The kits include the nucleic acids or vectors described herein. The kits may include additional agents (e.g., those discussed herein) that improve the efficacy of transfection or transformation.

[0151] method Also provided herein is a method for increasing and / or optimizing the effector function of an immune effector cell, comprising altering the size (e.g., height) of the extracellular portion of the CAR and / or the size (e.g., height) of the extracellular portion of the coreceptor to optimize the effector function of the immune effector cell upon contact with an APC. Specifically, the height of the extracellular antigen-binding domain of the CAR and / or the extracellular ligand-binding domain of the coreceptor is altered. The present invention also provides immune effector cells obtained or obtainable by this method. The effector function can be any effector function described herein.

[0152] Also provided herein is a method for identifying improved immune effector cells, the method comprising altering the size (e.g., height) of the extracellular portion of a CAR and / or the size (e.g., height) of the extracellular portion of a coreceptor, thereby altering the height of the extracellular antigen-binding domain of the CAR and / or the extracellular ligand-binding domain of the coreceptor, and determining whether immune effector cells expressing the modified CAR and / or modified coreceptor have improved effector function compared to immune effector cells expressing an unmodified CAR and / or unmodified coreceptor. The present invention also provides immune effector cells obtained or obtainable by this method.

[0153] The CAR can be a CAR described herein.

[0154] The co-receptor can be a co-receptor described herein. The co-receptor can be CD2.

[0155] The methods of the invention can involve modifying the size of the extracellular portion of the CAR and / or the size of the extracellular portion of the coreceptor, and thus the height of the extracellular antigen-binding domain of the CAR and / or the extracellular ligand-binding domain of the coreceptor, so that the intermembrane distance spanned by the complex of the CAR and the antigen is similar (e.g., within 5%, 10%, 15%, 20%, 25%, or 30% of, or the same) to the intermembrane distance spanned by the complex of the coreceptor and the ligand. The spanned intermembrane distance can be similar (e.g., within 5%, 10%, 15%, 20%, 25%, or 30% of, or the same) to the intermembrane distance spanned by the complex of the T cell receptor and the antigen.

[0156] The methods of the invention can include reducing the size (e.g., height) of the extracellular portion of the CAR, thereby reducing the height of the extracellular antigen-binding domain of the CAR.

[0157] The methods of the invention can include increasing the size (e.g., height) of the extracellular portion of the CAR, thereby increasing the height of the extracellular antigen-binding domain of the CAR from the membrane of the immune effector cell.

[0158] The methods of the invention can include reducing the size (eg, height) of the extracellular portion of the coreceptor, thereby reducing the extracellular ligand-binding domain of the coreceptor from the membrane of the immune effector cell.

[0159] The methods of the invention can include increasing the size (eg, height) of the extracellular portion of a coreceptor, thereby increasing the extracellular ligand-binding domain of the coreceptor from the membrane of the immune effector cell.

[0160] Methods of the invention can include increasing the size (e.g., height) of the extracellular portion of the CAR and decreasing the size (e.g., height) of the coreceptor, thereby increasing the height of the extracellular antigen-binding domain of the CAR from the membrane of the immune effector cell and decreasing the height of the extracellular ligand-binding domain of the coreceptor from the membrane of the immune effector cell.

[0161] Methods of the invention can include reducing the size (e.g., height) of the extracellular portion of the CAR and increasing the size (e.g., height) of the coreceptor, thereby reducing the height of the extracellular antigen-binding domain of the CAR from the membrane of the immune effector cell and increasing the height of the extracellular ligand-binding domain of the coreceptor from the membrane of the immune effector cell.

[0162] Determining the intermembrane distance to be spanned and the appropriate size (e.g., height) of the extracellular portion of a CAR and / or coreceptor of the invention, e.g., the height of the extracellular antigen-binding domain of a CAR and / or coreceptor of the invention from the membrane of an immune effector cell, is described herein.

[0163] The methods of the invention can include introducing a sequence in the extracellular portion of the CAR and / or coreceptor (e.g., the hinge of a CAR or the stalk of a coreceptor) that physically increases the height of the antigen-binding domain above the membrane of an immune effector cell. Suitable sequences that physically increase the height of the antigen-binding domain above the membrane of an immune effector cell are described herein.

[0164] The methods of the invention can include replacing the hinge of a CAR or the stalk of a coreceptor with a sequence that physically increases the height of the antigen-binding domain above the membrane of an immune effector cell. Suitable sequences that physically increase the height of the antigen-binding domain above the membrane of an immune effector cell are described herein.

[0165] The present invention also provides a method for identifying an improved immune effector cell, comprising determining whether the immune effector cell of the present invention has improved effector function when compared to an immune effector cell expressing a corresponding unmodified CAR and / or a corresponding unmodified receptor.

[0166] The methods of the invention can further include determining the level of effector function of immune effector cells, such as cell killing or cytokine production. Such methods are known in the art, as described herein.

[0167] Pharmaceutical Composition Also provided are compositions comprising immune effector cells or populations of immune effector cells of the invention. The immune effector cells or populations of immune effector cells can represent at least 1% of the total cells in the composition, e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% of the total cells in the composition. All cells in the composition can consist of or essentially consist of immune effector cells or populations of immune effector cells of the invention, i.e., no other cells are detectable in the composition.

[0168] The composition may be a pharmaceutical composition. The pharmaceutical composition may contain a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include aqueous carriers, diluents, or excipients. Examples of suitable carriers include: all aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, and solutes that make the composition isotonic with the blood of the intended recipient; aqueous and non-aqueous sterile suspensions which may contain suspending agents and thickening agents, dispersion media, antifungal and antibacterial agents, isotonic agents, and absorbents, etc. It will be understood that the compositions of the present invention may also contain other supplementary physiologically active agents.

[0169] This carrier is typically pharmaceutically "acceptable" in the sense that it is compatible with other components in this composition and is not harmful to the subject.Compositions include those suitable for parenteral administration, such as subcutaneous administration, intramuscular administration, intravenous administration and intradermal administration.This composition can be conveniently presented in unit dosage form and can be prepared by any method known in the field of pharmacy.Such method includes preparing carrier for association with isolated T cells.Generally, this composition is prepared by uniformly and intimately combining any active ingredient with liquid carrier.

[0170] This composition can be suitable for parenteral administration.In another embodiment, this composition is suitable for intravenous administration.The composition suitable for parenteral administration includes aqueous and non-aqueous isotonic sterile injection solution, which can contain antioxidant, buffer, bactericide and solute, and this composition is isotonic with the blood of intended recipient; and aqueous and non-aqueous sterile suspension, which can contain suspending agent and thickening agent.

[0171] The compositions described herein can be prepared by methods known in the art and are suitable for parenteral administration to mammals, particularly humans, comprising a therapeutically effective amount of the composition and one or more pharmaceutically acceptable carriers or diluents. The compositions contain at least about 1 x 10 6 ~Approx. 1×10 12The composition may comprise at least about 1 x 10 immune effector cells of the invention. 7 At least about 1 x 10 pieces 8 At least about 1 x 10 pieces 9 At least about 1 x 10 pieces 10 pieces, or at least about 1 x 10 11 The immune effector cells of the present invention may comprise:

[0172] The present disclosure also contemplates combinations of the compositions described herein with other active agents, and / or in addition to other treatment regimens or therapies, such as radiation therapy or surgery. When the compositions described herein are used in combination with known active agents, the combination may be administered sequentially (either consecutively or separated by periods of no treatment), simultaneously, or as an admixture.

[0173] Suitable anti-cancer agents will be known to those skilled in the art.

[0174] Combination treatment is also intended to include either treatment with a composition of the invention followed by a known treatment, or treatment with a known agent followed by treatment with a composition of the invention (e.g., as maintenance therapy).

[0175] For example, in the treatment of cancer, it is contemplated that the compositions of the invention may be administered in combination with alkylating agents (e.g., mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide cisplatin, or platinum-containing alkylating agents such as cisplatin, carboplatin, and oxaliplain), and antimetabolites (e.g., purine or pyrimidine analogs, or antifolates such as azathioprine and mercapto purines), anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, mitoxantrone, or anthracycline analogs), plant alkaloids (e.g., vinca alkaloids, or taxanes, e.g., vincristine, vinblastine, vinorelbine, vindesine, paclitaxel, or dotaxel), topoisomerase inhibitors (e.g., type I or type II topoisomerase inhibitors), podophyllotoxin, anticoagulants (e.g., etoposide or teniposide), tyrosine kinase inhibitors (e.g., imatinib mesylate, nilotinib, or dasatinib), adenosine receptor inhibitors (e.g., A2aR inhibitors, SCH58261, CPI-444, SYN115, ZM241385, FSPTP, or A2BR inhibitors, e.g., PSB-1115), adenosine receptor agonists (e.g., CCPA, IB-MECA, and CI-IB-MECA), checkpoint inhibitors ( including those in the PDL-l:PD-l axis), nivolumab, pembrolizumab, atezolizumab, BMS-936559, MEDI4736, MPDL33280A, or MSB0010718C, inhibitors of the CTLA-4 pathway (e.g., ipilimumab and tremelimumab), inhibitors of the TIM-3 pathway, or agonist monoclonal antibodies known to promote T cell function (including anti-OX40, such as MEDI6469, and anti-4-BB, such as PF-05082566).

[0176] The present invention also provides a kit or article of manufacture comprising the pharmaceutical composition described above.

[0177] The present invention also provides a kit for use in the above-described therapeutic applications, comprising: (a) a container holding a polypeptide, nucleic acid, vector, or pharmaceutical composition of the invention; and (b) A label or package insert showing instructions for use Also provided is a kit comprising:

[0178] Suitable containers include, for example, bottles, vials, syringes, blister packs, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container holds a therapeutic composition effective for treating a condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the therapeutic composition is used to treat the condition of choice. In some embodiments, the label or package insert includes instructions for use and indicates that the therapeutic or prophylactic composition can be used to treat cancer or other conditions described herein.

[0179] The kit may further comprise additional containers containing pharmaceutically acceptable buffers, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The kit may further include other materials desirable from a commercial and user standpoint, as would be known to those skilled in the art; suitable examples of this include other buffers, diluents, filters, needles, and syringes.

[0180] therapeutic use Also described herein is the use of a CAR, coreceptor, immune effector cell, or population of immune effector cells of the invention in methods of treatment of the human or animal body by therapy.

[0181] For example, a method for treating cancer in a subject is also provided, comprising administering to the subject an effective amount of the immune effector cells or a population of immune effector cells of the present invention.Thus, the present invention also provides a CAR, co-receptor, immune effector cell, or a population of immune effector cells of the present invention for use in a method for treating cancer.The present invention also provides the use of a CAR, co-receptor, immune effector cell, or a population of immune effector cells of the present invention for the manufacture of a drug for treating cancer.The present invention also provides the use of a CAR, co-receptor, immune effector cell, or a population of immune effector cells of the present invention for treating cancer.

[0182] Also provided is a method for performing adoptive cell therapy in a subject, comprising administering to the subject an effective amount of the immune effector cells or population of immune effector cells of the present invention. Thus, the present invention also provides a CAR, coreceptor, immune effector cell, or population of immune effector cells of the present invention for use in adoptive cell therapy. The present invention also provides the use of a CAR, coreceptor, immune effector cell, or population of immune effector cells of the present invention for the manufacture of a drug for adoptive cell therapy. The present invention also provides the use of a CAR, coreceptor, immune effector cell, or population of immune effector cells of the present invention for adoptive cell therapy.

[0183] The cancer can be any cancer, such as a solid cancer. The cancer can be a malignancy listed in Table 2. The cancer can be a hematological malignancy or a B-cell cancer.

[0184] The therapeutic uses and methods of treatment may comprise administering a therapeutically effective amount of an immune effector cell or population of immune effector cells.

[0185] Also provided is a method of formulating a composition for treating cancer, comprising admixing an immune effector cell or population of immune effector cells of the invention with an acceptable carrier to prepare the composition.

[0186] The subject may have already undergone cancer treatment, such as adoptive cell therapy.

[0187] The present methods of treatment and therapeutic uses may include, prior to treatment with the immune effector cells or population of immune effector cells of the present invention, determining whether the cancer expresses a target antigen that is specifically targeted by the immune effector cells or population of immune effector cells of the present invention.

[0188] The method may include selecting an immune effector cell or population of immune effector cells based on expression of a target antigen by the cancer, such that the immune effector cell or population of immune effector cells is specific for the cancer. The method may include transfecting or transforming the immune effector cell with a nucleic acid of the invention in response to information regarding expression of the target antigen by the cancer.

[0189] The therapeutic methods and uses described herein may include inhibiting a disease state (i.e., cancer) by, for example, preventing the progression of the disease state until a desired endpoint is reached and / or causing regression of the disease state. The therapeutic methods and uses of the present invention may include achieving a partial response, a complete response of the cancer. The therapeutic methods and uses of the present invention may achieve remission of the cancer.

[0190] The therapeutic methods and uses described herein may slow, stop, and / or reverse the growth of cancer. The therapeutic methods and uses of the present invention may reduce the size of cancer by at least 10%, for example, by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%.

[0191] Typically, the methods of treatment and therapeutic uses are directed to human subjects in need thereof. However, non-human animals, such as non-human mammals, are also contemplated. The non-human mammal may be a mouse, rabbit, sheep, pig, cow, cat, or dog.

[0192] The dose of the immune effector cells or population of immune effector cells can vary depending on the age and size of the subject, as well as the disease, condition, and route of administration. The immune effector cells or population of immune effector cells can be administered in a dose of about 1 x 10 6 pieces~approx. 1×10 12 The immune effector cells or population of immune effector cells may be administered at a dose of about 1 x 10 cells. 5 cells / kg ~ approx. 1 x 10 11 The antibody may be administered at a dose of 10 cells / kg body weight.

[0193] The immune effector cells or population of immune effector cells can be administered as a single dose. The immune effector cells or population of immune effector cells can be administered in a multiple dose regimen. For example, a first dose can be followed by a second or multiple subsequent doses. The second and subsequent doses can be spaced apart by an appropriate time. For example, the doses between each dose can be administered about once per week, about once per two weeks, about once per three weeks, about once per four weeks, or about once per month.

[0194] The immune effector cells or population of immune effector cells may be administered intravenously.

[0195] The immune effector cells or population of immune effector cells can be administered with one or more additional therapies, for example, with one or more additional therapeutic agents. The additional therapeutic agents can be anti-tumor agents. The additional therapeutic agents can be additional immune effector cells.

[0196] The combined administration of the immune effector cells or populations and additional therapeutic agents can be achieved in a number of different ways: All components can be administered together in a single composition, or each component can be administered separately as part of a combination therapy.

[0197] For example, the immune effector cells or populations of immune effector cells of the invention may be administered before, after, or simultaneously with an additional therapeutic agent, which may be chemotherapy, radiation therapy, and / or surgery.

[0198] Before administering the immune effector cell or population of immune effector cells of the present invention, the subject can undergo lymphodepletion.Lymphodepletion can be achieved by administering fludarabine, cyclophosphamide, and / or bendamustine to the subject.Lymphodepletion can be carried out for at least about 1 day, for example, for about 2 days or about 3 days.

[0199] The biological activity and / or therapeutic effect of the administered immune effector cell or population of immune effector cells can be measured by known methods, which can include, for example, imaging, such as magnetic resonance imaging.

[0200] Embodiments of the present invention 1. A method for optimizing effector function of immune effector cells, the immune effector cells comprising: - a chimeric antigen receptor (CAR) capable of binding to an antigen on an antigen-presenting cell (APC), the CAR comprising a fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain and the transmembrane domain are linked by a hinge; and - a coreceptor capable of binding to a ligand on the APC, the coreceptor comprising an extracellular ligand-binding domain and a transmembrane domain, the extracellular ligand-binding domain and the transmembrane domain being connected by a stalk. Including, the method comprises altering the height of the extracellular antigen binding domain of the CAR and / or the height of the extracellular ligand binding domain of the co-receptor from a membrane of the immune effector cell to optimize the effector function of the immune effector cell upon contact with the APC; Optionally, the effector function is cell killing. method.

[0201] 2. A method for identifying improved immune effector cells, the immune effector cells comprising: - a chimeric antigen receptor (CAR) capable of binding to an antigen on an antigen-presenting cell (APC), the CAR comprising a fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain and the transmembrane domain are linked by a hinge; and - a coreceptor capable of binding to a ligand on the APC, the coreceptor comprising an extracellular ligand-binding domain and a transmembrane domain, the extracellular ligand-binding domain and the transmembrane domain being connected by a stalk. wherein the method comprises: (a) altering the height of the extracellular antigen-binding domain of the CAR and / or the height of the extracellular ligand-binding domain of the coreceptor from the membrane of the immune effector cell; and (b) determining whether immune effector cells expressing the modified CAR and / or modified coreceptor have improved effector function when compared to immune effector cells expressing the unmodified CAR and / or unmodified coreceptor; Including, Optionally, the effector function is cell killing. method.

[0202] 3. The method of embodiment 1 or 2, comprising modifying the height of the extracellular antigen-binding domain of the CAR and / or the height of the extracellular ligand-binding domain of the coreceptor such that the intermembrane distance spanned by a CAR-antigen complex is similar to the intermembrane distance spanned by a coreceptor-ligand complex.

[0203] 4. (a) the intermembrane distance spanned by the CAR-antigen complex is within 5%, 10%, 15%, 20%, 25%, or 30% of the intermembrane distance spanned by the coreceptor-ligand complex, or is the same as the intermembrane distance spanned by the coreceptor-ligand complex; and / or (b) the intermembrane distance spanned by the CAR-antigen complex is comparable to (e.g., within 5%, 10%, 15%, 20%, 25%, or 30% of, or identical to) the intermembrane distance spanned by the complex of the T cell receptor and its peptide-MHC antigen; 4. The method of embodiment 3.

[0204] 5. The method of embodiment 3 or 4, wherein the spanned intermembrane distance is about 14 nm.

[0205] 6. (a) reducing the height of the extracellular antigen-binding domain of the CAR; (b) increasing the height of the extracellular antigen-binding domain of the CAR; (c) reducing the height of the extracellular ligand-binding domain of the coreceptor; (d) increasing the height of the extracellular ligand-binding domain of the coreceptor; (e) reducing the height of the extracellular antigen-binding domain of the CAR and increasing the height of the extracellular ligand-binding domain of the co-receptor; or (f) increasing the height of the extracellular antigen-binding domain of the CAR and decreasing the height of the extracellular ligand-binding domain of the coreceptor. 6. The method according to any one of embodiments 1 to 5, comprising:

[0206] 7. The method of any one of embodiments 1-6, wherein the height of the antigen-binding domain of the CAR from the membrane of the immune effector cell is similar to (e.g., within 5%, 10%, 15%, 20%, 25%, or 30% of, or the same as) the height of the extracellular antigen-binding domain of a T cell receptor from the membrane of the immune effector cell (e.g., about 7 nm).

[0207] 8. The method of any one of embodiments 1-7, wherein the height of the ligand-binding domain of the co-receptor from the membrane of the immune effector cell is about 7 nm.

[0208] 9. The method of any one of embodiments 1-8, comprising introducing into or replacing the hinge of the CAR and / or the stalk of the coreceptor with a sequence that physically increases the height of the ligand binding domain from the membrane of the immune effector cell.

[0209] 10. The sequence introduced into the hinge of the CAR is: (a) a fragment of the mucin-like extracellular sequence of CD43 set forth in SEQ ID NO: 62, e.g., 4 to 234 amino acids, e.g., 20 to 40 amino acids (e.g., set forth in any one of SEQ ID NOs: 53 to 61); (b) a folded polypeptide domain that is an immunoglobulin domain, e.g., an immunoglobulin constant domain or an FNIII domain; and / or (c) a fragment of the CD28 hinge set forth in SEQ ID NO: 10 or the CD8a hinge set forth in SEQ ID NO: 43, optionally the fragment being set forth in any one of SEQ ID NOs: 37-42, 46, 63, and 64. 10. The method of embodiment 9, comprising or consisting of:

[0210] 11. The method of embodiment 9, wherein the sequence introduced into the stalk of the coreceptor comprises or consists of a fragment of a mucin-like sequence or a fragment or derivative thereof, optionally wherein the mucin-like sequence is the mucin-like sequence of CD43 as set forth in SEQ ID NO: 62, and optionally wherein the fragment is 4 to 234 amino acid residues in length, e.g., any one of SEQ ID NOs: 53 to 61.

[0211] 12. The method of any one of embodiments 1 to 11, wherein the co-receptor is an adhesion receptor, such as CD2.

[0212] 13. The method of any one of embodiments 1-12, wherein the antigen is a peptide-MHC complex, CD19, mesothelin, BCMA, CD22, EGFR, or EGFRvIII, and optionally, the peptide in the peptide-MHC complex is a fragment of NY-ESO 1.

[0213] 14. Immune effector cells obtained or obtainable by a method according to any one of embodiments 1 to 13.

[0214] 15. A chimeric antigen receptor (CAR) capable of binding to an antigen on an antigen-presenting cell (APC), comprising a fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain and the transmembrane domain are linked by a hinge, the hinge comprising or consisting of a sequence that physically increases the height of the extracellular antigen-binding domain from the membrane of the immune effector cell, the sequence comprising a mucin-like sequence, one or more folded polypeptide domains, or a fragment of the CD28 hinge set forth in SEQ ID NO: 10 or the CD8a hinge set forth in SEQ ID NO: 43.

[0215] 16. A coreceptor capable of binding to a ligand on an APC, comprising an extracellular ligand-binding domain and a transmembrane domain, said extracellular ligand-binding domain and said transmembrane domain being linked by a stalk, said stalk comprising or consisting of a sequence that physically increases the height of said ligand-binding domain from the membrane of said immune effector cell, and optionally said stalk comprising a mucin-like sequence.

[0216] 17. An immune effector cell comprising a CAR according to embodiment 15 and / or a co-receptor according to embodiment 16.

[0217] 18. An immune effector cell, - a chimeric antigen receptor (CAR) capable of binding to an antigen on an antigen-presenting cell (APC), the CAR comprising a fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain and the transmembrane domain are linked by a hinge; and - a coreceptor capable of binding to a ligand on the APC, the coreceptor comprising an extracellular ligand-binding domain and a transmembrane domain, the extracellular ligand-binding domain and the transmembrane domain being connected by a stalk. Including, (a) the CAR and the co-receptor are each sized such that the intermembrane distance spanned by the CAR-antigen complex is comparable to the intermembrane distance spanned by the co-receptor-ligand complex; (b) the hinge of the CAR comprises or consists of a sequence that physically increases the height of the antigen-binding domain from the membrane of the immune effector cell, the sequence comprising a mucin-like sequence, one or more folded polypeptide domains, or a fragment of the CD28 hinge set forth in SEQ ID NO: 10 or the CD8a hinge set forth in SEQ ID NO: 43; and / or (c) the stalk of the coreceptor comprises a sequence that physically increases the height of the extracellular ligand-binding domain from the membrane of the immune effector cell. Immune effector cells.

[0218] 19. (a) the intermembrane distance spanned by the CAR-antigen complex is within 5%, 10%, 15%, 20%, 25%, or 30% of the intermembrane distance spanned by the coreceptor-ligand complex, or is the same as the intermembrane distance spanned by the coreceptor-ligand complex; and / or (b) the intermembrane distance spanned by the CAR-antigen complex or the coreceptor-ligand complex is comparable to (e.g., within 5%, 10%, 15%, 20%, 25%, or 30% of, or identical to) the intermembrane distance spanned by the complex of a T cell receptor and its corresponding peptide-MHC antigen; 19. The immune effector cell of embodiment 18.

[0219] 20. The immune effector cell of embodiment 18 or 19, wherein the hinge of the CAR comprises or consists of a sequence that physically increases the height of the antigen-binding domain from the membrane of the immune effector cell, said sequence comprising a mucin-like sequence, one or more folded polypeptide domains, or a fragment of the CD28 hinge set forth in SEQ ID NO: 10 or the CD8a hinge set forth in SEQ ID NO: 43.

[0220] 21. The immune effector cell of embodiment 18 or 19, wherein the stalk of the coreceptor comprises a sequence that physically increases the height of the extracellular ligand-binding domain from the membrane of the immune effector cell.

[0221] 22. (a) the hinge of the CAR comprises or consists of a sequence that physically increases the height of the antigen-binding domain from the membrane of the immune effector cell, the sequence comprising a mucin-like sequence, one or more folded polypeptide domains, or a fragment of the CD28 hinge set forth in SEQ ID NO: 10 or the CD8a hinge set forth in SEQ ID NO: 43; and (b) the stalk of the coreceptor comprises a sequence that physically increases the height of the extracellular ligand-binding domain from the membrane of the immune effector cell; 20. An immune effector cell according to embodiment 18 or 19.

[0222] 23. The immune effector cell of any one of embodiments 17 to 22, wherein the immune effector cell is a T cell, an NK cell, an NKT cell, a phagocyte, or a macrophage, and optionally, the T cell is a CD4-CD8+ T cell.

[0223] 24. A method for identifying an improved immune effector cell, comprising determining whether an immune effector cell of any one of embodiments 17-23 has improved effector function when compared to an immune effector cell expressing an unmodified CAR and / or an unmodified co-receptor.

[0224] 25. The immune effector cell of any one of embodiments 17 to 23, the CAR of embodiment 15, or the coreceptor of embodiment 16, wherein the immune effector cell, CAR, or coreceptor is for use in a method for treating cancer in a subject, and optionally the cancer is a hematological malignancy or a B-cell cancer.

[0225] others It should be understood that various uses of the disclosed CAR, immune effector cells, or pharmaceutical compositions of the present invention can be tailored to the particular needs of the art. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments of the present invention only and is not intended to be limiting.

[0226] Additionally, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise. So, for example, reference to "a CAR" includes two or more "CARs."

[0227] Furthermore, when reference is made herein to "≧x," this means greater than or equal to x. When reference is made herein to "≦x," this means less than or equal to x.

[0228] For the purposes of the present invention, to determine the percent identity of two sequences (e.g., two polynucleotide sequences or two polypeptide sequences), these sequences are aligned for optimal comparison (e.g., gaps can be introduced into the first sequence for optimal alignment with the second sequence). The nucleotide or amino acid residue at each position is then compared. If a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, the nucleotide or amino acid at this position is identical. The percent identity between two sequences is a function of the number of identical positions shared by these sequences (i.e., % identity = number of identical positions / total number of positions in the reference sequence × 100).

[0229] Typically, sequence comparison is performed over the length of the reference sequence. For example, if a user wants to determine whether a given ("test") sequence is 95% identical to SEQ ID NO: 3, SEQ ID NO: 3 is the reference sequence. To assess whether a sequence is at least 95% identical to SEQ ID NO: 3 (an example of a reference sequence), a person skilled in the art will perform alignment over the length of SEQ ID NO: 3 and determine how many positions in the test sequence are identical to SEQ ID NO: 3. If at least 95% of the positions are identical, the test sequence is at least 95% identical to SEQ ID NO: 3. If the sequence is shorter than SEQ ID NO: 3, gaps or missing positions should be considered as non-identical positions.

[0230] Those skilled in the art are aware of various computer programs that can be used to determine the homology or identity between two sequences.For example, the comparison of sequences and the determination of the percent identity between two sequences can be achieved using mathematical algorithms.In an embodiment, the percent identity between two amino acid sequences or nucleic acid sequences is determined using the Needleman and Wunsch (1970) algorithm that is incorporated into the GAP program of Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ), using either Blosum 62 matrix or PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6 or 4, and a length weight of 1, 2, 3, 4, 5 or 6.

[0231] "Specific" or "specifically binds" means that the antigen-binding region of a CAR binds to one or more antigenic determinants of a desired target antigen and does not bind to other polypeptides.For example, a CD19-specific CAR binds to the CD19 antigen but does not bind to antigens of different polypeptides, such as bovine serum albumin.A CAR can specifically bind to a target antigen when it binds with at least 10 times stronger affinity, preferably at least 100 times stronger affinity, compared to the binding to antigens of different polypeptides, such as bovine serum albumin.Methods for measuring binding affinity are known in the art.

[0232] All publications, patents, and patent applications cited herein (see above or below) are hereby incorporated by reference in their entirety.

[0233] The following examples illustrate the invention. [Example]

[0234] Example 1 - Identification of the mechanism responsible for the inability of CAR to utilize CD2 adhesion Determining the contribution of antigen receptor size and signaling to antigen sensitivity The inventors have generated a modified CAR (referred to herein as Fab CAR), a fusion protein in which an antibody-derived variable domain is conjugated to a TCR constant domain, followed by the CD28 transmembrane domain and then the TCR-ζ chain cytoplasmic region (Figure 2). Importantly, this construct omits the additional extracellular hinge that is often incorporated into CARs, making the CAR larger than the TCR.

[0235] The activity of the following receptors was tested: 1G4 TCR, STAR(D52N), eTruC(D52N), Fab-CAR(D52N), and CAR(D52N-CD28-28z) (Figure 3). The D52N single-chain variable fragment (scFv) binds to the same peptide-MHC complex recognized by the 1G4 TCR [the C9V variant (9V) of the NY-ESO-1157-165 peptide presented on HLA-A*02:01] (4).

[0236] These receptors were successfully expressed in Jurkat cells or primary human CD8+ T cells via lentivirus (see Figures 4, 5, and 6). Interestingly, Figure 4 shows that standard CAR (D52N-CD28-28z) and Fab-CAR (D52N-Fab-28z) are expressed (y-axis) without upregulating CD3 (x-axis). Thus, these receptors can be expressed independently of the TCR-CD3 complex, whereas this is not the case for the 1G4 TCR.

[0237] T2 target cells were loaded with the indicated concentrations of peptide antigens and then incubated with T cells expressing various antigen receptors and / or various sizes of CD2. Dose response and EC 50 The values ​​(antigen sensitivity; antigen concentration required to induce 50% of the maximal response) are shown in Figures 6A to 6C. Figure 7 shows cytokine production when primary T cells were used.

[0238] It can be seen that STARs, which have the same size and signal transduction mechanism as TCRs, have relatively high antigen sensitivity. In contrast, TruCs, which have the same signal transduction mechanism as TCRs but different sizes, show lower antigen sensitivity, indicating that similar sizes are required. Consistent with this and noteworthy, Fab-CARs, which are expressed independently of the TCR-CD3 complex and share a similar size to TCRs and STARs, achieved similar antigen sensitivity and cytokine production potential to TCRs and STARs, but higher antigen sensitivity and cytokine production potential than CARs and eTRuCs.

[0239] Thus, this data indicates that similar size, but not signaling mechanisms, is necessary to enable CARs to achieve the same high antigen sensitivity as TCRs. This data supports previous findings that T cell antigen recognition depends on the dimensions of the coreceptor-ligand complex (11), that the dimensions of the CD2 / ligand complex match those of the TCR / pMHC complex (14 nm) (3, 6), and that varying the dimensions of either the CD2 / ligand or TCR / pMHC complex (7) can be beneficial.

[0240] material and method Plasmid constructs Generation of lentiviral infectious particles was performed using a third-generation system containing the (i) pMD2.G (Addgene #12259), (ii) pRSV-Rev (Addgene #12253), and (iii) pMDLg / pRRE (Addgene #12251) packaging plasmids. Various lentiviral transfer vectors encoding receptors that bind to the NY-ESO-1 / HLA-A*02 pMHC complex were used. The coding sequences were contained in either the pLEX_307 (Addgene #41392) or pLEX307-NeoR (Addgene #134365) backbone.

[0241] receptor The 1G4 T cell receptor (TCR) has variable alpha (SEQ ID NO: 1) and constant alpha (SEQ ID NO: 2) domains, a P2A self-cleaving peptide (SEQ ID NO: 3) followed by variable beta (SEQ ID NO: 4) and constant beta (SEQ ID NO: 5) domains. The variable domains confer specificity.

[0242] D52N-CD28.H-28z CAR (also referred to herein as D52N-CD28-28z) has a leader sequence (SEQ ID NO: 6), the variable heavy domain of D52N scFv (SEQ ID NO: 7), a linker (SEQ ID NO: 8), the variable light domain of D52N scFv (SEQ ID NO: 9), a CD28-derived hinge (SEQ ID NO: 10), a CD28 transmembrane domain (SEQ ID NO: 11), a CD28 cytoplasmic domain (SEQ ID NO: 12), and a zeta chain (CD247) signaling tail (SEQ ID NO: 13).

[0243] A CAR similar to the D52N-CD28.H-28z CAR may be used, except for a CD8a hinge instead of a CD28 hinge. Such a CAR may have a leader sequence (SEQ ID NO: 6), the variable heavy domain of the D52N scFv (SEQ ID NO: 7), a linker (SEQ ID NO: 8), the variable light domain of the D52N scFv (SEQ ID NO: 9), a CD8a-derived hinge (SEQ ID NO: 43), a CD8a transmembrane domain (SEQ ID NO: 44), and a zeta chain (CD247) signaling tail (SEQ ID NO: 13). Alternative leader sequences may be used, for example, the GM-CSF leader sequence set forth in SEQ ID NO: 65.

[0244] D52N-ε-TRuC (also referred to herein as eTruC(D52N)) has a leader sequence (SEQ ID NO: 14), a D52N scFv (both heavy and light domains) (SEQ ID NO: 15), a glycine-serine linker (SEQ ID NO: 16), and the extracellular, helical, and cytoplasmic regions of human CD3E (SEQ ID NO: 17). As is known in the art, TRuC is a T cell receptor fusion construct (see, e.g., Reference 12).

[0245] D52N-STAR (also referred to herein as STAR(D52N)) has a leader sequence (SEQ ID NO: 14), the variable heavy domain of the D52N scFv (SEQ ID NO: 7), a murine constant alpha TCR domain (SEQ ID NO: 18), a P2A self-cleaving peptide (SEQ ID NO: 3), a leader sequence (SEQ ID NO: 19), the variable light domain of the D52N scFv (SEQ ID NO: 9), and a murine constant beta TCR domain (SEQ ID NO: 20). As is known in the art, STARs are synthetic T cell receptors and antigen receptors (see, e.g., Reference 13).

[0246] D52N-Fab-CAR has a leader sequence (SEQ ID NO: 14), the variable heavy domain of D52N scFv (SEQ ID NO: 7), an IgG1-CH1 domain (SEQ ID NO: 21), a linker (SEQ ID NO: 22), a CD28 transmembrane domain (SEQ ID NO: 11), a CD28 cytoplasmic domain (SEQ ID NO: 12), a zeta chain (CD247) signaling tail (SEQ ID NO: 13), a GSG stuffer (SEQ ID NO: 23), a P2A self-cleaving peptide (SEQ ID NO: 3), a leader sequence (SEQ ID NO: 14), the variable light domain of D52N scFv (SEQ ID NO: 9), an IgG1-CL domain (SEQ ID NO: 24), a linker (SEQ ID NO: 22), a CD28 transmembrane domain (SEQ ID NO: 11), a CD28 cytoplasmic domain (SEQ ID NO: 12), and a zeta chain (CD247) signaling tail (SEQ ID NO: 13).

[0247] Production of lentiviral supernatant 293T cells (ATCC CRL-3216) were plated in 6-well plates in 3 mL volumes to 50% confluency. Cells were allowed to attach overnight and transfected the following morning with a mixture of packaging plasmids (950 ng of pRSV-Rev, 370 ng of pMD2.G, 950 ng of pMDLg / pRRE, and 1000 ng of the corresponding lentiviral transfer plasmid). Transfection was performed using X-tremeGENE HP Transfection Reagent at a ratio of 1 μg of DNA to 3 μL of transfection reagent. 293T cells were then incubated for 48 hours at 37°C and 10% CO2 (v / v), and the lentiviral supernatant was filtered through a 0.45 μm cellulose acetate syringe filter. This supernatant was used to transduce either Jurkat cells or primary CD8+ T cells. Lentiviral supernatants were applied directly or pre-concentrated using Lenti-X concentration reagent (Takara Bio) according to the manufacturer's instructions. Stored supernatants were kept at -80°C.

[0248] cell The following cell lines were used: 293T cells (ATCC CRL-3216), T2 cells (ATCC CRL-1993), Jurkat E6.1 NFkB / eGFP cells (obtained by lentiviral transduction of the Jurkat E6-1 clone (ATCC TIB-152) with the pSIRV-NF-kB plasmid (Addgene #118093)), and Jurkat E6.1 TCRα-β cells.

[0249] Human leukocyte cones were obtained and CD8+ T cells were isolated. The isolated CD8+ T lymphocytes were mixed with human anti-CD3 / CD28 Dynabeads at a 1:1 cell-to-bead ratio. Cells were allowed to rest overnight, and the following morning, 1.5 million CD8+ T cells were transduced with the corresponding lentiviral supernatant encoding the receptor of interest. 72 hours after transduction, cells were subjected to either puromycin (pLEX_307 backbone) or G418 (pLEX307-NeoR backbone) selection.

[0250] T cell stimulation assay The T2 suspension cell line was used as an alternative APC, onto which the 9V NY-ESO-1 peptide was loaded. 4 T2 cells were seeded into a V-bottom 96-well plate in 110 μL of complete RPMI. Serial dilutions of 9V peptide were also prepared using complete RPMI. Then, 110 μL of diluted 9V peptide was added to the T2 cell suspension, and the cells were incubated for 90 minutes at 37°C and 5% CO2 (v / v). The T2 cells were then washed with complete RPMI and resuspended in 110 μL of this medium. A total of 30,000 T2 cells (equivalent to 100 μL) were transferred to a U-bottom 96-well plate. A total of 60,000 CD8+ T cells (100 μL) were then added to each well to establish a simple 2:1 effector:target ratio. The plate was gently centrifuged (15 g for 1 min) to promote contact between the T cells and APCs. Co-culture was performed for 20 hours, and the cells were incubated at 37°C and 5% CO2 (v / v).

[0251] Flow cytometry After the experiment was terminated, the cells were transferred to a V-bottom 96-well plate and centrifuged at 520 g for 5 minutes at 4°C. Cells were first stained with a fixable viability dye (Zombie Near-Infrared, 1:500 working dilution) in 50 μL of PBS. Samples were then stained with conjugated flow cytometry antibodies pre-diluted in 50 μL of PBS. Working dilutions ranged from 1:200 for commercial antibodies to 1:1000 for pMHC 9V tetramer. Cells were incubated for 30 minutes, washed, and resuspended in PBS containing 1% BSA. Sample acquisition was performed on a BD X-20 or Cytoflex cytometer, and analysis was performed in the FlowJo suite.

[0252] Notably, detection of multiple receptors was performed with fluorescent pMHC tetramers, which consisted of biotinylated, refolded 9V pMHC molecules complexed with PE-streptavidin. The fluorescent tetramers were prepared by vigorously mixing 66.6 μg of monomeric 9V pMHC and adding 10 μL of PE-streptavidin stepwise every 10 minutes (10 additions of PE-streptavidin over 100 minutes).

[0253] Cytokine measurements After the co-culture experiments, the supernatants were assayed for levels of IFN-γ and IL-2 using commercially available kits provided by Thermo Fisher Scientific.

[0254] Example 2 - Preparation of engineered adhesion receptors to enhance antigen sensitivity of CARs In this example, engineered adhesion receptor-ligand complexes of various dimensions are investigated to maximize their ability to enhance the CAR-antigen complex.

[0255] CD2 expansion variants are constructed and screened for their ability to affect susceptibility. A panel of extended CD2 constructs was prepared by inserting various fragments of the extracellular domain of the human mucin-like protein CD43 as spacers (Figure 9A). Either the 1G4 TCR or a standard CAR was transfected into the CD2 - It was expressed in the E6.1 Jurkat T cell line (Fig. 9B).

[0256] As expected, wild-type CD2 significantly increased the antigen sensitivity of TCR, whereas extension of CD2 by 4, 8, 20, 40, 50, 60, 80, 120, 160, or 234 amino acids from CD43 resulted in a gradual loss of antigen sensitivity (Figure 10). Similarly, the antigen sensitivity of STAR was significantly increased by wild-type CD2, whereas extension of CD2 by 4, 8, 20, 40, or 234 amino acids resulted in a gradual loss of antigen sensitivity (Figure 11).

[0257] In contrast, wild-type CD2 had only a modest effect on CAR antigen sensitivity, but CD2 extension increased antigen sensitivity, with the greatest increase achieved with intermediate-length CD2 constructs (CD2-CD43(40) or CD2-CD43(20)) (Figures 12 and 13).

[0258] Wild-type CD2 increased the antigen sensitivity of eTruC. CD2 expansion increased antigen sensitivity, with the greatest increase achieved with a medium-length CD2 construct (CD2-CD43(20)) (Figure 14). Based on the size of eTruC relative to the TCR and the results of CD2 expansion in combination with CAR, this result would be expected based on the inventors' findings.

[0259] Thus, this data indicates that CAR sensitivity can be increased by increasing the size of the auxiliary-ligand complex (e.g., CD2 / CD58 complex). It also confirms that the larger the size of the CAR-antigen complex, the more limited its ability to utilize CD2-CD58 (Figure 15). CD2-CD43(234) antagonizes TCR and CAR antigen recognition, due to excessive membrane separation for antigen binding.

[0260] material and method Preparation of elongated CD2 Extended CD2 variants were generated by fusing the extracellular portion of CD2 (SEQ ID NO: 49) to the extracellular portion of CD43 using a short linker sequence (GGGS; SEQ ID NO: 50), followed by fusing the CD2 transmembrane domain (SEQ ID NO: 51) to the CD2 intracellular domain (SEQ ID NO: 52). From the full-length sequence consisting of the entire CD43 extracellular portion, shorter variants were generated using site-directed mutagenesis to leave several amino acids from the C-terminus of the CD43 domain (e.g., SEQ ID NOs: 53-62). For example, CD2-CD43(20) contains the CD2 extracellular portion fused to 20 amino acids near the C-terminus of the CD43 extracellular portion. Extended CD2 variants are shown in SEQ ID NOs: 27-36, each of which also contains the CD2 signal peptide (SEQ ID NO: 47) and HA tag (SEQ ID NO: 48).

[0261] cell line Jurkat TCRα-β-cells were provided by Simon J. Davis (Oxford) and cultured in RPMI 1640 10% FBS (v / v) containing penicillin-streptomycin (100 U / mL and 100 mL, respectively) at 37°C and 5% CO .

[0262] The endogenous surface protein CD2 was knocked out using CRISPR / Cas9.

[0263] Co-culture with U87 cells 25,000 U87 cells were seeded into tissue-culture-treated flat-bottom 96-well plates and grown overnight. The next day, the medium was removed from the cells, and the cells were incubated with peptides at appropriate concentrations in complete DMEM (DMEM supplemented with 10% v / v FBS, 100 units / ml penicillin, and 100 μg / ml streptomycin) for 1 hour at 37°C.

[0264] The peptide-containing medium was then removed and 50,000 T cells were added per well. The co-cultures were then spun at 50×g for 2 minutes and incubated at 37° C. for 4 hours. After this period, a portion of the supernatant was removed for cytokine ELISA and stored at −20° C. EDTA was added to the remaining supernatant (final concentration 2.5 μM), and the cells were detached by pipetting.

[0265] Cells were stained for CD45 (clone HI30, dilution 1:200), CD69 (clone FN50, dilution 1:200), and 4-1BB (clone 4B4-1, dilution 1:200) in PBS 1% BSA, as well as PE-conjugated tetramer pMHC (dilution 1:500). Stained cells were either analyzed immediately or fixed with 1% formaldehyde in PBS and analyzed the next day. T cells were distinguished from U87 cells by CD45 staining and / or assessment of size and complexity. Single T cells were identified based on size, and subsequent analysis was performed on this population.

[0266] Example 3 - Effect of variable CD2 size on antigen sensitivity to tisagenlecleucel (Kymriah) This example examines the effect of variable size of CD2 on Kymriah, a clinically approved CAR that targets the surface antigen CD19. Kymriah expresses variable sizes of CD2, as well as CD2 - It was expressed in Jurkat T cells (Fig. 16A). Cells were sorted by corresponding expression levels (Fig. 16B).

[0267] To investigate antigen sensitivity in Kymriah, we used Nalm6 CombiCells to manipulate the surface levels of CD19 on B cells as described in reference 14. These cells express CD19 -Nalm6 CombiCells express the protein Spycatcher, which spontaneously forms a covalent bond with Spytag on its cell surface. By adding various concentrations of purified Spytag-CD19 in solution, we produced Nalm6 CombiCells with various levels of surface CD19 (Figure 16C).

[0268] Each Jurkat line was co-cultured with Nalm6 CombiCells loaded with various levels of CD19 (Figures 16D and 16E). Compared to CD2-deficient T cells, wild-type CD2 resulted in a significant improvement in antigen sensitivity (lower EC50), but increasing the size of CD2 by just four amino acids further improved antigen sensitivity. Further increases in CD2 size resulted in a gradual decrease in antigen sensitivity, with the longest variant exhibiting sensitivity similar to that of CD2-deficient Jurkat T cells.

[0269] Taken together, these results highlight that even modest expansion of CD2 can improve the antigen sensitivity of a CAR (Kymriah) targeting a folded antigen (CD19).

[0270] Production of Kymriah-expressing Jurkat T cells HEK 293T cells were seeded in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin in 6-well plates and reached 60-80% confluency the next day. Cells were transfected with 0.25 μg of pRSVRev (Addgene, 12253), 0.53 μg of pMDLg / pRRE (Addgene, 12251), 0.35 μg of pMD2.G (Addgene, 12259), and 0.8 μg of a transfer plasmid expressing the Kymriah CAR using a 5.8 X-tremeGENE HP (Roche). The medium was changed after 16 hours, and after a further 24 hours, the supernatant was collected by filtration through a 0.45 μm cellulose acetate filter. Supernatant from one well of a 6-well plate was used to transfect 1 million CD2 -Jurkat T cells were transduced and sorted by expression of Kymriah CAR, then further cultured at a density of 300,000 cells / ml.

[0271] Virus was prepared using HEK cells as described above. - Jurkat T cells were transduced with WT CD2 or one of six variable-sized CD2 molecules. Jurkat T cells were sorted according to the matching expression level of CD2 protein and then further cultured at a density of 300,000 cells / ml.

[0272] Conjugation of ligands to Nalm6 cells 30,000 Nalm6 CombiCells were seeded into a TC-coated 96-well round-bottom plate and incubated overnight at 37°C, 5% CO2. On the day of the experiment, the cells were transferred to a TC-coated 96-well V-bottom plate and spun down at 520g for 5 minutes. Spytag-CD19 ligand was diluted to the required concentration in complete RPMI (10% FCS, 1% penicillin-streptomycin). The existing medium was removed from the cells, and the diluted ligand was added in a volume of 50 μl and incubated for 40 minutes at 37°C, 5% CO2. The cells were then washed twice with complete RPMI.

[0273] Co-culture assay of Kymriah Jurkat T cells with Nalm6 CombiCells Kymriah Jurkat T cells were counted and washed once with complete RPMI. 50,000 Jurkat T cells in 200 μl of complete RPMI were added to Nalm6 CombiCells conjugated with Spytag-CD19 and transferred to a 96-well round-bottom plate. The cells were then incubated for 6 hours at 37°C and 5% CO2.

[0274] Flow cytometry-Spytag-CD19 detection Immediately after ligand conjugation and subsequent washing, Nalm6 CombiCells were transferred to a v-bottom plate and spun at 500 g for 5 minutes at 4° C. The cells were washed once with PBS-BSA 1% at 500 g for 5 minutes at 4° C. To detect the ligand, a fluorescently conjugated antibody against the protein of interest was diluted in PBS-BSA (1%) at a dilution of 1:200 and added to the cells in a volume of 50 μl. The cells were resuspended and incubated in the dark at 4° C. for 20 minutes. The cells were washed twice with PBS and resuspended in 60 μl of PBS before being run on a flow cytometer.

[0275] Flow Cytometry - Kymriah Jurkat Detection of T Cell Activation At the end of the stimulation assay, Kymriah Jurkat cells were transferred to a v-bottom plate and washed once with 200 μl PBS 1% BSA (500 g, 4°C, 5 min). An antibody against the activation marker CD69 was diluted 1:200 in PBS 1% BSA. 50 μl of this staining solution was added to the cells and incubated in the dark at 4°C for 20 min. The cells were washed twice with PBS, resuspended in 70 μl PBS, and then run on a flow cytometer. Nalm6 CombiCells could be distinguished from Kymriah Jurkat T cells using their unique GFP marker. Flow cytometry data were analyzed using FlowJo (BD Biosciences).

[0276] References

[0277] [Table 3]

[0278] Sequence Listing

[0279] [Table 4-1]

[0280]

Table 4-2

[0281]

Table 4-3

Claims

1. These are immune effector cells, - A chimeric antigen receptor (CAR) capable of binding to an antigen on an antigen-presenting cell (APC), wherein the CAR comprises a fusion protein including an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain and the transmembrane domain are hinged; and - A co-receptor capable of binding to a ligand on the APC, wherein the co-receptor comprises an extracellular ligand-binding domain and a transmembrane domain, and the extracellular ligand-binding domain and the transmembrane domain are linked by a stalk. Includes, (a) The stalk of the co-receptor comprises an array that physically increases the height of the extracellular ligand-binding domain from the membrane of the immune effector cell; (b) The hinge of the CAR comprises or consists of an array that physically increases the height of the antigen-binding domain from the membrane of the immunoeffector cell, the array comprising a mucin-like array, one or more folded polypeptide domains, or a fragment of the CD28 or CD8a hinge described in SEQ ID NO: 10 or SEQ ID NO: 43; and / or (c) The CAR and the co-receptor are each sized such that the intermembrane distance mediated by the CAR-antigen complex is approximately the same as the intermembrane distance mediated by the co-receptor-ligand complex. Immune effector cells.

2. The immunoeffector cell according to claim 1, wherein the co-receptor is CD2, L-selectin, α4 ​​integrin, LFA-1, CD28, PD-1, 4-1BB, or CD6.

3. The immune effector cell according to claim 1, wherein the co-receptor is CD2.

4. (a) The intermembrane distance spanned by the CAR-antigen complex is within 5%, 10%, 15%, 20%, 25%, or 30% of the intermembrane distance spanned by the co-receptor-ligand complex or the intermembrane distance spanned by the complex of the T cell receptor and its peptide-MHC antigen, or the same as thereto; (b) The intermembrane distance mediated by the CAR-antigen complex or the co-receptor-ligand complex is approximately 14 nm; (c) The height of the antigen-binding domain of the CAR from the membrane of the immune effector cell is within 5%, 10%, 15%, 20%, 25%, or 30% of the height of the extracellular antigen-binding domain of the T cell receptor from the membrane of the immune effector cell, or the same as such; (d) The extracellular antigen-binding domain of the CAR or the ligand-binding domain of the co-receptor is at a height of approximately 3 nm to 7 nm from the membrane of the immune effector cell, or at a height of approximately 7 nm to 47 nm from the membrane of the immune effector cell. The immune effector cells according to claim 1.

5. The immunoeffector cell according to claim 1, wherein the CAR comprises a CD28 hinge or a CD8a hinge.

6. (a) The hinge of the CAR is (i) Fragments of the mucin-like extracellular sequence of CD43 described in any one of sequence numbers 53-62; (ii) an immunoglobulin domain, an immunoglobulin constant domain, or an FNIIII domain; or (iii) Any one of sequence numbers 37-42, 46, 63, and 64 It includes or consists of; and / or (b) The stalk of the co-receptor contains or consists of a fragment of a mucin-like sequence of CD43 described in any one of SEQ ID NOs: 53 to 62. The immune effector cells according to claim 1.

7. (a) The CAR does not require endogenous TCR and / or CD3 for expression on the surface of the immunoeffector cells; (b) The CAR comprises an intracellular CD3ζ signaling domain; and / or (c) The CAR does not contain an intracellular CD2 signaling domain. The immune effector cells according to claim 1.

8. The immunoeffector cell according to claim 1, wherein the CAR is a homodimer or a heterodimer.

9. (a) The antigen is a peptide-MHC complex, CD19, mesothelin, BCMA, CD22, EGFR, or EGFRvIII; and / or (b) The ligand is CD58. The immune effector cells according to claim 1.

10. (a) The immune effector cells include modifications in their genome such as reducing the expression of endogenous coreceptors; and / or (b) The immune effector cells are T cells, NK cells, NKT cells, phagocytes, macrophages, or CD4-CD8+ T cells. The immune effector cells according to claim 1.

11. A chimeric antigen receptor (CAR) capable of binding to an antigen on an antigen-presenting cell (APC), comprising a fusion protein including an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain and the transmembrane domain are linked by a hinge, the hinge comprising or consisting of a sequence that physically increases the height of the extracellular antigen-binding domain from the membrane of the immune effector cell, the sequence comprising a mucin-like sequence, one or more folded polypeptide domains, or a fragment of the CD28 hinge described in SEQ ID NO: 10 or the CD8a hinge described in SEQ ID NO:

43.

12. A co-receptor capable of binding to a ligand on an antigen-presenting cell (APC), comprising an extracellular ligand-binding domain and a transmembrane domain, wherein the extracellular ligand-binding domain and the transmembrane domain are linked by a stalk, the stalk comprising or consisting of an array that physically increases the height of the ligand-binding domain from the membrane of the immunoeffector cell, the stalk comprising a mucin-like array.

13. A method for optimizing the effector function of immune effector cells, wherein the immune effector cells are - A chimeric antigen receptor (CAR) capable of binding to an antigen on an antigen-presenting cell (APC), wherein the CAR comprises a fusion protein including an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain and the transmembrane domain are hinged; and - A co-receptor capable of binding to a ligand on the APC, wherein the co-receptor comprises an extracellular ligand-binding domain and a transmembrane domain, and the extracellular ligand-binding domain and the transmembrane domain are linked by a stalk. Includes, The method includes optimizing the effector function of the immune effector cell upon contact with the APC by changing the height of the extracellular antigen-binding domain of the CAR and / or the extracellular ligand-binding domain of the co-receptor from the membrane of the immune effector cell. method.

14. The method according to claim 13, further comprising determining whether immune effector cells expressing a modified CAR and / or a modified co-receptor have improved effector function compared to immune effector cells expressing an unmodified CAR and / or an unmodified co-receptor.

15. (a) The method described above (i) This includes modifying the height of the extracellular antigen-binding domain of the CAR such that the intermembrane distance mediated by the CAR-antigen complex is approximately the same as the intermembrane distance mediated by the co-receptor-ligand complex; (ii) including modifying the height of the extracellular ligand-binding domain of the co-receptor such that the intermembrane distance mediated by the CAR-antigen complex is approximately the same as the intermembrane distance mediated by the co-receptor-ligand complex; and / or (iii) including modifying the height of the extracellular antigen-binding domain of the CAR and the extracellular ligand-binding domain of the co-receptor so that the intermembrane distance mediated by the CAR-antigen complex is approximately the same as the intermembrane distance mediated by the co-receptor-ligand complex; or (b) The intermembrane distance mediated by the CAR-antigen complex is within 5%, 10%, 15%, 20%, 25%, or 30% of the intermembrane distance mediated by the co-receptor-ligand complex or the intermembrane distance mediated by the T cell receptor and its corresponding antigen complex, or is the same as such. The method according to claim 13.

16. The method according to claim 13, comprising introducing a sequence into the hinge of the CAR and / or the stalk of the co-receptor that physically increases the height of the ligand-binding domain from the membrane of the immunoeffector cell, or replacing the hinge of the CAR and / or the stalk of the co-receptor with the sequence.

17. (a) The sequence introduced into the hinge in the CAR includes: (i) a fragment of the mucin-like extracellular sequence of CD43 described in any one of SEQ ID NOs. 53 to 62; (ii) an immunoglobulin constant domain or FNIIII domain; and / or (iii) a fragment of the CD28 hinge described in SEQ ID NOs. 10, a fragment of the CD8a hinge described in SEQ ID NOs. 43, or a fragment described in any one of SEQ ID NOs. 37 to 42, 46, 63, and 64; and / or (b) The sequence introduced into the stalk of the co-receptor comprises or consists of a fragment of the mucin-like sequence of CD43 described in any one of SEQ ID NOs. 53 to 62. The method according to claim 16.

18. The method according to claim 13, wherein the co-receptor is CD2, L-selectin, α4 ​​integrin, LFA-1, CD28, PD-1, 4-1BB, or CD6.

19. The method according to claim 18, wherein the co-receptor is CD2.

20. Immunoeffector cells obtained or obtainable by the method described in any one of claims 13 to 19.

21. A composition for use in a method for treating a target cancer, comprising immune effector cells according to any one of claims 1 to 10.

22. The composition according to claim 21, wherein the cancer is a hematological malignancy or a B-cell carcinoma.

23. A composition for use in a method of treating a target cancer, comprising the immune effector cells described in Claim 20.

24. The composition according to claim 23, wherein the cancer is a hematological malignant tumor or a B-cell carcinoma.

Images