Length-adapted chimeric antigen receptors and length-adapted helper receptors for improved CAR T cell activation

Abstract

The present invention relates to optimizing the effector function of an immune effector cell expressing a chimeric antigen receptor (CAR) co-localized with a co-receptor by ensuring that the membrane spacing spanned by a CAR-antigen complex is equivalent to the membrane spacing spanned by a co-receptor-ligand complex.

Description

Technical Field

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

[0002] T cells patrol the body, searching for antigens derived from infectious organisms or cancer cells. They use the T cell antigen receptor (TCR) to recognize peptide antigens on the major histocompatibility complex (pMHC). T cells have remarkable antigen sensitivity; they can be activated when they recognize only a single pMHC. This high sensitivity is important because infectious organisms and cancers use evasion mechanisms to reduce the amount of antigen presented to T cells.

[0003] An exciting new cancer therapy is to reprogram a patient’s T cells to target their cancer. This is accomplished by genetically engineering the T cells to express a chimeric antigen receptor (CAR). The CAR enables the patient’s T cells to recognize and kill cancer cells. This therapy has been approved for the treatment of B cell cancers. However, many patients relapse due to low levels of the target antigen expressed by their B cells. It is now clear that CARs have a serious defect in antigen sensitivity; CARs require 100-1000 times more antigen than TCRs to activate T cells. The mechanism of CAR’s defect in antigen sensitivity has not yet been elucidated.

[0004] Several attempts have been made to improve CARs, focusing on their intracellular signaling domains and / or targeting multiple different antigens. The inventors previously found that the defects in CAR sensitivity can be mainly attributed to the inability of CAR to utilize coreceptors (1).

[0005] There is an urgent need to improve the sensitivity of CARs to prevent cancer recurrence. More sensitive CARs will also allow CAR T cells to be used to treat more types of cancer.

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

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

[0008] Specifically, the inventors found that adjusting the size of the extracellular part of CAR, and then adjusting the height of the extracellular antigen binding domain of CAR, can lead to different antigen sensitivity, which can be demonstrated by the regulation of T cell effector function. Example 1 shows that the antigen sensitivity of CAR (using CD8a or CD28 hinge region between the extracellular antigen binding domain and the transmembrane domain) prepared according to the conventional design of the art is poor. On the other hand, compared with CAR prepared according to conventional design, CAR with a shorter hinge region, so that the extracellular antigen binding domain is shorter than the distance from the immune effector cell membrane, shows good antigen sensitivity, which is comparable to T cell receptor (TCR) and synthetic T cell receptor and antigen receptor (STAR), but higher than conventional CAR and ε-T cell receptor fusion construct (εTRuC). This shows that the extracellular size (rather than intracellular signaling) optimizes antigen recognition. In addition, smaller CARs do not bind to the TCR-CD3 complex, which is a favorable feature for clinical application purposes.

[0009] The inventors also showed that regulating the size of the extracellular part of the auxiliary receptor, and then regulating the height of the extracellular ligand binding domain of the auxiliary receptor (such as CD2) from the immune effector cell membrane, will lead to changes in the antigen sensitivity of CAR, which can be demonstrated by the regulation of T cell effector function. Example 2 shows that by increasing the height of the extracellular ligand binding domain of the auxiliary receptor (such as CD2), the antigen sensitivity of the CAR prepared according to the conventional design in the art can be improved. Therefore, by changing the size of the extracellular part to engineer the auxiliary receptor-ligand complex, the ability of the CAR-antigen complex can be maximized.

[0010] like Figure 1As shown in A, the co-receptor-ligand complex spans a membrane distance that matches the TCR-pMHC complex (e.g., 14 nm for the CD2-CD58 complex). However, this membrane distance does not apply to CARs (CARs) prepared according to conventional designs in the art. CON , i.e., a CAR comprising a CD28 hinge region or a CD8a hinge region) and a target antigen for optimization. The usefulness of the vsCAR and variable size co-receptors of the present invention will be described below.

[0011] For example, Figure 1 As shown in B, if CAR CON If the membrane distance spanned by the target antigen complex is larger than the membrane distance spanned by the co-receptor-ligand complex, CAR signal transduction will be weak ( Figure 1 B Left). To improve CAR signaling, a CAR CON The small size of vsCAR allows the membrane spacing spanned by the vsCAR-antigen complex to match that of the coreceptor-ligand complex (e.g., 14 nm for the CD2-CD58 complex (2, 3)). This results in optimal membrane alignment ( Figure 1 B right). Or, Figure 1 As shown in C, using a coreceptor of variable size that is larger than the corresponding endogenous coreceptor (e.g., an elongated CD2) can also achieve optimal membrane alignment and thus improve antigen recognition. Therefore, the membrane distance spanned by the coreceptor-ligand complex of variable size is similar to that of CAR. CON -Antigen complex matching.

[0012] For example, Figure 1 As shown in D, when the target antigen is large, it is difficult to optimize membrane alignment using endogenous co-receptor-ligand (such as CD2-CD58) complexes, because even a small-sized vsCAR may span too long a membrane distance to form a complex with the target antigen. In this case, vsCAR can be paired with co-receptors of variable size to optimize membrane alignment, thereby improving antigen recognition and CAR signaling.

[0013] The preparation of coreceptors of variable size can be achieved by modifying the size of the stalk between the extracellular ligand binding domain and the transmembrane domain, thereby determining the height of the coreceptor's extracellular ligand binding domain from the immune effector cell membrane. Figure 1As shown in E, a variety of sequences that can physically increase the height of the ligand binding domain from the immune effector cell membrane can be used (also referred to herein as non-flexible spacers). Therefore, the present invention provides a co-receptor comprising a stem region of variable size to control the height of the extracellular ligand binding domain of the co-receptor for optimizing the effector function of the immune effector cell triggered by CAR, wherein the co-receptor and the CAR are expressed on the immune effector cell.

[0014] The preparation of vsCAR can be carried out by modifying the size of the hinge region between the antigen binding domain and the transmembrane domain, thereby determining the height of the extracellular antigen binding domain of CAR from the immune effector cell membrane. Figure 1 F, a variety of sequences that physically increase the height of the ligand binding domain from the immune effector cell membrane (also referred to herein as non-flexible intervals) can be used. Therefore, the present invention provides a CAR comprising a variable size hinge region to control the height of the extracellular antigen binding domain of CAR for optimizing the effector function of immune effector cells expressing CAR.

[0015] In addition, the inventors have overcome a major problem in the field of CAR technology. Specifically, CARs that have entered the clinic and / or proven successful in clinical trials are usually targeted at membrane proximal epitopes expressed on APCs, compared to the antigenic sites of peptide-MHC complexes. This enables such CARs to utilize endogenous auxiliary receptors (such as CD2-CD58 complexes) to enhance the activation of T cells. The inventors now provide a mechanism by which CARs can successfully target membrane distal antigens expressed on APCs, such as peptide-MHC complexes themselves or larger antigens, while retaining the effect of auxiliary receptors to enhance T cell activation.

[0016] Therefore, the present invention provides an immune effector cell comprising:

[0017] - a chimeric antigen receptor (CAR) capable of binding to an antigen on an antigen presenting cell (APC), wherein the CAR comprises 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 connected by a hinge region; and

[0018] - a coreceptor capable of binding to a ligand on the APC, wherein the coreceptor comprises an extracellular ligand binding domain and a transmembrane domain, wherein the extracellular ligand binding domain and the transmembrane domain are connected by a stem region;

[0019] in:

[0020] (a) the coreceptor is an exogenous coreceptor and the stem region comprises a sequence that physically increases the height of the extracellular ligand binding domain from the immune effector cell membrane;

[0021] (b) the hinge region of the CAR comprises a sequence that physically increases the height of the antigen binding domain from the immune effector cell membrane, or consists of a sequence that physically increases the height of the antigen binding domain from the immune effector cell membrane, wherein the sequence comprises a mucin-like sequence, one or more folded polypeptide domains, or a fragment of CD28 as shown in SEQ ID NO: 10 or a fragment of the CD8a hinge region as shown in SEQ ID NO: 43; and / or

[0022] (c) The CAR and the coreceptor have respective sizes such that the membrane distance spanned by the CAR-antigen complex is comparable to the membrane distance spanned by the coreceptor-ligand complex.

[0023] 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, the 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 connected by a hinge region, wherein the hinge region comprises a sequence that physically increases the height of the antigen binding domain from the immune effector cell membrane, or is composed of a sequence that physically increases the height of the antigen binding domain from the immune effector cell membrane, wherein the sequence comprises a mucin-like sequence, one or more folded polypeptide domains, or a fragment of CD28 as shown in SEQ ID NO: 10 or a fragment of the CD8a hinge region as shown in SEQ ID NO: 43.

[0024] The present invention also provides an auxiliary receptor capable of binding to a ligand on an APC, the auxiliary receptor comprising an extracellular ligand binding domain and a transmembrane domain, wherein the extracellular ligand binding domain and the transmembrane domain are connected by a stem region, wherein the stem region comprises a sequence that physically increases the height of the ligand binding domain from the immune effector cell membrane, or is composed of a sequence that physically increases the height of the ligand binding domain from the immune effector cell membrane, optionally, wherein the stem region comprises a mucin-like sequence.

[0025] The present invention also provides an immune effector cell comprising or encoding the CAR and / or auxiliary receptor of the present invention.

[0026] The present invention also provides a method for preparing immune effector cells, comprising introducing a nucleic acid encoding the CAR and / or auxiliary receptor of the present invention into the immune effector cells.

[0027] The present invention also provides a method for optimizing the effector function of immune effector cells, wherein the immune effector cells comprise:

[0028] - a chimeric antigen receptor (CAR) capable of binding to an antigen on an antigen presenting cell (APC), wherein the CAR comprises 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 connected by a hinge region; and

[0029] - a coreceptor capable of binding to a ligand on the APC, wherein the coreceptor comprises an extracellular ligand binding domain and a transmembrane domain, wherein the extracellular ligand binding domain and the transmembrane domain are connected by a stem region;

[0030] Wherein, the method comprises modifying the height of the extracellular antigen binding domain of the CAR from the immune effector cell membrane and / or the height of the extracellular ligand binding domain of the auxiliary receptor from the immune effector cell membrane to optimize the effector function of the immune effector cell when contacting the APC.

[0031] The present invention also provides a method for identifying an improved immune effector cell, wherein the immune effector cell comprises:

[0032] - a chimeric antigen receptor (CAR) capable of binding to an antigen on an antigen presenting cell (APC), wherein the CAR comprises 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 connected by a hinge region; and

[0033] - a coreceptor capable of binding to a ligand on the APC, wherein the coreceptor comprises an extracellular ligand binding domain and a transmembrane domain, wherein the extracellular ligand binding domain and the transmembrane domain are connected by a stem region;

[0034] The method comprises:

[0035] (a) modifying the height of the extracellular antigen binding domain of the CAR from the immune effector cell membrane and / or the height of the extracellular ligand binding domain of the auxiliary receptor from the immune effector cell membrane; and

[0036] (b) determining whether the immune effector cells expressing the modified CAR and / or the modified coreceptor have improved effector function compared to immune effector cells expressing the unmodified CAR and / or the unmodified coreceptor.

[0037] 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 compared to an immune effector cell expressing an unmodified CAR and / or an unmodified co-receptor.

[0038] The present invention also provides an immune effector cell obtained or obtainable by the method of the present invention.

[0039] The present invention also provides a method for preparing an immune effector cell population for adoptive cell therapy, such as an in vivo or in vitro method, which comprises culturing the immune effector cells of the present invention to produce an immune effector cell population.

[0040] The present invention also provides a population of immune effector cells produced by the method of the present invention.

[0041] The present invention also provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of the immune effector cell or immune effector cell population of the present invention.

[0042] The present invention also provides use of the immune effector cell or immune effector cell population of the present invention in a method for treating cancer in a subject.

[0043] The present invention also provides use of the immune effector cell or immune effector cell population of the present invention in preparing a drug for treating cancer.

[0044] The present invention also provides use of the immune effector cell or immune effector cell population of the present invention for treating cancer.

[0045] The present invention also provides use of the immune effector cell or immune effector cell population of the present invention in adoptive cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1.Schematic diagram of the mechanism for optimizing antigen sensitivity through membrane alignment. (A) The membrane distance spanned by the co-receptor-ligand complex (such as CD2-CD58) (such as about 14nm) matches the TCR / pMHC binding. (B) In the case where the size of the standard CAR does not match the size of the co-receptor-ligand complex (such as CD2-CD58) (left figure), a hinge region of appropriate size can be selected to insert into the CAR to match this distance (right figure). For example, a variable-sized CAR (vsCAR) can be used so that the membrane distance spanned by the vsCAR-antigen complex matches the co-receptor-ligand complex, thereby achieving optimal membrane alignment and improving antigen recognition. (C) Alternatively, a variable-sized co-receptor (such as an elongated CD2) can be used so that the membrane distance spanned by the variable-sized co-receptor-ligand complex matches the conventional CAR-antigen complex, thereby achieving optimal alignment of the conventional CAR with the target antigen, thereby improving antigen recognition. (D) In ​​the case of large target antigens, it is difficult to optimize membrane alignment using naturally occurring co-receptor-ligand complexes (such as CD2-CD58), because even vsCAR may span a membrane spacing that is too large for the wild-type complex (left figure). In this case, vsCAR can be paired with co-receptors of variable size to optimize membrane alignment (right figure). (E) Design of co-receptors of variable size. The size of the co-receptor can be changed by using a non-flexible spacer (such as any one of SEQ ID NOs: 50 to 62) composed of different numbers of amino acid residues (such as 4, 8, 20, 40 or 234 amino acid residues) taken from CD43. See, for example, the elongated CD2 variants shown in SEQ ID NOs: 27 to 36. (F) Design of vsCAR. The shortest CAR contains a "mini", "micro", "nanoscale" or truncated hinge region from the therapeutic CD28 CAR (see, for example, SEQ ID NOs: 37, 41 and 42). The size of this CAR can be changed by using a non-flexible spacer (such as any one of SEQ ID NOs: 50 to 62) composed of different numbers of amino acid residues (such as 4, 8, 20, 40 or 234 amino acid residues) taken from CD43. For example, see SEQ ID NOs: 38 to 40, which can be used for hinge regions other than the hinge region of mini CD28.

[0047] Figure 2.Schematic diagram of 1G4 T cell receptor ("TCR"), D52N CAR ("CAR"), D52N Fab CAR ("Fab CAR"), and IgG1 antibody. 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. 1G4 TCR uses an endogenous CD3 component. D52N CAR is a CAR prepared according to a conventional design, containing a D52N single-chain variable fragment (scFv). D52N is an antigen binding domain that can recognize the same polypeptide antigen as that recognized by the 1G4 TCR. Conventional designs include, for example, D52N scFv, followed by a hinge region and a transmembrane domain (such as CD28 or CD8a), and an intracellular signaling domain (such as a CD28 cytoplasmic domain and a ζ chain (CD247) cytoplasmic domain). D52N FabCAR is a CAR of the present invention. It includes the variable heavy domain of D52N scFv coupled to IgG1-CH1 and the variable light domain of D52N scFv coupled to the IgG1-CL domain, each of the light and heavy domains are coupled to the CD28 transmembrane domain, the CD28 cytoplasmic domain and the ζ chain (CD247) signaling tail.

[0048] 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) Figure 2 As shown. The antigen receptors all recognized the same peptide antigen (SLLMWITQV; SEQ ID NO:66) presented on HLA-A*02:01. In the case of TCR, the variable domain of 1G4 TCR was used, while for the other receptors tested, the D52N variable domain from the 3M4E5 antibody was used (4, 5).

[0049] Figure 4 TCRα transduced with 1G4 TCR, D52N-CD28-28zCAR, and D52N-FabCAR-28z receptors - β - Surface CD3ε and surface antigen receptor expression profiles of Jurkat cells. E6.1 TCRα lentivirally transduced with the indicated constructs - β -Jurkat cells were stained. Tetramers of pMHC antigens detected the surface levels of each antigen receptor (y-axis), and UCHT antibodies detected surface CD3ε levels. CAR and Fab CAR constructs were detected on the cell surface, but there was no CD3ε upregulation, indicating that these receptors are CD3-independent, which is not the case for the 1G4 TCR.

[0050] 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 that were lentivirally transduced with the corresponding receptors and then antibiotic selected.

[0051] Figure 6 .Flow cytometry assessment of activation of primary CD8+T cells expressing different antigen receptors. T cells were incubated with T2 target cell line loaded with different concentrations of NY-ESO-1 9V peptide antigen for 20 hours before evaluating surface activation markers (A) 4-1BB and (B) CD25. (C) EC50 of three independent donors. EC50 is the concentration of antigen required to elicit 50% of the maximal response. Logarithmic transformation of EC50 values ​​of 4-1BB and CD25 readings were statistically analyzed using Dunnett's multiple comparison test (ns = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001).

[0052] Figure 7 Activation of primary CD8+ T cells expressing different antigen receptors was assessed by cytokine production. T cells were incubated with the T2 target cell line loaded with different concentrations of NY-ESO-1 9V peptide antigen for 20 hours before ELISA assessment of the cytokines IFN-g (upper row) and IL-2 (lower row). Data are from three independent donors.

[0053] Figure 8 .The antigen sensitivity of CAR can be improved by changing the size of the hinge region. (A) Schematic diagram of a conventional CAR with a CD28 hinge region (SEQ ID NO: 10) and a variable size CAR with a specified hinge region. The CD28 mini-hinge region contains a smaller hinge region (SEQ ID NO: 37) compared to the conventional CAR. The larger hinge region is generated by adding a specified size interval from CD43 (see SEQ ID NOs: 38 to 40). (B) Primary human CD8+ T cells expressing the specified antigen receptors were co-cultured with Nalm6 target cells pulsed with the specified concentration of peptide antigen. Surface expression of 4-1BB activation markers was measured after 5 hours. (C) Fitted EC50 of the specified antigen receptors.

[0054] Fig. 9 .Schematic diagram of the mechanism of optimizing antigen sensitivity by membrane alignment using variable-size coreceptors. (A) Design of variable-size coreceptors. In this study, the ligand-binding domain of the coreceptor is the extracellular domain (ectodomain) of CD2, extended by a mucin-like sequence fragment from the extracellular part of CD43. The transmembrane domain and the intracellular domain are taken from wild-type CD2. (B) Schematic diagram of the experimental system. Jurkat TCRα that does not express CD8 - β - T cells were subjected to CRISPR to remove endogenous CD2. Jurkat TCRα was first transduced with antigen receptor - β - CD2 - Then, CD2 WT or coreceptors of different variable sizes designed in (A) were used for transduction.

[0055] Fig.10 . Wild-type CD2 optimizes the antigen sensitivity of the T cell receptor. (A) Schematic diagram of the antigen receptor. (B, C) Surface CD69 of T cells co-cultured with the U87 target cell line pulsed with the indicated concentrations of peptide antigens. Representative dose response (B) and EC50 (C) of multiple experiments. Arrows indicate CD2 that optimizes antigen sensitivity.

[0056] Fig.11 . Wild-type CD2 optimizes the antigen sensitivity of STAR. (A) Schematic diagram of antigen receptor. (B, C) Surface CD69 of T cells co-cultured with U87 target cell line pulsed with peptide antigen at the indicated concentrations. Representative dose response (B) and EC50 (C) of multiple experiments. Arrows indicate CD2 with optimized antigen sensitivity.

[0057] Fig.12 .CD2-CD43(40) optimizes the antigen sensitivity of D52N-CD8a-zCAR. (A) Schematic diagram of antigen receptor. (B, C) Surface CD69 of T cells co-cultured with U87 target cell line pulsed with peptide antigen at the indicated concentrations. Representative dose response (B) and EC50 (C) of multiple experiments. Arrows indicate CD2 that optimizes antigen sensitivity.

[0058] Fig.13 .CD2-CD43(20) optimizes the antigen sensitivity of D52N-CD28-28zCAR. (A) Schematic diagram of antigen receptor. (B, C) Surface CD69 of T cells co-cultured with U87 target cell line pulsed with peptide antigen at the indicated concentrations. Representative dose response (B) and EC50 (C) of multiple experiments. Arrows indicate CD2 that optimizes antigen sensitivity.

[0059] Fig.14 .CD2-CD43(20) optimizes the antigen sensitivity of ε-TRuC(D52N). (A) Schematic diagram of antigen receptors. (B, C) Surface CD69 of T cells co-cultured with U87 target cell line pulsed with the indicated concentrations of peptide antigens. Representative dose response (B) and EC50 (C) of multiple experiments. Arrows indicate CD2 that optimizes antigen sensitivity.

[0060] Fig.15 .Lengthening the CD2-CD58 complex impairs antigen recognition by the TCR (top) but improves antigen recognition by the CAR (bottom), based on suboptimal and optimal membrane alignment, respectively.

[0061] Fig.16 .A. Jurkat T cells were transduced with FM63-CD8a-41BBz (Kymriah) chimeric antigen receptors. Surface levels were detected by GFP-Spycatcher fused to Spytag-CD19. B. Jurkat T cells expressing Kymriah were transduced with CD2 molecules of variable size, and cells with matching expression were sorted using anti-CD2 PE. C. Nalm6 CombiCells expressing Spycatcher were coupled to purified Spytag-CD19 at different concentrations and detected by flow cytometry. D. Representative dose responses of Kymriah CAR Jurkat T cells with the indicated variable size CD2 molecules. EN = Generalized measure of antigen sensitivity (EC50) in 3 independent experiments. A t-test was used to determine the p-value for the null hypothesis that the EC50 was the same for CD2 WT and other CD2 conditions, and a Sidak-Holm correction was performed for multiple comparisons. Abbreviations: * = p-value ≤ 0.05, ** = p-value ≤ 0.01, *** = p-value ≤ 0.01. DETAILED DESCRIPTION

[0062] Chimeric Antigen Receptor ( CAR )

[0063] The immune effector cells of the present invention include chimeric antigen receptors (CARs). CAR is a non-natural protein that includes an extracellular portion that includes an antigen-specific antigen binding domain that is connected to an intracellular portion via a hinge region and a transmembrane domain, and the intracellular portion includes one or more signaling molecules.

[0064] The immune effector cells of the present invention may comprise the CAR of the present invention. In this embodiment, the immune effector cells may further comprise the auxiliary receptor of the present invention.

[0065] The immune effector cells of the present invention may comprise a CAR prepared according to conventional design, also referred to herein as a conventional CAR or a CAR CON , as described in reference 6, comprising the CD28 hinge region shown in SEQ ID NO: 10 or the CD8a hinge region shown in SEQ ID NO: 43. In this embodiment, the immune effector cell further comprises the auxiliary receptor of the present invention.

[0066] Compared with conventional CAR, the CAR of the present invention has improved antigen sensitivity. The size of the CAR of the present invention is such that the membrane spacing spanned by the CAR-antigen complex is comparable to the membrane spacing spanned by the auxiliary receptor-ligand complex. The size (such as height) of the extracellular portion of CAR, especially the height of the extracellular antigen binding domain of CAR from the immune effector cell membrane, is very important for determining antigen sensitivity. The optimal size (such as height) of the extracellular portion of CAR, especially the optimal height of the extracellular antigen binding domain of CAR from the immune effector cell membrane, depends on the size (such as height) of the target antigen, auxiliary receptor and its ligand, which will be further described below.

[0067] The CAR of the present invention may comprise an extracellular antigen binding domain having a height comparable to (e.g., within ≤5% or ≤10%, or the same as) the height of the T cell receptor extracellular antigen binding domain from the immune effector cell membrane.

[0068] The extracellular antigen binding domain of the CAR of the present invention is about 7 nm high from the immune effector cell membrane.

[0069] The CAR of the present invention may comprise an extracellular antigen binding domain, which is higher than the height of the T cell receptor extracellular antigen binding domain from the immune effector cell membrane, for example, ≤15%, ≤20%, ≤25% or ≤30% higher.

[0070] The height of the extracellular antigen binding domain of the CAR of the present invention from the immune effector cell membrane may be higher than about 7nm but lower than about 47nm. The height of the extracellular antigen binding domain of the CAR of the present invention from the immune effector cell membrane may be>7nm, ≥10nm, ≥15nm, ≥20nm, ≥25nm, ≥30nm, ≥35nm, ≥40nm, ≥45nm. The height of the extracellular antigen binding domain of the CAR of the present invention from the immune effector cell membrane may be ≤45nm, ≤40nm, ≤35nm, ≤30nm, ≤25nm, ≤20nm, ≤15nm or ≤10nm.

[0071] The CAR of the present invention may comprise an extracellular antigen binding domain whose height is lower than the height of the T cell receptor extracellular antigen binding domain from the immune effector cell membrane, for example, ≤15%, ≤20%, ≤25% or ≤30% lower.

[0072] The height of the extracellular antigen binding domain of the CAR of the present invention from the immune effector cell membrane may be higher than about 3 nm but lower than about 7 nm. The height of the extracellular antigen binding domain of the CAR of the present invention from the immune effector cell membrane may be ≥3 nm, ≥4 nm, ≥5 nm or ≥6 nm. The height of the extracellular antigen binding domain of the CAR of the present invention from the immune effector cell membrane may be <7 nm, ≤6 nm, ≤5 nm or ≤4 nm.

[0073] The height of the extracellular antigen binding domain of the CAR of the present invention from the immune effector cell membrane can be determined according to routine methods in the art described herein.

[0074] The hinge region between the antigen binding domain and the transmembrane domain can determine the size (such as height) of the extracellular portion of the CAR of the present invention, in particular the height of the extracellular antigen binding domain. The inventors found that the antigen sensitivity of conventional CARs is poor. This is improved by adjusting (e.g., increasing or decreasing) the size of the CAR hinge region to optimize membrane alignment.

[0075] Therefore, the CAR of the present invention comprises a hinge region of a different size compared to conventional CARs, such that the CAR of the present invention may have a smaller or larger extracellular portion, and thus have a shorter or taller extracellular antigen binding domain than conventional CARs.

[0076] The CAR of the present invention does not comprise a hinge region consisting of: (a) the CD8a hinge region shown in SEQ ID NO:43 or (b) the CD28 hinge region shown in SEQ ID NO:10.

[0077] The hinge region of the CAR of the present invention comprises a sequence that physically increases the height of the antigen binding domain from the immune effector cell membrane, or consists of a sequence that physically increases the height of the antigen binding domain from the immune effector cell membrane.

[0078] The sequence can be a non-flexible sequence, for example, each block of amino acid residues in the sequence physically increases the height of the antigen binding domain from the immune effector cell membrane because the sequence itself does not compress or fold back. The block can contain one, two, three, four, five, six, seven, eight, nine, ten or more amino acid residues.

[0079] The sequence may be a bulky sequence that physically increases the height of the antigen binding domain from the immune effector cell membrane, for example by steric hindrance, thereby preventing the antigen binding domain from contacting the membrane.

[0080] The sequence may comprise or consist of a mucin-like sequence, one or more folded domains, or a fragment hinge region of the CD28 or CD8a hinge region shown in SEQ ID NOs: 10 and 43, respectively. The mucin-like peptide may be a fragment of the CD43 extracellular domain, 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.

[0081] The sequence that physically increases the height of the antigen binding domain from the immune effector cell membrane can be any suitable length. The length of the sequence can be at least 1 amino acid, such as at least 5, at least 10 or at least 20 amino acids. The length of the sequence can be 250 or less amino acids, 200 or less amino acids, 150 or less amino acids, or 100 or less amino acids, such as 80 or less amino acids, 60 or less amino acids, 40 or less amino acids, 30 or less amino acids, or 20 or less amino acids. The sequence length can be 1 to 40 amino acids, such as 2 to 30, 3 to 25, 4 to 20 or 5 to 15 amino acids.

[0082] The sequence that physically increases the height of the antigen binding domain from the immune effector cell membrane can be an immunoglobulin domain, such as an immunoglobulin constant domain, such as IgG1, IgG2, IgG3, IgG4, CD8 (such as CD8α hinge region) or the CH2 and CH3 regions of CD28. For example, with CAR CON In contrast, the CAR of the present invention can replace the hinge region of CD28 or CD8a with an immunoglobulin constant domain that is smaller in size than the hinge region.

[0083] In some cases, the sequence that physically increases the height of the antigen binding domain from the immune effector cell membrane is not an immunoglobulin domain.

[0084] The sequence that physically increases the height of the antigen binding domain from the immune effector cell membrane can be a fragment of the hinge region of CD28 shown in SEQ ID NO: 10. Preferably, the fragment retains the cysteine ​​residue at position 29 of SEQ ID NO: 10. Alternatively, the fragment may comprise a modification to position 29 of SEQ ID NO: 10, such as removing the cysteine ​​residue by substitution. For example, the fragment may consist of ≤20 consecutive amino acids from the C-terminus of SEQ ID NO: 10 or ≤30 consecutive amino acids from the N-terminus of SEQ ID NO: 10. The fragment may consist of ≤5, ≤10, ≤15, ≤20, ≤25 or ≤30 consecutive amino acids from SEQ ID NO: 10 and contain the cysteine ​​residue at position 29 of SEQ ID NO: 10. The fragment may consist of any one of the sequences shown in SEQ ID NO: 37, 41 or 42. For example, with a CAR containing the full-length SEQ ID NO: 10 CON In comparison, the CAR of the present invention may have a shorter CD28 hinge region.

[0085] The sequence that physically increases the height of the antigen binding domain from the immune effector cell membrane can be a fragment of the CD8a hinge region shown in SEQ ID NO: 43. Preferably, the fragment retains the cysteine ​​residue at position 27 of SEQ ID NO: 43. Alternatively, the fragment may include a modification to position 27 of SEQ ID NO: 43, such as removing the cysteine ​​residue by substitution. For example, the fragment may be composed of ≤30 or ≤25 consecutive amino acids from the C-terminus of SEQ ID NO: 43, or ≤30 consecutive amino acids from the N-terminus of SEQ ID NO: 43. The fragment may be composed of ≤5, ≤10, ≤15, ≤20, ≤25 or ≤30 consecutive amino acids from SEQ ID NO: 43, and contains a cysteine ​​residue at position 27 of SEQ ID NO: 43. The fragment may be composed of any one of the sequences shown in SEQ ID NO: 46, 63 and 64. For example, compared to a conventional CAR containing the full-length SEQ ID NO: 43, the CAR of the present invention may have a shorter CD8a hinge region.

[0086] The CAR of the present invention may include one or more (e.g., ≥2, ≥3, ≥4, ≥5, ≥6, ≥7, ≥8, ≥9 or 10) immunoglobulin domains, such as immunoglobulin constant domains. The CAR of the present invention may include no more than 10 immunoglobulin constant domains. Multiple immunoglobulin domains can be coupled in series or in parallel, for example, when the CAR of the present invention includes more than one polypeptide, each polypeptide may include one or more immunoglobulin domains, such as ≥2, ≥3, ≥4 or ≥5 immunoglobulin domains.

[0087] The sequence that physically increases the height of the antigen binding domain from the immune effector cell membrane can be a mucin-like sequence or a fragment or derivative thereof. The characteristics of the mucin-like sequence are that it is rich in protein, serine and threonine residues, and the serine and threonine residues are heavily O-glycosylated. The mucin-like sequence can be a mucin-like sequence of the extracellular portion of CD43, i.e., a mucin-like sequence as shown in SEQ ID NO:62. Mucin-like sequences from other proteins can also be used, such as MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC13, MUC15, MUC16, MUC17, MUC18, MUC20, MUC21, PSGL-1. The stem region of the surface protein of the mucin-like stem region can also be used, such as CD8a and CD28.

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

[0089] The CAR of the present invention can be a dimer, such as a heterodimer. For example, CAR may include two fusion proteins that form a dimer, and for each fusion protein, the antigen binding domain may be coupled to a folded polypeptide domain (such as an immunoglobulin constant domain), followed by a transmembrane domain (such as a CD28 transmembrane domain) and an intracellular signaling domain (such as a CD28 ζ chain signaling domain). For example, the first fusion protein may include a light chain variable domain as an antigen binding domain and a light chain constant domain as a folded polypeptide domain, and the second fusion protein may include a heavy chain variable domain as an antigen binding domain and a heavy chain constant domain as a folded polypeptide domain. Such CARs are actually like antibody Fabs coupled to transmembrane domains (such as CD28 transmembrane domains) and intracellular signaling domains (such as CD28 ζ chain signaling domains). Therefore, the extracellular portion of such CARs includes two immunoglobulin constant domains (one for each chain) and two immunoglobulin light chains (one for each chain), and its size (such as height) is comparable to that of a T cell receptor. For example, CAR can be Figure 2 This allows immune effector cells containing such CARs to interact strongly with APCs displaying the antigen, forming a CAR-antigen complex that spans an inter-membrane distance of approximately 14 nm.

[0090] The antigen binding domain of the CAR of the present invention can be scFv, monoclonal antibody (comprising 2 heavy chains and 2 light chains), polyclonal antibody, Fab, Fab', F(ab')2 fragment, heavy chain variable domain (VH) or nano antibody (VHH). The ScFv domain comprises the heavy chain variable domain (VH) and light chain variable domain (VL) of immunoglobulin and is connected by a short connecting peptide.

[0091] The CAR of the present invention may include more than one extracellular antigen binding domain, such as two extracellular antigen binding domains or three extracellular antigen binding domains. Two or more extracellular antigen binding domains can bind to different antigens, i.e., CAR can be bispecific or multispecific.

[0092] The antigen binding domain of the CAR of the present invention may be specific to any antigen (such as the antigens listed in Table 2). The antigen may be a peptide-MHC complex, CD19, mesothelin, BCMA, CD22, EGFR or EGFRvIII. For example, the peptide of the peptide-MHC complex may be a NY-ESO-1 peptide, i.e., a peptide derived from NY-ESO-1 proteolysis.

[0093] The transmembrane domain of the CAR of the present invention can be derived from naturally occurring transmembrane proteins, such as type I transmembrane proteins. The transmembrane domain spans a cell membrane, such as a cell membrane of a eukaryotic cell. After the extracellular antigen binding domain (such as scFv) binds to the ligand, the transmembrane domain transmits the activation signal to the cytoplasmic signal transduction domain. The transmembrane domain of the CAR of the present invention is generally the transmembrane domain of CD28. The transmembrane domain can be an α, β, δ or γ subunit of a T cell receptor, a transmembrane domain of CD3ε, CD3ζ, CD4, CD6, CD8α, CD28, CD86, OX-40, 4-1BB or CD40L (CD154). For example, when the immune effector cell is a NK cell, the transmembrane domain can be the transmembrane domain of CD8.

[0094] The intracellular signaling domain of the CAR of the present invention may include any activation domain in the art. The role of the activation domain is to activate immune effector cells after engaging with an extracellular domain (such as scFv). The intracellular signaling domain of the CAR of the present invention may include one or more of CD3ζ (ζ) activation domain, 4-1BB (CD137) activation domain, CD3ε (ε) activation domain, OX40 (CD134) activation domain, CD28 activation domain and / or CD27 activation domain.

[0095] The intracellular signaling domain may comprise a CD3 zeta (ζ) activation domain, e.g., as in the first generation CARs in the art.

[0096] The intracellular signaling domain may comprise a CD3 ζ (ζ) activation domain and a CD28 activation domain, also referred to as a CD28z domain, e.g., as in second generation CARs in the art.

[0097] The intracellular signaling domain may comprise a 4-1BB activation domain and a CD3 ζ (ζ) activation domain, also referred to as a 4-1BBz domain, e.g., as in the third generation CARs of the art.

[0098] The intracellular signaling domain may comprise 4-1BBz and CD28z domains, comprising CD3ζ, CD28 and 4-1BB activation domains.

[0099] The intracellular signaling domain may comprise a CD3ζ (ζ) activation domain alone, or a combination of a CD3ζ (ζ) activation domain and a CD28, CD27, OX-40 (CD134) and / or 4-1BB (CD137) domain.

[0100] Typically, the intracellular signaling domain does not comprise a CD2 domain, such as a CD2 intracellular signaling domain.

[0101] Other activation domains include IL-15Rα, CD2, CDS, ICAM-1, LTA-1, and ICOS, and can be used in combination with the above activation domains.

[0102] If the immune effector cell expressing the CAR is a phagocytic cell, the intracellular signaling domain may comprise the intracellular domain of Megf10 or FcRv.

[0103] The expression of the CAR of the present invention on the surface of immune effector cells can be independent of the endogenous TCR-CD3 complex. The expression of the CAR of the present invention on the surface of immune effector cells may not require endogenous TCR and / or CD3.

[0104] Coreceptors

[0105] The immune effector cell of the present invention includes a co-receptor, which is co-localized with CAR at the antigen presenting cell (APC) immune synapse on the cell membrane. The co-receptor can bind to the ligand on the antigen presenting cell and includes an extracellular ligand binding domain, a transmembrane domain and an intracellular signal transduction domain.

[0106] The immune effector cells of the present invention may comprise the co-receptors of the present invention, ie, the modified co-receptors. In this embodiment, the immune effector cells may also comprise the CAR of the present invention.

[0107] Compared with the corresponding endogenous auxiliary receptor, the auxiliary receptor of the present invention can provide improved antigen sensitivity of CAR expressed on the same immune effector cell. The size of the auxiliary receptor of the present invention makes the membrane spacing spanned by the auxiliary receptor-ligand complex comparable to the membrane spacing spanned by the CAR-antigen complex. Therefore, the size (such as height) of the extracellular part of the auxiliary receptor, especially the height of the extracellular ligand binding domain of the auxiliary receptor from the immune effector cell membrane, is very important for determining the antigen sensitivity of CAR expressed on the same immune effector cell. The optimal size (such as height) of the extracellular part of the auxiliary receptor, especially the optimal height of the extracellular ligand binding domain of the auxiliary receptor from the immune effector cell membrane, depends on the size (such as height) of its ligand, CAR and target antigen, as will be further described below.

[0108] The co-receptors of the invention may comprise an extracellular ligand binding domain that is higher than the height of the extracellular ligand binding domain of the corresponding endogenous co-receptor from the immune effector cell membrane, for example, ≤15%, ≤20%, ≤25% or ≤30% higher.

[0109] The height of the extracellular ligand binding domain of the auxiliary receptor of the present invention from the immune effector cell membrane may be greater than about 7nm but less than about 47nm. The height of the extracellular ligand binding domain of the auxiliary receptor of the present invention from the immune effector cell membrane may be>7nm,≥10nm,≥15nm,≥20nm,≥25nm,≥30nm,≥35nm,≥40nm,≥45nm. The height of the extracellular ligand binding domain of the auxiliary receptor of the present invention from the immune effector cell membrane may be≤45nm,≤40nm,≤35nm,≤30nm,≤25nm,≤20nm,≤15nm or≤10nm.

[0110] The co-receptors of the invention may comprise an extracellular ligand binding domain that is shorter than the extracellular ligand binding domain of the corresponding endogenous co-receptor on the immune effector cell membrane, for example, ≤15%, ≤20%, ≤25% or ≤30% shorter.

[0111] The height of the extracellular ligand binding domain of the auxiliary receptor of the present invention from the immune effector cell membrane may be greater than about 3nm but less than about 7nm. The height of the extracellular ligand binding domain of the auxiliary receptor of the present invention from the immune effector cell membrane may be ≥3nm, ≥4nm, ≥5nm or ≥6nm. The height of the extracellular ligand binding domain of the auxiliary receptor of the present invention from the immune effector cell membrane may be <7nm, ≤6nm, ≤5nm or ≤4nm.

[0112] The height of the extracellular ligand binding domain of the coreceptor of the present invention from the immune effector cell membrane can be determined according to routine methods in the art as described herein.

[0113] The stem region between the ligand binding domain and the transmembrane domain can determine the size (such as height) of the extracellular portion of the auxiliary receptor of the present invention, in particular the height of the extracellular ligand binding domain. The inventors have found that the antigen sensitivity of conventional CAR is poor. This can be improved by adjusting (such as increasing or decreasing) the size of the stem region of the auxiliary receptor (such as CD2) to optimize the membrane alignment of conventional CAR.

[0114] Thus, the co-receptors of the invention comprise a stem region that is of a different size than the corresponding endogenous co-receptors, such that the CARs of the invention may have a smaller or larger extracellular portion, and thus a shorter or taller extracellular ligand binding domain than the corresponding endogenous co-receptors.

[0115] The stem region of the coreceptor of the invention comprises or consists of a sequence that physically increases the height of the ligand binding domain from the immune effector cell membrane.

[0116] The sequence can be a non-flexible sequence, for example, each block of residues in the sequence physically increases the height of the ligand binding domain from the immune effector cell membrane because the sequence itself does not compress or fold back. The block can contain one, two, three, four, five, six, seven, eight, nine, ten or more residues.

[0117] The sequence can be a bulky sequence that physically increases the height of the ligand binding domain from the immune effector cell membrane by, for example, steric hindrance, thereby preventing the ligand binding domain from contacting the membrane. The sequence that physically increases the distance of the ligand binding domain from the immune effector cell membrane can be the hinge region of the CAR of the present invention as described above.

[0118] The sequence that physically increases the height of the ligand binding domain from the immune effector cell membrane can be a mucin-like sequence or a fragment or derivative thereof. The characteristics of the mucin-like sequence are that it is rich in protein, serine and threonine residues, and the serine and threonine residues are heavily O-glycosylated. The mucin-like sequence can be a mucin-like sequence of the extracellular portion of CD43, i.e., a mucin-like sequence as shown in SEQ ID NO:62. Mucin-like sequences from other proteins can also be used, such as MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC13, MUC15, MUC16, MUC17, MUC18, MUC20, MUC21, PSGL-1. The stem region of the surface protein of the mucin-like stem region can also be used, such as CD8a and CD28.

[0119] The mucin-like sequence may be a fragment of the CD43 extracellular domain shown in SEQ ID 62. For example, the fragment may be 4 to 234 amino acids (e.g. Figure 1 E). The fragment may consist of ≥5, ≥10, ≥20, ≥30, ≥40, ≥50, ≥50, ≥70, ≥80, ≥90, ≥100, ≥110, ≥120, ≥130, ≥140, ≥150, ≥160, ≥170, ≥180, ≥190, ​​≥200, ≥210, ≥220, ≥230 consecutive amino acids from the N-terminus or C-terminus of SEQ ID NO: 62. The fragment may consist of no more than 234 amino acids. 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 one of SEQ ID NOs: 53 to 62.

[0120] In some cases, the sequence that physically increases the height of the ligand binding domain from the immune effector cell membrane is not an immunoglobulin domain.

[0121] The co-receptor may be any of the receptors listed in Table 1. Thus, the ligand may be CD2, L-selectin, α4 ​​integrin, LFA-1, CD28, PD-1, 4-1BB, or CD6. The co-receptor may be an adhesion receptor. For example, the co-receptor may be CD2.

[0122] Table 1 - Examples of co-receptors and their ligands

[0123]

[0124] Thus, the ligand may be CD58, GLYCAM1, VCAM-1, ICAM-1, ICAM-2, ICAM-3, CD80, CD86, PD-L1, PD-L2, 4-1BBL, or CD166. The ligand may be CD58. Typically, the ligand on the APC is different from the antigen on the APC.

[0125] The co-receptor may comprise the extracellular ligand binding domain of CD2, and the ligand may be CD58. The co-receptor may be a modified CD2 according to the present invention, and the ligand may be CD58.

[0126] Membrane alignment and membrane spacing

[0127] Membrane alignment is influenced by the size of receptor and ligand complexes, such as CAR and co-receptors co-localized on immune effector cells, and their corresponding antigens and ligands on APCs. When the membrane distance spanned by the complex between CAR and target antigen is comparable to the membrane distance spanned by the complex between certain co-receptors and their ligands, the membrane alignment of CAR recognition of antigen is optimized.

[0128] The membrane distance spanned by the complex between the CAR and the target antigen is comparable to (e.g., within ≤5%, ≤10%, ≤15%, ≤20%, ≤25%, or ≤30%, or the same as) the membrane distance spanned by the complex between the coreceptor and the ligand.

[0129] The membrane spacing spanned by the complex between the CAR of the present invention and the target antigen can be comparable to (e.g., within ≤5%, ≤10%, ≤15%, ≤20%, ≤25% or ≤30%, or the same as) the membrane spacing spanned by the complex between the T cell receptor and its peptide-MHC antigen.

[0130] The membrane spacing spanned by the complex between the CAR of the present invention and its antigen may be about 6nm and 14nm. The membrane spacing spanned by the complex between the CAR of the present invention and its antigen may be ≥6nm, ≥7nm, ≥8nm, ≥9nm, ≥10nm, ≥11nm, ≥12nm or ≥13nm. The membrane spacing spanned by the complex between the CAR of the present invention and its antigen may be ≤14nm, ≤13nm, ≤12nm, ≤11nm, ≤10nm, ≤9nm, ≤8nm or ≤7nm.

[0131] The membrane spacing spanned by the complex between the CAR of the present invention and its antigen may be about 14nm and 54nm. The membrane spacing spanned by the complex between the CAR of the present invention and its antigen may be ≥15nm, ≥20nm, ≥25nm, ≥30nm, ≥35nm, ≥40nm, ≥45nm or ≥50nm. The membrane spacing spanned by the complex between the CAR of the present invention and its antigen may be ≤50nm, ≤45nm, ≤40nm, ≤35nm, ≤30nm, ≤25nm, ≤20nm or ≤15nm.

[0132] Likewise, the membrane spacing spanned by the complex between the co-receptor of the present invention and its ligand may be about 14 nm and 54 nm. The membrane spacing spanned by the complex between the co-receptor of the present invention and its ligand may be ≥15 nm, ≥20 nm, ≥25 nm, ≥30 nm, ≥35 nm, ≥40 nm, ≥45 nm, or ≥50 nm. The membrane spacing spanned by the complex between the co-receptor of the present invention and its ligand may be ≤50 nm, ≤45 nm, ≤40 nm, ≤35 nm, ≤30 nm, ≤25 nm, ≤20 nm, or ≤15 nm.

[0133] In one embodiment, an immune effector cell of the invention comprises a CAR of the invention (e.g., Figure 1 B), and the membrane distance spanned by the complex between the CAR of the present invention and the target antigen is comparable to the membrane distance spanned by the complex between the endogenous co-receptor and its ligand (e.g., within ≤5%, ≤10%, ≤15%, ≤20%, ≤25% or ≤30%, or the same). The CAR of the present invention has an appropriate height (h CAR ) of the extracellular antigen-binding domain, the height (h CAR ) can be determined based on the membrane distance (x) spanned by the complex between the endogenous coreceptor and its ligand and the size (e.g., height) of the antigen epitope targeted by the CAR on the APC membrane (h ant ) to determine. CAR Can be used with h CAR =xh antThe determined values ​​are comparable (e.g., within or the same as ≤5%, ≤10%, ≤15%, ≤20%, ≤25%, or ≤30%). CAR Can be obtained by xh ant Provided herein are examples of CARs of the invention having an extracellular antigen binding domain at an appropriate height from the immune effector cell membrane.

[0134] The immune effector cells of the present invention comprise CAR CON (i.e., a CAR comprising a CD28 hinge region or a CD8a hinge region) and an embodiment of a co-receptor of the invention (e.g., Figure 1 C) CAR CON The membrane distance spanned by the complex between the co-receptor and the target antigen is comparable to (e.g., within ≤5%, ≤10%, ≤15%, ≤20%, ≤25%, or ≤30%, or the same as) the membrane distance spanned by the complex between the co-receptor of the present invention and its ligand. The co-receptors of the present invention have an appropriate height (h acc ) of the extracellular ligand-binding domain, the height (h acc ) can be based on CAR CON The distance between the membrane and the target antigen (y) and the height of the ligand epitope (h lig ) to determine. acc Can be used with h acc =yh lig The determined values ​​are comparable (e.g., within or the same as ≤5%, ≤10%, ≤15%, ≤20%, ≤25%, or ≤30%). acc Can be obtained by h acc =yh lig Provided herein are examples of coreceptors of the invention having an extracellular ligand binding domain at an appropriate height from the immune effector cell membrane.

[0135] In embodiments where the immune effector cell comprises a CAR of the invention and a co-receptor of the invention (e.g., Figure 1 D), the membrane distance formed by the complex between the CAR of the present invention and the target antigen is comparable to the membrane distance spanned by the complex between the auxiliary receptor of the present invention and its ligand (e.g., within ≤5%, ≤10%, ≤15%, ≤20%, ≤25% or ≤30%, or the same). The CAR of the present invention has an appropriate height (h) from the immune effector cell membrane. CAR ) extracellular antigen binding domain, the auxiliary receptor of the present invention has an appropriate height (h acc ) of the extracellular ligand-binding domain, these two heights can be determined by considering the height of the antigen epitope (h ant ) and the height of the ligand epitope (hlig ) is determined, for example, by h CAR +h ant =h acc +h lig The present invention provides a method for determining the height (h) of the immune effector cell membrane. CAR ) and an example of a co-receptor of the present invention having an extracellular antigen binding domain with an appropriate height from the immune effector cell membrane.

[0136] Therefore, the present invention also relates to a method for determining the appropriate size (such as height) of the extracellular antigen binding domain of the CAR of the present invention and / or the appropriate size (such as height) of the extracellular ligand binding domain of the auxiliary receptor of the present invention.

[0137] The height of the membrane distance, extracellular antigen binding domain or extracellular ligand binding domain from the immune effector cell membrane of any membrane protein described herein can be determined according to conventional methods in the art. For example, (1) using theoretical methods, such as the methods used herein; (2) using existing structural information to predict the overall size, such as predicting from data in the protein database; (3) bioinformatics prediction tools based on protein amino acid sequences, such as using Alphafold to convert sequences into structures to estimate distances; or (4) microscopy, such as electron microscopy (7), immunofluorescence or quantum dot labeling. For example, the membrane distance spanned by the CD2-CD58 complex has been determined in references 3 and 6. The theoretical method used herein includes adding dimensions to the height of the known extracellular part of the protein, such as 3.7 nm for each immunoglobulin domain and / or 0.2 nm for each non-flexible amino acid (based on experimental data in reference 8). Electron microscopy can be used to directly measure the membrane distance. For example, T cells are incubated with APCs that present high concentrations of antigen to TCR or CAR. Direct measurements of membrane distance are taken at multiple locations of the contact interface (see references 7 and 9). Indirect methods can also be used, such as by comparing the activity of a CAR of the invention with a TCR, e.g., see the Examples below.

[0138] Immune effector cells

[0139] The immune effector cells of the present invention comprise CAR and / or the co-receptors of the present invention. The immune cells express the CAR and / or co-receptors of the present invention.

[0140] The immune effector cells of the present invention may comprise the CAR of the present invention. The CAR of the present invention may co-localize with endogenous co-receptors (such as endogenous CD2) at the immune synapses on the surface of the immune effector cells (such as Figure 1 B).

[0141] The immune effector cells of the present invention may include the auxiliary receptors of the present invention. The auxiliary receptors of the present invention may be combined with CAR CON (i.e., having a CD28 hinge region as shown in SEQ ID NO: 10 or a CD8a hinge region as shown in SEQ ID NO: 43) co-localizes at the immune synapse on the surface of immune effector cells (e.g., Figure 1 C).

[0142] The immune effector cell of the present invention may comprise the CAR of the present invention and the auxiliary receptor of the present invention. The CAR of the present invention may co-localize with the auxiliary receptor of the present invention at the immune synapse on the surface of the immune effector cell (e.g. Figure 1 D).

[0143] The immune effector cells of the present invention may be T cells, γδT cells, natural killer (NK) cells, NKT cells, induced pluripotent stem cell (iPSC)-derived NK cells (iPSC-NK), phagocytes or macrophages.

[0144] The immune effector cell can be a T cell. The T cell can be a CD8+ T cell or a cytotoxic T cell. The T cell can be a CD4-CD8+ T cell.

[0145] T cells can be CD4+T cells or helper T cells (TH cells), such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells or TFH cells. T cells can be regulatory T cells (Treg). T cells can be naive T cells, effector T cells, memory T cells, effector memory T cells, central memory T cells, stem cell memory T cells. T cells can be peripheral lymphocytes.

[0146] T cells can be expanded from PBMCs. T cells can be autologous to the subject to be administered. T cells can be allogeneic to the subject to be administered. T cells can be partially HLA mismatched to the subject to be administered.

[0147] NK cells can be cells of the NK92 cell line. NK cells can be isolated from plasma mononuclear cells (PBMC) of a subject to be treated or a healthy donor. NK cells can be isolated from umbilical cord blood. NK cells can be identified by CD34 + Differentiated from hematopoietic progenitor cells (HPCs).

[0148] Macrophages can differentiate into an "M1" phenotype. M1 macrophages express pro-inflammatory cytokines and have strong anti-tumor activity. Undifferentiated macrophages expressing the CAR described herein can be induced to differentiate into an M1 phenotype by culturing in the presence of an antigen.

[0149] The cell sources used according to the present invention are well known to those skilled in the art, and illustrative examples thereof include 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 one embodiment, the cells are derived from whole blood.

[0150] The immune effector cells of the present invention may be derived from autologous cells. The immune effector cells may be derived from allogeneic cells. The term "autologous" refers to any material derived from the same individual that is subsequently reintroduced into the individual. The term "allogeneic" refers to any material derived from a different individual of the same species as the individual into which the material is introduced. When two or more individuals are not identical in genes at one or more loci, they are said to be allogeneic to each other. In some aspects, allogeneic material from different individuals of the same species may be sufficiently different genetically to produce antigenic interactions.

[0151] The immune effector cells may comprise a nucleic acid as described herein. The immune effector cells may comprise a vector as described herein. The immune effector cells may comprise an RNA or RNA vector as described herein.

[0152] Immune effector cells can express specific CARs for one or more antigens.

[0153] Immune effector cells may express the CAR and / or accessory proteins of the invention permanently or transiently.

[0154] Immune effector cells may include modifications in their genome to reduce or eliminate the expression of endogenous co-receptors. Endogenous co-receptors may be any co-receptors described herein (as shown in Table 1). Endogenous co-receptors may be adhesion receptors, such as CD2. Genetic modification techniques for such modifications are well known in the art.

[0155] The present invention also provides immune effector cells obtained by any of the methods described herein.

[0156] The present invention further relates to a method for preparing an immune effector cell of the present invention or an immune effector cell group of the present invention. The method includes introducing a nucleic acid encoding the CAR and / or auxiliary receptor of the present invention into an immune effector cell, for example, by conversion (such as transfection or transduction).

[0157] The term "transduction" can be used to describe viral-mediated nucleic acid transfer. Viral vectors can be used to transduce cells with one or more constructs. Conventional viral-based expression systems can include herpes simplex virus (HSV) vectors, retroviruses, alpha-retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses (AAV) 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 well known in the art. The vector is preferably a vector as described herein. Immune effector cells can be transduced using any method known in the art. Transduction can be performed in vitro or ex vivo.

[0158] 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 transfection. Any vector capable of transfecting immune effector cells can be used, such as conventional plasmid DNA or RNA transfection, preferably mRNA transfection. Human artificial chromosomes and / or naked RNA can be used to transfect nucleic acid sequences or nucleic acid constructs into cells. For a description of human artificial chromosomes, see, for example, Kazuki et al., Mol. Ther. 19 (9): 1591-1601 (2011), and Kouprina et al., Expert Opinion on Drug Delivery 11 (4): 517-535 (2014). Other non-viral delivery systems include DNA plasmids, naked nucleic acids, and nucleic acids complexed with delivery vectors (such as liposomes). Non-viral delivery methods for nucleic acids include lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycationic or lipid:nucleic acid conjugates, naked DNA, naked RNA, artificial viral particles, and agents that enhance DNA uptake.

[0159] Nanoparticle delivery systems can be used to transfect immune effector cells with nucleic acid sequences. Such delivery systems include, but are not limited to, lipid-based systems, liposomes, micelles, microcapsules, and exosomes. For nanoparticles that can deliver RNA, see, for example, Alabie 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 acids (SNAs) TM ) constructs, nanocomplexes and other nanoparticles (especially gold nanoparticles) can also be considered as delivery methods for the nucleic acids or vectors of the present invention.

[0160] Immune effector cells can be transfected by electroporation. Electroporation is mRNA electroporation. This has the advantage of allowing transient expression of CAR and / or co-receptors.

[0161] The present invention also relates to a method for preparing an immune effector cell group (such as for adoptive cell therapy), such as an in vitro method, including culturing the immune effector cells of the present invention to produce immune effector cell groups. The present invention also provides immune effector cell groups obtained or obtainable by any method described herein. The present invention also provides immune effector cell groups expressing the CAR of the present invention and / or auxiliary receptors.

[0162] Immune effector cells induce effector functions of antigen presenting cells in contact therewith. Effector functions can be inhibition or reduction of immune effector cell activation (such as T cell activation), immune effector cell proliferation (such as T cell proliferation), cytolytic activity (such as apoptosis or cell death of APC), cell growth (such as cell growth of APC), and / or regulation of the release of cytokines (such as the release of proinflammatory cytokines). The method of measuring effector cell function is well known in the art. Immune effector cell activation can be measured by an increase in 4-1BB and / or CD25 surface expression and / or an increase in CD69 expression. Cytolytic activity can be measured by the death of target cells and / or the release of perforin and / or granzymes. The regulation of cytokine release can be an increase in the release of proinflammatory cytokines. Proinflammatory cytokines can be selected from GM-CSF, TNF-α, IL-2, IL-6, IL-1β and / or IFN-γ.

[0163] Surface expression of 4-1-BB and / or CD25 can be measured as a normalized percentage of maximum surface expression, e.g. Figure 6 and Figure 8 shown.

[0164] The release of pro-inflammatory cytokines, such as Figure 7 Preferably, the cytokines IL-2 and / or IFN-γ are measured.

[0165] The expression of CD69 can be measured as the percentage of CD69-positive cells. Figures 10 to 14 shown.

[0166] Compared to immune effector cells expressing conventional CARs comprising the same antigen binding domain, immune effector cells expressing the CAR of the present invention and / or the co-receptors of the present invention may have improved or optimized effector functions. Effector function is improved when immune effector cell activation increases, immune effector cell proliferation increases, cytolytic activity increases, APC proliferation slows or stops, pro-inflammatory cytokine release increases, and / or anti-inflammatory cytokine release decreases. As shown in the figure, effector function can be measured as EC 50 For comparison purposes. The effector function may increase by 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. When changing the size of CAR and / or co-receptors does not further improve the effector function, the effector function reaches the best. When the effector function is within + / -25% (e.g., + / -20%, + / -15%, + / -10% or + / -5%) of the best effector function, the effector function may be considered to be optimized.

[0167] Antigen presenting cells

[0168] The present invention is applicable to any antigen presenting cell (APC). APC can be a professional APC, such as a dendritic cell, a macrophage, a B cell or an epithelial cell. APC can be a non-professional APC, such as a fibroblast, a thymic epithelial cell, a thyroid epithelial cell, a glial cell, a pancreatic beta cell or a vascular endothelial cell. APC can be a tumor cell, such as the tumor cell provided in Table 2.

[0169] APC includes an antigen on its cell surface that can bind to the antigen binding domain of CAR (such as CAR of the present invention). Antigen can be any antigen, such as a tumor antigen (such as a tumor-associated antigen, a developmental tumor antigen and / or a new antigen), for example, as listed in Table 2. Antigen can be a peptide-MHC complex, CD19, mesothelin, BCMA, CD22, EGFR, EGFRvIII or NY-ESO-1.

[0170] Table 2 - Examples of CAR-targeted antigens and associated malignancies

[0171]

[0172]

[0173] Nucleic acids, vectors and host cells Also provided are one or more isolated nucleic acids encoding the CAR of the present invention and / or co-receptors. In some cases, the nucleic acids are present in more than one nucleic acid, but they are able to encode the CAR of the present invention and / or co-receptors together.

[0174] The nucleic acid encoding the CAR and / or auxiliary receptor of the present invention can be obtained by methods well known to those skilled in the art. For example, a DNA sequence encoding part or all of the antibody heavy chain and light chain can be synthesized from the corresponding amino acid sequence as needed.

[0175] The nucleic acid may be a DNA sequence. The nucleic acid may be an RNA sequence, such as mRNA. The vector may contain the nucleic acid.

[0176] The vector can be a viral vector. Conventional viral-based expression systems can include herpes simplex virus (HSV) vectors, retroviruses, alpha-retroviruses, lentiviruses, adenoviruses, adeno-associated viruses (AAV) 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 well known in the art.

[0177] The vector may be a cloning vector or an expression vector. Suitable vectors may be any vectors that can carry sufficient genetic information and allow expression of the polypeptide of the present invention.

[0178] The 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.

[0179] The general method of constructing vector, transfection method and culture method are well known to those skilled in the art. In this regard, reference can be made to "Current Protocols in Molecular Biology", 1999, FM Ausubel (ed), Wiley Interscience, New York and the Maniatis Manual published by Cold Spring Harbor Publishing.

[0180] Nucleic acid can be provided in the form of an expression cassette, which includes a control sequence operably connected to an insertion sequence, so as to allow the CAR and / or auxiliary receptor of the present invention to be expressed in vivo. Therefore, one or more expression cassettes are also provided, which encode one or more nucleic acids encoding the CAR and / or auxiliary receptor of the present invention. Accordingly, these expression cassettes are generally provided in a vector (such as a plasmid or a recombinant viral vector). Therefore, a vector encoding the CAR and / or auxiliary receptor of the present invention is also provided. In addition, a vector encoding the CAR and / or auxiliary receptor of the present invention is also provided.

[0181] The vector may be a human artificial chromosome. Human artificial chromosomes are described in, for example, Kazuki et al., Mol. Ther. 19(9): 1591-1601 (2011), and Kouprina et al., Expert Opinion on Drug Delivery 11(4): 517-535 (2014).

[0182] The vector can be a non-viral delivery system, such as a DNA plasmid, naked nucleic acid (such as naked RNA), and nucleic acid complexed with a delivery vehicle such as a liposome.

[0183] The nucleic acids, expression cassettes or vectors described herein can be introduced into a host cell, for example by transfection. Therefore, a host cell comprising one or more nucleic acids, expression cassettes or vectors of the present invention is also provided. The nucleic acids, expression cassettes or vectors described herein can be introduced into a host cell transiently or permanently, thereby allowing the 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, such as mammalian cells or insect cells; lower eukaryotic cells, such as yeast; or prokaryotic cells, such as bacterial cells. Specific examples of cells include mammalian HEK293, such as HEK293F, HEK293T, HEK293S or HEK Expi293F, CHO, HeLa, NS0 and COS cells, or any other cell line used herein.

[0184] 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 a host cell.

[0185] The polynucleotide or vector of the present invention can be an mRNA for administration to a patient, such as an mRNA vaccine. The patient's T cells can then express the CAR of the present invention and / or the auxiliary receptor of the present invention in vivo. Such mRNA molecules and related methods are described in reference 10.

[0186] Also provided is a kit suitable for transforming and / or transfecting immune effector cells or immune effector cell groups to generate immune effector cells or immune effector cell groups of the present invention. The kit comprises a nucleic acid or vector as described herein. The kit may comprise other agents that improve transfection or conversion efficiency, such as those discussed herein.

[0187] method

[0188] Also provided herein is a method for increasing and / or optimizing the effector function of immune effector cells, including modifying the size (such as height) of the extracellular portion of CAR and / or the size (such as height) of the extracellular portion of the auxiliary receptor, to optimize the effector function of immune effector cells when in contact with APC. Specifically, the height of the extracellular antigen binding domain of CAR and / or the height of the extracellular ligand binding domain of the auxiliary receptor are modified. The present invention also provides immune effector cells obtained or obtainable by the method. The effector function can be any effector function described herein.

[0189] Also provided herein is a method for identifying improved immune effector cells, including modifying the size (such as height) of the extracellular portion of CAR and / or the size (such as height) of the extracellular portion of the auxiliary receptor, so as to modify the height of the extracellular antigen binding domain of the CAR and / or the extracellular ligand binding domain of the auxiliary receptor;With determining whether the immune effector cells expressing modified CAR and / or modified auxiliary receptors have improved effector functions compared with the immune effector cells expressing unmodified CAR and / or modified auxiliary receptors. The present invention also provides immune effector cells obtained or obtainable by the method.

[0190] The CAR may be a CAR described herein.

[0191] The coreceptor may be a coreceptor as described herein. The coreceptor may be CD2.

[0192] The method of the present invention may include modifying the size of the extracellular portion of the CAR and / or the size of the extracellular portion of the auxiliary receptor, thereby modifying the height of the extracellular antigen binding domain of the CAR and / or the extracellular ligand binding domain of the auxiliary receptor, so that the membrane spacing spanned by the complex between the CAR and the antigen is comparable to the membrane spacing spanned by the complex between the auxiliary receptor and the ligand (e.g., within ≤5%, ≤10%, ≤15%, ≤20%, ≤25% or ≤30%, or the same). The membrane spacing spanned may be comparable to the membrane spacing spanned by the complex between the T cell receptor and the antigen (e.g., within ≤5%, ≤10%, ≤15%, ≤20%, ≤25% or ≤30%, or the same).

[0193] The methods of the present invention may 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.

[0194] The methods of the present invention may 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 immune effector cell membrane.

[0195] The methods of the invention may include reducing the size (eg, height) of the extracellular portion of a coreceptor, thereby reducing the height of the extracellular ligand binding domain of the coreceptor from the immune effector cell membrane.

[0196] The methods of the invention may include increasing the size (eg, height) of the extracellular portion of a coreceptor, thereby increasing the height of the extracellular ligand binding domain of the coreceptor from the immune effector cell membrane.

[0197] The method of the present invention may include increasing the size (e.g., height) of the extracellular portion of the CAR and reducing the size (e.g., height) of the auxiliary receptor, thereby increasing the height of the extracellular antigen binding domain of the CAR from the immune effector cell membrane and reducing the height of the extracellular ligand binding domain of the auxiliary receptor from the immune effector cell membrane.

[0198] The method of the present invention may include reducing the size (e.g., height) of the extracellular portion of the CAR and increasing the size (e.g., height) of the auxiliary receptor, thereby reducing the height of the extracellular antigen binding domain of the CAR from the immune effector cell membrane and increasing the height of the extracellular ligand binding domain of the auxiliary receptor from the immune effector cell membrane.

[0199] Determining the appropriate size (e.g., height) of the extracellular portion of a CAR and / or co-receptor of the invention (e.g., the height of the extracellular antigen binding domain of the CAR and / or co-receptor from the immune effector cell membrane) and the membrane spacing spanned are described herein.

[0200] The method of the present invention may include introducing a sequence that physically increases the height of the antigen binding domain from the immune effector cell membrane in the extracellular portion of the CAR and / or the auxiliary receptor, for example, in the hinge region of the CAR or the stem region of the auxiliary receptor. Suitable sequences that physically increase the height of the antigen binding domain from the immune effector cell membrane are described herein.

[0201] The method of the present invention may include replacing the hinge region of the CAR or the stem region of the auxiliary receptor with a sequence that physically increases the height of the antigen binding domain from the immune effector cell membrane. Suitable sequences that physically increase the height of the antigen binding domain from the immune effector cell membrane are described herein.

[0202] 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 compared to an immune effector cell expressing a corresponding unmodified CAR and / or a corresponding unmodified auxiliary receptor.

[0203] The methods of the present invention may also include determining the level of effector function of immune effector cells, such as cell killing or cytokine production. As described herein, such methods are well known in the art. Pharmaceutical composition

[0204] The present invention also provides compositions comprising immune effector cells or immune effector cell groups.Immune effector cells or immune effector cell groups can account for at least 1% of total cells in the composition, such as 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.Total cells in the composition can be composed of immune effector cells or immune effector cell groups of the present invention, or are substantially composed of immune effector cells or immune effector cell groups of the present invention, i.e., other cells are not detected in the composition.

[0205] 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 to 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 composition of the present invention may also include other auxiliary physiologically active agents.

[0206] The carrier is generally pharmaceutically "acceptable", i.e., compatible with the other ingredients in the composition and not harmful to the subject. The composition includes those suitable for parenteral administration, including subcutaneous administration, intramuscular administration, intravenous administration, and intradermal administration. The composition can be conveniently provided in unit dosage form and can be prepared by any method known in the pharmaceutical art. Such methods include preparing a carrier that is combined with isolated T cells. In general, the composition is prepared by uniformly and closely combining any active ingredient with a liquid carrier.

[0207] The composition can be used for parenteral administration. In another embodiment, the composition is suitable for intravenous administration. Compositions suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bactericides and solutes to make the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents.

[0208] The compositions described herein can be prepared in a manner known in the art and are suitable for parenteral administration to mammals, particularly humans, and contain a therapeutically effective amount of the composition and one or more pharmaceutically acceptable carriers or diluents. The composition may contain at least about 1x10 6 About 1x10 12 The composition may contain at least about 1x107 , at least about 1x10 8 , at least about 1x10 9 , at least about 1x10 10 Or at least about 1x10 11 The immune effector cells of the present invention.

[0209] The present disclosure also contemplates the combination of the compositions described herein with other active agents and / or with other treatment regimens or modalities (such as radiotherapy or surgery). When the compositions described herein are used in combination with known active agents, they can be administered sequentially (continuously or at intervals without treatment) or simultaneously or as a mixture.

[0210] Suitable anticancer agents are well known to those skilled in the art.

[0211] Combination therapies comprising treatment first with a composition of the invention followed by treatment with a known treatment, or treatment first with a known agent followed by treatment with a composition of the invention, for example, as a maintenance therapy, are also contemplated.

[0212] For example, in the treatment of cancer, it is contemplated that the compositions of the invention may be administered in combination with an alkylating agent (e.g., nitrogen mustard, cyclophosphamide, chlorambucil, ifosfamide cysplatin, or platinum-containing alkylating agents such as cisplatin, carboplatin, and oxaliplatin), an antimetabolite (e.g., purine or pyrimidine analogs or antifolates such as azathioprine and mercaptopurine), an anthracycline (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, mitoxantrone, or anthracycline analogs), an implantable cardiotonic agent (e.g., cytokines ... alkaloids (such as vinca alkaloids or taxanes, such as vincristine, vinblastine, vinorelbine, vindesine, paclitaxel, or docetaxel), topoisomerase inhibitors (such as type I topoisomerase inhibitors or type II topoisomerase inhibitors), podophyllotoxins (such as etoposide or teniposide), tyrosine kinase inhibitors (such as imatinib mesylate, nilotinib, or dasatinib), adenosine receptor inhibitors (such as A2aR inhibitors: SC H58261, CPI-444, SYN115, ZM241385, FSPTP or A2BR inhibitors such as PSB-1115), adenosine receptor agonists (such as CCPA, IB-MECA and CI-IB-MECA), checkpoint inhibitors (including those of PDL-1: PD-1 axis, nivolumab, pembrolizumab, atezolizumab, BMS-936559, MEDI4736, MPDL33280A or MSB0010718C), CTLA-4 pathway inhibitors (such as ipilimumab and tremelimumab), TIM-3 pathway inhibitors or agonist monoclonal antibodies known to promote T cell function (including anti-OX40 antibodies, such as MEDI6469; and anti-4-BB antibodies, such as PF-05082566).

[0213] The present invention also provides a kit or article comprising the above pharmaceutical composition.

[0214] The present invention also provides a kit for the above-mentioned therapeutic application, the kit comprising:

[0215] (a) a container containing the polypeptide, nucleic acid, vector or pharmaceutical composition of the present invention; and

[0216] (b) a label or package insert containing instructions for use.

[0217] Suitable containers include, for example, bottles, vials, syringes, blister packs, and the like. The container can be formed of a variety of materials such as glass or plastic. The container contains a therapeutic composition effective for treating the condition and may have a sterile access port (for example, the container may be an intravenous fluid bag or a vial with a stopper that can be pierced by a hypodermic needle). The label or package insert indicates that the therapeutic composition is used to treat the selected condition. In one embodiment, 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.

[0218] The kit may also include other containers containing pharmaceutically acceptable buffers, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may also include other materials that are desirable from a commercial and user perspective, which are well known to those skilled in the art, suitable examples of which include other buffers, diluents, filters, needles and syringes.

[0219] Therapeutic Uses

[0220] Also described herein are uses of the CARs, co-receptors, immune effector cells, or immune effector cell populations of the invention in methods of treating the human or animal body by therapy.

[0221] For example, the present invention also provides a method for treating cancer in a subject, the method comprising administering an effective amount of an immune effector cell or immune effector cell group of the present invention to the subject. Therefore, the present invention also provides the use of the CAR, auxiliary receptor, immune effector cell or immune effector cell group of the present invention for treating cancer. The present invention also provides the use of the CAR, auxiliary receptor, immune effector cell or immune effector cell group of the present invention in the preparation of a drug for treating cancer. The present invention also provides the use of the CAR, auxiliary receptor, immune effector cell or immune effector cell group of the present invention for treating cancer.

[0222] A method for adoptive cell therapy of a subject is also provided, the method comprising administering an effective amount of an immune effector cell or immune effector cell group of the present invention to the subject. Therefore, the present invention also provides the use of the CAR, auxiliary receptor, immune effector cell or immune effector cell group of the present invention for adoptive cell therapy. The present invention also provides the use of the CAR, auxiliary receptor, immune effector cell or immune effector cell group of the present invention in the preparation of a medicament for adoptive cell therapy. The present invention also provides the use of the CAR, auxiliary receptor, immune effector cell or immune effector cell group of the present invention in adoptive cell therapy.

[0223] The cancer may be any cancer, such as a solid cancer. The cancer may be a malignancy listed in Table 2. The cancer may be a hematological malignancy or a B-cell cancer.

[0224] Therapeutic uses and methods may comprise administering a therapeutically effective amount of an immune effector cell or a population of immune effector cells.

[0225] Also provided is a method for formulating a composition for treating cancer, wherein the method comprises mixing the immune effector cell or immune effector cell population of the present invention with an acceptable carrier to prepare the composition.

[0226] The subjects may have been previously treated for cancer, for example using adoptive cell therapy.

[0227] Therapeutic methods and uses may include determining whether a cancer expresses a target antigen specifically targeted by an immune effector cell or immune effector cell population of the invention prior to using the immune effector cell or immune effector cell population of the invention for treatment.

[0228] The method may include selecting immune effector cells or immune effector cell groups according to the expression of cancer target antigens, so that the immune effector cells or immune effector cell groups are specific to the cancer. The method may include transfecting or transforming immune effector cells with the nucleic acid of the present invention according to the expression information of the target antigen in the cancer.

[0229] The methods of treatment and uses described herein may include inhibiting a disease state (i.e., cancer), for example by preventing its development and / or causing regression of the disease state until a desired endpoint is reached. The methods of treatment and uses of the present invention may include achieving a partial response, a complete response to cancer. The methods of treatment and uses of the present invention may achieve remission of cancer.

[0230] The methods of treatment and uses described herein can slow cancer progression, stop cancer progression and / or reverse cancer progression. The methods of treatment and uses of the invention can reduce the size of a cancer by at least 10%, such as 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%.

[0231] Typically, the methods of treatment and 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 can be a rat, rabbit, sheep, pig, cow, cat or dog.

[0232] The dosage of immune effector cells or immune effector cell populations may vary depending on the age and size of the subject, as well as the disease, condition, and route of administration. The dosage of immune effector cells or immune effector cell populations may be about 1 x 10 6 About 1x10 12The immune effector cell or immune effector cell population may be administered in a dose of about 1 x 10 5 cells / kg body weight is about 1x10 11 cells / kg body weight.

[0233] The immune effector cells or immune effector cell groups can be administered in a single dose. The immune effector cells or immune effector cell groups can be administered in a multiple dose regimen. For example, after the initial dose, a second or multiple subsequent doses can be administered. The second dose and subsequent doses can be separated by an appropriate time. For example, the dose between doses can be administered approximately once a week, approximately once every two weeks, approximately once every three weeks, approximately once every four weeks, or approximately once a month.

[0234] The immune effector cells or population of immune effector cells can be administered intravenously.

[0235] The immune effector cell or immune effector cell population can be administered with one or more additional therapies (e.g., one or more additional therapeutic agents). The additional therapeutic agent can be an anti-tumor agent. The additional therapeutic agent can be an additional immune effector cell.

[0236] The combined administration of immune effector cells or cell populations with additional therapeutic agents can be achieved in a variety of different ways. All components can be administered together in a single composition. Each component can be administered separately as part of a combined therapy.

[0237] For example, the immune effector cells or immune effector cell populations of the invention can be administered before, after, or simultaneously with an additional therapeutic agent. The additional therapy can be chemotherapy, radiotherapy, and / or surgery.

[0238] Prior to administering the immune effector cells or immune effector cell populations of the present invention, the subject may undergo lymphodepletion. Lymphodepletion may be achieved by administering fludarabine, cyclophosphamide, and / or bendamustine to the subject. Lymphodepletion may be performed for at least about one day, such as about two days or about three days.

[0239] The biological activity and / or therapeutic efficacy of the administered immune effector cells or immune effector cell populations can be measured by known methods. For example, the method can include imaging, such as magnetic resonance imaging.

[0240] Embodiments of the present invention

[0241] 1. A method for optimizing the effector function of immune effector cells, wherein the immune effector cells comprise:

[0242] - a chimeric antigen receptor (CAR) capable of binding to an antigen on an antigen presenting cell (APC), wherein the CAR comprises 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 connected by a hinge region; and

[0243] - a coreceptor capable of binding to a ligand on the APC, wherein the coreceptor comprises an extracellular ligand binding domain and a transmembrane domain, wherein the extracellular ligand binding domain and the transmembrane domain are connected by a stem region;

[0244] Wherein, the method comprises modifying the height of the extracellular antigen binding domain of the CAR from the immune effector cell membrane and / or the height of the extracellular ligand binding domain of the auxiliary receptor from the immune effector cell membrane to optimize the effector function of the immune effector cell when in contact with the APC;

[0245] Optionally, wherein the effector function is cell killing.

[0246] 2. A method for identifying improved immune effector cells, wherein the immune effector cells comprise:

[0247] - a chimeric antigen receptor (CAR) capable of binding to an antigen on an antigen presenting cell (APC), wherein the CAR comprises 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 connected by a hinge region; and

[0248] - a coreceptor capable of binding to a ligand on the APC, wherein the coreceptor comprises an extracellular ligand binding domain and a transmembrane domain, wherein the extracellular ligand binding domain and the transmembrane domain are connected by a stem region;

[0249] The method comprises:

[0250] (a) modifying the height of the extracellular antigen binding domain of the CAR from the immune effector cell membrane and / or the height of the extracellular ligand binding domain of the auxiliary receptor from the immune effector cell membrane; and

[0251] (b) determining whether the immune effector cells expressing the modified CAR and / or the modified coreceptor have improved effector function compared to immune effector cells expressing unmodified CAR and / or unmodified coreceptor;

[0252] Optionally, wherein the effector function is cell killing.

[0253] 3. The method described in embodiment 1 or 2 includes modifying the height of the extracellular antigen binding domain of the CAR and / or the height of the extracellular ligand binding domain of the auxiliary receptor so that the membrane distance spanned by the CAR-antigen complex is equivalent to the membrane distance spanned by the auxiliary receptor-ligand complex.

[0254] 4. The method of embodiment 3, wherein:

[0255] (a) the membrane distance spanned by the CAR-antigen complex differs from the membrane distance spanned by the coreceptor-ligand complex by ≤5%, ≤10%, ≤15%, ≤20%, ≤25% or ≤30%, or is the same as the membrane distance spanned by the coreceptor-ligand complex; and / or

[0256] (b) The membrane distance spanned by the CAR-antigen complex is comparable to the membrane distance spanned by the T cell receptor and its peptide-MHC antigen complex (e.g., within ≤5%, ≤10%, ≤15%, ≤20%, ≤25% or ≤30%, or the same as it).

[0257] 5. The method of embodiment 3 or 4, wherein the transmembrane spacing is about 14 nm.

[0258] 6. The method of any of the preceding embodiments, comprising:

[0259] (a) reducing the height of the extracellular antigen binding domain of the CAR;

[0260] (b) increasing the height of the extracellular antigen binding domain of the CAR;

[0261] (c) reducing the height of the extracellular ligand binding domain of the coreceptor;

[0262] (d) increasing the height of the extracellular ligand binding domain of the coreceptor;

[0263] (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 coreceptor; or

[0264] (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 co-receptor.

[0265] 7. The method of any of the preceding embodiments, wherein the height of the antigen binding domain of the CAR from the immune effector cell membrane is comparable to (e.g., within ≤5%, ≤10%, ≤15%, ≤20%, ≤25% or ≤30%, or the same as) the height of the extracellular antigen binding domain of the T cell receptor from the immune effector cell membrane (e.g., about 7 nm).

[0266] 8. The method of any of the preceding embodiments, wherein the height of the ligand binding domain of the coreceptor from the immune effector cell membrane is about 7 nm.

[0267] 9. The method of any one of the preceding embodiments, comprising introducing a sequence that physically increases the height of the ligand binding domain from the immune effector cell membrane into the hinge region of the CAR and / or the stem region of the auxiliary receptor, or replacing the hinge region of the CAR and / or the stem region of the auxiliary receptor with the sequence.

[0268] 10. The method of embodiment 9, wherein the sequence introduced into the hinge region of the CAR comprises or consists of:

[0269] (a) a fragment of the mucin-like extracellular sequence of CD43 as shown in SEQ ID NO: 62, such as 4 to 234 amino acids, such as 20 to 40 amino acids (e.g., as shown in any one of SEQ ID NOs: 53 to 61);

[0270] (b) a folded polypeptide domain, which is an immunoglobulin domain, such as an immunoglobulin constant domain or an FNIII domain; and / or

[0271] (c) a fragment of the hinge region of CD28 as shown in SEQ ID NO:10 or a fragment of the hinge region of CD8a as shown in SEQ ID NO:43, optionally, wherein the fragment is shown in any one of SEQ ID NOs:37 to 42, 46, 63 and 64.

[0272] 11. The method described in embodiment 9, wherein the sequence introduced into the stem region of the auxiliary receptor comprises a fragment of a mucin-like sequence or a fragment or a derivative thereof, or consists of a fragment of a mucin-like sequence or a fragment or a derivative thereof, optionally wherein the mucin-like sequence is a mucin-like sequence of CD43 as shown in SEQ ID NO:62, optionally wherein the fragment has a length of 4 to 234 amino acid residues, such as any one of SEQ ID NOs:53 to 61.

[0273] 12. The method of any of the preceding embodiments, wherein the co-receptor is an adhesion receptor, such as CD2.

[0274] 13. The method of any of the preceding embodiments, wherein the antigen is a peptide-MHC complex, CD19, mesothelin, BCMA, CD22, EGFR or EGFRvIII, optionally wherein the peptide in the peptide-MHC complex is a fragment of NY-ESO 1.

[0275] 14. Immune effector cells obtained or obtainable by the method of any one of the preceding embodiments.

[0276] 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 connected by a hinge region, wherein the hinge region comprises a sequence that physically increases the height of the extracellular antigen binding domain from the immune effector cell membrane, or consists of a sequence that physically increases the height of the extracellular antigen binding domain from the immune effector cell membrane, wherein the sequence comprises a mucin-like sequence, one or more folded polypeptide domains, or a fragment of CD28 as shown in SEQ ID NO: 10 or a fragment of the CD8a hinge region as shown in SEQ ID NO: 43.

[0277] 16. A secondary receptor capable of binding to a ligand on an APC, comprising an extracellular ligand binding domain and a transmembrane domain, wherein the extracellular ligand binding domain and the transmembrane domain are connected by a stem region, wherein the stem region comprises a sequence that physically increases the height of the ligand binding domain from the immune effector cell membrane, or consists of a sequence that physically increases the height of the ligand binding domain from the immune effector cell membrane, optionally wherein the stem region comprises a mucin-like sequence.

[0278] 17. An immune effector cell comprising the CAR described in embodiment 15 and / or the auxiliary receptor described in embodiment 16.

[0279] 18. An immune effector cell comprising:

[0280] - a chimeric antigen receptor (CAR) capable of binding to an antigen on an antigen presenting cell (APC), wherein the CAR comprises 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 connected by a hinge region; and

[0281] - a coreceptor capable of binding to a ligand on the APC, wherein the coreceptor comprises an extracellular ligand binding domain and a transmembrane domain, wherein the extracellular ligand binding domain and the transmembrane domain are connected by a stem region;

[0282] in:

[0283] (a) the CAR and the coreceptor have respective sizes such that the membrane distance spanned by the CAR-antigen complex is comparable to the membrane distance spanned by the coreceptor-ligand complex;

[0284] (b) the hinge region of the CAR comprises a sequence that physically increases the height of the antigen binding domain from the immune effector cell membrane, or consists of a sequence that physically increases the height of the antigen binding domain from the immune effector cell membrane, wherein the sequence comprises a mucin-like sequence, one or more folded polypeptide domains, or a fragment of CD28 as shown in SEQ ID NO: 10 or a fragment of the CD8a hinge region as shown in SEQ ID NO: 43; and / or

[0285] (c) the stem region of the coreceptor comprises a sequence that physically increases the height of the extracellular ligand binding domain from the immune effector cell membrane.

[0286] 19. The immune effector cell of embodiment 18, wherein:

[0287] (a) the membrane distance spanned by the CAR-antigen complex is within ≤5%, ≤10%, ≤15%, ≤20%, ≤25% or ≤30% of the membrane distance spanned by the coreceptor-ligand complex, or is the same as the membrane distance spanned by the coreceptor-ligand complex; and / or

[0288] (b) the membrane 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%, or the same as) the membrane distance spanned by the complex between a T cell receptor and its corresponding peptide-MHC antigen.

[0289] 20. The immune effector cell of embodiment 18 or 19, wherein the hinge region of the CAR comprises, or consists of, a sequence that physically increases the height of the antigen binding domain from the immune effector cell membrane, wherein the sequence comprises a mucin-like sequence, one or more folded polypeptide domains, or a fragment of CD28 as shown in SEQ ID NO:10 or a fragment of the CD8a hinge region as shown in SEQ ID NO:43.

[0290] 21. The immune effector cell of embodiment 18 or 19, wherein the stem region of the auxiliary receptor comprises a sequence that physically increases the height of the extracellular ligand binding domain from the immune effector cell membrane.

[0291] 22. The immune effector cell of embodiment 18 or 19, wherein:

[0292] (a) the hinge region of the CAR comprises a sequence that physically increases the height of the antigen binding domain from the immune effector cell membrane, or consists of a sequence that physically increases the height of the antigen binding domain from the immune effector cell membrane, wherein the sequence comprises a mucin-like sequence, one or more folded polypeptide domains, or a fragment of CD28 as shown in SEQ ID NO: 10 or a fragment of the CD8a hinge region as shown in SEQ ID NO: 43; and

[0293] (b) the stem region of the coreceptor comprises a sequence that physically increases the height of the extracellular ligand binding domain from the immune effector cell membrane.

[0294] 23. The immune effector cell of any one of embodiments 17 to 22, wherein the immune effector cell is a T cell, a NK cell, a NKT cell, a phagocyte or a macrophage, optionally wherein the T cell is a CD4-CD8+ T cell.

[0295] 24. A method for identifying improved immune effector cells, comprising determining whether the immune effector cell of any one of embodiments 17 to 23 has improved effector function compared to an immune effector cell expressing an unmodified CAR and / or an unmodified auxiliary receptor.

[0296] 25. Use of the immune effector cell of any one of embodiments 17 to 23, the CAR of embodiment 15, or the auxiliary receptor of embodiment 16 for use in a method for treating cancer in a subject, optionally wherein the cancer is a hematological malignancy or a B-cell cancer.

[0297] other

[0298] It should be understood that the different applications of the CAR, immune effector cells or pharmaceutical compositions disclosed in the present invention can be customized according to the specific needs of the art. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments of the present invention and are not intended to be limiting.

[0299] Furthermore, as used in this specification and the appended claims, the singular indefinite articles ("a", "an") and the definite article ("the") include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "CAR" includes two or more "CARs".

[0300] Furthermore, when “≥x” is mentioned herein, this means equal to or greater than x; and when “≤x” is mentioned herein, this means less than or equal to x.

[0301] For the purposes of the present invention, in order to determine the percent identity of two sequences (such as two polynucleotide sequences or two polypeptide sequences), the sequences are aligned for optimal comparison purposes (for example, gaps can be introduced in the first sequence for optimal alignment with the second sequence). The nucleotides or amino acid residues at each position are then compared. When 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 that position is the same. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions in the reference sequence × 100).

[0302] Typically, sequence alignments are performed over the full length of a reference sequence. For example, if a user wishes to determine whether a given ("test") sequence is 95% identical to SEQ ID NO:3, then 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 technician will align over the full length of SEQ ID NO:3 and identify how many positions in the test sequence are identical to positions in 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 test sequence is shorter than SEQ ID NO:3, gaps or missing positions should be considered non-identical positions.

[0303] The skilled person is aware that there are different computer programs that can be used to determine the homology or identity between two sequences. For example, a mathematical algorithm can be used to complete the comparison of sequences and determine the percent identity between two sequences. In one embodiment, the percent identity between two amino acid or nucleic acid sequences is determined using the Needleman and Wunsch (1970) algorithm, which has been integrated into the GAP program in the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ), using a Blosum 62 matrix or a PAM250 matrix, with a gap weight of 16, 14, 12, 10, 8, 6 or 4, and a length weight of 1, 2, 3, 4, 5 or 6.

[0304] "Specificity" or "specific binding" refers to the binding of the antigen binding region of the CAR to one or more antigenic determinants of the desired target antigen, but not to other polypeptides. For example, a CD19-specific CAR binds to the CD19 antigen, but not to an antigen of a different polypeptide, such as bovine serum albumin. If the affinity of the CAR binding to the target antigen is at least 10 times stronger than the affinity of the CAR binding to an antigen of a different polypeptide, such as bovine serum albumin, and preferably at least 100 times stronger, then the CAR can be specifically bound to the target antigen. Methods for measuring binding affinity are well known in the art.

[0305] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0306] The following examples illustrate the invention.

[0307] Example 1 - Identification of potential mechanisms underlying the inability of CAR to utilize CD2 adhesion.

[0308] Determine the impact of antigen receptor size and signaling on antigen sensitivity

[0309] The inventors generated a modified CAR, referred to herein as Fab CAR. Fab-CAR is a fusion protein in which an antibody-derived variable domain is coupled to a TCR constant domain, followed by a CD28 transmembrane domain, and finally a TCR-ζ chain cytoplasmic region ( Figure 2 Importantly, this construct omits the extra extracellular hinge region that is typically incorporated into CARs, making them larger than TCRs.

[0310] The following receptors were tested for activity: 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 [the C9V variant (9V) of the NY-ESO-1157-165 peptide presented by HLA-A*02:01] that is recognized by the 1G4 TCR (4).

[0311] These receptors were successfully expressed in Jurkat cells or primary human CD8+ T cells by lentiviral transduction (see Figure 4 , Figure 5 and Figure 6 ). Interestingly, Figure 4 It is shown that the standard CAR (D52N-CD28-28z) and Fab-CAR (D52N-Fab-28z) are expressed (y-axis) without upregulating CD3 (x-axis). Therefore, these receptors can be expressed independently of the TCR-CD3 complex, which is not the case for the 1G4 TCR.

[0312] T2 target cells were loaded with the indicated concentrations of peptide antigens prior to incubation with T cells expressing different antigen receptors and / or CD2 of different sizes. 50 The values ​​(antigen sensitivity; antigen concentration required to elicit 50% of the maximal response) are shown in Figure 6 A to Figure 6 C. Figure 7 Cytokine production when primary T cells were used is shown.

[0313] As can be seen, STAR, which has the same size and signaling mechanism as TCR, has comparable antigen sensitivity. In contrast, TruC, which has the same signaling mechanism as TCR but a different size, exhibits lower antigen sensitivity, suggesting that comparable size is necessary. Consistent with this, it is striking that Fab-CAR, which is expressed independently of the TCR-CD3 complex and has a similar size to TCR and STAR, has similar antigen sensitivity and cytokine production to TCR and STAR, but higher antigen sensitivity and cytokine production compared to CAR and eTRuC.

[0314] Thus, these data suggest that comparable size rather than signaling mechanism is necessary for CARs to achieve the same high antigen sensitivity as TCRs. These data are supported by previous findings that T cell antigen recognition depends on the size of the coreceptor-ligand complex (11), matching the size of the CD2 / ligand complex to the size of the TCR / pMHC complex (14 nm) (3, 6), and varying the size of the CD2 / ligand or TCR / pMHC complex (7).

[0315] Materials and Methods

[0316] Plasmid constructs

[0317] Lentiviral infectious particles were generated using a third generation system that included: (i) pMD2.G (Addgene #12259), (ii) pRSV-Rev (Addgene #12253), and (iii) pMDLg / pRRE (Addgene #12251) packaging plasmids. Different lentiviral transfer vectors encoding receptors that bind to the NY-ESO-1 / HLA-A*02 pMHC complex were used. The coding sequence was contained in the pLEX_307 (Addgene #41392) or pLEX307-NeoR (Addgene #134365) backbone.

[0318] Receptors

[0319] The 1G4 T cell receptor (TCR) has a variable α (SEQ ID NO.1) domain and a constant α (SEQ ID NO.2) domain, a P2A self-cleaving peptide (SEQ ID NO.3), followed by a variable β (SEQ ID NO.4) domain and a constant β (SEQ ID NO.5) domain. The variable domain confers specificity.

[0320] D52N-CD28.H-28z CAR is also referred to herein as D52N-CD28-28z, which has: a leader sequence (SEQ ID NO.6), a variable heavy chain domain of D52N scFv (SEQ ID NO.7), a linker (SEQ ID NO.8), a variable light chain domain of D52N scFv (SEQ ID NO.9), a CD28-derived hinge region (SEQ ID NO.10), a CD28 transmembrane domain (SEQID NO.11), a CD28 cytoplasmic domain (SEQ ID NO.12) and a ζ chain (CD247) signaling tail (SEQ ID NO.13).

[0321] A CAR similar to the D52N-CD28.H-28z CAR but using a CD8a hinge region instead of a CD28 hinge region can be used. Such a CAR can have a leader sequence (SEQ ID NO.6), a variable heavy chain domain of a D52N scFv (SEQ ID NO.7), a linker (SEQ ID NO.8), a variable light chain domain of a D52N scFv (SEQ ID NO.9), a CD8a-derived hinge region (SEQ ID NO.43), a CD8a transmembrane domain (SEQ ID NO.44), and a ζ chain (CD247) signaling tail (SEQ ID NO.13). Another leader sequence can be used, for example, a GM-CSF leader sequence as shown in SEQ ID NO:65.

[0322] D52N-ε-TRuC is also referred to herein as eTruC (D52N), which has: a leader sequence (SEQ ID NO. 14), D52N scFv (both the heavy chain domain and the light chain domain) (SEQ ID NO. 15), a glycine-serine linker (SEQ ID NO. 16), and the extracellular region, helical region, and cytoplasmic region of human CD3E (SEQ ID NO. 17). As is well known in the art, TRuC is a T cell receptor fusion construct, e.g., see reference 12).

[0323] D52N-STAR is also referred to herein as STAR (D52N), which has: a leader sequence (SEQ ID NO. 14), a variable heavy chain domain of D52N scFv (SEQ ID NO. 7), a murine constant αTCR domain (SEQ ID NO. 18), a P2A self-cleavage peptide (SEQ ID NO. 3), a leader sequence (SEQ ID NO. 19), a variable light chain domain of D52N scFv (SEQ ID NO. 9) and a murine constant βTCR domain (SEQ ID NO. 20). As is well known in the art, STAR is a synthetic T cell receptor and antigen receptor, e.g., see reference 13).

[0324] D52N-Fab-CAR has: a leader sequence (SEQ ID NO.14), a variable heavy chain 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 ζ chain (CD247) signaling tail (SEQ ID NO.13), a GSG filler (SEQ ID NO.23), a P2A self-cleavage peptide (SEQ ID NO.3), a leader sequence (SEQ ID NO.14), a variable light chain 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 ζ chain (CD247) signaling tail (SEQ ID NO.13).

[0325] Production of lentiviral supernatant

[0326] 293T cells (ATCC CRL-3216) were seeded at 50% confluence in 6-well plates in a volume of 3 mL. The cells were allowed to attach overnight and transfected the next morning with a mixture of packaging plasmids (950 ng pRSV-Rev, 370 ng pMD2.G, 950 ng pMDLg / pRRE, and 1000 ng of the corresponding lentiviral transfer plasmid). Transfections were performed using X-tremeGENE HP transfection reagent at a ratio of 3 μL transfection reagent to 1 μg DNA. The 293T cells were then incubated at 37°C, 10% CO 2 Incubate at 40 °C (v / v) for 48 h and filter the lentiviral supernatant through a 0.45 μm cellulose acetate syringe filter. Use the supernatant to transduce Jurkat cells or primary CD8+ T cells. Lentiviral supernatant can be used directly or pre-concentrated using Lenti-X Concentration Reagent (TakaraBio) according to the manufacturer's instructions. Store the stored supernatant at -80 °C.

[0327] cell

[0328] 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 pSIRV-NF-kB plasmid (Addgene #118093) into Jurkat E6-1 clone (ATCC TIB-152)), Jurkat E6.1 TCRα-β-cells.

[0329] Human leukocyte cones were obtained and CD8+ T cells were isolated. The isolated CD8+ T lymphocytes were mixed with human anti-CD3 / CD28 immunomagnetic beads (Dynabead) at a ratio of 1:1 cells to beads. The cells were left to stand overnight and 1.5 million CD8+ T cells were transduced with the corresponding lentiviral supernatant encoding the target receptor the next morning. 72 hours after transduction, the cells were selected with puromycin (pLEX_307 backbone) or G418 (pLEX307-NeoR backbone).

[0330] T cell stimulation assay

[0331] T2 suspension cell line was used as a surrogate APC on which 9V NY-ESO-1 peptide was loaded. First, 3.3 x 10 4 T2 cells were seeded into a V-bottom 96-well plate with 110 μL of complete RPMI. Serial dilutions of 9V peptide were also prepared using complete RPMI. Then 110 μL of the diluted 9V peptide was added to the T2 cell suspension and incubated at 37°C and 5% CO. 2The cells were then incubated for 90 minutes under RPMI (v / v) conditions. The T2 cells were then washed with complete RPMI and resuspended in 110 μL of 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 (in a volume of 100 μL) were then added to each well to form a simple 2:1 effector cell to target cell ratio. The plate was gently centrifuged (15 g, min) to promote contact between T cells and APCs. The co-culture lasted for 20 hours, with the cells incubated at 37°C, 5% CO 2 Incubate under (v / v) conditions.

[0332] Flow cytometry

[0333] At the end of the experiment, 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 NIR, 1:500 working dilution) in a volume of 50 μL of PBS. Samples were then stained with conjugated flow cytometry antibodies pre-diluted in 50 μL 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. Samples were collected on a BD X-20 or Cytoflex flow cytometer and analyzed using the FlowJo kit.

[0334] It is noteworthy that the detection of multiple receptors was performed by fluorescent pMHC tetramers. The tetramers were composed of biotinylated, refolded 9V pMHC molecules complexed with PE streptavidin. The fluorescent tetramers were prepared by vigorously mixing 66.6 μg of 9V pMHC monomers with 10 μL of PE streptavidin added gradually every 10 minutes (10 additions of PE streptavidin in 100 minutes).

[0335] Cytokine measurement

[0336] After the co-culture experiment, the supernatant was analyzed for IFN-γ and IL-2 levels using commercial kits provided by Thermo Fisher Scientific.

[0337] Example 2 - Preparation of modified adhesion receptors to improve antigen sensitivity of CAR

[0338] This example investigates engineered adhesion receptor-ligand complexes of varying sizes to maximize their ability to enhance CAR-antigen complexes.

[0339] Lengthened variants of CD2 were constructed and screened for the ability to affect sensitivity.

[0340] A set of elongated CD2 constructs were prepared by inserting different fragments of the extracellular domain of the human mucin-like protein CD43 as spacers ( Fig. 9 A). 1G4 TCR or standard CAR was expressed in the CD2-E6.1 Jurkat T cell line, followed by transduction of different CD2 CAR constructs ( Fig. 9 B).

[0341] As expected, wild-type CD2 greatly improved the antigen sensitivity of the TCR, whereas the use of CD2 lengthened by 4, 8, 20, 40, 50, 60, 80, 120, 160, or 234 amino acids from CD43 resulted in a progressive loss of antigen sensitivity ( Fig.10 Similarly, the use of wild-type CD2 greatly improved the antigen sensitivity of STAR, while the use of CD2 extended by 4, 8, 20, 40, or 234 amino acids resulted in a gradual loss of antigen sensitivity ( Fig.11 ).

[0342] In sharp contrast, wild-type CD2 had only a minor effect on CAR antigen sensitivity, whereas lengthening CD2 improved antigen sensitivity, with the greatest improvement achieved with medium-length CD2 constructs (CD2-CD43(40) or CD2-CD43(20)). Fig.12 and Fig.13 ).

[0343] Wild-type CD2 increased the antigen sensitivity of eTruC. Lengthening CD2 increased antigen sensitivity, with the greatest improvement achieved with a medium-length CD2 construct (CD2-CD43(20)). Fig.14 This result is expected according to the inventors' studies, based on the size of eTruC relative to TCR, and the results of the combination of elongated CD2 and CAR.

[0344] Therefore, these data suggest that the sensitivity of CAR can be improved by increasing the size of the accessory-ligand complex (such as the CD2 / CD58 complex). It also confirms that it is the larger size of the CAR-antigen complex that limits its ability to utilize CD2-CD58 ( Fig.15 ). CD2-CD43(234) antagonizes TCR and CAR antigen recognition because the membranes are too far apart to allow antigen binding.

[0345] Materials and methods

[0346] Production of the extended CD2

[0347] The 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), and then fusing the CD2 transmembrane domain (SEQ ID NO: 51) and the CD2 intracellular domain (SEQ ID NO: 52). Several amino acids at the C-terminus of the CD43 domain were isolated by site-directed mutagenesis from the full-length sequence consisting of the entire CD43 extracellular portion, thereby generating shorter variants (e.g., SEQ ID NOs: 53 to 62). For example, CD2-CD43 (20) contains the extracellular portion of CD2 fused to 20 amino acids proximal to the C-terminus of the extracellular portion of CD43. SEQ ID NOs: 27 to 36 provide extended CD2 variants, each of which also includes a CD2 signal peptide (SEQ ID NO: 47) and an HA tag (SEQ ID NO: 48).

[0348] Cell lines

[0349] Jurkat TCRα-β cells were a gift from Simon J. Davis (Oxford University) 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. 2 cultured under the conditions of .

[0350] Endogenous surface protein CD2 was knocked out using CRISPR / Cas9.

[0351] Co-culture with U87 cells

[0352] 25000 U87 cells were seeded into tissue culture treated flat bottom 96 well plates and cultured overnight. The next day, the medium was removed from the cells and they were then incubated with appropriate concentrations of peptides in complete DMEM (DMEM supplemented with 10% v / v FBS, 100 units / ml penicillin and 100 μg / ml streptomycin) at 37°C for 1 hour.

[0353] The peptide-containing medium was then removed and 50,000 T cells were added to each well. The co-cultures were then spun at 50 x g for 2 minutes and incubated at 37°C for 4 hours. At the end of the incubation period, a portion of the supernatant was removed for cytokine ELISA and stored at -20°C. EDTA (final concentration 2.5 μM) was added to the remaining supernatant and the cells were detached by pipetting.

[0354] Cells were stained in PBS containing 1% BSA for detection of CD45 (clone HI30, dilution 1:200), CD69 (clone FN50, dilution 1:200) and 4-1BB (clone 4B4-1, dilution 1:200) staining, as well as PE-coupled tetrameric pMHC (dilution 1:500). Stained cells were analyzed immediately or fixed with PBS containing 1% formaldehyde and analyzed the next day.

[0355] T cells were distinguished from U87 cells by CD45 staining and / or assessment of size and complexity. Individual T cells were identified based on size and subsequent analysis of this population was performed.

[0356] Example 3 - Effect of CD2 of different sizes on the antigen sensitivity of tisagenlecleucel (Kymriah)

[0357] This example tests the effect of variable size CD2 on Kymriah, a clinically approved CAR targeting the surface antigen CD19. Kymriah has a CD2 - Expression in Jurkat T cells ( Fig.16 A), while also expressing CD2 of variable size. Cells were sorted based on matching expression levels ( Fig.16 B).

[0358] To test the antigen sensitivity of Kymriah, CD19 levels on the surface of B cells were manipulated using Nalm6 CombiCells as described in reference 14. These cells are CD19 - The Spycatcher protein that spontaneously forms a covalent bond with the Spytag is expressed on the cell surface. By adding different concentrations of purified Spytag-CD19 to the solution, Nalm6CombiCells with different surface CD19 levels can be generated ( Fig.16 C).

[0359] Each Jurkat strain was co-cultured with Nalm6 CombiCells loaded with different levels of CD19 ( Fig.16 D. Fig.16 E). Compared with T cells lacking CD2, studies have found that wild-type CD2 can significantly improve antigen sensitivity (EC 50 However, increasing the size of CD2 by 4 amino acids further increased antigen sensitivity. Further increases in CD2 size resulted in a gradual decrease in antigen sensitivity, with the longest CD2 variant having a sensitivity similar to that of Jurkat T cells lacking CD2.

[0360] Taken together, these results highlight that even modest lengthening of CD2 can improve the antigen sensitivity of CARs (Kymriah) targeting a folded antigen (CD19).

[0361] Production of Jurkat T cells expressing Kymriah

[0362] HEK 293T cells were seeded in 6-well plates containing DMEM supplemented with 10% FBS and 1% penicillin / streptomycin to reach 60-80% confluence on the second day. Cells were transfected with 0.25pRSVRev (Addgene, 12253), 0.53μgpMDLg / pRRE (Addgene, 12251), 0.35μg pMD2.G (Addgene, 12259) and 0.8μg of the transport plasmid expressing Kymriah CAR using 5.8X-tremeGENE HP (Roche). The medium was replaced after 16 hours, and the supernatant was filtered with a 0.45μm cellulose acetate filter after another 24 hours of culture. The supernatant from one well of a 6-well plate was used to transfect 1 million CD2 - Jurkat T cells. Jurkat T cells expressing Kymriah CAR were sorted and further cultured at a density of 300,000 cells / ml.

[0363] Virus was prepared using HEK cells as described above. CD2 expressing Kymriah was transduced with WT CD2 or one of six CD2 molecules of variable size. - Jurkat T cells. Jurkat T cells were sorted based on their CD2 protein expression levels and then further cultured at a density of 300,000 cells / ml.

[0364] Coupling of ligands to Nalm6 cells

[0365] 30,000 Nalm6 CombiCells were seeded into TC-coated 96-well round-bottom plates and incubated at 37°C, 5% CO 2 Incubate overnight. On the day of the experiment, transfer the cells to a TC-coated 96-well V-bottom plate and centrifuge at 520 g for 5 minutes. Dilute the Spytag-CD19 ligand to the desired concentration in complete RPMI (10% FCS, 1% penicillin-streptomycin). Remove the existing culture medium from the cells, add 50 μl of the diluted ligand, and incubate at 37°C, 5% CO 2 Incubate for 40 minutes. Then wash cells twice with complete RPMI.

[0366] Co-culture assay of Kymriah Jurkat T cells and Nalm6 CombiCells

[0367] Kymriah Jurkat T cells were counted and washed once in complete RPMI. 50,000 Jurkat T cells in 200 μl complete RPMI were added to Nalm6 CombiCells coupled to Spytag-CD19 and transferred to a 96-well round-bottom plate. The cells were then incubated at 37°C, 5% CO 2 Incubate for 6 hours.

[0368] Flow cytometry-detection of Spytag-CD19

[0369] After ligand coupling and subsequent washing, Nalm6 CombiCells were transferred to a V-bottom plate and spun at 500 g for 5 min at 4 °C. Cells were washed with 1% PBS-BSA at 500 g for 5 min at 4 °C. For ligand detection, fluorescently conjugated antibodies against the target protein were diluted 1:200 in 1% PBS-BSA and added to the cells in a volume of 50 μl. Cells were resuspended and incubated at 4 °C in the dark for 20 min. Cells were washed twice in PBS, resuspended in 60 μl PBS, and analyzed on a flow cytometer.

[0370] Flow cytometry-detection of KymriahJurkatT cell activation

[0371] After the stimulation test, Kymriah Jurkat cells were transferred to a V-bottom plate and washed once with 200 μl of PBS containing 1% BSA (500g, 4°C, 5 minutes). Antibodies against the activation marker CD69 were diluted 1:200 in PBS containing 1% BSA. 50 μl of staining solution was added to the cells and incubated at 4°C in the dark for 20 minutes. The cells were washed twice with PBS and resuspended in 70 μl PBS and analyzed on a flow cytometer. Nalm6 CombiCells can be distinguished from Kymriah Jurkat T cells by their inherent GFP marker. Flow cytometry data were analyzed using FlowJo (BD Biosciences).

[0372] References

[0373] 1 Burton et al. (2021) Inefficient exploitation of accessory receptors reduces the sensitivity of chimeric antigen receptors. bioRxiv (preprint).

[0374] 2 van der Merwe et al.(1995)Current Biology 5:74-84.

[0375] 3 Demetriou,et al.(2020)Nature Immunology 21:1232-1243.

[0376] 4 Maus et al.,(2017)Mol Ther Oncolytics 11;3:1-9.

[0377] 5 Stewart-Jones et al.,(2009)106(14)5784-5788.

[0378] 6 Kershaw et al.(2013)Nature Reviews Cancer,13(8):525-41

[0379] 7 Choudhuri et al.,(2005)Nature,436(7050):578-82.

[0380] 8 Cyster et al.,(1991)EMBO J.10(4):893-902.

[0381] 9 Milstein et al., (2008) J Biol Chem 283(49): 34414-34422.

[0382] 10 Rurik et al.,(2022)Science 375(6576):91-96.

[0383] 11 Wild et al.(1999)J Exp Med,190(1):31-41.

[0384] 12 Baeuerle et al.(2019)Nature communications,10:2087.

[0385] 13 Liu et al.(2021)Science Translational Medicine,13(586):eabb5191.

[0386] 14 Patel et al.,(2023)bioRxiv,2023-06 15.545075

[0387] (https: / / doi.org / 10.1101 / 2023.06.15.545075)

[0388] Sequence Listing

[0389]

[0390]

[0391]

Claims

1. An immune effector cell, comprising: - a chimeric antigen receptor (CAR) capable of binding to an antigen on an antigen presenting cell (APC), wherein the CAR comprises 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 connected by a hinge region; and - a coreceptor capable of binding to a ligand on the APC, wherein the coreceptor comprises an extracellular ligand binding domain and a transmembrane domain, wherein the extracellular ligand binding domain and the transmembrane domain are connected by a stem region; in: (a) the stem region of the coreceptor comprises a sequence that physically increases the height of the extracellular ligand binding domain from the immune effector cell membrane; (b) the hinge region of the CAR comprises a sequence that physically increases the height of the antigen binding domain from the immune effector cell membrane, or consists of a sequence that physically increases the height of the antigen binding domain from the immune effector cell membrane, wherein the sequence comprises a mucin-like sequence, one or more folded polypeptide domains, or a fragment of CD28 as shown in SEQ ID NO: 10 or a fragment of the CD8a hinge region as shown in SEQ ID NO: 43; and / or (c) The CAR and the coreceptor have respective sizes such that the membrane distance spanned by the CAR-antigen complex is comparable to the membrane distance spanned by the coreceptor-ligand complex.

2. The immune effector cell of claim 1, wherein the membrane distance spanned by the CAR-antigen complex differs from the membrane distance spanned by the auxiliary receptor-ligand complex by ≤5%, ≤10%, ≤15%, ≤20%, ≤25% or ≤30%, or is the same as the membrane distance spanned by the auxiliary receptor-ligand complex.

3. The immune effector cell of claim 1 or 2, wherein the membrane spacing spanned by the CAR-antigen complex or the co-receptor-ligand complex is comparable to (e.g., within ≤5%, ≤10%, ≤15%, ≤20%, ≤25%, or ≤30%, or the same as) the membrane spacing spanned by the complex between a T cell receptor and its peptide-MHC antigen.

4. The immune effector cell of any of the preceding claims, wherein the membrane spacing spanned is about 14 nm.

5. The immune effector cell of any one of the preceding claims, wherein the height of the antigen binding domain of the CAR from the immune effector cell membrane is comparable to the height (e.g., about 7 nm) of the extracellular antigen binding domain of the T cell receptor from the immune effector cell membrane (e.g., within ≤5%, ≤10%, ≤15%, ≤20%, ≤25% or ≤30%, or the same as thereof).

6. The immune effector cell of any one of the preceding claims, wherein the extracellular antigen binding domain of the CAR is at a height of about 3 nm to 7 nm from the immune effector cell membrane.

7. The immune effector cell of any one of the preceding claims, wherein the antigen binding domain of the CAR is at a height of about 7 nm to 47 nm from the immune effector cell membrane.

8. The immune effector cell of any of the preceding claims, wherein the ligand binding domain of the coreceptor is at a height of about 3 nm to 7 nm from the immune effector cell membrane.

9. The immune effector cell of any of the preceding claims, wherein the ligand binding domain of the coreceptor is at a height of about 7 nm to 47 nm from the immune effector cell membrane.

10. The method of any of the preceding claims, wherein the height of the ligand binding domain of the coreceptor from the immune effector cell membrane is about 7 nm.

11. The immune effector cell of any of the preceding claims, wherein the co-receptor is an endogenous co-receptor.

12. The immune effector cell of any one of the preceding claims, wherein the CAR comprises a CD28 hinge region or a CD8a hinge region.

13. The immune effector cell of any of the preceding claims, wherein the hinge region of the CAR comprises, or consists of, a sequence that physically increases the height of the antigen binding domain from the immune effector cell membrane, wherein the sequence comprises a mucin-like sequence, one or more folded polypeptide domains, or a fragment of CD28 as shown in SEQ ID NO: 10 or a fragment of the CD8a hinge region as shown in SEQ ID NO:

43.

14. The immune effector cell of claims 1 to 12, wherein the stem region of the coreceptor comprises a sequence that physically increases the height of the extracellular ligand binding domain from the immune effector cell membrane.

15. The immune effector cell of any one of claims 1 to 12, wherein the immune effector cell comprises: (a) a coreceptor having a stem region comprising a sequence that physically increases the height of the extracellular ligand binding domain from the immune effector cell membrane; and (b) a CAR having a hinge region, wherein the hinge region comprises a sequence that physically increases the height of the antigen binding domain from the immune effector cell membrane, or consists of a sequence that physically increases the height of the antigen binding domain from the immune effector cell membrane, wherein the sequence comprises a mucin-like sequence, one or more folded polypeptide domains, or a fragment of CD28 as shown in SEQ ID NO: 10 or a fragment of the CD8a hinge region as shown in SEQ ID NO:

43.

16. The immune effector cell of any one of the preceding claims, wherein the hinge region of the CAR comprises or consists of composition: (a) a fragment of the mucin-like extracellular sequence of CD43 as shown in SEQ ID NO: 62, such as 4 to 234 amino acids, such as 20 to 40 amino acids (e.g., as shown in any one of SEQ ID NOs: 53 to 61); (b) a folded polypeptide domain, which is an immunoglobulin domain, such as an immunoglobulin constant domain or an FNIII domain; and / or (c) a fragment of the hinge region of CD28 as shown in SEQ ID NO:10 or a fragment of the hinge region of CD8a as shown in SEQ ID NO:43, optionally, wherein the fragment is shown in any one of SEQ ID NOs:37 to 42, 46, 63 and 64.

17. The immune effector cell of any one of claims 1 to 10 and 12 to 16, wherein the stem region of the coreceptor comprises or consists of a fragment of a mucin-like sequence.

18. The immune effector cell of claim 17, wherein the mucin-like sequence is a mucin-like sequence of CD43 as shown in SEQ ID NO: 62, such as 4 to 234 amino acids, as shown in any one of SEQ ID NOs: 53 to 61.

19. The immune effector cell of any of the preceding claims, wherein the co-receptor is an adhesion receptor.

20. The immune effector cell of claim 19, wherein the adhesion receptor is CD2.

21. The immune effector cell of any one of the preceding claims, in: (a) the expression of the CAR on the surface of the immune effector cell does not require endogenous TCR and / or CD3; (b) the CAR comprises an intracellular CD3ζ signaling domain; and / or (c) the CAR does not comprise an intracellular CD2 signaling domain.

22. The immune effector cell of any one of the preceding claims, wherein the CAR is a dimer, e.g., a homodimer or a heterodimer.

23. The immune effector cell of any one of the preceding claims, in: (a) the antigen is a peptide-MHC complex, CD19, mesothelin, BCMA, CD22, EGFR or EGFRvIII, optionally, wherein the peptide in the peptide-MHC complex is a fragment of NY-ESO 1; (b) the ligand is CD58; and / or (c) the antigen is different from the ligand.

24. 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 connected by a hinge region, wherein the hinge region comprises a sequence that physically increases the height of the extracellular antigen binding domain from the immune effector cell membrane, or consists of a sequence that physically increases the height of the extracellular antigen binding domain from the immune effector cell membrane, wherein the sequence comprises a mucin-like sequence, one or more folded polypeptide domains, or a fragment of CD28 as shown in SEQ ID NO: 10 or a fragment of the CD8a hinge region as shown in SEQ ID NO:

43.

25. The CAR of claim 21, as defined in any one of claims 1 to 23.

26. A secondary receptor capable of binding to a ligand on an APC, comprising an extracellular ligand binding domain and a transmembrane domain, wherein the extracellular ligand binding domain and the transmembrane domain are connected by a stem region, wherein the stem region comprises a sequence that physically increases the height of the ligand binding domain from the immune effector cell membrane, or is composed of a sequence that physically increases the height of the ligand binding domain from the immune effector cell membrane, optionally wherein the stem region comprises a mucin-like sequence.

27. The coreceptor of claim 26, as defined in any one of claims 1 to 23.

28. An immune effector cell comprising or encoding the CAR and / or co-receptor described in any one of claims 24 to 27.

29. The immune effector cell of any one of claims 1 to 23 and 28, wherein the immune effector cell comprises a modification in its genome to reduce expression of the endogenous co-receptor.

30. The immune effector cell of any one of claims 1 to 23, 28 and 29, wherein the immune effector cell is a T cell, a NK cell, a NKT cell, a phagocyte or a macrophage, optionally wherein the T cell is a CD4-CD8+ T cell.

31. A method for preparing immune effector cells, comprising introducing a nucleic acid encoding a CAR and / or an auxiliary receptor described in any one of claims 24 to 27 into an immune effector cell.

32. A method for optimizing the effector function of immune effector cells, wherein the immune effector cells comprise: - a chimeric antigen receptor (CAR) capable of binding to an antigen on an antigen presenting cell (APC), wherein the CAR comprises 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 connected by a hinge region; and - a coreceptor capable of binding to a ligand on the APC, wherein the coreceptor comprises an extracellular ligand binding domain and a transmembrane domain, wherein the extracellular ligand binding domain and the transmembrane domain are connected by a stem region; in, The method includes modifying the height of the extracellular antigen binding domain of the CAR from the immune effector cell membrane and / or the height of the extracellular ligand binding domain of the auxiliary receptor from the immune effector cell membrane to optimize the effector function of the immune effector cell when in contact with the APC.

33. A method for identifying improved immune effector cells, wherein the immune effector cells comprise: - a chimeric antigen receptor (CAR) capable of binding to an antigen on an antigen presenting cell (APC), wherein the CAR comprises 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 connected by a hinge region; and - a coreceptor capable of binding to a ligand on the APC, wherein the coreceptor comprises an extracellular ligand binding domain and a transmembrane domain, wherein the extracellular ligand binding domain and the transmembrane domain are connected by a stem region; The method include: (a) modifying the height of the extracellular antigen binding domain of the CAR from the immune effector cell membrane and / or the height of the extracellular ligand binding domain of the auxiliary receptor from the immune effector cell membrane; and (b) determining whether the immune effector cells expressing the modified CAR and / or the modified coreceptor have improved effector function compared to immune effector cells expressing the unmodified CAR and / or the unmodified coreceptor.

34. The method of claim 32 or 33, comprising modifying the height of the extracellular antigen binding domain of the CAR so that the membrane distance spanned by the CAR-antigen complex is comparable to the membrane distance spanned by the auxiliary receptor-ligand complex.

35. The method of claim 32 or 33, comprising modifying the height of the extracellular ligand binding domain of the auxiliary receptor so that the membrane distance spanned by the CAR-antigen complex is comparable to the membrane distance spanned by the auxiliary receptor-ligand complex.

36. The method of claim 32 or 33, comprising modifying the height of the extracellular antigen binding domain of the CAR and the height of the extracellular ligand binding domain of the auxiliary receptor so that the membrane distance spanned by the CAR-antigen complex is equivalent to the membrane distance spanned by the auxiliary receptor-ligand complex.

37. The method of claim 36, in: (a) the membrane distance spanned by the CAR-antigen complex differs from the membrane distance spanned by the coreceptor-ligand complex by ≤5%, ≤10%, ≤15%, ≤20%, ≤25% or ≤30%, or is the same as the membrane distance spanned by the coreceptor-ligand complex; and / or (b) the membrane distance spanned by the CAR-antigen complex is comparable to the membrane distance spanned by the complex between the T cell receptor and its corresponding antigen (e.g., within ≤5%, ≤10%, ≤15%, ≤20%, ≤25% or ≤30%, or the same as thereof).

38. The method of any one of claims 32 to 37, include: (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 coreceptor; 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 co-receptor.

39. The method of any one of claims 32 to 38, comprising introducing a sequence that physically increases the height of the ligand binding domain from the immune effector cell membrane into the hinge region of the CAR and / or the stem region of the auxiliary receptor, or replacing the hinge region of the CAR and / or the stem region of the auxiliary receptor with a sequence that physically increases the height of the ligand binding domain from the immune effector cell membrane.

40. The method of claim 39, wherein the sequence introduced into the hinge region of the CAR comprises or consists of composition: (a) a fragment of the mucin-like extracellular sequence of CD43 as shown in SEQ ID NO: 62, such as 4 to 234 amino acids, such as 20 to 40 amino acids (e.g., as shown in any one of SEQ ID NOs: 53 to 61); (b) a folded polypeptide domain, which is an immunoglobulin domain, such as an immunoglobulin constant domain or an FNIII domain; and / or (c) a fragment of the hinge region of CD28 as shown in SEQ ID NO:10 or a fragment of the hinge region of CD8a as shown in SEQ ID NO:43, optionally, wherein the fragment is shown in any one of SEQ ID NOs:37 to 42, 46, 63 and 64.

41. The method of claim 39, wherein the sequence introduced into the stem region 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 a mucin-like sequence of CD43 as shown in SEQ ID NO: 62, optionally wherein the fragment has a length of 4 to 234 amino acid residues, such as any one of SEQ ID NOs: 53 to 61.

42. The method of any one of claims 32 to 41, wherein the CAR, the co-receptor and / or the immune effector cell are as defined in any one of claims 1 to 30.

43. A method for identifying an improved immune effector cell, comprising determining whether the immune effector cell of any one of claims 1 to 23 and 28 to 30 has improved effector function compared to an immune effector cell expressing an unmodified CAR and / or an unmodified co-receptor.

44. The method of any one of claims 32 to 43, wherein the effector function is cell killing.

45. Immune effector cells obtained or obtainable by the method of any one of claims 32 to 44.

46. ​​A method for preparing an immune effector cell population for adoptive cell therapy, such as an ex vivo method, comprising culturing the immune effector cell of any one of claims 1 to 23, 28 to 30 and 45 to produce an immune effector cell population.

47. A population of immune effector cells produced by the method of claim 46.

48. A method of treating cancer in a subject, comprising administering to the subject an effective amount of the immune effector cell of any one of claims 1 to 23, 28 to 30 and 45 or the immune effector cell population of claim 47.

49. The method of claim 48, wherein the cancer is a hematological malignancy or a B-cell cancer.

50. Use of the immune effector cell of any one of claims 1 to 23, 28 to 30 and 45, or the population of immune effector cells of claim 47, in a method of treating cancer in a subject, optionally wherein the cancer is a hematological malignancy or a B-cell cancer.

51. Use of the immune effector cell of any one of claims 1 to 23, 28 to 30 and 45 or the immune effector cell population of claim 47 in the preparation of a medicament for treating cancer, optionally wherein the cancer is a hematological malignancy or a B-cell cancer.

52. Use of the immune effector cell of any one of claims 1 to 23, 28 to 30 and 45 or the immune effector cell population of claim 47 for treating cancer, optionally wherein the cancer is a hematological malignancy or a B-cell cancer.

53. Use of the immune effector cell of any one of claims 1 to 23, 28 to 30 and 45 or the immune effector cell population of claim 47 for adoptive cell therapy.

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