WO2025140265A1

WO2025140265A1 - Combination of chimeric antigen receptor and dap10 in cell therapy

Info

Publication number: WO2025140265A1
Application number: PCT/CN2024/142173
Authority: WO
Inventors: Qian CUI; Hong Wang; Qiong Wu; Yong Zheng; Jijie Gu
Original assignee: Wuxi Biologics Shanghai Co Ltd; Wuxi Biologics Ireland Ltd
Current assignee: Wuxi Biologics Shanghai Co Ltd; Wuxi Biologics Ireland Ltd
Priority date: 2023-12-26
Filing date: 2024-12-25
Publication date: 2025-07-03
Anticipated expiration: 2026-06-26

Abstract

Provided are expression constructs encoding chimeric antigen receptor and DAP10 recombinant polypeptide, engineered immune cells, and methods of use thereof. Further provided are activation and expansion of cells for therapeutic uses, especially for chimeric antigen receptor-based immune cell immunotherapy.

Description

COMBINATION OF CHIMERIC ANTIGEN RECEPTOR AND DAP10 IN CELL THERAPY

CROSS-REFERENCING

This application claims the benefit of International application PCT/CN2023/141856, filed on December 26, 2023, which is incorporated by reference in its entirety.
SEQUENCE LISTING

The instant application contains a sequence listing which has been submitted electronically and is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to chimeric antigen receptor constructs, engineered immune cells, and methods of use thereof. The present disclosure further relates to activation and expansion of cells for therapeutic uses, especially for chimeric antigen receptor-based immune cell immunotherapy.

BACKGROUND

Cancer is a group of diseases characterized by abnormal cell growth, capable of invading and spreading to other parts of the body. Despite significant advancements in cancer therapy, it remains a leading cause of death. Harnessing the potential of the immune system has become a key focus in cancer treatment, leading to the development of NK, T, and γδT cell therapies. While chimeric antigen receptor (CAR) modified immune cells have shown promising effects against certain cancers, their efficacy in most tumors remains inadequate.

T lymphocytes employed in CAR-T therapy primarily refer to CD3⁺ T cells, with CD4⁺ and CD8⁺ T cells being the most prominent subsets. The application of chimeric antigen receptor modified T cells has yielded remarkable clinical responses, resulting in the approval of six CAR T-cell therapies by the FDA since 2017, with hundreds of clinical trials registered.

NK cells, a distinct subpopulation of lymphocytes, were first identified in 1975. These cells originate from CD34⁺ hematopoietic progenitor cells through down-regulation of CD34 and up-regulation of CD56. An advantage of NK cell therapy over T cell therapy is that NK cells can be readily available off the shelf. Numerous NK therapeutics, including natural NK cells and CAR-NK cells, have been employed in clinical trials, showcasing remarkable results. Although no NK cell therapeutic has received FDA approval thus far, clinical experiments have demonstrated promising outcomes.

Gamma delta (γδ) T cells constitute a subset of CD3+ T cells distinguished by the expression of Vγ (γ-2, 3, 4, 5, 8, and 9) and Vδ (δ-1, 2, 3, 4, 5, 6, 7, and 8) chains, which form a heterodimeric γδ T cell receptor (TCR) . In contrast to conventional αβ T cells that rely on MHC (Major Histocompatibility Complex) -dependent antigen processing for target recognition, γδ T cells typically recognize target cells in an MHC-independent manner. Accumulating evidence indicates that both subtypes of γδ T cells play critical roles in tumor immune surveillance and anti-tumor immune responses.

Tumor immune escape represents a major hurdle in cancer therapy, including CAR therapies. Escape mechanisms encompass various factors such as CAR target downregulation, major histocompatibility complex (MHC) defects, and the presence of local immunosuppressive microenvironments. While the precise target recognition of CARs ensures their safety as therapeutic agents, target downregulation could otherwise be a significant limiting factor.

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. The ligands of NKG2D 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. Such features make NKG2D one of the most important activators in immune cells, playing a significant role in natural anti-tumor processes.

In human cells, the signal mediated by NKG2D is specifically transmitted through DAP10. DAP10, also known as Hematopoietic Cell Signal Transducer, is a transmembrane signaling adaptor containing an YxxM motif in its cytoplasmic domain. The signal transduction is accomplished through the assembly of two DAP10 dimers and one NKG2D receptor. The presence of a pair of aspartic acid residues near the center of the transmembrane domain of the DAP10 dimer, along with the conserved arginine in the NKG2D transmembrane domain, is necessary and sufficient for the assembly of the signal transfer process.

To address this limitation, the present invention utilizes DAP10 to enhance immune cell function by providing an additional signaling pathway alongside CAR activation. An improved immune cell therapy and related nucleic acid constructs utilizing the interaction of NKG2D and DAP10 are provided.

SUMMARY

In the present application, recombinant polypeptides (also designated herein as “adaptor polypeptide” ) comprising a DAP-10 transmembrane (TM) domain and an intracellular signaling domain in various formats are designed, the expression of which enable immune cells to trigger a robust immune response against various cancers via binding to NKG2D. Further, by combining these adaptor polypeptides with chimeric antigen receptors (CARs) targeting the desired antigens in an expression construct, the inventors discover that the immune cells expressing the CAR polypeptide and the adaptor polypeptide exhibit a dual specificity against cancer cells both in vitro and in vivo, and the combination of the DAP10 TM-comprising adaptor polypeptide with the CAR polypeptide can inhibit or eliminate cancer cells that express the antigens more efficiently.

In one aspect, the present disclosure provides an expression construct, comprising a first nucleic acid sequence that encodes a chimeric antigen receptor (CAR) polypeptide and a second nucleic acid sequence that encodes a recombinant polypeptide comprising a DAP10 transmembrane region, wherein
the DAP10 transmembrane region is derived from human DAP10 and can interact with NKG2D, and
the first nucleic acid sequence is separated from the second nucleic acid sequence by a nucleotide
sequence encoding a cleavable linker.

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

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

In some embodiments, the recombinant polypeptide comprises an intracellular region comprising 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 at least 85%, 90%or 95%identical to SEQ ID No: 10.

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

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

In some embodiments, the recombinant polypeptide comprises an affinity tag located in the extracellular region, such as at the N terminal of the transmembrane region. More specifically, the affinity tag may be located between the signal peptide and the hinge region.

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

In some embodiments, the intracellular region further comprises one or more costimulatory signaling domains derived from any of CD28, 4-1BB, 2B4, 2B4·ITSM2, CD27, OX40, CD30, CD40, CD3, LFA-1, ICOS (CD278) , NTBA, CD2, CD7, LIGHT, NKG2C, B7-H3, ligands of CD83 and combinations thereof.

In some embodiments, the intracellular region further comprises a primary signaling domain. The primary signaling domain may be derived from CD3ζ, optionally the primary signaling domain comprises the amino acid sequence of SEQ ID No: 5 or an amino acid sequence at least 85%, 90%or 95%identical to SEQ ID No: 5.

In some embodiments, the recombinant polypeptide comprises, from N terminal to C terminal:
(a) DAP10 signal peptide (SP) , DAP10 hinge region, DAP10 transmembrane region (TM) and a costimulatory
signaling domain, optionally a primary signaling domain;
(b) DAP10 SP, DAP10 TM and a costimulatory signaling domain, optionally a primary signaling domain;
(c) CD8 SP, DAP10 hinge region, DAP10 TM and a costimulatory signaling domain, optionally a primary
signaling domain; or
(d) CD8 SP, DAP10 TM and a costimulatory signaling domain, optionally a primary signaling domain.

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

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

In some embodiments, the cleavable linker is selected from P2A, E2A, F2A, T2A peptide, an Internal Ribosomal Entry Site (IRES) sequence and a functional variant thereof.

In some embodiments, the recombinant polypeptide comprises or consists of the amino acid sequence of any of SEQ ID Nos: 23-32 or an amino acid sequence at least 80% (e.g. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%) identical to any of SEQ ID Nos: 23-32.

In some embodiments, the CAR polypeptide comprises an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain. In some embodiments, the extracellular antigen binding domain is selected from a single-chain Fv (scFv) , a Fab, a Fab’ , a F (ab’ ) 2, an Fv, minibody, a diabody, a single-domain antibody (sdAb) or VHH domain, such as a scFv, optionally the extracellular antigen binding domain is targeted to GPC3, CD19 or CD33, e.g. the scFv targeting GPC3 comprises the amino acid sequence of SEQ ID No: 1, the scFv targeting CD19 comprises the amino acid sequence of SEQ ID No: 13, the scFv targeting CD33 comprises the amino acid sequence of SEQ ID No: 14.

In some embodiments, the transmembrane domain of the CAR polypeptide is derived from any of CD8, ICOS, CD4, CD28, CD137, CD80, CD86, CD152 and PD1. In some embodiments, the transmembrane domain of the CAR polypeptide is derived from CD8 or CD28, optionally the transmembrane domain comprises the amino acid sequence of SEQ ID No: 3 or 22 or an amino acid sequence at least 85%, 90%or 95%identical to SEQ ID No: 3 or 22.

In some embodiments, the primary intracellular signaling domain of the CAR polypeptide is derived from CD3ζ, optionally the primary intracellular signaling domain comprises the amino acid sequence of SEQ ID No: 5 or an amino acid sequence at least 85%, 90%or 95%identical to SEQ ID No: 5.

In some embodiments, the co-stimulatory signaling domain of the CAR polypeptide is derived from a co-stimulatory molecule selected from CD28, 4-1BB, 2B4 (e.g. 2B4·ITSM2) , CD27, OX40, CD30, CD40, CD3, LFA-1, ICOS (CD278) , NTBA, CD2, CD7, LIGHT, NKG2C, B7-H3, ligands of CD83 and combinations thereof. In some embodiments, the co-stimulatory signaling domain of the CAR polypeptide is derived from 4-1BB, 2B4 or CD28, optionally, the co-stimulatory signaling domain comprises any of the amino acid sequences of SEQ ID Nos: 4, 11-12 and 21 or an amino acid sequence at least 85%, 90%or 95%identical to any of SEQ ID Nos: 4, 11-12 and 21.

In some embodiments, the CAR polypeptide further comprises:
a hinge domain located between the extracellular antigen binding domain and the transmembrane domain,
optionally the hinge domain is 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.

In some embodiments, the CAR polypeptide comprises or consists of the amino acid sequence of any of SEQ ID Nos: 33-38 or an amino acid sequence at least 80% (e.g. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%) identical to any of SEQ ID Nos: 33-38.

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.

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

In some embodiments, the second cleavable linker is selected from P2A, E2A, F2A, T2A peptide, IRES sequence and a functional variant thereof.

The positions of the first, the second and the third (if present) nucleic acid sequences in the expression construct may be switched. In some embodiments, the expression construct comprises, from 5’ to 3’:
(a) the first nucleic acid sequence, the second nucleic acid sequence and the third nucleic acid sequence;
(b) the first nucleic acid sequence, the third nucleic acid sequence and the second nucleic acid sequence;
(c) the second nucleic acid sequence, the first nucleic acid sequence and the third nucleic acid sequence;
(d) the second nucleic acid sequence, the third nucleic acid sequence and the 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.

In some embodiments, the three nucleic acid sequences are in operably linkage and are separated by a nucleotide sequence encoding a self-cleavable peptide (such as P2A, E2A, F2A or T2A) or an IRES sequence.

In one aspect, the present disclosure provides a recombinant polypeptide comprising a DAP10 transmembrane region derived from human DAP10 and can interact with NKG2D, and one or more intracellular signaling domain. In some embodiments, the present disclosure provides a recombinant polypeptide encoded by the second nucleic acid sequence of the expression construct as disclosed herein.

In one aspect, the present disclosure provides a vector comprising the expression construct as disclosed herein. The vector may be a viral vector such as an adenoviral vector, adeno-associated viral vector, lentiviral vector or retroviral vector, or a non-viral vector such as a plasmid, liposome, nanoparticle, lipid or combination thereof.

In one aspect, the present disclosure provides an engineered immune cell comprising or expressing the expression construct or the vector as disclosed herein.

In some embodiments, the immune cell is a natural killer (NK) cell, T cell, gamma delta T cells, invariant NKT (iNKT) cell, B cell, macrophage, MSCs, or dendritic cell. In some embodiments, the NK cell is derived from cord blood, peripheral blood, induced pluripotent stem cells, human embryonic stem cells, bone marrow, or from a cell line.

In one aspect, the present disclosure provides a population of the immune cell as disclosed herein, wherein the cells present in a suitable medium.

In one aspect, the present disclosure provides a pharmaceutical composition comprising the engineered immune cells as disclosed herein, and a pharmaceutically acceptable carrier.

In one aspect, the present disclosure provides a method for preventing or treating a cancer in a subject in need thereof, comprising administering an effective amount of the immune cells or the expression construct as disclosed herein to the subject. The immune cells may be allogeneic or autologous with respect to the subject.

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

In one aspect, the present disclosure provides the expression construct or the immune cells as disclosed herein for use in treating a tumor in a subject in need thereof.

In one aspect, the present disclosure provides use of the expression construct or the engineered immune cell as disclosed herein in the manufacture of a medicament for treating cancer in a subject. The subject may be a mammal such as human or non-human animals.

In one aspect, the present disclosure provides a method for activating an immune cell, comprising expressing the expression construct as disclosed herein in the immune cell, wherein the activation occurs in response to the CAR engaging a corresponding target molecule. In some embodiments, the immune cell, or a plurality thereof, are introduced to a subject in need thereof; and the activation occurs in the subject.

Other features, objects, and advantages will be apparent from the disclosure that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a structural diagram of the CAR polypeptide and the adaptor polypeptide as disclosed herein.

FIG. 2 shows the proliferation of T cells transfected with structures 1-8.

FIG. 3 shows the expression of structure 1-8 and CD4/CD8 percentage of T cell.

FIG. 4 shows the expression of MICA/B and GPC3 in hepG2 and sk-hep1. (A) MICA/B expression in hepG2 (a) and sk-hep1 (b) ; (B) GPC3 expression in hepG2 (a) and sk-hep1 (b) .

FIG. 5 shows CAR-T cell cytotoxicity against hepG2 cells (co-culture 2 days) .

FIG. 6 shows CAR-T cell cytotoxicity against sk-hep1 cells (co-culture 1 day) .

FIG. 7 shows CAR-T cell cytotoxicity against sk-hep1 cells (co-culture 2 days) .

FIG. 8 shows the proliferation of NK cells transfected with structures 1-7.

FIG. 9 shows the expression of structures 1-7 in NK cells.

FIG. 10 shows CAR-NK cell cytotoxicity against hepG2 cells.

FIG. 11 shows CAR-NK cell cytotoxicity against sk-hep1 cells.

FIG. 12 shows the proliferation of γδT cells transfected with structures 1, 5, 6.

FIG. 13 shows the subtype and structures’ expression of γδT cells.

FIG. 14 shows CAR-γδT cell cytotoxicity against hepG2 cells.

FIG. 15 shows CAR-γδT cell cytotoxicity against sk-hep1 cells.

FIG. 16 shows the subtype and structures’ expression of T cells transfected with structure 1, 2 and 9-12 in Example 4.

FIG. 17 shows the subtype and structures’ expression of NK cells transfected with structure 1, 2 and 9-12 in Example 4.

FIG. 18 shows the subtype and structures’ expression of γδT cells transfected with structure 9, 11 and 13 in Example 4.

FIG. 19 shows the activation of γδT cells by hepG2.

FIG. 20 shows CAR-T cell (structure 1, 2 and 9-12) cytotoxicity against hepG2.

FIG. 21 shows CAR-T cell (structure 1, 2 and 9-12) cytotoxicity against sk-hep1.

FIG. 22 shows CAR-NK cell (structure 1, 2 and 9-12) cytotoxicity against sk-hep1.

FIG. 23 shows CAR-γδT cell (structure 9, 13) cytotoxicity against sk-hep1.

FIG. 24 shows the proliferation of NK cells transfected with structures 16-22.

FIG. 25 shows the CAR expression in CD56⁺ cells.

FIG. 26 shows CAR-NK cell cytotoxicity against hepG2.

FIG. 27 shows CAR-NK cell cytotoxicity against sk-hep1.

FIG. 28 shows the proliferation of NK cells transfected with structures 23-27.

FIG. 29 shows expression of the structures 23-27 of NK cells.

FIG. 30 shows CAR-NK cell cytotoxicity against hepG2.

FIG. 31 shows CAR-NK cell cytotoxicity against sk-hep1.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms “a” , “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a protein” includes a plurality of proteins; reference to “acell” includes mixtures of cells, and the like. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “comprise” as well as other equivalents, such as “contain" and “include” is not limiting. In addition, ranges provided in the specification and appended claims include both end points and all points between the end points.

As used herein, the term “DNAX-activating protein 10” or “DAP10” refers to the transmembrane adaptor protein for natural killer group 2, member D (NKG2D) . DAP10 protein is present in lymphoid and myeloid cells of mammals whose exact sequence might vary based on the species, isoform and from individual to individual. The membrane localization and signal transduction of NKG2D depend on DAP10 protein. Human DAP10 protein may 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 is amino acids 1-18, the hinge region is amino acids 19-48, the transmembrane region is amino acids 49-69 and the intracellular region is amino acids 70-93. Within the DAP10 cytoplasmic region, an SH2 domain-binding site was capable of recruiting the p85 subunit of the phosphatidylinositol 3-kinase, providing for NKG2D-dependent transduction. Amino acid sequences of human DAP10 may be represented by the predominant polypeptide sequence UnitProt Q9UBK5 and NCBI accession NP 055081.1 and AF072845; however, different isoforms and variants may exist.

As used herein, the term “DAP10 transmembrane domain” encompasses the transmembrane domain of human DAP10 and any homologous variants thereof which can harness NKG2D receptor. The presence of a transmembrane region in the adaptor polypeptide which can interact with NKG2D is crucial for its function.

The term “hinge region” , as used herein, refers to a short sequence that may be present at the N terminal of the transmembrane region in the adaptor polypeptide or the CAR polypeptide to increase their structural flexibility. The hinge region may consist of at least 2 (e.g., 5, 10 or more) amino acids which result in a flexible or semi-flexible linkage between the transmembrane domain and the signal peptide in a single polypeptide molecule.

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 serves as a primary activating receptor wherein ligand binding triggers cytotoxicity and cytokine production. NKG2D provides costimulation through an associated adapter molecule, DAP10, which recruits phosphatidylinositol-3 kinase. In mice, NKG2D also associates with DAP12, which recruits protein tyrosine kinases. In humans, NKG2D is expressed by NK cells, γδ T cells and CD8⁺ αβ T cells, and CD4⁺ T cells under certain pathological conditions (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, activated CD8⁺ 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 proteins (ULBPs) in humans, and retinoic acid early transcript-1, minor histocompatibility antigen H-60 and murine ULBP-like transcript-1 in mice. For example, NKG2D-DAP10 receptor complexes may activate NK and T-cell responses against MICA-bearing tumors.

As used herein, the term “chimeric antigen receptor” or “CAR” refers to a recombinant polypeptide construct comprising an extracellular antigen binding moiety that provides the antigen-specificity, a transmembrane domain that anchors the CAR in the cellular plasma membrane, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. Immune cells, in particular T and NK lymphocytes, can be genetically modified to express CARs inserted into their plasma membrane. If such a CAR modified immune cell encounters other cells or tissue structures expressing or being decorated with the appropriate target of the CAR’s antigen binding moiety, upon binding of the binding moiety to the target antigen, the CAR modified immune cell is cross-linked to the target. Cross-linking leads to an induction of signal pathways via the CAR signaling chains, which will change the biologic properties of the CAR engrafted immune cell. The domains in the CAR construct may be in the same polypeptide chain, e.g., comprise a chimeric fusion protein. Alternatively, the domains in the CAR construct may be not contiguous with each other, e.g., are in different polypeptide chains, e.g., as provided in a split CAR.

The term “intracellular signaling domain” , as used herein, refers to an intracellular portion of a CAR that can generate a signal that promotes an immune effector function of the CAR containing cell, e.g., a CAR-T cell or CAR-expressing NK cell. Examples of immune effector function, e.g., in a CAR-T cell or CAR-expressing NK cell, include cytolytic activity and helper activity, including the secretion of cytokines. The intracellular signaling domain may transduce the effector function signal and directs the cell to perform a specialized function. While the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. The intracellular signaling domain may comprise a primary intracellular signaling domain and optionally further comprises co-stimulatory signaling domain.

The term “primary intracellular signaling domain” refers to cytoplasmic signaling sequence that acts in a stimulatory manner to induce immune effector functions. The primary intracellular signaling domain may contain a signaling motif known as immunoreceptor tyrosine-based activation motif, or ITAM. An “ITAM” as used herein, is a conserved protein motif that is generally present in the tail portion of signaling molecules expressed in many immune cells. ITAMs within signaling molecules are important for signal transduction within the cell, which is mediated at least in part by phosphorylation of tyrosine residues in the ITAM following activation of the signaling molecule. ITAMs may also function as docking sites for other proteins involved in signaling pathways.

The term “co-stimulatory signaling domain” refers to the intracellular portion of a costimulatory molecule. The intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment thereof. Costimulatory molecules are the cognate binding partner on immune cell (e.g. NK and T cells) that specifically bind with a costimulatory ligand, thereby mediating a costimulatory response by the immune cells, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response. Costimulatory molecules include, but are not limited to an a MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins) , activating NK cell receptors, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CDl la/CD18) , 4-1BB (CD137) , B7-H3, CDS, ICAM-1, ICOS (CD278) , GITR, BAFFR, LIGHT, HVEM (LIGHTR) , KIRDS2, SLAMF7, NKp80 (KLRF1) , NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, 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, CD 19a, and a ligand that specifically binds with CD83.

The term “immunoreceptor tyrosine-switch motif (ITSM) ” , as used herein, refers to a motif identified in the cytoplasmic regions of the signaling lymphocyte activation molecule (SLAM) family receptors, which are immunoglobulin-like type I transmembrane receptors that are present on cells throughout the hematopoietic system and are mostly engaged in homotypic interactions. For example, 2B4 belongs to the SLAM-related receptors and contains 4 immunoreceptor tyrosine-based switch motifs (ITSMs) in its cytoplasmic tail. In some cases, the ITSM within the cytoplasmic tail of 2B4 is sufficient for 2B4-mediated NK-cell activation.

As used herein, a “self-cleaving peptide” refers to an oligopeptide that allow multiple proteins to be encoded as polyproteins, which dissociate into component proteins upon translation, such as 2A linkers. Use of the term “self-cleaving” is not intended to imply a proteolytic cleavage reaction. Various self-cleaving or 2A linkers are known to those of skill in the art, including, without limitation, those found in members of the Picornaviridae virus family, e.g., foot-and-mouth disease virus (FMDV) , equine rhinitis A virus (ERAV0, Thosea asigna virus (TaV) , and porcine tescho virus-1 (PTV-1) ; and carioviruses such as Theilovirus and encephalomyocarditis viruses. 2A linkers derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as “F2A, ” “E2A, ” “P2A, ” and “T2A, ” respectively. The P2A linker may have a sequence that is 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%identical to the amino acid sequence of SEQ ID No: 17. The T2A linker may have a sequence that is 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%identical to the amino acid sequence of SEQ ID No: 18.

The terms “operably linked” refer to a juxtaposition, with or without a spacer or linker, of two or more biological sequences of interest in such a way that they are in a relationship permitting them to function in an intended manner. When used with respect to polypeptides, it is intended to mean that the polypeptide sequences are linked in such a way that permits the linked product to have the intended biological function. For example, the transmembrane region of the adaptor polypeptide may be “operably linked to” the intracellular region by a direct linkage or indirect linkage via a linker sequence, as long as the two parts can function normally. The term may also be used with respect to polynucleotides. For one instance, when a polynucleotide encoding a CAR polypeptide is operably linked to a polynucleotide encoding the adaptor polypeptide, it is intended to mean that the polynucleotide sequences are linked in such a way that permits normal expression of the polypeptides from the nucleic acid molecule.

The term “antibody” as used herein refers to a protein, or polypeptide sequence derived from an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be polyclonal or monoclonal, multiple or single chain, or intact immunoglobulins, and may be derived from natural sources or from recombinant sources. Antibodies can be tetramers of immunoglobulin molecules.

The term “antigen-binding domain” as used herein refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not comprise an intact native antibody structure. Examples of antigen-binding domain include, without limitation, a variable domain, a variable region, a single variable domain (i.e. VHH) , a nanobody, a domain antibody, a diabody, a Fab, a Fab', a F (ab') 2, an Fv fragment, a single chain Fv fragment (scFv) , a disulfide stabilized Fv fragment (dsFv) , a (dsFv) 2, a bispecific dsFv (dsFv-dsFv') , a disulfide stabilized diabody (ds diabody) , a multispecific antibody, a camelized single domain antibody, and a bivalent domain antibody. An antigen-binding domain is capable of binding to the same antigen to which the parent antibody binds. More detailed formats of antigen-binding domain are described in Spiess et al, (2015) Molecular Immunology 67: 95-106, and Brinkman et al., mAbs, 9 (2) , pp. 182–212 (2017) , which are incorporated herein by their entirety.

The term "complementarity determining region" or "CDR" , as used herein, refers to the sequences of amino acids within antibody variable domains which confer antigen specificity and binding affinity. For example, in general, there are three CDRs in each heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3) . The extent of CDRs and the framework region can be precisely identified using methodology known in the art, for example, by the Kabat definition, the definitions at Dr. Martin’s website, the Chothia definition, the AbM definition, the EU definition, and the contact definition, all of which are well known in the art. See, e.g., Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth 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 on 31 July 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 U S A. 1969 May; 63 (1) : 78-85; and Almagro, J. Mol. Recognit. 17: 132-143 (2004) . See also hgmp. mrc. ac. uk and bioinf. org. uk/abs. Correspondence or alignments between numberings according to different definitions can for example 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. Dev Comp Immunol. (2003) 27: 55–77) . In some instances, the scheme for identification of a particular CDR or CDRs is specified, such as the CDR as defined by the IMGT, Kabat, AbM, Chothia, or Contact method. One or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering. See, e.g., Deschacht et al., 2010. J Immunol 184: 5696-704 for an exemplary numbering for VHH domains according to Kabat. In other cases, the particular amino acid sequence of a CDR is given. It should be noted CDR regions may also be defined by a combination of various numbering systems, e.g., a combination of Kabat and Chothia numbering systems, a combination of Kabat and AbM numbering systems, or a combination of Kabat and IMGT numbering systems. Therefore, the term such as “a CDR as set forth in a specific VH, VL or VHH” includes any CDRs as defined by the exemplary CDR numbering systems described above, but is not limited thereby. Once a variable region (e.g., a VHH domain, a VH or VL domain) is given, those skilled in the art would understand that CDRs within the region can be defined by different numbering systems or combinations thereof.

The term “immune cell” as used herein refers to a cell that is part of the immune system and helps the body fight infections and other diseases. Immune cells include natural killer cells, invariant NK cells, NK T cells, T cells of any kind (e.g., regulatory T cells, CD4. sup. + T cells, CD8. sup. + T cells, or gamma-delta 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 (iPSC) cells) . CAR triggering in effector immune cells such as immune CD4⁺ and CD8⁺ T cells will activate typical effector functions like secretion of lytic compounds and cytokines which will eventually lead to the killing of the respective target cell. The adoptive transfer of immune cells engineered with CARs is currently considered as a highly promising therapeutic option for treatment of otherwise incurable malignant, infectious or autoimmune diseases.

The term “cancer” as used herein refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include but are not limited to, stomach cancer, pancreatic cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like. The terms "tumor" and "cancer" are used interchangeably herein.

The terms “patient” , “subject” , “individual” and the like are used interchangeably herein, and refer to any animal, amenable to the methods described herein. In certain nonlimiting embodiments, the patient, subject or individual is a human.
DAP10 and its interaction with NKG2D

DNAX-activating protein of 10 kDa (DAP10) is the adaptor molecule that associates with the cell surface, cytotoxic receptor natural killer group 2 member D (NKG2D) . NKG2D is an activating immune receptor which regulates both innate and adoptive immune responses. NKG2D is abundantly present on all NK cells, CD8⁺ T cells, subsets of γδ T cells and some autoreactive CD4⁺ 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 a number of structurally different MHC-I-like ligands. NKG2D ligands have in common that under homeostatic conditions their expression is generally low, but high expression in tumor or under stress. In humans, the NKG2D ligands are MICA, MICB, and six members of the ULBP family.

NKG2D lacks a signaling motif in its cytoplasmic domain. Signaling via NKG2D depends on its association with DAP10, a transmembrane adaptor molecule containing the sequence “YINM” , which signals via recruitment of phosphatidylinositol 3-kinase and Grb2 (growth factor receptor-bound protein 2) (Pedro R, et al. (2009) J Biol Chem. 284 (24) : 16463-16472) . The DAP10 adaptor molecule, capable of promoting and stabilizing the surface membrane expression of NKG2D (Wu, J., et al., (1999) ) . Accordingly, it is herein recognized that methodologies capable of modulating NKG2D and/or DAP10 expression and/or associated signaling pathways are of therapeutic interest.

The human NKG2D receptor assembles with the DAP10 signaling dimer, with one NKG2D homodimer paired with a DAP10 dimer by formation of two salt bridges between conserved transmembrane (TM) arginine residues (Garrity, D. et al. (2005) PNAS USA 102 (21) : 7641-7646) . The DAP10 dimer carries a pair of aspartic acid residues close to the center of the transmembrane (TM) domains, and these residues interact with the conserved arginine in the TM sequence of NKG2D for assembly with the DAP10 dimer. Thus, the NKG2D homodimer associates with the DAP10 adaptor molecule in its transmembrane domain to form a hexameric structure which can initiate signaling cascades.

As mentioned, the DAP10 dimer is a disulfide-linked homodimer. An exemplary amino acid sequence of the wild-type human DAP10 polypeptide is shown below as SEQ ID No: 20. The DAP10 cytoplasmic domain comprises a tyrosine-based motif (YINM) , as can be found at residues 86-89 of SEQ ID No:20. This YINM motif is similar to the motif in CD28, which provides for costimulatory signaling in conjunction with the immunoreceptor tyrosine-based activation motif (ITAM) -based TCR/CD3 complex in T cells. DAP10 further comprises a ubiquitinylation site that encompasses the lysine at amino acid 84 of the DAP10 protein sequence (SEQ ID No: 20) . Ligand stimulation of NKG2D on NK cells results in the ubiquitylation of DAP10, which is required for the endocytosis and degradation of the NKG2D-DAP10 complex (see e.g., Molfetta, R., et al. (2014) Eur. J. Immunol. 44, 2761-2770) .
Adaptor polypeptides comprising DAP10 TM domain

In one aspect, the disclosure provides a recombinant polypeptide (also designated as adaptor polypeptide) comprising: (i) a transmembrane (TM) domain from DAP10 or a functional variant thereof that can interact with NKG2D; and optionally one or more of the following: (ii) a signal peptide; (iii) a hinge region; (iv) an intracellular region. The intracellular region may comprise one or more costimulatory signaling domains (e.g., derived from 4-1BB, DAP10, OX40, 2B4, ICOS, CD28) and/or a primary signaling domain. The recombinant polypeptide as disclosed herein may be used as CAR adaptor to modulate and/or mute signaling through one or more receptors with which they associate.

In some embodiments, the adaptor polypeptide comprises a full length human DAP10 amino acid sequence, e.g. as shown in SEQ ID No: 20. Human DAP10 protein may be divided into four regions from N terminal to C terminal: a signal peptide, a hinge region, a transmembrane region and an intracellular region. The delineation of different regions in human DAP10 has been well-established in the art. Within the four regions, the transmembrane (TM) region is crucial to the interaction between DAP10 and NKG2D and is an indispensable portion in the adaptor polypeptide. The TM region is preferably from or derived from human DAP10, whereas the signal peptide, the hinge region and the intracellular region may be derived from a variety of molecules other than DAP10 as long as they can perform the desired functions. The adaptor polypeptide may comprise all the 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 to the amino acid sequence of SEQ ID No: 20. For example, a homologue of human DAP10 amino acid sequence may comprise amino acid substitutions at positions corresponding to K84 and/or Y86.

In some embodiments, the adaptor polypeptide may comprise a functional portion of full length DAP10 amino acid sequence. For example, the adaptor polypeptide may comprise only the transmembrane (TM) region derived from human DAP10, while the intracellular region, the signal peptide and the hinge region are absent. In some other embodiments, the DAP10 may comprise the transmembrane (TM) region derived from human DAP10 and an intracellular region, while the signal peptide and the hinge region are absent. Specifically, the TM region derived from human DAP10 may comprise the amino acid sequence as shown in SEQ ID No: 9 or a functional variant thereof that can interact with NKG2D. The functional variant may have an amino acid sequence having at least 85%, 90%, 95%, 97%, or 99%identity to SEQ ID No: 9. The intracellular region may comprise the intracellular domain (ICD) of human DAP10 or a functional variant thereof. The intracellular region may comprise the amino acid sequence as shown in SEQ ID No: 10 or an amino acid sequence having at least 85%, 90%, 95%, 97%, or 99%identity to SEQ ID No: 10. In some embodiments, the intracellular region further comprises one or more costimulatory signaling domains derived from e.g. 4-1BB, OX40, 2B4, ICOS, CD28. In some embodiments, the intracellular region further comprises a primary signaling domain, e.g. a CD3ζ signaling domain.

In some embodiments, the adaptor polypeptide further comprises a signal peptide at the N terminal. The signal peptide may be derived from DAP10, T cell surface expressed receptor (such as CD8, CD28 and TCR) , NK cell surface expressed receptor (2B4, CD16, NKP30, NKP44, NKP46) , or IgG. In some embodiments, the signal peptide may comprise a DAP10 signal peptide comprising the amino acid sequence as shown in SEQ ID No: 6 or an amino acid sequence at least 85%, 90%or 95%identical to SEQ ID No: 6. In some other embodiments, the signal peptide may comprise a CD8 signal peptide comprising the amino acid sequence as shown in SEQ ID No: 19 or an amino acid sequence at least 85%, 90%or 95%identical to SEQ ID No: 19.

In some embodiments, the adaptor polypeptide further comprises a hinge region at the N terminal of the TM region. For example, the hinge region may be located between the signal peptide and the TM region. The hinge region may be any linker that can provide a desired flexibility to the adaptor polypeptide. The hinge region may be derived from human DAP10, other molecules such as CD8, CD28 or other immune cell surface expressed receptor. In some embodiments, the hinge region comprises the amino acid sequence as shown in SEQ ID No: 8 or an amino acid sequence at least 85%, 90%or 95%identical to SEQ ID No: 8.

Specifically, the adaptor polypeptide may comprise, from N terminal to C terminal, a signal peptide operably linked to the TM region and the TM region operably linked to the intracellular region (SP-TM-Intracellular) . In some embodiments, the adaptor polypeptide further comprises a hinge region at the N terminal. Specifically, the adaptor polypeptide may comprise, from N terminal to C terminal, a hinge region operably linked to the TM region and the TM region operably linked to the intracellular region (Hinge-TM-Intracellular) . In some embodiments, the adaptor polypeptide comprises a signal peptide and a hinge region at the N terminal. Specifically, the adaptor polypeptide comprises, from N terminal to C terminal, a signal peptide operably linked to a hinge region, a hinge region operably linked to the TM region and the TM region operably linked to the intracellular region (SP-Hinge-TM-Intracellular) .

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

In some embodiments, the adaptor polypeptide may comprise an affinity tag for purification, such as an HA tag, in the extracellular region. In some embodiments, the affinity tag is inserted between the signal peptide and the hinge region of the adaptor polypeptide. For example, the adaptor polypeptide comprises, from N terminal to C terminal, a signal peptide comprising SEQ ID No: 6 or 19, a HA tag comprising SEQ ID No: 7, a hinge region comprising SEQ ID No: 8, a transmembrane domain comprising SEQ ID No: 9 and an intracellular region comprising SEQ ID No: 10. As would be appreciated by a person in the art, the affinity tag may be inserted elsewhere in the extracellular region of the adaptor polypeptide, e.g. at the N terminal of the signal peptide or at the C terminal of the hinge region. In some embodiments, a HA tag replaces the hinge region of the adaptor polypeptide, e.g. the adaptor polypeptide comprises, from N terminal to C terminal, a signal peptide comprising SEQ ID No: 6, a HA tag comprising SEQ ID No: 7, a transmembrane region comprising SEQ ID No: 9 and an intracellular region comprising SEQ ID No: 10.

In some embodiments, the intracellular region comprises one or more costimulatory domains selected from or derived from e.g., a MHC class I molecule, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins) , activating NK cell receptors, BTLA, a Toll ligand receptor, and the like. The costimulatory domain 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, CDl la, CDl lb, CDl lc, CDl ld, IL2Rp, IL2y, 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 (CD1 la/CD18) , FcR, FcyRI, FcyRII, 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 a combinations thereof. In some embodiments, in addition to DAP10 intracellular domain, the intracellular region comprises a costimulatory domain selected from 4-IBB, 2B4 (e.g. 2B4·ITSM2) , ICOS, CD28, OX40, and CD27 costimulatory domains.

The adaptor polypeptide may include 1, 2, 3 or more costimulatory domains. When more than one costimulatory domain is included, the costimulatory domains may have same or different amino acid sequences. In some embodiments, the intracellular region comprises one 4-1BB costimulatory domain. The 4-1BB costimulatory domain may comprise the amino acid sequence as shown in SEQ ID No: 4 or an amino acid sequence having at least one, at least two, or at least three or more modifications compared to SEQ ID No: 4. In some embodiments, the 4-1BB costimulatory domain is substantially similar (e.g. at least 85%, 90%or 95%identical) to the 4-1BB costimulatory domain comprising SEQ ID No: 4. In some embodiments, the intracellular region comprises one 2B4 costimulatory domain. The 2B4 costimulatory domain may comprise the amino acid sequence as shown in SEQ ID No: 11 or an amino acid sequence having at least one, at least two, or at least three or more modifications compared to SEQ ID No: 11. In some embodiments, the 2B4 costimulatory domain is substantially similar (e.g. at least 85%, 90%or 95%identical) to the 2B4 costimulatory domain comprising SEQ ID No: 11. In some embodiments, the 2B4 costimulatory domain is the 2B4·ITSM2 costimulatory domain which consists of the first two ITSM motifs. The 2B4·ITSM2 costimulatory domain may comprise the 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·ITSM2 costimulatory domain is substantially similar (e.g. at least 85%, 90%or 95%identical) to SEQ ID No: 12. In some embodiments, the intracellular region comprises one CD28 costimulatory domain. The CD28 costimulatory domain may comprise the amino acid sequence as shown in SEQ ID No: 21 or an amino acid sequence having at least one, at least two, or at least three or more modifications compared to SEQ ID No: 21. In some embodiments, the CD28 costimulatory domain is substantially similar (e.g. at least 85%, 90%or 95%identical) to SEQ ID No: 21. In some embodiments, the intracellular region comprises a combination of 2B4 costimulatory domain and 4-1BB costimulatory domain.

In addition to costimulatory signaling domain (s) , the adaptor polypeptide may further comprise an intracellular primary signaling domain. In some embodiments, the adaptor polypeptide comprises a costimulatory signaling domain (s) and a primary signaling domain (s) . In some other embodiments, the adaptor polypeptide comprises only a primary signaling domain (s) and does not comprise a costimulatory signaling domain. The intracellular primary signaling domain may increase proliferation, persistence, and/or cytotoxic activity of the host cell (e.g., NK cell, NKT cell, y6 cell, etc. ) harboring the polypeptide as herein disclosed. For example, in some embodiments, the intracellular primary signaling domain (s) comprise CD3ζ, repeat (e.g., 2-5) DAP10 YINM motifs, signaling domains derived from LFA-1, DAP12, FcRy, FcRP, CD3y, CD36, CD3s, CD79a, CD79b, CD5, CD22, FcsRI, CD66d, and the like. The intracellular primary signaling domain may specifically be selected from a group consisting of CD3ζ, DAP12, LFA-1 and CD3t, or combinations thereof. The intracellular region may include a plurality (e.g., 2, 3, 4, or more) of intracellular primary signaling domains. In a case where more than one intracellular primary signaling domain is included, they may comprise different amino acid sequences.

In some embodiments, an intracellular primary signaling domain comprises a CD3ζ signaling domain. The CD3ζ signaling domain may comprise the 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 modifications compared to SEQ ID No: 5. In some embodiments, the CD3ζ signaling domain is substantially similar (e.g. at least 85%, 90%or 95%identical) to the amino acid sequence of SEQ ID No: 5. In some embodiments, the intracellular region comprises a 4-1BB costimulatory domain and an CD3ζ intracellular signaling domain, or a 2B4 costimulatory domain and an CD3ζ intracellular signaling domain.

In some embodiments, the adaptor polypeptide comprises or consists of the DAP10 TM region and the 2B4 costimulatory domain. In some embodiments, the adaptor polypeptide comprises or consists of the DAP10 TM region and the 4-1BB costimulatory domain. In some embodiments, the adaptor polypeptide comprises or consists of the DAP10 TM region and the CD28 costimulatory domain. In some embodiments, the adaptor polypeptide comprises or consists of the DAP10 TM region and 2B4·ITSM (the first two ITSM motifs of 2B4) costimulatory domain. In some embodiments, the adaptor polypeptide comprises or consists of the DAP10 TM region, 2B4 costimulatory domain and 4-1BB costimulatory domain. In some embodiments, the adaptor polypeptide comprises or consists of the DAP10 TM region, 2B4 costimulatory domain and CD3ζintracellular signaling domain. In some embodiments, the adaptor polypeptide comprises or consists of the DAP10 TM region, 4-1BB costimulatory domain and CD3ζ intracellular signaling domain. The adaptor polypeptide disclosed above may further comprise a DAP10 intracellular domain.

In some embodiments, the adaptor polypeptide comprises, from N-terminus to 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 costimulatory domain (as shown in structures 2, 9-12, 17, 27) ;
(b) 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 4-1BB costimulatory domain (as shown in structures 3, 18, 19);
(c) 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 costimulatory domain and the 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 costimulatory domain and the CD3ζ intracellular signaling 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 signaling 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 costimulatory 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 costimulatory domain (as shown in structure 8) ;
(h) 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 2B4 costimulatory domain (as shown in structure 24) ;
(i) the signal peptide of CD8, the TM region of human DAP10, the intracellular region of human DAP10, and
the 2B4 costimulatory 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 2B4·ITSM2 costimulatory domain (as shown in structure 26) ; optionally, an affinity tag such as a HA tag is inserted between the signal peptide and the hinge region.

In some embodiments, the adaptor polypeptide comprises, from N-terminus to C-terminus:
(j) the signal peptide of CD8, an HA tag, the TM region of human DAP10, the intracellular region of human
DAP10, and the 2B4 costimulatory domain (as shown in structure 25) .

The costimulatory domains and CD3ζ intracellular signaling domain as described in the above structures can be exchanged with a variety of other costimulatory domains and intracellular signaling domains commonly used in CAR construction.

In some embodiments, the adaptor polypeptide can comprise one or more mutations, for example one or more mutations (e.g., one or more substitutions, additions, or deletions) in DAP10 regions and/or the costimulatory domain (s) and/or the primary signaling domain (s) .

The adaptor polypeptide may be designed with modulated/muted attributes (e.g., by way of the one or more mutations) and/or added signaling attributes (e.g., by way of the C-terminal fusion) and expressed in a host cell to promote a favorable balance of signaling pathways upon receptor-target engagement (e.g., NKG2D engagement of an extracellular target ligand) , which may serve to address the problem of low or lost expression of the target (i.e., antigen escape) . Accordingly, the adaptor polypeptides disclosed herein provide for improved functional properties including but not limited to altered (e.g., enhanced) cytolytic, proliferative, survival and/or costimulatory properties that are elicited upon engagement with the ligands of receptors that partner with DAP10 (e.g., widely expressed ligands of NKG2D) . The precise composition of the adaptor polypeptides can be designed based on a given disease indication and in some examples on pairing with the specificity and signaling components of other receptor (s) that are present on the same cells. As one illustrative example, immunosuppressive signals within a tumor microenvironment (TME) may inhibit anti-tumor immune cell responses through inhibitory receptors, and it is within the scope of this disclosure that via the use of a DAP10-comprising polypeptide such an inhibitory output may be switched to an immunostimulatory one.

In some embodiments, the adaptor polypeptides may function at least in part to stabilize cell surface receptor (s) (e.g., NKG2D) with which they associate. To “stabilize” cell surface receptor (s) as disclosed herein means to decrease a rate at which said cell surface receptors are endocytosed or otherwise removed from the surface of the cell, as compared to the rate at which said cell surface receptors are otherwise removed under similar circumstances in absence of the DAP10-comprising adaptor polypeptide herein described. Encompassed within the scope of receptor stabilization herein includes positive feedback mechanisms through which adaptor polypeptide signaling results in increased cell surface expression of the NKG2D with which endogenous DAP10 and the DAP10-comprising adaptor associate.
Engineered receptor polypeptides

One aspect of the present disclosure provides a combination of the adaptor polypeptide as described above with an engineered receptor polypeptide. The engineered receptor polypeptide generally comprises an extracellular antigen binding domain, and optionally an intracellular signaling domain. Exemplary engineered receptors include, but are not limited to, chimeric antigen receptor (CAR) , engineered NK-cell receptor (NKCR) , engineered T-cell receptor (TCR) , and T-cell antigen coupler (TAC) receptor. The engineered receptor may comprise an extracellular antigen binding domain that specifically binds to an antigen (e.g., GPC3, CD19 or CD33) , a transmembrane domain, and an intracellular signaling region. The intracellular signaling region can comprise a primary intracellular signaling domain and/or a co-stimulatory signaling domain. The engineered receptor polypeptide can be encoded by a heterologous polynucleotide operably linked to a promoter (such as a constitutive promoter or an inducible promoter) .

The engineered receptor polypeptide can comprise one or more specific binding domains that target at least one tumor antigen, and one or more intracellular effector domains, such as one or more primary intracellular signaling domains and/or co-stimulatory signaling domains.

In some embodiments, the present disclosure provides chimeric antigen receptors (CARs) that specifically bind to one or more antigens (e.g., GPC3, CD19 or CD33) . The CAR may comprise: (a) an extracellular antigen binding domain that binds to an antigen or with antigenic specificity; (b) a transmembrane domain; and (c) an intracellular signaling domain.

The antigenic specificity can be directed to any suitable antigen or epitope, for example, one that is exogenous antigen, endogenous antigen, autoantigen, neoantigen, viral antigen or tumor antigen. An exogenous antigen enters a body by inhalation, ingestion or injection, and can be presented by the antigen-presenting cells (APCs) by endocytosis or phagocytosis and form MHC II complex. An endogenous antigen is generated within normal cells as a result of cell metabolism, intracellular viral or bacterial infection, which can form MHC I complex. An autoantigen (e.g. peptide, DNA or RNA, etc. ) is recognized by the immune system of a patient suffering from autoimmune diseases, whereas under normal condition, this antigen should not be the target of the immune system. A neoantigen is entirely absent from the normal body, and is generated because of a certain disease, such as tumor or cancer. In certain embodiments, the antigen is associated with a certain disease (e.g. tumor or cancer, autoimmune diseases, infectious and parasitic diseases, cardiovascular diseases, neuropathies, neuropsychiatric conditions, injuries, inflammations, coagulation disorder) . In certain embodiments, the antigen is associated with immune system (e.g. immunological cells such as B cell, T cell, NK cells, macrophages, etc. ) .

In certain embodiments, one antigenic specificity is directed to an immune-related antigen or an epitope thereof. Examples of an immune-related antigen include a natural killer cell (NK cell) specific receptor molecule and/or a T-cell specific receptor molecule. The T-cell specific receptor molecule allows a T cell to bind to and, if additional signals are present, to be activated by and respond to an epitope/antigen presented by another cell called the antigen-presenting cell or APC. The T-cell specific receptor molecule can be TCR, CD3, CD28, CD134 (also termed OX40) , 4-1BB, CD5, and CD95 (also known as the Fas receptor) . Examples of a NK cell specific receptor molecule include CD16, a low affinity Fc receptor, as well as NKG2D, and CD2.

In certain embodiments, one antigenic specificity is directed to a tumor-associated antigen or an epitope thereof. The term “tumor associated antigen” refers to an antigen that is or can be presented on a tumor cell surface and that is located on or within tumor cells. In some embodiments, the tumor associated antigens can be presented only by tumor cells and not by normal, i.e. non-tumor cells. In some other embodiments, the tumor associated antigens can be exclusively expressed on tumor cells or may represent a tumor specific mutation compared to non-tumor cells. In some other embodiments, the tumor associated antigens can be found in both tumor cells and non-tumor cells, but is overexpressed on tumor cells when compared to non-tumor cells or are accessible for antibody binding in tumor cells due to the less compact structure of the tumor tissue compared to non-tumor tissue. In some embodiments the tumor associated antigen is located on the vasculature of a tumor. Illustrative examples of a tumor associated surface antigen are described in PCT/CN2018/106766 (WO 2019/057122) and are incorporated herein by reference.

In certain embodiments, one antigenic specificity is directed to an antigen or an epitope thereof 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 (amutant form of EGFR) , HER2, HER3, DLL3, BCMA, Sialyl-Lea, 5T4, ROR1, melanoma-associated chondroitin sulfate proteoglycan, mesothelin, folate receptor 1, VEGF receptor, EpCAM, HER2/neu, HER3/neu, G250, CEA, MAGE, proteoglycans, VEGF, FGFR, alphaVbeta3-integrin, HLA, HLA-DR, ASC, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD11, CD13, CD14, CD21, CD23, CD24, CD28, CD30, CD37, CD40, CD41, CD44, CD52, CD64, c-erb-2, CALLA, MHCII, CD44v3, CD44v6, p97, ganglioside GM1, GM2, GM3, GD1a, GD1b, GD2, GD3, GT1b, GT3, GQ1, NY-ESO-1, NFX2, SSX2, SSX4 Trp2, gp100, tyrosinase, Muc-1, telomerase, survivin, G250, p53, CA125 MUC, Wue 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.

The extracellular antigen binding domain of CAR may exist in a variety of forms, including for example, a single-domain antibody (sdAb) or VHH domain, a single chain variable fragment (scFv) , a Fab, a Fab', a F (ab) '2, a F (ab) '3, an Fv, a bis-scFv, a (scFv) 2, a minibody, a diabody, a triabody, a tetrabody, an intrabody, a disulfide stabilized Fv protein (dsFv) , a unibody, a nanobody, an affibody, a DARPin, a monobody, an adnectin, an alphabody, or a designed binder. The antigen-binding domain can be single domain antibodies (e.g., VHH domains) , such as a camelid, shark, chimeric, human, or humanized single domain antibodies (e.g., VHH domains) , or scFvs comprising VH and VL regions derived from parental antibodies. The CARs as disclosed herein may comprise an antigen binding domain comprising a scFv. The CARs as disclosed herein may comprise an antigen binding domain comprising one or more VHHs. The VHHs can be fused to each other directly via peptide bonds, or via peptide linkers.

The CAR may be monospecific or multi-specific (such as bispecific) , monovalent or multi-valent (such as bivalent) , tandem CAR or split CAR. In some embodiments, the CARs are multi-specific (such as bispecific) CARs comprising one or more antigen-binding domains that have different antigen binding specificities. Depending on the desired antigen to be targeted, the CARs as disclosed herein can be engineered to include the appropriate antigen-binding domain that specifically targets the desired antigen.

The CAR may be a monospecific CAR or multivalent CAR. In some embodiments, the CAR may be a single-GPC3 CAR comprising an extracellular antigen binding domain that comprises an anti-GPC3 scFv targeting the GPC3 antigen. The single-GPC3 specific CAR may comprise an antigen binding domain comprising the amino acid sequence of SEQ ID No: 1. In some embodiments, the CAR may be a single-CD19 CAR comprising an extracellular antigen binding domain that comprises an anti-CD19 scFv targeting the CD19 antigen. The single-GPC3 specific CAR may comprise an antigen binding domain comprising the amino acid sequence of SEQ ID No: 13. In some embodiments, the CAR may be a single-CD33 CAR comprising an extracellular antigen binding domain that comprises an anti-CD33 scFv targeting the CD33 antigen. The single-CD33 specific CAR may comprise an antigen binding domain comprising the amino acid sequence of SEQ ID No: 14.

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

In certain embodiments, the parental antibody is a monoclonal antibody. A monoclonal antibody can be produced by various methods known in the art, for example, hybridoma technology, recombinant method, phage display, or any combination thereof. Exemplary methods of producing antibodies are described in WO 2019/057122 and are incorporated herein by reference.

The parental antibodies described herein can be further modified, for example, to graft the CDR sequences to a different framework or scaffold, to substitute one or more amino acid residues in one or more framework regions, to replace one or more residues in one or more CDR regions for affinity maturation, and so on. These can be accomplished by a person skilled in the art using conventional techniques.

The parental antibody can also be a therapeutic antibody known in the art, for example those approved by FDA for therapeutic or diagnostic use, or those under clinical trial for treating a condition, or those in research and development. Polynucleotide sequences and protein sequences for the variable regions of known antibodies can be obtained from public databases such as, www. ncbi. nlm nih gov/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 the therapeutic antibodies disclosed in WO 2019/057122, incorporated herein entirely by reference. In some embodiments, the parental antibody deriving the antigen-binding domain of the CAR as disclosed herein is an anti-GPC3 antibody, an anti-CD33 antibody or an anti-CD19 antibody.

The CARs of the present application may further comprise a transmembrane domain that can be directly or indirectly fused to the extracellular antigen binding region. The transmembrane domain may be derived either from a natural source or from a synthetic source. Different from the transmembrane region of the adaptor polypeptide which is preferably derived from human DAP10 for interaction with NKG2D, the “transmembrane domain” of the CAR polypeptide may be any protein structure that is thermodynamically stable in a cell membrane, for example, a eukaryotic cell membrane. Transmembrane domains compatible for use in the CARs described herein may be obtained from a naturally occurring protein. Alternatively, it can be a synthetic, non-naturally occurring protein segment, e.g., a hydrophobic protein segment that is thermodynamically stable in a cell membrane.

The transmembrane domain of the CAR as disclosed herein may comprise a transmembrane domain chosen from the transmembrane domain of an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CDl la, CD18) , ICOS (CD278) , 4-1BB (CD137) , GITR, CD40, BAFFR, HVEM (LIGHTR) , SLAMF7, NKp80 (KLRFl) , CD160, CD19, IL-2R beta, IL-2R gamma, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226) , SLAMF4 (CD244, 2B4) , CD84, CD96 (Tactile) , CEACAM1, CRT AM, Ly9 (CD229) , CD160 (BY55) , PSGL1, CDIOO (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 a molecule selected from the group consisting of CD8, CD4, CD28, CD137, CD80, CD86, CD152 and PD1.

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%identical to the amino acid sequence set forth in SEQ ID No: 3 or 22.

Transmembrane domains for use in the CARs described herein can also comprise at least a portion of a synthetic, non-naturally occurring protein segment. The transmembrane domain may be a synthetic, non-naturally occurring alpha helix or beta sheet. In some embodiments, the protein segment is at least approximately 20 amino acids, e.g., 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 in U.S. Patent No. 7,052,906 B1 and PCT Publication No. WO 2000/032776 A2, the relevant disclosures of which are incorporated by reference herein.

In some embodiments, the intracellular signaling region of the CAR polypeptide comprises one or more costimulatory domains selected from or derived from e.g., a MHC class I molecule, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins) , activating NK cell receptors, BTLA, a Toll ligand receptor, and the like. The costimulatory domain 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, CDl la, CDl lb, CDl lc, CDl ld, IL2Rp, IL2y, 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 (CD1 la/CD18) , FcR, FcyRI, FcyRII, 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 a combinations thereof. In some embodiments, the intracellular signaling region comprises a costimulatory domain selected from 4-IBB, 2B4 (e.g. 2B4·ITSM2) , ICOS, CD28, OX40, and CD27 costimulatory domains.

The intracellular signaling region may include 1, 2, 3 or more costimulatory domains. When more than one costimulatory domain is included, the costimulatory domains may have same or different amino acid sequences. In some embodiments, the intracellular signaling region comprises one 4-1BB costimulatory domain. The 4-1BB costimulatory domain may comprise the amino acid sequence as shown in SEQ ID No: 4 or an amino acid sequence having at least one, at least two, or at least three or more modifications compared to SEQ ID No: 4. The 2B4 costimulatory domain may comprise the amino acid sequence as shown in SEQ ID No: 11 or an amino acid sequence having at least one, at least two, or at least three or more modifications compared to SEQ ID No: 11. In some embodiments, the 2B4 costimulatory domain is the 2B4·ITSM2 costimulatory domain which consists of the first two ITSM motifs. The 2B4·ITSM2 costimulatory domain may comprise the 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 2B4 costimulatory domain and 4-1BB costimulatory domain.

In addition to costimulatory signaling domain (s) , the adaptor polypeptide may further comprise an intracellular primary signaling domain. In some embodiments, the intracellular signaling region of the CAR polypeptide comprises one costimulatory signaling domain and one primary signaling domain. In some other embodiments, the intracellular signaling region comprises only a primary signaling domain and does not comprise a costimulatory signaling domain. The intracellular primary signaling domain may increase proliferation, persistence, and/or cytotoxic activity of the host cell (e.g., NK cell, NKT cell, y6 cell, etc. ) harboring the polypeptide as herein disclosed. For example, in some embodiments, the intracellular primary signaling domain (s) comprise CD3ζ, repeat (e.g., 2-5) DAP10 YINM motifs, signaling domains derived from LFA-1, DAP12, FcRy, FcRP, CD3y, CD36, CD3s, CD79a, CD79b, CD5, CD22, FcsRI, CD66d, and the like. The intracellular primary signaling domain may specifically be selected from a group consisting of CD3ζ(CD3ζ) , DAP12, LFA-1 and CD3t, or combinations thereof. The intracellular region may include a plurality (e.g., 2, 3, 4, or more) of intracellular primary signaling domains. In a case where more than one intracellular primary signaling domain is included, they may comprise different amino acid sequences.

In some embodiments, an intracellular primary signaling domain comprises a CD3ζ signaling domain. The CD3ζ signaling domain may comprise the 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 modifications compared to SEQ ID No: 5. In some embodiments, the intracellular signaling region comprises or consists of a 4-1BB costimulatory domain and an CD3ζ intracellular signaling domain, or a 2B4 costimulatory domain and an CD3ζ intracellular signaling domain. In some embodiments, the intracellular signaling region comprises or consists of a 4-1BB costimulatory domain, a 2B4 costimulatory domain or a CD3ζ intracellular signaling domain.

When expressed from a host cell, the CAR polypeptide and the adaptor polypeptide as disclosed herein may be separately expressed in two amino acid chains. The nucleic acid sequence encoding the CAR polypeptide and the nucleic acid sequence encoding the adaptor polypeptide are intervened by a cleavable linker including an IRES sequence. The IRES element promotes efficient interaction of the mRNA with the ribosome and allows for internal ribosome entry.
Constructs combining the CAR polypeptide and adaptor polypeptide

One aspect of the present disclosure provides a combination of the adaptor polypeptide and a CAR polypeptide. In some embodiments, the present disclosure provides a nucleic acid construct comprising a first nucleic acid sequence encoding the CAR polypeptide operably linked to a second nucleic acid sequence encoding the adaptor polypeptide. The nucleic acid sequence encoding the CAR polypeptide may be present in the same nucleic acid construct as the nucleic acid sequence encoding the adaptor polypeptide, although in other cases they may be on separate nucleic acid constructs.

Specifically, the adaptor polypeptide may comprise the DAP10 derived transmembrane region as described herein and optionally an intracellular region, and the CAR polypeptide may comprise 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 of the CAR polypeptide may comprise a primary intracellular signaling domain and/or a co-stimulatory signaling domain. The adaptor polypeptide may further comprise a primary intracellular signaling domain at the C terminal of the co-stimulatory signaling domain. Both the CAR polypeptide and the adaptor polypeptide may comprise more than one co-stimulatory signaling domains.

In some embodiments, the first nucleic acid sequence is operably 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 nucleic acid sequence and the second nucleic acid sequence 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 comprise a self-cleavage sequence such as a 2A self-cleaving sequence (e.g., T2A, P2A, E2A, F2A) which can induce ribosomal skipping during translation of the adaptor polypeptide. In some embodiments, the cleavable linker is a furin sequence. In some embodiments, the linker is an IRES sequence. After subjecting to translation, the nucleic acid construct expresses two polypeptide chains wherein the first polypeptide chain comprises the CAR polypeptide, and the second polypeptide chain comprises the adaptor polypeptide as disclosed herein.

In some embodiments, the linker may comprise both a 2A self-cleaving sequence and a furin sequence, e.g., the furin cleavage sequence is at the N terminal to the 2A self-cleaving sequence. Additional linker “GSG” or “SGSG” and the like may also be used to improve cleavage efficiency. In some embodiments, the nucleic acid sequence encoding the linker may comprise an IRES site which initiates separate translations of the adaptor polypeptide and the CAR polypeptide.

The nucleic acid construct may be a DNA or RNA. In some embodiments, the nucleic acid construct is an expression cassette comprised in a vector. In some other embodiments, the nucleic acid construct is a vector, such as a plasmid, a phagemid, a phage derivative, a cosmid, a transposon, a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.

Expression constructs or vectors useful in the present disclosure may comprise (in a 5’ -to-3’ direction) a eukaryotic transcriptional promoter operably linked to a protein-coding sequence, a transcriptional termination/polyadenylation sequence, and optionally splice signals including intervening sequences. In some embodiments, the expression construct comprises, in a 5’ -to-3’ direction, a promoter operably linked to the first nucleic acid sequence, followed by the second nucleic acid sequence. In some other embodiments, the expression construct comprises, in a 5’ -to-3’ direction, a promoter operably linked to the second nucleic acid sequence, followed by the first nucleic acid sequence. The promoters and enhancers that control the transcription of protein encoding genes in eukaryotic cells may be comprised of multiple genetic elements. A promoter used herein may include constitutive, inducible, and tissue-specific promoters, for example. In cases wherein the vector is utilized for the generation of cancer therapy, a promoter may be effective under conditions of hypoxia.

In some embodiments, the expression constructs provided herein comprise a promoter to drive both the expression of the CAR and the adaptor polypeptide. In some other embodiments, the transcription of the first nucleic acid sequence and the second nucleic acid sequence are under the control of separate promoters. A promoter generally comprises a sequence that functions to position 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 promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation. Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. To bring a coding sequence “under the control of’ a promoter, one positions the 5'end of the transcription initiation site of the transcriptional reading frame “downstream” of (i.e., 3'of) the chosen promoter. The “upstream” promoter stimulates transcription of the DNA and promotes expression of the encoded RNA.

The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, for example, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription. A promoter may or may not be used in conjunction with an “enhancer, ” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

Non-limiting examples of promoters include early or late viral promoters, such as, SV40 early or late promoters, cytomegalovirus (CMV) immediate early promoters, Rous Sarcoma Virus (RSV) early promoters; eukaryotic cell promoters, such as, e.g, beta actin promoter, GADPH promoter, metallothionein promoter; and concatenated response element promoters, such as cyclic AMP response element promoters (ere) , serum response element promoter (sre) , phorbol ester promoter (TP A) and response element promoters (tre) near a minimal TATA box. It is also possible to use human growth hormone promoter sequences (e.g., the human growth hormone minimal promoter described ataccession no. X05244, nucleotide 283-341) or a mouse mammary tumor promoter (available from the ATCC, Cat. No. ATCC 45007) . In certain embodiments, the promoter is CMV IE, dectin-1, dectin-2, human CD1 lc, F4/80, SM22, RSV, SV40, Ad MLP, beta-actin, MHC class I or MHC class II promoter, however any other promoter that is useful to drive expression of the therapeutic gene is applicable to the practice of the present disclosure.

In some embodiments, methods of the disclosure also concern enhancer sequences, i.e., nucleic acid sequences that increase a promoter’s activity and that have the potential to act in cis, and regardless of their orientation, even over relatively long distances (up to several kilobases away from the target promoter) . However, enhancer function is not necessarily restricted to such long distances as they may also function in close proximity to a given promoter.
Constructs combining the CAR polypeptide, adaptor polypeptide and cytokine

In some aspects, the present disclosure provides a combination of the adaptor polypeptide, a CAR polypeptide and a cytokine (s) . In some embodiments, one or more cytokines are present in the same expression construct as the CAR and adaptor polypeptide, although in other cases they may be in separate expression constructs. In some embodiments, one or more cytokines are co-expressed from the same expression construct as the CAR and adaptor polypeptide. One or more cytokines may be produced as a separate polypeptide from the CAR and adaptor polypeptide. The cytokine can be selected from but are not limited to IL-15, IL-2, IL-4, IL-7, IL-9, IL-21, IL-23, including wild-type or variants thereof.

In some embodiments, the cytokine used in the combination is Interleukin-15 (IL-15) , including wild-type or mutated versions thereof. IL-15 may be employed because, for example, it is tissue restricted and only under pathologic conditions is it observed at any level in the serum, or systemically. IL-15 possesses several attributes that are desirable for adoptive therapy. IL-15 is a homeostatic cytokine that induces development and cell proliferation of natural killer cells, promotes the eradication of established tumors via alleviating functional suppression of tumor-resident cells, and inhibits activation-induced cell death. In addition to IL-15, other cytokines are envisioned. These include, but are not limited to, cytokines, chemokines, and other molecules that contribute to the activation and proliferation of cells used for human application. As one example, the cytokine is IL-15, IL-12, IL-2, IL-18, IL-21, IL-7, or combination thereof. NK cells expressing IL-15 may be utilized and are capable of continued supportive cytokine signaling, which is useful for their survival post-infusion.

Specifically, the CAR polypeptide may comprise 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, and the adaptor polypeptide may comprise the DAP10 derived TM region as described herein and an intracellular signaling region. The intracellular regions of the CAR polypeptide and the adaptor polypeptide may comprise a primary intracellular signaling domain and/or a co-stimulatory signaling domain. Both the CAR polypeptide and the adaptor polypeptide may comprise more than one a co-stimulatory signaling domains.

In some embodiments, the nucleic acid construct comprises a first nucleic acid sequence encoding the CAR polypeptide, a second nucleic acid sequence encoding the adaptor polypeptide and a third nucleic acid sequence encoding the cytokine polypeptide. In such cases, the expression of the CAR polypeptide, the adaptor polypeptide and the cytokine polypeptide may or may not be regulated by the same regulatory element (s) . Further, the cytokine encoding nucleic acid sequence may or may not be comprised in the same nucleic acid construct.

The three nucleic acid sequences may be operably linked via a nucleotide sequence encoding a linker including one or more self-cleavage sequences, or one or more sequences cleaved by an endogenous protease, or an IRES sequence. For example, the self-cleavage sequence may be a 2A self-cleaving sequence (e.g., T2A, P2A, E2A, F2A) , the sequence cleaved by an endogenous protease may be a furin sequence. In some embodiments, the linker may comprise both a 2A self-cleaving sequence and a furin sequence, e.g., the furin cleavage sequence is at the N terminal to the 2A self-cleaving sequence. Additional linker “GSG” or “SGSG” and the like may also be used to improve cleavage efficiency.

In some specific embodiments, the nucleic acid construct comprises, in 5’ to 3’ direction:
(a) the first nucleic acid sequence operably linked to the second nucleic acid sequence via a first linker-
encoding sequence, and the second nucleic acid sequence operably linked to the third nucleic acid sequence via a second linker-encoding sequence;
(b) the first nucleic acid sequence operably linked to the third nucleic acid sequence via a first linker-
encoding sequence, and the third nucleic acid sequence operably linked to the second nucleic acid sequence via a second linker-encoding sequence;
(c) the second nucleic acid sequence operably linked to the first nucleic acid sequence via a first linker-
encoding sequence, and the first nucleic acid sequence operably linked to the third nucleic acid sequence via a second linker-encoding sequence;
(d) the second nucleic acid sequence operably linked to the third nucleic acid sequence via a first linker-
encoding sequence, and the third nucleic acid sequence operably linked to the first nucleic acid sequence via a second linker-encoding sequence;
(e) the third nucleic acid sequence operably linked to the first nucleic acid sequence via a first linker-
encoding sequence, and the first nucleic acid sequence operably linked to the second nucleic acid sequence via a second linker-encoding sequence; or
(f) the third nucleic acid sequence operably linked to the second nucleic acid sequence via a first linker-
encoding sequence, and the second nucleic acid sequence operably linked to the first nucleic acid sequence via a second linker-encoding sequence.

The first, the second and the third linker encoding sequences may be the same or different from each other. The nucleic acid construct may be a DNA or RNA. In some embodiments, the nucleic acid construct is an expression cassette comprised in a vector. In some other embodiments, the nucleic acid construct is a vector, such as a plasmid, a phagemid, a phage derivative, a cosmid, a transposon, a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.
Peptide linkers

Different domains within the CAR polypeptides and the adaptor polypeptide may be linked to each other via peptide linkers.

For example, each peptide linker in a CAR may have the same or different length and/or sequence depending on the structural and/or functional features of the various domains. Each peptide linker may be selected and optimized independently. The length, the degree of flexibility and/or other properties of the peptide linker (s) used in the CARs may have some influence on properties, including but not limited to the affinity, specificity or avidity for one or more particular antigens or epitopes. For example, longer peptide linkers may be selected to ensure that two adjacent domains do not sterically interfere with one another. A short peptide linker may be disposed between the transmembrane domain and the intracellular signaling domain of a CAR. A peptide linker comprises flexible residues (such as glycine and serine) so that the adjacent domains are free to move relative to each other. For example, a glycine-serine doublet can be a suitable peptide linker.

The peptide linker can be of any suitable length. In some embodiments, 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 long. The peptide linker may be no more than 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 long. The length of the peptide linker may be any of about 1 amino acid to about 10 amino acids, about 1 amino acids 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 long, 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.

The peptide linker may have a naturally occurring sequence, or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of heavy chain only antibodies may be used as the linker. See, for example, WO1996/34103. In some embodiments, the peptide linker is a flexible linker. 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.
Intracellular signaling domain

The adaptor polypeptides and the CAR polypeptides as disclosed herein may comprise one or more intracellular signaling domains. The intracellular signaling domain may refer to a primary intracellular signaling domain or a co-stimulatory signaling domain. The intracellular signaling domain is responsible for activation of at least one of the normal effector functions of the immune cell expressing the CARs and the adaptor polypeptides. The term “effector function” refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Thus, the term “intracellular signaling domain” refers to the intracellular portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire cytoplasmic signaling domain can be employed, in many cases it is not necessary to use the entire chain.

The intracellular signaling domain may comprise a primary intracellular signaling domain of an immune cell. The intracellular signaling domain may consist essentially of a primary intracellular signaling domain of an immune cell. Exemplary ITAM-containing primary cytoplasmic signaling sequences include those derived from 4-1BB, 2B4, CD3ζ, FcR gamma (FcεRIγ) , FcR beta (Fc Epsilon Rib) , CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. The intracellular signaling domain may consist of the cytoplasmic signaling domain of CD3ζ. The primary intracellular signaling domain may be a cytoplasmic signaling domain of wildtype CD3ζ. The primary intracellular signaling domain is a functional mutant of the cytoplasmic signaling domain of CD3ζ containing one or more mutations. The primary intracellular signaling domain of wildtype CD3ζ may comprise the amino acid sequence of SEQ ID No: 5, or an amino acid sequence at least 85%, 90%or 95%identical to SEQ ID No: 5.

Many immune cells require co-stimulation, in addition to stimulation of an antigen-specific signal, to promote cell proliferation, differentiation and survival, as well as to activate effector functions of the cell. The CAR polypeptide and/or the adaptor polypeptide as disclosed herein may comprise at least one co-stimulatory signaling domain. The co-stimulatory signaling domain described herein can be a cytoplasmic signaling domain from a co-stimulatory protein, which transduces a signal and modulates responses mediated by immune cells, such as T cells, NK cells, macrophages, neutrophils, or eosinophils. “Co-stimulatory signaling domain” can be the cytoplasmic portion of a co-stimulatory molecule.

The intracellular signaling domain of the CAR polypeptide and/or the adaptor polypeptide may comprise a single co-stimulatory signaling domain. The intracellular signaling domain may comprise two or more (such as about any of 2, 3, 4, or more) co-stimulatory signaling domains, e.g., two or more of the same co-stimulatory signaling domains, or two or more co-stimulatory signaling domains from different co-stimulatory proteins. The intracellular signaling domain may comprise a primary intracellular signaling domain (such as cytoplasmic signaling domain of 4-1BB, 2B4, CD28) and one or more co-stimulatory signaling domains. The one or more co-stimulatory signaling domains and the primary intracellular signaling domain (such as cytoplasmic signaling domain of CD3ζ) may be fused to each other via optional peptide linkers. The primary intracellular signaling domain, and the one or more co-stimulatory signaling domains may be arranged in any suitable order. One or more co-stimulatory signaling domains are located between the transmembrane domain and the primary intracellular signaling domain (such as cytoplasmic signaling domain of CD3ζ) . Multiple co-stimulatory signaling domains may provide additive or synergistic stimulatory effects.

Activation of a co-stimulatory signaling domain in a host cell (e.g., an immune cell) may induce the cell to increase or decrease the production and secretion of cytokines, phagocytic properties, proliferation, differentiation, survival, and/or cytotoxicity. The co-stimulatory signaling domain of any co-stimulatory molecule may be compatible for use in the CAR polypeptid and/or the adaptor polypeptides described herein. The type (s) of co-stimulatory signaling domain is selected based on factors such as the type of the immune cells in which the effector molecules would 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 use in theCAR polypeptide and/or the adaptor polypeptide can be the cytoplasmic signaling domain of co-stimulatory proteins, including, without limitation, 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) ; 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-alpha/TNF-beta, OX40/TNFRSF4, OX40 Ligand/TNFSF4, RELT/TNFRSF19L, TACI/TNFRSF13B, TL1A/TNFSF15, TNF-alpha, 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 alpha 4/CD49d, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/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.

In some embodiments, the co-stimulatory signaling domains are selected from the group consisting 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, ligands of CD83 and combinations thereof.

Also within the scope of the present disclosure are variants of any of the co-stimulatory signaling domains described herein, such that the co-stimulatory signaling domain is capable of modulating the immune response of the immune cell. The co-stimulatory signaling domains may comprise up to 10 amino acid residue variations (e.g., 1, 2, 3, 4, 5, or 8) as compared to a wild-type counterpart. Such co-stimulatory signaling domains comprising one or more amino acid variations may be referred to as variants. Mutation of amino acid residues of the co-stimulatory signaling domain may result in an increase in signaling transduction and enhanced stimulation of immune responses relative to co-stimulatory signaling domains that do not comprise the mutation. Mutation of amino acid residues of the co-stimulatory signaling domain may result in a decrease in signaling transduction and reduced stimulation of immune responses relative to co-stimulatory signaling domains that do not comprise the mutation.
Hinge region

A hinge domain is an amino acid segment that is generally found between two domains of a protein and may allow for flexibility of the protein and movement of one or both of the domains relative to one another. The CAR polypeptide and the adaptor polypeptide of the present application may comprise a hinge domain that is located between the extracellular domain and the transmembrane domain. Any amino acid sequence that provides such flexibility and movement of the extracellular antigen binding domain relative to the transmembrane domain of the effector molecule can be used. In some embodiments, the hinge domain of the adaptor polypeptide comprises a DAP10 derived hinge sequence, e.g. as shown in SEQ ID No: 8.

The hinge domain may contain about 10-100 amino acids, e.g., about any one of 15-75 amino acids, 20-50 amino acids, or 30-60 amino acids. The hinge domain may be at least about any one of 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 in length.

The hinge domain may be a hinge domain of a naturally occurring protein (e.g. an immunoglobulin) . Hinge domains of any protein known in the art to comprise a hinge domain are compatible for use in the chimeric receptors described herein. The hinge domain may be at least a portion of a hinge domain of a naturally occurring protein and confers flexibility to the chimeric receptor. In some embodiments, the hinge domain is derived from CD8α. The hinge domain may be a portion of the hinge domain of CD8α, e.g., a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8α. The hinge domain of CD8α may comprise an amino acid sequence of SEQ ID No: 2, or at least 85%, 90%or 95%identical to the amino acid sequence of SEQ ID No: 2.

Hinge domains of antibodies, such as an IgG, IgA, IgM, IgE, or IgD antibodies, are also compatible for use in the pH-dependent chimeric receptor systems described herein. The hinge domain may be the hinge domain that joins the constant domains CH1 and CH2 of an antibody. The hinge domain may be of an antibody and comprises the hinge domain of the antibody and one or more constant regions of the antibody. The hinge domain may comprise the hinge domain of an antibody and the CH3 constant region of the antibody. The hinge domain may comprise the hinge domain of an antibody and the CH2 and CH3 constant regions of the antibody. The antibody may be an IgG, IgA, IgM, IgE, or IgD antibody. The antibody may be an IgG1, IgG2, IgG3, or IgG4 antibody. The hinge region may comprise the hinge region and the CH2 and CH3 constant regions of an IgG1 antibody. The hinge region may comprise the hinge region and the CH3 constant region of an IgG1 antibody.

Non-naturally occurring peptides may also be used as hinge domains for the CAR polypeptide and adaptor polypeptides described herein. The hinge domain located between the C-terminus of the extracellular ligand-binding domain of an Fc receptor and the N-terminus of the transmembrane domain, or between the signal peptide and the transmembrane domain may be a peptide linker, such as a (GxS) n linker, wherein x and n, independently can be an integer between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more.
Nucleic Acids and Vectors

The present disclosure also provides nucleic acid molecules comprising a first nucleic acid sequence encoding the adaptor polypeptide, a second nucleic acid sequence encoding the CAR polypeptide and optionally a third nucleic acid sequence encoding the cytokine polypeptide which are operably linked. The nucleic acid molecule may be provided as a messenger RNA transcript or as a DNA.

In one aspect, provided herein is a nucleic acid molecule comprising: (1) a nucleic acid sequence encoding the CAR polypeptide, such as a GPC3 targeting CAR polypeptide, a CD19 targeting CAR polypeptide or a CD33 targeting CAR polypeptide as disclosed herein; (2) a nucleic acid sequence encoding the adaptor polypeptide as disclosed herein. Optionally, the two nucleic acid sequences are linked by a nucleic acid sequence encoding a self-cleavable peptide (such as P2A, E2A, F2A or T2A) or IRES sequence. In some further embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a cytokine polypeptide.

The nucleic acid sequences coding for the desired polypeptides can be obtained using recombinant methods known in the art, using standard techniques. For example, the sequence of interest can be produced synthetically or cloned.

The present disclosure also provides vectors in which a nucleic acid sequence or nucleic acid construct as disclosed herein is inserted. The nucleic acid sequences encoding the CAR polypeptide, the adaptor polypeptide and optionally the cytokine polypeptide may be comprised in the same vector. The nucleic acid sequences may be encoded by a single nucleic molecule in the same frame. Vectors derived from retroviruses are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. A retroviral vector may be, e.g., a gamma retroviral vector.

A gamma retroviral vector may include, e.g., a promoter, a packaging signal (ψ) , a primer binding site (PBS) , one or more (e.g., two) long terminal repeats (LTR) , and a transgene of interest, e.g., a gene encoding a CAR. A gamma retroviral vector may lack viral structural gens such as gag, pol, and env. Exemplary gammaretroviral vectors include Murine Leukemia Virus (MLV) , Spleen-Focus Forming Virus (SFFV) , and Myeloproliferative Sarcoma Virus (MPSV) , and vectors derived therefrom. Other gamma retroviral vectors are described, e.g., in Tobias Maetzig et al., "Gammaretroviral Vectors: Biology, Technology and Application" Viruses. 2011 Jun; 3 (6) : 677-713.

In some embodiments, the expression of natural or synthetic nucleic acids encoding CARs and adaptor polypeptides is typically achieved by operably linking a nucleic acid encoding the CAR polypeptide and/or a nucleic acid encoding the adaptor polypeptide to a promoter, and incorporating the construct into an expression vector. The vectors can be suitable for replication and integration eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

The expression constructs of the present disclosure may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties. In another embodiment, the disclosure provides a gene therapy vector.

The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY) , and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193) .

A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In one embodiment, lentivirus vectors are used.

Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

The vector may also contain a selectable marker gene or a reporter gene to select cells expressing the CAR from the population of host cells transfected through lentiviral vectors. Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences to enable expression in the host cells. For example, the vector may contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the nucleic acid sequences.
Engineered Immune Cells

In one aspect, provided herein is an engineered immune cell expressing the CAR and the adaptor polypeptide, the nucleic acid or the vector as disclosed herein. Accordingly, the disclosure provides an engineered immune cell, e.g., a NK cell, a T cell, a γδT cell, and methods of their use for adoptive therapy. The immune cells, including NK cells, may be derived from cord blood (including pooled cord blood from multiple sources) , peripheral blood, induced pluripotent stem cells (iPSCs) , hematopoietic stem cells (HSCs) , bone marrow, or a mixture thereof. The NK cells may be derived from a cell line such as, but not limited to, NK-92 cells, for example. The NK cell may be a cord blood mononuclear cell, such as a CD56⁺ NK cell.

“Immune cells” are immune cells that can perform immune effector functions. Examples of immune cells include peripheral blood mononuclear cells (PBMC) , natural killer (NK) cells, monocytes, cytotoxic T cells, T helper cells, γδT cells, neutrophils, and eosinophils.

In some embodiments, the immune cells are NK cells. The immune cells can be established cell lines, for example, NK-92 cells. In some 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^-, or combinations thereof. The T cells may produce IL-2, TFN, and/or TNF upon expressing the CAR or DAP10 adaptor polypeptide and binding to the target cells, such as CLDN18.2⁺ or GUCY2C⁺ tumor cells. The T cells may lyse specific target cells upon expressing the CAR or DAP10 adaptor polypeptide and binding to the target cells.

In some embodiments, the immune cells are differentiated from a stem cell, such as a hematopoietic stem cell, a pluripotent stem cell, an iPS, or an embryonic stem cell.

The engineered immune cells as disclosed herein may be prepared by introducing the expression construct or vector as disclosed herein into the immune cells, such as NK cells. Methods of introducing vectors or isolated nucleic acids into a mammalian cell are known in the art. The vectors described can be transferred into an immune cell by physical, chemical, or biological methods.

Physical methods for introducing the vector into an immune cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. The vector may be introduced into the cell by electroporation. Biological methods for introducing the vector into an immune cell include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian, e.g., human cells. Chemical means for introducing the vector into an immune cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro is a liposome (e.g., an artificial membrane vesicle) .

The transduced or transfected immune cell may be propagated ex vivo after introduction of the vector or isolated nucleic acid. In some embodiments, the transduced or transfected immune cell is cultured to propagate for at least about any of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days or 21 days. The transduced or transfected immune cell may be further evaluated or screened to select the engineered mammalian cell.

Reporter genes may be used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al. FEBS Letters 479: 79-82 (2000) ) . Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.

Other methods to confirm the presence of the nucleic acid encoding the polypeptides in the engineered immune cell, include, for example, molecular biological assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; biochemical assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological methods (such as ELISAs and Western blots) .

In some embodiments, the immune cells are NK cells. Prior to expansion and genetic modification of the NK cells, a source of NK cells can be obtained from an individual. NK cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. NK cells can be obtained from an allogeneic or an autologous donor. The NK cells can be partially or entirely purified, or not purified, and expanded ex vivo. Methods and compositions for ex vivo expansion include, without limitation, those described in Becker et al., (2016) Cancer Immunol. Immunother. 65 (4) : 477-84. The expansion may be performed before or after, or before and after, a chimeric DAP10 adaptor polypeptide is introduced into the NK cell (s) . Briefly and without limitation, expansion of NK cells can include the use of engineered feeder cells, cytokine cocktails (e.g., IL-2, IL-15) , and/or aAPCs (Cortes-Selva, D et al., (2021) Trends Pharmacol Sci. 42 (1) : 45-59) .

In some embodiments, the immune cells are T cells. T cells may be isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL gradient or by counterflow centrifugal elutriation. A specific subpopulation of T cells, such as CD3⁺, CD28⁺, CD4⁺, CD8⁺, CD45RA⁺, and CD45RO⁺T cells, can be further isolated by positive or negative selection techniques. T cells may be obtained from a patient directly following treatment. In this regard, it has been observed that following certain cancer treatments, in particular treatments with drugs that damage the immune system, shortly after treatment during the period when patients would normally be recovering from the treatment, the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo. Likewise, following ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced engraftment and in vivo expansion. Thus, it is contemplated within the context of the present disclosure to collect blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery phase. Further, mobilization (for example, mobilization with GM-CSF) and conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time following therapy. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

Following genetic modification with the vector (s) , the NK cells may be immediately infused or may be stored. In certain aspects, following genetic modification, the cells may be propagated for days, weeks, or months ex vivo as a bulk population within about 1, 2, 3, 4, 5 days or more following gene transfer into cells. In a further aspect, the transfectants are cloned and a clone demonstrating presence of a single integrated or episomally maintained expression cassette or plasmid, and expression of the CAR and adaptor polypeptide is expanded ex vivo. The clone selected for expansion demonstrates the capacity to specifically recognize and lyse CAR-expressing target cells. The recombinant immune cells may be expanded by stimulation with IL-2, or other cytokines that bind the common gamma-chain (e.g., IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, and others) . The recombinant immune cells may be expanded by stimulation with artificial antigen presenting cells.
In a further aspect, the genetically modified cells may be cryopreserved.

Whether prior to or after genetic modification of the T cells or NK cells with the nucleic acid constructs described herein, the T cells or NK cells can be activated and expanded generally using methods as described, for example, in U.S. Pat. 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.
Pharmaceutical compositions

Further provided by the present application are pharmaceutical compositions comprising any one of the engineered immune cells as described herein, and a pharmaceutically acceptable carrier. Pharmaceutical compositions can be prepared by mixing the engineered immune cells having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) ) . The pharmaceutical composition may be in the form of lyophilized formulations or aqueous solutions.

Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants including ascorbic acid, methionine, Vitamin E, sodium metabisulfite; preservatives, isotonicifiers, stabilizers, metal complexes (e.g. Zn-protein complexes) ; chelating agents such as EDTA and/or non-ionic surfactants.

In order for the pharmaceutical compositions to be used for in vivo administration, they must be sterile. The pharmaceutical composition may be rendered sterile by filtration through sterile filtration membranes. The pharmaceutical compositions herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accordance with known and accepted methods, such as by single or multiple bolus or infusion over a long period of time in a suitable manner, e.g., injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, topical administration, inhalation or by sustained release or extended-release means.
Methods and Uses

In one aspect, the present disclosure provides a method for treating a tumor in a subject in need thereof, comprising administering an effective amount of the engineered immune cells as disclosed herein to the subject. The CAR expressed in the immune cells may be designed for targeting the cancer to be treated. Such methods and uses include therapeutic methods and uses, for example involving administration of the molecules, cells, or compositions containing the same, to a subject having a disease, condition, or disorder expressing or associated with abnormal antigen expression. In some embodiments, the subject has a GPC3 related cancer.

The present application further relates to methods and compositions for use in cell immunotherapy. In some embodiments, the cell immunotherapy is for treating cancer. Any of the nucleic acids and engineered immune cells described herein may be used in the method of treating cancer. The engineered immune cells such as NK cells described herein may be useful for treating tumors having antigen loss escape mutations, and for reducing resistance to existing therapies. In some embodiments, the methods and compositions described herein may be used for treating other diseases that are associated with GPC3.

The engineered immune cell may be autologous. The engineered immune cell may be allogenic. In some embodiments, the cancer is a solid cancer, including but not limited to, gastric cancer, esophageal cancer, pancreatic ductal cancer, lung cancer such as non-small cell lung cancer (NSCLC) , ovarian cancer, colorectal cancer, liver cancer, head and neck cancer, gallbladder cancer and its metastasis.

The methods are applicable to cancers of all stages, including early stage, advanced stage and metastatic cancer. The methods described herein may be used as a first therapy, second therapy, third therapy, or combination therapy with other types of cancer therapies known in the art, such as chemotherapy, surgery, radiation, gene therapy, immunotherapy, bone marrow transplantation, stem cell transplantation, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, radio-frequency ablation or the like, in an adjuvant setting or a neoadjuvant setting.

Administration of the pharmaceutical compositions may be carried out in any convenient manner, including by injection, ingestion, transfusion, implantation or transplantation. The compositions may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intravenously, or intraperitoneally. In some embodiments, the pharmaceutical composition is administered systemically. The pharmaceutical composition may be administered to an individual by infusion, such as intravenous infusion. Infusion techniques for immunotherapy are known in the art (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676 (1988) ) . In some embodiments, the pharmaceutical composition is administered to an individual by intradermal or subcutaneous injection. The compositions may be administered by intravenous injection. The compositions may be injected directly into a tumor, or a lymph node. The pharmaceutical composition may be administered locally to a site of tumor, such as directly into tumor cells, or to a tissue having tumor cells.

Dosages and desired drug concentration of pharmaceutical compositions of the present disclosure may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary artisan. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W. “The Use of Interspecies Scaling in Toxicokinetics” In Toxicokinetics and New Drug Development, Yacobi et al., Eds, Pergamon Press, New York 1989, pp. 42-46. It is within the scope of the present application that different formulations will be effective for different treatments and different disorders, and that administration intended to treat a specific organ or tissue may necessitate delivery in a manner different from that to another organ or tissue.
Combination Therapy

The engineered immune cells or pharmaceutical composition as described herein may be used in combination with other known agents and therapies. Administered "in combination" , as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. The delivery of one treatment may be still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as "simultaneous" or "concurrent delivery" . In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

The engineered immune cells described herein and the at least one additional therapeutic agent can be administered simultaneously, in the same or in separate compositions, or sequentially. For sequential administration, the cells expressing the CAR and adaptor polypeptide described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed.

The CAR therapy and/or other therapeutic agents, procedures or modalities can be administered during periods of active disorder, or during a period of remission or less active disease. The CAR therapy can be administered before the other treatment, concurrently with the treatment, post-treatment, or during remission of the disorder.

In further aspects, the engineered immune cells described herein may be used in a treatment regimen in combination with surgery, cytokines, radiation, or chemotherapy such as Cytoxan, fludarabine, histone deacetylase inhibitors, demethylating agents, or peptide vaccine, such as that described in Izumoto et al.2008 J Neurosurg 108: 963-971.
Summary of the sequences

Appended to the instant application is a sequence listing comprising a number of amino acid sequences. The following Table A provides a summary of the included sequences.

Table A.

EXAMPLES
Example 1. Enhanced cytotoxicity of CAR+DAP10-ICD transfected T cells compared to T cells
transfected with CAR alone
1.1 Expression of CAR+DAP10-ICD structure in expanded T cells

In this experiment, we utilized GC33, a chimeric antigen receptor (CAR) targeting GPC3 (as disclosed in patent US20070190599A1) , a tumor-associated antigen (TAA) expressed in liver cancer, to construct CAR-modified immune cells. Retroviruses carrying various CAR constructs were produced, including:
CAR (full) (Structure 1)
CAR (full) + DAP10 + 2B4 (Structure 2)
CAR (full) + DAP10 + 41BB (Structure 3)
CAR (full) + DAP10 + 2B4 + 41BB (Structure 4)
CAR (full) + DAP10 + 2B4 + CD3ζ (Structure 5)
CAR (full) + DAP10 + 4-1BB + CD3ζ (Structure 6)
CAR (full) + DAP10 + 2B4·ITSM2 (Structure 7)
CAR (full) + DAP10 + CD28 (ICD) (Structure 8)

Table 1. Structures of nucleic acid molecules encoding the CAR polypeptide and the adaptor polypeptide (the proteins deriving the SP, hinge, TM and ICD regions are listed)

Retrovirus was produced and titer was determined with 293T cells. To generate CAR (full) +DAP10 + ICD T cells, primary human T cells were also isolated from PBMCs obtained from healthy donors. T cells were stimulated with T Cell TransAct (Miltenyi Biotec) . After 3 days of expansion in the presence of recombinant IL-2, T cells were transduced with retrovirus under the help of transduction enhancer Vectofusin-1 (Miltenyi Biotec) . Then, transduced T cells were expanded in the presence of recombinant IL-2, and used for function assay or cryopreserved around 7～12 days after transduction. Activated T cells were transduced with the retroviruses. This transduction process resulted in the expression of the desired CAR (full) +DAP10+ICD T cells.
1.2 Transduction efficiency and effect on cell proliferation determination

On day 5 after transduction, the 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 ℃ for 1 hour with either anti-CD4 (Biolegend, 300537) and anti-CD8 (Biolegend, 301008) antibodies or GPC3-FITC protein (Acro Biosystems, GPC3-H82E5, for CAR detection) along with anti-HA antibody (Invitrogen, 26183, for DAP10 identification) . Following incubation, the cells were washed with 1%BSA-PBS (w/v) , and then washed again and resuspended in 1%BSA-PBS for flow cytometry analysis. The data obtained were analyzed using FlowJo software.

Figure 2 shows the proliferation of CAR-T cells with different structures. Figure 3 presents the CD4/CD8 percentage, CAR expression, and DAP10 expression of the CAR-T cells. The results demonstrated that the inclusion of DAP10+ICD had minimal impact on CAR-T cell expansion in culture. Moreover, the CAR-T cells used in this experiment exhibited an appropriate CD4/CD8 proportion, and structures 1-8 were all efficiently produced with high transduction efficiency.
1.3 CAR-T cell activation and cytotoxicity

To evaluate the cytotoxicity of the modified T cells, we used two liver cancer cell lines: hepG2, which exhibits high expression of both GPC3 and MICA/B, and sk-hep1, which has minimal expression of GPC3 but high expression of MICA/B. The expression of MICA/B and GPC3 was shown in Figure 4.

For the cytotoxicity assay, both hepG2 and sk-hep1 cells stably expressing GFP were pre-seeded in a 96-well plate 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 the green fluorescence signal using the IncuCyte automated live cell imaging system. The results are presented in Figure 5-7, demonstrating that the inclusion of the DAP10+ICD structure enhances the cytotoxicity of CAR-T cells towards CAR-target expressing and non-expressing cell lines.
Example 2: Enhanced cytotoxicity of CAR+DAP10-ICD transfected NK cells compared to T cells
transfected with CAR alone
2.1 Expression of CAR+DAP10-ICD structure in expanded NK cells

In this experiment, we selected GPC3 as the target for CAR therapy against liver cancer. Retroviruses carrying various CAR constructs were produced, including:
CAR (full) (Structure 1)
CAR (full) + DAP10 + 2B4 (Structure 2)
CAR (full) + DAP10 + 41BB (Structure 3)
CAR (full) + DAP10 + 2B4 + 41BB (Structure 4)
CAR (full) + DAP10 + 2B4 + CD3ζ (Structure 5)
CAR (full) + DAP10 + 4-1BB + CD3ζ (Structure 6)
CAR (full) + DAP10 + 2B4·ITSM2 (Structure 7)

To generate CAR (full) + 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 5 to 6 days of expansion, NK cells were transduced with retrovirus pre-coated on RetroNectin. Then, transduced NK cells were expanded in the presence of recombinant IL-2, and used for function assay or cryopreserved around 7～10 days after transduction. This transduction process resulted in the expression of the desired CAR (full) +DAP10+ICD NK cells.
2.2 Transduction efficiency and effect on cell proliferation determination

On day 3 after transduction, feeder cells were supplied as the E: T=2: 1 to re-activate the NK cell. On day 6 after transduction, the CAR-NK cells were counted using an NC250 cell counter, and 1E5 cells were transferred per well based on the cell count. The CAR-NK cells were then incubated at 4 ℃ for 1 hour with either anti-CD56-violet (Biolegend. 318328) , GPC3-FITC protein (Acro Biosystems, GPC3-H82E5, for CAR detection) along with anti-HA antibody (Invitrogen, 26183, for DAP10 identification) . Following incubation, the cells were washed with 1%BSA-PBS (w/v) , and then washed again and resuspended in 1%BSA-PBS for flow cytometry analysis. The data obtained were analyzed using FlowJo software.

Figure 8 shows the proliferation of CAR-NK cells with different structures. Figure 9 presents the CAR expression, and DAP10 expression in CD56⁺ cells. The results demonstrated that the inclusion of DAP10+ICD had minimal impact on CAR-NK cell expansion. Moreover, structures 1-7 were all efficiently produced with high transduction efficiency.
2.3 CAR-NK cell activation and cytotoxicity

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

For the cytotoxicity assay, both hepG2 and sk-hep1 cells transfected with GFP were pre-seeded in a 96-well plate 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 the green fluorescence signal using the IncuCyte automated live cell imaging system. The results are presented in Figure 10 and Figure 11, demonstrating that the inclusion of the DAP10+ICD structure enhances the cytotoxicity of CAR-NK cells towards CAR-target non-expressing cell lines.
Example 3: Enhanced cytotoxicity of CAR+DAP10-ICD transfected γδT cells compared to T cells
transfected with CAR alone
3.1 Expression of CAR+DAP10-ICD structure in expanded γδT cells

In this experiment, we selected GPC3 as the target for CAR therapy against liver cancer. Retroviruses carrying various CAR constructs were produced, including:
CAR (full) (Structure 1)
CAR (full) + DAP10 + 2B4 + CD3ζ (Structure 5)
CAR (full) + DAP10 + 4-1BB + CD3ζ (Structure 6)

To generate CAR (full) + 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 7 to 9 days of expansion, γδT cells were transduced with retrovirus. Then, transduced γδT cells were expanded in the presence of recombinant IL-2, and used for function assay or cryopreserved around 7～10 days after transduction. This transduction process resulted in the expression of the desired CAR (full) +DAP10+ICD γδT cells.
3.2 Transduction efficiency and effect on cell proliferation determination

On day 6 after transduction, the 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 ℃for 1 hour with either anti-γδT1 and anti-γδT2 or GPC3-FITC protein (Acro Biosystems, GPC3-H82E5, for CAR detection) along with anti-HA antibody (Invitrogen, 26183, for DAP10 identification) . Following incubation, the cells were washed with 1%BSA-PBS (w/v) , and then washed again and resuspended in 1%BSA-PBS for flow cytometry analysis. The data obtained were analyzed using FlowJo software.

Figure 12 shows the proliferation of CAR-γδT cells with different structures. Figure 13 presents the γδT1/γδT2 percentage, CAR expression, and DAP10 expression of the CAR-γδT cells. The results demonstrated that the inclusion of DAP10+ICD had minimal impact on CAR-γδT cell expansion. Moreover, the CAR-γδT cells used in this experiment exhibited an appropriate γδT1/γδT2 proportion, and structures 1, 5, 6 were all efficiently produced with high transduction efficiency.
3.3 CAR-γδT cell activation and cytotoxicity

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

For the cytotoxicity assay, both hepG2 and sk-hep1 cells transfected with GFP were pre-seeded in a 96-well plate and allowed to adhere for 24 hours. CAR-γδT cells were then added to each well at different E:T ratios. The number of viable cells was assessed by monitoring the green fluorescence signal using the IncuCyte automated live cell imaging system. The results are presented in Figure 14 (cytotoxicity on hepG2) and Figure 15 (cytotoxicity on sk-hep1) , demonstrating that the inclusion of the DAP10+ICD structure enhances the cytotoxicity of CAR-γδT cells towards CAR-target non-expressing cell lines.
Example 4: The function of DAP10-ICD could be combined with various CAR structures
4.1 Expression of CAR+DAP10-ICD structure in expanded immune cells

In this experiment, immune cells isolation and expansion is the same as in Examples 1-3. We still selected GPC3 as the target for CAR therapy against liver cancer. Retroviruses carrying various CAR constructs were produced, 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 the CAR polypeptide and the adaptor polypeptide (the proteins deriving the SP, hinge, TM and ICD regions are listed)

To generate CAR + DAP10 -ICD immune cells, activated cells were transduced with the retroviruses. This transduction process resulted in the expression of the desired CAR +DAP10+ICD immune cells.
4.2 Transduction efficiency and effect on cell proliferation determination

The transduction efficiency was detected as described in Examples 1-3. Figure 16 to Figure 18 shows the proliferation of immune cells with different structures. The results demonstrated that structures in 4.1 were all efficiently produced with high transduction efficiency.
4.3 Immune cell activation and cytotoxicity

To evaluate the cytotoxicity of the modified cells, we used two liver cancer cell lines: hepG2, which exhibits high expression of both GPC3 and MICA/B, and sk-hep1, which has minimal expression of GPC3 but high expression of MICA/B.

For the CAR-γδT activation experiment, the activation marker CD107a was selected. 1E5 target cells of each type were transferred per well and co-cultured with CAR-T cells at an effector-to-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 an additional 4 hours of co-culture. CD107a was detected using APC anti-human CD107a (Biolegend, 328620) . The results are presented in Figure 19, which indicates that γδT cells transfected with CAR+DAP10-ICD can be efficency activated by targetting cell lines. For the cytotoxicity assay, the results are presented in Figure 20 to Figure 23, demonstrating that the change of CAR-ICD structure did not affect the function of DAP10-ICD. hepG2 and sk-hep1 can activate modified immune cells with DAP10-ICD structure and be killed, while it is not the case with immune cells with only CAR.
Example 5: DAP10-ICD structure works well with cytokine modification
5.1 Expression of CAR+DAP10-ICD structure in expanded NK cells

NK cells isolation and expansion is the same as in Example 2. We selected GPC3 as the target for CAR therapy against liver cancer. Retroviruses carrying various CAR constructs were produced, 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, IL5RF is a membrane bound form of IL-15.

Table 4. Structures of nucleic acid molecules encoding the CAR polypeptide and the adaptor polypeptide (the proteins deriving the SP, hinge, TM and ICD regions are listed)

To generate CAR + DAP10 + ICD NK cells, activated NK cells were transduced with the retroviruses. This transduction process resulted in the expression of the desired CAR +DAP10+ICD NK cells.
5.2 Transduction efficiency and effect on cell proliferation determination

The transduction efficiency and proliferation were detected as described in Example 2. Figure 24 shows the proliferation of CAR-NK cells with different structures. Figure 25 presents the CAR expression in CD56⁺ cells. The results demonstrated that the inclusion of DAP10+ICD had minimal impact on CAR-NK cell expansion. Moreover, structures 16-22 were all efficiently produced with high transduction efficiency.
5.3 NK cell cytotoxicity

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

Experiments were done as described in Example 2. The results are presented in Figure 26 and Figure 27, which indicates DAP10-ICD structure works well with IL15 expression
Example 6: Full DAP10 structure is not essential for the DAP10-ICD structure function
6.1 Expression of CAR+DAP10-ICD structure in expanded NK cells

NK cells 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 various CAR constructs were produced, 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 a secreted and soluble form of IL-15.
Table 5. Structures of nucleic acid molecules encoding the CAR polypeptide and the adaptor polypeptide
(the proteins deriving the SP, hinge, TM and ICD regions are listed)

To generate CAR + DAP10 + ICD NK cells, activated NK cells were transduced with the retroviruses. This transduction process resulted in the expression of the desired CAR (full) +DAP10+ICD NK cells.
6.2 Transduction efficiency and effect on cell proliferation determination

The transduction efficiency and proliferation were detected as described in Example 2. Figure 28 shows the proliferation of CAR-NK cells with different structures. Figure 29 presents the CAR expression in CD56⁺ cells. The results demonstrated that the inclusion of DAP10+ICD had minimal impact on CAR-NK cell expansion. Moreover, structures 23-27 were all efficiently produced with high transduction efficiency.
6.3 NK cell cytotoxicity

Experiments were done as described in Example 2. The results are presented in Figure 30 and 31, which indicates that changing DAP10 SP or DAP10 hinge did not affect the function of DAP10-ICD structure. The result is unanimous to the previous reports that NKG2D and DAP10 construct trimer through their transmembrane structure.

Claims

An expression construct, comprising a first nucleic acid sequence that encodes a chimeric antigen receptor (CAR) polypeptide and a second nucleic acid sequence that encodes a recombinant polypeptide comprising a DAP10 transmembrane region, wherein

the DAP10 transmembrane region is derived from human DAP10 and can interact with NKG2D, and

the first nucleic acid sequence is separated from the second nucleic acid sequence by a nucleotide sequence encoding a cleavable linker.
The expression construct of claim 1, wherein the DAP10 transmembrane region comprises an amino acid sequence at least 90%identical to SEQ ID No: 9.
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.
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 at least 85%, 90%or 95%identical to SEQ ID No: 10.
The expression construct of claim 3 or 4, wherein the signal peptide is derived from human DAP10, T cell surface expressed receptor (such as CD8, CD28 and TCR) , NK cell surface expressed receptor (2B4, CD16, NKP30, NKP44, NKP46) , or IgG.
The expression construct of any of claims 3-5, wherein the hinge region is derived from human DAP10, TCR or an immunoglobulin.
The expression construct of any of claims 3-6, wherein the affinity tag is located at the N terminal of the DAP10 transmembrane region.
The expression construct of any of claims 3-7, wherein the recombinant polypeptide comprises, from N terminal to C terminal, the DAP10 transmembrane region and the intracellular region.
The expression construct of claim 8, wherein the recombinant polypeptide comprises, from N terminal to C terminal, the signal peptide, the hinge region, the DAP10 transmembrane region and the intracellular region, optionally with an affinity tag (such as HA tag) between the signal peptide and the hinge region.
The expression construct of claim 8, wherein the recombinant polypeptide comprises, from N terminal to C terminal, the signal peptide, the DAP10 transmembrane region and the intracellular region, optionally with an affinity tag (such as HA tag) between the signal peptide and DAP10 transmembrane region.
The expression construct of any of claims 3-10, wherein the intracellular region further comprises one or more costimulatory signaling domains derived from any of CD28, 4-1BB, 2B4, CD27, OX40, CD30, CD40, CD3, LFA-1, ICOS (CD278) , NTBA, CD2, CD7, LIGHT, NKG2C, B7-H3, ligands of CD83 and combinations thereof.
The expression construct of any of claims 3-11, wherein the intracellular region further comprises a primary signaling domain.
The expression construct of claim 12, wherein the primary signaling domain is derived from CD3ζ, optionally the primary signaling domain comprises the amino acid sequence of SEQ ID No: 5 or an amino acid sequence at least 85%, 90%or 95%identical to SEQ ID No: 5.
The expression construct of any one of claims 1-13, wherein the recombinant polypeptide comprises, from N terminal to C terminal:

(a) DAP10 signal peptide (SP) , DAP10 hinge region, DAP10 transmembrane region (TM) and a costimulatory signaling domain, optionally a primary signaling domain;

(b) DAP10 SP, DAP10 TM and a costimulatory signaling domain, optionally a primary signaling domain;

(c) CD28 SP, DAP10 hinge region, DAP10 TM and a costimulatory signaling domain, optionally a primary signaling domain; or

(d) CD28 SP, DAP10 TM and a costimulatory signaling domain, optionally a primary signaling domain.
The expression construct of claim 14, wherein the recombinant polypeptide comprises, from N terminal to C terminal:

(a) DAP10 signal peptide (SP) , DAP10 hinge region, DAP10 transmembrane region (TM) , DAP10 intracellular domain (ICD) and 2B4 costimulatory signaling domain;

(b) DAP10 SP, DAP10 hinge region, DAP10 TM, DAP10 ICD and 4-1BB costimulatory signaling domain;

(c) DAP10 SP, DAP10 hinge region, DAP10 TM, DAP10 ICD, 2B4 costimulatory signaling domain and 4-1BB costimulatory signaling domain;

(d) DAP10 SP, DAP10 hinge region, DAP10 TM, DAP10 ICD, 2B4 costimulatory signaling domain and CD3ζ signaling domain;

(e) DAP10 SP, DAP10 hinge region, DAP10 TM, DAP10 ICD, 4-1BB costimulatory signaling domain and CD3ζ signaling domain;

(f) DAP10 SP, DAP10 hinge region, DAP10 TM, DAP10 ICD and 2B4·ITSM2 costimulatory signaling domain;

(g) DAP10 SP, DAP10 hinge region, DAP10 TM, DAP10 ICD and CD28 costimulatory signaling domain;

(h) CD8 SP, DAP10 hinge region, DAP10 TM, DAP10 ICD and 2B4 costimulatory signaling domain;

(i) CD8 SP, DAP10 TM, DAP10 ICD and 2B4 costimulatory signaling domain; or

(j) CD8 SP, DAP10 hinge region, DAP10 TM, DAP10 ICD and 2B4·ITSM2 costimulatory signaling domain.
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.
The expression construct of any of claims 1-16, wherein the cleavable linker is selected from P2A, E2A, F2A, T2A peptide, an IRES sequence and a functional variant thereof.
The expression construct of any of claims 1-17, wherein the CAR polypeptide comprises an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, and the extracellular antigen binding domain is selected from a single-chain Fv (scFv) , a Fab, a Fab’, a F (ab’) 2, an Fv, minibody, a diabody, a single-domain antibody (sdAb) or VHH domain, such as a scFv.
The expression construct of claim 18, wherein the extracellular antigen binding domain is a scFv targeting GPC3, CD19 or CD33, e.g. the scFv targeting GPC3 comprises the amino acid sequence of SEQ ID No: 1.
The expression construct of any of claims 18-19, wherein the transmembrane domain of the CAR polypeptide is derived from any of CD8, ICOS, CD4, CD28, CD137, CD80, CD86, CD152 and PD1.
The expression construct of claim 20, wherein the transmembrane domain of the CAR polypeptide is derived from CD8 or CD28, optionally the transmembrane domain comprises the amino acid sequence of SEQ ID No: 3 or 22.
The expression construct of claim 18, wherein the intracellular signaling domain of the CAR polypeptide comprises a primary intracellular signaling domain, a co-stimulatory signaling domain or both.
The expression construct of claim 22, wherein the primary intracellular signaling domain of the CAR polypeptide is derived from CD3ζ, optionally the primary intracellular signaling domain comprises the amino acid sequence of SEQ ID No: 5 or an amino acid sequence at least 85%, 90%or 95%identical to SEQ ID No: 5.
The expression construct of any of claims 22-23, wherein the co-stimulatory signaling domain of the CAR polypeptide is derived from a co-stimulatory molecule selected from CD28, 4-1BB (CD137) , 2B4, CD27, OX40, CD30, CD40, CD3, LFA-1, ICOS (CD278) , NTBA, CD2, CD7, LIGHT, NKG2C, B7-H3, ligands of CD83 and combinations thereof.
The expression construct of claim 24, wherein the co-stimulatory signaling domain of the CAR polypeptide is derived from 4-1BB, 2B4 or CD28, optionally, the co-stimulatory signaling domain comprises any of the amino acid sequences of SEQ ID Nos: 4, 11-12 and 21 or an amino acid sequence at least 85%, 90%or 95%identical to any of SEQ ID Nos: 4, 11-12 and 21.
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 the hinge domain is 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.
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.
The expression construct of claim 27, wherein the cytokine polypeptide comprises a 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.
The expression construct of claim 28, wherein the second cleavable linker is selected from P2A, E2A, F2A, T2A peptide, IRES sequence and a functional variant thereof.
A vector comprising the expression construct of any of claims 1-29.
The vector of claim 30, wherein the vector is a viral vector such as an adenoviral vector, adeno-associated viral vector, lentiviral vector or retroviral vector, or a non-viral vector such as a plasmid, liposome, nanoparticle, lipid or combination thereof.
An engineered immune cell comprising the expression construct of any of claims 1-29, or the vector of claim 30 or 31.
The engineered immune cell of claim 32, which the immune cell is a natural killer (NK) cell, T cell, gamma delta T cells, invariant NKT (iNKT) cell, B cell, macrophage, MSCs, or dendritic cell.
The immune cell of claim 33, wherein the NK cell is derived from cord blood, peripheral blood, induced pluripotent stem cells, human embryonic stem cells, bone marrow, or from a cell line.
A population of the immune cell of any of claims 32-34, wherein the cells present in a suitable medium.
A recombinant polypeptide encoded by the second nucleic acid sequence of the expression construct of any of claims 1-29.
A pharmaceutical composition comprising the engineered immune cells of any one of claims 32-34, and a pharmaceutically acceptable carrier.
A method for preventing or treating a cancer in a subject in need thereof, comprising administering an effective amount of the immune cells of any one of claims 32-34 or the expression construct of any of claims 1-29 to the subject, optionally the immune cells are allogeneic with respect to the subject.
The method of claim 38, wherein the cancer is selected from multiple myeloma (MM) , acute myeloid leukemia (AML) , acute lymphoblastic leukemia (ALL) , gliomas, breast cancer, cervical cancer, prostate cancer, kidney cancer, gastric cancer, esophageal cancer, pancreatic ductal cancer, lung cancer such as non-small cell lung cancer (NSCLC) , ovarian cancer, colorectal cancer, liver cancer, head and neck cancer, gallbladder cancer.
The immune cells of any one of claims 32-34 or the expression construct of any of claims 1-29 for use in treating a cancer in a subject in need thereof.