Combination of chimeric antigen receptors with dap10 in cell therapy

Abstract

Expression constructs encoding chimeric antigen receptors and DAP10 recombinant polypeptides, engineered immune cells, and methods of use thereof are provided. Further provided are methods for activation and expansion of cells for therapeutic use, in particular for chimeric antigen receptor-based immune cell immunotherapy.

Description

[0001] Cross-references

[0002] This application claims the benefit of international application PCT / CN2023 / 141856, filed on December 26, 2023, the entire contents of which are incorporated herein by reference.

[0003] sequence list

[0004] This application includes a sequence list, which has been filed electronically and whose entire contents are hereby incorporated herein by reference. Technical Field

[0005] This disclosure relates to chimeric antigen receptor constructs, engineered immune cells, and methods of using them. This disclosure further relates to the activation and expansion of cells for therapeutic uses, particularly for chimeric antigen receptor-based immunocellular immunotherapy. Background Technology

[0006] Cancer is a group of diseases characterized by abnormal cell growth that can invade and spread to other parts of the body. Despite significant advances in cancer treatment, it remains one of the leading causes of death. Unleashing the potential of the immune system has become a central focus of cancer treatment, thus promoting the development of NK, T, and γδT cell therapies. While chimeric antigen receptor (CAR)-modified immune cells have shown encouraging effects against some cancers, their efficacy in most tumors remains unsatisfactory.

[0007] The T lymphocytes used in CAR-T therapy mainly refer to CD3 cells. + T cells, including CD4 + and CD8 + T cells are the most prevalent subset. The application of chimeric antigen receptor-modified T cells has yielded significant clinical responses, leading to FDA approval of six CAR-T cell therapies since 2017 and the registration of hundreds of clinical trials.

[0008] NK cells are a unique subset of lymphocytes, first identified in 1975. These cells originate from CD34. + Hematopoietic progenitor cells are formed by downregulating CD34 and upregulating CD56. One advantage of NK cell therapy compared to T-cell therapy is that NK cells are readily available as off-the-shelf formulations. Many NK cell therapies, including natural NK cells and CAR-NK cells, have been used in clinical trials and have shown promising results. Although no NK cell therapy has yet received FDA approval, clinical trials have demonstrated promising outcomes.

[0009] γδ T cells constitute a subset of CD3+ T cells, characterized by the expression of Vγ (γ-2, 3, 4, 5, 8, and 9) chains and Vδ (δ-1, 2, 3, 4, 5, 6, 7, and 8) chains, which form heterodimeric γδ T cell receptors (TCRs). In contrast to conventional αβ T cells, which rely on MHC (major histocompatibility complex)-dependent antigen processing for target recognition, γδ T cells typically recognize target cells in an MHC-independent manner. Increasing evidence suggests that both subtypes of γδ T cells play crucial roles in tumor immune surveillance and antitumor immune responses.

[0010] In cancer treatment, including CAR therapy, tumor immune escape represents a major obstacle. Escape mechanisms involve multiple factors, such as CAR target downregulation, major histocompatibility complex (MHC) deficiency, and the presence of a local immunosuppressive microenvironment. While precise target recognition of CARs ensures their safety as therapeutic agents, target downregulation can be a significant limiting factor in other situations.

[0011] NKG2D is an activating receptor expressed on the surface of immune cells, including T cells, NK cells, and γδT cells. Unlike CARs or antibodies, NKG2D does not have a single unique ligand. NKG2D ligands include, but are not limited to, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. These ligands are specifically expressed in tumor or stressed tissues. These characteristics make NKG2D one of the most important activators in immune cells, playing a crucial role in natural antitumor processes.

[0012] In human cells, NKG2D-mediated signals are specifically transmitted via DAP10. DAP10, also known as a hematopoietic signal transducer, is a transmembrane signal transduction adaptor containing the YxxM motif in its cytoplasmic domain. Signal transduction is achieved through the assembly of two DAP10 dimers with an NKG2D receptor. A pair of aspartic acid residues near the center of the transmembrane domain of the DAP10 dimer, along with the conserved arginine residue in the NKG2D transmembrane domain, are necessary and sufficient for the assembly of the signal transduction process.

[0013] To overcome this limitation, this invention utilizes DAP10 to enhance immune cell function by providing an additional signaling pathway simultaneously with CAR activation. This disclosure provides improved immunocellular therapies utilizing the interaction between NKG2D and DAP10, and related nucleic acid constructs. Invention Overview

[0015] In this application, various forms of recombinant peptides (also referred to herein as "adaptor peptides") containing the DAP-10 transmembrane (TM) domain and intracellular signal transduction domain were designed. Their expression enables immune cells to trigger potent immune responses against various cancers by binding to NKG2D. Furthermore, by combining these adaptor peptides with chimeric antigen receptors (CARs) targeting desired antigens in the expression constructs, the inventors discovered that immune cells expressing both CAR peptides and adaptor peptides exhibit dual specificity against cancer cells, both in vitro and in vivo. Moreover, the combination of adaptor peptides containing DAP10™ with CAR peptides can more effectively inhibit or eliminate cancer cells expressing these antigens.

[0016] On one hand, this disclosure provides an expression construct comprising a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) polypeptide and a second nucleic acid sequence encoding a recombinant polypeptide containing a DAP10 transmembrane region, wherein

[0017] The DAP10 transmembrane region originates from human DAP10 and can interact with NKG2D, and

[0018] The first nucleic acid sequence is separated from the second nucleic acid sequence by a nucleotide sequence encoding a cleavable adapter.

[0019] In some embodiments, the transmembrane region of DAP10 contains an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID No: 9.

[0020] In some implementations, the recombinant polypeptide further comprises one or more of a signal peptide, a hinge region, an intracellular region, and an affinity tag.

[0021] In some embodiments, the recombinant polypeptide includes an intracellular region comprising an intracellular domain (ICD) of DAP10 derived from human DAP10, optionally comprising an amino acid sequence SEQ ID No: 10 or an amino acid sequence having at least 85%, 90% or 95% identity with SEQ ID No: 10.

[0022] In some embodiments, the recombinant polypeptide comprises a signal peptide (SP) derived from human DAP10, T cell surface expressed receptors (such as CD8, CD28, and TCR), NK cell surface expressed receptors (such as 2B4, CD16, NKP30, NKP44, and NKP46), or IgG. In some embodiments, the SP region comprises a DAP10 SP containing an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with SEQ ID No: 6. In some embodiments, the SP region comprises a CD8 SP containing an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with SEQ ID No: 19.

[0023] In some embodiments, the recombinant polypeptide comprises a hinge region derived from human DAP10, TCR, or immunoglobulin. In some embodiments, the hinge region is derived from DAP10 and comprises an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with SEQ ID No: 8.

[0024] In some embodiments, the recombinant polypeptide includes an affinity tag located in an extracellular region, such as the N-terminus of a transmembrane region. More specifically, the affinity tag may be located between the signal peptide and the hinge region.

[0025] In some embodiments, the recombinant polypeptide comprises a DAP10 transmembrane region and an intracellular region from the N-terminus to the C-terminus. In some embodiments, the recombinant polypeptide comprises a signal peptide, a hinge region, a DAP10 transmembrane region, and an intracellular region from the N-terminus to the C-terminus, optionally with an affinity tag (such as an HA tag) between the signal peptide and the hinge region. In some embodiments, the recombinant polypeptide comprises a signal peptide, a DAP10 transmembrane region, and an intracellular region from the N-terminus to the C-terminus, optionally with an affinity tag (such as an HA tag) between the signal peptide and the DAP10 transmembrane region.

[0026] In some implementations, the intracellular region further includes one or more co-stimulatory signaling domains derived from any of the following: CD28, 4-1BB, 2B4, 2B4 Ligands and combinations thereof of ITSM2, CD27, OX40, CD30, CD40, CD3, LFA-1, ICOS (CD278), NTBA, CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0027] In some embodiments, the intracellular region further comprises a primary signal transduction domain. This primary signal transduction domain may be derived from CD3ζ, and optionally, it comprises the amino acid sequence SEQ ID No: 5 or an amino acid sequence having at least 85%, 90%, or 95% identity with SEQ ID No: 5.

[0028] In some embodiments, the recombinant polypeptide comprises, from the N-terminus to the C-terminus: (a) DAP10 signal peptide (SP), DAP10 hinge region, DAP10 transmembrane region (TM) and co-stimulatory signal transduction domain, optionally including primary signal transduction domain; (b) DAP10 SP, DAP10 TM and costimulatory signal transduction domains, optionally including primary signal transduction domains; (c) CD8 SP, DAP10 hinge region, DAP10 TM, and co-stimulatory signal transduction domain, optionally including the primary signal transduction domain; or (d) CD8 SP, DAP10 TM and co-stimulatory signal transduction domains, optionally including primary signal transduction domains.

[0029] In some embodiments, the recombinant polypeptide comprises, from the N-terminus to the C-terminus: (a) DAP10 signal peptide (SP), DAP10 hinge region, DAP10 transmembrane region (TM), DAP10 intracellular domain (ICD) and 2B4 co-stimulatory signal transduction domain; (b) DAP10 SP, DAP10 hinge region, DAP10 TM, DAP10 ICD and 4-1BB co-stimulatory signal transduction domains; (c) DAP10 SP, DAP10 hinge region, DAP10 TM, DAP10 ICD, 2B4 costimulatory signal transduction domain and 4-1BB costimulatory signal transduction domain; (d) DAP10 SP, DAP10 hinge region, DAP10 TM, DAP10 ICD, 2B4 co-stimulatory signal transduction domain and CD3ζ signal transduction domain; (e) DAP10 SP, DAP10 hinge region, DAP10 TM, DAP10 ICD, 4-1BB co-stimulatory signal transduction domain and CD3ζ signal transduction domain; (f) DAP10 SP, DAP10 hinge region, DAP10 TM, DAP10 ICD and 2B4·ITSM2 co-stimulatory signal transduction domains; (g) DAP10 SP, DAP10 hinge region, DAP10 TM, DAP10 ICD and CD28 co-stimulatory signal transduction domains; (h) CD8 SP, DAP10 hinge region, DAP10 TM, DAP10 ICD and 2B4 co-stimulatory signal transduction domain; (i) CD8 SP, DAP10 TM, DAP10 ICD, and 2B4 co-stimulatory signal transduction domains; or (j) CD8 SP, DAP10 hinge region, DAP10 TM, DAP10 ICD and 2B4·ITSM2 co-stimulatory signal transduction domains.

[0030] In some embodiments, DAP10 SP contains the amino acid sequence SEQ ID No: 6. In some embodiments, CD8 SP contains the amino acid sequence SEQ ID No: 19. In some embodiments, the DAP10 hinge region contains the amino acid sequence SEQ ID No: 8. In some embodiments, DAP10 TM contains the amino acid sequence SEQ ID No: 9. In some embodiments, DAP10 ICD contains the amino acid sequence SEQ ID No: 10.

[0031] In some implementations, the cleavable linker is selected from P2A, E2A, F2A, T2A peptides, internal ribosome entry site (IRES) sequences and their functional variants.

[0032] In some embodiments, the recombinant polypeptide comprises or consists of the following amino acid sequences: any of the amino acid sequences in SEQ ID No: 23-32 or an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with any of the amino acid sequences in SEQ ID No: 23-32.

[0033] In some embodiments, the CAR peptide comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain. In some embodiments, the extracellular antigen-binding domain is selected from single-chain Fv (scFv), Fab, Fab', F(ab')2, Fv, microantibodies, diabody antibodies, single-domain antibodies (sdAb), or VHH domains, such as scFv. Optionally, the extracellular antigen-binding domain targets GPC3, CD19, or CD33. For example, a scFv targeting GPC3 contains the amino acid sequence SEQ ID No: 1, a scFv targeting CD19 contains the amino acid sequence SEQ ID No: 13, and a scFv targeting CD33 contains the amino acid sequence SEQ ID No: 14.

[0034] In some embodiments, the transmembrane domain of the CAR peptide is derived from any one of CD8, ICOS, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the transmembrane domain of the CAR peptide is derived from CD8 or CD28, and optionally, the transmembrane domain comprises the amino acid sequence SEQ ID No: 3 or 22 or an amino acid sequence having at least 85%, 90%, or 95% identity with SEQ ID No: 3 or 22.

[0035] In some embodiments, the primary intracellular signal transduction domain of the CAR peptide is derived from CD3ζ, and optionally, the primary intracellular signal transduction domain comprises the amino acid sequence SEQ ID No: 5 or an amino acid sequence having at least 85%, 90% or 95% identity with SEQ ID No: 5.

[0036] In some embodiments, the co-stimulatory signaling domain of the CAR peptide is derived from a co-stimulatory molecule selected from CD28, 4-1BB, 2B4 (e.g., 2B4). Ligands of ITSM2), CD27, OX40, CD30, CD40, CD3, LFA-1, ICOS (CD278), NTBA, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and combinations thereof. In some embodiments, the co-stimulatory signal transduction domain of the CAR peptide is derived from 4-1BB, 2B4, or CD28, and optionally, the co-stimulatory signal transduction domain comprises any one of the amino acid sequences SEQ ID No: 4, 11-12, and 21 or an amino acid sequence having at least 85%, 90%, or 95% identity with any one of SEQ ID No: 4, 11-12, and 21.

[0037] In some implementations, the CAR peptide further comprises: A hinge domain, located between the extracellular antigen-binding domain and the transmembrane domain, optionally derived from CD8 or CD28; and / or The signal peptide is located at the N-terminus of the extracellular antigen-binding domain, and optionally, the signal peptide is derived from CD8.

[0038] In some embodiments, the CAR polypeptide comprises or consists of the following amino acid sequences: any of the amino acid sequences in SEQ ID No: 33-38 or an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity with any of the amino acid sequences in SEQ ID No: 33-38.

[0039] In some embodiments, the expression construct further comprises a third nucleic acid sequence encoding a cytokine polypeptide (e.g., wild-type IL-15, IL-2, IL-4, IL-7, IL-21, IL-23 or variants thereof), wherein the third nucleic acid sequence is separated from the second nucleic acid sequence or the first nucleic acid sequence by a nucleotide sequence encoding a second cleavable linker.

[0040] In some embodiments, the cytokine polypeptide comprises membrane-bound IL-15 having the amino acid sequence SEQ ID No: 15 or soluble IL-15 having the amino acid sequence SEQ ID No: 16.

[0041] In some implementations, the second cleavable linker is selected from P2A, E2A, F2A, T2A peptide, IRES sequence, and functional variants thereof.

[0042] The positions of the first, second, and third (if present) nucleic acid sequences in the expression construct can be interchanged. In some implementations, the expression construct contains from 5' to 3': (a) First nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence; (b) First nucleic acid sequence, third nucleic acid sequence, and second nucleic acid sequence; (c) Second nucleic acid sequence, first nucleic acid sequence and third nucleic acid sequence; (d) Second nucleic acid sequence, third nucleic acid sequence, and first nucleic acid sequence; (e) The third nucleic acid sequence, the first nucleic acid sequence, and the second nucleic acid sequence; or (f) The third nucleic acid sequence, the second nucleic acid sequence, and the first nucleic acid sequence.

[0043] In some implementations, the three nucleic acid sequences are operatively linked together and separated by nucleotide sequences encoding self-cleaving peptides (such as P2A, E2A, F2A, or T2A) or IRES sequences.

[0044] On one hand, this disclosure provides a recombinant polypeptide comprising a DAP10 transmembrane region derived from human DAP10 and capable of interacting with NKG2D, and one or more intracellular signal transduction domains. In some embodiments, this disclosure provides a recombinant polypeptide encoded by a second nucleic acid sequence of an expression construct as disclosed herein.

[0045] On one hand, this disclosure provides a vector comprising the expression construct as disclosed herein. The vector may be a viral vector, such as an adenovirus vector, adeno-associated virus vector, lentiviral vector, or retroviral vector, or a non-viral vector, such as a plasmid, liposome, nanoparticle, lipid, or a combination thereof.

[0046] On the one hand, this disclosure provides an engineered immune cell that contains or expresses expression constructs or vectors as disclosed herein.

[0047] In some implementations, the immune cells are natural killer (NK) cells, T cells, γδ T cells, invariant NKT (iNKT) cells, B cells, macrophages, MSCs, or dendritic cells. In some implementations, the NK cells are derived from umbilical cord blood, peripheral blood, induced pluripotent stem cells, human embryonic stem cells, bone marrow, or cell lines.

[0048] On the one hand, this disclosure provides populations of immune cells as disclosed herein, wherein the cells are present in a suitable medium.

[0049] In one aspect, this disclosure provides a pharmaceutical composition comprising engineered immune cells as disclosed herein and a pharmaceutically acceptable carrier.

[0050] In one aspect, this disclosure provides a method for preventing or treating cancer in a subject with this need, comprising administering to the subject an effective amount of an immune cell or expression construct as disclosed herein. In the case of the subject, the immune cell may be allogeneic or autologous.

[0051] The cancer may be a solid tumor or a blood cancer. In some implementations, the cancer is selected from multiple myeloma (MM), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), glioma, breast cancer, cervical cancer, prostate cancer, kidney cancer, stomach cancer, esophageal cancer, pancreatic ductal carcinoma, lung cancer such as non-small cell lung cancer (NSCLC), ovarian cancer, colorectal cancer, liver cancer, head and neck cancer, and gallbladder cancer.

[0052] On the one hand, this disclosure provides expression constructs or immune cells, as disclosed herein, for the treatment of tumors in subjects in need of such treatment.

[0053] On the one hand, this disclosure provides the use of expression constructs or engineered immune cells, as disclosed herein, in the preparation of medicaments for treating cancer in subjects. Subjects may be mammals, such as humans or non-human animals.

[0054] In one aspect, this disclosure provides a method for activating immune cells, comprising expressing an expression construct as disclosed herein in the immune cells, wherein activation occurs in response to binding of a CAR to a corresponding target molecule. In some embodiments, immune cells or multiple immune cells are introduced into a subject in need of this method; and the activation occurs in the subject.

[0055] Other features, purposes, and beneficial effects will become apparent from the following disclosure. Attached Figure Description

[0056] Figure 1 A schematic diagram of the structure of the CAR peptide and the linker peptide as disclosed herein is shown.

[0057] Figure 2 The proliferation of T cells transfected with structures 1-8 is shown.

[0058] Figure 3 The expression of structures 1-8 and the percentage of CD4 / CD8 in T cells are shown.

[0059] Figure 4 The expression of MICA / B and GPC3 in hepG2 and sk-hep1 is shown. (A) Expression of MICA / B in hepG2 (a) and sk-hep1 (b); (B) Expression of GPC3 in hepG2 (a) and sk-hep1 (b).

[0060] Figure 5 The cytotoxicity of CAR-T cells against hepG2 cells (co-cultured for 2 days) was demonstrated.

[0061] Figure 6 The cytotoxicity of CAR-T cells against sk-hep1 cells was demonstrated (co-cultured for 1 day).

[0062] Figure 7 The cytotoxicity of CAR-T cells against sk-hep1 cells was demonstrated (co-cultured for 2 days).

[0063] Figure 8 The proliferation of NK cells transfected with structures 1-7 is shown.

[0064] Figure 9 The expression of structures 1-7 in NK cells is shown.

[0065] Figure 10 The study demonstrated the cytotoxicity of CAR-NK cells against hepG2 cells.

[0066] Figure 11 The study demonstrated the cytotoxicity of CAR-NK cells against sk-hep1 cells.

[0067] Figure 12 The proliferation of γδT cells transfected with structures 1, 5, and 6 is shown.

[0068] Figure 13 The subtypes and structural expression of γδT cells are shown.

[0069] Figure 14 The study demonstrated the cytotoxicity of CAR-γδT cells against hepG2 cells.

[0070] Figure 15 The study demonstrated the cytotoxicity of CAR-γδT cells against sk-hep1 cells.

[0071] Figure 16 The subtypes of T cells transfected with structures 1, 2, and 9-12 in Example 4 and the expression of the structures are shown.

[0072] Figure 17 The subtypes of NK cells transfected with structures 1, 2, and 9-12 in Example 4 and the expression of the structures are shown.

[0073] Figure 18 The subtypes of γδT cells transfected with structures 9, 11, and 13 in Example 4 and the expression of the structures are shown.

[0074] Figure 19 The activation of γδT cells by hepG2 was demonstrated.

[0075] Figure 20 The cytotoxicity of CAR-T cells (structures 1, 2 and 9-12) against hepG2 was demonstrated.

[0076] Figure 21 The cytotoxicity of CAR-T cells (structures 1, 2, and 9-12) against sk-hep1 was demonstrated.

[0077] Figure 22 The cytotoxicity of CAR-NK cells (structures 1, 2 and 9-12) against sk-hep1 was demonstrated.

[0078] Figure 23 The cytotoxicity of CAR-γδT cells (structures 9 and 13) against sk-hep1 was demonstrated.

[0079] Figure 24 The proliferation of NK cells transfected with structures 16-22 is shown.

[0080] Figure 25 CD56 is shown + CAR expression in cells.

[0081] Figure 26 The study demonstrated the cytotoxicity of CAR-NK cells against hepG2.

[0082] Figure 27 The study demonstrated the cytotoxicity of CAR-NK cells against sk-hep1.

[0083] Figure 28 The proliferation of NK cells transfected with structures 23-27 is shown.

[0084] Figure 29 The expression of structures 23-27 in NK cells is shown.

[0085] Figure 30 The study demonstrated the cytotoxicity of CAR-NK cells against hepG2.

[0086] Figure 31 The study demonstrated the cytotoxicity of CAR-NK cells against sk-hep1. Invention Details

[0088] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Furthermore, unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. More specifically, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context explicitly specifies otherwise. Thus, for example, a reference to “a protein” includes multiple proteins; a reference to “a cell” includes a mixture of cells, and so on. In this application, unless otherwise stated, the use of “or” means “and / or.” Furthermore, the use of the term “comprise” and other equivalent terms such as “contain” and “include” is not restrictive. Additionally, the scope provided in this specification and the appended claims includes both endpoints and all points between the endpoints.

[0089] As used herein, the term "DNAX activating protein 10" or "DAP10" refers to the transmembrane adaptor protein of Natural Killer Group 2 Member D (NKG2D). The DAP10 protein is present in mammalian lymphocytes and myeloid cells, and its exact sequence may vary depending on species, subtype, and individual differences. Membrane localization and signal transduction of NKG2D depend on the DAP10 protein. The human DAP10 protein can be divided into four regions: a signal peptide, a hinge region, a transmembrane region, and a cytoplasmic region. As shown in the polypeptide sequence UniProt Q9UBK5, the signal peptide consists of amino acids 1-18, the hinge region of amino acids 19-48, the transmembrane region of amino acids 49-69, and the intracellular region of amino acids 70-93. Within the cytoplasmic region of DAP10, the SH2 domain binding site recruits the p85 subunit of phosphatidylinositol 3-kinase, thereby providing NKG2D-dependent transduction. The amino acid sequence of human DAP10 can be represented by the dominant polypeptide sequence UnitProt Q9UBK5 and NCBI accession numbers NP 055081.1 and AF072845; however, different subtypes and variants may exist.

[0090] As used herein, the term "DAP10 transmembrane domain" encompasses the transmembrane domain of human DAP10 as well as any of its homologous variants capable of recruiting the NKG2D receptor. The presence of a transmembrane region in the adaptor peptide capable of interacting with NKG2D is crucial for its function.

[0091] As used herein, the term "hinge region" refers to a short sequence that may be present at the N-terminus of a transmembrane region in a linker peptide or CAR peptide to enhance its structural flexibility. A hinge region may consist of at least two (e.g., 5, 10, or more) amino acids, forming a flexible or semi-flexible bond between the transmembrane domain and the signal peptide within a single peptide molecule.

[0092] As used herein, the term “NKG2D” or “NKG2D receptor” refers to a transmembrane protein belonging to the NKG2 family of C-type lectin-like receptors. NKG2D acts as the primary activating receptor, where ligand binding triggers cytotoxicity and cytokine production. NKG2D provides co-stimulatory effects through the associated adaptor molecule DAP10, which recruits phosphatidylinositol-3 kinase. In mice, NKG2D also associates with DAP12, which recruits protein tyrosine kinase. In humans, NKG2D is activated by NK cells, γδ T cells, and CD8+ cells. + αβ T cell expression, and under specific pathological conditions, CD4 +T cell expression (Stanjanovic A. et al. (2018) Front. Immunol. 23: 1-15). In mice, NKG2D is expressed by NK cells, NK1.1+ T cells, γδ T cells, and activated CD8+ T cells. + Expression on αβ T cells and activated macrophages. NKG2D specifically recognizes different stress-induced major histocompatibility complex (MHC) class I molecule-like structures, including MICA, MICB, and UL16-binding protein (ULBP) in humans, and retinoic acid early transcript-1, minor histocompatibility antigen H-60, and murine ULBP-like transcript-1 in mice. For example, the NKG2D-DAP10 receptor complex can activate NK cell and T cell responses to combat tumors carrying MICA.

[0093] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a recombinant polypeptide construct comprising an extracellular antigen-binding moiety providing antigen specificity, a transmembrane domain anchoring the CAR to the cell membrane, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. Immune cells, particularly T lymphocytes and NK lymphocytes, can be genetically modified to express a CAR inserted into their plasma membrane. If such a CAR-modified immune cell encounters another cell or tissue structure expressing or carrying a suitable target with the antigen-binding moiety of the CAR, the CAR-modified immune cell cross-links with the target antigen after the binding moiety binds to the target antigen. This cross-linking induces the induction of signaling pathways via the CAR signaling chain, which alters the biological characteristics of the CAR-implanted immune cell. The domains in the CAR construct may be located on the same polypeptide chain, for example, constituting a chimeric fusion protein. Alternatively, the domains in the CAR construct may be discontinuous, for example, located on different polypeptide chains, as provided, for example, as in the form of a split CAR.

[0094] As used herein, the term "intracellular signaling domain" refers to the intracellular portion of a CAR that can generate signals that promote immune effector functions in CAR-containing cells (e.g., CAR-T cells or CAR-expressing NK cells). Examples of immune effector functions, such as those in CAR-T cells or CAR-expressing NK cells, include lytic activity and helper activities, including cytokine secretion. Intracellular signaling domains can transduce effector function signals and direct cells to perform specific functions. While the entire intracellular signaling domain can be utilized, in many cases it is not necessarily required to use the whole chain. Intracellular signaling domains may include primary intracellular signaling domains and optionally further include co-stimulatory signaling domains.

[0095] The term "primary intracellular signaling domain" refers to a cytoplasmic signaling sequence that functions in a stimulatory manner to induce immune effector functions. A primary intracellular signaling domain may contain a signaling motif known as an immune receptor tyrosine-based activation motif, or ITAM. As used herein, "ITAM" is a conserved protein motif commonly found at the tail of signaling molecules expressed in many immune cells. The ITAM within a signaling molecule is important for intracellular signal transduction, which is at least partially mediated by phosphorylation of tyrosine residues in the ITAM following activation of the signaling molecule. The ITAM can also serve as a docking site for other proteins involved in signaling pathways.

[0096] The term "co-stimulatory signaling domain" refers to the intracellular portion of a co-stimulatory molecule. Intracellular signaling domains can contain the entire intracellular portion of the molecule from which they originate, the entire native intracellular signaling domain, or functional fragments thereof. Co-stimulatory molecules are like-minded binding partners on immune cells (e.g., NK and T cells) that specifically bind to co-stimulatory ligands, thereby mediating co-stimulatory responses of immune cells, such as, but not limited to, proliferation. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are essential for an effective immune response. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal transduction lymphocyte activating molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, and HVEM. (LIGHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD4 9f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and ligands that specifically bind to CD83.

[0097] As used herein, the term "immunoreceptor tyrosine switching motif (ITSM)" refers to a motif identified in the cytoplasmic region of the SLAM family of receptors, which are immunoglobulin-like type I transmembrane receptors present on the cell surface throughout the hematopoietic system and primarily involved in isotype interactions. For example, 2B4 belongs to the SLAM-associated receptor family and contains four immunoreceptor tyrosine-based switching motifs (ITSMs) in its cytoplasmic tail. In some cases, the ITSMs within the 2B4 cytoplasmic tail are sufficient for 2B4-mediated NK cell activation.

[0098] As used herein, “self-cleaving peptide” refers to an oligopeptide such as a 2A linker that allows multiple proteins to be encoded into a polyprotein, which dissociates post-translationally into component proteins. The use of the term “self-cleaving” is not intended to imply the occurrence of a proteolytic cleavage reaction. Various self-cleaving linkers or 2A linkers are known to those skilled in the art, including but not limited to linkers present in members of the Picornaviridae virus family, such as foot-and-mouth disease virus (FMDV), equine rhinitis virus type A (ERAV), TaV, and PTV-1; and carioviruses such as Theilovirus and encephalomyocarditis virus. 2A linkers derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as “F2A,” “E2A,” “P2A,” and “T2A,” respectively. The sequence of the P2A linker may have at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence SEQ ID No: 17. The sequence of the T2A linker may have at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence SEQ ID No: 18.

[0099] The term "operably linked" refers to the juxtaposition of two or more biological sequences of interest (with or without spacers or adapters) in such a manner that they are in a relationship that allows them to function in the intended way. When used with peptides, it is intended to indicate that the peptide sequences are linked in such a way that the linked product is allowed to have the intended biological function. For example, the transmembrane region of an adaptor peptide can be "operably linked" to an intracellular region either directly or indirectly via an adapter sequence, provided that both portions function normally. The term can also be used with polynucleotides. As an example, when a polynucleotide encoding a CAR peptide is operably linked to a polynucleotide encoding an adaptor peptide, this is intended to indicate that these polynucleotide sequences are linked in such a way that the peptide can be normally expressed from the nucleic acid molecule.

[0100] As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that binds specifically to an antigen. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and can be derived from natural or recombinant sources. Antibodies can be tetramers of immunoglobulin molecules.

[0101] As used herein, the term "antigen-binding domain" refers to an antibody fragment consisting of an antibody moiety containing one or more CDRs, or any other antibody fragment that binds an antigen but does not contain the complete structure of a native antibody. Examples of antigen-binding domains include, but are not limited to, variable domains, variable regions, single variable domains (VHHs), nanobodies, domain antibodies, bispecific antibodies, Fab, Fab', F(ab')2, Fv fragments, single-chain Fv fragments (scFv), disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bispecific antibodies (ds bispecific antibodies), multispecific antibodies, camel-derived single-domain antibodies, and bivalent domain antibodies. An antigen-binding domain can bind to the same antigen that binds to the parent antibody. A more detailed form of the antigen-binding domain is described in Spiess et al., (2015) Molecular Immunology 67: 95-106 and Brinkman et al., mAbs, 9(2), pp. 182–212 (2017), the full contents of which are incorporated herein by reference.

[0102] As used herein, the term "complementarity-determining region" or "CDR" refers to the amino acid sequence within the variable domain of an antibody that confers antigen specificity and binding affinity. For example, typically, three CDRs exist within each heavy chain variable domain (e.g., HCDR1, HCDR2, and HCDR3), and three CDRs also exist within each light chain variable domain (LCDR1, LCDR2, and LCDR3). The extent of CDRs and frame regions can be precisely identified using methods known in the art, such as those defined by Kabat, Dr. Martin's website, Chothia, AbM, EU, and contact, all of which are well-known in the art. See, for example, Kabat, EA et al. (1991). Sequences of Proteins of Immunological Interest, 5th Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Martin A. “Antibody bioinformatics website of Dr. Andrew Martin's lab at UCL”, last updated July 31, 2018; Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948; Edelman et al., Proc Natl Acad Sci US A. May 1969; 63(1):78-85; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs. For example, correspondences or comparisons between numberings according to different definitions can be found at http: / / www.imgt.org / (see also Giudicelli V et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. (1997) 25:206–11; Lefranc MP et al. Unique database numbering system for immunogenetic analysis. Immunol Today (1997) 18:509; and Lefranc MP et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. DevComp Immunol. (2003) 27:55–77). In some cases, a scheme is specified for identifying one or more specific CDRs, such as CDRs defined by IMGT, Kabat, AbM, Chothia, or Contact methods. One or more positions of the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by the Kabat number.See, for example, Deschacht et al., 2010. J Immunol 184: 5696-704, which discloses exemplary numbering of the VHH domain according to Kabat. In other cases, specific amino acid sequences of the CDR are given. It should be noted that the CDR region can also be defined by combinations of various numbering systems, such as combinations of the Kabat and Chothia numbering systems, combinations of the Kabat and AbM numbering systems, or combinations of the Kabat and IMGT numbering systems. Therefore, terms such as “CDR shown in a particular VH, VL, or VHH” include, but are not limited to, any CDR defined by the exemplary CDR numbering systems described above. Once a variable region (e.g., the VHH domain, VH, or VL domain) is given, those skilled in the art will understand that the CDR within that region can be defined by different numbering systems or combinations thereof.

[0103] As used in this article, the term "immune cells" refers to cells that are part of the immune system and assist the body in fighting infections and other diseases. Immune cells include natural killer cells, homeostatic NK cells, NK T cells, and any type of T cell (e.g., regulatory T cells, CD4+). + T cells, CD8 + T cells (or γδ T cells), B cells, monocytes, granulocytes, myeloid cells, neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, and / or stem cells (e.g., mesenchymal stem cells (MSCs) or induced pluripotent stem cells (iPSCs)). Effector immune cells such as immune CD4+ cells... + and CD8 + In T cells, CAR triggering activates typical effector functions, such as the secretion of cleaving compounds and cytokines, which ultimately leads to the killing of corresponding target cells. Adoptive transfer of CAR-engineered immune cells is currently considered a promising therapeutic option for treating malignant diseases, infectious diseases, or autoimmune diseases that are incurable by other methods.

[0104] As used herein, the term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. This article describes examples of various cancers, including but not limited to stomach cancer, pancreatic cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, and lung cancer. The terms "tumor" and "cancer" are used interchangeably in this article.

[0105] The terms “patient,” “subject,” “individual,” etc., are used interchangeably herein and refer to any animal suitable for the methods described herein. In some non-limiting embodiments, the patient, subject, or individual is a human.

[0106] DAP10 and its interaction with NKG2D

[0107] The 10 kDa DNAX activating protein (DAP10) is an adaptor molecule that associates with the cytotoxic receptor Natural Killer Group 2 Member D (NKG2D) on the cell surface. NKG2D is an activating immune receptor that regulates both innate and adaptive immune responses. NKG2D is abundant in all NK cells and CD8 cells. + T cells, γδ T cell subsets, and some autoreactive CD4+ cells + On T cells, NKG2D is a key trigger for some forms of NK cell-mediated cytotoxicity (Daniel B et al. (2003) Nat Immunol. 4(6):557-64). The molecular structure of NKG2D allows it to bind to several structurally different MHC-I class-like ligands. A common feature of NKG2D ligands is that their expression is generally low under homeostatic conditions, but higher under tumor or stress conditions. In humans, NKG2D ligands are six members of the MICA, MICB, and ULBP families.

[0108] NKG2D lacks signal transduction motifs in its cytoplasmic domain. Signal transduction via NKG2D depends on its association with DAP10, a transmembrane adaptor molecule containing the sequence “YINM” that transduces signals by recruiting phosphatidylinositol 3-kinase and Grb2 (growth factor receptor-binding protein 2) (Pedro R et al. (2009) J Biol Chem. 284(24):16463-16472). The DAP10 adaptor molecule can promote and stabilize the surface membrane expression of NKG2D (Wu, J. et al., (1999)). Therefore, this paper proposes a method that can regulate NKG2D and / or DAP10 expression and / or related signal transduction pathways, which has therapeutic significance.

[0109] The human NKG2D receptor assembles with the DAP10 signaling dimer, with one NKG2D homodimer pairing with one DAP10 dimer, forming two salt bridges between conserved transmembrane (TM) arginine residues (Garrity, D. et al. (2005) PNAS USA 102(21): 7641-7646). The DAP10 dimer contains a pair of aspartic acid residues near the center of the transmembrane (TM) domain, and these residues interact with conserved arginine residues in the NKG2D TM sequence, thereby assembling with the DAP10 dimer. Therefore, the NKG2D homodimer associates with the DAP10 linker molecule in its transmembrane domain to form a hexamer structure, which can initiate the signal transduction cascade.

[0110] As described above, the DAP10 dimer is a disulfide-linked homodimer. An exemplary amino acid sequence of the wild-type human DAP10 polypeptide is shown in SEQ ID No: 20 below. The cytoplasmic domain of DAP10 contains a tyrosine-based motif (YINM), as shown at residues 86-89 of SEQ ID No: 20. This YINM motif is similar to the motif in CD28, which provides co-stimulatory signaling in T cells together with the TCR / CD3 complex based on the immune receptor tyrosine activation motif (ITAM). DAP10 further contains a ubiquitination site containing lysine at amino acid 84 of the DAP10 protein sequence (SEQ ID No: 20). NKG2D ligand stimulation on NK cells leads to DAP10 ubiquitination, which is essential for the endocytosis and degradation of the NKG2D-DAP10 complex (see, for example, Molfetta, R. et al. (2014) Eur. J. Immunol. 44, 2761-2770).

[0111] Linker peptides containing the DAP10™ domain

[0112] In one aspect, this disclosure provides a recombinant polypeptide (also known as an adaptor polypeptide) comprising: (i) a transmembrane (TM) domain derived from DAP10 or a functional variant thereof capable of interacting with NKG2D; and optionally one or more of the following: (ii) a signal peptide; (iii) a hinge region; and (iv) an intracellular region. The intracellular region may contain one or more co-stimulatory signal transduction domains (e.g., derived from 4-1BB, DAP10, OX40, 2B4, ICOS, CD28) and / or primary signal transduction domains. The recombinant polypeptide disclosed herein can be used as a CAR adaptor to modulate and / or silence signal transduction via one or more receptors associated with it.

[0113] In some embodiments, the adaptor polypeptide comprises the full-length amino acid sequence of human DAP10, for example, the amino acid sequence shown in SEQ ID No: 20. The human DAP10 protein can be divided into four regions from the N-terminus to the C-terminus: a signal peptide, a hinge region, a transmembrane region, and an intracellular region. The definition of the different regions in human DAP10 is well-established in the art. Of these four regions, the transmembrane (TM) region is crucial for the interaction between DAP10 and NKG2D and is an integral part of the adaptor polypeptide. The TM region is preferably derived from or derived from human DAP10, while the signal peptide, hinge region, and intracellular region can be derived from various molecules other than DAP10, provided they can perform the desired function. The adaptor polypeptide may comprise all four regions of human DAP10. In some embodiments, the adaptor polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 97%, or 99% identity with the amino acid sequence SEQ ID No: 20. For example, homologs of the human DAP10 amino acid sequence may include amino acid substitutions at positions corresponding to K84 and / or Y86.

[0114] In some embodiments, the adaptor peptide may comprise a functional portion of the full-length DAP10 amino acid sequence. For example, the adaptor peptide may comprise only the transmembrane (TM) region derived from human DAP10, without the intracellular region, signal peptide, and hinge region. In some other embodiments, DAP10 may comprise both the transmembrane (TM) region and the intracellular region derived from human DAP10, without the signal peptide and hinge region. Specifically, the TM region derived from human DAP10 may comprise the amino acid sequence shown in SEQ ID No: 9 or a functional variant thereof capable of interacting with NKG2D. The amino acid sequence of this functional variant may have at least 85%, 90%, 95%, 97%, or 99% identity with SEQ ID No: 9. The intracellular region may comprise the intracellular domain (ICD) of human DAP10 or a functional variant thereof. This intracellular region may comprise the amino acid sequence shown in SEQ ID No: 10 or an amino acid sequence having at least 85%, 90%, 95%, 97%, or 99% identity with SEQ ID No: 10. In some embodiments, the intracellular region further comprises one or more co-stimulatory signaling domains derived from, for example, 4-1BB, OX40, 2B4, ICOS, or CD28. In some embodiments, the intracellular region further comprises primary signaling domains, such as the CD3ζ signaling domain.

[0115] In some embodiments, the adaptor polypeptide further comprises a signal peptide at its N-terminus. This signal peptide may be derived from DAP10, T cell surface-expressed receptors (such as CD8, CD28, and TCR), NK cell surface-expressed receptors (such as 2B4, CD16, NKP30, NKP44, and NKP46), or IgG. In some embodiments, the signal peptide may comprise a DAP10 signal peptide containing the amino acid sequence shown in SEQ ID No: 6 or an amino acid sequence having at least 85%, 90%, or 95% identity with SEQ ID No: 6. In some other embodiments, the signal peptide may comprise a CD8 signal peptide containing the amino acid sequence shown in SEQ ID No: 19 or an amino acid sequence having at least 85%, 90%, or 95% identity with SEQ ID No: 19.

[0116] In some embodiments, the adaptor peptide further includes a hinge region at the N-terminus of the TM region. For example, this hinge region may be located between the signal peptide and the TM region. The hinge region can be any linker that provides the required flexibility to the adaptor peptide. The hinge region may be derived from human DAP10, other molecules such as CD8, CD28, or other immune cell surface expressed receptors. In some embodiments, the hinge region comprises an amino acid sequence as shown in SEQ ID No: 8 or an amino acid sequence having at least 85%, 90%, or 95% identity with SEQ ID No: 8.

[0117] Specifically, the adaptor polypeptide may include a signal peptide operatively linked to a TM region and a TM region operatively linked to an intracellular region from its N-terminus to its C-terminus (SP-TM-intracellular). In some embodiments, the adaptor polypeptide further includes a hinge region at its N-terminus. Specifically, the adaptor polypeptide may include a hinge region operatively linked to a TM region and a TM region operatively linked to an intracellular region from its N-terminus to its C-terminus (hinge-TM-intracellular). In some embodiments, the adaptor polypeptide includes a signal peptide and a hinge region at its N-terminus. Specifically, the adaptor polypeptide includes a signal peptide operatively linked to a hinge region, a hinge region operatively linked to a TM region, and a TM region operatively linked to an intracellular region from its N-terminus to its C-terminus (SP-hinge-TM-intracellular).

[0118] In some specific embodiments, the linker polypeptide comprises a transmembrane region and an intracellular region, wherein the transmembrane region comprises an amino acid sequence SEQ ID No: 9 or an amino acid sequence substantially similar to (e.g., at least 85%, 90%, or 95% identity) of SEQ ID No: 9, and the intracellular region comprises an amino acid sequence SEQ ID No: 10 or an amino acid sequence substantially similar to (e.g., at least 85%, 90%, or 95% identity) of SEQ ID No: 10. In some specific embodiments, the linker polypeptide comprises a transmembrane region, an intracellular region, a signal peptide, and optionally a hinge region, wherein the transmembrane region comprises an amino acid sequence of SEQ ID No: 9 or substantially similar to (e.g., at least 85%, 90%, or 95% identity) with SEQ ID No: 9; the intracellular region comprises an amino acid sequence of SEQ ID No: 10 or substantially similar to (e.g., at least 85%, 90%, or 95% identity) with SEQ ID No: 10; the signal peptide comprises an amino acid sequence of SEQ ID No: 6 or 19 or substantially similar to (e.g., at least 85%, 90%, or 95% identity) with SEQ ID No: 6 or 19; and the hinge region comprises an amino acid sequence of SEQ ID No: 7 or 8 or substantially similar to (e.g., at least 85%, 90%, or 95% identity) with SEQ ID No: 7 or 8. In some specific embodiments, the linker polypeptide includes a signal peptide, a hinge region, a transmembrane region, and an intracellular region, wherein the signal peptide includes SEQ ID No: 6 or 19, the hinge region includes SEQ ID No: 7 or 8, the transmembrane region includes SEQ ID No: 9, and the intracellular region includes SEQ ID No: 10.

[0119] In some embodiments, the adaptor peptide may contain an affinity tag for purification, such as an HA tag, in its extracellular region. In some embodiments, the affinity tag is inserted between the signal peptide and the hinge region of the adaptor peptide. For example, the adaptor peptide comprises a signal peptide, an HA tag, a hinge region, a transmembrane domain, and an intracellular region from its N-terminus to its C-terminus, wherein the signal peptide comprises SEQ ID No: 6 or 19, the HA tag comprises SEQ ID No: 7, the hinge region comprises SEQ ID No: 8, the transmembrane domain comprises SEQ ID No: 9, and the intracellular region comprises SEQ ID No: 10. Those skilled in the art will understand that the affinity tag can be inserted at other locations in the extracellular region of the adaptor peptide, for example, at the N-terminus of the signal peptide or at the C-terminus of the hinge region. In some embodiments, the HA tag replaces the hinge region of the linker polypeptide. For example, the linker polypeptide includes a signal peptide, an HA tag, a transmembrane region, and an intracellular region from the N-terminus to the C-terminus, wherein the signal peptide includes SEQ ID No: 6, the HA tag includes SEQ ID No: 7, the transmembrane region includes SEQ ID No: 9, and the intracellular region includes SEQ ID No: 10.

[0120] In some implementations, the intracellular region includes one or more co-stimulatory domains selected from or derived from, for example, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal transduction lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, etc. The co-stimulatory domains described herein may be derived from the group consisting of: CD244 (2B4), 2B4 ITSM2, CD137 (4-1BB), CD28, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, B7-H3, CEACAM1, CRTAM, CD2, CD3C, CD4, CD7, CD8a, CD8p, CD1 1a, CD11b, CD11c, CD11d, IL2Rβ, IL2γ, IL7Ra, IL4R, IL7R, IL15R, IL21R, CD18, CD19, CD19a, CD27, CD29, CD30, CD40, CDS, CD49a, CD49D, CD49f, CD54 (ICAM), CD69, CD70, CD80, CD83, CD84, CD86, CD96 (Tactile), CD100(SEMA4D), CD103, CD134 (OX40), CD152 (CTLA-4), CD160 (BY55), CD162 (SELPLG), CD270(HVEM), CD226 (DNAM1), CD229 (Ly9), CD278 (ICOS), ICAM-1, LFA-1 (CD11a / CD18), FcR, FcγRI, FcγRII, Fc7R.HI, LAT, NKG2C, SLP76, TRIM, ZAP70, GITR, BAFFR, LTBR, ​​LAT, GADS, LIGHT, HVEM (LIGHTR), KIRDS2, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, NKG2C, NKG2D, IA4, VLA-1, VLA-6, SLAM (SLAMF1, CD150, IPO-3), SLAMF4, SLAMF6 (NTB-A, Lyl08), SLAMF7, SLAMF8 (BLAME), SLP-76, PAG / Cbp, NKp80 (KLRF1), NKp44, NKp30, NKp46, BTLA, JAML, CD150, PSGL1, TSLP, TNFR2, TRANCE / RANKL, or combinations thereof. In some embodiments, in addition to the DAP10 intracellular domain, the intracellular region also contains a subset selected from 4-IBB, 2B4 (e.g., 2B4). The co-stimulatory domains include ITSM2), ICOS, CD28, OX40, and CD27.

[0121] The linker polypeptide may include one, two, three, or more co-stimulatory domains. When more than one co-stimulatory domain is included, the co-stimulatory domains may have the same or different amino acid sequences. In some embodiments, the intracellular region includes a 4-1BB co-stimulatory domain. The 4-1BB co-stimulatory domain may include the amino acid sequence shown in SEQ ID No: 4 or have at least one, at least two, or at least three or more modified amino acid sequences compared to SEQ ID No: 4. In some embodiments, the 4-1BB co-stimulatory domain is substantially similar to the 4-1BB co-stimulatory domain including SEQ ID No: 4 (e.g., at least 85%, 90%, or 95% identity). In some embodiments, the intracellular region includes a 2B4 co-stimulatory domain. The 2B4 co-stimulatory domain may include the amino acid sequence shown in SEQ ID No: 11 or have at least one, at least two, or at least three or more modified amino acid sequences compared to SEQ ID No: 11. In some embodiments, the 2B4 co-stimulatory domain is substantially similar to the 2B4 co-stimulatory domain comprising SEQ ID No: 11 (e.g., at least 85%, 90%, or 95% identity). In some embodiments, the 2B4 co-stimulatory domain is 2B4. The ITSM2 co-stimulatory domain is composed of the first two ITSM motifs. This 2B4 The ITSM2 co-stimulatory domain may contain an amino acid sequence as shown in SEQ ID No: 12 or an amino acid sequence having at least one, at least two, or at least three or more modifications compared to SEQ ID No: 12. In some embodiments, the 2B4 The ITSM2 co-stimulatory domain is substantially similar to SEQ ID No: 12 (e.g., at least 85%, 90%, or 95% identity). In some embodiments, the intracellular region comprises a CD28 co-stimulatory domain. This CD28 co-stimulatory domain may comprise the amino acid sequence shown in SEQ ID No: 21 or an amino acid sequence having at least one, at least two, or at least three or more modified amino acid sequences compared to SEQ ID No: 21. In some embodiments, the CD28 co-stimulatory domain is substantially similar to SEQ ID No: 21 (e.g., at least 85%, 90%, or 95% identity). In some embodiments, the intracellular region comprises a combination of a 2B4 co-stimulatory domain and a 4-1BB co-stimulatory domain.

[0122] In addition to one or more co-stimulatory signaling domains, the adaptor peptide may further comprise intracellular primary signaling domains. In some embodiments, the adaptor peptide comprises one or more co-stimulatory signaling domains and one or more primary signaling domains. In some other embodiments, the adaptor peptide comprises only one or more primary signaling domains without co-stimulatory signaling domains. Intracellular primary signaling domains may increase the proliferation, persistence, and / or cytotoxic activity of host cells (e.g., NK cells, NKT cells, γδ cells, etc.) carrying peptides disclosed herein. For example, in some embodiments, one or more intracellular primary signaling domains comprise CD3ζ, repeating (e.g., 2-5) DAP10 YINM motifs, and signaling domains derived from LFA-1, DAP12, FcRγ, FcRP, CD3γ, CD36, CD3ε, CD79a, CD79b, CD5, CD22, FcεRI, CD66d, etc. Intracellular primary signaling domains may specifically be selected from the group consisting of CD3ζ, DAP12, LFA-1, and CD3t, or combinations thereof. Intracellular regions may include multiple (e.g., 2, 3, 4, or more) intracellular primary signaling domains. In cases where more than one domain is included, these intracellular primary signaling domains may contain different amino acid sequences.

[0123] In some embodiments, the intracellular primary signaling domain comprises a CD3ζ signaling domain. This CD3ζ signaling domain may comprise the amino acid sequence shown in SEQ ID No: 5 or an amino acid sequence having at least one, at least two, or at least three or more modifications compared to SEQ ID No: 5. In some embodiments, the CD3ζ signaling domain is substantially similar to the amino acid sequence SEQ ID No: 5 (e.g., at least 85%, 90%, or 95% identity). In some embodiments, the intracellular region comprises a 4-1BB co-stimulatory domain and a CD3ζ intracellular signaling domain, or comprises a 2B4 co-stimulatory domain and a CD3ζ intracellular signaling domain.

[0124] In some embodiments, the adaptor peptide comprises or consists of a DAP10™ region and a 2B4 co-stimulatory domain. In some embodiments, the adaptor peptide comprises or consists of a DAP10™ region and a 4-1BB co-stimulatory domain. In some embodiments, the adaptor peptide comprises or consists of a DAP10™ region and a CD28 co-stimulatory domain. In some embodiments, the adaptor peptide comprises a DAP10™ region and a 2B4... The ITSM (the first two ITSM motifs of 2B4) co-stimulatory domains or are composed thereof. In some embodiments, the adaptor peptide comprises or is composed of the DAP10™ region, the 2B4 co-stimulatory domain, and the 4-1BB co-stimulatory domain. In some embodiments, the adaptor peptide comprises or is composed of the DAP10™ region, the 2B4 co-stimulatory domain, and the CD3ζ intracellular signal transduction domain. In some embodiments, the adaptor peptide comprises or is composed of the DAP10™ region, the 4-1BB co-stimulatory domain, and the CD3ζ intracellular signal transduction domain. The adaptor peptides disclosed above may further comprise the DAP10 intracellular domain.

[0125] In some embodiments, the linker polypeptide comprises, from the N-terminus to the C-terminus: (a) The signal peptide of human DAP10, the hinge region of human DAP10, the TM region of human DAP10, the intracellular region of human DAP10, and the 2B4 co-stimulatory domain (as shown in structures 2, 9-12, 17, and 27). (b) The signal peptide, hinge region, TM region, intracellular region and 4-1BB co-stimulatory domain of human DAP10 (as shown in structures 3, 18 and 19). (c) Signal peptide of human DAP10, hinge region of human DAP10, TM region of human DAP10, intracellular region of human DAP10, 2B4 costimulatory domain and 4-1BB costimulatory domain (as shown in structures 4 and 20). (d) The signal peptide of human DAP10, the hinge region of human DAP10, the TM region of human DAP10, the intracellular region of human DAP10, the 2B4 co-stimulatory domain and the CD3ζ intracellular signal transduction domain (as shown in structures 5 and 21). (e) The signal peptide of human DAP10, the hinge region of human DAP10, the TM region of human DAP10, the intracellular region of human DAP10, the 4-1BB costimulatory domain and the CD3ζ intracellular signal transduction domain (as shown in structures 6 and 22). (f) The signal peptide of human DAP10, the hinge region of human DAP10, the TM region of human DAP10, the intracellular region of human DAP10, and the 2B4·ITSM2 co-stimulatory domain (as shown in Structure 7). (g) The signal peptide of human DAP10, the hinge region of human DAP10, the TM region of human DAP10, the intracellular region of human DAP10, and the CD28 co-stimulatory domain (as shown in Structure 8). (h) CD8 signal peptide, hinge region of human DAP10, TM region of human DAP10, intracellular region of human DAP10 and 2B4 co-stimulatory domain (as shown in structure 24). (i) The CD8 signal peptide, the TM region of human DAP10, the intracellular region of human DAP10, and the 2B4 co-stimulatory domain (as shown in Structure 25); or (j) The signal peptide of CD8, the hinge region of human DAP10, the TM region of human DAP10, the intracellular region of human DAP10, and the 2B4ITSM2 co-stimulatory domain (as shown in Structure 26). Optionally, an affinity tag, such as an HA tag, can be inserted between the signal peptide and the hinge region.

[0126] In some embodiments, the linker polypeptide comprises, from the N-terminus to the C-terminus, a CD8 signal peptide, an HA tag, a TM region of human DAP10, an intracellular region of human DAP10, and a 2B4 co-stimulatory domain (as shown in Structure 25).

[0127] The co-stimulatory domains and CD3ζ intracellular signal transduction domains described in the above structure can be replaced with various other co-stimulatory domains and intracellular signal transduction domains commonly used in CAR construction.

[0128] In some implementations, the adaptor peptide may contain one or more mutations, such as one or more mutations (e.g., one or more substitutions, additions, or deletions) in the DAP10 region and / or one or more co-stimulatory domains and / or one or more primary signaling domains.

[0129] Adaptor peptides can be engineered to possess regulated / silencing properties (e.g., through one or more mutations) and / or signaling-enhancing properties (e.g., through C-terminal fusion) and expressed in host cells to promote a favorable balance of signaling pathways during receptor-target binding (e.g., NKG2D binding to an extracellular target ligand), which may help address issues of low or absent target expression (i.e., antigen escape). Therefore, the adaptor peptides disclosed herein offer improved functional properties, including but not limited to alterations (e.g., enhancement) in cell lysis, proliferation, survival, and / or co-stimulatory properties induced upon binding to receptor ligands paired with DAP10 (e.g., widely expressed ligands of NKG2D). The precise composition of adaptor peptides can be engineered based on a given disease indication and, in some examples, on the specificity and signaling components of other receptors present on the same cells. As an illustrative example, immunosuppressive signals within the tumor microenvironment (TME) can suppress anti-tumor immune cell responses via inhibitory receptors, and the conversion of such inhibitory output into immunostimulatory output by using a peptide containing DAP10 is also within the scope of this disclosure.

[0130] In some embodiments, the adaptor peptide can at least partially function to stabilize cell surface receptors (e.g., NKG2D) associated with it. As disclosed herein, "stabilizing" a cell surface receptor means reducing the rate at which said cell surface receptor is endocytotic or otherwise removed from the cell surface, determined by comparison with the rate at which said cell surface receptor is otherwise removed in the absence of the DAP10-containing adaptor peptide described herein. The receptor stabilization scope covered herein includes positive feedback mechanisms through which adaptor peptide signaling results in increased cell surface expression of NKG2D associated with endogenous DAP10 and the adaptor peptide containing said DAP10.

[0131] Engineered receptor peptides

[0132] One aspect of this disclosure provides a combination of the linker peptide described above with an engineered receptor peptide. The engineered receptor peptide typically includes an extracellular antigen-binding domain and optionally an intracellular signaling domain. Exemplary engineered receptors include, but are not limited to, chimeric antigen receptors (CARs), engineered NK cell receptors (NKCRs), engineered T cell receptors (TCRs), and T cell antigen-coupled device (TAC) receptors. The engineered receptor may include an extracellular antigen-binding domain that specifically binds to an antigen (e.g., GPC3, CD19, or CD33), a transmembrane domain, and an intracellular signaling domain. The intracellular signaling domain may include a primary intracellular signaling domain and / or a co-stimulatory signaling domain. The engineered receptor peptide may be encoded by a heteropolynucleotide operatively linked to a promoter (e.g., a constitutive or inducible promoter).

[0133] Engineered receptor peptides may include one or more specific binding domains that target at least one tumor antigen, as well as one or more intracellular effector domains, such as one or more primary intracellular signaling domains and / or co-stimulatory signaling domains.

[0134] In some embodiments, this disclosure provides a chimeric antigen receptor (CAR) that specifically binds to one or more antigens (e.g., GPC3, CD19, or CD33). The CAR may comprise: (a) an extracellular antigen-binding domain that binds to or is antigen-specific; (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0135] Antigen specificity can target any suitable antigen or epitope, such as exogenous antigens, endogenous antigens, autoantigens, neoantigens, viral antigens, or tumor antigens. Exogenous antigens enter the body through inhalation, ingestion, or injection and can be presented by antigen-presenting cells (APCs) and form MHC class II complexes via endocytosis or phagocytosis. Endogenous antigens are produced within normal cells due to cellular metabolism, intracellular viral or bacterial infection, and can form MHC class I complexes. Autoantigens (e.g., peptides, DNA, or RNA) are recognized by the immune system of patients with autoimmune diseases, whereas under normal circumstances, such antigens should not be targets of the immune system. Neoantigens are completely absent from the normal body but are produced due to a disease such as a tumor or cancer. In some embodiments, the antigen is associated with a disease (e.g., tumor or cancer, autoimmune disease, infectious and parasitic disease, cardiovascular disease, neuropathy, neuropsychiatric condition, injury, inflammation, coagulation disorder). In some embodiments, the antigen is associated with the immune system (e.g., immune cells such as B cells, T cells, NK cells, macrophages, etc.).

[0136] In some implementations, an antigen is specific to an immune-associated antigen or its epitope. Examples of immune-associated antigens include natural killer cell (NK cell)-specific receptor molecules and / or T cell-specific receptor molecules. T cell-specific receptor molecules allow T cells to bind to epitopes / antigens presented by another cell type called antigen-presenting cells or APCs, and allow these T cells to be activated and respond to them if additional signals are present. T cell-specific receptor molecules can be TCR, CD3, CD28, CD134 (also known as OX40), 4-1BB, CD5, and CD95 (also known as Fas receptors). Examples of NK cell-specific receptor molecules include the low-affinity Fc receptor CD16, as well as NKG2D and CD2.

[0137] In some embodiments, an antigen is specifically targeted at a tumor-associated antigen or its epitope. The term "tumor-associated antigen" refers to an antigen presented on or potentially presented on the surface of tumor cells and located on or within tumor cells. In some embodiments, the tumor-associated antigen may be presented only by tumor cells and not by normal cells, i.e., non-tumor cells. In some other embodiments, the tumor-associated antigen may be expressed only on tumor cells or may represent a tumor-specific mutation compared to non-tumor cells. In some other embodiments, the tumor-associated antigen may be present in both tumor cells and non-tumor cells, but it is overexpressed on tumor cells compared to non-tumor cells, or it may be more readily bound to antibodies in tumor cells because the structure of tumor tissue is less compact compared to non-tumor tissue. In some embodiments, the tumor-associated antigen is located on the vascular system of the tumor. Illustrative examples of tumor-associated surface antigens are described in PCT / CN2018 / 106766 (WO 2019 / 057122) and are incorporated herein by reference.

[0138] In some embodiments, an antigen is specifically targeted against an antigen or epitope selected from the group consisting of: GPC3, PSMA, CD3, CD19, CD20, 4-1BB (CD137), OX40 (CD134), CD16, CD47, CD22, CD33, CD38, CD123, CD133, CEA, cdH3, EpCAM, epidermal growth factor receptor (EGFR), EGFRvIII (EGFR mutations), HER2, HER3, DLL3, BCMA, sialylated Lea, 5T4, ROR1, melanoma-associated chondroitin sulfate proteoglycan, mesothelin, folate receptor 1, VEGF receptor, EpCAM, HER2 / neu, HER3 / neu, G250, CEA, MAGE, proteoglycan, VEGF, FGFR, αVβ3-integrin, HLA, HLA-DR, ASC, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD11, CD13, CD14, CD2 1. CD23, CD24, CD28, CD30, CD37, CD40, CD41, CD44, CD52, CD64, c-erb-2, CALLA, MHCII, CD44v3, CD44v6, p97, gangliosides GM1, GM2, GM3, GD1a, GD1b, GD2, GD3, GT1b, GT3, GQ1, NY-ESO-1, NFX2, SSX2, SSX4, Trp2, gp100, tyrosinase, Muc-1, telomerase, susceptin, G250, p53, CA125 MUC, Wu antigen, Lewis Y antigen, HSP-27, HSP-70, HSP-72, HSP-90, Pgp, MCSP, EpHA2, cell surface targets GC182, GT468 or GT512, IL-17, IL-20, IL-13 and IL-4.

[0139] The extracellular antigen-binding domain of a CAR can exist in a variety of forms, including, for example, a single-domain antibody (sdAb) or VHH domain, a single-chain variable fragment (scFv), Fab, Fab', F(ab)'2, F(ab)'3, Fv, bisscFv, (scFv)2, microantibodies, bispecific antibodies, triabody antibodies, tetrabody antibodies, intracellular antibodies, disulfide-stabilized Fv proteins (dsFv), unibody antibodies, nanobodies, affinity molecules, DARPin, monobody antibodies, adnectin, alphabody antibodies, or designed conjugates. The antigen-binding domain can be a single-domain antibody (e.g., a VHH domain), such as a camel-derived, shark-derived, chimeric, human, or humanized single-domain antibody (e.g., a VHH domain), or it can be a scFv containing VH and VL regions derived from a parent antibody. The CAR disclosed herein may contain an antigen-binding domain containing scFv. The CAR disclosed herein may contain an antigen-binding domain comprising one or more VHHs. These VHHs may be fused directly to each other via peptide bonds or via peptide linkers.

[0140] CARs can be monospecific or multispecific (e.g., bispecific), monovalent or multivalent (e.g., bivalent), tandem CARs, or split CARs. In some embodiments, the CAR is a multispecific (e.g., bispecific) CAR that includes one or more antigen-binding domains with different antigen-binding specificities. Depending on the desired antigen to be targeted, the CAR disclosed herein can be engineered to include suitable antigen-binding domains that specifically target the desired antigen.

[0141] The CAR can be a monospecific CAR or a multivalent CAR. In some embodiments, the CAR can be a mono-GPC3-specific CAR containing an extracellular antigen-binding domain of an anti-GPC3 scFv targeting the GPC3 antigen. The mono-GPC3-specific CAR can contain an antigen-binding domain containing the amino acid sequence of SEQ ID No: 1. In some embodiments, the CAR can be a mono-CD19-specific CAR containing an extracellular antigen-binding domain of an anti-CD19 scFv targeting the CD19 antigen. The mono-CD19-specific CAR can contain an antigen-binding domain containing the amino acid sequence of SEQ ID No: 13. In some embodiments, the CAR can be a mono-CD33-specific CAR containing an extracellular antigen-binding domain of an anti-CD33 scFv targeting the CD33 antigen. The mono-CD33-specific CAR can contain an antigen-binding domain containing the amino acid sequence of SEQ ID No: 14.

[0142] The antigen-binding domain of a CAR can be derived from a parental antibody. The parental antibody can be any type of antibody, including, for example, fully human antibodies, humanized antibodies, or animal antibodies (e.g., mouse, rat, rabbit, sheep, bovine, dog antibodies, etc.). The parental antibody can be a monoclonal antibody or a polyclonal antibody.

[0143] In some embodiments, the parent antibody is a monoclonal antibody. Monoclonal antibodies can be produced by various methods known in the art, such as hybridoma technology, recombinant methods, phage display, or any combination thereof. Exemplary methods for producing antibodies are described in WO 2019 / 057122 and are incorporated herein by reference.

[0144] The parent antibody described herein can be further modified, for example, to transplant the CDR sequence onto different frames or scaffolds, to replace one or more amino acid residues in one or more frame regions, to replace one or more residues in one or more CDR regions to achieve affinity maturation, and so on. These can be achieved by those skilled in the art using conventional techniques.

[0145] Parental antibodies can also be therapeutic antibodies known in the art, such as FDA-approved antibodies for therapeutic or diagnostic purposes, antibodies undergoing clinical trials for the treatment of a condition, or antibodies in the development stage. Polynucleotide sequences and protein sequences of known antibody variable regions are available from public databases such as www.ncbi.nlm.nihgov / entrez- / query.fcgi; www.atcc.org / phage / hdb.html; www.sciquest.com / ; www.abcam.com / ; www.antibodyresource.com / onlinecomp.html. Examples of therapeutic antibodies include, but are not limited to, those disclosed in WO 2019 / 057122, which are incorporated herein by reference in their entirety. In some embodiments, the parental antibody used to derive the antigen-binding domain of a CAR as disclosed herein is an anti-GPC3 antibody, an anti-CD33 antibody, or an anti-CD19 antibody.

[0146] The CAR of this application may further include a transmembrane domain that can be directly or indirectly fused to an extracellular antigen-binding region. The transmembrane domain may be of natural or synthetic origin. Unlike the transmembrane region of a linker polypeptide preferably derived from human DAP10 to interact with NKG2D, the “transmembrane domain” of the CAR polypeptide can be any protein structure that is thermodynamically stable in a cell membrane, such as a eukaryotic cell membrane. The transmembrane domain compatible with the CAR described herein can be derived from naturally occurring proteins. Alternatively, it can be a synthetic, non-naturally occurring protein segment, such as a hydrophobic protein segment that is thermodynamically stable in a cell membrane.

[0147] The transmembrane domains of CARs disclosed herein may include transmembrane domains selected from the following: 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, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), CD160, CD19, IL-2Rβ, IL-2Rγ, IL-7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11 d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100(SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. In some embodiments, the transmembrane domain is derived from molecules selected from the group consisting of: CD8, CD4, CD28, CD137, CD80, CD86, CD152, and PD1.

[0148] For example, the transmembrane domain may be derived from CD8 or CD28. In some embodiments, the transmembrane domain is a transmembrane domain of CD8. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID No: 3 or 22. The transmembrane domain may comprise an amino acid sequence having at least 85%, 90%, or 95% identity with the amino acid sequence shown in SEQ ID No: 3 or 22.

[0149] The transmembrane domain used in the CAR described herein may also comprise at least a portion of a synthetic, non-naturally occurring protein segment. The transmembrane domain may be a synthetic, non-naturally occurring α-helix or β-sheet. In some embodiments, the protein segment comprises at least about 20 amino acids, for example, at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids. Examples of synthetic transmembrane domains are known in the art, for example, those disclosed in U.S. Patent No. 7,052,906 B1 and PCT Publication No. WO 2000 / 032776 A2, the relevant disclosures of which are incorporated herein by reference.

[0150] In some embodiments, the intracellular signal transduction region of the CAR peptide includes one or more co-stimulatory domains selected from or derived from, for example, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal transduction lymphocyte activating molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, etc. The costimulatory domains in this article may originate from the following groups: CD244 (2B4), 2B4 ITSM2, CD137 (4-1BB), CD28, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, B7-H3, CEACAM1, CRTAM, CD2, CD3C, CD4, CD7, CD8a, CD8p, CD11a, CD11b, CD11c, CD11d, IL2Rβ, IL2γ, IL7Ra, IL4R, IL7R, IL15R, IL21R, CD18, CD19, CD19a, CD27, CD29, CD30, CD40, CDS, CD49a, CD49D, CD49f, CD54 (ICAM), CD69, CD70, CD80, CD83, CD84, CD86, CD96 (Tactile), CD100(SEMA4D), CD103, CD134 (OX40), CD152 (CTLA-4), CD160 (BY55), CD162 (SELPLG), CD270(HVEM), CD226 (DNAM1), CD229 (Ly9), CD278 (ICOS), ICAM-1, LFA-1 (CD11a / CD18), FcR, FcγRI, FcγRII, Fc7R.HI, LAT, NKG2C, SLP76, TRIM, ZAP70, GITR, BAFFR, LTBR, ​​LAT, GADS, LIGHT, HVEM (LIGHTR), KIRDS2, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, NKG2C, NKG2D, IA4, VLA-1, VLA-6, SLAM (SLAMF1, CD150, IPO-3), SLAMF4, SLAMF6 (NTB-A, Lyl08), SLAMF7, SLAMF8 (BLAME), SLP-76, PAG / Cbp, NKp80 (KLRF1), NKp44, NKp30, NKp46, BTLA, JAML, CD150, PSGL1, TSLP, TNFR2, TRANCE / RANKL, or combinations thereof.In some implementations, the intracellular signal transduction region includes a co-stimulatory domain selected from 4-IBB, 2B4 (e.g., 2B4 ITSM2), ICOS, CD28, OX40, and CD27 co-stimulatory domains.

[0151] Intracellular signal transduction regions may include one, two, three, or more co-stimulatory domains. When more than one co-stimulatory domain is included, the co-stimulatory domains may have the same or different amino acid sequences. In some embodiments, the intracellular signal transduction region includes a 4-1BB co-stimulatory domain. The 4-1BB co-stimulatory domain may include the amino acid sequence shown in SEQ ID No:4 or have at least one, at least two, or at least three or more modified amino acid sequences compared to SEQ ID No:4. The 2B4 co-stimulatory domain may include the amino acid sequence shown in SEQ ID No:11 or have at least one, at least two, or at least three or more modified amino acid sequences compared to SEQ ID No:11. In some embodiments, the 2B4 co-stimulatory domain is 2B4. The ITSM2 co-stimulatory domain is composed of the first two ITSM motifs. This 2B4 The ITSM2 costimulatory domain may comprise an amino acid sequence as shown in SEQ ID No: 12 or an amino acid sequence having at least one, at least two, or at least three or more modifications compared to SEQ ID No: 12. In some embodiments, the intracellular region comprises a combination of a 2B4 costimulatory domain and a 4-1BB costimulatory domain.

[0152] In addition to one or more co-stimulatory signaling domains, CAR peptides may further include intracellular primary signaling domains. In some embodiments, the intracellular signaling region of the CAR peptide includes one co-stimulatory signaling domain and one primary signaling domain. In some other embodiments, the intracellular signaling region includes only the primary signaling domain and does not include the co-stimulatory signaling domain. The intracellular primary signaling domain may increase the proliferation, persistence, and / or cytotoxic activity of host cells (e.g., NK cells, NKT cells, γδ cells, etc.) carrying peptides as disclosed herein. For example, in some embodiments, one or more intracellular primary signaling domains include CD3ζ, repeating (e.g., 2-5) DAP10 YINM motifs, and signaling domains derived from LFA-1, DAP12, FcRγ, FcRP, CD3γ, CD36, CD3ε, CD79a, CD79b, CD5, CD22, FcεRI, CD66d, etc. Intracellular primary signaling domains can be specifically selected from the group consisting of CD3ζ, DAP12, LFA-1, and CD3t, or combinations thereof. Intracellular regions may include multiple (e.g., 2, 3, 4, or more) intracellular primary signaling domains. In cases where more than one intracellular primary signaling domain is included, they may contain different amino acid sequences.

[0153] In some embodiments, the intracellular primary signal transduction domain comprises a CD3ζ signal transduction domain. This CD3ζ signal transduction domain may comprise an amino acid sequence as shown in SEQ ID No: 5 or an amino acid sequence having at least one, at least two, or at least three or more modified amino acid sequences compared to SEQ ID No: 5. In some embodiments, the intracellular signal transduction region comprises or consists of a 4-1BB co-stimulatory domain and a CD3ζ intracellular signal transduction domain, or comprises or consists of a 2B4 co-stimulatory domain and a CD3ζ intracellular signal transduction domain. In some embodiments, the intracellular signal transduction region comprises or consists of a 4-1BB co-stimulatory domain, a 2B4 co-stimulatory domain, or a CD3ζ intracellular signal transduction domain.

[0154] When expressed from host cells, CAR peptides and adaptor peptides, as disclosed herein, can be expressed in two separate amino acid chains. A cleavable linker, including an IRES sequence, can be inserted between the nucleic acid sequence encoding the CAR peptide and the nucleic acid sequence encoding the adaptor peptide. The IRES element facilitates efficient interaction between the mRNA and the ribosome and allows for internal ribosome entry.

[0155] Constructs composed of CAR peptides and linker peptides

[0156] One aspect of this disclosure provides a combination of a linker peptide and a CAR peptide. In some embodiments, this disclosure provides a nucleic acid construct comprising a first nucleic acid sequence encoding a CAR peptide, the first nucleic acid sequence being operatively linked to a second nucleic acid sequence encoding a linker peptide. The nucleic acid sequence encoding the CAR peptide may be present in the same nucleic acid construct as the nucleic acid sequence encoding the linker peptide, although in other cases they may be present in separate nucleic acid constructs.

[0157] Specifically, the adaptor peptide may include a transmembrane region derived from DAP10 as described herein and optionally an intracellular region, and the CAR peptide may include an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain that specifically binds to antigens (e.g., GPC3, CD19, or CD33). The intracellular signal transduction domain of the CAR peptide may include a primary intracellular signal transduction domain and / or a co-stimulatory signal transduction domain. The adaptor peptide may further include a primary intracellular signal transduction domain located at the C-terminus of the co-stimulatory signal transduction domain. Both the CAR peptide and the adaptor peptide may include more than one co-stimulatory signal transduction domain.

[0158] In some embodiments, the first nucleic acid sequence is operatively linked to the second nucleic acid sequence via a sequence encoding a linker such as a cleavable peptide linker or an IRES sequence. In some embodiments, the first and second nucleic acid sequences are separated by a sequence encoding a linker such as a cleavable peptide linker or an IRES sequence. For example, the cleavable peptide linker may contain a self-cleaving sequence, such as a 2A self-cleaving sequence (e.g., T2A, P2A, E2A, F2A), which can induce ribosome jumping during the translation of the linker polypeptide. In some embodiments, the cleavable linker is a furin protease sequence. In some embodiments, the linker is an IRES sequence. After translation, the nucleic acid construct expresses two polypeptide chains, wherein the first polypeptide chain contains a CAR polypeptide, and the second polypeptide chain contains a linker polypeptide as disclosed herein.

[0159] In some embodiments, the adapter may contain both a 2A self-cleaving sequence and a furin protease sequence, for example, the furin protease cleaving sequence being located at the N-terminus of the 2A self-cleaving sequence. Additional adapters such as "GSG" or "SGSG" may also be used to improve cleavage efficiency. In some embodiments, the nucleic acid sequence encoding the adapter may contain an IRES site, which initiates the translation of the adaptor peptide and the CAR peptide, respectively.

[0160] The nucleic acid construct can be DNA or RNA. In some embodiments, the nucleic acid construct is an expression cassette contained in a vector. In other embodiments, the nucleic acid construct is a vector, such as a plasmid, phage particle, phage derivative, granule, transposon, retroviral vector, lentiviral vector, adenovirus vector, or adeno-associated virus vector.

[0161] Expression constructs or vectors that can be used in this disclosure may include (in a 5' to 3' orientation) a eukaryotic transcription promoter operatively linked to a protein-coding sequence, a transcription termination / polyadenylation sequence, and optional splicing signals, including intercalation sequences. In some embodiments, the expression construct includes a promoter operatively linked to a first nucleic acid sequence in a 5' to 3' orientation, followed by a second nucleic acid sequence. In some other embodiments, the expression construct includes a promoter operatively linked to a second nucleic acid sequence in a 5' to 3' orientation, followed by a first nucleic acid sequence. Promoters and enhancers that control the transcription of protein-coding genes in eukaryotic cells may consist of multiple genetic elements. For example, promoters used herein may include constitutive, inducible, and tissue-specific promoters. In cases where the vector is used to generate cancer therapies, the promoter may be effective under hypoxic conditions.

[0162] In some embodiments, the expression constructs provided herein include a promoter to drive the expression of both the CAR and the linker polypeptide. In other embodiments, transcription of the first and second nucleic acid sequences is controlled by separate promoters. Promoters typically contain sequences that locate the start site for RNA synthesis. One example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoters of mammalian terminal deoxynucleotidyl transferase genes and SV40 late genes, discrete elements covering the start site itself help to fix the start site. Additional promoter elements regulate the frequency of transcription initiation. Typically, these are located upstream of the start site, although some promoters have been shown to also contain functional elements downstream of the start site. To bring the coding sequence under the “control” of the promoter, the 5’ end of the transcription start site of the transcription reading frame is placed “downstream” (i.e., the 3’ end) of the selected promoter. The “upstream” promoter stimulates DNA transcription and promotes the expression of the encoded RNA.

[0163] The spacing between promoter elements is typically flexible, allowing promoter function to be preserved when elements are reversed or moved relative to each other. For example, in the tk promoter, the spacing between promoter elements can increase to 50 bp before activity begins to decline. Depending on the promoter, individual elements can function synergistically or independently to activate transcription. Promoters may or may not be used with "enhancers," which are cis-regulatory sequences involved in the transcriptional activation of nucleic acid sequences.

[0164] Non-limiting examples of promoters include early or late viral promoters, such as the SV40 early or late promoter, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus (RSV) early promoter; eukaryotic cell promoters, such as the β-actin promoter, the GADPH promoter, and the metallothionein promoter; and cascade response element promoters, such as the cyclic AMP response element promoter (cre), the serum response element promoter (sre), the phorbol ester promoter (TPA), and the response element promoter near the minimal TATA box (tre). Human growth hormone promoter sequences (e.g., the minimal human growth hormone promoter described in GenBank®, accession number X05244, nucleotides 283-341) or mouse mammary tumor promoters (available from ATCC, catalog number ATCC 45007) may also be used. In some embodiments, the promoter is CMV IE, dectin-1, dectin-2, human CD11c, F4 / 80, SM22, RSV, SV40, Ad MLP, β-actin, MHC class I or MHC class II promoters; however, any other promoter that can be used to drive the expression of therapeutic genes is suitable for the practice of this disclosure.

[0165] In some embodiments, the methods of this disclosure also involve enhancer sequences, i.e., nucleic acid sequences that increase promoter activity and have cis-acting potential, regardless of their orientation, even spanning relatively long distances (up to several thousand bases from the target promoter). However, the function of enhancers is not necessarily limited to such long distances, as they can also function in close proximity to a given promoter.

[0166] A construct composed of CAR peptides, linker peptides, and cytokines

[0167] In some aspects, this disclosure provides combinations of adaptor peptides, CAR peptides, and cytokines (one or more). In some embodiments, one or more cytokines are present in the same expression construct containing the CAR and the adaptor peptide, although in other cases they may be present in separate expression constructs. In some embodiments, one or more cytokines are co-expressed by the same expression construct containing the CAR and the adaptor peptide. One or more cytokines may be produced as peptides separate from the CAR and the adaptor peptide. Cytokines may be selected from, but are not limited to, IL-15, IL-2, IL-4, IL-7, IL-9, IL-21, and IL-23, including wild-type or variants thereof.

[0168] In some implementations, the cytokine used in the combination is interleukin-15 (IL-15), including its wild-type or mutant forms. For example, IL-15 can be used because it is tissue-restricted and can only be observed in serum or systemically at any level under pathological conditions. IL-15 possesses several properties required for adoptive therapy. IL-15 is a homeostatic cytokine that induces the development and proliferation of natural killer cells, promotes the eradication of established tumors by mitigating the functional suppression of tumor-resident cells, and inhibits activation-induced cell death. In addition to IL-15, other molecules are included, including but not limited to cytokines, chemokines, and other molecules that contribute to cell activation and proliferation for human applications. For example, cytokines include IL-15, IL-12, IL-2, IL-18, IL-21, IL-7, or combinations thereof. NK cells expressing IL-15 can be used, and these cells are capable of sustained supportive cytokine signaling, which is useful for their survival after infusion.

[0169] Specifically, the CAR peptide may contain an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain that specifically binds to antigens (e.g., GPC3, CD19, or CD33), and the adaptor peptide may contain a DAP10-derived TM region and an intracellular signal transduction region as described herein. The intracellular regions of both the CAR peptide and the adaptor peptide may contain a primary intracellular signal transduction domain and / or a co-stimulatory signal transduction domain. Both the CAR peptide and the adaptor peptide may contain more than one co-stimulatory signal transduction domain.

[0170] In some implementations, the nucleic acid construct comprises a first nucleic acid sequence encoding a CAR peptide, a second nucleic acid sequence encoding an adaptor peptide, and a third nucleic acid sequence encoding a cytokine peptide. In this case, the expression of the CAR peptide, adaptor peptide, and cytokine peptide may or may not be regulated by the same regulatory elements. Furthermore, the cytokine-encoding nucleic acid sequence may or may not be included in the same nucleic acid construct.

[0171] These three nucleic acid sequences can be operatively linked by encoding a nucleotide sequence of an adapter, which includes one or more self-cleaving sequences, one or more sequences cleaved by an endogenous protease, or an IRES sequence. For example, the self-cleaving sequence may be a 2A self-cleaving sequence (e.g., T2A, P2A, E2A, F2A), and the sequence cleaved by the endogenous protease may be a furin protease sequence. In some embodiments, the adapter may contain both a 2A self-cleaving sequence and a furin protease sequence, for example, the furin protease cleaving sequence located at the N-terminus of the 2A self-cleaving sequence. Additional adapters such as "GSG" or "SGSG" can also be used to improve cleavage efficiency.

[0172] In some specific implementation schemes, the nucleic acid construct includes the following components in the 5' to 3' orientation: (a) A first nucleic acid sequence is operatively ligated to a second nucleic acid sequence via a first adapter coding sequence, and the second nucleic acid sequence is operatively ligated to a third nucleic acid sequence via a second adapter coding sequence; (b) The first nucleic acid sequence is operatively ligated to the third nucleic acid sequence via a first adapter coding sequence, and the third nucleic acid sequence is operatively ligated to the second nucleic acid sequence via a second adapter coding sequence; (c) The second nucleic acid sequence is operatively ligated to the first nucleic acid sequence via the first adapter coding sequence, and the first nucleic acid sequence is operatively ligated to the third nucleic acid sequence via the second adapter coding sequence; (d) The second nucleic acid sequence is operatively ligated to the third nucleic acid sequence via the first adapter coding sequence, and the third nucleic acid sequence is operatively ligated to the first nucleic acid sequence via the second adapter coding sequence; (e) A third nucleic acid sequence is operatively ligated to a first nucleic acid sequence via a first adapter coding sequence, and a first nucleic acid sequence is operatively ligated to a second nucleic acid sequence via a second adapter coding sequence; or (f) The third nucleic acid sequence is operatively ligated to the second nucleic acid sequence via the first adapter coding sequence, and the second nucleic acid sequence is operatively ligated to the first nucleic acid sequence via the second adapter coding sequence.

[0173] The coding sequences for the first, second, and third linkers may be the same or different from each other. The nucleic acid construct may be DNA or RNA. In some embodiments, the nucleic acid construct is an expression cassette contained in a vector. In some other embodiments, the nucleic acid construct is a vector, such as a plasmid, phage particle, phage derivative, granule, transposon, retroviral vector, lentiviral vector, adenovirus vector, or adeno-associated virus vector.

[0174] peptide linkers

[0175] Different domains within CAR peptides and linker peptides can be interconnected via peptide linkers.

[0176] For example, each peptide linker in a CAR can have the same or different lengths and / or sequences, depending on the structural and / or functional characteristics of the individual domains. Each peptide linker can be selected and optimized independently. The length, flexibility, and / or other properties of the peptide linkers used in a CAR can have some influence on the properties, including but not limited to affinity, specificity, or avidity to one or more specific antigens or epitopes. For example, longer peptide linkers can be selected to ensure that two adjacent domains do not spatially interfere with each other. Short peptide linkers can be placed between the transmembrane domains and intracellular signaling domains of the CAR. Peptide linkers contain flexible residues (e.g., glycine and serine) that allow adjacent domains to move freely relative to each other. For example, a glycine-serine dinucleotide can be a suitable peptide linker.

[0177] The peptide linker can have any suitable length. In some embodiments, the length of the peptide linker is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids. The length of the peptide linker can not exceed about 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or fewer amino acids. The length of the peptide linker can be any of the following: about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids.

[0178] Peptide linkers can have naturally occurring or non-natural sequences. For example, a sequence derived from the hinge region of a heavy-chain-only antibody can be used as a linker. See, for example, WO1996 / 34103. In some embodiments, peptide linkers are flexible linkers. Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n, (GGGS)n, and (GGGGS)n, where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art.

[0179] Intracellular signal transduction domains

[0180] The adaptor peptides and CAR peptides disclosed herein may contain one or more intracellular signaling domains. Intracellular signaling domains may refer to primary intracellular signaling domains or co-stimulatory signaling domains. Intracellular signaling domains are responsible for activating at least one normal effector function of immune cells expressing CAR and adaptor peptides. The term "effector function" refers to a specific function of a cell. For example, the effector function of T cells may be lytic activity or helper activity, including the secretion of cytokines. Therefore, the term "intracellular signaling domain" refers to the intracellular portion of a protein that transduces effector function signals and directs the cell to perform a specific function. While the entire cytoplasmic signaling domain can often be utilized, in many cases, it is not necessarily necessary to use the entire chain.

[0181] Intracellular signal transduction domains may include primary intracellular signal transduction domains of immune cells. Intracellular signal transduction domains may consist substantially of primary intracellular signal transduction domains of immune cells. Exemplary primary cytoplasmic signal transduction sequences containing ITAM include sequences derived from: 4-1BB, 2B4, CD3ζ, FcRγ (FcεRIγ), FcRβ (FcεRIβ), CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0182] In some implementations, the primary intracellular signal transduction domain is derived from CD3ζ. The intracellular signal transduction domain may consist of the cytoplasmic signal transduction domain of CD3ζ. The primary intracellular signal transduction domain may be the cytoplasmic signal transduction domain of wild-type CD3ζ. The primary intracellular signal transduction domain may be a functional mutant of the CD3ζ cytoplasmic signal transduction domain, containing one or more mutations. The primary intracellular signal transduction domain of wild-type CD3ζ may contain the amino acid sequence of SEQ ID No: 5 or an amino acid sequence having at least 85%, 90%, or 95% identity with SEQ ID No: 5.

[0183] In addition to stimulating antigen-specific signals, many immune cells require co-stimulation to promote cell proliferation, differentiation, and survival, as well as to activate effector functions. CAR peptides and / or adaptor peptides disclosed herein may contain at least one co-stimulatory signaling domain. The co-stimulatory signaling domain described herein may be a cytoplasmic signaling domain derived from a co-stimulatory protein, which transduces signals and regulates responses mediated by immune cells such as T cells, NK cells, macrophages, neutrophils, or eosinophils. The "co-stimulatory signaling domain" may be the cytoplasmic portion of a co-stimulatory molecule.

[0184] The intracellular signaling domain of the CAR peptide and / or adaptor peptide may contain a single costimulatory signaling domain. The intracellular signaling domain may contain two or more (e.g., about two, three, four, or more) costimulatory signaling domains, such as two or more identical costimulatory signaling domains, or two or more costimulatory signaling domains from different costimulatory proteins. The intracellular signaling domain may contain a primary intracellular signaling domain (e.g., the cytoplasmic signaling domain of 4-1BB, 2B4, or CD28) and one or more costimulatory signaling domains. One or more costimulatory signaling domains and the primary intracellular signaling domain (e.g., the cytoplasmic signaling domain of CD3ζ) may be fused to each other via an optional peptide linker. The primary intracellular signaling domain and one or more costimulatory signaling domains may be arranged in any suitable order. One or more costimulatory signaling domains may be located between the transmembrane domain and the primary intracellular signaling domain (e.g., the cytoplasmic signaling domain of CD3ζ). Multiple co-stimulatory signal transduction domains can provide superimposed or synergistic stimulation.

[0185] Activation of costimulatory signaling domains in host cells (e.g., immune cells) can induce increased or decreased cellular production and secretion of cytokines, phagocytic properties, proliferation, differentiation, survival, and / or cytotoxicity. The costimulatory signaling domain of any costimulatory molecule may be suitable for the CAR peptides and / or adaptor peptides described herein. The type of costimulatory signaling domain is selected based on a variety of factors, such as the type of immune cell in which the effector molecule will be expressed (e.g., T cells, NK cells, macrophages, neutrophils, or eosinophils) and the desired immune effector function (e.g., ADCC effect).Examples of co-stimulatory signaling domains for CAR peptides and / or linker peptides can be cytoplasmic signaling domains of co-stimulatory proteins, including but not limited to members of the B7 / CD28 family (e.g., B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7-DC, and PDCD6); and members of the TNF superfamily (e.g., 4-1BB / TNFSF9 / CD137, 4-1BB ligand / TNFSF9, BAFF / BLyS / TNFSF13B, BAFF). R / TNFRSF13C, CD27 / TNFRSF7, CD27 ligand / TNFSF7, CD30 / TNFRSF8, CD30 ligand / TNFSF8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, Lymphotoxin-α / TNF-β, OX40 / TNFRSF4, OX40 ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNF-α and TNF RII / TNFRSF1B); members of the SLAM family (e.g., 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, and SLAM / CD150); and any other co-stimulatory molecules such as CD2, CD7, CD53, CD82 / Kai-1, CD90 / Thy1, CD96, CD160, CD200, CD300a / LMIR1, HLA Class I, HLA-DR, Ikaros, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function-associated antigen-1 (LFA-1), and NKG2C.

[0186] In some implementations, the co-stimulatory signal transduction domain is selected from the group consisting of ligands of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), ICOS (CD278), NTBA, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and combinations thereof.

[0187] Any variant of the costimulatory signaling domain described herein is also within the scope of this disclosure, provided that the costimulatory signaling domain can modulate the immune response of immune cells. The costimulatory signaling domain may contain up to 10 amino acid residue variations (e.g., 1, 2, 3, 4, 5, or 8) compared to its wild-type counterpart. Such a costimulatory signaling domain containing one or more amino acid variations is referred to as a variant. Mutations in amino acid residues of the costimulatory signaling domain may result in increased signal transduction and enhanced immune response stimulation, relative to a costimulatory signaling domain without mutations. Mutations in amino acid residues of the costimulatory signaling domain may also result in decreased signal transduction and weakened immune response stimulation, relative to a costimulatory signaling domain without mutations.

[0188] Hinge area

[0189] A hinge domain is an amino acid segment that typically resides between two domains of a protein and allows the protein to be flexible and for one or both domains to move relative to each other. The CAR peptides and adaptor peptides of this application may comprise a hinge domain located between an extracellular domain and a transmembrane domain. Any amino acid sequence that provides this flexibility and movement relative to the transmembrane domain of an effector molecule for the extracellular antigen-binding domain can be used. In some embodiments, the hinge domain of the adaptor peptide comprises a DAP10-derived hinge sequence, for example, as shown in SEQ ID No: 8.

[0190] The hinge domain can contain approximately 10-100 amino acids, for example, approximately 15-75 amino acids, 20-50 amino acids, or 30-60 amino acids. The length of the hinge domain can be any of at least approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids.

[0191] The hinge domain can be a hinge domain of a naturally occurring protein (e.g., an immunoglobulin). The hinge domain of any protein known in the art that contains a hinge domain is suitable for the chimeric receptor described herein. The hinge domain can be at least a portion of a hinge domain in a naturally occurring protein and imparts flexibility to the chimeric receptor. In some embodiments, the hinge domain is derived from CD8α. The hinge domain can be a portion of a CD8α hinge domain, for example, a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of a CD8α hinge domain. The CD8α hinge domain can contain the amino acid sequence SEQ ID No: 2 or an amino acid sequence having at least 85%, 90%, or 95% identity with SEQ ID No: 2.

[0192] Hinge domains of antibodies such as IgG, IgA, IgM, IgE, or IgD antibodies are also suitable for the pH-dependent chimeric receptor systems described herein. The hinge domain can be a hinge domain connecting the antibody's constant domains CH1 and CH2. The hinge domain can be derived from the antibody and includes the antibody's hinge domain and one or more constant regions of the antibody. The hinge domain can include the antibody's hinge domain and the antibody's CH3 constant region. The hinge domain can include the antibody's hinge domain and the antibody's CH2 and CH3 constant regions. The antibody can be an IgG, IgA, IgM, IgE, or IgD antibody. The antibody can be an IgG1, IgG2, IgG3, or IgG4 antibody. The hinge region can include the hinge region of an IgG1 antibody as well as the CH2 and CH3 constant regions. The hinge region can include the hinge region of an IgG1 antibody and the CH3 constant region.

[0193] Non-naturally occurring peptides can also be used as hinge domains for the CAR peptides and linker peptides described herein. Hinge domains located between the C-terminus of the extracellular ligand-binding domain and the N-terminus of the transmembrane domain, or between the signal peptide and the transmembrane domain, can be peptide linkers, such as (GxS)n linkers, where x and n can each independently be integers from 3 to 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more.

[0194] Nucleic acid and vector

[0195] This disclosure also provides a nucleic acid molecule comprising a first nucleic acid sequence encoding an adaptor polypeptide, a second nucleic acid sequence encoding a CAR polypeptide, and optionally a third nucleic acid sequence encoding a cytokine polypeptide, these nucleic acid sequences being operatively linked together. The nucleic acid molecule may be provided in the form of a messenger RNA transcript or DNA.

[0196] In one aspect, this document provides a nucleic acid molecule comprising: (1) a nucleic acid sequence encoding a CAR polypeptide as disclosed herein, said CAR polypeptide being, for example, a CAR polypeptide targeting GPC3, a CAR polypeptide targeting CD19, or a CAR polypeptide targeting CD33; and (2) a nucleic acid sequence encoding an adapter polypeptide as disclosed herein. Optionally, the two nucleic acid sequences are linked together by a nucleic acid sequence encoding a self-cleaving peptide (such as P2A, E2A, F2A, or T2A) or an IRES sequence. In some further embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a cytokine polypeptide.

[0197] The nucleic acid sequence encoding the desired polypeptide can be obtained using recombination methods known in the art and employing standard techniques. For example, the sequence of interest can be generated through synthesis or cloning.

[0198] This disclosure also provides vectors in which nucleic acid sequences or constructs as disclosed herein are inserted. Nucleic acid sequences encoding CAR peptides, adaptor peptides, and optionally cytokine peptides can be contained within the same vector. The nucleic acid sequences may be encoded by a single nucleic acid molecule within the same reading frame. Vectors derived from retroviruses are suitable tools for achieving long-term gene transfer because they allow for the long-term stable integration of transgenes and their amplification in daughter cells. Retroviral vectors can be, for example, gamma retroviral vectors.

[0199] Gamma retroviral vectors may include, for example, a promoter, a packaging signal (ψ), a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTRs), and a transgene of interest, such as a gene encoding a CAR. Gamma retroviral vectors may lack viral structural genes, such as gag, pol, and env. Exemplary gamma retroviral vectors include murine leukemia virus (MLV), spleen focal formation virus (SFFV), and myeloproliferative sarcoma virus (MPSV), and vectors derived therefrom. For a description of other gamma retroviral vectors, see, for example, Tobias Maetzig et al., "Gammaretroviral Vectors: Biology, Technology and Application" Viruses. June 2011; 3(6): 677-713.

[0200] In some implementations, the expression of natural or synthetic nucleic acids encoding CAR and adaptor peptides is typically achieved by operatively linking the nucleic acid encoding the CAR peptide and / or the nucleic acid encoding the adaptor peptide to a promoter and incorporating the construct into an expression vector. The vector is suitable for replication and integration in eukaryotes. A typical cloning vector contains transcription and translation terminators, a start sequence, and a promoter, all of which can be used to regulate the expression of the desired nucleic acid sequence.

[0201] Using standard gene delivery protocols, the expression constructs of this disclosure can also be used for nucleic acid immunotherapy and gene therapy. Gene delivery methods are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, the entire contents of which are incorporated herein by reference. In another embodiment, this disclosure provides a gene therapy vector.

[0202] Nucleic acids can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors, including but not limited to plasmids, phage particles, phage derivatives, animal viruses, and entrapments. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0203] In addition, expression vectors can be provided to cells in the form of viral vectors. Viral vector technology is well known in the art and has been described, for example, in the following literature: Sambrook et al., 2012, MOLECULARCLONING: A LABORATORY MANUAL, Volumes 1-4, Cold Spring Harbor Press, NY), and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, a suitable vector contains at least one origin of replication that is functional in at least one organism, a promoter sequence, a useful restriction endonuclease site, and one or more selectable markers (e.g., WO01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0204] Various virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the recipient's cells, either in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0205] Other promoter elements, such as enhancers, regulate the frequency of transcription initiation. These are typically located 30–110 bp upstream of the start site, although some promoters have been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is generally flexible, allowing promoter function to be preserved when elements are flipped or moved relative to each other. In the thymidine kinase (TK) promoter, the spacing between promoter elements can increase to 50 bp before activity begins to decline. Depending on the promoter, individual elements can function synergistically or independently to activate transcription.

[0206] Vectors can also contain selectable marker genes or reporter genes to select CAR-expressing cells from a host cell population transfected via lentiviral vectors. Appropriate regulatory sequences can be added flanking the selectable markers and reporter genes to achieve expression in host cells. For example, vectors can contain transcription and translation terminators, initiation sequences, and promoters, all of which can be used to regulate the expression of nucleic acid sequences.

[0207] Engineered immune cells

[0208] On the one hand, this document provides an engineered immune cell that expresses CARs and adaptor peptides, nucleic acids, or vectors as disclosed herein. Accordingly, this disclosure provides an engineered immune cell, such as NK cells, T cells, γδT cells, and a method for using them in adoptive therapy. Immune cells, including NK cells, can be derived from umbilical cord blood (including combined umbilical cord blood from multiple sources), peripheral blood, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), bone marrow, or mixtures thereof. For example, NK cells can be derived from cell lines such as, but not limited to, NK-92 cells. NK cells can be umbilical cord blood mononuclear cells, such as CD56. + NK cells.

[0209] "Immune cells" refer to immune cells that can perform immune effector functions. Examples of immune cells include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, helper T cells, γδ T cells, neutrophils, and eosinophils.

[0210] In some embodiments, the immune cells are NK cells. The immune cells can be established cell lines, such as NK-92 cells. In other embodiments, the immune cells are T cells. The T cells may be αβ T cells or γδ T cells. The T cells may be CD4+. + / CD8 - CD4 - / CD8 + CD4 + / CD8 +CD4 - / CD8 - Type or combination thereof. T cells express CAR or DAP10 adaptor peptides and interact with target cells such as CLDN18.2. + Or GUCY2C + After binding with tumor cells, they can produce IL-2, TFN, and / or TNF. T cells, after expressing CAR or DAP10 adaptor peptides and binding to specific target cells, can lyse the target cells.

[0211] In some implementations, immune cells are derived from stem cells, such as hematopoietic stem cells, pluripotent stem cells, iPS cells, or embryonic stem cells.

[0212] The engineered immune cells disclosed herein can be prepared by introducing expression constructs or vectors as disclosed herein into immune cells, such as NK cells. Methods for introducing vectors or isolated nucleic acids into mammalian cells are known in the art. The described vectors can be transferred into immune cells by physical, chemical, or biological methods.

[0213] Physical methods for introducing vectors into immune cells include calcium phosphate precipitation, liposome transfection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York. Vectors can be introduced into cells via electroporation. Biological methods for introducing vectors into immune cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian cells, such as human cells. Chemical means of introducing vectors into immune cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, microbeads, and lipid-based systems, including oil-in-water emulsions, micelles, hybrid micelles, and liposomes. An exemplary colloidal system used as a medium for in vitro delivery is a liposome (e.g., an artificial membrane vesicle).

[0214] After the introduction of a vector or isolated nucleic acid, the transduced or transfected immune cells can proliferate in vitro. In some embodiments, the transduced or transfected immune cells are cultured to allow them to proliferate for at least about 1, 2, 3, 4, 5, 6, 7, 10, 12, 15, or 21 days. The transduced or transfected immune cells can be further evaluated or screened to select engineered mammalian cells.

[0215] Reporter genes can be used to identify potentially transfected cells and to assess the functionality of regulatory sequences. Generally, a reporter gene is a gene that is absent or not expressed in the recipient organism or tissue, and it encodes a polypeptide whose expression exhibits easily detectable properties, such as enzymatic activity. Reporter gene expression is measured at an appropriate time after DNA is introduced into recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al. FEBS Letters 479: 79-82 (2000)). Suitable expression systems are well-known and can be prepared using known techniques or obtained commercially.

[0216] Other methods for confirming the presence of nucleic acids encoding peptides in engineered immune cells include, for example, molecular biological assays well known to those skilled in the art, such as Southern blotting and Northern blotting, RT-PCR, and PCR; and biochemical assays, such as detecting the presence or absence of a specific peptide, for example by immunological methods (such as ELISA or Western blotting).

[0217] In some implementations, the immune cells are NK cells. The source of NK cells can be obtained from an individual before amplification and genetic modification. NK cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. NK cells can be obtained from allogeneic or autologous donors. NK cells can be partially or completely purified, or unpurified, and amplified in vitro. Methods and compositions for in vitro amplification include, but are not limited to, those described in Becker et al., (2016) CancerImmunol. Immunother. 65(4): 477-84. Amplification can be performed before or after the introduction of the chimeric DAP10 linker peptide into NK cells, or both before and after introduction. In short, and not limited to, NK cell expansion can include the use of engineered feeder cells, combinations of cytokines (e.g., IL-2, IL-15) and / or aAPC (Cortes-Selva, D et al., (2021) Trends Pharmacol Sci. 42(1): 45-59).

[0218] In some implementations, the immune cells are T cells. T cells can be isolated from peripheral blood lymphocytes by lysing red blood cells and removing monocytes, for example, by PERCOLL gradient centrifugation or by countercurrent centrifugation washing. Specific T cell subsets, such as CD3, can be further isolated using positive or negative selection techniques. + CD28 + CD4 + CD8 + CD45RA + and CD45RO + T cells. T cells can be obtained directly from the patient after treatment. In this regard, it has been observed that, shortly after certain cancer treatments, particularly those with drugs that impair the immune system, the quality of T cells obtained at this time—typically during the recovery period from such treatment—may be optimal or enhanced in terms of their ex vivo expansion capacity. Similarly, these cells may be in a preferred state after ex vivo manipulation using the methods described herein, thereby facilitating enhanced transplantation and in vivo expansion. Therefore, in the context of this disclosure, the collection of blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, is covered during this recovery phase. Furthermore, mobilization (e.g., mobilization using GM-CSF) and conditioning protocols can be employed to create a state in the subject that favors the repopulation, recycling, regeneration, and / or expansion of specific cell types, particularly within a time window defined post-treatment. Illustrated cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0219] After gene modification using a vector, NK cells can be immediately infused or stored. In some cases, following gene modification, cells can be proliferated in vitro as a bulk population for days, weeks, or months after gene transfer, approximately 1, 2, 3, 4, 5 days, or longer. In other cases, transfectants are cloned, and clones that meet the following criteria are amplified in vitro: they exhibit the presence of a single integrated or free expression cassette or plasmid, and express CAR and adaptor peptides. Clones selected for amplification demonstrate the ability to specifically recognize and lyse target-expressing cells. Recombinant immune cells can be amplified by stimulation with IL-2 or other cytokines binding to a common γ chain (e.g., IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, etc.). Recombinant immune cells can also be amplified by stimulation with artificial antigen-presenting cells. Furthermore, genetically modified cells can be cryopreserved.

[0220] Whether before or after genetic modification of T cells or NK cells using the nucleic acid constructs described herein, methods such as those described in the following literature can typically be used to activate and expand T cells or NK cells: U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.

[0221] Pharmaceutical Composition

[0222] This application further provides pharmaceutical compositions comprising any of the engineered immune cells described herein and a pharmaceutically acceptable carrier. The pharmaceutical compositions can be prepared by mixing engineered immune cells of desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). The pharmaceutical compositions can be in the form of lyophilized formulations or aqueous solutions.

[0223] Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the doses and concentrations used and include buffers, antioxidants including ascorbic acid, methionine, vitamin E, sodium metabisulfite; preservatives, isotonic agents, stabilizers, metal complexes (e.g., zinc-protein complexes); chelating agents such as EDTA and / or nonionic surfactants.

[0224] For pharmaceutical compositions to be used in vivo, they must be sterile. Sterility can be achieved by filtration through a sterile filter membrane. Typically, the pharmaceutical compositions described herein are placed in containers with sterile access points, such as intravenous solution bags or vials with stoppers that can be punctured by a hypodermic needle.

[0225] The route of administration should conform to known and recognized methods, such as single or multiple bolus injections or infusions over a longer period of time, for example, via subcutaneous, intravenous, intraperitoneal, intramuscular, intra-arterial, intralesional, or intra-articular routes, local application, inhalation, or administration via sustained or controlled release.

[0226] Methods and uses

[0227] In one aspect, this disclosure provides a method for treating a tumor in a subject with this need, comprising administering to the subject an effective amount of engineered immune cells as disclosed herein. The CAR expressed in the immune cells may be engineered to target the cancer to be treated. Such methods and uses include therapeutic methods and uses, such as those involving administering molecules, cells, or compositions containing them to a subject suffering from a disease, condition, or symptom associated with the expression of an aberrant antigen. In some embodiments, the subject suffers from GPC3-related cancer.

[0228] This application further relates to methods and compositions for cellular immunotherapy. In some embodiments, cellular immunotherapy is used to treat cancer. Any of the nucleic acids and engineered immune cells described herein can be used in methods for treating cancer. Engineered immune cells described herein, such as NK cells, can be used to treat tumors with antigen-deficient escape mutations and to reduce resistance to existing therapies. In some embodiments, the methods and compositions described herein can be used to treat other diseases related to GPC3.

[0229] Engineered immune cells can be autologous. Engineered immune cells can be allogeneic. In some implementations, the cancer is a solid cancer, including but not limited to gastric cancer, esophageal cancer, pancreatic ductal carcinoma, lung cancer such as non-small cell lung cancer (NSCLC), ovarian cancer, colorectal cancer, liver cancer, head and neck cancer, gallbladder cancer and its metastases.

[0230] The methods described herein are applicable to cancer at all stages, including early, advanced, and metastatic cancer. The methods described herein can be used as first-line, second-line, or third-line therapy, or in combination with other types of cancer therapies known in the art, whether in adjuvant or neoadjuvant therapy phases, such as chemotherapy, surgery, radiotherapy, gene therapy, immunotherapy, bone marrow transplantation, stem cell transplantation, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, radiofrequency ablation, etc.

[0231] The drug composition can be administered in any convenient manner, including injection, ingestion, infusion, implantation, or transplantation. The composition can be administered to a patient via arterial, subcutaneous, intradermal, intratumoral, intralymphatic, intramedullary, intramuscular, intravenous, or intraperitoneal routes. In some embodiments, the drug composition is administered systemically. The drug composition can be administered to an individual via infusion, such as intravenous infusion. Infusion techniques for immunotherapy are known in the art (see, for example, Rosenberg et al., New Eng.J. of Med. 319: 1676 (1988)). In some embodiments, the drug composition is administered to an individual via intradermal or subcutaneous injection. The composition can be administered via intravenous injection. The composition can be injected directly into a tumor or lymph node. The drug composition can be applied locally to a tumor site, such as directly into tumor cells or into tissue containing tumor cells.

[0232] The dosage and desired drug concentration of the pharmaceutical compositions disclosed herein may vary depending on the intended specific use. Determining the appropriate dosage or route of administration is entirely within the skill of a person skilled in the art. Animal studies have provided reliable guidance for determining effective dosages for human treatment. Interspecies extrapolation of effective dosages can be performed according to the principles outlined in Mordenti, J. and Chappell, W., “The Use of Interspecies Scaling in Toxicokinetics,” in Toxicokinetics and New Drug Development, Yacobi et al., Pergamon Press, New York, 1989, pp. 42-46. Within the scope of this application, different formulations will be effective for different treatments and different conditions, and administration intended for the treatment of a specific organ or tissue may require delivery in a different manner than delivery to another organ or tissue.

[0233] combination therapy

[0234] Engineered immune cells or pharmaceutical compositions as described herein can be used in combination with other known agents and therapies. As used herein, “combined” administration means the delivery of two (or more) different treatments to a subject during the course of a condition, for example, after the subject has been diagnosed with the condition and before the condition is cured or eliminated, or before treatment is discontinued for other reasons. When the delivery of the second treatment begins, the delivery of the first treatment may still be in progress, resulting in overlap in administration. This is sometimes referred to herein as “simultaneous” or “parallel” delivery. In other embodiments, the delivery of one treatment begins only after the delivery of another treatment has ended. In some embodiments under either condition, the treatment is more effective due to combined administration. For example, the second treatment is more effective when combined than when the first treatment is administered in the absence of the first treatment; for example, an equivalent effect can be observed with less of the second treatment, or the second treatment alleviates symptoms to a greater extent, or similar effects are observed with the first treatment. In some embodiments, the delivery results in a greater degree of symptom or other condition-related parameter reduction than when one of the treatments is delivered in the absence of the other treatment. The effects of the two treatments can be partially additive, completely additive, or greater than the additive effect. Delivery allows the effect of the first treatment to remain detectable when the second treatment is delivered.

[0235] The engineered immune cells described herein and at least one additional therapeutic agent can be administered simultaneously, either in the same composition or in separate compositions, or sequentially. For sequential administration, cells expressing the CAR and the linker peptide described herein can be administered first, followed by the additional agent, or the order of administration can be reversed.

[0236] CAR therapy and / or other therapeutic agents, procedures, or modalities can be administered during periods of active disease, or during periods of remission or low disease activity. CAR therapy can be administered before, concurrently with, after, or during periods of remission of other treatments.

[0237] In other respects, the engineered immune cells described herein can be used in treatment regimens in combination with surgery, cytokines, radiotherapy, or chemotherapy, such as the use of cyclophosphamide (Cytoxan), fludarabine, histone deacetylase inhibitors, demethylating agents, or peptide vaccines, as described, for example, in Izumoto et al. 2008 J Neurosurg 108:963-971.

[0238] Sequence Summary

[0239] This application is accompanied by a sequence listing containing multiple amino acid sequences. Table A below provides a summary of the included sequences.

[0240] Table A

[0241] Example

[0242] Example 1. T cells transfected with CAR+DAP10-ICD showed enhanced cytotoxicity compared to T cells transfected with CAR alone.

[0243] 1.1 Expression of CAR+DAP10-ICD structure in expanded T cells

[0244] In this experiment, we used a chimeric antigen receptor (CAR) GC33 targeting GPC3 (as disclosed in patent US20070190599A1) to construct CAR-modified immune cells, where GPC3 is a tumor-associated antigen (TAA) expressed in liver cancer. Retroviruses carrying different CAR constructs were prepared, including: CAR (Full Length) (Structure 1) CAR (full length) + DAP10 + 2B4 (structure 2) CAR (full length) + DAP10 + 41BB (structure 3) CAR (full length) + DAP10 + 2B4 + 41BB (structure 4) CAR (full length) + DAP10 + 2B4 + CD3ζ (structure 5) CAR (full length) + DAP10 + 4-1BB + CD3ζ (structure 6) CAR (full length) + DAP10 + 2B4·ITSM2 (structure 7) CAR (full length) + DAP10 + CD28 (ICD) (Structure 8) Table 1. Structures of nucleic acid molecules encoding CAR peptides and adaptor peptides (listing proteins from which SP, hinge, TM, and ICD regions are derived)

[0245] Retroviruses were prepared and their titers were determined using 293T cells. Primary human T cells were isolated from PBMCs obtained from healthy donors to generate CAR (full-length) + DAP10 + ICD T cells. T cells were stimulated using T cell TransAct (Miltenyi Biotec). After expansion for 3 days in the presence of recombinant IL-2, T cells were transduced with retroviruses with the assistance of the transduction enhancer Vectofusin-1 (Miltenyi Biotec). The transduced T cells were then expanded in the presence of recombinant IL-2 and used for functional assays or cryopreservation approximately 7–12 days post-transduction. Activated T cells were then transduced with retroviruses. This transduction process achieved the desired expression of CAR (full-length) + DAP10 + ICD T cells.

[0246] 1.2 Transduction efficiency and its effect on cell proliferation assay

[0247] On day 5 post-transduction, CAR-T cells were counted using an NC250 cell counter, and 1E5 cells were transferred per well based on the cell count. The CAR-T cells were then incubated at 4°C for 1 hour with anti-CD4 antibody (Biolegend, 300537) and anti-CD8 antibody (Biolegend, 301008) or GPC3-FITC protein (Acro Biosystems, GPC3-H82E5, for CAR detection) and anti-HA antibody (Invitrogen, 26183, for DAP10 identification). After incubation, the cells were washed with 1% BSA-PBS (w / v), then washed again and resuspended in 1% BSA-PBS for flow cytometry analysis. Data were analyzed using FlowJo software.

[0248] Figure 2 The proliferation of CAR-T cells with different structures was shown. Figure 3 The results show the CD4 / CD8 ratio, CAR expression, and DAP10 expression in CAR-T cells. The results indicate that the inclusion of DAP10+ICD has minimal impact on the expansion of CAR-T cells in the culture. Furthermore, the CAR-T cells used in this experiment exhibited an appropriate CD4 / CD8 ratio, and structures 1-8 were efficiently generated with high transduction efficiency.

[0249] 1.3 CAR-T cell activation and cytotoxicity

[0250] To evaluate the cytotoxicity of modified T cells, we used two liver cancer cell lines: hepG2, which showed high expression of both GPC3 and MICA / B, and sk-hep1, which had extremely low GPC3 expression but high MICA / B expression. The expression levels of MICA / B and GPC3 are shown below. Figure 4 As shown.

[0251] For cytotoxicity assays, stably GFP-expressing hepG2 and sk-hep1 cells were pre-seeded in 96-well plates and allowed to adhere for 24 hours. CAR-T cells were then added to each well at different E:T ratios. The number of viable cells was assessed by monitoring green fluorescence signals using the IncuCyte automated live-cell imaging system. Results were shown... Figure 5-7 The results showed that the inclusion of the DAP10+ICD structure enhanced the cytotoxicity of CAR-T cells against both CAR-target and non-CAR-target cell lines.

[0252] Example 2: NK cells transfected with CAR+DAP10-ICD showed enhanced cytotoxicity compared to NK cells transfected with CAR alone.

[0253] 2.1 Expression of CAR+DAP10-ICD structure in expanded NK cells

[0254] In this experiment, we selected GPC3 as the target for CAR therapy against liver cancer. Retroviruses carrying different CAR constructs were prepared, including: CAR (Full Length) (Structure 1) CAR (full length) + DAP10 + 2B4 (structure 2) CAR (full length) + DAP10 + 41BB (structure 3) CAR (full length) + DAP10 + 2B4 + 41BB (structure 4) CAR (full length) + DAP10 + 2B4 + CD3ζ (structure 5) CAR (full length) + DAP10 + 4-1BB + CD3ζ (structure 6) CAR (full length) + DAP10 + 2B4·ITSM2 (structure 7) To generate CAR (full-length) + DAP10 + ICD NK cells, primary human NK cells were isolated from PBMCs (peripheral blood mononuclear cells) obtained from healthy donors. NK cells were stimulated with feeder cells (K562-mbIL21-41BBL) in the presence of recombinant IL-2. After expansion for 5–6 days, NK cells were transduced using a retrovirus pre-coated with RetroNectin. The transduced NK cells were then expanded in the presence of recombinant IL-2 and used for functional assays or cryopreservation approximately 7–10 days post-transduction. This transduction process achieved the desired expression of CAR (full-length) + DAP10 + ICD NK cells.

[0255] 2.2 Transduction efficiency and its effect on cell proliferation assay

[0256] On day 3 post-transduction, feeder cells were provided at an E:T ratio of 2:1 to reactivate NK cells. On day 6 post-transduction, CAR-NK cells were counted using an NC250 cell counter, and 1E5 cells were transferred per well based on the cell count. CAR-NK cells were then incubated at 4°C for 1 hour with anti-CD56-violet (Biolegend. 318328), GPC3-FITC protein (AcroBiosystems, GPC3-H82E5, for CAR detection), and anti-HA antibody (Invitrogen, 26183, for DAP10 identification). After incubation, cells were washed with 1% BSA-PBS (w / v), then washed again and resuspended in 1% BSA-PBS for flow cytometry analysis. Data were analyzed using FlowJo software.

[0257] Figure 8 The proliferation of CAR-NK cells with different structures was shown. Figure 9 CD56 was showcased + CAR and DAP10 expression in cells were studied. The results showed that the DAP10+ICD had minimal impact on CAR-NK cell expansion. Furthermore, structures 1-7 were all efficiently produced with high transduction efficiency.

[0258] 2.3 CAR-NK cell activation and cytotoxicity

[0259] To evaluate the cytotoxicity of modified NK cells, we used two hepatocellular carcinoma cell lines: hepG2, which showed high expression of both GPC3 and MICA / B, and sk-hep1, which had low GPC3 expression but high MICA / B expression.

[0260] For cytotoxicity assays, GFP-transfected hepG2 and sk-hep1 cells were pre-seeded in 96-well plates and allowed to adhere for 24 hours. CAR-NK cells were then added to each well at different E:T ratios. The number of viable cells was assessed by monitoring green fluorescence signals using the IncuCyte automated live-cell imaging system. Results were shown... Figure 10 and Figure 11 The results showed that the inclusion of the DAP10+ICD structure enhanced the cytotoxicity of CAR-NK cells against cell lines that do not express CAR targets.

[0261] Example 3: Compared with T cells transfected with CAR alone, γδT cells transfected with CAR+DAP10-ICD showed enhanced cytotoxicity.

[0262] 3.1 Expression of CAR+DAP10-ICD structure in expanded γδT cells

[0263] In this experiment, we selected GPC3 as the target for CAR therapy against liver cancer. Retroviruses carrying different CAR constructs were prepared, including: CAR (Full Length) (Structure 1) CAR (full length) + DAP10 + 2B4 + CD3ζ (structure 5) CAR (full length) + DAP10 + 4-1BB + CD3ζ (structure 6) To generate CAR (full-length) + DAP10 + ICD γδT cells, primary human γδT cells were isolated from PBMCs (peripheral blood mononuclear cells) obtained from healthy donors. γδT cells were stimulated with feeder cells (K562-mbIL21-41BBL) in the presence of recombinant IL-2. After expansion for 7–9 days, γδT cells were transduced using retroviruses. The transduced γδT cells were then expanded in the presence of recombinant IL-2 and used for functional assays or cryopreservation approximately 7–10 days post-transduction. This transduction process achieved the desired expression of CAR (full-length) + DAP10 + ICD γδT cells.

[0264] 3.2 Transduction efficiency and its effect on cell proliferation assay

[0265] On day 6 post-transduction, CAR-γδT cells were counted using an NC250 cell counter, and 1E5 cells were transferred per well based on the cell count. The CAR-γδT cells were then incubated at 4°C for 1 hour with anti-γδT1 and anti-γδT2 or GPC3-FITC protein (AcroBiosystems, GPC3-H82E5, for CAR detection) and anti-HA antibody (Invitrogen, 26183, for DAP10 identification). After incubation, the cells were washed with 1% BSA-PBS (w / v), then washed again and resuspended in 1% BSA-PBS for flow cytometry analysis. Data were analyzed using FlowJo software.

[0266] Figure 12 The proliferation of CAR-γδT cells with different structures was shown. Figure 13 The percentage of γδT1 / γδT2, CAR expression, and DAP10 expression in CAR-γδT cells were shown. The results indicate that the inclusion of DAP10+ICD had minimal impact on CAR-γδT cell proliferation. Furthermore, the CAR-γδT cells used in this experiment exhibited an appropriate γδT1 / γδT2 ratio, and structures 1, 5, and 6 were efficiently generated with high transduction efficiency.

[0267] 3.3 CAR-γδT cell activation and cytotoxicity

[0268] To evaluate the cytotoxicity of modified γδT cells, we used two hepatocellular carcinoma cell lines: hepG2, which showed high expression of both GPC3 and MICA / B, and sk-hep1, which had low GPC3 expression but high MICA / B expression.

[0269] For cytotoxicity assays, GFP-transfected hepG2 and sk-hep1 cells were pre-seeded in 96-well plates and allowed to adhere for 24 hours. Then, CAR-γδT cells were added to each well at different E:T ratios. The number of viable cells was assessed by monitoring green fluorescence signals using the IncuCyte automated live-cell imaging system. Results were shown... Figure 14 (Cytotoxicity to hepG2) and Figure 15 In (cytotoxicity against sk-hep1), it was shown that the inclusion of the DAP10+ICD structure enhanced the cytotoxicity of CAR-γδT cells against cell lines that do not express CAR targets.

[0270] Example 4: The DAP10-ICD can be combined with various CAR structures.

[0271] 4.1 Expression of CAR+DAP10-ICD structure in expanded immune cells

[0272] In this experiment, the isolation and expansion of immune cells were the same as in Examples 1-3. We again chose GPC3 as the target for CAR therapy against liver cancer. Retroviruses carrying different CAR constructs were prepared, including: CAR(CD28TM+4-1BB+CD3ζ ICD) (Structure 1) CAR(CD28TM+4-1BB+CD3ζ ICD) + DAP10 + 2B4 (Structure 2) CAR(CD8TM+4-1BB+CD3ζ ICD) + DAP10 + 2B4 (Structure 9) CAR(CD8TM+2B4+CD3ζ ICD) + DAP10 + 2B4 (structure 10) CAR(CD8TM+CD3ζ ICD) + DAP10 + 2B4 (Structure 11) CAR(CD8TM+4-1BB ICD) + DAP10 + 2B4 (Structure 12) CAR(CD8TM+4-1BB+CD3ζ ICD) (Structure 13) Table 2. Structures of nucleic acid molecules encoding CAR peptides and adaptor peptides (listing proteins from which SP, hinge, TM, and ICD regions are derived)

[0273] To generate CAR+DAP10-ICD immune cells, retroviral transduction was used to activate cells. This transduction process achieved the expression of the desired CAR+DAP10+ICD immune cells.

[0274] 4.2 Transduction efficiency and its effect on cell proliferation assay

[0275] The transduction efficiency was tested as described in Examples 1-3. Figures 16 to 18 The proliferation of immune cells with different structures was shown. The results indicate that the structures in 4.1 were all efficiently produced with high transduction efficiency.

[0276] 4.3 Immune cell activation and cytotoxicity

[0277] To evaluate the cytotoxicity of the modified cells, we used two liver cancer cell lines: hepG2, which showed high expression of both GPC3 and MICA / B, and sk-hep1, which had very low GPC3 expression but high MICA / B expression.

[0278] For the CAR-γδT activation assay, the activation marker CD107a was selected. 1E5 target cells of each cell type were transferred to each well and co-cultured with CAR-T cells, with an effector-target (E:T) ratio of 1:0.5 for hepG2. After 1 hour of co-culture, brefeldin A (Biolegend, 420601) and monensin (Biolegend, 420701) were added to the system, followed by another 4 hours of co-culture. CD107a was detected using APC anti-human CD107a (Biolegend, 328620). Results are as follows: Figure 19 As shown, this indicates that γδT cells transfected with CAR+DAP10-ICD can be efficiently activated through targeted cell lines. For cytotoxicity assays, the results are as follows: Figures 20 to 23 As shown, the changes in CAR-ICD structure do not affect the function of DAP10-ICD. hepG2 and sk-hep1 can activate and kill modified immune cells with DAP10-ICD structure, but this does not occur in immune cells with only CAR.

[0279] Example 5: DAP10-ICD structure and cytokine modification are well compatible

[0280] 5.1 Expression of CAR+DAP10-ICD structure in expanded NK cells

[0281] The isolation and expansion of NK cells were the same as in Example 2. We selected GPC3 as the target for CAR therapy against liver cancer. Retroviruses carrying different CAR constructs were prepared, including: pMSCV-GC33-IL15RF (Structure 16) pMSCV-GC33-BBz-DAP10-2B4-IL5RF (Structure 17) pMSCV-GC33-2B4z-DAP10-41BB-IL5RF (Structure 18) pMSCV-GC33-z-DAP10-41BB-IL5RF (Structure 19) pMSCV-GC33-z-DAP10-2B4-41BB-IL5RF (Structure 20) pMSCV-GC33-BB-DAP10-2B4z-IL5RF (Structure 21) pMSCV-GC33-2B4-DAP10-41BBz-IL5RF (Structure 22) “z” refers to CD3z, and IL5RF is the membrane-bound form of IL-15.

[0282] Table 3. Structures of nucleic acid molecules encoding CAR peptides and adaptor peptides (listing proteins from which SP, hinge, TM, and ICD regions are derived)

[0283] To generate CAR+DAP10+ICD NK cells, activated NK cells were transduced using a retrovirus. This transduction process achieved the desired expression of CAR+DAP10+ICD NK cells.

[0284] 5.2 Transduction efficiency and its effect on cell proliferation assay

[0285] As described in Example 2, the transduction efficiency and proliferation were detected. Figure 24 The proliferation of CAR-NK cells with different structures was shown. Figure 25 CD56 was showcased + CAR expression in cells. The results showed that the DAP10+ICD had minimal impact on CAR-NK cell expansion. Furthermore, structures 16-22 were efficiently produced with high transduction efficiency.

[0286] 5.3 NK cell cytotoxicity

[0287] To evaluate the cytotoxicity of the modified cells, we used two liver cancer cell lines: hepG2, which showed high expression of both GPC3 and MICA / B, and sk-hep1, which had low GPC3 expression but high MICA / B expression.

[0288] The experiment was conducted as described in Example 2. The results showed... Figure 26 and Figure 27 The results indicate that the DAP10-ICD structure works well with IL15 expression.

[0289] Example 6: The full-length DAP10 structure is not essential for the functionality of the DAP10-ICD structure.

[0290] 6.1 Expression of CAR+DAP10-ICD structure in expanded NK cells

[0291] The NK cell isolation and expansion procedures were the same as in Example 2. We selected GPC3 as the target for CAR therapy against liver cancer. Retroviruses carrying different CAR constructs were prepared, including: pMSCV-GC33-BBz-sIL15 (Structure 23) pMSCV-GC33-BBz-DAP10-2B4-sIL15 (Structure 24) pMSCV-GC33-BBz-DAP10(ECDdel)-2B4-sIL15 (Structure 25) pMSCV-GC33-BBz-DAP10-2B4(ITSM2)-sIL15 (Structure 26) pMSCV-GC33-BBz-DAP10(SP)-2B4-sIL15 (Structure 27) sIL15 is the secretory soluble form of IL-15.

[0292] Table 4. Structures of nucleic acid molecules encoding CAR peptides and adaptor peptides (listing proteins from which SP, hinge, TM, and ICD regions are derived).

[0293] To generate CAR+DAP10+ICD NK cells, activated NK cells were transduced using a retrovirus. This transduction process achieved the desired expression of CAR (full-length)+DAP10+ICD NK cells.

[0294] 6.2 Transduction efficiency and its effect on cell proliferation assay

[0295] As described in Example 2, the transduction efficiency and proliferation were detected. Figure 28 The proliferation of CAR-NK cells with different structures was shown. Figure 29 CD56 was showcased + CAR expression in cells. The results showed that the DAP10+ICD had minimal impact on CAR-NK cell expansion. Furthermore, structures 23-27 were efficiently produced with high transduction efficiency.

[0296] 6.3 NK cell cytotoxicity

[0297] To evaluate the cytotoxicity of the modified cells, we used two liver cancer cell lines: hepG2, which showed high expression of both GPC3 and MICA / B, and sk-hep1, which had low GPC3 expression but high MICA / B expression.

[0298] The experiment was conducted as described in Example 2. The results are as follows: Figure 30 and 31 As shown, this indicates that altering the DAP10 SP or DAP10 hinge does not affect the functionality of the DAP10-ICD structure. This result is consistent with previous reports that NKG2D and DAP10 form trimers through their transmembrane structures.

Claims

1. An expression construct comprising a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) polypeptide and a second nucleic acid sequence encoding a recombinant polypeptide containing a DAP10 transmembrane region, wherein: The DAP10 transmembrane region is derived from human DAP10 and can interact with NKG2D. The first nucleic acid sequence is separated from the second nucleic acid sequence by a nucleotide sequence encoding a cleavable adapter.

2. The expression construct of claim 1, wherein the DAP10 transmembrane region comprises an amino acid sequence having at least 90% identity with SEQ ID No:

9.

3. The expression construct of claim 1 or 2, wherein the recombinant polypeptide further comprises one or more of a signal peptide, a hinge region, an intracellular region, and an affinity tag.

4. The expression construct of claim 3, wherein the intracellular region comprises a DAP10 intracellular domain (ICD) derived from human DAP10, optionally, the DAP10 ICD comprises the amino acid sequence of SEQ ID No: 10 or an amino acid sequence having at least 85%, 90% or 95% identity with SEQ ID No:

10.

5. The expression construct of claim 3 or 4, wherein the signal peptide is derived from human DAP10, T cell surface expression receptors (such as CD8, CD28 and TCR), NK cell surface expression receptors (2B4, CD16, NKP30, NKP44, NKP46) or IgG.

6. The expression construct according to any one of claims 3-5, wherein the hinge region is derived from human DAP10, TCR, or immunoglobulin.

7. The expression construct of any one of claims 3-6, wherein the affinity tag is located at the N-terminus of the transmembrane region of the DAP10.

8. The expression construct of any one of claims 3-7, wherein the recombinant polypeptide comprises the DAP10 transmembrane region and the intracellular region from the N-terminus to the C-terminus.

9. The expression construct of claim 8, wherein the recombinant polypeptide comprises, from the N-terminus to the C-terminus, the signal peptide, the hinge region, the DAP10 transmembrane region, and the intracellular region, optionally having an affinity tag (such as an HA tag) between the signal peptide and the hinge region.

10. The expression construct of claim 8, wherein the recombinant polypeptide comprises, from the N-terminus to the C-terminus, the signal peptide, the DAP10 transmembrane region, and the intracellular region, optionally having an affinity tag (such as an HA tag) between the signal peptide and the DAP10 transmembrane region.

11. The expression construct of any one of claims 3-10, wherein the intracellular region further comprises one or more co-stimulatory signal transduction domains derived from any of the following: ligands of CD28, 4-1BB, 2B4, CD27, OX40, CD30, CD40, CD3, LFA-1, ICOS (CD278), NTBA, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and combinations thereof.

12. The expression construct of any one of claims 3-11, wherein the intracellular region further comprises a primary signal transduction domain.

13. The expression construct of claim 12, wherein the primary signal transduction domain is derived from CD3ζ, and optionally, the primary signal transduction domain comprises the amino acid sequence of SEQ ID No: 5 or an amino acid sequence having at least 85%, 90% or 95% identity with SEQ ID No:

5.

14. The expression construct of any one of claims 1-13, wherein the recombinant polypeptide comprises, from the N-terminus to the C-terminus: (a) DAP10 signal peptide (SP), DAP10 hinge region, DAP10 transmembrane region (TM) and co-stimulatory signal transduction domain, optionally including primary signal transduction domain; (b) DAP10 SP, DAP10 TM and costimulatory signaling domains, optionally including primary signaling domains; (c) CD28 SP, DAP10 hinge region, DAP10 TM, and co-stimulatory signal transduction domain, optionally including the primary signal transduction domain; or (d) CD28 SP, DAP10 TM and co-stimulatory signal transduction domains, optionally including primary signal transduction domains.

15. The expression construct of claim 14, wherein the recombinant polypeptide comprises, from the N-terminus to the C-terminus: (a) DAP10 signal peptide (SP), DAP10 hinge region, DAP10 transmembrane region (TM), DAP10 intracellular domain (ICD) and 2B4 co-stimulatory signal transduction domain; (b) DAP10 SP, DAP10 hinge region, DAP10 TM, DAP10 ICD and 4-1BB co-stimulatory signal transduction domains; (c) DAP10 SP, DAP10 hinge region, DAP10 TM, DAP10 ICD, 2B4 costimulatory signal transduction domain and 4-1BB costimulatory signal transduction domain; (d) DAP10 SP, DAP10 hinge region, DAP10 TM, DAP10 ICD, 2B4 co-stimulatory signal transduction domain and CD3ζ signal transduction domain; (e) DAP10 SP, DAP10 hinge region, DAP10 TM, DAP10 ICD, 4-1BB co-stimulatory signal transduction domain and CD3ζ signal transduction domain; (f) DAP10 SP, DAP10 hinge region, DAP10 TM, DAP10 ICD and 2B4·ITSM2 co-stimulatory signal transduction domains; (g) DAP10 SP, DAP10 hinge region, DAP10 TM, DAP10 ICD and CD28 co-stimulatory signal transduction domain; (h) CD8 SP, DAP10 hinge region, DAP10 TM, DAP10 ICD and 2B4 co-stimulatory signal transduction domain; (i) CD8 SP, DAP10 TM, DAP10 ICD, and 2B4 co-stimulatory signal transduction domains; or (j) CD8 SP, DAP10 hinge region, DAP10 TM, DAP10 ICD and 2B4·ITSM2 co-stimulatory signal transduction domains.

16. The expression construct of claim 14 or 15, wherein the DAP10 SP comprises the amino acid sequence of SEQ ID No: 6, the CD8 SP comprises the amino acid sequence of SEQ ID No: 19, the DAP10 hinge region comprises the amino acid sequence of SEQ ID No: 8, the DAP10 TM comprises the amino acid sequence of SEQ ID No: 9, and / or the DAP10 ICD comprises the amino acid sequence of SEQ ID No:

10.

17. The expression construct of any one of claims 1-16, wherein the cleavable linker is selected from P2A, E2A, F2A, T2A peptide, IRES sequence and functional variants thereof.

18. The expression construct of any one of claims 1-17, wherein the CAR polypeptide comprises an extracellular antigen-binding domain, a transmembrane domain and an intracellular signal transduction domain, and the extracellular antigen-binding domain is selected from single-chain Fv (scFv), Fab, Fab', F (ab')2, Fv, microantibodies, bispecific antibodies, single-domain antibodies (sdAb), or VHH domains, such as scFv.

19. The expression construct of claim 18, wherein the extracellular antigen-binding domain is a scFv targeting GPC3, CD19, or CD33, for example, the GPC3-targeting scFv comprising the amino acid sequence of SEQ ID No:

1.

20. The expression construct of any one of claims 18-19, wherein the transmembrane domain of the CAR peptide is derived from any one of CD8, ICOS, CD4, CD28, CD137, CD80, CD86, CD152 and PD1.

21. The expression construct of claim 20, wherein the transmembrane domain of the CAR peptide is derived from CD8 or CD28, and optionally, the transmembrane domain comprises the amino acid sequence of SEQ ID No: 3 or 22.

22. The expression construct of claim 18, wherein the intracellular signal transduction domain of the CAR peptide comprises a primary intracellular signal transduction domain, a co-stimulatory signal transduction domain, or both.

23. The expression construct of claim 22, wherein the primary intracellular signal transduction domain of the CAR peptide is derived from CD3ζ, optionally, the primary intracellular signal transduction domain comprises the amino acid sequence of SEQ ID No: 5 or an amino acid sequence having at least 85%, 90% or 95% identity with SEQ ID No:

5.

24. The expression construct of any one of claims 22-23, wherein the co-stimulatory signal transduction domain of the CAR peptide is derived from ligands selected from co-stimulatory molecules including CD28, 4-1BB (CD137), 2B4, CD27, OX40, CD30, CD40, CD3, LFA-1, ICOS (CD278), NTBA, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and combinations thereof.

25. The expression construct of claim 24, wherein the co-stimulatory signal transduction domain of the CAR peptide is derived from 4-1BB, 2B4, or CD28, optionally, the co-stimulatory signal transduction domain comprises an amino acid sequence of any one of SEQ ID No: 4, 11-12, and 21 or an amino acid sequence having at least 85%, 90%, or 95% identity with any one of SEQ ID No: 4, 11-12, and 21.

26. The expression construct of any one of claims 17-25, wherein the CAR polypeptide further comprises: A hinge domain, located between the extracellular antigen-binding domain and the transmembrane domain, optionally derived from CD8 or CD28; and / or A signal peptide located at the N-terminus of the extracellular antigen-binding domain, optionally, the signal peptide is derived from CD8.

27. The expression construct of any one of claims 1-26, further comprising a third nucleic acid sequence encoding a cytokine polypeptide (e.g., wild-type IL-15, IL-2, IL-4, IL-7, IL-21, IL-23 or variants thereof), wherein the third nucleic acid sequence is separated from the second nucleic acid sequence or the first nucleic acid sequence by a nucleotide sequence encoding a second cleavable linker.

28. The expression construct of claim 27, wherein the cytokine polypeptide comprises membrane-bound IL-15 having the amino acid sequence of SEQ ID No: 15 or soluble IL-15 having the amino acid sequence of SEQ ID No:

16.

29. The expression construct of claim 28, wherein the second cleavable linker is selected from P2A, E2A, F2A, T2A peptide, IRES sequence and functional variants thereof.

30. A vector comprising the expression construct of any one of claims 1-29.

31. The vector of claim 30, wherein the vector is a viral vector, such as an adenovirus vector, an adeno-associated virus vector, a lentiviral vector, or a retroviral vector, or a non-viral vector, such as a plasmid, liposome, nanoparticle, lipid, or a combination thereof.

32. An engineered immune cell comprising the expression construct of any one of claims 1-29, or the vector of claim 30 or 31.

33. The engineered immune cell of claim 32, wherein the immune cell is a natural killer (NK) cell, a T cell, a γδ T cell, an invariant NKT (iNKT) cell, a B cell, a macrophage, an MSC, or a dendritic cell.

34. The immune cells of claim 33, wherein the NK cells are derived from umbilical cord blood, peripheral blood, induced pluripotent stem cells, human embryonic stem cells, bone marrow, or cell lines.

35. A population of immune cells according to any one of claims 32-34, wherein said cells are present in a suitable medium.

36. A recombinant polypeptide encoded by a second nucleic acid sequence of the expression construct according to any one of claims 1-29.

37. A pharmaceutical composition comprising the engineered immune cells and a pharmaceutically acceptable carrier as described in any one of claims 32-34.

38. A method for preventing or treating cancer in a subject in need, comprising administering to the subject an effective amount of an immune cell of any one of claims 32-34 or an expression construct of any one of claims 1-29, optionally wherein the immune cell is allogeneic relative to the subject.

39. The method of claim 38, wherein the cancer is selected from multiple myeloma (MM), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), glioma, breast cancer, cervical cancer, prostate cancer, kidney cancer, gastric cancer, esophageal cancer, pancreatic ductal carcinoma, lung cancer such as non-small cell lung cancer (NSCLC), ovarian cancer, colorectal cancer, liver cancer, head and neck cancer, and gallbladder cancer.

40. The immune cell of any one of claims 32-34 or the expression construct of any one of claims 1-29, for use in treating cancer in subjects in need.

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