Enhanced CAR-T cell therapy using antigen presenting cells

Abstract

A cancer therapy comprising both a population of genetically engineered T cells expressing chimeric antigen receptors (CARs) and a population of antigen presenting cells (APCs) that enhance the efficacy of the T cells expressing the CARs.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of the filing date of International Application No. PCT / CN2022 / 080943, filed on March 15, 2022, the entire contents of which are incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy created on March 13, 2023 is named 112126-0046-70006WO01_SEQ.XML and is 196,714 bytes in size. Background Art

[0005] Adoptive cell transfer therapy is an immunotherapy involving ex vivo expansion of immune cells, which can be modified to express chimeric antigen receptors (CARs) that specifically target cells expressing specific antigens (e.g., tumor-associated antigens (TAAs)). CAR T cell therapy has shown promising therapeutic effects in treating certain types of cancer. However, its application is often limited by toxicity, for example, unexpected increases in cytokine levels (known as cytokine release syndrome), which may lead to death of the recipient. Morgan et al., Molecular Therapy 18(4): 843-851, 2010. In addition, modified immune cells may not be able to expand well in the patient, may not survive long enough in the body, and may be sensitive to the cytotoxic environment caused by their own in vivo activity.

[0006] Antigen presenting cells (APCs) are immune cells that mediate cellular immune responses by processing and presenting antigens for recognition by certain lymphocytes, such as T cells. Classical APCs include dendritic cells, macrophages, Langerhans cells, and B cells. Summary of the invention

[0007] The present disclosure is based at least in part on the development of using antigen presenting cells to enhance the therapeutic efficacy of CAR T cells. Without being bound by theory, the co-use of antigen presenting cells disclosed herein is expected to enhance the in vivo expansion of CAR-T cells, thereby improving clinical efficacy.

[0008] Therefore, in some aspects, the present disclosure features a method for treating a tumor, the method comprising administering to a subject in need: (a) an effective amount of a population of genetically engineered T cells expressing one or more chimeric antigen receptors (CARs); and (b) an effective amount of antigen presenting cells (APCs). In some embodiments, the subject may be a human patient with a solid tumor. In other embodiments, the subject may be a human patient with a hematopoietic cancer, such as acute myeloid leukemia (AML).

[0009] In some embodiments, the genetically engineered T cells may express a bispecific CAR (e.g., containing a single fusion polypeptide or two fusion polypeptides), the bispecific CAR including a first antigen binding portion specific to a tumor associated antigen (TAA) and a second antigen binding portion specific to CD19 or B cell maturation antigen (BCMA). In some cases, the TAA is different from CD19 or BCMA. In other embodiments, the genetically engineered T cells may express a T cell receptor (TCR) and a CAR specific to TAA, the CAR including an antigen binding portion specific to CD19 or BCMA. The APC used in the methods disclosed herein may express CD19, BCMA, or a combination thereof. In some embodiments, the APC may further express TAA.

[0010] In some embodiments, the genetically engineered T cells used in any of the methods disclosed herein can further express cytokines, such as antagonists of cytokines capable of activating an immune response. Examples include, but are not limited to, interleukin-1 (IL-1), interleukin-1 (IL-2), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-1 (IL-9), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), interleukin-23 (IL-23), interleukin-24 (IL-24), interleukin-33 (IL-33), interleukin-36 (IL-36), GM-CSF, interferon gamma (IFNγ), and chemokine (CC motif) ligand 19 (CCL19). In some examples, the antagonist of the cytokine can be a fusion polypeptide, which includes a binding portion of the cytokine (e.g., a receptor or a binding fragment thereof) and an immune activating cytokine (e.g., IL-2, IL-7, IL-9, IL-10, IL-12, IL-15, IL-18, IL-21, IL-23, IL-24, IL-36, IL-33 and CCL19).

[0011] In one example, the fusion polypeptide includes a binding portion of IFNγ fused to IL-18, such as a binding portion disclosed herein. Without being bound by theory, this fusion protein is expected to enhance CAR-T cell efficacy through the IL-18 portion, while inhibiting cytokine toxicity mediated by IFNγ through the anti-IFNγ portion in the fusion protein. In a specific example, the binding portion of IFNγ is an anti-IFNγ scFv, and the anti-IFNγ scFv preferably includes the amino acid sequence of SEQ ID NO: 55. Alternatively or additionally, IL-18 may include the amino acid sequence of SEQ ID NO: 53. In one example, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 56.

[0012] In some embodiments, the genetically engineered T cells used in any of the methods disclosed herein may express a bispecific CAR. For example, the bispecific CAR may include a first antigen binding portion specific to the TAA and a second antigen binding portion specific to CD19. Genetically engineered T cells expressing such bispecific CARs can be used together with APCs expressing the CD19 and optionally the TAA. In other examples, the bispecific CAR includes a first antigen binding portion specific to the TAA and a second antigen binding portion specific to the BCMA. Genetically engineered T cells expressing such bispecific CARs can be used together with APCs expressing the BCMA and optionally the TAA.

[0013] In some cases, the bispecific CAR disclosed herein may include a fusion polypeptide comprising the first antigen binding portion and the second antigen binding portion. In some instances, the first antigen binding portion and the second antigen may be connected by a peptide linker. In some instances, the first antigen binding portion and / or the second antigen binding portion may be in a single-chain variable fragment (scFv) format. Alternatively, the first antigen binding portion and / or the second antigen binding portion may be in a single domain antibody (VHH) format. The bispecific CAR may further include an intracellular signaling domain, which may include one or more signaling domains (e.g., a co-stimulatory domain and a cytoplasmic signaling domain); and a hinge domain and a transmembrane domain that optionally connect the antigen binding portion to the intracellular signaling domain. In some specific instances, the bispecific CAR may be Figure 2A The format described in .

[0014] In some cases, the bispecific CAR may include a first fusion polypeptide comprising the first antigen binding portion and a second fusion polypeptide comprising the second antigen binding portion. In some instances, the first antigen binding portion and / or the second antigen binding portion may be or be in a single chain variable fragment (scFv) format. Alternatively, the first antigen binding portion and / or the second antigen binding portion may be in a single domain antibody (VHH) format. In yet another example, the first antigen binding portion may be in a single chain variable fragment (scFv) or in a single domain antibody (VHH) format. The second antigen binding portion may be an extracellular domain of a ligand that binds to the TAA. In some instances, the bispecific CAR construct may be Figure 3A In some specific examples, the first antigen binding moiety is the extracellular domain of CD27, which binds to CD70, and the second antigen binding moiety is specific for BCMA. Genetically engineered T cells expressing this pair of bispecific CARs can be used to treat AML, optionally in combination with APCs expressing BCMA and optionally CD70.

[0015] In some examples, the first fusion polypeptide and the second fusion polypeptide include an intracellular signaling domain, which may include one or more signaling domains (e.g., a co-stimulatory signaling domain and a cytoplasmic signaling domain); and optionally a hinge domain and a transmembrane domain connecting the antigen binding portion and the intracellular domain.

[0016] In some specific examples, the bispecific CAR includes one or more scFv fragments specific to CD19, BCMA or TAA as disclosed herein. Alternatively or additionally, the bispecific CAR may include one or more VHH fragments specific to CD19, BCMA or the TAA as disclosed herein. Exemplary scFv fragments and / or VHH fragments for constructing the bispecific CAR construct disclosed herein are provided below. See also the sequence table, all of which are within the scope of the present disclosure.

[0017] (a) a scFv fragment specific for BCMA (anti-BCMA scFv), the anti-BCMA scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL). In some cases, the VH of the anti-BCMA scFv comprises the amino acid sequence of SEQ ID NO: 14, and the VL of the anti-BCMA scFv comprises the amino acid sequence of SEQ ID NO: 15. In a specific example, the anti-BCMA scFv comprises the amino acid sequence of SEQ ID NO: 16.

[0018] (b) an scFv fragment specific for CD19 (anti-CD19 scFv), the anti-CD19 scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein optionally the VH of the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 59, and the VL of the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 60, preferably wherein the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 61 or 62;

[0019] (c) a scFv fragment specific for Meso (anti-Meso scFv), the anti-Meso scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL). In some cases, the VH of the anti-Meso scFv comprises the amino acid sequence of SEQ ID NO: 11, and the VL of the anti-Meso scFv comprises the amino acid sequence of SEQ ID NO: 12. In one example, the anti-Meso scFv comprises the amino acid sequence of SEQ ID NO: 13.

[0020] (d) a scFv fragment specific for HER2 (anti-HER2 scFv), the anti-HER2 scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL). In some cases, the VH of the anti-HER2 scFv comprises the amino acid sequence of SEQ ID NO: 17, and the VL of the anti-HER2 scFv comprises the amino acid sequence of SEQ ID NO: 18. In one example, the anti-HER2 scFv comprises the amino acid sequence of SEQ ID NO: 19.

[0021] (e) a scFv fragment specific for GPC3 (anti-GPC3 scFv), the anti-GPC3 scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL). In some cases, the VH of the anti-GPC3 scFv comprises the amino acid sequence of SEQ ID NO: 20, and the VL of the anti-GPC3 scFv comprises the amino acid sequence of SEQ ID NO: 21. In one example, the anti-BCMA scFv comprises the amino acid sequence of SEQ ID NO: 22.

[0022] (f) a scFv fragment specific for Claudin 18.2 (anti-Claudin 18.2scFv), the anti-Claudin18.2scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL). In some cases, the VH of the anti-Claudin18.2scFv comprises the amino acid sequence of SEQ ID NO: 23, and the VL of the anti-Claudin18.2scFv comprises the amino acid sequence of SEQ ID NO: 24. In one example, the anti-Claudin18.2scFv comprises the amino acid sequence of SEQ ID NO: 25.

[0023] (g) an anti-CD33 VHH comprising the amino acid sequence of SEQ ID NO: 42; and

[0024] (h) an anti-CD123 VHH comprising the amino acid sequence of any one of SEQ ID NOs: 47-52;

[0025] (i) an anti-HER2 VHH comprising the amino acid sequence of SEQ ID NO: 69 or 72;

[0026] (j) an anti-Claudin 18.2 VHH comprising the amino acid sequence of SEQ ID NO: 75 or 78;

[0027] (k) an anti-mesothelin VHH comprising the amino acid sequence of SEQ ID NO: 81 or 84;

[0028] (1) an anti-PSMA VHH comprising the amino acid sequence of SEQ ID NO: 87 or 90;

[0029] (m) an anti-GPC3 VHH comprising the amino acid sequence of SEQ ID NO: 93; or

[0030] (n) an anti-EGFR VHH comprising the amino acid sequence of SEQ ID NO:96.

[0031] In some cases, the bispecific CAR for the present disclosure is combined with Meso and BCMA. The anti-Meso / BCMA bispecific CAR may include a fusion polypeptide. In one example, the anti-Meso / BCMA bispecific CAR includes an amino acid sequence of 26 (with a signal peptide) or 27 (without a signal peptide). In another example, the anti-Meso / BCMA bispecific CAR includes an amino acid sequence of 107 (with a signal peptide) or 108 (without a signal peptide). In another example, the anti-Meso / BCMA bispecific CAR includes an amino acid sequence of 109 (with a signal peptide) or 110 (without a signal peptide). Alternatively, the anti-Meso / BCMA bispecific CAR may include two CAR polypeptides independently, one binding to mesothelin and the other binding to BCMA. In one example, the anti-meso CAR polypeptide includes an amino acid sequence of SEQ ID NO: 82 (with a signal peptide) or 83 (without a signal peptide). In another example, the anti-meso CAR polypeptide comprises an amino acid sequence of SEQ ID NO: 85 (with a signal peptide) or 86 (without a signal peptide). The anti-BCMA CAR polypeptide may comprise an amino acid sequence of SEQ ID NO: 38 (with a signal peptide) or 39 (without a signal peptide). Alternatively, the anti-BCMA CAR polypeptide may comprise an amino acid sequence of SEQ ID NO: 125 (with a signal peptide) or 126 (without a signal peptide).

[0032] In some cases, the bispecific CAR for the present disclosure is combined with HER2 and BCMA. Anti-HER2 / BCMA bispecific CAR may include a fusion polypeptide. In one example, the anti-HER2 / BCMA bispecific CAR includes an amino acid sequence of 28 (with a signal peptide) or 29 (without a signal peptide). In another example, the anti-HER2 / BCMA bispecific CAR includes an amino acid sequence of 99 (with a signal peptide) or 100 (without a signal peptide). In another example, the anti-HER2 / BCMA bispecific CAR includes an amino acid sequence of 101 (with a signal peptide) or 102 (without a signal peptide). Alternatively, the anti-HER2 / BCMA bispecific CAR may include two separate CAR polypeptides, one combined with HER2 and the other combined with BCMA. In one example, the anti-HER2 CAR polypeptide includes an amino acid sequence of SEQ ID NO:70 (with a signal peptide) or 71 (without a signal peptide). In another example, the anti-HER2 CAR polypeptide includes an amino acid sequence of SEQ ID NO: 73 (with a signal peptide) or 74 (without a signal peptide). The anti-BCMA CAR polypeptide may include an amino acid sequence of SEQ ID NO: 38 (with a signal peptide) or 39 (without a signal peptide). Alternatively, the anti-BCMA CAR polypeptide may include an amino acid sequence of SEQ ID NO: 125 (with a signal peptide) or 126 (without a signal peptide).

[0033] In some cases, the bispecific CAR for the present disclosure is combined with GPC3 and BCMA. The anti-GPC3 / BCMA bispecific CAR may include a fusion polypeptide. In one example, the anti-GPC3 / BCMA bispecific CAR includes an amino acid sequence of 30 (with a signal peptide) or 31 (without a signal peptide). In another example, the anti-GPC3 / BCMA bispecific CAR includes an amino acid sequence of 115 (with a signal peptide) or 116 (without a signal peptide). Alternatively, the anti-GPC3 / BCMA bispecific CAR may include two separate CAR polypeptides, one binding to GPC3 and the other binding to BCMA. In one example, the anti-GPC3CAR polypeptide includes an amino acid sequence of SEQ ID NO: 94 (with a signal peptide) or 95 (without a signal peptide). The anti-BCMA CAR polypeptide may include an amino acid sequence of SEQ ID NO: 38 (with a signal peptide) or 39 (without a signal peptide). Alternatively, the anti-BCMA CAR polypeptide may include the amino acid sequence of SEQ ID NO: 125 (with a signal peptide) or 126 (without a signal peptide).

[0034] In some cases, the bispecific CAR for the present disclosure is combined with Claudin 18.2 and BCMA. Anti-Claudin 18.2 / BCMA bispecific CAR may include a fusion polypeptide. In one example, the anti-Claudin18.2 / BCMA bispecific CAR includes an amino acid sequence of 32 (with a signal peptide) or 33 (without a signal peptide). In another example, the anti-Claudin 18.2 / BCMA bispecific CAR includes an amino acid sequence of 103 (with a signal peptide) or 104 (without a signal peptide). In another example, the anti-Claudin 18.2 / BCMA bispecific CAR includes an amino acid sequence of 105 (with a signal peptide) or 106 (without a signal peptide). Alternatively, the anti-Claudin 18.2 / BCMA bispecific CAR may include two separate CAR polypeptides, one combined with Claudin 18.2 and the other combined with BCMA. In one example, the anti-Claudin 18.2CAR polypeptide includes an amino acid sequence of SEQ ID NO: 76 (with a signal peptide) or 77 (without a signal peptide). In another example, the anti-Claudin 18.2CAR polypeptide includes an amino acid sequence of SEQ ID NO: 79 (with a signal peptide) or 80 (without a signal peptide). The anti-BCMA CAR polypeptide may include an amino acid sequence of SEQ ID NO: 38 (with a signal peptide) or 39 (without a signal peptide). Alternatively, the anti-BCMACAR polypeptide may include an amino acid sequence of SEQ ID NO: 125 (with a signal peptide) or 126 (without a signal peptide).

[0035] In some cases, the bispecific CAR for the present disclosure is combined with CD19 and BCMA. Anti-CD19 / BCMA bispecific CAR may include a fusion polypeptide. In one example, the anti-CD19 / BCMA bispecific CAR includes an amino acid sequence of 57 (with a signal peptide) or 58 (without a signal peptide). Alternatively, the anti-CD19 / BCMA bispecific CAR may include two separate CAR polypeptides, one combined with CD19 and the other combined with BCMA.

[0036] In some cases, the bispecific CAR for the present disclosure binds to CD70 and BCMA. The anti-CD70 / BCMA bispecific CAR may include a fusion polypeptide. Alternatively, the anti-CD70 / BCMA bispecific CAR may include two separate CAR polypeptides, one binding to CD70 and the other binding to BCMA. In one example, the anti-CD70 CAR polypeptide includes an amino acid sequence of SEQ ID NO: 36 (with a signal peptide) or 37 (without a signal peptide). The anti-BCMA CAR polypeptide may include an amino acid sequence of SEQ ID NO: 38 (with a signal peptide) or 39 (without a signal peptide). Alternatively, the anti-BCMA CAR polypeptide may include an amino acid sequence of SEQ ID NO: 125 (with a signal peptide) or 126 (without a signal peptide).

[0037] In some cases, the bispecific CAR for the present disclosure is combined with PSMA and BCMA. Anti-PSMA / BCMA bispecific CAR may include a fusion polypeptide. In one example, the anti-PSMA / BCMA bispecific CAR includes an amino acid sequence of 111 (with a signal peptide) or 112 (without a signal peptide). In another example, the anti-PSMA / BCMA bispecific CAR includes an amino acid sequence of 113 (with a signal peptide) or 114 (without a signal peptide). Alternatively, the anti-PSMA / BCMA bispecific CAR may include two separate CAR polypeptides, one combined with PSMA and the other combined with BCMA. In one example, the anti-PSMA CAR polypeptide includes an amino acid sequence of SEQ ID NO: 88 (with a signal peptide) or 89 (without a signal peptide). In another example, the anti-PSMACAR polypeptide includes an amino acid sequence of SEQ ID NO: 91 (with a signal peptide) or 92 (without a signal peptide). The anti-BCMA CAR polypeptide may include an amino acid sequence of SEQ ID NO: 38 (with a signal peptide) or 39 (without a signal peptide). Alternatively, the anti-BCMA CAR polypeptide may include the amino acid sequence of SEQ ID NO: 125 (with a signal peptide) or 126 (without a signal peptide).

[0038] In some cases, the bispecific CAR for the present disclosure is combined with EGFR and BCMA. The anti-EGFR / BCMA bispecific CAR may include a fusion polypeptide. In one example, the anti-EGFR / BCMA bispecific CAR includes an amino acid sequence of 117 (with a signal peptide) or 118 (without a signal peptide). Alternatively, the anti-EGFR / BCMA bispecific CAR may include two separate CAR polypeptides, one binding to EGFR and the other binding to BCMA. In one example, the anti-EGFR CAR polypeptide includes an amino acid sequence of SEQ ID NO: 97 (with a signal peptide) or 98 (without a signal peptide). The anti-BCMA CAR polypeptide may include an amino acid sequence of SEQ ID NO: 38 (with a signal peptide) or 39 (without a signal peptide). Alternatively, the anti-BCMA CAR polypeptide may include an amino acid sequence of SEQ ID NO: 125 (with a signal peptide) or 126 (without a signal peptide).

[0039] In other specific examples, the bispecific CAR includes a first antigen binding moiety and a second antigen binding moiety, the first antigen binding moiety is the extracellular domain of CD27, the CD27 is combined with CD70, and the second antigen binding moiety is specific to BCMA. In some cases, the anti-BCMA portion may include a scFv fragment (anti-BCMA scFv) specific for BCMA, and the anti-BCMA scFv includes a heavy chain variable region (VH) and a light chain variable region (VL). In some instances, the VH of the anti-BCMA scFv includes SEQ ID NO:14 amino acid sequence, and the VL of the anti-BCMA scFv includes SEQ ID NO:15 amino acid sequence. In one example, the anti-BCMA scFv includes SEQ ID NO:16 amino acid sequence. In a specific example, the anti-BCMACAR includes SEQ ID NO:38 or 39 amino acid sequence. In another specific example, the anti-BCMA CAR polypeptide may include an amino acid sequence of SEQ ID NO: 125 (with a signal peptide) or 126 (without a signal peptide). Alternatively or additionally, the extracellular domain of CD27 may include an amino acid sequence of SEQ ID NO: 34 or 35.

[0040] In yet other cases, the population of genetically engineered T cells may express a T cell receptor (TCR) and a CAR that is specific for a TAA, the CAR comprising an antigen binding portion that is specific for CD19 or BCMA. In some instances, the CAR comprises the antigen binding portion that is specific for CD19. The genetically engineered T cells expressing the TCR and the CAR can be used in conjunction with an APC that expresses CD19 and, optionally, the TAA. In other instances, the CAR may include an antigen binding portion that is specific for BCMA. The genetically engineered T cells expressing the TCR and the CAR can be used in conjunction with an APC that expresses BCMA and, optionally, the TAA. In some instances, the antigen binding portion is in the form of a single-chain variable fragment (scFv) or in the form of a single domain antibody (VHH). In some specific instances, the TCR and the CAR that are specific for the TAA may be in the form of Figure 4A In one example, the T cell receptor (TCR) can be specific for NY-ESO-1, and the TCR optionally includes a TCR alpha chain and a TCR beta chain, wherein the TCR alpha chain includes the amino acid sequence of SEQ ID NO: 40, and the TCR beta chain includes the amino acid sequence of SEQ ID NO: 41.

[0041] In some examples, the TCR disclosed herein may be a complex comprising a first fusion polypeptide comprising an antigen binding portion of CD33 and a second fusion polypeptide comprising an antigen binding portion of CD123. In some cases, the first fusion polypeptide may further include a transmembrane fragment of CD3δ, and the second fusion polypeptide may further include a transmembrane fragment of CD3γ. Alternatively, the first fusion polypeptide may further include a transmembrane fragment of CD3γ, and the second fusion polypeptide may further include a transmembrane fragment of CD3δ. In some examples, the transmembrane fragments of CD3δ and CD3γ do not contain the intracellular domains of CD3δ and CD3γ. In some specific examples, TCR-based bispecific CARs and co-expressed CARs may be presented. Figure 5A The format described in .

[0042] In some examples, such T cell receptors (TCRs) include a modified CD3δ chain and a modified CD3γ chain, wherein the modified CD3δ chain and the modified CD3γ chain together include a first antigen binding portion (anti-CD33 portion) specific for CD33 and a second antigen binding portion (anti-CD123 portion) specific for CD123. In some cases, the modified CD3δ chain includes an extracellular domain and a transmembrane domain of CD3δ fused to the anti-CD33 portion, and the modified CD3γ chain includes an extracellular domain and a transmembrane domain of CD3γ fused to the anti-CD123 portion, or vice versa. In specific examples:

[0043] (a) the modified CD3 δ chain comprises the amino acid sequence of SEQ ID NO: 45;

[0044] (b) the modified CD3γ chain comprises the amino acid sequence of SEQ ID NO: 46;

[0045] (c) the anti-CD33 portion is an anti-CD33 VHH, the anti-CD33 VHH optionally comprising the amino acid sequence of SEQ ID NO: 42; and / or

[0046] (d) the anti-CD123 portion is an anti-CD123 VHH, which optionally comprises the amino acid sequence of any one of SEQ ID NOs: 47-52.

[0047] Alternatively or additionally, the antigen binding portion of the CAR that is specific for CD19 comprises VH and VL, the VH comprises SEQ ID NO: 59, and the VL comprises SEQ ID NO: 60. In some examples, the antigen binding portion of the CAR that is specific for CD19 is a scFv fragment that comprises the amino acid sequence of SEQ ID NO: 61 or 62.

[0048] Alternatively or additionally, the antigen binding portion specific for BCMA in the CAR includes VH and VL, the VH includes SEQ ID NO: 14, and the VL includes SEQ ID NO: 15. In some examples, the antigen binding portion specific for BCMA in the CAR is a scFv fragment, and the scFv fragment includes the amino acid sequence of SEQ ID NO: 16. In a specific example, the CAR is an anti-BCMA CAR, and the anti-BCMA CAR includes the amino acid sequence of SEQ ID NO: 38 or 39. In other specific examples, the anti-BCMA CAR polypeptide may include the amino acid sequence of SEQ ID NO: 125 (with a signal peptide) or 126 (without a signal peptide).

[0049] Any of the CARs disclosed herein may further include an intracellular signaling domain, which may include one or more signaling domains (e.g., a costimulatory signaling domain and a cytoplasmic signaling domain); and a hinge domain and a transmembrane domain that optionally connect the antigen binding portion and the intracellular domain. In some examples, the CAR includes the hinge domain, which may belong to a CD8, CD28, CD4, CD3 or IgG molecule. Alternatively or additionally, the CAR may include a transmembrane domain, which may be from CD3, CD4, CD8, CD27 or CD28. In some examples, the CAR includes an intracellular signaling domain, which includes a costimulatory signaling domain and a cytoplasmic signaling domain. Exemplary signaling domains used as components of the intracellular signaling domain in any of the CARs disclosed herein include signaling domains from CD3, FcR, DAP12, 41BB, OX40, CD28, CD27, ICOS, IL-2R, IL-7R, IL-9R, IL-10R, IL-12R, IL18R, IL-21R or IL-23R or a combination thereof. In one example, the intracellular signaling domain includes a costimulatory domain of 4-1BB, an IL2Rb signaling domain, and a CD3 zeta signaling domain. In some specific examples, the costimulatory domain of 4-1BB includes an amino acid sequence of SEQ ID NO: 8, the IL2Rb signaling domain includes an amino acid sequence of SEQ ID NO: 9, and / or the CD3 zeta signaling domain includes an amino acid sequence of SEQ ID NO: 10.

[0050] In some embodiments, the colony of genetically engineered T cells includes tumor infiltrating T cells (TIL). In some embodiments, the colony of genetically engineered T cells is autologous to the subject. Alternatively, the colony of genetically engineered T cells is allogeneic to the subject.

[0051] In some embodiments, the APC cells include immune cells, stem cells or tumor cells. In some instances, the immune cells may be, but are not limited to, T cells, natural killer (NK) cells, tumor infiltrating lymphocytes, dendritic cells, macrophages, B cells, neutrophils, eosinophils, basophils, mast cells, myeloid-derived suppressor cells and / or mesenchymal stem cells. In some instances, the stem cells may be mesenchymal stem cells. In some instances, the tumor cells may be K562 cells. In some cases, the APC is a native cell expressing CD19 and / or BCMA and optionally the TAA. Alternatively, the APC is genetically engineered to express the CD19 and / or the BCMA and optionally the TAA. In some cases, the APC is derived from peripheral blood cells, umbilical cord blood cells, induced pluripotent stem cells (iPSC) or immune cell lines. In some instances, the APC is autologous to the subject. Alternatively, the APC is allogeneic to the subject.

[0052] Any of the APCs disclosed herein can be genetically engineered to further express membrane-bound stimulatory cytokines. Examples include IL-10, IL-18, IL-15, IL-9, or IL-21. See the examples provided in the sequence table below.

[0053] Further, the present disclosure provides a kit for treating cancer, the kit comprising: (a) any one of the populations of genetically engineered T cells disclosed herein; and (b) any one of the APCs disclosed herein. Also within the scope of the present disclosure are such kits for cancer treatment, and the use of the kits for preparing a medicament for cancer therapy.

[0054] Any of the genetically engineered T cells expressing bispecific CAR or TCR and CAR are within the scope of the present disclosure. Any of the genetically engineered APC cells disclosed herein are also within the scope of the present disclosure.

[0055] The details of one or more embodiments of the invention are set forth in the following description. Other features or advantages of the invention will become apparent from the following drawings and detailed description of several embodiments and from the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The following drawings form part of this specification and are included to further illustrate certain aspects of the present disclosure, which may be better understood by reference to the drawings in combination with the detailed description of specific embodiments presented herein.

[0057] Figure 1 is a schematic diagram depicting antigen presenting cells expressing CD19 and / or BCMA.

[0058] Figures 2A-2C Contains a graph showing the anti-tumor activity of genetically engineered T cells expressing a bispecific chimeric antigen receptor (CAR) targeting BCMA and a tumor-specific antigen (TAA). Figure 2A : Depicted

[0059] Schematic diagram of a bispecific CAR in a single fusion polypeptide format, in which two antigen binding moieties are repeated in tandem. One antigen binding moiety may be specific to a tumor-associated antigen (TAA), for example, mesothelin, GPC3, Claudin 18.2, HER2, PSMA, or EGFR. Another antigen binding moiety may be specific to CD19 or BCMA. The CAR polypeptide may further include a hinge domain and a transmembrane domain (e.g., from CD8) and an intracellular signaling domain (e.g., a 4-1BB costimulatory domain, a truncated IL2Rb signaling domain, and a CD3ζ signaling domain). Figure 2B : A graph showing the efficiency of lysis of BCMA+MM1S cells by genetically engineered T cells expressing a tandem bispecific CAR targeting BCMA and one of the TAAs as indicated. Figure 2C : Graph showing the efficiency of TAA lysis of tumor cells expressing TAAs by genetically engineered T cells expressing a tandem bispecific CAR targeting BCMA and one of the TAAs as indicated.

[0060] Figure 3A-3B Contains a graph showing the anti-tumor activity of genetically engineered T cells expressing two CAR constructs. Figure 3A: A schematic diagram of a bispecific CAR in a two-chain format, each including an antigen binding portion, is depicted. An antigen binding portion can be an extracellular domain of a ligand of a tumor-associated antigen, for example, an extracellular domain of a ligand CD27 of CD70. The extracellular domain of the ligand can be fused to an intracellular signaling domain, such as a CD3ζ signaling domain. Another antigen binding portion may be specific to CD19 or BCMA. CAR polypeptides including this type may further include a hinge domain and a transmembrane domain (e.g., from CD8) and an intracellular signaling domain (e.g., a 4-1BB costimulatory domain, a truncated IL2Rb signaling domain, and a CD3ζ signaling domain). Figure 3B : A graph showing the tumor cell lysis efficiency of genetically engineered T cells expressing a CAR targeting CD70 and a CAR targeting BCMA against various tumor cells as indicated.

[0061] Figure 4A-4B Contains graphs showing the anti-tumor activity of engineered T cells expressing TCRs specific for TAAs and CARs targeting BCMA or CD19. Figure 4A : A schematic diagram of a genetically engineered T cell expressing a TCR specific for a TAA such as NY-ESO-1 and a CAR comprising an antigen binding portion of CD19 or BCMA is depicted. The CAR may further include a hinge domain and a transmembrane domain (e.g., from CD8) and an intracellular signaling domain (e.g., a 4-1BB costimulatory domain, a truncated IL2Rb signaling domain, and a CD3ζ signaling domain). Figure 4B : A graph showing the tumor cell lysis efficiency of genetically engineered T cells expressing NY-ESO-1-specific TCR and CAR targeting BCMA against various tumor cells as indicated.

[0062] Figures 5A-5C Contains a graph showing the anti-tumor activity of engineered T cells expressing TCR-based bispecific CARs and separate CAR constructs targeting BCMA or CD19. Figure 5A: A schematic diagram of a genetically engineered T cell expressing a modified TCR specific for TAA and a CAR comprising an antigen binding portion of CD19 or BCMA is depicted. The modified TCR includes a first chain and a second chain, wherein the first chain includes an antigen binding portion of the first TAA (e.g., a VHH targeting CD33), and the second chain includes an antigen binding portion of the second TAA (e.g., a VHH targeting CD123). Each VHH can be fused to a fragment of CD3δ or CD3γ that does not contain an intracellular domain. CAR can further include a hinge domain and a transmembrane domain (e.g., from CD8) and an intracellular signaling domain (e.g., a 4-1BB costimulatory domain, a truncated IL2Rb signaling domain, and a CD3ζ signaling domain). Figure 5B : Graph showing the cytotoxicity of genetically engineered T cells expressing TCR-based bispecific CAR targeting CD33 and CD123 and anti-BCMACAR against RPMI8226 B lymphocytes. Figure 5C : A graph showing the cytotoxicity of genetically engineered T cells expressing bispecific CARs based on TCR targeting CD33 and CD123 and anti-BCMACAR against Molm13 human leukemia cells. Numbers 1-6 at the x-axis correspond to anti-CD123 VHH1-anti-CD123 VHH6 listed in the sequence table.

[0063] Figures 6A-6C Contains a graph showing that co-culture with antigen presenting cells (APCs) enhances CAR-T cell expansion. Fig. 6A : Genetically engineered T cells expressing bispecific CAR targeting CD70 and BCMA were co-cultured with K562 cells, NK92 cells expressing GFP / BCMA, or NK92 cells expressing CD19 / BCMA. Figure 6B : Genetically engineered T cells expressing bispecific anti-NY-ESO-1 TCR / anti-BCMA CAR were co-cultured with K562 cells, NK92 cells expressing GFP / BCMA, or NK92 cells expressing CD19 / BCMA. Figure 6C : Genetically engineered T cells expressing bispecific CAR targeting CD19 and BCMA were co-cultured with K562 cells expressing GFP / BCMA or NK92 cells expressing CD19 / BCMA.

[0064] Figures 7A-7D Contains graphs showing in vivo amplified cytotoxicity against target cells of CAR-T cells co-administered with APCs. Fig. 7A: A graph showing the in vitro expansion of CAR-T cells co-cultured with K562, MM1S, or NK92 cells expressing CD19 / BCMA as indicated. Figure 7B : Graph showing the in vitro cytotoxicity of CAR-T cells against various target cells as indicated. Figure 7C : Graph showing the in vivo expansion of CAR-T cells co-administered with antigen presenting cells. Fig.7D : A graph showing the levels of CD123+, CD33+, and CD123+CD33+ cells in human MM patients treated with both CAR-T cells and APCs. DETAILED DESCRIPTION

[0065] The clinical success of CAR-T therapy in treating blood cancers depends on robust CAR-T cell expansion in patients, which may be driven by the interaction between the CAR expressed on the CAR-T cells and the antigen presented on the tumor cells to which the CAR binds. However, initial clinical trials of solid tumors with CAR-T therapy have shown very limited expansion of CAR-T cells in patients. It has been suggested that this observation may be due to the limited presence of target solid tumor antigens in the local tumor area and their unavailability in the systemic blood circulation.

[0066] To address this problem, the present disclosure is intended to use antigen presenting cells (APCs) that simultaneously display one or more target antigens with CAR-T cells that target at least one of such antigens. Subject to theoretical constraints, such APCs are expected to be present in the blood circulation, which will promote CAR-T cell proliferation and expansion, thereby enhancing infiltration into disease tissues such as solid tumor tissues, and thus having a higher therapeutic efficacy for target diseases (e.g., solid tumors).

[0067] Therefore, described herein are compositions of genetically engineered T cells and antigen presenting cells expressing CD19 and / or BCMA and optionally TAA for treating solid tumors and hematopoietic cancers such as AML, including TCRs expressing chimeric antigen receptors (CAR) or targeting specific antigens (e.g., tumor-associated antigens or TAAs). As described herein, antigen presenting cells successfully enhance the expansion of CAR-T cells both in vitro and in vivo. Therefore, the combined cancer therapy provided herein is expected to be more effective compared to the treatment including only CAR-T cells.

[0068] I. Genetically Engineered T Cells

[0069] In some aspects, provided herein is a bispecific CAR and tumor-associated antigen (TAA) expressing specificity to CD19 or BCMA or a T cell receptor (e.g., modified, also referred to as a chimeric antigen receptor based on TCR) expressing specificity to TAA and a genetically engineered T cell having specificity to CD19 or BCMA. Such genetically engineered T cells can be used together with antigen presenting cells (APCs) expressing CD19 and / or BCMA, optionally as well as TAAs to improve therapeutic efficacy.

[0070] CAR disclosed herein is an artificial (non-naturally occurring) receptor that has binding specificity to a target antigen of interest (e.g., a tumor cell antigen) and is capable of triggering an immune response in immune cells expressing such a target antigen when bound to the target antigen. CAR typically includes an extracellular antigen binding domain fused to at least one intracellular signaling domain. Cartellieri et al., Journal of Biomedicine and Biotechnology (J Biomed Biotechnol) 2010:956304, 2010. As disclosed herein, a chimeric receptor based on TCR can be an artificial TCR or an engineered TCR that specifically transplants an artificial antigen to a target antigen.

[0071] (A) Genetically engineered T cells expressing bispecific CARs

[0072] In certain embodiments, provided herein is a genetically engineered T cell expressing a bispecific CAR. As disclosed herein, a single-chain fusion polypeptide or a bispecific CAR comprising multiple chains (e.g., two chains) is a CAR or CAR complex that can be combined with two different antigens or antigenic epitopes.

[0073] In some examples, the bispecific CAR comprises a single polypeptide, which may include (a) an extracellular antigen binding domain comprising a first antigen binding portion and a second antigen binding portion; (b) a hinge / transmembrane domain; and (c) one or more intracellular signaling domains (e.g., including a co-stimulatory signaling domain and one or more cytoplasmic signaling domains). The two antigen binding portions may be arranged in tandem repeats. For example, see Figure 2A .

[0074] In other examples, the bispecific CAR disclosed herein may include two separate CAR polypeptides. The first polypeptide (CAR 1) includes an extracellular domain comprising a first antigen binding portion and an intracellular signaling domain (e.g., including a co-stimulatory signaling domain and one or more cytoplasmic signaling domains). The second polypeptide (CAR 2) includes an extracellular domain comprising a second antigen binding portion and an intracellular signaling domain (e.g., including a co-stimulatory signaling domain and one or more cytoplasmic signaling domains). The first polypeptide or the second polypeptide or both may further include a hinge domain and a transmembrane domain. In some examples, one of the two CAR polypeptides (e.g., CAR 1) does not contain a hinge domain and a transmembrane domain, and the other polypeptide (e.g., CAR 2) includes such a hinge domain and a transmembrane domain. For example, see Figure 3A .

[0075] (a) Extracellular domain

[0076] The extracellular domain in the bispecific CAR disclosed herein comprises one or two antigen binding moieties as disclosed herein.

[0077] In some cases, the first antigen binding portion and / or the second antigen binding portion can be in a single chain variable fragment (scFv) format, in which the antibody heavy chain variable region (VH) and the antibody light chain variable region (VL) are linked, optionally through a flexible peptide linker. The scFv binding portion can have a VH-VL orientation (N-terminal to C-terminal). Alternatively, the scFv binding portion can have a VL-VH orientation.

[0078] In other cases, the first antigen binding moiety and / or the second antigen binding moiety can be a single domain antibody fragment (e.g., a heavy chain antibody or VHH only). In some cases, one of the first antigen binding moieties can be a scFv fragment, and the other antigen binding domain can be a VHH.

[0079] In a bispecific CAR, the first antigen binding moiety binds to CD19 or BCMA and the second antigen binding moiety binds to a tumor-associated antigen (TAA).

[0080] CD19 is a B lymphocyte antigen expressed in B lineage cells. It is reported that CD19 acts as an adaptor protein to recruit cytoplasmic signaling proteins to the membrane. It also acts in the CD19 / CD21 complex to reduce the threshold of B cell receptor signaling. CD19 is a well-characterized receptor in the art. For example, the amino acid sequence of human CD19 is provided in the sequence table below.

[0081] The anti-CD19 antigen binding portion in any of the bispecific antibodies disclosed herein can be derived from anti-CD19 antibodies or anti-CD19 CARs as known in the art. For example, the anti-CD19 antigen binding portion can be derived from the anti-CD19 antibody FMC-63. Alternatively, the anti-CD19 antigen binding portion can be derived from tisagenlecleucel or brexucabtageneautoleucel. The anti-CD19 antigen binding portion in the bispecific antibodies disclosed herein can have the same heavy chain and light chain complementary determining regions (CDRs) or the same VH and VL fragments as the antigen binding portions of anti-CD19 antibodies or anti-CD19 CARs known in the art. Alternatively, it can include one or more variants as disclosed herein. In one example, the anti-CD19 portion for preparing an anti-CD19 CAR can be derived from FMC63 (e.g., having the same heavy chain and light chain CDRs as FMC63 or having the same heavy chain variable region and light chain variable region).

[0082] BCMA (B cell maturation antigen) is a cell surface receptor that recognizes B cell activation factor (BAFF). This receptor is preferentially expressed in mature B lymphocytes. It is reported that BCMA may be important for B cell development and autoimmune response. BCMA is used as a drug target for the treatment of multiple myeloma. This receptor has been well characterized in the art. For example, the amino acid sequence of human BCMA is provided in the sequence table below.

[0083] The anti-BCMA antigen binding portion in any of the bispecific antibodies disclosed herein can be derived from an anti-BCMA antibody or an anti-BCMA CAR as known in the art. For example, the anti-BCMA antigen binding portion can be derived from the anti-BCMA antibody provided in the sequence table below. The anti-BCMA antigen binding portion in the bispecific antibodies disclosed herein may have the same heavy chain and light chain complementary determining regions (CDRs) or the same VH and VL fragments as provided in the sequence table. Alternatively, it may include one or more variants as disclosed herein. In some specific instances, the anti-BCMA antigen binding portion may be a scFv fragment, for example, an anti-BCMA scFv provided in the sequence table.

[0084] Tumor-associated antigens (TAAs) refer to antigens produced by tumor cells. In some instances, TAAs are tumor-specific antigens—antigens expressed only by tumor cells or antigens expressed by tumor cells at a level much higher than that expressed by non-cancer cells. In some cases, the TAAs disclosed herein are not CD19 or BCMA. Non-limiting examples of TAAs include 5T4, CD2, CD3, CD5, CD7, CD20, CD22, CD30, CD33, CD38, CD70, CD123, CD133, CD171, CEA, CS1, BAFF-R, PSMA, PSCA, desmoglein (Dsg3), HER-2, FAP, FSHR, NKG2D, GD2, EGFRVIII, mesothelin, ROR1, MAGE, MUC1, MUC16, GPC3, Lewis Y, HER2, Claudin 18.2, and VEGFRII.

[0085] In some examples, the TAA may be mesothelin, HER2, Claudin 18.2, GPC3, or EGFR. Exemplary antibodies that bind to these TAAs are provided in the sequence table. The anti-TAA antigen binding portion in the bispecific antibody disclosed herein may have the same heavy chain and light chain complementary determining regions (CDRs) or the same VH and VL fragments as the antigen binding portion provided in the sequence table. Alternatively, it may comprise one or more variants as disclosed herein. In some specific examples, the anti-TAA antigen binding portion may be a scFv fragment, for example, an anti-meso scFv, an anti-HER2 scFv, an anti-GPC3 scFv, or an anti-Claudin 18.2 scFv provided in the sequence table. In other specific examples, the anti-TAA antigen binding portion may be a VHH fragment, for example, an anti-HER2 VHH, an anti-meso VHH, an anti-Claudin 18.2 VHH, an anti-PSMA VHH, an anti-GPC3 VHH, or an anti-EGFR VHH provided in the sequence table.

[0086] In some examples, the TAA can be CD33 or CD 123. The antigen binding portion specific for CD33 or CD 123 can be a VHH fragment, for example, a fragment provided in the sequence listing.

[0087] In other examples, the TAA is a cell surface receptor, e.g., an immune cell receptor. In such cases, the antigen binding portion specific for the TAA can be the extracellular domain of a ligand specific for the receptor / TAA. For example, when the TAA is CD70, the antigen binding portion can be the extracellular domain of CD27, a ligand for CD70.

[0088] (b) Intracellular signaling domain

[0089] The single-chain bispecific CAR or CAR 1 / CAR 2 of the dual-chain bispecific CAR disclosed herein further includes an intracellular signaling domain. In some embodiments, the intracellular signaling domain may include at least one costimulatory signaling domain and at least one cytoplasmic signaling domain.

[0090] Exemplary costimulatory signaling domains can be derived from suitable immune receptors, e.g., OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, and DAP12. Therefore, CAR can have a costimulatory domain derived from 4-1BB, OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, and DAP12, or any combination thereof. In some instances, the costimulatory domain for the bispecific CAR disclosed herein can be derived from the costimulatory receptor 4-1BB (also referred to as CD137), e.g., from human 4-1BB. Examples are provided in the sequence table.

[0091] The intracellular signaling domain in the bispecific CAR disclosed herein may also include one or more cytoplasmic signaling domains, for example, including ITAM (e.g., ITAM1, ITAM2 and / or ITAM3 of CD3ζ as provided in the sequence table below). Examples include CD3ζ signaling domains, interleukin 2 receptor β subunit (IL-2Rβ) cytoplasmic signaling domains, or combinations thereof. See the amino acid sequences of the exemplary CD3ζ signaling domains and truncated IL-2Rβ cytoplasmic signaling domains in the sequence table, which can be used alone or in combination in the bispecific antibodies disclosed herein. In some examples, the intracellular signaling domain may include a 4-1BB costimulatory domain and an ITAM motif from CD3ζ, such as ITAM3 of CD3ζ.

[0092] (c) Hinge domain and transmembrane domain

[0093] The bispecific CAR polypeptide disclosed herein may contain a hinge domain and a transmembrane domain between the extracellular domain and the intracellular signaling domain. The hinge segment may be any oligopeptide or polypeptide, so as to provide flexibility for CAR or its domain, or to prevent steric hindrance of CAR or its domain. In some embodiments, the hinge segment may include up to 300 amino acids (e.g., 10 to 100 amino acids, or 5 to 20 amino acids). In some embodiments, one or more hinge domains may be included in other regions of CAR. In some instances, the hinge domain may belong to CD28, CD8, IgD or IgG, such as IgG1 or IgG4. See U.S. Patent No. 10,160,794. In a specific example, the hinge domain may be a CD8 hinge domain. Other hinge domains may be used.

[0094] The membrane-spanning segment can be a hydrophobic alpha helix across the membrane. As used herein, "membrane-spanning domain" refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. The membrane-spanning domain can provide stability for CARs containing such domains. In some embodiments, the membrane-spanning domain can be obtained from suitable cell surface receptors, for example, α, β or ζ chains of T cell receptors, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD271, TNFRSF19 and killer cell immunoglobulin-like receptors (KIR). In some instances, the membrane-spanning domain can be a CD8 membrane-spanning domain.

[0095] Exemplary hinge and transmembrane domains of CD8 are provided in the sequence listing, which can be used to construct any of the bispecific CARs disclosed herein.

[0096] (d) Exemplary bispecific CARs

[0097] The bispecific CAR for the present disclosure may include one or more components or functional variants thereof provided in the sequence table. Functional variants of the reference CAR components listed in the sequence table (e.g., antigen binding portion, hinge / transmembrane domain, costimulatory signaling domain, and cytoplasmic signaling domain, etc.) may share at least 85% sequence identity (e.g., at least 90%, at least 95%, at least 97%, at least 98% or higher) and retain substantially the same function as the reference CAR component.

[0098] The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 87:2264-68, 1990, as modified by Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-77, 1993. Such algorithms are incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al., J. Mol. Biol., 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program (score = 50, wordlength = 3) to obtain amino acid sequences homologous to a protein molecule of interest. When there are gaps between the two sequences, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST programs and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0099] In some examples, one or more components used in any of the bispecific CARs may comprise one or more conservative amino acid residue substitutions relative to the reference CAR components provided in the sequence listing.

[0100] As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein to which the amino acid substitution is made. Variants can be prepared according to methods for changing polypeptide sequences known to those of ordinary skill in the art, such as those found in references for compiling such methods, such as, for example, Molecular Cloning: A Laboratory Manual, edited by J. Sambrook et al., 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989 or Current Protocols in Molecular Biology, edited by FM Ausubel et al., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions between amino acids in the following groups: ((a) A→G, S; (b) R→K, H; (c) N→Q, H; (d) D→E, N; (e) C→S, A; (f) Q→N; (g) E→D, Q; (h) G→A; (i) H→N, Q; (j) I→L, V; (k) L→I, V; (l) K→R, H; (m) M→L, I, Y; (n) F→Y, M, L; (o) P→A; (p) S→T; (q) T→S; (r) W→Y, F; (s) Y→W, F; and (t) V→I, L).

[0101] The sequence table provides examples of bispecific CARs with specificity for Meso / BCMA, HER2 / BCMA, GPC3 / BCMA, CD19 / BCMA, CD70 / BCMA, Claudin 18.2 / BCMA, PSMA / BCMA, and EGFR / BCMA. An exemplary double-chain bispecific CAR is also provided in the sequence table, one chain binds to CD70, and the other chain binds to BCMA. Any of these examples, as well as nucleic acids encoding them and host cells expressing such are within the scope of the present disclosure. In other cases, a double-chain bispecific CAR (split bispecific CAR) may include (a) a CAR polypeptide with specificity for HER2, Claudin 18.2, mesothelin, PSMA, GPC3, CD19, CD70, or EGFR provided in the sequence table (e.g., including a scFv antigen binding portion or a VHH antigen binding portion), and (b) a CAR polypeptide with specificity for BCMA also provided in the sequence table.

[0102] (B) Genetically engineered T cells expressing modified T cell receptors and CARs

[0103] In some aspects, provided herein are genetically engineered T cells expressing a T cell receptor (TCR) (e.g., an engineered TCR) specific for a TAA and a CAR specific for BCMA or CD19.

[0104] In some cases, the TCR includes a TCR α chain and a TCR β chain, which form a complex for recognizing a TAA or an antigenic peptide thereof presented by an MHC complex. See, for example Figure 4A An exemplary TCRα / TCRβ pair specific for NY-ESO-1 is provided in the sequence listing. Such a TCR may be a natural TCR molecule, for example, from a naturally occurring T cell specific for a tumor antigen. Alternatively, the TCR may be an engineered one to which specificity for a TAA is grafted.

[0105] In other examples, the TCR comprises a TCR alpha chain and a TCR beta chain complexed with a CD3 complex comprising a modified CD3 delta chain and a modified CD3 gamma chain. See, e.g. Figure 5A . The modified CD3 δ chain may include a truncated CD3 δ chain without an intracellular domain fused to a first antigen binding portion having specificity for a first TAA. The modified CD3 γ chain may include a truncated CD3 γ chain without an intracellular domain fused to a second antigen binding portion having specificity for a second TAA. The first and second TAA may be any TAA known in the art or disclosed herein. In some cases, the first TAA is different from the second TAA. In other cases, the first TAA is the same as the second TAA. The first antigen binding portion or the second antigen binding portion may be a scFv fragment. Alternatively, the first antigen binding portion or the second antigen binding portion may be a single domain antibody, such as a VHH. In some examples, the first antigen binding portion and the second antigen binding portion are VHH fragments.

[0106] In some examples, the first TAA is CD33 and the second TAA is CD123, or vice versa. The first antigen binding portion and / or the second antigen binding portion may be a VHH fragment. Examples of VHH fragments specific for CD33 or CD123 are provided in the sequence table, any of which can be used to prepare the modified TCR disclosed herein.

[0107] Any of the TAA-specific TCRs disclosed herein can be co-expressed with a CAR specific for CD19 or BCMA in genetically engineered T cells. See, e.g. Figure 4Aand 5A Any of the anti-CD19 or anti-BCMACAR disclosed herein can be used in such genetically engineered T cells. See the examples in the sequence listing.

[0108] (C) Additional modifications to genetically engineered T cells

[0109] Any of the genetically engineered T cells expressing bispecific CARs or TAA-specific TCRs and CARs disclosed herein can be further engineered to express antagonists of cytokines, such as antagonists of cytokines that can activate immune responses. Antagonists as used herein refer to molecules that can inhibit or eliminate the biological activity of target cytokines to a meaningful extent, for example, inhibit or eliminate at least 20%, 50%, 70%, 85%, 90% or more.

[0110] Exemplary target cytokines include, but are not limited to, interleukin-1 (IL-1), interleukin-1 (IL-2), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-1 (IL-9), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), interleukin-23 (IL-23), interleukin-24 (IL-24), interleukin-33 (IL-33), interleukin-36 (IL-36), GM-CSF, interferon gamma (IFNγ), and chemokine (CC motif) ligand 19 (CCL19).

[0111] In some embodiments, the antagonist of the target cytokine can be an antibody that can bind to the target cytokine and inhibit its biological activity. Typical antibody molecules include a heavy chain variable region (VH) and a light chain variable region (VL), and the VH and the VL are generally involved in antigen binding. The VH and VL regions can be further subdivided into hypervariable regions, also referred to as "complementarity determining regions" ("CDRs"), interspersed with more conservative regions, which are referred to as "framework regions" ("FRs"). Each VH and VL is composed of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The scope of the framework region and CDR can be accurately identified using methods known in the art, such as by Kabat definition, Chothia definition, AbM definition and / or contact definition, all of which are well known in the art. See, e.g., Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., USD Department of Health and Human Services, NIH Publication No. 91-3242; Chothia et al. (1989) Nature 342:877; Chothia, C. et al. (1987) J. Molecular Biology 196:901-917; Al-lazikani et al. (1997) J. Molecular Biology 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also the antibody rules described in the Human Genome Mapping Project Resources of the Medical Research Council of the United Kingdom and the Bioinformatics and Computational Biology group website at University College London.

[0112] Antagonistic antibodies disclosed herein can be in any suitable format.Antibodies as used herein (used interchangeably in the plural form) are immunoglobulin molecules that can specifically bind to target cytokines as disclosed herein by at least one antigen recognition site located in the variable region of an immunoglobulin molecule.As used herein, the term "antibody" not only encompasses complete (e.g., full-length) antibodies and heavy chain antibodies (e.g., alpaca heavy chain IgG antibodies), but also encompasses antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single chains (scFv), single domain antibodies (sdAb; VHH) (also referred to as nanobodies), mutants thereof, fusion proteins including antibody portions, humanized antibodies, chimeric antibodies, bifunctional antibodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configurations of immunoglobulin molecules including antigen recognition sites of desired specificity, the modified configurations comprising glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies include antibodies of any class, such as IgD, IgE, IgG, IgA or IgM (or its subclass), and antibodies do not need to belong to any particular class. According to the antibody amino acid sequence of the constant domain of its heavy chain, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several classes in these classes can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains corresponding to the immunoglobulins of different classes are respectively referred to as α, δ, ε, γ and μ. The subunit structure and three-dimensional configuration of the immunoglobulins of different classes are well known.

[0113] In certain embodiments, the antibody "binding" to a target cytokine as described herein can specifically bind to a target cytokine.Antibodies that "specifically bind" (used interchangeably herein) to a target or epitope are well known terms in the art, and methods for determining such specific binding are also well known in the art.If a molecule reacts or associates with a specific target antigen more frequently, more quickly, for a longer duration, and / or with greater affinity than the molecule with an alternative target, the molecule is referred to as exhibiting "specific binding".If an antibody binds to a target cytokine more than the antibody with other substances with greater affinity, with higher affinity, more easily, and / or with longer duration, the antibody "specifically binds" to the target cytokine.For example, an antibody that specifically (or preferentially) binds to a target cytokine is an antibody that binds to this cytokine more than it binds to other epitopes in other cytokines or target cytokines with greater affinity, with higher affinity, more easily, and / or with longer duration. It should also be understood by reading this definition that, for example, an antibody that specifically binds to a first target cytokine may or may not specifically bind or preferentially bind to a second target cytokine. Thus, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. Typically, but not necessarily, reference to binding means preferential binding.

[0114] In some embodiments, the antagonist antibodies of target cytokines as described herein have a suitable binding affinity for the target cytokine or its antigenic epitope. As used herein, "binding affinity" refers to the apparent association constant or KA. KA is the reciprocal of the dissociation constant (KD). The binding affinity (KD) of the antagonist antibodies described herein for the target cytokine or its antigenic epitope can be at least 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10M or lower. Increased binding affinity corresponds to a decreased KD. The higher affinity binding of an antibody to a first target cytokine relative to a second target cytokine can be indicated by a KA (or a smaller numerical KD) for binding to the first target cytokine that is higher than the KA (or numerical KD) for binding to the second target cytokine. In this case, the antibody is specific for the first target cytokine relative to the second target cytokine. In some embodiments, the antagonist antibodies described herein have a higher binding affinity (higher KA or smaller KD) for the mature form of the target cytokine compared to the binding affinity for the precursor form of the target cytokine or another protein (e.g., a cytokine in the same family as the target cytokine). The difference in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000 or 10 5 times.

[0115] Binding affinity (or binding specificity) can be determined by various methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using fluorescence). Exemplary conditions for evaluating binding affinity are in HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% (v / v) surfactant P20). These techniques can be used to measure the concentration of bound binding protein as a function of the concentration of target protein. The concentration of bound binding protein ([bound]) is generally related to the concentration of free target protein ([free]) by the following equation:

[0116] [Bound] = [Free] / (Kd + [Free])

[0117] However, precise determination of KA is not always necessary, as sometimes it is sufficient to obtain a quantitative measure of affinity, to obtain a qualitative measure of affinity, or to obtain an inference of affinity, for example, by a functional assay, such as activity in an in vitro or in vivo assay, which is proportional to KA, for example, as determined using methods such as ELISA or FACS analysis, and can therefore be used for comparison, such as determining whether a higher affinity is, for example, 2-fold.

[0118] In some embodiments, the antagonist antibodies as described herein can bind and inhibit at least 50% (e.g., 60%, 70%, 80%, 90%, 95% or more) signal transduction mediated by the target cytokine. The inhibitory activity of the antagonist antibodies described herein can be determined by conventional methods known in the art.

[0119] The antibodies described herein can be murine, rat, human or any other source (including chimeric or humanized antibodies). Such antibodies are non-naturally occurring, e.g., will not be produced in animals without human action (e.g., immunizing such animals with a desired antigen or fragment thereof).

[0120] Any antibody in the antibodies described herein can be monoclonal or polyclonal. "Monoclonal antibody" refers to a homogeneous antibody population, and "polyclonal antibody" refers to a heterogeneous antibody population. These two terms do not limit the source of the antibody or the way in which the antibody is prepared.

[0121] In one example, the antibody used in the methods described herein is a humanized antibody. Humanized antibodies refer to the form of non-human (e.g., mouse) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments containing the minimum sequence derived from non-human immunoglobulins. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies), wherein the residues from the complementary determining regions (CDRs) of the recipient are replaced by residues from the CDRs of non-human species (donor antibodies) with desired specificity, affinity, and ability, such as mice, rats, or rabbits. In some cases, the Fv framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. In addition, humanized antibodies can include residues that are neither found in the recipient antibody nor found in the CDR or framework sequences imported but are included to further refine and optimize antibody performance. Generally, humanized antibodies will include substantially all variable domains in at least one and typically two variable domains, wherein all or substantially all CDR regions correspond to the region of non-human immunoglobulins, and all or substantially all FR regions are regions of human immunoglobulin consensus sequences. Humanized antibodies will also optimally include at least a portion of an immunoglobulin constant region or domain (Fc), typically at least a portion of a human immunoglobulin. The antibody may have a modified Fc region as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five and / or six) that change about the original antibody, also referred to as "derived from" one or more CDRs from the original antibody. Humanized antibodies may also relate to affinity maturation.

[0122] In other embodiments, the antagonist of the target cytokine can include a binding portion of the target cytokine, which can be fused to an immune-activating cytokine (e.g., IL-2, IL-7, IL-9, IL-10, IL-12, IL-15, IL-18, IL-21, IL-23, IL-24, IL-36, IL-33, and CCL19). The binding portion can be an antigen binding fragment (e.g., scFv fragment or VHH fragment) of an antibody specific for the target cytokine. Alternatively, the binding portion can be a soluble receptor that binds to the target cytokine.

[0123] In some instances, the fusion antagonist includes the binding portion of IFNγ fused to IL-18. The binding portion of IFNγ (for example, antibodies such as scFv fragments combined with IFNγ) can be fused to the N-terminus of IL-18. Alternatively, the binding portion of IFNγ (for example, antibodies such as scFv fragments combined with IFNγ) can be fused to the C-terminus of IL-18. In some cases, the binding portion of IFNγ (for example, antibodies such as scFv fragments combined with IFNγ) and IL-18 part can be connected by a peptide linker. Alternatively, the two parts can be directly connected. An example of such fusion protein is provided in the sequence table below.

[0124] II. Antigen Presenting Cells (APC)

[0125] The present disclosure also provides a population of antigen presenting cells (APCs) expressing CD19 and / or BCMA. See, e.g., Figure 1 Such APCs may also express TAAs, as disclosed herein.

[0126] Antigen presenting cells are cells that display antigens or antigenic peptides for T cell recognition through major histocompatibility complex (MHC) molecules (MHC I or MHC II molecules) on the cell surface. Typical APCs include immune cells, such as immune cells, which are optionally T cells, natural killer (NK) cells, tumor infiltrating lymphocytes, dendritic cells, macrophages, B cells, neutrophils, eosinophils, basophils, mast cells, myeloid-derived suppressor cells and / or mesenchymal stem cells. Any of these immune cells or a combination thereof can be used in the present disclosure.

[0127] In some cases, the APC disclosed herein can be genetically engineered to further express one or more membrane-bound stimulatory cytokines. Examples of such stimulatory cytokines include, but are not limited to, mIL-10, mIL-18, mIL-15, mIL-9, and mIL-21. The amino acid sequences of such exemplary membrane-bound stimulatory cytokines are provided in the sequence table below.

[0128] In some embodiments, the APCs used in the present disclosure may be naturally occurring APC cells expressing CD19 and / or BCMA and optionally TAAs. Alternatively, the APCs may be genetically engineered to express one or more of the antigens of CD19, BCMA and optionally TAAs.

[0129] In some examples, the APC used in the methods disclosed herein can be a universal APC prepared from an NK cell line, such as NK92-MI cells. For example, an expression vector carrying a transgenic gene encoding CD19, BCMA or TAA can be introduced into NK cells by conventional methods, and transfected cells expressing the target antigen can be isolated. In some cases, NK cells that stably express the target antigen can be determined by conventional methods.

[0130] In some examples, the APC can be an immune cell, such as a T cell, a natural killer (NK) cell, a tumor infiltrating lymphocyte, a dendritic cell, a macrophage, a B cell, a neutrophil, an eosinophil, a basophil, a mast cell, or a myeloid-derived suppressor cell.

[0131] In some examples, the APCs can be stem cells, for example, mesenchymal stem cells.

[0132] In some examples, the APC can be a tumor cell (eg, a tumor cell line such as K562 cells).

[0133] III. Methods for preparing genetically engineered immune cells and APCs

[0134] Genetically engineered T cells and antigen presenting cells can be prepared from immune cells that can be derived from a suitable source. Examples include, but are not limited to, immune cell populations obtained from donors (such as healthy human donors). In some instances, immune cells can be derived from PBMCs. Alternatively, immune cells can be derived from stem cells (e.g., adult stem cells, non-human embryonic stem cells, more specifically non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells). In some instances, immune cells can be derived from the differentiation of induced pluripotent cells (iPSC) colonies.

[0135] Suitable immune cells include but are not limited to T cells, NK cells, tumor infiltrating lymphocytes, dendritic cells, macrophages, B cells, neutrophils, eosinophils, basophils, mast cells, myeloid-derived suppressor cells, mesenchymal stem cells, precursors thereof, or combinations thereof. T cells may be selected from the group consisting of inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or helper T lymphocytes. In some embodiments, T cells may be derived from the group consisting of CD4+ T lymphocytes and CD8+ T lymphocytes.

[0136] Gene modification disclosed herein, including any knock-in transgene encoding any of the bispecific CAR disclosed herein, modified TCR and / or cytokine antagonists, can be introduced into suitable immune cells by conventional methods and / or methods described herein. Typically, this method will involve delivering genetic material to suitable immune cells to down-regulate the expression of target endogenous inflammatory proteins, express a cytokine antagonist of interest, or express an immunosuppressive cytokine of interest.

[0137] In order to produce knock-in of one or more bispecific CARs, modified TCRs and cytokine antagonists described herein, the coding sequence of any one of the bispecific CARs, modified TCRs and cytokine antagonists described herein can be cloned into a suitable expression vector (e.g., including but not limited to lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated vectors, PiggyBac transposon vectors and Sleeping Beauty transposon vectors), and introduced into host immune cells using conventional recombinant techniques. Sambrook et al., Molecular Cloning: Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press. As a result, the modified immune cells of the present disclosure may include one or more exogenous nucleic acids encoding at least one bispecific CAR or its chain, at least one modified TCR and / or at least one cytokine antagonist. In some cases, one or more transgenics may be integrated into the genome of the cell to achieve stable expression. In some cases, transgenics may not be integrated into the genome of the cell.

[0138] The exogenous nucleic acid including the coding sequence of bispecific CAR or its chain, modified TCR and / or cytokine antagonist may further include a suitable promoter, which may be operably connected to the coding sequence. As used herein, a promoter refers to a nucleotide sequence (site) on a nucleic acid, and RNA polymerase may bind to the nucleotide sequence to initiate transcription of the coding DNA (e.g., for cytokine antagonists) into mRNA, which is then translated into the corresponding protein (i.e., gene expression). When the promoter is in the correct functional position and orientation relative to the coding sequence to control ("drive") the transcription initiation and expression (to produce the corresponding protein molecule) of the coding sequence, the promoter is considered to be "operably connected" to the coding sequence. In some cases, the promoter described herein may be constitutive, which initiates transcription independent of other regulatory factors. In some cases, the promoter described herein may be inducible, which depends on the regulatory factor used for transcription. Exemplary promoters include but are not limited to ubiquitin, RSV, CMV, EF1α and PGK1. In one example, one or more nucleic acids encoding one or more antagonists of one or more inflammatory cytokines as described herein, operably linked to one or more suitable promoters, can be introduced into immune cells by conventional methods to drive expression of the one or more antagonists.

[0139] In addition, the exogenous nucleic acid described herein may further contain, for example, one or more of the following: a selectable marker gene, such as a neomycin gene for selecting stable or transient transfectants in mammalian cells; an enhancer / promoter sequence from the immediate early gene of human CMV for high-level transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma replication origin and ColE1 for appropriate episomal replication; a universal multiple cloning site; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable methods for producing vectors containing transgenes are well known and available in the art. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press.

[0140] In some cases, the combination of bispecific CAR or its chain, modified TCR and / or cytokine antagonists as described herein can be constructed in a polycistronic manner in an expression cassette so that a variety of cytokine antagonists are used as separate polypeptides. In some instances, an internal ribosome entry site can be inserted between two coding sequences to achieve this goal. Alternatively, a nucleotide sequence encoding a self-cleavage peptide (e.g., T2A or P2A) can be inserted between two coding sequences. The exemplary design of such a polycistronic expression cassette is provided in the sequence table below.

[0141] An immune cell population comprising any one or a combination of the modified immune cells described herein can be prepared by introducing one or more of the knockout modifications disclosed herein into a host immune cell population.

[0142] In some cases, after the previous modification event and before the next modification event, one or more modifications are introduced into the host cell in a sequential manner without separating and / or enriching the modified cells. In that case, the resulting immune cell colony can be heterogeneous, including cells carrying different modifications or different combinations of modifications. This immune cell colony can also include unmodified immune cells. Relative to the total number of host immune cells, the level of each modification event occurring in the immune cell colony can be controlled by inducing the amount of genetic material of this modification. See also above discussion.

[0143] In other cases, after the first modification event and before the second modification event, the modified immune cells can be isolated and enriched. This method will produce a substantially homogeneous population of immune cells carrying all knock-in modifications and / or knock-out modifications introduced into the cells.

[0144] IV. Therapeutic Applications

[0145] Any of the genetically engineered T cells described herein can be used for adoptive immune cell therapy for the treatment of target diseases, such as solid tumors or hematopoietic cancers (e.g., chronic lymphocytic leukemia or CLL), used together with any of the APCs disclosed herein to enhance therapeutic efficacy. In some cases, the methods provided herein can also be used to treat immune disorders, such as autoimmune diseases (e.g., systemic lupus erythematosus when genetically engineered T cells express anti-CD19 CAR or anti-BCMACAR), or for the treatment of infectious diseases (e.g., using the genetically engineered T cells expressing one or more targeting one or more CARs derived from antigens of pathogens such as viruses or bacteria).

[0146] To practice the treatment methods described herein, an effective amount of genetically engineered T cells as disclosed herein can be administered to a subject in need of treatment by an appropriate route (e.g., intravenous infusion), along with an effective amount of APCs. It is also within the scope of the present disclosure that genetically engineered T cells and / or APCs can be mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition prior to administration.

[0147] As used herein, the term "effective amount" refers to the amount of each active agent required for the treatment effect of the subject, either alone or in combination with one or more active agents. As recognized by those skilled in the art, the effective amount varies according to the specific condition treated, the severity of the condition, individual patient parameters including age, physical condition, size, sex and weight, treatment duration, route of administration, excipient use, co-use with other active agents (if any), and similar factors within the knowledge and expertise of health practitioners. The amount to be administered depends on the subject to be treated, including the ability of the individual immune system to produce a cell-mediated immune response, for example. The precise amount of the active ingredient to be administered depends on the judgment of the practitioner. However, those skilled in the art can easily determine a suitable dosage range.

[0148] As used herein, the term "treat" refers to the application or administration of a composition comprising one or more active agents to a subject suffering from a target disease, a symptom of a target disease, or a susceptibility to a target disease, with the purpose of curing, rehabilitating, alleviating, mitigating, altering, remedying, ameliorating, improving, or affecting the disease, a symptom of a disease, or a susceptibility to a disease.

[0149] The genetically engineered T cells, APCs, or both can be autologous to the subject, that is, the cells are obtained from a subject in need of treatment, modified to express one or more cytokine antagonists described herein, to express a CAR construct and / or an exogenous TCR or a combination thereof, or to express an antigen of CD19 and / or BCMA. The resulting modified cells can then be administered to the same subject. Administration of autologous cells to a subject can reduce rejection of donor cells compared to administration of non-autologous cells.

[0150] Alternatively, the genetically engineered T cells, APCs, or both can be allogeneic cells, i.e., the cells are obtained from a first subject, modified as described herein, and administered to a second subject that is different from the first subject but of the same species. For example, allogeneic immune cells can be derived from a human donor and administered to a human recipient that is different from the donor.

[0151] The subject to be treated can be a mammal (e.g., a human, mouse, pig, cow, rat, dog, guinea pig, rabbit, hamster, cat, goat, sheep or monkey). The subject may have cancer, such as a solid tumor or a hematopoietic cancer, such as AML. In some cases, the subject has a cancer involving cancer cells expressing a target antigen, such as CD19, BCMA, HER2, mesothelin, GPC3, claudin18.2, PSMA, CD70 and / or EGFR. The corresponding CAR-T cells can be selected according to the expression of the target antigen in the subject to be treated.

[0152] In some examples, genetically engineered T cells expressing bispecific CARs that can bind to CD19 or BCMA and a TAA, such as HER2, mesothelin, GPC3, claudin18.2, PSMA, CD70, or EGFR, can be used together with APCs expressing CD19 and / or BCMA and, optionally, a TAA to treat solid tumors.

[0153] In other examples, genetically engineered T cells expressing a TAA-specific TCR (e.g., specific for NY-ESO-1) and a CAR specific for BCMA can be used together with APCs expressing BCMA and / or CD19 and, optionally, a TAA to treat solid tumors.

[0154] In some examples, genetically engineered T cells expressing a bispecific CAR capable of binding to CD70 and BCMA can be used together with APCs expressing BCMA and / or CD19 and optionally CD70 to treat AML.

[0155] Alternatively, genetically engineered T cells expressing a modified TCR capable of binding to CD33 and CD123 and a CAR specific for BCMA can be used together with APCs expressing BCMA and / or CD19, optionally CD33 and / or CD123, to treat AML.

[0156] An effective amount of genetically engineered T cells and APCs can be administered to a human patient in need of treatment by a suitable route, such as intravenous infusion. In some cases, about 1 x 10 6 About 1x 10 8 CAR+T cells and / or APC cells are administered to human patients (e.g., leukemia patients, lymphoma patients, or multiple myeloma patients). In some instances, human patients can receive multiple doses of genetically engineered immune cells. For example, a patient can receive two doses of immune cells for two consecutive days. In some cases, the first dose is the same as the second dose. In other cases, the first dose is less than the second dose, and vice versa.

[0157] The CAR-T cell / APC combination therapy disclosed herein can be used in combination with other types of therapies for cancer (such as chemotherapy, surgery, radiation, gene therapy, etc.). Such therapies can be administered simultaneously or sequentially (in any order) with the immunotherapy described herein. When co-administered with another therapeutic agent, the appropriate therapeutically effective dose of each agent can be reduced due to additive or synergistic effects.

[0158] In some examples, prior to the CAR-T / APC therapy disclosed herein, the subject undergoes a suitable anti-cancer therapy (e.g., a therapy disclosed herein) to reduce tumor burden. For example, a subject (e.g., a human cancer patient) may undergo chemotherapy (e.g., including a single chemotherapeutic agent or a combination of two or more chemotherapeutic agents) at a dose that significantly reduces tumor burden. In some cases, chemotherapy can reduce the subject's total white blood cell count to less than 10. 8 / L, for example, less than 10 7 / L. The tumor burden of the patient after initial anticancer therapy and / or after the CAR-T cell / APC therapy disclosed herein can be monitored by conventional methods. If the patient exhibits a high growth rate of cancer cells after initial anticancer therapy and / or after CAR-T / APC therapy, the patient can be subjected to a new round of chemotherapy to reduce the tumor burden, followed by any CAR-T therapy as disclosed herein.

[0159] Non-limiting examples of other anti-cancer therapeutics that can be used in combination with the modified immune cells described herein include, but are not limited to, immune checkpoint inhibitors (e.g., PDL1, PD1 and CTLA4 inhibitors), anti-angiogenic agents (e.g., TNP-470, platelet factor 4, thrombospondin-1, tissue inhibitors of metalloproteinases, prolactin, angiostatin, endostatin, bFGF soluble receptor, transforming growth factor β, interferon α, soluble KDR and FLT-1 receptors, and placenta proliferator-activated protein kinase); VEGF antagonists (e.g., anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments); chemotherapeutic compounds.Exemplary chemotherapeutic compounds include pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, capecitabine, gemcitabine, and cytarabine); purine analogs (e.g., fludarabine); folate antagonists (e.g., mercaptopurine and thioguanine); antiproliferative or antimitotic agents, such as vinca alkaloids. microtubule disrupting agents such as taxanes (e.g., paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones, and vinorelbine, as well as epidipodophyllotoxins; DNA damaging agents (e.g., actinomycin, amsacrine, anthracycline, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, carbosporin, (cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethyhnelamine, oxaliplatin, iphosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide, and etoposide).

[0160] In some embodiments, radiation or radiation and chemotherapy are used in combination with a cell population comprising the modified immune cells described herein. Additional useful agents and therapies may be found in Physician's Desk Reference, 59th Supplement, (2005), Thomson PDR, Montvale NJ; Gennaro et al., eds., Remington's The Science and Practice of Pharmacy, 20th Supplement, (2000), Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., eds., Harrison's Principles of Internal Medicine, 15th Supplement, (2001), McGraw Hill, New York; Berkow et al., eds., The Merck Manual of Diagnosis and Therapy. Therapy, (1992), Merck Research Laboratories, Rahway NJ.

[0161] V. Kits for therapeutic use or preparation of modified immune cells

[0162] The present disclosure also provides kits for any target disease described herein involving the genetically engineered T cells and APCs described herein, as well as kits for preparing modified immune cells as described herein.

[0163] The kit for therapeutic use as described herein may include one or more containers including a population of genetically engineered T cells or a population of APCs, each of which can be formulated to form a pharmaceutical composition. In some embodiments, the kit may additionally include instructions for using genetically engineered T cells and APCs in any of the methods described herein. The included instructions may include a description of administering a cell population or a pharmaceutical composition comprising such a cell population to a subject to achieve the desired activity in the subject. The kit may further include a description of selecting a subject suitable for treatment based on identifying whether the subject needs treatment. In some embodiments, the instructions include a description of administering genetically engineered T cells and APCs or a pharmaceutical composition comprising such a cell population to a subject in need of treatment.

[0164] Instructions related to the use of genetically engineered T cells and APCs as described herein or pharmaceutical compositions comprising such generally contain information about the dosage, dosing regimen, and route of administration for the intended treatment. The container may be a unit dose, bulk package (e.g., multi-dose package), or subunit dose. The instructions provided in the kit of the present disclosure are generally written instructions on a label or package insert. The label or package insert indicates that the pharmaceutical composition is used to treat a disease or condition in a subject, delay the onset of a disease or condition in a subject, and / or alleviate a disease or condition in a subject.

[0165] The kits provided herein are packaged in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, etc. It is also contemplated that packaging used in combination with a specific device (such as an inhaler, a nasal application device, or an infusion device) is provided. The kit may have a sterile inlet port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle). The container may also have a sterile inlet port. At least one active agent in the pharmaceutical composition is a population of genetically engineered T cells and APCs.

[0166] The kit may optionally provide additional components such as buffers and explanatory information. Typically, the kit includes a container and a label or package insert on or associated with the container. In some embodiments, the present disclosure provides an article of manufacture comprising the contents of the above-described kit.

[0167] Also provided herein is a kit for preparing a modified immune cell or APC as described herein. This kit may include one or more containers, each containing a reagent for introducing a knock-in modification into an immune cell or APC. Alternatively or additionally, the kit may include one or more exogenous nucleic acids for expressing any one of the bispecific CAR, TCR and / or cytokine antagonists as described herein, and a reagent for delivering exogenous nucleic acids to host immune cells. This kit may further include instructions for performing the desired modification on the host immune cell.

[0168] General techniques

[0169] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are fully explained in the following literature, such as: Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait ed. 1984); Methods in Molecular Biology (Humana Press); Cell Biology: A Laboratory Notebook (JE Cellis ed., 1989) Academic Press; Animal Cell Culture (RI Freshney ed., 1987); Introduction to Cell and Tissue Culture (JPather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (JP Cellis ed., 1998) Academic Press; Procedures" (A. Doyle, JB Griffiths and DG Newell, ed. 1993-8) John Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, ed.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, ed. 1987); Current Protocols in Molecular Biology (FMAusubel et al., ed., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., ed., 1994); Current Protocols in Immunology (J.E. Coligan et al., ed., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, ed., Oxford University Press, 1994); =University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999)); The Antibodies (M. Zanetti and JD Capra, eds.; Harwood Academic Publishers, 1995); DNA Cloning: A practical approach, Volumes I and II (DN Glover, ed., 1985); Nucleic Acid Hybridization (BD Hames and SJ Higgins, eds. (1985)); Transcription and Translation (BD Hames and SJ Higgins, eds. (1984)); Animal Cell Culture (RI Freshney, ed. (1986)); Immobilized Cells and Enzymes (IRL Press, (1986)); and B.Perbal, A Practical Guide To Molecular Cloning (1984); FM Ausubel et al. (eds.).

[0170] Without further elaboration, it is believed that those skilled in the art can utilize the present invention to the greatest extent based on the above description. Therefore, the following specific examples should be interpreted as being merely illustrative and not limiting the remainder of the present disclosure in any way. All disclosures cited herein are incorporated by reference for the purpose or subject matter of this reference.

[0171] Example 1 :Preparation of universal antigen presenting cells (APCs) expressing CD19 and BCMA

[0172] NK92-MI cells were cultured under conventional conditions. A lentiviral expression vector including coding sequences for BCMA and CD19 was constructed by recombinant technology in a bicistronic format, in which the BCMA gene and the CD19 gene were linked by a nucleotide sequence encoding the T2A peptide. Figure 1 The amino acid sequences of BCMA, CD19 and T2A are provided in the sequence table below. The lentiviral expression vector is introduced into NK92-MI cells, and NK92-MI cells expressing both BCMA and CD19 surface proteins are enriched and can be used as universal APCs to enhance CAR-T cell efficacy.

[0173] Example 2 : Preparation and characterization of genetically engineered CAR-T cells for solid tumor therapy

[0174] The lentiviral vector designed to express the bispecific CAR is constructed by traditional recombinant technology. The bispecific CAR includes a first scFv that binds to CD19, HER2, mesothelin, GPC3 or Claudin 18.2, and a second scFv that binds to BCMA. In addition to the antigen binding portion, each of the bispecific CARs also contains the hinge domain and transmembrane domain of CD8, and the intracellular signaling domain of IL2Rb signaling-CD3ζ truncated by 4-1BB. The amino acid sequences of Meso / BCMA, GPC3 / BCMA, HER2 / BCMA and Claudin18.2 / BCMA are provided in the sequence table below. See also Figure 2A .

[0175] Primary T cells from healthy donors are activated by anti-CD3 / CD28 beads (Thermo Scientific). One day later, T cells are transduced with a lentiviral vector encoding one of the above-mentioned bispecific CARs. Transduced cells are amplified and tested for CD3 expression by FACS analysis, and CD3+ populations are gated for further analysis. The expression of bispecific CARs can be analyzed by conventional methods. For example, CAR expression is analyzed by flow cytometry using a biotinylated primary antibody that recognizes the antibody fragment in CAR and fluorescein-labeled streptavidin.

[0176] The functionality of bispecific CAR-T cells is analyzed by co-culturing CAR-T cells with target APCs or target tumor cells to evaluate CAR-T cell proliferation and / or cytotoxicity. More specifically, one of the above-mentioned bispecific CARs (see also the sequence listing and Figure 2A ) and genetically engineered T cells co-expressing anti-IFNG scFv (see also sequence list) were co-cultured with GFP-expressing target cells MM1S and TAA-expressing target cells at a 1:3 ratio (E:T) to evaluate killing activity. Different TAA-targeting VHHs as indicated in the bispecific anti-TAA / BCMACAR were tested. Figure 2B In, HER2 and 2D3 refer to VHHs targeting TAA HER2; 182-19 and 182-6 refer to VHHs targeting TAA Claudin 18.2; Meso2 and Meso1 refer to VHHs targeting TAA mesothelin; PSMA363 and PSMA362 refer to VHHs targeting TAA PSMA; GPC3 refers to VHHs targeting TAA GPC3; EGFR refers to VHHs targeting TAA EGFR. Figure 2C In the figure, SKOV3 refers to HER2+ tumor target; AGS-Claudin18.2 refers to AGS cells expressing Claudin 18.2; Aspc1 refers to mesothelin+ tumor cells; LnCap refers to PSMA+ tumor cells; Huh7 refers to GPC3+ tumor cells; Panc1 refers to EGFR+ tumor cells. After 3 days, the remaining GFP+ tumor cells were counted by flow cytometry to determine the percentage of tumor cells killed by CART cells. The results showed that each of the bispecific anti-TAA / BCMA CARs showed effective killing of BCMA+MM1S cells, as well as effective killing of the corresponding TAA-expressing tumor cells. Figure 2B and Figure 2C .

[0177] Example 3 : Preparation of genetically engineered CAR-T cells for the treatment of AML

[0178] Lentiviral vectors designed to express bispecific CARs are constructed by conventional recombinant techniques. The bispecific CARs include two separate polypeptides, each of which includes a binding portion for an antigen of interest. The first polypeptide includes the extracellular domain of CD27 (a truncated CD27 without the intracellular domain) fused to the intracellular signaling domain of CD3ζ. CAR1 (binds to CD70, also known as anti-CD70 CAR), such as Figure 3A The second polypeptide includes an anti-BCMA scFv, the hinge and transmembrane domains of CD8, and the intracellular signaling domain of IL2Rb signaling-CD3ζ truncated by 4-1BB (SEQ ID NO: 38). See Figure 3A CAR2 depicted in FIG.

[0179] The amino acid sequences of CAR1 and CAR2 are provided in the sequence listing below. The coding sequences of CAR1 and CAR2 are in a bicistronic format in a lentiviral vector, and the coding sequences are connected by a nucleotide sequence encoding a T2A peptide linker.

[0180] Primary T cells from healthy donors are activated by anti-CD3 / CD28 beads (Thermo Scientific). One day later, T cells are transduced with a lentiviral vector encoding the above-mentioned double-chain bispecific CAR. Transduced cells are amplified and tested for CD3 expression by FACS analysis, and CD3+ colonies are gated for further analysis. The expression of bispecific CAR can be analyzed by conventional methods.

[0181] Expressing the above-described bispecific anti-CD70 / BCMA CAR (see also Figure 3B ) were co-cultured with GFP-expressing target cells RPMI8226, U937, and Molm13 at a 1:3 ratio (E:T) to evaluate their target cell killing activity. After 3 days, the remaining GFP+ tumor cells were counted by flow cytometry to determine the percentage of tumor cells killed by genetically engineered CAR-T cells. The results showed that bispecific anti-CD70 / BCMA CAR-T cells showed effective killing of BCMA+RMPI8226 cells, CD123+U937 cells, and CD123+ / CD33+Molm13 cells. Figure 3B .

[0182] Example 4 : Preparation of genetically engineered TCR-T cells for cancer therapy

[0183] A lentiviral vector designed to express a TCR receptor specific for the tumor antigen NY-ESO-1 and a CAR specific for BCMA (SEQ ID NO: 38) was constructed by conventional recombinant technology. Figure 4A . The TCR specific for NY-ESO-1 comprises a TCR alpha chain and a TCR beta chain, the amino acid sequences of which are provided in the sequence table below. The coding sequences of the two chains are in a bicistronic format in a lentiviral vector, and the coding sequences are connected by a nucleotide sequence encoding a T2A peptide linker. The amino acid sequence of the anti-BCMA CAR is also provided in the sequence table. The coding sequence of the anti-BCMA CAR is connected to the coding sequence of the TCR beta chain by a nucleotide sequence encoding a P2A peptide linker.

[0184] Primary T cells from healthy donors are activated by anti-CD3 / CD28 beads (Thermo Scientific). One day later, T cells are transduced with a lentiviral vector encoding the above-mentioned TCR and anti-BCMACAR. Transduced cells are amplified and tested for CD3 expression by FACS analysis, and CD3+ colonies are gated for further analysis. The expression of bispecific CAR is analyzed by conventional methods.

[0185] Expressing the bispecific anti-TAA TCR / BCMACAR as described above (see also the sequence listing and Figure 4A ) were co-cultured with GFP-expressing target cells RPMI8226 and Nalm6-NYESO1 at a 1:3 ratio (E:T) to evaluate the killing activity. After 3 days, the remaining GFP+ tumor cells were counted by flow cytometry to determine the percentage of tumor cells killed by CART cells. Figure 4B The results shown indicate that the anti-bispecific anti-TAA TCR / BCMACAR showed effective killing effects on BCMA+RMPI8226 cells and Nalm6-NYESO1.

[0186] Example 5: Preparation and characterization of genetically engineered T cells expressing bispecific TCR and anti-BCMA CAR for AML treatment

[0187] A recombinant vector designed to express CD33 (e.g., Figure 5A TAA1 in ) and CD123 (e.g., Figure 5A A lentiviral vector comprising a bispecific TCR receptor specific for TAA2 in SEQ ID NO: 125 and a CAR specific for BCMA (SEQ ID NO: 125). Figure 5A. The bispecific TCR comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a VHH having specificity for CD33 fused to a CD3δ fragment (without an intracellular domain), and the second polypeptide comprises a VHH having specificity for CD123 fused to a CD3γ fragment (without an intracellular domain). The amino acid sequences of anti-CD33 VHH and anti-CD123 VHH as well as CD3δ and CD3γ fragments are provided in the sequence table. The coding sequences of the two polypeptides are connected by a nucleotide sequence encoding a T2A peptide linker. The amino acid sequence of anti-BCMA CAR (SEQ ID NO: 125) is also provided in the sequence table. The coding sequence of anti-BCMA CAR is connected to the coding sequence of one of the polypeptides by a nucleotide sequence encoding a P2A peptide linker.

[0188] Primary T cells from healthy donors are activated by anti-CD3 / CD28 beads (Thermo Scientific). One day later, T cells are transduced with a lentiviral vector encoding the above-mentioned bispecific TCR complex and anti-BCMA CAR. Transduced cells are amplified and tested for CD3 expression by FACS analysis, and CD3+ populations are gated for further analysis. The expression of bispecific CAR can be analyzed by conventional methods.

[0189] Expressing the above-described bispecific TCR complex and anti-BCMA CAR (see also Figure 5A )'s genetically engineered T cells and GFP-expressing target cells RPMI8226 and Molm13 were co-cultured with a 1:3 ratio (E:T) to evaluate killing activity. After 3 days, the remaining GFP+ tumor cells were counted by flow cytometry to determine the percentage of tumor cells killed by genetically engineered T cells. The results show that anti-CD123 / CD33 CAR (each of the 6 anti-CD123 VHH candidates listed in the sequence table) shows a killing efficiency similar to BCMA+RMPI8226 cells. Figure 5B Compared with other bispecific TCRs containing anti-CD123 VHH, genetically engineered T cells expressing bispecific TCR and anti-CD123 VHH3 showed relatively high killing efficiency against CD123+ / CD33+Molm13 cells. Figure 5C .

[0190] Example 6: Co-culture with antigen presenting cells enhances CAR-T cell expansion

[0191] Genetically engineered T cells expressing a bispecific CAR targeting CD70 and BCMA as disclosed in Example 3 above, an anti-NY-ESO-1 TCR / anti-BCMA CAR as disclosed in Example 4 above, and a bispecific anti-CD19 VHH / anti-BCMA CAR as disclosed in Example 2 above were co-cultured with K562 cells, or K562 cells expressing GFP / BCMA, NK92 cells, NK92 cells expressing GFP / BCMA, or NK92 cells expressing CD19 / BCMA to evaluate the effect of APC cells on CAR-T cell expansion. After 3 days, CAR+ cells were analyzed by flow cytometry, and Figures 6A-6C The results presented indicate that co-culture with APC cells expressing CD19 and / or BCMA effectively enhanced the expansion of all genetically engineered T cells tested herein.

[0192] Example 7: In vivo anti-tumor efficacy of co-administration of CAR-T cells and antigen presenting cells

[0193] According to the disclosure in Example 5 above, genetically engineered T cells and CAR constructs targeting BCMA (SEQ ID NO: 125) co-expressing TCR-based bispecific CARs targeting CD123 (anti-CD123 VHH3) and CD33 were prepared. The structural information of TCR-based bispecific CARs and anti-BCMA CARs (comprising 4-1BB costimulatory signaling domains and CD3z-ITAM3 signaling domains) is provided in the sequence table below. See also Figure 5A CAR-T cells were co-cultured with target cells K562, MM1S, or NK-92 expressing CD19 and BCMA. After 3 days, the percentage of CAR was analyzed by flow cytometry. Fig. 7A As shown, effective CAR-T cell expansion was observed when co-cultured with BCMA+MM1S cells or NK-92 expressing CD19 and BCMA compared with co-culture with K562 control cells.

[0194] CAR-T cells were co-cultured with GFP-expressing target cells K562, MM1S, CD123+U937, or CD123+ / CD33+Molm13 at a 1:3 ratio (E:T). After 3 days, the remaining GFP+ tumor cells were counted by flow cytometry to determine the percentage of tumor cells killed by CART cells. Figure 7BAs shown, the anti-CD123 / CD33 CAR with #3 anti-CD123 VHH candidate has effective killing effect on BCMA+MM1S and CD123+ / CD33+Molm13 cells. Surprisingly, compared with non-transduced mock T cells, CART cells did not show effective killing of CD123+U937 cells, indicating that the TCR-based bispecific anti-CD123 / CD33CAR is more powerful in killing dual-positive CD123+ / CD33+ targets than single-positive CD123 targets.

[0195] CAR-T cells and APCs expressing CD19 and / or BCMA were used to treat patients with relapsed / refractory AML. Patients were infused with 0.6 x 10 8 CAR-T cells, and 1.2 x 10 8 NK92-CD19 / BCMA cells. A 4.65% change in the percentage of CAR was observed on day 15, indicating that CAR-T cells expanded in vivo. Since the TCR-based bispecific anti-CD123 / CD33 CAR lacks co-stimulatory signaling, this expansion may be attributed to the co-expressed anti-BCMA CAR stimulated by APCNK-92-CD19 / BCMA. Figure 7C In summary, the results suggest that in vivo CAR-T expansion can be self-enhanced by co-administration of BCMA-expressing APCs.

[0196] 17 days after CART infusion, bone marrow cells were analyzed by flow cytometry. The results showed that the levels of CD123+ cells (2.1%) and CD33+ cells (4.32%) were low, and the level of CD123+ / CD33+ double-positive cells was much lower (0.45%). Fig.7D These results indicate that the TCR-based bispecific anti-CD123 / CD33 CAR is more effective in killing CD123+ / CD33+ double-positive cells compared to CD123+ or CD33+ single-positive cells. This result suggests that CAR-T therapy has a superior safety profile in the treatment of AML.

[0197] Sequence Listing

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214] Other embodiments

[0215] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature for the same, equivalent or similar purpose. Therefore, unless otherwise expressly stated, each feature disclosed is merely an example of a series of general equivalent or similar features.

[0216] Through the above description, those skilled in the art can easily determine the essential characteristics of the present invention, and without departing from the spirit and scope of the present invention, various changes and modifications can be made to the present invention to make it suitable for various uses and conditions. Therefore, other embodiments are also within the scope of the claims.

[0217] Equivalent

[0218] Although various inventive embodiments have been described and illustrated herein, a person of ordinary skill in the art will readily conceive of various other devices and / or structures for performing the functions described herein and / or obtaining the results and / or one or more advantages described herein, and each of such changes and / or modifications is considered to be within the scope of the inventive embodiments described herein. More generally, a person of ordinary skill in the art will readily understand that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that actual parameters, dimensions, materials, and / or configurations will depend on one or more specific applications used using the teachings of the present invention. Using only routine experiments, a person of ordinary skill in the art will recognize or be able to determine many equivalents of the specific inventive embodiments described herein. Therefore, it should be understood that the foregoing embodiments are presented only by way of example, and within the scope of the appended claims and their equivalents, embodiments of the present invention may be practiced in a manner different from that specifically described and claimed. The inventive embodiments of the present disclosure relate to each individual feature, system, article, material, kit, and / or method described herein. In addition, if any combination of two or more such features, systems, articles, materials, kits, and / or methods is not mutually inconsistent, such features, systems, articles, materials, kits, and / or methods are included within the scope of the present disclosure.

[0219] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0220] All references, patents, and patent applications disclosed herein are hereby incorporated by reference with respect to the subject matter for which each is cited, in some cases in their entirety.

[0221] Unless clearly indicated to the contrary, the indefinite articles "a" and "an" as used in this specification and claims should be understood to mean "at least one".

[0222] As used herein in the specification and claims, the phrase "and / or" should be understood to mean "either or both" of the elements so combined, i.e., elements that are present in combination in some cases and separately in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more elements" of the elements so combined. In addition to the elements specifically identified by the "and / or" clause, other elements may optionally be present, whether related or unrelated to those specifically identified. Thus, as a non-limiting example, in one embodiment, when used in conjunction with open language such as "including", a reference to "A and / or B" may refer only to A (optionally including elements other than B); in another embodiment, only to B (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0223] As used herein in this specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating the items in the list, "or" or "and / or" should be interpreted as inclusive, that is, including at least one element in many elements or element lists, but also including more than one element and optionally other unlisted items. Only explicitly pointing out the opposite term such as "only one" or "just one" or when used in the claims, "consisting of" refers to including exactly one element in many elements or element lists. In general, when there is an exclusive term before, such as "any one", "one of...", "only one in..." or "just one in...", the term "or" used in this article should only be interpreted as indicating exclusive alternatives (that is, "one or another, not two"). When used in the claims, "essentially consisting of" should have the common meaning as used in the field of patent law.

[0224] As used herein in this specification and claims, the phrase "at least one" with respect to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one element of each element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows that elements other than the elements specifically identified in the list of elements referred to by the phrase "at least one" may optionally be present, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, in one embodiment, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently "at least one of A and / or B") may refer to at least one optionally containing more than one A, without B (and optionally containing elements other than B); in another embodiment, may refer to at least one optionally containing more than one B, without A (and optionally containing elements other than A); in yet another embodiment, may refer to at least one optionally containing more than one A, and at least one optionally containing more than one B (and optionally containing other elements); etc.

[0225] It should also be understood that in any method claimed herein that includes more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order of the steps or actions of the method as recited unless explicitly stated to the contrary.

Claims

1. A method for treating a tumor, the method comprising administering to a subject in need thereof: (a) an effective amount of a population of genetically engineered T cells expressing one or more chimeric antigen receptors (CARs); and (b) an effective amount of antigen presenting cells (APCs); in: (i) the genetically engineered T cells express a bispecific CAR, wherein the bispecific CAR comprises a first antigen binding portion specific for a tumor associated antigen (TAA) and a second antigen binding portion specific for CD19 or BCMA; or (ii) the genetically engineered T cells express a T cell receptor (TCR) specific for the TAA and a CAR, wherein the CAR comprises an antigen binding portion specific for CD19 or BCMA; and wherein the APC expresses (i) CD19 and / or BCMA, and optionally (ii) the TAA.

2. The method of claim 1, wherein the genetically engineered T cells further express an antagonist of a cytokine, optionally wherein the cytokine is selected from the group consisting of interleukin-1 (IL-1), interleukin-1 (IL-2), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-1 (IL-9), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), interleukin-23 (IL-23), interleukin-24 (IL-24), interleukin-33 (IL-33), interleukin-36 (IL-36), GM-CSF, interferon gamma (IFNγ) and chemokine (CC motif) ligand 19 (CCL19).

3. The method of claim 2, wherein the antagonist of the cytokine is a fusion polypeptide comprising a binding portion of the cytokine and an immune-activating cytokine, optionally wherein the immune-activating cytokine is selected from the group consisting of IL-2, IL-7, IL-9, IL-10, IL-12, IL-15, IL-18, IL-21, IL-23, IL-24, IL-36, IL-33 and CCL19.

4. The method of claim 3, wherein the fusion polypeptide comprises a binding portion of IFNγ fused to IL-18; Optionally wherein the binding portion of IFNγ is an anti-IFNγ scFv, the anti-IFNγ scFv preferably comprises the amino acid sequence of SEQ ID NO: 55; and / or optionally wherein the IL-18 comprises the amino acid sequence of SEQ ID NO: 53; Preferably, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:

56.

5. The method according to any one of claims 1 to 4, wherein the subject is a human patient with a solid tumor or a hematopoietic cancer; optionally wherein the hematopoietic cancer is acute myeloid leukemia (AML).

6. The method of any one of claims 1 to 5, wherein the genetically engineered T cells express the bispecific CAR of (i), and wherein: (a) the bispecific CAR comprises the first antigen binding portion having specificity for the TAA and the second antigen binding portion having specificity for the CD19, and the APC expresses the CD19 and optionally the TAA; or (b) the bispecific CAR comprises the first antigen binding portion having specificity for the TAA and the second antigen binding portion having specificity for the BCMA, and the APC expresses the BCMA and optionally the TAA.

7. The method of claim 6, wherein the bispecific CAR comprises a fusion polypeptide comprising the first antigen binding portion and the second antigen binding portion; optionally wherein the first antigen binding portion and the second antigen binding portion are connected by a peptide linker.

8. The method of claim 7, wherein the first antigen binding moiety, the second antigen binding moiety, or both are in a single chain variable fragment (scFv) format or in a single domain antibody (VHH) format.

9. The method of claim 7 or claim 8, wherein the bispecific CAR further comprises an intracellular domain comprising one or more signaling domains; and optionally a hinge domain and a transmembrane domain connecting the antigen binding portion and the intracellular domain.

10. The method of claim 9, wherein the bispecific CAR comprises the hinge domain, which optionally belongs to a CD8, CD28, CD4, CD3 or IgG molecule.

11. A method according to claim 9 or claim 10, wherein the bispecific CAR includes the transmembrane domain, which optionally belongs to CD3, CD4, CD8, CD27 or CD28.

12. The method of any one of claims 9 to 11, wherein the intracellular domain comprises a co-stimulatory signaling domain and a cytoplasmic signaling domain.

13. The method of any one of claims 9 to 12, wherein the intracellular domain comprises a signaling domain of CD3, FcR, DAP12, 41BB, OX40, CD28, CD27, ICOS, IL-2R, IL-7R, IL-9R, IL-10R, IL-12R, IL18R, IL-21R, or IL-23R, or a combination thereof; Optionally, the intracellular domain comprises a co-stimulatory domain of 4-1BB, an IL2Rb signaling domain, and a CD3 zeta signaling domain; preferably, the co-stimulatory domain of 4-1BB comprises the amino acid sequence of SEQ ID NO: 8, the IL2Rb signaling domain comprises the amino acid sequence of SEQ ID NO: 9, and / or the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO:

10.

14. The method of any one of claims 1 to 6, wherein the genetically engineered T cells express the bispecific CAR of (i), and wherein the bispecific CAR comprises a first fusion polypeptide comprising the first antigen binding portion and a second fusion polypeptide comprising the second antigen binding portion.

15. The method of claim 14, wherein the first antigen binding moiety, the second antigen binding moiety, or both are in a single chain variable fragment (scFv) format or in a single domain antibody (VHH) format.

16. The method of claim 14, wherein the first antigen binding moiety is in a single chain variable fragment (scFv) or in a single domain antibody (VHH) format, and wherein the second antigen binding moiety is the extracellular domain of a ligand that binds to the TAA.

17. The method of any one of claims 1 to 16, wherein the bispecific CAR comprises one or more of: (a) an scFv fragment specific for BCMA (anti-BCMA scFv), the anti-BCMA scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein optionally the VH of the anti-BCMA scFv comprises the amino acid sequence of SEQ ID NO: 14, and the VL of the anti-BCMA scFv comprises the amino acid sequence of SEQ ID NO: 15, preferably wherein the anti-BCMA scFv comprises the amino acid sequence of SEQ ID NO: 16; (b) an scFv fragment specific for CD19 (anti-CD19 scFv), the anti-CD19 scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein optionally the VH of the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 59, and the VL of the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 60, preferably wherein the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 61 or 62; (c) a scFv fragment specific for Meso (anti-Meso scFv), the anti-Meso scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein optionally the VH of the anti-Meso scFv comprises the amino acid sequence of SEQ ID NO: 11, and the VL of the anti-Meso scFv comprises the amino acid sequence of SEQ ID NO: 12, preferably wherein the anti-Meso scFv comprises the amino acid sequence of SEQ ID NO: 13; (d) a scFv fragment specific for HER2 (anti-HER2 scFv), the anti-HER2 scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein optionally the VH of the anti-HER2 scFv comprises the amino acid sequence of SEQ ID NO: 17, and the VL of the anti-HER2 scFv comprises the amino acid sequence of SEQ ID NO: 18, preferably wherein the anti-HER2 scFv comprises the amino acid sequence of SEQ ID NO: 19; (e) a scFv fragment specific for GPC3 (anti-GPC3 scFv), the anti-GPC3 scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein optionally the VH of the anti-GPC3 scFv comprises the amino acid sequence of SEQ ID NO: 20, and the VL of the anti-GPC3 scFv comprises the amino acid sequence of SEQ ID NO: 21, preferably wherein the anti-BCMA scFv comprises the amino acid sequence of SEQ ID NO: 22; (f) an scFv fragment specific for Claudin 18.2 (anti-Claudin 18.2 scFv), the anti-Claudin18.2 scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein optionally the VH of the anti-Claudin 18.2 scFv comprises the amino acid sequence of SEQ ID NO: 23, and the VL of the anti-Claudin 18.2 scFv comprises the amino acid sequence of SEQ ID NO: 24, preferably wherein the anti-Claudin 18.2 scFv comprises the amino acid sequence of SEQ ID NO: 25; (g) an anti-CD33 VHH comprising the amino acid sequence of SEQ ID NO: 42; and (h) an anti-CD123 VHH comprising the amino acid sequence of any one of SEQ ID NOs: 47-52; (i) an anti-HER2 VHH comprising the amino acid sequence of SEQ ID NO: 69 or 72; (j) an anti-Claudin 18.2 VHH comprising the amino acid sequence of SEQ ID NO: 75 or 78; (k) an anti-mesothelin VHH comprising the amino acid sequence of SEQ ID NO: 81 or 84; (1) anti-PSMA VHH, wherein the anti-PSMA VHH comprises the amino acid sequence of SEQ ID NO: 87 or 90; (m) an anti-GPC3 VHH comprising the amino acid sequence of SEQ ID NO: 93; and (n) an anti-EGFR VHH comprising the amino acid sequence of SEQ ID NO:

96.

18. The method of claim 16, wherein the first antigen binding moiety is the extracellular domain of CD27, which binds to CD70, and wherein the second antigen binding moiety is specific for BCMA; Optionally wherein the extracellular domain of CD27 comprises the amino acid sequence of SEQ ID NO: 34 or 35; and / or Optionally, the antigen binding portion specific for BCMA comprises a scFv fragment specific for BCMA (anti-BCMA scFv), the anti-BCMA scFv comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein optionally the VH of the anti-BCMA scFv comprises the amino acid sequence of SEQ ID NO: 14, and the VL of the anti-BCMA scFv comprises the amino acid sequence of SEQ ID NO: 15, preferably wherein the anti-BCMA scFv comprises the amino acid sequence of SEQ ID NO:

16.

19. The method of any one of claims 14 to 18, wherein each of the first fusion polypeptide and the second fusion polypeptide comprises an intracellular domain comprising one or more signaling domains; and optionally a hinge domain and a transmembrane domain connecting the antigen binding portion and the intracellular domain.

20. The method of claim 19, wherein each of the first fusion polypeptide and the second fusion polypeptide comprises the hinge domain, optionally belonging to a CD8, CD28, CD4, CD3 or IgG molecule.

21. The method of claim 19 or claim 20, wherein each of the first fusion polypeptide and the second fusion polypeptide comprises the transmembrane domain, optionally belonging to CD3, CD4, CD8, CD27 or CD28.

22. The method of any one of claims 19 to 21, wherein the intracellular domain comprises a co-stimulatory signaling domain and a cytoplasmic signaling domain.

23. The method of any one of claims 19 to 22, wherein the intracellular domain comprises a signaling domain of CD3, FcR, DAP12, 41BB, OX40, CD28, CD27, ICOS, IL-2R, IL-7R, IL-9R, IL-10R, IL-12R, IL18R, IL-21R, or IL-23R, or a combination thereof; Optionally, the intracellular domain comprises a co-stimulatory domain of 4-1BB, an IL2Rb signaling domain, and a CD3 zeta signaling domain; preferably, the co-stimulatory domain of 4-1BB comprises the amino acid sequence of SEQ ID NO: 8, the IL2Rb signaling domain comprises the amino acid sequence of SEQ ID NO: 9, and / or the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO:

10.

24. The method of claim 1, wherein the population of genetically engineered T cells expresses a bispecific CAR that binds to: (a) Meso and BCMA, wherein the bispecific CAR optionally comprises an amino acid sequence of SEQ ID NO: 26 or 27, an amino acid sequence of SEQ ID NO: 107 or 108, or an amino acid sequence of SEQ ID NO: 109 or 110; (b) HER2 and BCMA, wherein the bispecific CAR optionally comprises the amino acid sequence of SEQ ID NO: 28 or 29, the amino acid sequence of SEQ ID NO: 99 or 100, or the amino acid sequence of SEQ ID NO: 101 or 102; (c) GPC3 and BCMA, wherein the bispecific CAR optionally comprises the amino acid sequence of SEQ ID NO: 30 or 31 or the amino acid sequence of SEQ ID NO: 115 or 116; (d) Claudin 18.2 and BCMA, wherein the bispecific CAR optionally comprises an amino acid sequence of SEQ ID NO: 32 or 33, an amino acid sequence of SEQ ID NO: 103 or 104, or an amino acid sequence of SEQ ID NO: 105 or 106; (e) CD19 and BCMA, wherein the bispecific CAR optionally comprises the amino acid sequence of SEQ ID NO: 57 or 58; (f) PSMA and BCMA, wherein the bispecific CAR optionally comprises the amino acid sequence of SEQ ID NO: 111 or 112 or the amino acid sequence of SEQ ID NO: 113 or 114; (g) EGFR and BCMA, wherein the bispecific CAR optionally comprises the amino acid sequence of SEQ ID NO: 117 or 118; (h) CD70 and BCMA, which optionally includes a first CAR polypeptide specific for CD70 and a second CAR polypeptide specific for BCMA, preferably wherein the first CAR polypeptide includes the amino acid sequence of SEQ ID NO: 36 or 37, and / or the second CAR polypeptide includes the amino acid sequence of SEQ ID NO: 38 or 39 or the amino acid sequence of SEQ ID NO: 125 or 126; (i) HER2 and BCMA, wherein the bispecific CAR optionally comprises a first CAR polypeptide specific for HER2 and a second CAR polypeptide specific for BCMA; preferably wherein the CAR polypeptide specific for HER2 comprises the amino acid sequence of SEQ ID NO: 70 or 71 or the amino acid sequence of SEQ ID NO: 73 or 74, and the CAR polypeptide specific for BCMA comprises the amino acid sequence of SEQ ID NO: 38 or 39 or the amino acid sequence of SEQ ID NO: 125 or 126; (j) Claudin 18.2 and BCMA, wherein the bispecific CAR optionally comprises a first CAR polypeptide specific for Claudin 18.2 and a second CAR polypeptide specific for BCMA; preferably wherein the CAR polypeptide specific for Claudin 18.2 comprises an amino acid sequence of SEQ ID NO: 76 or 77 or an amino acid sequence of SEQ ID NO: 79 or 80; and the CAR polypeptide specific for BCMA comprises an amino acid sequence of SEQ ID NO: 38 or 39 or an amino acid sequence of SEQ ID NO: 125 or 126; (k) mesothelin and BCMA, wherein the bispecific CAR optionally comprises a first CAR polypeptide specific for mesothelin and a second CAR polypeptide specific for BCMA; preferably wherein the CAR polypeptide specific for mesothelin comprises the amino acid sequence of SEQ ID NO: 82 or 83 or the amino acid sequence of SEQ ID NO: 85 or 86; and the CAR polypeptide specific for BCMA comprises the amino acid sequence of SEQ ID NO: 38 or 39 or the amino acid sequence of SEQ ID NO: 125 or 126; (l) PSMA and BCMA, wherein the bispecific CAR optionally comprises a first CAR polypeptide specific for PSMA and a second CAR polypeptide specific for BCMA; preferably wherein the CAR polypeptide specific for PSMA comprises the amino acid sequence of SEQ ID NO: 88 or 89 or the amino acid sequence of SEQ ID NO: 91 or 92, and the CAR polypeptide specific for BCMA comprises the amino acid sequence of SEQ ID NO: 38 or 39 or the amino acid sequence of SEQ ID NO: 125 or 126; (m) GPC3 and BCMA, wherein the bispecific CAR optionally comprises a first CAR polypeptide specific for GPC3 and a second CAR polypeptide specific for BCMA; preferably wherein the CAR polypeptide specific for GPC3 comprises the amino acid sequence of SEQ ID NO: 94 or 95, and the CAR polypeptide specific for BCMA comprises the amino acid sequence of SEQ ID NO: 38 or 39 or the amino acid sequence of SEQ ID NO: 125 or 126; or (n) EGFR and BCMA, wherein the bispecific CAR optionally includes a first CAR polypeptide specific for EGFR and a second CAR polypeptide specific for BCMA; preferably, wherein the CAR polypeptide specific for EGFR includes the amino acid sequence of SEQ ID NO: 97 or 98, and the CAR polypeptide specific for BCMA includes the amino acid sequence of SEQ ID NO: 38 or 39 or the amino acid sequence of SEQ ID NO: 125 or 126.

25. The method of any one of claims 1 to 5, wherein the population of genetically engineered T cells expresses the T cell receptor (TCR) specific for the TAA, and wherein the CAR comprises an antigen binding portion specific for CD19 or BCMA.

26. The method of claim 25, wherein the CAR comprises the antigen binding portion that is specific for CD19, and the APC expresses CD19 and optionally the TAA; or wherein the CAR comprises the antigen binding portion that is specific for BCMA, and the APC expresses BCMA and optionally the TAA.

27. The method of claim 26, wherein the antigen binding portion is in a single chain variable fragment (scFv) format or in a single domain antibody (VHH) format.

28. The method of any one of claims 25 to 27, wherein the CAR further comprises an intracellular domain comprising one or more signaling domains; and optionally a hinge domain and a transmembrane domain connecting the antigen binding portion and the intracellular domain.

29. The method of claim 28, wherein the CAR comprises the hinge domain, which optionally belongs to a CD8, CD28, CD4, CD3 or IgG molecule.

30. A method according to claim 28 or claim 29, wherein the CAR comprises the transmembrane domain, which optionally belongs to CD3, CD4, CD8, CD27 or CD28.

31. The method of any one of claims 28 to 30, wherein the intracellular domain comprises a co-stimulatory signaling domain and a cytoplasmic signaling domain.

32. The method of any one of claims 28 to 31, wherein the intracellular domain comprises a signaling domain of CD3, FcR, DAP12, 41BB, OX40, CD28, CD27, ICOS, IL-2R, IL-7R, IL-9R, IL-10R, IL-12R, IL18R, IL-21R or IL-23R or a combination thereof; optionally wherein the intracellular domain comprises a co-stimulatory domain of 4-1BB, an IL2Rb signaling domain, and a CD3 zeta signaling domain; preferably wherein the co-stimulatory domain of 4-1BB comprises the amino acid sequence of SEQ ID NO: 8, the IL2Rb signaling domain comprises the amino acid sequence of SEQ ID NO: 9, and / or the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO:

10.

33. The method of any one of claims 25 to 32, wherein the T cell receptor (TCR) is specific for NY-ESO-1, the TCR optionally comprising a TCR alpha chain comprising the amino acid sequence of SEQ ID NO: 40 and a TCR beta chain comprising the amino acid sequence of SEQ ID NO:

41.

34. The method of any one of claims 25 to 32, wherein the T cell receptor (TCR) comprises a modified CD3 δ chain and a modified CD3 γ chain, the modified CD3 δ chain and the modified CD3 γ chain together comprising a first antigen binding portion specific for CD33 (anti-CD33 portion) and a second antigen binding portion specific for CD123 (anti-CD123 portion); Optionally wherein the modified CD3δ chain comprises the extracellular domain and the transmembrane domain of CD3δ fused to the anti-CD33 portion, and the modified CD3γ chain comprises the extracellular domain and the transmembrane domain of CD3γ fused to the anti-CD123 portion, or vice versa.

35. The method of claim 34, wherein: (a) the modified CD3 δ chain comprises the amino acid sequence of SEQ ID NO: 45; (b) the modified CD3γ chain comprises the amino acid sequence of SEQ ID NO: 46; (c) the anti-CD33 portion is an anti-CD33 VHH, the anti-CD33 VHH optionally comprising the amino acid sequence of SEQ ID NO: 42; and / or (d) the anti-CD123 portion is an anti-CD123 VHH, which optionally comprises the amino acid sequence of any one of SEQ ID NOs: 47-52.

36. A method according to any one of claims 25 to 35, wherein the antigen binding portion in the CAR that is specific for CD19 comprises VH and VL, the VH comprises SEQ ID NO: 59, and the VL comprises SEQ ID NO: 60; optionally wherein the antigen binding portion in the CAR that is specific for CD19 is a scFv fragment, and the scFv fragment comprises the amino acid sequence of SEQ ID NO: 61 or 62.

37. A method according to any one of claims 25 to 35, wherein the antigen-binding portion specific for BCMA in the CAR comprises VH and VL, the VH comprises SEQ ID NO: 14, and the VL comprises SEQ ID NO: 15; optionally wherein the antigen-binding portion specific for BCMA in the CAR is a scFv fragment, and the scFv fragment comprises the amino acid sequence of SEQ ID NO: 16; preferably wherein the CAR is an anti-BCMA CAR, and the anti-BCMA CAR comprises the amino acid sequence of SEQ ID NO: 38 or 39 or the amino acid sequence of SEQ ID NO: 125 or 126.

38. A method according to any one of claims 25 to 37, wherein the TCR is a complex comprising a first fusion polypeptide comprising an antigen binding portion of CD33 and a second fusion polypeptide comprising an antigen binding portion of CD123, wherein one of the first fusion polypeptides further comprises a transmembrane fragment of CD3δ and the other further comprises a transmembrane fragment of CD3γ; and optionally wherein the transmembrane fragments of CD3δ and CD3γ do not contain the intracellular domains of CD3δ and CD3γ.

39. The method of any one of claims 25 to 38, wherein the population of genetically engineered T cells comprises tumor infiltrating T cells (TILs).

40. The method of any one of claims 1 to 39, wherein the population of genetically engineered T cells is autologous to the subject.

41. The method of any one of claims 1 to 40, wherein the population of genetically engineered T cells is allogeneic to the subject.

42. The method of any one of claims 1 to 41, wherein the APC cells comprise immune cells, stem cells or tumor cells, optionally wherein the immune cells are T cells, natural killer (NK) cells, tumor infiltrating lymphocytes, dendritic cells, macrophages, B cells, neutrophils, eosinophils, basophils, mast cells, myeloid-derived suppressor cells and / or mesenchymal stem cells; optionally wherein the stem cells are mesenchymal stem cells, and / or optionally wherein the tumor cells are K562 cells.

43. The method of claim 42, wherein the APCs are genetically engineered to express the CD19 and / or the BCMA and optionally the TAA.

44. The method of claim 42 or claim 43, wherein the APCs are derived from peripheral blood cells, umbilical cord blood cells, induced pluripotent stem cells (iPSCs), or immune cell lines.

45. The method of any one of claims 42 to 44, wherein the APC is genetically engineered to further express a membrane-bound stimulatory cytokine.

46. ​​The method of claim 45, wherein the membrane-bound stimulatory cytokine is IL-10, IL-18, IL-15, IL-9 or IL-21.

47. The method of any one of claims 1 to 46, wherein the APCs are autologous to the subject.

48. The method of any one of claims 1 to 46, wherein the APCs are allogeneic to the subject.

49. A kit for treating cancer, the kit comprising: (a) a population of genetically engineered T cells as set forth in any one of claims 1 to 41; and (b) the APC as set forth in any one of claims 1, 6, 26, and 42 to 48.

50. A population of genetically engineered T cells, comprising genetically engineered T cells expressing: (i) a bispecific CAR comprising a first antigen binding portion specific for a tumor-associated antigen (TAA) and a second antigen binding portion specific for CD19 or BCMA; or (ii) a T cell receptor (TCR) specific for the TAA, and the CAR includes an antigen binding portion specific for CD19 or BCMA.

51. A population of genetically engineered T cells according to claim 50, wherein the genetically engineered T cells express the bispecific CAR of (i), which is shown in any one of claims 6 to 24.

52. The population of genetically engineered T cells of claim 50, wherein the genetically engineered T cells express the TCR of (ii), which is set forth in any one of claims 25 to 38.

53. A population of genetically engineered antigen presenting cells (APCs), wherein the APCs express CD19 and / or BCMA, and wherein the APCs are further genetically engineered to express a membrane-bound stimulatory cytokine.

54. The population of genetically engineered APCs as set forth in any one of claims 42 to 46.

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