Engineered immune cells

Abstract

The present invention relates to chimeric antigen receptors (chimeric antigen receptors), engineered immune cells (e.g., NK cells, NKT cells, and T cells), and methods of use thereof. The chimeric antigen receptors provided herein can enhance the killing activity of engineered NK cells.

Description

Cross-references

[0001] This application claims priority to U.S. Provisional Application No. 63 / 376,931 filed on September 23, 2022, and this application incorporates its entire disclosure by reference. Gene sequence list

[0002] This application includes a computer-readable gene sequence list, which is submitted with this application in XML file format, and this application includes its entire contents by reference. The gene sequence list XML file submitted with this application is named "14767-001-228_SEQLISTING.xml", which was created on September 10, 2023, and has a file size of 215,150 bytes. 1. Technical Field

[0003] The present application relates to chimeric antigen receptors (CARs), engineered immune cells (e.g., NK cells, NKT cells, and T cells), and methods of use thereof. The chimeric antigen receptors provided herein can enhance the killing activity of engineered NK cells. 2. Background technology

[0004] Immunotherapy has become a promising cancer treatment method. It has been shown that T cells expressing chimeric antigen receptors (CARs) are effective against liquid tumors, but have only very limited effects on solid tumors (Porter et al. (2011) Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med. 365 (8): 725-33; Ahmed et al. (2015) Autologous HER2 CMV bispecific CAR T cells are safe and demonstrate clinical benefit for glioblastoma in a Phase I trial. J Immunother Cancer. 3 (Suppl 2): ​​O11; Fesnak et al. (2016) Engineered T cells: the promise and challenges of cancer immunotherapy. Nat Rev Cancer. 16 (9): 566-81).

[0005] Natural killer (NK) cells are an essential part of the natural immune system and constitute the first line of defense against tumors (Campbell et al. (2013) Natural killer cell biology: an update and future directions. J Allergy Clin Immunol. 132(3): 536-544). Due to the unique biological properties of NK cells, chimeric antigen receptor-engineered NK cells (chimeric antigen receptor-NK) have become more attractive. First, chimeric antigen receptor-NK cells have higher safety than chimeric antigen receptor-T cells, including reduced risk of cytokine release syndrome (CRS) and neurotoxicity, and reduced risk of graft-versus-host disease (GVHD) (Liu et al. (2020) Use of CAR-Transduced Natural Killer Cells in CD19-Positive Lymphoid Tumors. NEngl J Med. 382(6):545-553; Lupo et al. (2019) Natural Killer Cells as Allogeneic Effectors in Adoptive Cancer Immunotherapy. Cancers (Basel). 11(6):769). Second, the multifunctionality of chimeric antigen receptor-NK cells enables them to kill target tumor cells in a chimeric antigen receptor-specific manner.In addition, NK cells display potent cytotoxicity against tumor cells through many constitutively expressed activating receptors (e.g., NCR, NKG2D, DNAM-1, activated KIR (KIR2DS1, KIR2DS4, KIR2DL4), CD16-mediated antibody-dependent cellular cytotoxicity (ADCC), and TRAIL- or FasL-induced apoptosis (Oei et al. (2018) Intrinsic Functional Potential of NK-Cell Subsets Constrains Retargeting Driven by Chimeric Antigen Receptors. Cancer Immunol Res. 6(4):467-480; Sun et al. (2015) NK cell receptor imbalance and NK cell dysfunction in HBV infection and hepatocellular carcinoma. Cell Mol Immunol. 12(3):292-302; Wu et al. (2019) Role of ADAM17 as a regulatory checkpoint of CD16A in NK cells and as a potential target for cancer immunotherapy. J Leukoc Biol. 105(6):1297-1303). Third, chimeric antigen receptor-NK cells are already available as off-the-shelf products, eliminating the need for personalized and patient-specific products.Since the risk of alloreactivity and GVHD is low, it is potentially possible to use various product sources of allogeneic chimeric antigen receptor-NK cells, including NK cell lines, peripheral blood mononuclear cells (PBMCs) and pluripotent stem cells (iPSCs) (Shimasaki et al. (2020) NK cells for cancer immunotherapy. Nat Rev Drug Discov. 19(3): 200-218; Ueda et al. (2020) Non-clinical efficacy, safety and stable clinical cell processing of induced pluripotent stem cell-derived anti-glypican-3 chimeric antigen receptor-expressing natural killer / innate lymphoid cells. Cancer Sci. 111(5): 1478-1490).

[0006] Most NK chimeric antigen receptors, including the latest CAR-NK studied in clinical trials, use T cell-derived chimeric antigen receptors, such as ScFv-CD8a hinge-CD28 TM and ICD-CD3ζ. It has been reported that NK-specific chimeric antigen receptors are better than T chimeric antigen receptors expressed in NK cells (Li et al. (2018) Human iPSC-Derived Natural Killer Cells Engineered with Chimeric Antigen Receptors Enhance Anti-tumor Activity. Cell Stem Cell. 23(2): 181-192.e5). 3. Summary of the invention

[0007] One aspect of the present application provides a chimeric antigen receptor (CAR). In certain embodiments, the chimeric antigen receptor (CAR) provided herein comprises: (i) an extracellular antigen-binding domain; (ii) a transmembrane domain; and (iii) comprising an intracellular signaling domain derived from the intracellular domain of the first polypeptide, wherein the intracellular domain derived from the first polypeptide is linked to the C-terminus of the transmembrane domain directly or via a peptide linker, and The first polypeptide is DAP10 or DAP12.

[0008] In certain embodiments, provided herein is a chimeric antigen receptor (CAR) comprising: (i) an extracellular antigen-binding domain; (ii) comprises a transmembrane domain derived from the transmembrane domain of a second polypeptide; and (iii) comprising an intracellular signaling domain derived from the intracellular domain of the first polypeptide, wherein the second polypeptide is different from the first polypeptide, wherein the intracellular domain derived from the first polypeptide is linked to the C-terminus of the transmembrane domain derived from the second polypeptide directly or via a peptide linker, and wherein the first polypeptide is DNAM1.

[0009] In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain derived from a second polypeptide, wherein the second polypeptide is different from the first polypeptide, and wherein the intracellular domain derived from the first polypeptide is linked directly or via a peptide linker to the C-terminus of the transmembrane domain derived from the second polypeptide.

[0010] In certain embodiments, the intracellular signaling domain of the CAR further comprises an intracellular domain derived from a third polypeptide, wherein the third polypeptide is different from the first polypeptide.

[0011] In certain embodiments, the second polypeptide and the third polypeptide are the same polypeptide.

[0012] In certain embodiments, the CAR further comprises a hinge domain between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain.

[0013] In certain embodiments, the hinge domain of the CAR comprises a hinge domain derived from the second polypeptide.

[0014] In certain embodiments, the second polypeptide or the third polypeptide is a signaling lymphocyte activation molecule family (SLAMF) receptor, CD2, CD28H, DNAM1, CD28, 4-1BB, OX40, KIR-S, NKG2C or NKG2E. In certain embodiments, the SLAMF receptor is SLAMF4 (CD244, 2B4), SLAMF3 (CD229, Ly9), SLAMF6 (CD352, NTB-A, SF2000), SLAMF5 (CD84), SLAMF7 (CD319, CRACC, CS1), SLAMF1 (CD150), SLAMF2 (CD48, FimH), SLAMF8 (CD353) or SLAMF9 (CD84H1, SF2001). In certain embodiments, the KIR-S is KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, or KIR3DS1.

[0015] In certain embodiments, the intracellular signaling domain of the CAR further comprises an intracellular domain derived from CD3ζ.

[0016] In certain embodiments, the intracellular signal domain of the CAR further includes an intracellular domain derived from SLAM associated protein (SAP).In certain embodiments, the intracellular signal domain of the CAR further includes an intracellular domain derived from Ewing's (Ewing's) sarcoma activated transcript-2 (EAT2).

[0017] In certain embodiments, the intracellular domain derived from CD3ζ is directly or via a peptide linker connected to the intracellular domain derived from SAP or the intracellular domain derived from EAT2. In certain embodiments, the intracellular domain derived from CD3ζ is connected to the intracellular domain derived from SAP or the intracellular domain derived from EAT2 via P2A, T2A or E2A.

[0018] In certain embodiments, the extracellular antigen binding domain of the CAR comprises a scFv. In certain embodiments, the extracellular antigen binding domain of the CAR binds to a tumor antigen or a fragment thereof.

[0019] In certain embodiments, the first polypeptide is DAP10.

[0020] In certain embodiments, the CAR comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:20.

[0021] In certain embodiments, the CAR comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:86.

[0022] In certain embodiments, the CAR comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:87.

[0023] In certain embodiments, the CAR comprises SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144 or SEQ ID NO:145, or an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144 or SEQ ID NO:145.

[0024] In certain embodiments, the CAR comprises from N-terminus to C-terminus: (i) an extracellular antigen-binding domain; (ii) comprises a transmembrane domain derived from the transmembrane domain of the second polypeptide; (iii) an intracellular signaling domain comprising (iii)-(a) derived from the intracellular domain of DAP10; (iii)-(b) derived from the intracellular domain of CD3ζ; and (iii)-(c) an intracellular domain derived from SAP or an intracellular domain derived from EAT2, wherein these intracellular domains within the intracellular signaling domain are linked to each other directly or via one or more peptide linkers, and wherein the extracellular antigen-binding domain, the transmembrane domain and the intracellular domain are linked to each other directly or via one or more peptide linkers.

[0025] The second aspect of the present application provides engineered immune cells. In certain embodiments, provided herein are engineered immune cells comprising the aforementioned CAR, wherein the engineered immune cells are NK cells, NKT cells or T cells.

[0026] In certain embodiments, provided herein is an engineered immune cell comprising a CAR, wherein the CAR comprises: (i) an extracellular antigen-binding domain; (ii) a transmembrane domain; and (iii) an intracellular signaling domain, and Wherein the engineered immune cell further comprises an exogenously introduced polynucleotide encoding an intracellular domain derived from a SLAM-associated protein (SAP), and wherein the engineered immune cell is a NK cell, a NKT cell or a T cell.

[0027] In certain embodiments, provided herein is an engineered immune cell comprising a CAR, wherein the CAR comprises: (i) an extracellular antigen-binding domain; (ii) a transmembrane domain; and (iii) an intracellular signaling domain, and wherein the engineered immune cell further comprises an exogenously introduced polynucleotide encoding an intracellular domain derived from Ewing's sarcoma activated transcript-2 (EAT2), and wherein the engineered immune cell is a NK cell, a NKT cell or a T cell.

[0028] In certain embodiments, the polynucleotide encodes the CAR in the engineered immune cell, and wherein the intracellular signaling domain of the CAR in the engineered immune cell comprises the intracellular domain derived from SAP or the intracellular domain derived from EAT2.

[0029] In certain embodiments, the intracellular signaling domain of the CAR in the engineered immune cell comprises an intracellular domain derived from a first polypeptide, and wherein the first polypeptide is DAP10, DNAM1, CD28H, CD2, DAP12, CD28, or OX40.

[0030] In certain embodiments, the intracellular domain of the first polypeptide is linked to the C-terminus of the transmembrane domain directly or via a peptide linker.

[0031] In certain embodiments, the transmembrane domain of the CAR in the engineered immune cell comprises a transmembrane domain derived from a second polypeptide.

[0032] In certain embodiments, the intracellular signaling domain of the CAR in the engineered immune cell comprises an intracellular domain derived from a third polypeptide.

[0033] In certain embodiments, the second polypeptide or the third polypeptide is a signaling lymphocyte activation molecule family (SLAMF) receptor, CD2, CD28H, DNAM1, CD28, 4-1BB, OX40, KIR-S, NKG2C or NKG2E. In certain embodiments, the SLAMF receptor is SLAMF4 (CD244, 2B4), SLAMF3 (CD229, Ly9), SLAMF6 (CD352, NTB-A, SF2000), SLAMF5 (CD84), SLAMF7 (CD319, CRACC, CS1), SLAMF1 (CD150), SLAMF2 (CD48, FimH), SLAMF8 (CD353) or SLAMF9 (CD84H1, SF2001). In certain embodiments, the KIR-S is KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, or KIR3DS1.

[0034] In certain embodiments, the intracellular signaling domain of the CAR in the engineered immune cell comprises an intracellular domain derived from CD3ζ.

[0035] In certain embodiments, the intracellular signaling domain of the CAR in the engineered immune cell further comprises an intracellular domain derived from a SLAM-associated protein (SAP) or an intracellular domain derived from Ewing's sarcoma activated transcript-2 (EAT2), and wherein the intracellular domain derived from CD3ζ is directly or via a peptide linker connected to the intracellular domain derived from SAP or the intracellular domain derived from EAT2. In certain embodiments, the intracellular domain derived from CD3ζ is connected to the intracellular domain derived from SAP or the intracellular domain derived from EAT2 via P2A, T2A or E2A.

[0036] In certain embodiments, the extracellular antigen binding domain of the CAR in the engineered immune cell comprises a scFv. In certain embodiments, the extracellular antigen binding domain of the CAR binds to a tumor antigen or a fragment thereof.

[0037] In certain embodiments, the CAR in the engineered immune cell comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:20.

[0038] In certain embodiments, the CAR in the engineered immune cell comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:86.

[0039] In certain embodiments, the CAR in the engineered immune cell comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:87.

[0040] In certain embodiments, the CAR in the engineered immune cell comprises the sequence of SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144 or SEQ ID NO:145 are amino acid sequences that are at least 90%, at least 95% or 100% identical to the amino acid sequences of SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144 or SEQ ID NO:145.

[0041] In certain embodiments, the engineered immune cells are NK cells.

[0042] Another aspect of the present application provides polypeptides. In certain embodiments, the polypeptides herein comprise CARs provided herein.

[0043] In certain embodiments, the polypeptide provided herein comprises a chimeric antigen receptor (CAR) and an intracellular domain derived from a SLAM-associated protein (SAP), wherein the CAR comprises: (i) an extracellular antigen-binding domain; (ii) a transmembrane domain; and (iii) an intracellular signaling domain, and wherein the CAR is linked to an intracellular domain derived from SAP via a peptide linker.

[0044] In certain embodiments, provided herein is a polypeptide comprising a chimeric antigen receptor (CAR) and an intracellular domain derived from Ewing's sarcoma activated transcript-2 (EAT2), wherein the CAR comprises: (i) an extracellular antigen-binding domain; (ii) a transmembrane domain; and (iii) an intracellular signaling domain, and wherein the CAR is linked to an intracellular domain derived from SAP via a peptide linker.

[0045] In certain embodiments, said CAR is linked to said intracellular domain derived from SAP or said intracellular domain derived from EAT2 via P2A, T2A or E2A.

[0046] In certain embodiments, the polypeptide comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:86 or SEQ ID NO:87.

[0047] In certain embodiments, the polypeptide comprises SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ : The invention relates to an amino acid sequence that is at least 90%, at least 95% or 100% identical to an amino acid sequence of SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144 or SEQ ID NO:145.

[0048] On the other hand, polynucleotides are provided herein. In certain embodiments, the polynucleotides provided herein encode the CAR provided herein or the polypeptide provided herein.

[0049] In another aspect, provided herein are vectors. In certain embodiments, provided herein are vectors comprising a polynucleotide provided herein.

[0050] In another aspect, provided herein are pharmaceutical compositions. In certain embodiments, provided herein are pharmaceutical compositions comprising: (i) a CAR provided herein, an engineered immune cell provided herein, a polypeptide provided herein, a polynucleotide provided herein, or a vector provided herein; and (ii) a pharmaceutically acceptable excipient.

[0051] In another aspect, provided herein are methods for treating a disease or condition in an individual. In certain embodiments, provided herein are methods for treating a disease or condition in an individual comprising: administering to the individual an effective amount of an engineered immune cell provided herein or a pharmaceutical composition provided herein. In certain embodiments, the disease or condition is cancer.

[0052] In another aspect, provided herein is a method for engineering an immune cell. In certain embodiments, provided herein is a method for engineering an immune cell comprising: introducing a polynucleotide provided herein, a vector provided herein, or a polypeptide provided herein into the immune cell. 4. Description of the drawings

[0053] Figures 1A to 1F This indicates the functional impairment of natural NKG2D in NKG2D-CAR-NK. Figure 1A : CAR constructs of CAR0 and CAR2, and expression of CAR on primary human NK cells. Figures 1B to 1C : Expression of NKG2D and 2B4 on CAR0-NK and CAR2-NK cells. **P<0.01, ***P<0.001. Figure 1D : Analysis of CAR-NK cell killing after 10 hours of culture. If CD19 is present on 293T, CAR2 killing is extremely high, but if CD19 is absent, CAR2 killing ability is significantly reduced through NKG2D stimulation, and the same situation occurs in HUH7 cells. ***p<0.001. Figure 1E : Duolink signal between CAR+DAP10 and NKG2D+DAP10 in CAR-NK cells stimulated by 293T-CD19-MICA. The four images on the left show the Duolink PLA signal of bright spots, and the four images on the right show the nuclei stained with DAPI. The cells with large nuclei are NK cells, while the target cells have smaller nuclei. Figure 1F :like Figure 1E Calculation of the number of Duolink PLA signals in , ****P<0.0001.

[0054] FIG. 2A to FIG. 2E This demonstrates the complete signaling of NK cells and the anti-tumor activity mediated by 2B4-CAR. Figure 2A : CAR construct design with ScFv against CD19 or GPC3. Figure 2B: FACS analysis of the phenotypes of 293T, 293T-CD19, NALM6 and HUH7. CD19 expressed in 293T-CD19 but not in 293T-WT (left), CD19 expressed on NALM6 cells (middle) and GPC3 expressed on HUH7 cells (right). Figure 2C : CAR-NK92 cells kill target cells 293T, 293T-CD19 (upper, 4 hours), NALM6 (middle, 20 hours), HUH7 (lower, 6 hours) at the specified effector: target ratio. The average percentage of specific target cell lysis ± SEM is shown. Figure 2D and Figure 2E : In the analysis of CAR-NK92 cells after stimulation by 293T, 293T-CD19 and NALM6, CD107a ( Figure 2D ) and IFNg( Figure 2E ) are expressed as FACS. Data were normalized to the percentage of CAR-NK92 cells treated with medium and are shown as mean ± SEM. *P<0.05, ***p<0.001.

[0055] FIG. 3A to FIG. 3C Demonstrating a complete signaling CAR enhanced by SAP. Figure 3A : Design of CAR constructs with SAP. Figure 3B : Analysis of signal-enhanced NKG2D CAR and 2B4 CAR-NK cell killing after 4 hours of culture. Figure 3C : IFNg production and degranulation of CAR-NK cells after stimulation of designated target cells. *P<0.05, **P<0.01, ***P<0.001.

[0056] 4A to 4D This indicates that 2B4-DAP10-CD3Z CAR-NK has normal NKG2D function. Figure 4A :CAR construct design. Figure 4B : Expression of NKG2D and 2B4 on CAR-NK cells. Figure 4C : The interaction between NKG2D and DAP10 was reduced on NKG2DCAR-NK cells, but normal in 2B4 CAR-NK cells. ****P<0.0001, ns means not significant. Figure 4D : Shows the killing analysis of target cells killed by CAR-NK cell co-culture for 10 hours.

[0057] FIG. 5A to FIG. 5D This indicates that csCAR can effectively overcome the HLA-E barrier. Figure 5A :CAR construct design. Figure 5B : HLA-E expression on target cells of 293T-CD19, 293T-CD19-HLA-E and NALM6 cells. Figure 5C : NKG2A expression on CAR-NK cells. Figure 5D : CAR-NK killing analysis of 293T-CD19, 293T-CD19-HLA-E after 4 hours of culture and NALM6 cells after 20 hours of culture.

[0058] Fig. 6A Figure H illustrates the domains of NK-CAR in NK activation and killing. Fig. 6A and Fig. 6E : Vector constructs encoding CD19-specific -CAR2 and -CAR4 with domain deficiencies of DAP10 or CD3Z, and different positions of DAP10 (A), and the DAP10 of CAR4, CAR5, CAR6, CAR8 was repositioned to generate CAR4B, CAR5B, CAR6B and CAR8B. Figure 6B : NK cells were cultured with 293T or 293T-CD19 target cells at the indicated effector:target ratios after 4 hours of culture. Mean percentages of specific target cell lysis ± SEM are shown. Figure 6C and Fig.6D : In the analysis of CAR-NK92 cells after stimulation with 293T, 293T-CD19 and NALM6, IFNg( Figure 6C ) and CD107a( Fig.6D ) are expressed as FACS. Data were normalized to the percentage of CAR-NK92 cells treated with medium and are shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001. Fig. 6F : NK cells were cultured with target cells of NALM6 at the indicated effector:target ratios after 20 hours of culture. Mean percentages of specific target cell lysis ± SEM are shown. Figure 6G and Figure 6H : In the analysis of CAR-NK92 cells after stimulation with 293T, 293T-CD19 and NALM6, and IFNg( Figure 6H ) and CD107a( Figure 6G ) expressed as FACS. Data were normalized to the percentage of CAR-NK92 cells treated with medium, and mean values ​​are shown. ±SEM. *P<0.05, **P<0.01.

[0059] 7A to 7E This demonstrates a broader selection of SLAMF and non-SLAMF csCARs in NK cell activation and killing. Fig. 7A:CAR construct design. Figure 7B : Analysis of the killing of 293T-CD19 and 293T-CD19-HLA-E by CAR-NK cells after 4 hours of culture and NALM6 after 20 hours of culture. Figure 7C : IFNg production and degranulation of CAR-NK cells stimulated by target cells. Fig.7D : The killing analysis of NALM6 by CAR16B-NK cells after 20 hours of culture was better than that by CAR16-NK cells. Fig. 7E : IFNg production and degranulation of CAR16B-NK cells stimulated by target cells, *P<0.05, **P<0.01, ***P<0.001, ns means not significant.

[0060] Figure 8 Several SLMAF family genes are described to be expressed on NK cells, excluding SLAMF2 (CD48), which does not have an intracellular signaling domain. 5. Specific implementation methods

[0061] Provided herein are CAR, engineered immune cells (such as NK cells, NKT cells and T cells), related polypeptides, related polynucleotides and carriers, related pharmaceutical compositions, methods for treating diseases and methods for engineering immune cells (such as NK cells, NKT cells and T cells). In certain embodiments, provided herein are CAR (see Section 5.2), which includes an extracellular antigen binding domain (see Section 5.2.2), a transmembrane domain (see Section 4.2.3), an intracellular signaling domain (see Section 5.2.4), an optional hinge domain (see Section 5.2.5) and one or more optional peptide linkers (see Section 5.2.6). In certain embodiments, provided herein are engineered immune cells (see Section 5.3). In certain embodiments, provided herein are engineered immune cells, which include CAR as provided in Section 5.2. In certain embodiments, provided herein are engineered immune cells, which include any CAR known in the art and polynucleotides encoding exogenous introduction of intracellular domains derived from SAP or EAT2. In certain embodiments, provided herein is an engineered immune cell, which expresses any CAR known in the art and an intracellular domain derived from SAP or EAT2. In certain embodiments, provided herein is an engineered immune cell, which includes a CAR comprising an intracellular domain derived from SAP or EAT2. In certain embodiments, provided herein is a polypeptide (see Section 5.4). In certain embodiments, provided herein is a polypeptide comprising a CAR provided in Section 5.2. In certain embodiments, provided herein is a polypeptide comprising an intracellular domain of a CAR known in the art and derived from SAP or EAT2 and CAR is connected to an intracellular domain derived from SAP or EAT2 via a flexible linker. In certain embodiments, provided herein is a polynucleotide (see Section 5.5). In certain embodiments, provided herein is a carrier (see Section 5.5). In certain embodiments, provided herein is a pharmaceutical composition comprising (i) a CAR provided in Section 5.2, an engineered immune cell provided in Section 5.3, a polypeptide provided in Section 5.4, a polynucleotide provided in Section 5.5, or a vector provided in Section 5.5; and (ii) a pharmaceutically acceptable excipient (see Section 5.6). In certain embodiments, provided herein is a method for engineering immune cells (see Section 5.7). In certain embodiments, provided herein is an exemplary analysis for preparing, using, and measuring the compositions provided herein (see Section 5.9).

[0062] In certain embodiments, the engineered immune cells provided herein are NK cells, NKT cells, or T cells.

[0063] In certain embodiments, the CAR provided herein can enhance the killing activity of engineered immune cells. In certain embodiments, the CAR provided herein can enhance the killing activity of engineered NK cells.

[0064] In certain embodiments, CAR provided herein includes SLAMF or non-SLAMF hinge / transmembrane domain / intracellular domain (ICD), ICD of DAP10 and CD3ζ signal domain to mediate robust antigen-specific NK signal transduction. In certain embodiments, CAR provided herein includes DAP10 directly or via a peptide linker connected to a transmembrane domain. In certain embodiments, CAR provided herein includes signal enhancing polypeptide SAP or EAT2. In certain embodiments, engineered immune cells derived from human peripheral blood cells significantly enhance anti-tumor activity compared with T-CAR or reported NKG2D CAR (including 2B4 CAR, CD229 CAR, NTB-A CAR, CD84 CAR, CRACC CAR, CD28H CAR, CD2 CAR, DNAM1 CAR). In certain embodiments, it is not limited to any particular theory, and the CAR provided can effectively block the inhibitory signal transduction stimulated by HLA-E, thereby overcoming the inhibitory receptor signal transduction barrier during NK cell activation, thereby resulting in more effective NK cell function and further enhanced anti-tumor activity.

[0065] NKG2D CAR is the most potent NK-CAR reported to date, which consists of scFv-CD8 hinge-NKG2DTM-2B4ICD-CD3ζ. NKG2D CAR uses only the transmembrane domain (TM) of NKG2D, which can recruit DAP10 to the CAR tail and act as a co-stimulator and as a natural NKG2D receptor trigger signal (Li et al. (2018) Human iPSC-Derived Natural Killer Cells Engineered with ChimericAntigen Receptors Enhance Anti-tumor Activity.Cell Stem Cell.23(2):181-192.e5). However, without being limited to any particular theory, this situation will occupy DAP10 and cause defective recruitment of DAP10 to natural NKG2D, so that the function of natural NKG2D is impaired. Without being limited to any particular theory, this phenomenon has been identified and confirmed in studies related to CAR provided herein. Without being limited to any particular theory, multifunctional NK cells can be activated by a variety of activating receptors, and many tumor cells express NKG2D ligands, such as MICA / MICB and ULBP1-3, so maintaining the full function of NKG2D is extremely important for tumor cell clearance. Without being limited to any particular theory, we designed a novel NK CAR different from NKG2D CAR.

[0066] Without being limited to any particular theory, in order to control NK cell responses, a balanced integration of activation and inhibition is required. Without being limited to any particular theory, the synergistic activation of activating receptor pairs may be sufficient to trigger the activation of NK cells, such as 2B4 and NKG2D, or 2B4 and DNAM-1. Without being limited to any particular theory, this synergy is sufficient to overcome the inhibitory signaling of inhibitory receptors via c-Cbl or SHP-dependent inhibition. Without being limited to any particular theory, among the activating receptors, 1) 2B4 signaling is Fyn-dependent and Syk-independent, which can phosphorylate the aptamer SLP-76 on Tyr113; 2) NKG2D uses the tail of the immunoreceptor tyrosine tail (ITT)-like motif to recruit the aptamer molecules DAP10 and DNAM-1 to induce Syk-independent phosphorylation of SLP-76 on Tyr128; and 3) CD16 is signaled via ITAM, resulting in Syk-dependent phosphorylation of SLP-76 on both Tyr113 and Tyr128. Without being limited to any particular theory, the origin of the design concept of our novel NK CAR is to provide sufficient and complete activation signals for the full activation of NK cells, and to overcome the transmission barrier of inhibitory receptor signals (Long et al. (2013) Controlling natural killer cell responses: integration of signals for activation and inhibition. Annu Rev Immunol. 31: 227-58).

[0067] Without being limited to any particular theory, the cytoplasmic tails of SLAM family (SLAMF) molecules, such as 2B4, CD229, NTB-A, CD84, and CRACC, contain an immunoreceptor tyrosine-based switch motif (ITSM) for signal transduction. Without being limited to any particular theory, the phosphorylated tyrosine of ITSM can bind to SHP, and the SHP provides an inhibitory signal to NK cells. Without being limited to any particular theory, phosphorylated ITSM can also recruit SLAM-associated protein (SAP) and Ewing's sarcoma activated transcript-2 (EAT-2) (Ma et al. (2007) Regulation of cellular and humoral immune responses by the SLAM and SAP families of molecules. Annu Rev Immunol. 25: 337-79; Claus et al. (2008) Regulation of NK cell activity by 2B4, NTB-A and CRACC. Front Biosci. 13: 956-65). Without being bound to any particular theory, ITSM-bound SAP and EAT2 molecules can recruit the Src-family kinase Fyn, which then phosphorylates phosphatase C-γ or Vav-1 (Chen et al. (2004) Molecular dissection of 2B4 signaling: implications for signal transduction by SLAM-related receptors. Mol Cell Biol. 24(12):5144-56; Eissmann et al. (2006) Molecular analysis of NTB-A signaling: a role for EAT-2 in NTB-A-mediated activation of human NK cells. J Immunol. 177(5):3170-7). Without being bound by any particular theory, the relative amounts of SAP and SLAMF receptors determine the direction of effector cells; insufficient SAP results in a dominant inhibitory signal and increased ligand density, or expression of SLAMF receptors can limit SAP expression and lead to inhibition of effector cell function (Waggoner et al. (2012) Evolving role of 2B4 / CD244 in T and NK cell responses during virus infection. Front Immunol. 3:377).Without being bound to any particular theory, a sufficient amount of SAP can competitively bind to the pTyr of ITSM in the SLAMF tail, then prevent ITSM from binding to the inhibitory signal SHP, allowing Fyn molecules to be recruited and leading to the activation of SLP-76 via phosphorylation at Tyr113. 5.1 Definitions

[0068] The techniques and procedures described or referred to herein include conventional methods that are understood and / or commonly used by those skilled in the art, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition, 2001); Current Protocols in Molecular Biology (Ausubel et al., ed., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed., 2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed., 2010); and Antibody Engineering, Vol. 1 and Vol. 2 (Kontermann and Dübel ed., 2nd edition, 2010) described in the widely used methods. Unless otherwise defined herein, the technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill. For the purpose of interpreting this specification, the following description of the terms may be applicable as appropriate, and the terms used in the singular also include the plural, and vice versa. In the event that the description as a term is set forth conflicts with any document incorporated herein by reference, the description as a term is set forth below shall control.

[0069] Herein, the terms "antibody", "immunoglobulin" or "Ig" are used interchangeably and in the broadest sense, specifically, as described below, covering (for example) monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length or intact monoclonal antibodies), antibody compositions with multi-epitope or mono-epitope specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies, as long as they exhibit the desired biological activity) (which are formed by at least two intact antibodies), single-chain antibodies and fragments thereof (e.g., domain antibodies). Antibodies can be human, humanized, chimeric and / or affinity matured, as well as antibodies from other species (e.g., mouse, rabbit, rhea, etc.). The term "antibody" is intended to include polypeptide products of B cells within the immunoglobulin class of polypeptides that are capable of binding to a specific molecular antigen and are composed of two identical polypeptide chain pairs, wherein each chain pair has one heavy chain (about 50 to 70 kDa) and one light chain (about 25 kDa), each amino terminal portion of each chain comprises a variable region of about 100 to about 130 or more amino acids, and each carboxyl terminal portion of each chain comprises a constant region. For example, see Antibody Engineering (Borrebaeck, ed., 2nd ed., 1995); and Kuby, Immunology (3rd ed., 1997). Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, single domain antibodies comprising from camelid species (e.g., llamas or alpacas) or humanized variants thereof, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen binding fragments) of any of the above, i.e., a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment is derived. Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fv fragments, diantibodies, triantibodies, tetraantibodies, and miniantibodies. In particular, the antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as antigen binding domains or molecules containing antigen binding sites that bind to antigens (e.g., one or more CDRs of antibodies). For example, these antibody fragments can be found in Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995); Huston et al., 1993, Cell Biophysics 22: 189-224; Plückthun and Skerra, 1989, Meth. Enzymol. 178: 497-515; and Day, Advanced Immunochemistry (2nd ed. 1990). The antibodies provided herein can be any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecules. The antibodies can be agonistic antibodies or antagonistic antibodies. The antibodies can be neither agonistic nor antagonistic.

[0070] An "antigen" is a structure to which an antibody can selectively bind. A target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide. In certain embodiments, an antigen is associated with a cell, for example, is present on or within a cell.

[0071] A "complete" antibody is one that includes an antigen binding site as well as CL and at least heavy chain constant regions CH1, CH2, and CH3. The constant region may include a human constant region or an amino acid sequence variant thereof. In certain embodiments, the complete antibody has one or more effector functions.

[0072] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising VH and VL antibody domains connected to a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0073] The term "heavy chain antibody" or "HCAb" refers to a functional antibody comprising a heavy chain but lacking the light chain typically found in a 4-chain antibody. Camelids (eg, camels, llamas, or alpacas) are known to produce HCAbs.

[0074] As used herein, "single domain antibody" or "sdAb" refers to a single monomeric variable antibody domain, and it is capable of binding to an antigen (eg, a single domain antibody that binds to CD 19). A single domain antibody includes a VHH domain as described herein. Examples of single domain antibodies include, but are not limited to, antibodies that naturally lack light chains (e.g., from camelid species (e.g., llamas, etc.), single domain antibodies derived from common 4-chain antibodies, engineered antibodies, and single domain scaffolds that are not derived from antibodies. Single domain antibodies can be derived from any species, including, but not limited to, mice, humans, camels, llamas, goats, rabbits, and cows. For example, single domain antibodies can be derived from camelid species, such as antibodies produced in camels, llamas, dromedaries, alpacas, and guanacos as described herein. Other species besides Camelidae can also produce heavy chain antibodies that naturally lack light chains; VHHs derived from these other species are also within the scope of the present invention. In some embodiments, the single domain antibodies (e.g., VHHs) provided herein have a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. As described herein, single domain antibodies can be genetically fused or chemically bound to another molecule (e.g., an agent). Single domain antibodies can be part of a larger binding molecule (e.g., a multispecific antibody or a chimeric antigen receptor).

[0075] The terms "binds" and "binding" refer to interactions between molecules, including, for example, to form a complex. The interactions may be, for example, non-covalent interactions, including hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. A complex may also include a combination of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of all non-covalent interactions between a single antigen binding site on an antibody and a single epitope of a target molecule (e.g., an antigen) is the affinity of the antibody or functional fragment for that epitope. The dissociation rate (k) of a binding molecule (e.g., an antibody) from a monovalent antigen is the affinity of the antibody or functional fragment for that epitope. off ) and the association rate (k on ) ratio (k off / k on ) is the dissociation constant K D , which is inversely related to affinity. K D The lower the value, the higher the affinity of the antibody. D The value changes depending on k on and k off Both. The dissociation constant K of the antibodies provided herein DCan use any method provided herein or any other method well known to those skilled in the art to measure.The affinity at one binding site does not usually reflect the true intensity of the interaction between antibody and antigen.When a complex antigen (e.g., multivalent antigen) containing multiple repeated epitopes contacts an antibody containing multiple binding sites, the interaction of antibody and antigen at one site will increase the possibility of the reaction at a second site.The intensity of these multiple interactions between multivalent antibodies and antigens is called avidity.

[0076] In conjunction with the binding molecules described herein, terms such as "binds to", "specifically binds to" and similar terms are also used interchangeably herein and refer to a binding molecule that specifically binds to an antigen binding domain of an antigen (e.g., a polypeptide). A binding molecule or antigen binding domain that binds to or specifically binds to an antigen can be detected, for example, by immunoassays, Or other technical identification known to those skilled in the art. In some embodiments, the binding molecule or antigen binding domain binds to or specifically binds to the antigen, at which point it binds to the antigen with a higher affinity than any cross-reactive antigen, and can be determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Typically, the specific or selective reaction is at least twice the background signal or noise, and can be greater than 10 times the background. For a discussion of binding specificity, see, for example, Fundamental Immunology 332-36 (Paul ed., 2nd edition, 1989). In certain embodiments, the degree of binding of a binding molecule or antigen binding domain to a "non-target" protein is less than about 10% of the binding of the binding molecule or antigen binding domain to its specific target antigen, for example, as determined by fluorescence activated cell sorting (FACS) analysis or RIA. The binding molecule or antigen binding domain that binds to the antigen includes those that can bind to the antigen with sufficient affinity so that the binding molecule can be used as a therapeutic agent and / or diagnostic agent, for example, in terms of targeted antigens. In certain embodiments, the binding molecule or antigen binding domain that binds to the antigen has a dissociation constant (K) of less than or equal to 1 μM, 800 nM, 600 nM, 550 nM, 500 nM, 300 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM. D ). In certain embodiments, the binding molecule or antigen binding domain binds to an epitope of an antigen that is conserved in antigens from different species.

[0077] In certain embodiments, these binding molecules or antigen binding domains may comprise "chimeric" sequences, wherein a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of these chains is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of these antibodies, as long as it exhibits the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-55). Chimeric sequences may comprise humanized sequences.

[0078] In certain embodiments, these binding molecules or antigen binding domains may include a portion of a "humanized" form of a non-human (e.g., camelid, murine, non-human primate) antibody comprising a sequence from a human immunoglobulin (e.g., a recipient antibody), wherein the native CDR residues are replaced with residues of the corresponding CDRs of non-human species (e.g., donor antibodies) with the desired specificity, affinity, and ability, such as camelid, mouse, rat, rabbit, or non-human primate. In some examples, one or more FR region residues of the human immunoglobulin sequence are replaced with corresponding non-human residues. In addition, humanized antibodies may include residues not found in the recipient antibody or the donor antibody. Making these modifications can further refine antibody performance. A humanized antibody heavy chain or light chain may include substantially all structures of at least one or more variable regions, wherein all or substantially all CDRs correspond to corresponding portions of non-human immunoglobulins, and all or substantially all FRs are corresponding portions of human immunoglobulin sequences. In certain embodiments, the humanized antibody comprises at least a portion of an immunoglobulin constant region (Fc) that is typically a human immunoglobulin. For further details, see: Jones et al., Nature 321:522-25 (1986); Riechmann et al., Nature 332:323-29 (1988); Presta, Curr. Op. Struct. Biol. 2:593-96 (1992); Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-89 (1992); U.S. Pat. Nos. 6,800,738, 6,719,971, 6,639,055, 6,407,213, and 6,054,297.

[0079] In certain embodiments, these binding molecules or antigen binding domains may comprise a portion of a "fully human antibody" or "human antibody", wherein these terms are used interchangeably herein and refer to antibodies comprising human variable regions and / or human constant regions. Binding molecules may comprise single domain antibody sequences. In specific embodiments, these terms refer to antibodies comprising variable regions and constant regions of human origin. In certain embodiments, "fully human" antibodies may also comprise antibodies that bind to polypeptides and are encoded by nucleic acid sequences that are naturally occurring somatic variants of human germline immunoglobulin nucleic acid sequences. The term "fully human antibody" comprises antibodies having variable and constant regions corresponding to human germline immunoglobulin sequences, as described by Kabat et al. (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition, US Department of Health and Human Services, NIH publication number 91-3242). "Human antibodies" are those having amino acid sequences corresponding to antibodies produced by humans and / or have been prepared using any of the techniques for preparing human antibodies. Specifically, this definition of a human antibody excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol. 227: 381 (1991); Marks et al., J. Mol. Biol. 222: 581 (1991)) and yeast display libraries (Chao et al., Nature Protocols 1: 755-68 (2006)). Preparation of human monoclonal antibodies can also be used for Cole et al., Monoclonal Antibodies and Cancer Therapy 77 (1985); Boerner et al., J. Immunol. 147 (1): 86-95 (1991); and van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001).Human antibodies can be prepared by administering antigen to transgenic animals (e.g., mice) that have been modified to produce these antibodies in response to antigen challenge, but whose endogenous loci have been disabled (e.g., see Jakobovits, Curr. Opin. Biotechnol. 6(5):561-66 (1995); Brüggemann and Taussing, Curr. Opin. Biotechnol. 8(4):455-58 (1997); and the XENOMOUSE of U.S. Pat. Nos. 6,075,181 and 6,150,584). TM Technology). For human antibodies generated via human B cell hybridoma technology, see also, e.g., Li et al., Proc. Natl. Acad. Sci. USA 103:3557-62 (2006).

[0080] In certain embodiments, these binding molecules or antigen binding domains may comprise a portion of a "recombinant human antibody", wherein the phrase includes human antibodies prepared, expressed, created or isolated by recombinant methods, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from an animal (e.g., mouse or cow) that has been transfected with genes and / or chromosomes for human immunoglobulin genes (see, e.g., Taylor, LD et al., Nucl. Acids Res. 20: 6287-6295 (1992)), or antibodies prepared, expressed, created or isolated by any other method that involves splicing human immunoglobulin gene sequences to other DNA sequences. These recombinant human antibodies may have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242). However, in certain embodiments, these recombinant human antibodies have undergone in vitro mutagenesis (or in vivo somatic mutagenesis when an animal transgene of a human Ig sequence is used) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies, while derived from and related to human germline VH and VL sequences, are sequences that do not naturally exist within the human antibody germline repertoire in vivo.

[0081] In certain embodiments, these binding molecules or antigen binding domains may comprise a portion of a "monoclonal antibody", wherein in this article, the term refers to an antibody obtained from a substantially homologous antibody population, for example, the individual antibodies contained in the population are substantially the same, but there may be a minimal amount of mutations that may occur naturally or common post-translational modifications, such as amino acid isomerization or deamidation, methionine oxidation, and asparagine or glutamine deamidation, and each monoclonal antibody generally recognizes a single epitope on an antigen. In a specific embodiment, in this article, a "monoclonal antibody" is an antibody produced by a single hybridoma or other cell. The term "monoclonal" is not limited to any particular method for preparing antibodies. For example, monoclonal antibodies that can be used in the present invention can be prepared by hybridoma methodology first described by Kohler et al. in Nature 256:495 (1975) or can be prepared using recombinant DNA methods in bacteria or eukaryotic animals or plant cells (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using techniques such as those described in Clackson et al., Nature 352:624-28 (1991) and Marks et al., J. Mol. Biol. 222:581-97 (1991). Other methods for preparing pure cell lines and thereby expressing monoclonal antibodies are also widely known. See, for example, Short Protocols in Molecular Biology (Ausubel et al., ed., 5th edition, 2002).

[0082] A typical 4-chain antibody unit is a heterotetrameric glycoprotein consisting of two identical light chains (L) and two identical heavy chains (H). In the case of IgG, the 4-chain unit is generally about 150,000 daltons. Each L chain is connected to the H chain by a covalent disulfide bond, and the two H chains are connected to each other by one or more disulfide bonds (depending on the configuration of the H chain). Each H and L chain also has regularly spaced intrachain disulfide bonds. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) of each α and γ chain and four CH domains of μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus, followed by a constant domain (CL) at its other end. Align VL with VH, and align CL with the first constant domain (CH1) of the heavy chain. It is generally believed that specific amino acid residues form an interface between the light chain variable domain and the heavy chain variable domain. The pairing of VH and VL together forms a single antigen binding site. For the structure and properties of different classes of antibodies, see Basic and Clinical Immunology 71 (Stites et al., eds., 8th edition, 1994); and Immunobiology (Janeway et al., eds., 5th edition, 2001).

[0083] The term "Fab" or "Fab region" refers to the region of an antibody that binds to an antigen. As is well known, IgG typically comprises two Fab regions, each located on one of the two arms of the Y-shaped IgG structure. Each Fab region typically consists of one variable region and one constant region for each of the heavy chain and the light chain. More specifically, the variable region and constant region of the heavy chain in the Fab region are the VH and CH1 regions, and the variable region and constant region of the light chain in the Fab region are the VL and CL regions. The VH, CH1, VL and CL in the Fab region can be arranged in various ways to confer antigen binding ability of the present invention. For example, the VH and CH1 regions can be on one polypeptide, and the VL and CL regions can be on separate polypeptides, similar to the Fab regions of known IgG. Alternatively, the VH, CH1, VL and CL regions can all be on the same polypeptide and oriented in different orders, as described in more detail in the next section.

[0084] The terms "variable region", "variable domain", "V region" or "V domain" refer to a portion of an antibody's light or heavy chain that is generally located at the amino terminus of a light or heavy chain and has 120 to 130 amino acids in a heavy chain and about 100 to 110 amino acids in length in a light chain and is used for the binding and specificity of each particular antibody to its particular antigen. The heavy chain variable region may be referred to as "VH". The light chain variable region may be referred to as "VL". The term "variable" refers to the fact that certain segments of the variable region vary greatly in sequence in an antibody. The V region mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110 amino acid span of the variable region. Instead, the V region includes a less variable region (e.g., relatively unchanged) called a framework region (FR) of about 15 to 30 amino acids, and a shorter more variable "hypervariable region" (e.g., with extreme variability) of about 9 to 12 amino acids in length, the latter separating the former. The variable regions of the heavy and light chains each contain four FRs, most of which adopt a β-folded configuration, connected by three hypervariable regions, forming a ring connection, or in some cases, forming part of a β-folded structure. The hypervariable regions in each chain are held together and in close proximity by the FRs, and together with the hypervariable regions from other chains, promote the formation of the antigen binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest (5th edition, 1991)). The constant region is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) to participate in the action of the antibody. The variable region has great sequence differences between different antibodies. In a specific embodiment, the variable region is a human variable region.

[0085] The term "variable region residue numbering according to Kabat" or "amino acid position numbering as in Kabat" and variations thereof refer to the use of the aforementioned antibody numbering system of Kabat et al. for the numbering of heavy chain variable regions or light chain variable regions. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortened FRs or CDRs of the variable domain, or inserted into FRs or CDRs of the variable domain. For example, the heavy chain variable domain may contain a single amino acid insertion after residue 52 (Kabat residue 52a), and three inserted residues after residue 82 (e.g., Kabat residues 82a, 82b, and 82c, etc.). The Kabat numbering of residues can be determined for a given antibody by alignment at regions of sequence homology of antibodies with "standard" Kabat numbering sequences. When referring to residues in the variable domain (roughly residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system (such as the Kabat system described above) is generally used. When referring to residues in the constant region of an immunoglobulin heavy chain, the "EU numbering system" or "EU index" (e.g., the EU index of Kabat et al., described above) is generally used. The "EU index as in Kabat" refers to the residue numbering of the human IgG1EU antibody. Other numbering systems include those described by AbM, Chothia, Contact, IMGT, and Ahon.

[0086] When referring to antibodies, the term "heavy chain" refers to a polypeptide chain of about 50 to 70 kDa, wherein the amino terminal portion comprises a variable region of about 120 to 130 or more amino acids, and the carboxyl terminal portion comprises a constant region. Based on the amino acid sequence of the heavy chain constant region, the constant region may be one of five different types (e.g., isotypes) called α, δ, ε, γ, and μ. Different heavy chains have different sizes: α, δ, and γ contain about 450 amino acids, while μ and ε contain about 550 amino acids. When combined with a light chain, these different types of heavy chains produce five common classes (e.g., isotypes) of antibodies, namely IgA, IgD, IgE, IgG, and IgM, as well as four subclasses comprising IgG, i.e., IgG1, IgG2, IgG3, and IgG4.

[0087] When referring to antibodies, the term "light chain" refers to a polypeptide chain of about 25 kDa, wherein the amino terminal portion comprises a variable region of about 100 to 110 or more amino acids, and the carboxyl terminal portion comprises a constant region. The approximate length of a light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, there are two different types, called kappa or lambda.

[0088] Herein, the terms "hypervariable region", "HVR", "complementarity determining region" and "CDR" are used interchangeably. "CDR" refers to one of the three hypervariable regions (H1, H2 or H3) within the non-framework region of the β-sheet framework of the VH of an immunoglobulin (Ig or antibody), or one of the three hypervariable regions (L1, L2 or L3) within the non-framework region of the β-sheet framework of the VL of an antibody. Therefore, CDRs are variable region sequences interspersed within framework region sequences.

[0089] CDR regions are well known to those skilled in the art and are defined by common numbering systems. For example, the Kabat complementarity determining regions (CDRs) are based on sequence variability and are most commonly used (see the system of Kabat et al., supra). In contrast, Chothia refers to the position of the structural loops (see Chothia and Lesk, J. Mol. Biol. 196:901-17 (1987)). When the Kabat numbering convention is used, the end of the Chothia CDR-H1 loop varies between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable region represents a compromise region between the Kabat CDR and the Chothia structural loop and is used by Oxford Molecular's AbM antibody modeling software (see Antibody Engineering Volume 2 (Kontermann and Dübel eds., 2nd edition, 2010)). The "contact" hypervariable region is based on analysis of existing complex crystal structures. Another universal numbering system that has been developed and widely used is the ImMunoGeneTics (IMGT) Information System. (Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003)). IMGT is an integrated information system dedicated to immunoglobulins (IG), T cell receptors (TCR) and major histocompatibility complex (MHC) of humans and other vertebrates. When CDRs are mentioned in this article, it refers to the amino acid sequence and position within the light chain or heavy chain. Because the "position" of CDRs within the structure of the immunoglobulin variable domain is maintained among species and exists in the structure of so-called loops, CDR and framework residues are easily identified in a numbering system that aligns variable domain sequences based on structural features. This information can be used to graft or replace CDR residues from an immunoglobulin from one species into a receptor framework, usually from a human antibody. Another numbering system (AHon) has been developed by Honegger and Plückthun, J. Mol. Biol. 309:657-70 (2001). The correspondence between numbering systems, including Kabat numbering and the IMGT unique numbering system, is well known to those skilled in the art (see Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra). Residues from these hypervariable regions or CDRs, etc. are exemplified in Table 1 below. Table 1. Exemplary CDRs according to various numbering systems

[0090] The boundaries of specific CDRs may vary with different recognition schemes. Therefore, unless otherwise specified, the term "CDR" and "complementarity determining region" of a given antibody or its region (e.g., variable region), and the individual CDRs (e.g., CDR-H1, CDR-H2) of an antibody or its region should be understood to include complementary determining regions as defined by any one of the known schemes described above. In some instances, a method for identifying specific one or more CDRs is specified, such as the CDRs defined by the IMGT, Kabat, Chothia, or Contact methods. In other cases, a specific amino acid sequence of CDR is provided. It should be noted that CDR regions may also be defined using a combination of various numbering systems, such as a combination of Kabat and Chothia numbering systems, or a combination of Kabat and IMGT numbering systems. Therefore, for example, the term described in "specific VH or VHH", and "CDR" includes any CDR1 as defined by the above-mentioned exemplary CDR numbering system, but is not limited thereto. Once a variable region (eg, VHH, VH or VL) is provided, one skilled in the art will appreciate that the CDRs within that region may be defined by different numbering systems or combinations thereof.

[0091] The hypervariable regions may comprise the following "expanded hypervariable regions": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH.

[0092] The term "constant region" or "constant domain" refers to the carboxyl terminal portion of the light chain and heavy chain that is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions (e.g., interaction with Fc receptors). The term refers to a portion of an immunoglobulin molecule that has a more stable amino acid sequence relative to the rest of the immunoglobulin (the variable region containing the antigen binding site). The constant region may contain the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0093] The term "framework" or "FR" refers to the variable region residues that flank the CDRs. FR residues are present, for example, in chimeric antibodies, humanized antibodies, human antibodies, domain antibodies (e.g., single domain antibodies), diabodies, linear antibodies, and bispecific antibodies. FR residues are those variable domain residues other than hypervariable region residues or CDR residues.

[0094] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including, for example, a native sequence Fc region, a recombinant Fc region, and a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the Fc region of a human IgG heavy chain is generally defined as extending from an amino acid residue at position Cys226 or Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during the production or purification of the antibody, or by recombinant engineering of a nucleic acid encoding the heavy chain of the antibody. Thus, a composition of a complete antibody may include an antibody population in which all K447 residues are removed, an antibody population in which the K447 residue is not removed, and an antibody population having a mixture of antibodies containing or not containing the K447 residue. A "functional Fc region" has an "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding, CDC, Fc receptor binding, ADCC, phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and the like. These effector functions generally require the Fc region to be evaluated in combination with a binding region or binding domain (e.g., an antibody variable region or domain), or can be evaluated using various assays known to those skilled in the art. A "variant Fc region" comprises at least one amino acid modification (e.g., substitution, addition, or deletion) that differs from the amino acid sequence of a native sequence Fc region. In certain embodiments, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or the Fc region of a parent polypeptide, for example, about 1 to about 10 amino acid substitutions, or about 1 to about 5 amino acid substitutions in the native sequence Fc region or the Fc region of a precursor polypeptide. The variant Fc region herein may have at least about 80% homology, or at least about 90% homology, for example, at least about 95% homology, to the native sequence Fc region and / or the Fc region of a parent polypeptide.

[0095] In this article, "epitope" is a term in the art, referring to a local region of an antigen to which a binding molecule (e.g., an antibody comprising a single domain antibody sequence) can specifically bind. An epitope may be a linear epitope or a conformational, non-linear, or discontinuous epitope. In the case of a polypeptide antigen, an epitope may be a continuous amino acid of a polypeptide (a "linear" epitope) or an epitope may comprise amino acids from two or more discontinuous regions of a polypeptide (a "conformational, non-linear, or discontinuous" epitope). It will be appreciated by those skilled in the art that, in general, a linear epitope may or may not be dependent on a secondary, tertiary, or quaternary structure. For example, in some embodiments, a binding molecule is bound to a group of amino acids, regardless of whether it is folded in a native three-dimensional protein structure. In other embodiments, the binding molecule requires the amino acid residues that make up the epitope to display a specific conformation (e.g., bending, twisting, turning, or folding) to recognize and bind to the epitope.

[0096] A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of an antigen to which it binds. In some embodiments, a blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of an antigen.

[0097] An "agonist" or activating antibody is an antibody that enhances or initiates signaling of an antigen to which it binds. In some embodiments, an agonist antibody causes or activates signaling in the absence of a natural ligand.

[0098] "Amino acid sequence identity percentage (%)" and "homology" of peptide, polypeptide or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences or introducing gaps if necessary to achieve maximum sequence identity, and excluding any stabilizing substitutions as sequence identity. For the purpose of determining amino acid sequence identity, various alignment methods in the art can be used, for example, using existing public computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN TM (DNASTAR) and other software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum match over the full length of the sequences being compared.

[0099] Herein, "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that can be used to specifically graft one or more antigens to immune effector cells (e.g., NK cells, NKT cells, and T cells). Some CARs are also referred to as "artificial T cell receptors," "chimeric T cell receptors," or "chimeric immune receptors." In some embodiments, the CAR comprises an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain of T cells, NKT cells, or NK cells and / or other receptors specifically for one or more antigens (e.g., tumor antigens). "CAR-T cells" refer to T cells expressing CAR. "CAR-NKT cells" refer to NKT cells expressing CAR. "CAR-NK cells" refer to NK cells expressing CAR.

[0100] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be a straight or branched chain, it may comprise modified amino acids, and it may be interrupted by non-amino acids. These terms also include amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. Also included in the definition are, for example, polypeptides containing one or more amino acid analogs, including but not limited to non-natural amino acids, as well as other modifications known in the art. It should be understood that because the polypeptides of the present invention may be based on antibodies or other members of the immunoglobulin superfamily, in certain embodiments, a "polypeptide" may appear as a single chain or two or more associated chains.

[0101] Herein, "polynucleotide" or "nucleic acid" used interchangeably refers to a polymer of nucleotides of any length, including DNA and RNA. These nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or analogs thereof, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may include modified nucleotides, such as methylated nucleotides and their analogs. Herein, "oligonucleotide" refers to a shorter, generally single-stranded synthetic polynucleotide of general length, but not necessarily less than about 200 nucleotides. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides may be equivalently applicable to, or completely applicable to, oligonucleotides. Cells producing the binding molecules of the present invention may include parent hybridoma cells, as well as bacteria and eukaryotic host cells into which nucleic acids encoding antibodies have been introduced. Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of the nascent RNA transcript is called the transcription direction; on the DNA chain, in the same sequence as the RNA transcript, the part located upstream of the 5' end of the RNA transcript is called the "upstream sequence"; and the part located downstream of the 3' end of the RNA transcript is called the "downstream sequence".

[0102] "Isolated nucleic acid" is a nucleic acid substantially separated from other genomic DNA sequences or proteins or complexes (such as ribosomes or polymerases), for example, separated from RNA, DNA or mixed nucleic acids, which are naturally accompanied by native sequences. "Isolated" nucleic acid molecules are separated from other nucleic acid molecules present in the natural source of nucleic acid molecules. In addition, when produced by recombinant technology, "isolated" nucleic acid molecules (such as cDNA molecules) are substantially free of other cell materials or culture media, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a specific embodiment, one or more nucleic acid molecules encoding single-domain antibodies or antibodies as described herein are separated or purified. The term includes nucleic acid sequences that have been removed from their natural production environment, and includes recombinant or cloned DNA isolates, and chemically synthesized analogs, or analogs synthesized by heterologous systems. A substantially pure molecule may include an isolated form of the molecule. Specifically, "isolated" nucleic acid molecules encoding CAR or sdAb described herein are nucleic acid molecules identified and separated from at least one contaminating nucleic acid molecule, which are usually combined with the contaminating nucleic acid molecules in their production environment.

[0103] The term "control sequence" refers to DNA sequences necessary for expression of an operably linked coding sequence in a particular host organism. Suitable control sequences for prokaryotes include, for example, a promoter, an optional operator sequence, and a ribosome binding site. It is known that eukaryotic cells utilize promoters, polyadenylation, and enhancers.

[0104] As used herein, the term "operably linked" and similar phrases (e.g., genetically fused) when referring to nucleic acids or amino acids refer to the operable connection of nucleic acid sequences or amino acid sequences so that they are in a functional relationship with each other. For example, operably linked promoters, enhancer elements, open reading frames, 5' and 3' UTRs, and terminator sequences can achieve accurate production of nucleic acid molecules (e.g., RNA). In some embodiments, operably linked nucleic acid elements result in transcription of the open reading frame and the production of the final polypeptide (i.e., expression of the open reading frame). As another example, in an operably linked peptide chain, its functional domains are arranged at appropriate distances to impart the intended function of each domain.

[0105] The term "vector" refers to a substance for carrying or containing a nucleic acid sequence, including, for example, a nucleic acid sequence encoding a binding molecule (e.g., an antibody) described herein, to introduce the nucleic acid sequence into a host cell. Suitable vectors for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include a selection sequence, or may include a marker for stable integration into the chromosome of a host cell. In addition, these vectors may include one or more selectable marker genes, and suitable expression control sequences. The selectable marker genes that may be included may, for example, provide resistance to antibiotics or toxins, supplement nutrition for nutrient deficiency defects, or supply key nutrients not present in the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, etc., which are common knowledge in the art. When two or more nucleic acid molecules are to be co-expressed (e.g., antibody heavy and light chains or antibody VH and VL), the two nucleic acid molecules may be inserted into a single expression vector, or separate expression vectors. For single vector expression, the encoding nucleic acid can be operably linked to a common expression control sequence, or to different expression control sequences, such as an inducible promoter and a constitutive promoter. The introduction of nucleic acid molecules into host cells can be confirmed using methods well known in the art. These methods include, for example, nucleic acid analysis, such as Northern blot or polymerase chain reaction amplification (PCR) of mRNA, immunoblotting for gene product expression, or other suitable analytical methods for testing the expression of the introduced nucleic acid sequence or its corresponding gene product. It will be understood by those skilled in the art that the nucleic acid molecules are expressed in sufficient quantities to produce the desired product; and it will be further understood that the degree of expression can be optimized using methods well known in the art to obtain sufficient expression.

[0106] As used herein, the term "host" refers to an animal, such as a mammal (eg, a human).

[0107] As used herein, the term "host cell" refers to a specific individual cell that can be transfected with a nucleic acid molecule and the offspring or potential offspring of such a cell. The offspring of such a cell may differ from the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in the offspring or the nucleic acid molecule is integrated into the host cell genome.

[0108] As used herein, the term "autologous" refers to any material derived from the same individual that is later reintroduced into that individual.

[0109] "Allogeneic" refers to a transplant derived from a different individual of the same species.

[0110] As used herein, the term "transfected" or "transformed" or "transduced" refers to the process of transfecting or introducing a heterologous polynucleotide into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with a heterologous polynucleotide. The cell includes the primary target cell and its progeny. In certain embodiments, the heterologously introduced polynucleotide is not a polynucleotide naturally present in the cell (e.g., an engineered immune cell).

[0111] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the United States Pharmacopeia, the European Pharmacopeia or other generally recognized pharmacopeia for use in animals, or more specifically in humans.

[0112] "Excipient" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, solvent or encapsulating material. Excipients include, for example, encapsulating materials or additives, such as absorption accelerators, antioxidants, binders, buffers, carriers, coating agents, diluents, disintegrants, emulsifiers, synergists, fillers, flavoring agents, humectants, lubricants, perfumes, preservatives, propellants, release agents, sterilants, sweeteners, solubilizers, wetting agents and mixtures thereof. The term "excipient" may also refer to a diluent, adjuvant (e.g., Freunds' adjuvant (complete or incomplete)) or vehicle.

[0113] In some embodiments, the excipient is a pharmaceutically acceptable excipient. Examples of pharmaceutically acceptable excipients include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid; low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®. TM , polyethylene glycol (PEG) and PLURONICS TM Other examples of pharmaceutically acceptable excipients can be found in Remington and Gennaro, Remington's Pharmaceutical Sciences (18th edition, 1990).

[0114] As used herein, the term "pharmaceutically acceptable" when referring to a carrier means that the carrier, diluent or excipient is not toxic or otherwise deleterious (i.e., the material can be administered to an individual without causing undesirable biological effects) and is compatible with the other components of the composition. The term "carrier" refers to a diluent, adjuvant, excipient or vehicle used to administer the therapeutic agent. These pharmaceutical carriers can be sterile liquids, such as saline solutions. When the pharmaceutical composition is administered intravenously, saline solutions can be carriers. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. If necessary, the composition may also contain a minimal amount of a wetting agent or emulsifier or a pH buffer.

[0115] In some embodiments, the excipient is a sterile liquid, such as water and oil, which may be derived from petroleum, animal, plant or synthetic sources (e.g., peanut oil, soybean oil, mineral oil, sesame oil and the like). When the composition (e.g., pharmaceutical composition) is administered intravenously, water is an exemplary excipient. Saline solutions and aqueous dextrose and glycerol solutions may also be used as liquid excipients, particularly for injectable solutions. Excipients may also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol and the like. If desired, the composition may also contain a minimum amount of a wetting agent or emulsifier or pH buffer. The composition may be in the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release formulation and the like. Oral compositions (including formulations) can include standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.

[0116] Compositions (including pharmaceutical compounds) may contain binding molecules (eg, antibodies) in isolated or purified form together with a suitable amount of excipients.

[0117] As used herein, the term "effective amount" or "therapeutically effective amount" refers to the amount of a single domain antibody or therapeutic molecule comprising an agent, as well as the amount of a single domain antibody or pharmaceutical composition provided herein, sufficient to result in the desired result.

[0118] The terms "subject" and "patient" are used interchangeably. In certain embodiments herein, the subject is a mammal, such as a non-primate or a primate (e.g., a human). In specific embodiments, the subject is a human. In one embodiment, the subject is a mammal, such as a human diagnosed with a disease or disorder. In another embodiment, the subject is a mammal, such as a human at risk of developing a disease or disorder.

[0119] "Administer" or "administration" refers to the act of physically delivering a substance into a patient's body by injection or otherwise, when it is present outside the body, such as by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other method of physical delivery described herein or known in the art.

[0120] As used herein, the term "treat" (treat / treatment / treating) refers to a reduction or improvement in the progression, severity and / or duration of a disease or condition resulting from the administration of one or more therapies. Treatment can be determined by assessing whether there is a reduction, alleviation and / or alleviation of one or more symptoms associated with the underlying condition, such as an observed improvement in a patient, although the patient may still be suffering from the underlying condition. The term "treatment" includes both management and improvement of a disease. The term "manage" (manage / managing / management) refers to the beneficial effects that an individual obtains from a therapy, which does not necessarily result in a cure of the disease.

[0121] The terms "prevent", "preventing" and "prevention" refer to reducing the likelihood of the onset or recurrence of a disease, disorder, condition or related symptoms (eg, diabetes or cancer).

[0122] In this article, "delaying" the development of cancer refers to delaying, hindering, slowing down, delaying, stabilizing and / or postponing the development of the disease. This delay can be a variable length of time, depending on the history of the disease and / or the individual being treated. Those skilled in the art will appreciate that sufficient or significant delays may actually include prevention, i.e., preventing the individual from developing the disease. The method of "delaying" the development of cancer is to reduce the likelihood of the disease developing within a given time frame and / or reduce the severity of the disease within a given time frame when compared to not using the method. These comparisons are typically based on clinical studies and use a statistically significant number of individuals. The development of cancer can be detected by standard methods, including, but not limited to, computerized axial tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation tests, arterial imaging, or tissue biopsy. Development may also refer to initially undetectable cancer progression, and includes occurrence, recurrence, and onset.

[0123] The terms "about" or "approximately" mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.

[0124] In this disclosure and in the claims of the present disclosure, the singular forms "a", "an" and "the" include the plural forms unless the context clearly indicates otherwise.

[0125] It should be understood that wherever in this document, when the term "comprising" is used to describe an embodiment, other embodiments with the same contents but described with "consisting of" and / or "consisting essentially of" are also provided. It should also be understood that wherever in this document, when the phrase "consisting essentially of" is used to describe an embodiment, other embodiments with the same contents but described with "consisting of" are also provided.

[0126] In the phrase “between A and B” or “between A to B”, the term “between” refers to a range that includes both A and B.

[0127] In this document, in the phrase "A and / or B", the term "and / or" refers to situations including both A and B, A or B, A (alone), and B (alone), etc. Similarly, in the phrase "A, B, and / or C", the term "and / or" refers to situations including the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0128] It is known in the art that, except for signal peptides (SP), polypeptide sequences and fragments thereof can be used in reverse form. Various polypeptide sequences as provided herein (e.g., amino acid sequences of hinge domains, transmembrane domains, or intracellular domains of polypeptides) can be used in reverse form. In other words, it should be understood that wherever an amino acid sequence is provided herein, its reverse form is also provided in the present application. As a clear illustration, an example is that the amino acid sequence of the intracellular domain (ICD) of DAP10 is LCARPRRSPAQEDGKVYINMPGRG (SEQ ID NO: 20), and the reverse form of the amino acid sequence of the intracellular domain (ICD) of DAP10 is GRGPMNIYVKGDEDQAPSRRPRACL (SEQ ID NO: 177). Similarly, the amino acid sequence of SAP is SEQ ID NO: 86, and its reverse form is SEQ ID NO: 178. The amino acid sequence of EAT2 is SEQ ID NO: 87, and its reverse form is SEQ ID NO: 179. 5.2 Chimeric Antigen Receptor (CAR)

[0129] One aspect of the present application provides a chimeric antigen receptor (CAR). In certain embodiments, the chimeric antigen receptor (CAR) provided herein comprises: (i) an extracellular antigen-binding domain; (ii) a transmembrane domain; and (iii) comprising an intracellular signaling domain derived from the intracellular domain of the first polypeptide, wherein the intracellular domain derived from the first polypeptide is linked to the C-terminus of the transmembrane domain directly or via a peptide linker, and The first polypeptide is DAP10 or DAP12.

[0130] In certain embodiments, provided herein is a chimeric antigen receptor (CAR) comprising: (i) an extracellular antigen-binding domain; (ii) comprises a transmembrane domain derived from the transmembrane domain of a second polypeptide; and (iii) comprising an intracellular signaling domain derived from the intracellular domain of the first polypeptide, wherein the second polypeptide is different from the first polypeptide, wherein the intracellular domain derived from the first polypeptide is linked to the C-terminus of the transmembrane domain derived from the second polypeptide directly or via a peptide linker, and wherein the first polypeptide is DNAM1.

[0131] In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain derived from a second polypeptide, wherein the second polypeptide is different from the first polypeptide, and wherein the intracellular domain derived from the first polypeptide is linked directly or via a peptide linker to the C-terminus of the transmembrane domain derived from the second polypeptide.

[0132] In certain embodiments, the intracellular signaling domain of the CAR further comprises an intracellular domain derived from a third polypeptide, wherein the third polypeptide is different from the first polypeptide.

[0133] In certain embodiments, the second polypeptide and the third polypeptide are the same polypeptide.

[0134] In certain embodiments, the CAR further comprises a hinge domain between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain.

[0135] In certain embodiments, the hinge domain of the CAR comprises a hinge domain derived from the second polypeptide.

[0136] In certain embodiments, the second polypeptide or the third polypeptide is a signaling lymphocyte activation molecule family (SLAMF) receptor, CD2, CD28H, DNAM1, CD28, 4-1BB, OX40, KIR-S, NKG2C or NKG2E. In certain embodiments, the SLAMF receptor is SLAMF4 (CD244, 2B4), SLAMF3 (CD229, Ly9), SLAMF6 (CD352, NTB-A, SF2000), SLAMF5 (CD84), SLAMF7 (CD319, CRACC, CS1), SLAMF1 (CD150), SLAMF2 (CD48, FimH), SLAMF8 (CD353) or SLAMF9 (CD84H1, SF2001). In certain embodiments, the KIR-S is KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, or KIR3DS1.

[0137] In certain embodiments, the intracellular signaling domain of the CAR further comprises an intracellular domain derived from CD3ζ.

[0138] In certain embodiments, the intracellular signal domain of the CAR further includes an intracellular domain derived from SLAM associated protein (SAP).In certain embodiments, the intracellular signal domain of the CAR further includes an intracellular domain derived from EAT2.

[0139] In certain embodiments, the intracellular domain derived from CD3ζ is directly or via a peptide linker connected to the intracellular domain derived from SAP or the intracellular domain derived from EAT2. In certain embodiments, the intracellular domain derived from CD3ζ is connected to the intracellular domain derived from SAP or the intracellular domain derived from EAT2 via P2A, T2A or E2A.

[0140] In certain embodiments, the extracellular antigen binding domain of the CAR comprises a scFv. In certain embodiments, the extracellular antigen binding domain of the CAR binds to a tumor antigen or a fragment thereof.

[0141] In certain embodiments, the first polypeptide is DAP10.

[0142] In certain embodiments, the CAR comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:20.

[0143] In certain embodiments, the CAR comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:86.

[0144] In certain embodiments, the CAR comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:87.

[0145] In certain embodiments, the CAR comprises SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144 or SEQ ID NO:145, or an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144 or SEQ ID NO:145.

[0146] In certain embodiments, the CAR comprises from N-terminus to C-terminus: (i) an extracellular antigen-binding domain; (ii) comprising a transmembrane domain derived from the transmembrane domain of the second polypeptide; (iii) an intracellular signaling domain comprising (iii)-(a) derived from the intracellular domain of DAP10; (iii)-(b) derived from the intracellular domain of CD3ζ; and (iii)-(c) an intracellular domain derived from SAP or an intracellular domain derived from EAT2, wherein these intracellular domains within the intracellular signaling domain are linked to each other directly or via one or more peptide linkers, and wherein the extracellular antigen-binding domain, the transmembrane domain and the intracellular domain are linked to each other directly or via one or more peptide linkers. 5.2.1 Exemplary CAR

[0147] In certain embodiments, a CAR provided herein comprises, from N-terminus to C-terminus: (i) an extracellular antigen-binding domain; (ii) an optional hinge domain, which may be, but is not limited to, a hinge domain derived from a second polypeptide; (iii) comprises a transmembrane domain derived from the transmembrane domain of a second polypeptide; (iv) an optional peptide linker, which may be, but is not limited to, a peptide linker provided herein; (v) an intracellular signaling domain comprising: (v)-(a) derived from the intracellular domain of a first polypeptide (DAP10, DNAM1 or DAP12); (v)-(b) optionally, derived from the intracellular domain of a third polypeptide; (v)-(c) optionally, derived from the intracellular domain of CD3ζ; (v)-(d) optionally, a peptide linker, which may be, but is not limited to, P2A, T2A or E2A; (v)-(e) are, as appropriate, derived from the intracellular domain of SAP or derived from the intracellular domain of EAT2.

[0148] In certain embodiments, the second polypeptide is different from the first polypeptide. In certain embodiments, the third polypeptide is different from the first polypeptide. In certain embodiments, the second polypeptide and the third polypeptide are the same polypeptide. In certain embodiments, the second polypeptide and the third polypeptide are different polypeptides. In certain embodiments, the second polypeptide and the third polypeptide are signaling lymphocyte activation molecule family (SLAMF) receptor, CD2, CD28H, DNAM1, CD28, 4-1BB, OX40, KIR-S, NKG2C or NKG2E. In certain embodiments, the SLAMF receptor is SLAMF4 (CD244, 2B4), SLAMF3 (CD229, Ly9), SLAMF6 (CD352, NTB-A, SF2000), SLAMF5 (CD84), SLAMF7 (CD319, CRACC, CS1), SLAMF1 (CD150), SLAMF2 (CD48, FimH), SLAMF8 (CD353) or SLAMF9 (CD84H1, SF2001). In certain embodiments, the KIR-S is KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5 or KIR3DS1.

[0149] In certain embodiments, the CAR comprises SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144 or SEQ ID NO:145.

[0150] In certain embodiments, the CAR provided herein comprises one or more elements as shown in Table 2. In certain embodiments, the CAR provided herein comprises elements as shown in Table 2, and at the same time has one or more peptide connectors between certain elements (e.g., between a hinge domain and a transmembrane domain, between a transmembrane domain and an intracellular signaling domain, and between various intracellular domains of different polypeptides derived from an intracellular signaling domain). In certain embodiments, the symbol "-" in Table 2 represents direct or via a peptide connector connection, for example, via a peptide connector connection provided in Section 5.2.6. Table 2. Exemplary CAR constructs 5.2.2 Extracellular antigen binding domain

[0151] In certain embodiments, the extracellular antigen binding domain of CAR provided herein recognizes and binds to the specific binding element on the relevant target.Non-limiting examples of extracellular antigen binding domains include single-chain variable fragments (scFv), nano antibodies, ligands of homologous receptors, native receptors for targets, and small peptide chains.In certain embodiments, the extracellular antigen binding domain of CAR includes scFv.In certain embodiments, the extracellular antigen binding domain of CAR binds to tumor antigens or fragments thereof.

[0152] In certain embodiments, the extracellular antigen binding domain of CAR binds to GPC3 or a fragment thereof. In certain embodiments, the extracellular antigen binding domain of CAR binds to a polypeptide comprising an amino acid sequence of SEQ ID NO: 92. In certain embodiments, the extracellular antigen binding domain of CAR comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence of SEQ ID NO: 170. In certain embodiments, the extracellular antigen binding domain of CAR comprises an amino acid sequence of SEQ ID NO: 170. In certain embodiments, the extracellular antigen binding domain of CAR binds to GPC3, and its scFv comprises VH CDR1-3 and VL CDR1-3, and the scFv comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence of SEQ ID NO: 170. In certain embodiments, the extracellular antigen binding domain of CAR binds to GPC3, and its scFv comprises VH CDR1-3 and VLCDR1-3, and the scFv comprises the amino acid sequence of SEQ ID NO: 170. In certain embodiments, the extracellular antigen binding domain of CAR binds to GPC3 and comprises VH CDR1 comprising the amino acid sequence of SEQ ID NO: 171, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 172, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 173, VL CDR 1 comprising the amino acid sequence of SEQ ID NO: 174, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 175, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 176.

[0153] In certain embodiments, the extracellular antigen binding domain of CAR binds to CD19 or a fragment thereof. In certain embodiments, the extracellular antigen binding domain of CAR binds to a polypeptide comprising the amino acid sequence of SEQ ID NO: 93. In certain embodiments, the extracellular antigen binding domain of CAR comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 163. In certain embodiments, the extracellular antigen binding domain of CAR comprises an amino acid sequence of SEQ ID NO: 163. In certain embodiments, the extracellular antigen binding domain of CAR binds to CD19 and comprises VH CDR1-3 and VL CDR1-3 in scFv, the scFv comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 163. In certain embodiments, the extracellular antigen binding domain of CAR binds to CD19, and its scFv comprises the amino acid sequence of SEQ ID NO: 163, and comprises VH CDR1-3 and VL CDR 1-3. In certain embodiments, the extracellular antigen binding domain of CAR binds to CD19, and comprises VH CDR1 comprising the amino acid sequence of SEQ ID NO: 164, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 165, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 166, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 167, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 168, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 169.

[0154] A variety of antigens known in the art can be targeted using CAR, and these antigens are also suitable for use in the present application. Tumor antigens are proteins produced by tumor cells that can elicit an immune response, particularly a T cell-mediated immune response. Exemplary tumor antigens include, but are not limited to, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, HER2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2 protein, CD22, insulin growth factor-I (IGF-I), IGF-II, IGF-I receptor, and mesothelin.

[0155] In some embodiments, the tumor antigen comprises one or more antigen cancer epitopes associated with a malignant tumor. Malignant tumors express many proteins that can be used as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens, such as MART-1, tyrosinase and gp100 in melanoma and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to transformation-related molecule groups (e.g., oncogenes HER2 / Neu / ErbB-2). Another group of target antigens is tumor-fetal antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins constitute true tumor-specific immunoglobulin antigens that are unique to individual tumors. In addition to CD19, B-cell differentiation antigens (e.g., CD20 and CD37) are other candidate antigens for target antigens in B-cell lymphomas.

[0156] In some embodiments, the tumor antigen is a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSA is specific to tumor cells and does not appear on other cells in the body. TAA-related antigens are not specific to tumor cells, but rather they are expressed on normal cells under conditions that fail to induce a state of immune tolerance to the antigen. The expression of antigens on tumors can occur under conditions that enable the immune system to react to the antigen. TAA may be an antigen that is expressed and cannot react on normal cells during fetal development when the immune system is immature, or it may be an antigen that is usually present at a very low level on normal cells, but is expressed at a much higher level on tumor cells.

[0157] Non-limiting examples of TSA or TAA antigens include: differentiation antigens, such as MART-1 / MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2; and tumor-specific multi-lineage antigens, such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; over-expressed embryonic antigens, such as CEA and alpha-fetoprotein (AFP); over-expressed oncogenes and mutated tumor suppressor genes, such as p53, Ras, HER2 / neu; unique tumor antigens arising from chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as Epstein Barr virus antigen EBVA, human immunodeficiency virus (HIV) antigen CD32a, hepatitis B virus (HBV) antigen HbsAg, and human papillomavirus (HPV) antigens E6 and E7.

[0158] Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23HI, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA15-3\CA27.29\BCAA, CA195, CA242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS 1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, CLDN18.1, CLDN18.2, CLDN18.6, FAP and TPS. In some more specific embodiments, the one or more additional antigens are selected from the following group of antigens: CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFRvIII, GD-2, NY-ESO-1, MAGE A3 and glycolipid F77. 5.2.3 Transmembrane domain

[0159] In certain embodiments, the CAR provided herein comprises a transmembrane domain that is linked directly or via a hinge domain provided herein to an extracellular antigen binding domain provided herein.

[0160] In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain derived from a second polypeptide, wherein the second polypeptide is different from the first polypeptide, and wherein the intracellular domain derived from the first polypeptide is linked directly or via a peptide linker to the C-terminus of the transmembrane domain derived from the second polypeptide.

[0161] In certain embodiments, the second polypeptide is a signaling lymphocyte activation molecule family (SLAMF) receptor, CD2, CD28H, DNAM1, CD28, 4-1BB, OX40, KIR-S, NKG2C or NKG2E. In certain embodiments, the third polypeptide is a signaling lymphocyte activation molecule family (SLAMF) receptor, CD2, CD28H, DNAM1, CD28, 4-1BB, OX40, KIR-S, NKG2C or NKG2E. In certain embodiments, the SLAMF receptor is SLAMF4 (CD244, 2B4), SLAMF3 (CD229, Ly9), SLAMF6 (CD352, NTB-A, SF2000), SLAMF5 (CD84), SLAMF7 (CD319, CRACC, CS1), SLAMF1 (CD150), SLAMF2 (CD48, FimH), SLAMF8 (CD353) or SLAMF9 (CD84H1, SF2001). In certain embodiments, the KIR-S is KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5 or KIR3DS1.

[0162] In certain embodiments, the transmembrane domain of CAR includes a transmembrane domain derived from SLAMF4 (CD244, 2B4). In certain embodiments, the transmembrane domain of CAR includes a transmembrane domain derived from SLAMF3 (CD229, Ly9). In certain embodiments, the transmembrane domain of CAR includes a transmembrane domain derived from SLAMF6 (CD352, NTB-A, SF2000). In certain embodiments, the transmembrane domain of CAR includes a transmembrane domain derived from SLAMF7 (CD319, CRACC, CS1). In certain embodiments, the transmembrane domain of CAR includes a transmembrane domain derived from SLAMF1 (CD150). In certain embodiments, the transmembrane domain of CAR includes a transmembrane domain derived from SLAMF2 (CD48, FimH). In certain embodiments, the transmembrane domain of CAR includes a transmembrane domain derived from SLAMF8 (CD353). In certain embodiments, the transmembrane domain of CAR includes a transmembrane domain derived from SLAMF9 (CD84H1, SF2001). In certain embodiments, the transmembrane domain of CAR includes a transmembrane domain derived from SLAMF5 (CD84). In certain embodiments, the transmembrane domain of CAR includes a transmembrane domain derived from NKG2D. In certain embodiments, the transmembrane domain of CAR includes a transmembrane domain derived from CD28H. In certain embodiments, the transmembrane domain of CAR includes a transmembrane domain derived from DNAM1. In certain embodiments, the transmembrane domain of CAR includes a transmembrane domain derived from CD2.

[0163] In certain embodiments, the transmembrane domain of CAR includes SLAMF4 (CD244, 2B4), SLAMF3 (CD229, Ly9), SLAMF6 (CD352, NTB-A, SF2000), SLAMF5 (CD84), SLAMF7 (CD319, CRACC, CS1), SLAMF1 (CD150), SLAMF2 (CD48, FimH), SLAMF8 (CD353), SLAMF9 (CD84H1, SF2001), CD8, CD28, 4-1BB, OX40, 2B4, CD2, CD28H, DNAM1, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, NKG2C or NKG2E transmembrane domain (TD). In certain embodiments, the transmembrane domain of the CAR comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:56, SEQ ID NO:60, SEQ ID NO:64, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:80, or SEQ ID NO:84. In certain embodiments, the transmembrane domain of the CAR comprises the amino acid sequence of SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:56, SEQ ID NO:60, SEQ ID NO:64, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:80, or SEQ ID NO:84.

[0164] The transmembrane domain may be derived from a natural source or a synthetic source. In this article, "transmembrane domain" refers to any protein structure on a cell membrane, preferably on a eukaryotic cell membrane, that is thermodynamically stable. The transmembrane domain of the CAR described herein may be obtained from a naturally occurring protein. Alternatively, it may be a synthetic non-naturally occurring protein fragment, for example, a thermodynamically stable hydrophobic protein fragment on a cell membrane.

[0165] Transmembrane domains are classified based on the three-dimensional structure of the transmembrane domain. For example, the transmembrane domain can form an alpha helix, a complex of more than one alpha helix, a beta-barrel, or any other stable structure that can cross the phospholipid bilayer structure of the cell. In addition, the transmembrane domain can also be classified based on the topological structure of the transmembrane domain, including the number of channels that the transmembrane domain crosses the cell membrane and the orientation of the protein. For example, a single-pass membrane protein crosses the cell membrane once, and a multi-pass membrane protein crosses the cell membrane at least twice (for example, 2, 3, 4, 5, 6, 7 times or more times). Membrane proteins can be defined as type I, type II, or type III based on the topological structure of its end and transmembrane segment relative to the inside and outside of the cell. Type I membrane proteins have a single transmembrane segment, and its orientation makes the N-terminus of the protein present on the cell side of the lipid bilayer of the cell, and the C-terminus of the protein present on the cytoplasmic side. Type II membrane proteins also have a single transmembrane segment, but the orientation makes the C-terminus of the protein present on the cell side of the lipid bilayer of the cell, and the N-terminus of the protein present on the cytoplasmic side. Type III membrane proteins have multiple transmembrane segments and can be further divided into subclasses based on the number of transmembrane segments and the orientation of the N- and C-termini.

[0166] In some embodiments, the transmembrane domain of the CAR described herein is derived from a type I single-pass membrane protein. In some embodiments, the transmembrane domain from a multi-pass membrane protein may also be applicable to the CAR described herein. The multi-pass membrane protein may comprise a composite α-helix or β-folded structure (at least 2, 3, 4, 5, 6, 7 or more). In some embodiments, the N-terminus and C-terminus of the multi-pass membrane protein are present on opposite sides of the lipid bilayer, for example, the N-terminus of the protein is present on the cytoplasmic side of the lipid bilayer and the C-terminus of the protein is present on the extracellular side.

[0167] In some embodiments, the transmembrane domain of CAR comprises a member selected from the group consisting of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL-2Rβ, IL-2Rγ, IL-7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 ld, ITGAE, CD103, ITGAL, CDlla, 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, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D and / or NKG2C α, β, ζ chain transmembrane domain, however, the intracellular domain of CAR includes an intracellular domain derived from a first polypeptide, and the first polypeptide is different from the peptide chains listed in this paragraph.

[0168] The transmembrane domain of CAR herein may also include at least a portion of a non-natural synthetic protein fragment. In some embodiments, the transmembrane domain is a non-natural synthetic alpha helix or beta fold. In some embodiments, the protein fragment is at least about 20 amino acids, for example, at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids. Examples of synthetic transmembrane domains are known in the art, for example, in U.S. Patent No. 7,052,906 and PCT Publication No. WO2000 / 032776, the relevant disclosures of which are incorporated herein by reference. 5.2.4 Intracellular signaling domain

[0169] In certain embodiments, the intracellular signal domain of CAR provided herein includes an intracellular domain derived from a first polypeptide, and the first polypeptide is DAP10, DNAM1 or DAP12. In certain embodiments, the intracellular signal domain of CAR provided herein includes the intracellular domain of DAP10, DNAM1 or DAP12. In certain embodiments, the first polypeptide is DAP10. In certain embodiments, the intracellular signal domain of CAR provided herein includes an intracellular domain derived from DAP10. In certain embodiments, the intracellular signal domain of CAR provided herein includes the intracellular domain of DAP10 (DAP10 ICD). In certain embodiments, the intracellular domain derived from the first polypeptide is directly or via a peptide connector provided herein connected to the C-terminus of the transmembrane domain.

[0170] In certain embodiments, the first polypeptide can activate PI3K at the cell membrane. In certain embodiments, the intracellular domain derived from the first polypeptide (i.e., DAP10, DNAM1 or DAP12) is connected to the C-terminus of the transmembrane domain via a peptide connector. In certain embodiments, the length of the peptide connector between the C-terminus of the intracellular domain derived from the first polypeptide and the transmembrane domain is less than 2,3,4,5,6,7,8,9 or 10 amino acids, so that the intracellular domain derived from the first polypeptide is close to the C-terminus of the transmembrane domain. In certain embodiments, the length of the peptide connector between the C-terminus of the intracellular domain derived from the first polypeptide and the transmembrane domain is so that the intracellular domain derived from the first polypeptide is close to the C-terminus of the transmembrane domain, and / or close to the cell membrane of engineered immune cells. In certain embodiments, the length of the peptide connector between the C-terminus of the intracellular domain derived from the first polypeptide and the transmembrane domain is less than 2,3,4,5,6,7,8,9 or 10 amino acids, so that the intracellular domain derived from the first polypeptide can activate PI3K at the cell membrane. In certain embodiments, the length of the peptide linker between the intracellular domain derived from the first polypeptide and the C-terminus of the transmembrane domain is such that the intracellular domain derived from the first polypeptide can activate PI3K at the cell membrane. In certain embodiments, the peptide linker between the intracellular domain derived from the first polypeptide and the C-terminus of the transmembrane domain is GGGS (SEQ ID NO: 153). In certain embodiments, the peptide linker between the intracellular domain derived from the first polypeptide and the C-terminus of the transmembrane domain is one or more DNA restriction endonuclease sites. Exemplary DNA restriction endonuclease sites include, but are not limited to, NotI, BglII, BamhI, EcoRI, NheI, XhoI, Pad, Sad, SacII, XbaI, SalI, and ClaI.

[0171] In certain embodiments, the intracellular signaling domain of CAR comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 53 or SEQ ID NO: 21. In certain embodiments, the intracellular signaling domain of CAR comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the intracellular signaling domain of CAR comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the intracellular signaling domain of CAR comprises an amino acid sequence of SEQ ID NO: 20.

[0172] In certain embodiments, the intracellular signal domain of CAR further comprises an intracellular domain derived from a third polypeptide, wherein the third polypeptide is different from the first polypeptide. In certain embodiments, the second polypeptide and the third polypeptide are the same polypeptide. In certain embodiments, the second polypeptide and the third polypeptide are different polypeptides. In certain embodiments, the third polypeptide is a signaling lymphocyte activation molecule family (SLAMF) receptor, CD2, CD28H, DNAM1, CD28, 4-1BB, OX40, KIR-S, NKG2C or NKG2E. In certain embodiments, the SLAMF receptor is SLAMF4 (CD244, 2B4), SLAMF3 (CD229, Ly9), SLAMF6 (CD352, NTB-A, SF2000), SLAMF5 (CD84), SLAMF7 (CD319, CRACC, CS1), SLAMF1 (CD150), SLAMF2 (CD48, FimH), SLAMF8 (CD353) or SLAMF9 (CD84H1, SF2001). In certain embodiments, the KIR-S is KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5 or KIR3DS1.

[0173] In some embodiments, the intracellular signaling domain of CAR further includes an intracellular domain derived from SLAMF4 (CD244, 2B4), SLAMF3 (CD229, Ly9), SLAMF6 (CD352, NTB-A, SF2000), SLAMF5 (CD84), SLAMF7 (CD319, CRACC, CS1), SLAMF1 (CD150), SLAMF2 (CD48, FimH), SLAMF8 (CD353), SLAMF9 (CD84H1, SF2001), CD8, CD28, 4-1BB, OX40, 2B4, CD2, CD28H, DNAM1, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, NKG2C or NKG2E. In some embodiments, the intracellular signaling domain of CAR further includes an intracellular domain (ICD) of SLAMF4 (CD244, 2B4), SLAMF3 (CD229, Ly9), SLAMF6 (CD352, NTB-A, SF2000), SLAMF5 (CD84), SLAMF7 (CD319, CRACC, CS1), SLAMF1 (CD150), SLAMF2 (CD48, FimH), SLAMF8 (CD353), SLAMF9 (CD84H1, SF2001), CD8, CD28, 4-1BB, OX40, 2B4, CD2, CD28H, DNAM1, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, NKG2C or NKG2E. In certain embodiments, the intracellular signaling domain of the CAR comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 79, or SEQ ID NO: 83.In certain embodiments, the intracellular signaling domain of the CAR comprises the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 79, or SEQ ID NO: 83.

[0174] In certain embodiments, the intracellular signal domain of CAR further includes an intracellular domain derived from CD3ζ. In certain embodiments, the intracellular signal domain of CAR further includes an intracellular domain of CD3ζ (CD3ζICD). In certain embodiments, the intracellular signal domain of CAR comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:19. In certain embodiments, the intracellular signal domain of CAR comprises an amino acid sequence of SEQ ID NO:19.

[0175] In certain embodiments, the intracellular signal domain of CAR further includes an intracellular domain derived from a SLAM-associated protein (SAP). In certain embodiments, the intracellular signal domain of CAR includes an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:86. In certain embodiments, the intracellular signal domain of CAR includes an amino acid sequence of SEQ ID NO:86.

[0176] In certain embodiments, the intracellular signaling domain of CAR further includes an intracellular domain derived from EAT2. In certain embodiments, the intracellular signaling domain of CAR comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 87. In certain embodiments, the intracellular signaling domain of CAR comprises an amino acid sequence of SEQ ID NO: 87.

[0177] In certain embodiments, the intracellular signaling domain of CAR further includes a peptide linker derived from P2A, T2A or E2A. In certain embodiments, the intracellular signaling domain of CAR further includes P2A, T2A or E2A. In certain embodiments, the intracellular signaling domain of CAR comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. In certain embodiments, the intracellular signaling domain of CAR comprises an amino acid sequence of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. In certain embodiments, the intracellular signaling domain of CAR further includes a peptide linker derived from P2A. In certain embodiments, the intracellular signaling domain of CAR further includes P2A. In certain embodiments, the intracellular signaling domain of CAR comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the intracellular signaling domain of CAR comprises an amino acid sequence of SEQ ID NO: 1.

[0178] In certain embodiments, the intracellular domain derived from CD3 ζ is directly or via a peptide linker connected to the intracellular domain derived from SAP or the intracellular domain derived from EAT2. In certain embodiments, the intracellular domain derived from CD3 ζ is connected to the intracellular domain derived from SAP or the intracellular domain derived from EAT2 via P2A, T2A or E2A.

[0179] In certain embodiments, without being limited to any particular theory, the intracellular domain derived from SAP or the intracellular domain derived from EAT2 can promote CAR to trigger a full activation signal and completely overcome inhibitory receptor signaling.

[0180] In certain embodiments, the intracellular signaling domain of CAR includes a main signaling domain and one or more costimulatory signaling domains. In certain embodiments, the main signaling domain and one or more costimulatory signaling domains are directly connected to each other or connected to each other via a peptide linker.

[0181] In certain embodiments, the intracellular signal domain of CAR includes a major signal domain comprising an intracellular domain derived from CD3 ζ. In certain embodiments, the intracellular signal domain of CAR includes a major signal domain comprising an intracellular domain of CD 3 ζ (CD3 ζ ICD). In certain embodiments, the intracellular signal domain of CAR includes a major signal domain comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 19. In certain embodiments, the intracellular signal domain of CAR includes a major signal domain comprising an amino acid sequence of SEQ ID NO: 19.

[0182] In certain embodiments, the intracellular signaling domain of CAR includes a costimulatory signaling domain comprising an intracellular domain of a first polypeptide derived from DAP10, DNAM1 or DAP12. In certain embodiments, the intracellular signaling domain of CAR includes a costimulatory signaling domain comprising an intracellular domain of DAP10, DNAM1 or DAP12. In certain embodiments, the intracellular signaling domain of CAR includes a costimulatory signaling domain comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 20, SEQ ID NO: 53 or SEQ ID NO: 21. In certain embodiments, the intracellular signaling domain of CAR includes a costimulatory signaling domain comprising an amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 53 or SEQ ID NO: 21. In certain embodiments, the intracellular signaling domain of CAR includes a costimulatory signaling domain comprising an intracellular domain derived from DAP10. In certain embodiments, the intracellular signaling domain of CAR includes a costimulatory signaling domain comprising an intracellular domain of DAP10 (DAP10ICD). In certain embodiments, the intracellular signaling domain of CAR includes a costimulatory signaling domain comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the intracellular signaling domain of CAR includes a costimulatory signaling domain comprising an amino acid sequence of SEQ ID NO: 20.

[0183] In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling domain comprising an intracellular domain derived from a third polypeptide, wherein the third polypeptide is different from the first polypeptide. In certain embodiments, the intracellular signaling domain of CAR includes a co-stimulatory signaling domain comprising an intracellular domain derived from SLAMF4 (CD244, 2B4), SLAMF3 (CD229, Ly9), SLAMF6 (CD352, NTB-A, SF2000), SLAMF5 (CD84), SLAMF7 (CD319, CRACC, CS1), SLAMF1 (CD150), SLAMF2 (CD48, FimH), SLAMF8 (CD353), SLAMF9 (CD84H1, SF2001), CD8, CD28, 4-1BB, OX40, 2B4, CD2, CD28H, DNAM1, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, NKG2C or NKG2E. In certain embodiments, the intracellular signaling domain of CAR includes a costimulatory signaling domain comprising an intracellular domain (ICD) of SLAMF4 (CD244, 2B4), SLAMF3 (CD229, Ly9), SLAMF6 (CD352, NTB-A, SF2000), SLAMF5 (CD84), SLAMF7 (CD319, CRACC, CS1), SLAMF1 (CD150), SLAMF2 (CD48, FimH), SLAMF8 (CD353), SLAMF9 (CD84H1, SF2001), CD8, CD28, 4-1BB, OX40, 2B4, CD2, CD28H, DNAM1, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, NKG2C or NKG2E. In certain embodiments, the intracellular signaling domain of the CAR includes a co-stimulatory signaling domain comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 79, or SEQ ID NO: 83.In certain embodiments, the intracellular signaling domain of the CAR includes a co-stimulatory signaling domain comprising an amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 79, or SEQ ID NO: 83.

[0184] In certain embodiments, the intracellular signaling domain of CAR includes a costimulatory signaling domain comprising an intracellular domain derived from SAP or EAT2. In certain embodiments, the intracellular signaling domain of CAR includes a costimulatory signaling domain comprising SAP or EAT. In certain embodiments, the intracellular signaling domain of CAR includes a costimulatory signaling domain comprising an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:86 or SEQ ID NO:87. In certain embodiments, the intracellular signaling domain of CAR includes a costimulatory signaling domain comprising an amino acid sequence of SEQ ID NO:86 or SEQ ID NO:87. 5.2.5 Hinge domain

[0185] The CAR provided herein may include a hinge domain between the extracellular antigen binding domain and the transmembrane domain. In certain embodiments, the CAR further includes a hinge domain between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain. In certain embodiments, the hinge domain of CAR includes a hinge domain derived from a second polypeptide provided herein.

[0186] In some embodiments, the hinge domain is derived from SLAMF4 (CD244, 2B4), SLAMF3 (CD229, Ly9), SLAMF6 (CD352, NTB-A, SF2000), SLAMF5 (CD84), SLAMF7 (CD319, CRACC, CS1), SLAMF1 (CD150), SLAMF2 (CD48, FimH), SLAMF8 (CD353), SLAMF9 (CD84H1, SF2001), CD8, CD28, 4-1BB, OX40, 2B4, CD2, CD28H, DNAM1, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, NKG2C or NKG2E. In some embodiments, the hinge domain is the hinge domain of SLAMF4 (CD244, 2B4), SLAMF3 (CD229, Ly9), SLAMF6 (CD352, NTB-A, SF2000), SLAMF5 (CD84), SLAMF7 (CD319, CRACC, CS1), SLAMF1 (CD150), SLAMF2 (CD48, FimH), SLAMF8 (CD353), SLAMF9 (CD84H1, SF2001), CD8, CD28, 4-1BB, OX40, 2B4, CD2, CD28H, DNAM1, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, NKG2C or NKG2E. In certain embodiments, the hinge domain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:47, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:59, SEQ ID NO:63, SEQ ID NO:67, SEQ ID NO:71, SEQ ID NO:75, SEQ ID NO:81, or SEQ ID NO:85.In certain embodiments, the hinge domain comprises the amino acid sequence of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:47, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:59, SEQ ID NO:63, SEQ ID NO:67, SEQ ID NO:71, SEQ ID NO:75, SEQ ID NO:81, or SEQ ID NO:85.

[0187] Hinge domains are stretches of amino acids generally found between two domains of a protein that can provide flexibility to the protein and allow one or both of the domains to move relative to each other. Any amino acid sequence that can provide such flexibility and allow the extracellular antigen binding domain to move relative to the transmembrane domain of the effector molecule can be used.

[0188] The hinge domain may contain about 10 to 100 amino acids, for example, about 15 to 75 amino acids, 20 to 50 amino acids, or any range of 30 to 60 amino acids. In some embodiments, the hinge domain may be at least about 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.

[0189] In some embodiments, the hinge domain is a hinge domain of a naturally occurring protein. The hinge domain of any protein comprising a hinge domain known in the art is suitable for use in CAR provided herein. In some embodiments, the hinge domain is at least a portion of a hinge domain of a naturally occurring protein, and imparts flexibility to a chimeric receptor. In some embodiments, the hinge domain is a portion of a hinge domain of CD8 α, for example, a fragment of at least 15 consecutive amino acids of a hinge domain containing CD8 α (e.g., 20, 25, 30, 35, or 40).

[0190] The hinge domain of an antibody (e.g., IgG, IgA, IgM, IgE, or IgD antibody) is also suitable for use in the CAR described herein. In some embodiments, the hinge domain is a hinge domain connecting the constant domains CH1 and CH2 of an antibody. In some embodiments, the hinge domain is an antibody and comprises a hinge domain of an antibody and one or more constant regions of an antibody. In some embodiments, the hinge domain comprises a hinge domain of an antibody and a CH3 constant region of an antibody. In some embodiments, the hinge domain comprises a hinge domain of an antibody and CH2 and CH3 constant regions of an antibody. In some embodiments, the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the hinge region comprises a hinge region and CH2 and CH3 constant regions of an IgG1 antibody. In some embodiments, the hinge region comprises a hinge region and CH3 constant regions of an IgG1 antibody.

[0191] Non-naturally occurring peptide chains can also be used as hinge domains of the chimeric receptors described herein. In some embodiments, the hinge domain between the C-terminus of the extracellular ligand binding domain of the Fc receptor and the N-terminus of the transmembrane domain is a peptide linker, such as a (GxS)n linker, wherein x and n can independently be integers between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more. 5.2.6 Peptide Linkers

[0192] In certain embodiments, the CAR provided herein comprises one or more peptide linkers. In certain embodiments, the length of the peptide linker is less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. In certain embodiments, the peptide linker is GGGS (SEQ ID NO: 153). In certain embodiments, the peptide linker comprises one or more DNA restriction endonuclease sites. Exemplary DNA restriction endonuclease sites include, but are not limited to, NotI, BglII, BamhI, EcoRI, NheI, XhoI, PacI, SacI, SacII, XbaI, SalI and ClaI.

[0193] In certain embodiments, CAR provided herein is included in a peptide connector between a transmembrane domain and an intracellular signal domain. In certain embodiments, the intracellular domain derived from the first polypeptide (ie, DAP10, DNAM1, CD28H, CD2, DAP12, CD28 or OX40) is connected to the C-terminus of the transmembrane domain via a peptide connector. In certain embodiments, the peptide connector between the C-terminus of the intracellular domain derived from the first polypeptide and the transmembrane domain is less than 2,3,4,5,6,7,8,9 or 10 amino acids long so that the peptide connector between the intracellular domain derived from the first polypeptide approaches the C-terminus of the transmembrane domain. In certain embodiments, the peptide connector between the C-terminus of the intracellular domain derived from the first polypeptide and the transmembrane domain is GGGS (SEQ ID NO: 153). In certain embodiments, the peptide connector between the C-terminus of the intracellular domain derived from the first polypeptide and the transmembrane domain is one or more DNA restriction endonuclease sites. Exemplary DNA restriction endonuclease sites include, but are not limited to, NotI, BglII, BamhI, EcoRI, NheI, Xhol, Pad, Sad, SacII, XbaI, SalI, and ClaI.

[0194] In certain embodiments, CAR provided herein includes one or more peptide linkers in the intracellular signal domain. In certain embodiments, one or more peptide linkers in the intracellular signal domain are between the intracellular domains derived from polypeptides. In certain embodiments, the intracellular domain derived from CD3ζ is connected to the intracellular domain derived from SAP or the intracellular domain derived from EAT2 via a peptide linker.

[0195] Each peptide connexon in CAR may have the same or different lengths and / or sequences, depending on the structural and / or functional characteristics of the single domain antibody and / or various domains. Each peptide connexon can be independently selected and optimized. The length, flexibility and / or other properties of the peptide connexon used for CAR may have some effects on the affinity, specificity or antibody affinity of one or more specific antigens or epitopes, and are not limited thereto. For example, a longer peptide connexon may be selected to ensure that the two adjacent domains do not interfere with each other in space. In some embodiments, a shorter peptide connexon may be arranged between the transmembrane domain and the intracellular signaling domain of CAR. In some embodiments, the peptide connexon comprises flexible residues (eg, glycine and serine) so that adjacent domains can move freely relative to each other. For example, a glycine-serine doublet structure may be a suitable peptide connexon.

[0196] The peptide linker can be any suitable length. In some embodiments, the length of the peptide linker is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids. In some embodiments, the length of the peptide linker is 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 less amino acids. In some embodiments, the peptide linker has a length of about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or any of about 1 amino acid to about 100 amino acids.

[0197] 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 only the heavy chain of an antibody may be used as a linker. See WO1996 / 34103. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include (but are not limited to) glycine polymers (G) n , glycine-serine polymers (including (GS) n 、(GSGGS) n 、(GGGS) n and (GGGGS) n , wherein n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Exemplary peptide linkers are listed in the following table. Table 3. Exemplary peptide linkers SEQ ID NO: sequence 146 <![CDATA[(GS) n , n is an integer including 1, 2, 3, 4, 5 and 6. ]]> 147 <![CDATA[(GSGGS) n , n is an integer including 1, 2, 3, 4, 5 and 6. ]]> 148 <![CDATA[(GGGS) n , n is an integer including 1, 2, 3, 4, 5 and 6. ]]> 149 <![CDATA[(GGGGS) n , n is an integer including 1, 2, 3, 4, 5 and 6. ]]> 150 GS 151 GSGS 152 GSGSGS 153 GGGS 154 GGGSGGGS 155 GSGGS 156 GSGGSGSGGS 157 GSGGGS 158 GGGGS 159 GGGGSGGGGS 160 GGR 161 GGGGSGGGGSGGGGGGSGSGGGGS 162 GGRRGGGS

[0198] Other linkers known in the art, for example, as described in WO2016014789, WO2015158671, WO2016102965, US20150299317, WO2018067992, US7741465, Colcher et al., J. Nat. Cancer Inst. 82: 1191-1197 (1990), and Bird et al., Science 242: 423-426 (1988) may also be included in the CAR provided herein, the contents of which are incorporated herein by reference. 5.3 Engineered immune cells

[0199] On the other hand, provided herein is an engineered immune cell. In certain embodiments, provided herein is an engineered immune cell comprising a CAR provided herein.

[0200] In certain embodiments, provided herein is an engineered immune cell comprising a CAR, wherein the CAR comprises: (i) an extracellular antigen-binding domain; (ii) a transmembrane domain; and (iii) an intracellular signaling domain, and Wherein the engineered immune cell further comprises an exogenously introduced polynucleotide encoding an intracellular domain derived from a SLAM-associated protein (SAP), and wherein the engineered immune cell is a NK cell, a NKT cell or a T cell.

[0201] In certain embodiments, provided herein is an engineered immune cell comprising a CAR, wherein the CAR comprises: (i) an extracellular antigen-binding domain; (ii) a transmembrane domain; and (iii) an intracellular signaling domain, and wherein the engineered immune cell further comprises an exogenously introduced polynucleotide encoding an intracellular domain derived from Ewing's sarcoma activated transcript-2 (EAT2), and wherein the engineered immune cell is a NK cell, a NKT cell or a T cell.

[0202] In certain embodiments, the polynucleotide encodes a CAR in an engineered immune cell, and wherein the intracellular signaling domain of the CAR in the engineered immune cell comprises the intracellular domain derived from SAP or the intracellular domain derived from EAT2.

[0203] In certain embodiments, the intracellular signaling domain of the CAR in the engineered immune cell comprises an intracellular domain derived from a first polypeptide, and wherein the first polypeptide is DAP10, DNAM1, CD28H, CD2, DAP12, CD28, or OX40.

[0204] In certain embodiments, the intracellular domain of the first polypeptide is linked to the C-terminus of the transmembrane domain directly or via a peptide linker.

[0205] In certain embodiments, the transmembrane domain of the CAR in the engineered immune cell includes a transmembrane domain derived from a second polypeptide. In certain embodiments, the intracellular signaling domain of the CAR in the engineered immune cell includes an intracellular domain derived from a third polypeptide. In certain embodiments, the second polypeptide or the third polypeptide is a signaling lymphocyte activation molecule family (SLAMF) receptor, CD2, CD28H, DNAM1, CD28, 4-1BB, OX40, KIR-S, NKG2C or NKG2E. In certain embodiments, the SLAMF receptor is SLAMF4 (CD244, 2B4), SLAMF3 (CD229, Ly9), SLAMF6 (CD352, NTB-A, SF2000), SLAMF5 (CD84), SLAMF7 (CD319, CRACC, CS1), SLAMF1 (CD150), SLAMF2 (CD48, FimH), SLAMF8 (CD353) or SLAMF9 (CD84H1, SF2001). In certain embodiments, the KIR-S is KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5 or KIR3DS1.

[0206] In certain embodiments, the intracellular signal domain of the CAR in the engineered immune cell includes an intracellular domain derived from CD3 ζ.In certain embodiments, the intracellular signal domain of the CAR in the engineered immune cell further includes an intracellular domain derived from SLAM associated proteins (SAP) or an intracellular domain derived from EAT2, and wherein the intracellular domain derived from CD3 ζ is directly or via a peptide linker connected to the intracellular domain derived from SAP or the intracellular domain derived from EAT2.In certain embodiments, the intracellular domain derived from CD3 ζ is connected to the intracellular domain derived from SAP or the intracellular domain derived from EAT2 via P2A, T2A or E2A.

[0207] In certain embodiments, the extracellular antigen binding domain of the CAR in the engineered immune cell comprises a scFv. In certain embodiments, the extracellular antigen binding domain of the CAR binds to a tumor antigen or a fragment thereof.

[0208] In certain embodiments, the CAR in the engineered immune cell comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:20.

[0209] In certain embodiments, the CAR in the engineered immune cell comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:86.

[0210] In certain embodiments, the CAR in the engineered immune cell comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:87.

[0211] In certain embodiments, the CAR in the engineered immune cell comprises the sequence of SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: : The invention relates to an amino acid sequence that is at least 90%, at least 95% or 100% identical to an amino acid sequence of SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144 or SEQ ID NO:145.

[0212] In certain embodiments, the engineered immune cells are NK cells. 5.3.1 Exemplary Engineered Immune Cells

[0213] In certain embodiments, the engineered immune cells provided herein comprise a CAR comprising: (i) an extracellular antigen-binding domain; (ii) a transmembrane domain; and (iii) an intracellular signaling domain, and Wherein the engineered immune cell further comprises an exogenously introduced polynucleotide encoding an intracellular domain derived from SLAM-associated protein (SAP) or Ewing's sarcoma activated transcript-2 (EAT2), and wherein the engineered immune cell is a NK cell, a NKT cell or a T cell.

[0214] In certain embodiments, the engineered immune cells provided herein include any CAR known in the art and polynucleotides derived from the exogenous introduction of the intracellular domain of coding SLAM-associated proteins (SAP). In certain embodiments, the engineered immune cells provided herein include any CAR known in the art and polynucleotides encoding the exogenous introduction of SLAM-associated proteins (SAP). In certain embodiments, the engineered immune cells provided herein include any CAR known in the art and polynucleotides encoding the exogenous introduction of SEQ ID NO:86 amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences. In certain embodiments, the engineered immune cells provided herein include any CAR known in the art and polynucleotides encoding the exogenous introduction of SEQ ID NO:86 amino acid sequence.

[0215] In certain embodiments, the engineered immune cells provided herein include any CAR known in the art and an exogenously introduced polynucleotide encoding an intracellular domain derived from Ewing's sarcoma activation transcript-2 (EAT2). In certain embodiments, the engineered immune cells provided herein include any CAR known in the art and an exogenously introduced polynucleotide encoding Ewing's sarcoma activation transcript-2 (EAT2). In certain embodiments, the engineered immune cells provided herein include any CAR known in the art and an exogenously introduced polynucleotide encoding an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:87. In certain embodiments, the engineered immune cells provided herein include any CAR known in the art and an exogenously introduced polynucleotide encoding an amino acid sequence of SEQ ID NO:87.

[0216] In certain embodiments, any CAR known in the art and the intracellular domain derived from SLAM-associated protein (SAP) are expressed in the engineered immune cells provided herein. In certain embodiments, any CAR known in the art and SAP are expressed in the engineered immune cells provided herein. In certain embodiments, any CAR known in the art and SEQ ID NO:86 amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical amino acid sequence are expressed in the engineered immune cells provided herein. In certain embodiments, any CAR known in the art and SEQ ID NO:86 amino acid sequence are expressed in the engineered immune cells provided herein.

[0217] In certain embodiments, any CAR known in the art and the intracellular domain derived from Ewing's sarcoma activated transcript-2 (EAT2) are expressed in the engineered immune cells provided herein. In certain embodiments, any CAR known in the art and EAT2 are expressed in the engineered immune cells provided herein. In certain embodiments, any CAR known in the art and an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 87 are expressed in the engineered immune cells provided herein. In certain embodiments, any CAR known in the art and the amino acid sequence of SEQ ID NO: 87 are expressed in the engineered immune cells provided herein.

[0218] In certain embodiments, the polynucleotide encoding the intracellular domain derived from SLAM-associated protein (SAP) or the intracellular domain derived from Ewing's sarcoma activated transcript-2 (EAT2) also encodes CAR. In certain embodiments, the CAR and the intracellular domain derived from SAP are encoded by the same polynucleotide. In certain embodiments, the CAR and the intracellular domain derived from EAT2 are encoded by the same polynucleotide.

[0219] In certain embodiments, the intracellular domain derived from SLAM associated protein (SAP) or the intracellular domain derived from EAT2 is a part of the CAR in the engineered immune cell. In certain embodiments, the engineered immune cell provided herein includes CAR, and the CAR includes from N-terminus to C-terminus: (i) an extracellular antigen-binding domain; (ii) an optional hinge domain, which may be, but is not limited to, a hinge domain derived from a second polypeptide; (iii) optionally, comprises a transmembrane domain derived from the transmembrane domain of a second polypeptide; (iv) optionally, a peptide linker, which may be, but is not limited to, a peptide linker provided herein; (v) an intracellular signaling domain comprising an intracellular domain derived from SAP or an intracellular domain derived from EAT2.

[0220] In certain embodiments, the intracellular signaling domain of the CAR in the engineered immune cell further comprises one or more elements, which are (v)-(a) an intracellular domain derived from a first polypeptide such as DAP10, DNAM1, CD28H, CD2, DAP12, CD28 or OX40; (v)-(b) derived from the intracellular domain of a third polypeptide; (v)-(c) derived from the intracellular domain of CD3ζ; and (v)-(d) A peptide linker, which may be, but is not limited to, P2A, T2A or E2A.

[0221] In certain embodiments, the second polypeptide is different from the first polypeptide. In certain embodiments, the third polypeptide is different from the first polypeptide. In certain embodiments, the second polypeptide and the third polypeptide are the same polypeptide. In certain embodiments, the second polypeptide and the third polypeptide are different polypeptides. In certain embodiments, the second polypeptide or the third polypeptide is a signaling lymphocyte activation molecule family (SLAMF) receptor, CD2, CD28H, DNAM1, CD28, 4-1BB, OX40, KIR-S, NKG2C or NKG2E. In certain embodiments, the SLAMF receptor is SLAMF4 (CD244, 2B4), SLAMF3 (CD229, Ly9), SLAMF6 (CD352, NTB-A, SF2000), SLAMF5 (CD84), SLAMF7 (CD319, CRACC, CS1), SLAMF1 (CD150), SLAMF2 (CD48, FimH), SLAMF8 (CD353) or SLAMF9 (CD84H1, SF2001). In certain embodiments, the KIR-S is KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5 or KIR3DS1.

[0222] In certain embodiments, the engineered immune cells are NK cells. In certain embodiments, the engineered immune cells are NKT cells. In certain embodiments, the engineered immune cells are T cells, such as CAR-T, TIL, TCR-T, γ-δT, and double negative DN-T. 5.4 Peptides

[0223] In another aspect, polypeptides are provided herein. In certain embodiments, a polypeptide is provided herein comprising a CAR provided herein.

[0224] In certain embodiments, provided herein is a polypeptide comprising a chimeric antigen receptor (CAR) and an intracellular domain derived from a SLAM-associated protein (SAP), wherein the CAR comprises: (i) an extracellular antigen-binding domain; (ii) a transmembrane domain; and (iii) an intracellular signaling domain, and wherein the CAR is linked to an intracellular domain derived from SAP via a peptide linker.

[0225] In certain embodiments, provided herein is a polypeptide comprising a chimeric antigen receptor (CAR) and an intracellular domain derived from EAT2, wherein the CAR comprises: (i) an extracellular antigen-binding domain; (ii) a transmembrane domain; and (iii) an intracellular signaling domain, and wherein the CAR is linked to an intracellular domain derived from EAT2 via a peptide linker.

[0226] In certain embodiments, said CAR is linked to said intracellular domain derived from SAP or said intracellular domain derived from EAT2 via P2A, T2A or E2A.

[0227] In certain embodiments, the polypeptide comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:86 or SEQ ID NO:87.

[0228] In certain embodiments, the polypeptide comprises SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ : The invention relates to an amino acid sequence that is at least 90%, at least 95% or 100% identical to an amino acid sequence of SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144 or SEQ ID NO:145. 5.5 Polynucleotides and vectors

[0229] On the other hand, polynucleotides are provided herein. In certain embodiments, a polynucleotide is provided herein, encoding a CAR provided herein. In certain embodiments, a polynucleotide is provided herein, encoding a polypeptide provided herein.

[0230] In certain embodiments, the polynucleotide encodes an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:86 or SEQ ID NO:87.

[0231] In certain embodiments, the polynucleotide encodes the sequence of SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125 NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144 or SEQ ID NO:145 are amino acid sequences that are at least 90%, at least 95% or 100% identical to the amino acid sequences of SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144 or SEQ ID NO:145.

[0232] The polynucleotides of the present invention may be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and may be double-stranded or single-stranded, and if single-stranded, may be coding strands or non-coding strands (antisense). In some embodiments, the polynucleotide is in the form of cDNA. In some embodiments, the polynucleotide is a synthetic polynucleotide.

[0233] The present application further relates to variants of polynucleotides described herein, wherein the variant encodes (for example) a fragment, analog and / or derivative of a CAR provided herein or a polypeptide provided herein. In certain embodiments, the present application provides polynucleotides, including polynucleotides having at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, and in some embodiments, at least about 96%, 97%, 98% or 99% identical nucleotide sequences. In this article, the phrase "a polynucleotide having a nucleotide sequence at least (for example) 95%'same' as a reference nucleotide sequence" refers to a polynucleotide having the same nucleotide sequence as the reference sequence, but the polynucleotide sequence may include up to 5 point mutations in every 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides of the reference sequence may be lacking or replaced by another nucleotide, or up to 5% of the total nucleotides of the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' end of the reference nucleotide sequence, or anywhere between these ends, interspersed individually among the nucleotides of the reference sequence, or in one or more contiguous groups within the reference sequence.

[0234] Polynucleotide variants may contain changes in coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants contain changes that form silent substitutions, additions, or deletions, but do not change the properties or activity of the encoded polypeptide. In some embodiments, polynucleotide variants include silent substitutions that result in unchanged amino acid sequences of polypeptides (due to degradation of genetic coding). Polynucleotide variants may be produced for a variety of reasons, for example, to express with optimized codons of a particular host (i.e., changing the codons of human mRNA to preferred codons of a bacterial host, such as E. coli). In some embodiments, polynucleotide variants include at least one silent mutation in a non-coding region or coding region of a sequence.

[0235] In some embodiments, the formation of polynucleotide variants is used to regulate or alter the expression (or degree of expression) of the encoded polypeptide. In some embodiments, the formation of polynucleotide variants is used to increase the expression of the encoded polypeptide. In some embodiments, polynucleotide variants are generated to reduce the expression of the encoded polypeptide. In some embodiments, the polynucleotide variants increase the expression of the encoded polypeptide compared to the parental polynucleotide sequence. In some embodiments, the polynucleotide variants reduce the expression of the encoded polypeptide compared to the parental polynucleotide sequence.

[0236] In another aspect, provided herein are vectors. In certain embodiments, provided herein are vectors comprising a polynucleotide provided herein.

[0237] In one embodiment, the polynucleotide can be incorporated into a recombinant expression vector. The present application provides a recombinant expression vector comprising any one of the polynucleotides provided herein. In this article, the term "recombinant expression vector" refers to an oligonucleotide or polynucleotide construct that can be genetically modified to express mRNA, protein, polypeptide or peptide by host cells when the construct comprises a nucleotide sequence encoding mRNA, protein, polypeptide or peptide, and the vector is in contact with the cell under conditions sufficient to express mRNA, protein, polypeptide or peptide in the cell. Herein, as a whole, the vector is not naturally produced; however, part of the vector may be naturally produced. The recombinant expression vector may contain any type of nucleotides, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and may contain natural, non-natural or altered nucleotides. The recombinant expression vector may include a connection of naturally occurring or non-naturally occurring nucleotides, or two types of connections. Non-naturally occurring or altered nucleotides, or connections between nucleotides do not hinder the transcription or replication of the vector. 5.6 Pharmaceutical compositions

[0238] In another aspect, provided herein are pharmaceutical compositions. In certain embodiments, provided herein are pharmaceutical compositions comprising (i) a CAR provided herein, an engineered immune cell provided herein, a polypeptide provided herein, a polynucleotide provided herein, or a vector provided herein; and (ii) a pharmaceutically acceptable excipient.

[0239] In certain embodiments, provided herein is a pharmaceutical composition comprising (i) the CAR provided herein, and (ii) a pharmaceutically acceptable excipient.

[0240] In certain embodiments, provided herein is a pharmaceutical composition comprising (i) the engineered immune cells provided herein; and (ii) a pharmaceutically acceptable excipient.

[0241] In certain embodiments, provided herein is a pharmaceutical composition comprising (i) a polypeptide as provided herein; and (ii) a pharmaceutically acceptable excipient.

[0242] In certain embodiments, provided herein is a pharmaceutical composition comprising (i) a polynucleotide as provided herein; and (ii) a pharmaceutically acceptable excipient.

[0243] In certain embodiments, provided herein is a pharmaceutical composition comprising (i) a vector as provided herein; and (ii) a pharmaceutically acceptable excipient.

[0244] In certain embodiments, a pharmaceutical composition is provided herein, comprising a CAR provided herein, an engineered immune cell provided herein, a polypeptide provided herein, a polynucleotide provided herein, or a vector provided herein. In certain embodiments, a pharmaceutical composition is provided herein, comprising a CAR provided herein. In certain embodiments, a pharmaceutical composition is provided herein, comprising an engineered immune cell provided herein. In certain embodiments, a pharmaceutical composition is provided herein, comprising a polypeptide provided herein. In certain embodiments, a pharmaceutical composition is provided herein, comprising a polynucleotide provided herein. In certain embodiments, a pharmaceutical composition is provided herein, comprising a vector provided herein.

[0245] In certain embodiments, a pharmaceutical composition is provided herein, comprising an effective amount of CAR provided herein, an effective amount of engineered immune cells provided herein, an effective amount of polypeptides provided herein, an effective amount of polynucleotides provided herein, or an effective amount of vectors provided herein. In certain embodiments, a pharmaceutical composition is provided herein, comprising an effective amount of CAR provided herein. In certain embodiments, a pharmaceutical composition is provided herein, comprising an effective amount of engineered immune cells provided herein. In certain embodiments, a pharmaceutical composition is provided herein, comprising an effective amount of polypeptides provided herein. In certain embodiments, a pharmaceutical composition is provided herein, comprising an effective amount of polynucleotides provided herein. In certain embodiments, a pharmaceutical composition is provided herein, comprising an effective amount of vectors provided herein.

[0246] In one embodiment, each component is "pharmaceutically acceptable" in the sense that it is compatible with the other ingredients of the pharmaceutical formulation and is suitable for use in contact with the tissues or organs of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications commensurate with a reasonable benefit / risk ratio. See Lippincott Williams and Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th edition; Rowe et al., eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd edition; Ash and Ash, eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd Edition; Gibson, ed.; CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, the pharmaceutically acceptable excipient is nontoxic to the cells or mammals exposed thereto at the doses and concentrations employed. In some embodiments, the pharmaceutically acceptable excipient is an aqueous pH buffered solution.

[0247] In particular embodiments, the term "excipient" may also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), carrier, or vehicle. Pharmaceutical excipients may be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Saline solutions and aqueous dextrose and glycerol solutions may also be used as liquid excipients. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, and the like. If desired, the composition may also contain a minimum amount of a wetting or emulsifying agent or a pH buffer. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. Examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA. These compositions contain a prophylactically or therapeutically effective amount of an active ingredient provided herein (e.g., in purified form) together with a suitable amount of an excipient so as to provide a form suitable for administration to a patient. The formulation should suit the mode of administration.

[0248] In some embodiments, the choice of excipient depends, in part, on the particular cells, binding molecules and / or antibodies and / or method of administration. Thus, there are a variety of suitable formulations.

[0249] Generally, 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, isotonic agents, stabilizers, metal complexing agents (e.g., Zn-protein complexing agents), chelating agents (e.g., EDTA) and / or non-ionic surfactants.

[0250] Buffers can control the pH to be within the range that is optimal for therapeutic effectiveness, especially when stability and pH are related. Suitable buffers for use in the present invention include both organic and inorganic acids and their salts. For example, citrates, phosphates, succinates, tartrates, fumarates, gluconates, oxalates, lactates, acetates. In addition, buffers can include histidine and trimethylamine salts (e.g., Tris).

[0251] Preservatives may be added to retard microbial growth. Suitable preservatives for use in the present invention include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; alkyldimethylbenzylamine halides (e.g., chlorides, bromides, iodides), benzethonium chloride; thimerosal, phenol, butyl alcohol or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol and m-cresol.

[0252] Osmotic agents (sometimes referred to as "stabilizers") can be used to adjust or maintain the osmotic properties of the liquid in the composition. When large charged biomolecules (such as proteins and antibodies) are used, they are often referred to as "stabilizers" because they can interact with the charged groups of the amino acid side chains, thereby reducing the potential for intermolecular and intramolecular interactions. Exemplary osmotic agents include polyols, triols or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0253] Other exemplary excipients include: (1) bulking agents, (2) solubilizing agents, (3) stabilizers, and (4) agents that prevent denaturation or adhesion to the container wall. These excipients include: polysaccharide alcohols (see above for enumeration); amino acids, such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamine, threonine, etc.; organic sugars or sugar alcohols, such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbitol, xylose, ribose, ribitol, inosinose, inositol, galactose, galactitol, glycerol, cyclic alcohols (e.g., inositol), polyethylene glycol, ; sulfur-containing reducing agents such as urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose); disaccharides (e.g., lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

[0254] Nonionic surfactants or detergents (also called "wetting agents") can be used to help dissolve the therapeutic agent and protect the therapeutic protein from agitation-induced aggregation. They also allow the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. Suitable nonionic surfactants include, for example, polysorbates (20, 40, 60, 65, 80, etc.), polyoxamers (184, 188, etc.), Polyols, Polyoxyethylene sorbitan monoether ( -20, -80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glyceryl monostearate, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents include alkyl dimethyl benzylamine chloride or benzethonium chloride.

[0255] In order for the pharmaceutical composition to be used for in vivo administration, it is preferably sterile. The pharmaceutical composition can be sterilized by filtration through a sterile filtration membrane. The pharmaceutical composition herein can generally be placed in a container with a sterile access port (e.g., an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle).

[0256] The administration route can be a known and widely accepted method, such as one or more administrations via pills, or long-term infusion via a suitable means, such as injection or infusion via subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, topical administration, inhalation or via sustained release or extended release.

[0257] In another embodiment, the pharmaceutical composition can be provided by a controlled release or sustained release system. In one embodiment, a pump can be used to achieve controlled release or sustained release (see Sefton, Crit. Ref. Biomed. Eng. 14: 201-40 (1987); Buchwald et al., Surgery 88: 507-16 (1980); and Saudek et al., N. Engl. J. Med. 321: 569-74 (1989)). In another embodiment, polymeric materials can be used to achieve controlled or sustained release of a prophylactic or therapeutic agent (e.g., a fusion protein as described herein) or a composition provided herein (see Medical Applications of Controlled Release (Langer and Wise, eds., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball, eds., 1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61-126 (1983); Levy et al., Science 228:190-92 (1985); During et al., Ann. Neurol. 25:351-56 (1989); Howard et al., J. Neurosurg. 71:105-12 (1989); U.S. Pat. Nos. 5,679,377, 5,916,597, 5,912,015, 5,989,463, and 5,128,326; PCT Publication Nos. WO99 / 15154 and WO99 / 20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactic acid (PLA), poly(lactic acid-co-glycolide) (PLGA), and polyorthoesters. In one embodiment, the polymer used in the sustained release formulation is inert, free of leachable impurities, storage stable, sterile and biodegradable. In another embodiment, the controlled or sustained release system can be placed adjacent to a specific target tissue (e.g., nasal passages or lungs), so only a portion of the systemic dose is required (see Goodson, Medical Applications of Controlled Release Vol. 2, 115-38 (1984)). Controlled release systems can also be found in the discussion of Langer, Science 249: 1527-33 (1990). Any technique known to those skilled in the art can be used to produce sustained release formulations comprising one or more agents as described herein (see U.S. Pat. No. 4,526,938, PCT Publication Nos. WO91 / 05548 and WO96 / 20698, Ning et al., Radiotherapy & Oncology 39: 179-89 (1996); Song et al., PDA J. of Pharma. Sci. & Tech. 50: 372-97 (1995); Cleek et al., Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24: 853-54 (1997); and Lam et al., Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24: 759-60 (1997)).

[0258] The active ingredient can also be placed in microcapsules, which can be prepared by coacervation technology or interfacial polymerization technology, for example, hydroxymethylcellulose or gelatin microcapsules and poly (methyl methacrylate) microcapsules, or by using colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or by using macroemulsions. These techniques can be found in Remington's Pharmaceutical Sciences, 18th edition.

[0259] Various delivery compositions and delivery systems are prior art and can be used for the therapeutic agents provided herein, including but not limited to encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing single domain antibodies or therapeutic molecules provided herein, nucleic acid molecule constructs as part of retroviruses or other vectors, etc.

[0260] In some embodiments, the pharmaceutical compositions provided herein comprise binding molecules and / or cells (e.g., therapeutically effective or preventively effective doses) for treating or preventing a disease or condition. In some embodiments, the therapeutic or preventive efficacy is monitored by periodic assessment of the treated individual. For repeated administration over a period of several days or longer, depending on the condition, the treatment will be repeated until the disease symptoms are suppressed. However, other effective dosage regimens may also be determined. 5.7 Treatment methods and uses

[0261] In another aspect, provided herein are methods of treating a disease or condition in an individual. In certain embodiments, provided herein are methods of treating a disease or condition in an individual, comprising administering to the individual an effective amount of an engineered immune cell provided herein or a pharmaceutical composition provided herein.

[0262] In certain embodiments, the method of treating a disease or condition in an individual comprises administering to the individual an effective amount of an engineered immune cell provided herein. In certain embodiments, the method of treating a disease or condition in an individual comprises administering to the individual an effective amount of a pharmaceutical composition provided herein.

[0263] As described herein, the engineered immune cells provided herein are suitable for cell therapy, for example, for treating cancer. In certain embodiments, treatment with the engineered immune cells provided herein or the pharmaceutical compositions provided herein has one, two or more of the following therapeutic effects: (1) reducing the severity, progression, spread and / or frequency of one or more symptoms, (2) eliminating one or more symptoms and / or their root causes, (3) preventing the occurrence of one or more symptoms and / or their root causes, and (4) ameliorating or remedying damage. In certain embodiments, treatment includes therapeutic measures and preventive or inhibitory measures for a condition, disease or disorder.

[0264] These methods and uses include methods of treatment and uses, for example, involving administering molecules, cells, or compositions containing them to individuals suffering from a disease, condition, or illness. In some embodiments, molecules, cells, and / or compositions are administered in effective amounts to achieve treatment of a disease or condition. The uses include the use of engineered immune cells provided herein or pharmaceutical compositions provided herein in these methods and treatments, and in the preparation of medicaments for use in these methods of treatment. In some embodiments, these methods are performed by administering engineered immune cells or pharmaceutical compositions provided herein to individuals suffering from or suspected of having a disease or condition. In some embodiments, these methods achieve treatment of a disease or condition in an individual.

[0265] In some embodiments, the treatments provided herein result in a complete or partial improvement or reduction of a disease or condition or symptom, adverse effect or result, or phenotype associated therewith. The therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological results of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. These terms include but do not imply a complete cure of the disease, or the complete elimination of any or all symptoms or results.

[0266] Herein, in some embodiments, the treatment provided herein can delay the development of a disease or condition, for example, delay, hinder, slow down, delay, stabilize, inhibit and / or postpone the progression of a disease (e.g., cancer). This delay can be a variable length of time, depending on the history of the disease and / or the individual being treated. It will be appreciated by those skilled in the art that a sufficient or significant delay can actually include prevention, because the individual no longer develops the disease or condition. For example, advanced cancer (e.g., progression of metastasis) can be delayed.

[0267] In some embodiments, the methods comprise administering to an individual, tissue, or cell (eg, one having, at risk for, or suspected of having a disease or disorder) an engineered immune cell provided herein or a pharmaceutical composition provided herein.

[0268] In some embodiments, the subject to which the engineered immune cells provided herein or the pharmaceutical compositions provided herein are administered is a primate, such as a human. The subject may be male or female and may be of any suitable age, including infants, adolescents, teenagers, adults, and elderly subjects. In some instances, the subject is a validated animal model for disease, recipient cell therapy, and / or assessment of toxicity outcomes.

[0269] The engineered immune cells provided herein or the pharmaceutical compositions provided herein can be delivered by any suitable method, for example, by injection, for example, intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjunctival injection (subconjectval), subconjunctival injection (subconjuntival), Tenon's injection (Tenon's), retrobulbar injection, peribulbar injection, or post-scleral delivery. In some embodiments, administration is parenteral, intrapulmonary, and intranasal, and if required for local treatment, administration can be intralesional. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

[0270] Regarding the dosage of the preventive or therapeutic agent provided herein, the effective dose for effectively preventing and / or treating a disease or condition can be determined by standard clinical techniques. The effective dose can be obtained by extrapolation of a dose-response curve derived from an in vitro or animal model test system. When used for the prevention or treatment of a disease, the appropriate dose of the binding molecule or cell may depend on the type of disease or condition to be treated, the type of binding molecule, the severity and cause of the disease or condition (regardless of whether the therapeutic agent is given for prevention or treatment purposes), previous therapy, the patient's clinical history and the response to the agent, and the discretion of the attending physician. In some embodiments, the composition, molecule, and cell are appropriately given to the patient in one or a series of treatment courses.

[0271] In some embodiments, regarding engineered immune cells, an amount of about 1 million to about 100 billion cells may be administered to an individual and / or an amount of the cells may be administered per kilogram of body weight. In some embodiments, a pharmaceutical composition comprises any of the engineered immune cells described herein, wherein the pharmaceutical composition is administered at a dose of at least about 10 per kilogram of body weight of the individual. 4 , 10 5 , 10 6 , 10 7 , 10 8 or 10 9 The dosage may vary depending on the disease or condition and / or the patient and / or other conditions being treated.

[0272] In some embodiments, the pharmaceutical composition is administered once. In some embodiments, the pharmaceutical composition is administered multiple times (e.g., any of 2, 3, 4, 5, 6 or more times). In some embodiments, the pharmaceutical composition is administered once or multiple times during a dosing cycle. The dosing cycle may be 1, 2, 3, 4, 5 or more weeks, or 1, 2, 3, 4, 5 or more months. The optimal dosage and treatment regimen for a particular patient may be determined by a person skilled in the art of medicine based on monitoring the patient's disease signs.

[0273] In some embodiments, the engineered immune cells provided herein or the pharmaceutical compositions provided herein are administered as part of a combination therapy, e.g., simultaneously or sequentially in any order with another therapeutic intervention (e.g., another antibody or engineered cell or receptor or agent, e.g., a cytotoxic agent or a therapeutic agent).

[0274] In some embodiments, the engineered immune cells provided herein or the pharmaceutical compositions provided herein are co-administered with one or more additional therapeutic agents, or are administered simultaneously or sequentially in any order in combination with another therapeutic intervention. In some cases, cells are co-administered with another therapy within a sufficiently close time frame so that the cell population can enhance the effect of one or more additional therapeutic agents, and vice versa. In some embodiments, cells or antibodies are administered before one or more additional therapeutic agents. In some embodiments, cells or antibodies are administered after one or more additional therapeutic agents.

[0275] In other embodiments, the methods or uses provided herein are used to prevent a disease or condition. In some embodiments, the disease or condition is a CD19-related disease or condition. In some embodiments, the disease or condition is a GPC3-related disease or condition.

[0276] In certain embodiments, the disease or condition is cancer. The method of treatment or use can be determined according to the cancer to be treated. For example, in certain embodiments, the cancer is a hematological malignancy or leukemia. In certain embodiments, the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), myelodysplasia, myelodysplastic syndrome, acute T-lymphoblastic leukemia, or acute promyelocytic leukemia, chronic myelomonocytic leukemia, or the myeloid blast phase of chronic myeloid leukemia.

[0277] Other exemplary cancers that can be treated with the engineered immune cells provided herein or the pharmaceutical compositions provided herein include: breast cancer, ovarian cancer, esophageal cancer, bladder cancer or gastric cancer, salivary duct cancer, salivary duct cancer, invasive forms of lung or uterine cancer (e.g., endometrial serous carcinoma). In some other embodiments, the cancer is brain cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, rectal cancer, kidney cancer, gastric cancer, testicular cancer or uterine cancer.In other embodiments, the cancer is squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, neuroblastoma, sarcoma (e.g., angiosarcoma or chondrosarcoma), laryngeal cancer, parotid cancer, biliary cancer, thyroid cancer, acral lentigo melanoma, actinic keratosis, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous cell carcinoma, anal canal cancer, anal cancer, anorectal cancer, astrocytoma, vestibular gland cancer, basal cell carcinoma, biliary cancer (biliary cancer), bone cancer, bone marrow cancer, bronchial cancer, bronchial adenocarcinoma, carcinoid, bile duct cancer (cholangiocarcinoma), chondrosarcoma, choroid plexus papilloma / carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue cancer, cystadenoma, digestive system cancer, duodenal cancer, endocrine system cancer, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, endothelial cell carcinoma, ependymal carcinoma, epidermal cell carcinoma, Ewing's sarcoma, eye and orbital cancer, female genital cancer, focal nodular hyperplasia, gallbladder cancer, gastric antrum cancer, gastric fundus cancer, gastrinoma, glioblastoma, glucagonoma, heart cancer, hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenocarcinoma, hepatic adenomatosis, hepatobiliary cancer, hepatocellular carcinoma, Hodgkin's disease disease), ileal cancer, insulinoma, intraepithelial neoplasia, epithelial-mesenchymal squamous cell neoplasia, intrahepatic bile duct cancer, invasive squamous cell carcinoma, jejunal cancer, joint cancer, Kaposi's sarcoma, pelvic cancer, large cell carcinoma, colorectal cancer, leiomyosarcoma, lentiform malignant melanoma, lymphoma, male genital cancer, malignant melanoma, malignant mesothelioma, medulloblastoma, medullary epithelioma, meningeal cancer, mesothelioma, metastatic cancer, oral cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal passage cancer, nervous system cancer, neuroepithelial gland carcinoma nodular melanoma, non-epithelial skin cancer, non-Hodgkin's lymphoma, oat cell carcinoma, oligodendroglial carcinoma, oral cancer, osteosarcoma , papillary serous adenocarcinoma, penile cancer, pharyngeal cancer, pituitary tumor, plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell carcinoma, respiratory cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, sinus cancer, skin cancer, small cell carcinoma, small intestine cancer, smooth muscle cancer, soft tissue cancer, somatostatin-secreting tumor, spinal cancer, squamous cell carcinoma, rhabdomyocarcinoma, submesothelial carcinoma, superficial spreading melanoma, T-cell leukemia, tongue cancer, undifferentiated carcinoma, ureteral cancer, urethral cancer, bladder cancer, urinary system cancer, cervical cancer, uterine corpus cancer, uveal melanoma, vaginal cancer, verrucous carcinoma, VIPoma, vulvar cancer, well-differentiated carcinoma or Wilms tumor.

[0278] In some other embodiments, the cancer to be treated is a non-Hodgkin's lymphoma, such as a B-cell lymphoma or a T-cell lymphoma. In certain embodiments, the non-Hodgkin's lymphoma is a B-cell lymphoma, such as diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, or primary central nervous system (CNS) lymphoma. In certain other embodiments, the non-Hodgkin's lymphoma is a T-cell lymphoma, such as precursor T-lymphoblastic lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, angioimmunoblastic T-cell lymphoma, extranodal natural killer / T-cell lymphoma, enteropathy-type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma, or peripheral T-cell lymphoma.

[0279] In certain embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is liver cancer. 5.8 Methods of Engineering Immune Cells

[0280] In another aspect, provided herein is a method of engineering an immune cell. In certain embodiments, provided herein is a method of engineering an immune cell, comprising introducing a polynucleotide provided herein, a vector provided herein, or a polypeptide provided herein into an immune cell.

[0281] In certain embodiments, a method of engineering an immune cell is provided herein, comprising introducing a polynucleotide provided herein into an immune cell. In certain embodiments, a method of engineering an immune cell is provided herein, comprising introducing a vector provided herein into an immune cell. In certain embodiments, a method of engineering an immune cell is provided herein, comprising introducing a polypeptide provided herein into an immune cell.

[0282] In certain embodiments, the immune cell is a NK cell, a NKT cell or a T cell. In certain embodiments, the immune cell is a NK cell.

[0283] In some embodiments, there is provided herein a method for engineering immune cells, comprising introducing the polynucleotides provided herein into immune cells. In some embodiments, the method further comprises selecting immune cells for expressing CAR, which is encoded by polynucleotides or vectors provided herein. In some embodiments, the method further comprises selecting immune cells for expressing SAP or EAT2, which are encoded by polynucleotides or vectors provided herein. 5.9 Analysis

[0284] In practice, the embodiments provided herein can be applied to molecular biology, microbiology and immunology techniques well known to those skilled in the art. These techniques are fully explained in the literature. Examples of particularly suitable literature include: Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999); Glover, ed., DNA Cloning, Volumes I and II (1985); Freshney, ed., Animal Cell Culture: Immobilized Cells and Enzymes (IRL Press, 1986); Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes, Protein Purification: Principles and Practice (Springer Verlag, NY, 2nd edition, 1987); and / or Janeway et al., Immunobiology (Garland Science, 7th edition, 2008).

[0285] In certain embodiments, once the engineered immune cells are administered to a mammal (e.g., a human), the biological activity of the engineered immune cell population can be measured by any of a number of known methods. Assessment parameters include: in vivo, specific binding of engineered or natural immune cells to antigens can be assessed by imaging; or in vitro, by ELISA or flow cytometry. In certain embodiments, the ability of engineered immune cells to destroy target cells can be assessed by any suitable method known in the art, such as using Kochenderfer et al., J. Immunotherapy, 32 (7): 689-702 (2009), and Herman et al., J. Immunological Methods, 285 (1): 25-40 (2004) in cytotoxicity analysis method to measure. In certain embodiments, the biological activity of engineered immune cells can also be measured by analyzing the expression and / or secretion of certain cytokines (e.g., CD107a, IFNγ, IL-2, and TNF). In some aspects, biological activity is measured by evaluating clinical outcomes (e.g., reduction in tumor burden or load). 5.9.1 Cells, cell culture and cloning

[0286] Any cell known in the art can be used herein, for example, NK92 cells, NALM6 cells, 293T cells, HUH7 cells, human primary NK cells and K562-CD137L cells. Human primary NK cells can be isolated from the peripheral blood of healthy human donors. Any known cloning technology can be used herein. 5.9.2 In vitro killing and degranulation analysis

[0287] Various known methods in the art can be used to evaluate the killing activity of engineered immune cells. For example, as previously described, 293T, 293T-CD19, NALM6 and Huh7 target cells can be genetically modified and stably express Luc2-GFP (Zhang et al., (2021) Ectopic activation of the miR-200c-EpCAM axis enhances antitumor Tcell responses in models of adoptive cell therapy. Sci Transl Med. 13 (611): eabg4328). To evaluate the killing activity, as shown in the accompanying drawings, CAR-NK cells and target cells can be co-cultured at different ratios for different times. D- luciferin can be used for the measurement of fluorescence emission spectra. Target cells without NK cells can be used as 100% live cell controls. The killing ability can be determined based on normalized spectra and background subtraction, and the equation can be used: Killing rate (%) = (1-(exp / 100% active control))*100 (Zhang et al. (2021) Ectopic activation of the miR-200c-EpCAM axis enhances antitumor T cell responses in models of adoptive cell therapy. Sci Transl Med. 13(611):eabg4328).

[0288] The degranulation activity of engineered immune cells can be evaluated using various known methods in the art. For example, to evaluate the degranulation activity, a fluorescently bound anti-CD107a antibody can be added to the NK-target co-culture, and after cultivation, the cells are washed with FACS buffer and stained with anti-CD56 antibodies. CD107a+NK cells stained with CD56 can be quantitatively analyzed by flow cytometry analysis (Beckman Coulter CytoFLEX LX). 5.9.3 Flow cytometric analysis of intracellular cytokines

[0289] The production of IFN can be evaluated using various methods known in the art. For example, to evaluate the production of IFN, live NK cells can be co-cultured with target cells in a U-shaped bottom 96-well plate after centrifugation. Brefeldin A can be added to the cells and cultured. Cell surface markers can be stained, washed, and fixed with IC fixation buffer (eBioscience), then washed, and stained with cytokine antibodies and permeation buffer (eBioscience), and then flow cytometry analysis can be performed. 5.9.4 Duolink PLA Analysis

[0290] PLA analysis can be performed using the In Situ Duolink PLA Red Kit (Sigma-Aldrich) according to the manufacturer's instructions. NK cells can be adhered to poly-l-lysine glass, and then target cells can be added and cultured. Non-adherent cells can be removed by washing with PBS. Slides can be fixed and stained with anti-GFP (rabbit IgG) and anti-DAP10 (mouse IgG), washed, and stained with anti-rabbit and anti-mouse PLA probes, followed by probe DNA ligation, amplification, and fluorescence staining and observation using confocal microscopy. 5.9.5 Retroviral transduction

[0291] Any known method for NK cell transduction can be used herein. For example, for NK cell transduction, Phoenix A cell expression vector can be transfected together with packaging vector GagPol using calcium ion method. Filtered viral supernatant, polybrene and IL-2 can be mixed with NK cell line or primary NK cells expanded for 3 days in 6-well plates. 6-well plates can be centrifuged. Transduced NK cell line can be cultured with corresponding culture medium for in vitro experiments, and transduced primary NK cells can be co-cultured with inactivated K562-CD137L and recombinant IL-2 for in vitro verification of mice in subsequent steps, and adoptive cell therapy experiments. 6. Examples 6.1 Example 1: Exemplary Structure of Novel Complete Signaling NK-CAR (NK-csCAR) Using SLMAF Gene or Non-SLAMF Gene ScFv-SLAMF hinge-SLAMF TM-DAP10ICD-SLAMFICD-CD3Z-P2A-SAP or EAT2ScFv-non-SLAMF hinge-SLAMF TM-DAP10ICD-non-SLAMF ICD-CD3Z-P2A-SAP or EAT2

[0292] As shown above, exemplary elements of the novel complete signaling NK-CAR (NK-csCAR) include:

[0293] ScFv, to specifically recognize the target antigen;

[0294] SLAMF (e.g. 2B4, CD229, NTB-A, CRACC, CD84) or non-SLAMF to provide hinge and transmembrane (TM); herein, the term "non-SLAMF" refers to polypeptides other than SLAMF, such as CD2, CD28H, DNAM1.

[0295] DAP10ICD provides the first co-stimulatory signal. This ICD is directly anchored in the cell's inner membrane, and the orientation of the DAP10ICD is unexpectedly critical to enhance the effectiveness of activation signaling;

[0296] ICD of the same SLAMF or non-SLAMF gene to provide a second co-stimulatory signal;

[0297] CD3ζ, to provide a third signal to phosphorylate SLP-76 on both Tyr113 and Tyr128 via a pZAP70 / pSyk-dependent pathway;

[0298] As a signal enhancer, without being limited to a specific theory, SAP or EAT2 can act as a fourth signal to eliminate the effect of inhibitory receptor signaling by competitively binding to the ITSM tail of SLAMF and prevent SHP from binding to pTry and recruiting Fyn, thereby ensuring complete activation signal transduction and completely overcoming the inhibitory receptor signaling barrier.

[0299] We demonstrated that NK cells can be effectively activated by the natural hinge and transmembrane domains of selected molecules. Unexpectedly, the first co-stimulatory signal provided by the ICD of DAP10 is necessary. Its anchoring position on the inner membrane is particularly critical for the unexpected effect of effectively enhancing the activation signal. Unexpectedly, the second co-stimulatory signal and the third signal CD3ζ also play an important role in the effective triggering of the CAR signal. Unexpectedly, SAP or EAT2 signal enhancers can significantly enhance the activation of NK cells and help overcome the inhibitory receptor signaling barrier stimulated by HLA-I. Through complete signal design, we have identified a series of excellent CAR candidates with unexpected effects, including: effectively activating NK cell function and showing complete resistance to inhibitory receptor signaling. 6.2 Example 2: Exemplary Materials and Methods 6.2.1 Cells

[0300] All cells were cultured at 37°C and 5% CO2. NK92 cells were cultured in RPMI-1640 supplemented with 2mM L-glutamine (Sigma), 1.5g / L sodium bicarbonate (Sigma), 0.2mM inositol (Sigma), 0.1mM 2-mercaptoethanol (Sigma), 0.02mM folic acid (Sigma), 100IU / mL recombinant IL-2, 12.5% ​​horse serum (Sigma) and 12.5% ​​fetal bovine serum (FBS, Sigma). NALM6 cells were cultured in complete RPMI-1640 supplemented with 10mM HEPES, 2mM L-glutamine, 1x non-essential amino acids, 1mM sodium pyruvate, 50μM 2-mercaptoethanol and 10% FBS. 293T and HUH7 cells were cultured in complete Dulbecco's Modified Eagle Medium DMEM with 10% FBS.

[0301] Human primary NK cells were isolated from the peripheral blood of healthy human donors using an NK cell negative selection kit (STEMCELL Technologies Inc.). Freshly isolated NK cells were cultured with paraformaldehyde (PFA) fixed K562-CD137L cells (1:1) and 100 IU / mL IL-2 at 37°C and 5% CO2 in serum-free OpTmizer T cell expansion medium (Gibco) with 10% human IL-2 preparation (Hemagen Diagnostics). On day 3, NK cells were used for genetic modification. Cultured NK cells were used 2 weeks after genetic modification. 6.2.2 Vector constructs

[0302] The CAR construct was cloned into the BglII / XhoI of the MSCV retroviral vector using ThermoFisher Scientific synthetic primers and long DNA fragments. The CAR backbone with ScFv recognizing CD19 or GPC3 was generated based on the design shown in Figures 1 to 7, and the gene sequences used in the experiment are shown in Table 4. The P2A sequence used for SAP expression after CAR is GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 1). Table 4. Gene sequences used in the experiments 6.2.3 In vitro killing and degranulation analysis

[0303] As previously described, 293T, 293T-CD19, NALM6, and Huh7 target cells were genetically modified and stably expressed Luc2-GFP (Zhang et al. (2021) Ectopic activation of the miR-200c-EpCAM axis enhances antitumor T cell responses in models of adoptive cell therapy. Sci Trans lMed. 13(611): eabg 4328). To evaluate the killing activity, CAR-NK cells and target cells were co-cultured at different ratios at 37 ° C for different times as shown in the accompanying drawings. 0.75 mg / mL D-luciferin can be used to measure the fluorescence emission spectrum. Target cells that do not contain NK cells can be used as a 100% live cell control. Based on the normalized spectra and background subtraction, the killing ability can be calculated using the equation: Killing rate (%) = (1-(exp / 100% active control))*100 (Zhang et al. (2021) Ectopic activation of the miR-200c-EpCAM axis enhances antitumor T cell responses in models of adoptive cell therapy. Sci Transl Med. 13(611):eabg4328).

[0304] To assess degranulation, 2 μg / ml fluorescently conjugated anti-CD107a antibody was added to the NK-target co-culture and incubated for 2 hours at 37°C, after which the cells were washed with FACS buffer containing 1 mM EDTA and stained with anti-CD56 antibody. CD56-stained CD107a+ NK cells were quantified by flow cytometry analysis (Beckman Coulter CytoFLEX LX). 6.2.4 Flow cytometric analysis of intracellular cytokines

[0305] To evaluate IFN production, live NK cells were centrifuged at 300 g for 3 minutes and co-cultured with target cells (1:1) in U-bottom 96-well plates and incubated for 2 hours at 37° C. Brefeldin A was added to the cells at a final concentration of 5 μg / ml and incubated for an additional 4 hours at 37° C. Cell surface markers were stained, washed and fixed with IC fixation buffer (eBioscience), washed and stained with cytokine antibodies and permeabilization buffer (eBioscience), and then analyzed by flow cytometry. 6.2.5Duolink PLA

[0306] PLA analysis was performed using the In Situ Duolink PLA Red Kit (Sigma-Aldrich) according to the manufacturer's instructions. CAR0 (empty vector with GFP expression) or CAR2-GFP fusion expressing NK cells were adhered to poly-l-lysine glass, and then target cells were added and incubated at 37°C for 30 minutes. Non-adherent cells were removed by washing with PBS. The slides were fixed and stained with anti-GFP (rabbit IgG) and anti-DAP10 (mouse IgG), washed, and stained with anti-rabbit and anti-mouse PLA probes, followed by probe DNA ligation, amplification, and then fluorescent staining and observation using confocal microscopy. 6.2.6 Retroviral transduction

[0307] For NK cell transduction, Phoenix A cells were transfected with the expression vector together with the packaging vector GagPol using the calcium ion method. The viral supernatant after 0.45μm filtration, 8μg / ml polybrene and IL-2 (100IU / mL) were mixed with the NK cell line or primary NK cells expanded for 3 days in a 6-well plate. The 6-well plate was centrifuged at 1400xg and 35°C for 2 hours. After the transduced NK cell line was cultured with the corresponding culture medium, it was used for in vitro experiments. After the transduced primary NK cells were co-cultured with inactivated K562-CD137L and recombinant IL-2 (100IU / mL) for 14 days, they were used for in vitro verification and adoptive cell therapy experiments in mice in the next step. 6.3 Example 3: Dysfunction of natural NKG2D in NKG2D-CAR NK cells

[0308] NKG2D is an activating receptor expressed by NK cells, which alone cannot trigger the activation of NK cells (Long et al. (2013) Controlling natural killer cell responses: integration of signals for activation and inhibition. Annu Rev Immunol. 31: 227-58). Tumor cells usually express various ligands of NK activation receptors. For example, 293T cells not only express MICA / B (NKG2D) ligands, but also express NKp30 ligands (Flaig et al. (2004) Cutting edge: NTB-A activates NK cells via homophilic interaction. J Immunol. 172 (11): 6524-7), and the activation of NK cells requires the coordination of multiple activating receptors.

[0309] We used an empty vector as CAR0 and the NKG2D CAR as CAR2 ( Figure 1A ), the data showed that CAR2NK cells expressed lower levels of NKG2D, but the same levels of 2B4 ( Figures 1B to 1C ). The CAR based on the NK-specific NKG2D-TM is more effective than the T-CAR expressed on NK cells. However, the expression of NKG2D CAR reduces the expression of natural NKG2D in NK cells, see report (Li et al. (2018) Human iPSC-Derived Natural Killer Cells Engineered with Chimeric Antigen Receptors Enhance Anti-tumor Activity. Cell Stem Cell. 23 (2): 181-192. e5), which indicates that the expression of NKG2D CAR can affect the function of natural NKG2D.

[0310] To observe the function of constitutive natural NKG2D, we used different target cells, such as 293T cells and HUH7-MICA cells. 293T cells (no CD19 expression) express NKG2D ligands, such as MICA / MICB. HUH7 cells (no CD19 expression) do not have constitutive MICA / MICB expression, so we established a HUH7 cell line that stably expresses MICA / MICB.

[0311] When 293T-CD19-MICA target cells were used, CAR2-NK cells showed excellent killing ability ( Figure 1D ). When 293T-MICA was used, although CD19 expression was absent on target cells, CAR0-NK and CAR2-NK cells were still able to kill these cells to a lower degree; the expression of CAR2 on NK cells reduced the triggering of killing compared to CAR0. The same situation was observed when Huh7-MICA was used as a target cell ( Figure 1D ). This may be at least partly attributed to the lower NKG2D expression in CAR2 NK cells and may also be related to the occupancy of signaling ligands by NKG2D-CAR, such as Figure 1E to Figure 1F as shown in .

[0312] The NKG2D receptor does not have an intracellular signaling domain. It recruits the ligand DNAX-activated protein 10 (DAP10) and transmits signals through the YxxM motif (Eagle et al. (2009) Beyond Stressed Self: Evidence for NKG2D Ligand Expression on Healthy Cells. Curr Immunol Rev. 5 (1): 22-34). Duolink proximity ligation analysis (PLA) is a technique for observing molecular interactions. When the distance between two molecules is less than 30nm, a signal will be displayed. We used CAR0 NK cells expressing GFP as a control, and used NK cells expressing CAR2-GFP fusion molecules, co-cultured with target 293T-CD19-MICA cells, and performed PLA analysis between GFP and DAP10. DAP10 is recruited to CAR2 and is effectively stimulated by CD19. When NK cells expressing NKG2D-GFP were used, PLA analysis between GFP and DAP10 showed that the expression of CAR2 significantly reduced the recruitment of DAP10 to natural NKG2D ( Figure 1E and 1F ). The data indicate that CAR2 recruits DAP10 for signaling and triggering NK cell activation, while at the same time, it occupies DAP10 resources, resulting in less DAP10 available for natural NKG2D activation.

[0313] Figures 1A to 1E The following results were confirmed: 1. NKG2D-CAR-NK cells expressed lower levels of NKG2D, but the same levels of 2B4. 2. NKG2D-CAR-NK cells had lower natural NKG2D function. 3. NKG2D had lower interaction with DAP10 in NKG2D-CAR NK cells. 6.4 Example 4: Anti-tumor activity mediated by 2B4-CAR mediated by complete signaling of NK cells

[0314] Without being limited by theory, we designed a new complete signaling CAR for NK cells based on NK cell biology. 2B4 (CD244) is an activating receptor expressed by NK cells (Agresta et al. (2018) The Emerging Role of CD244 Signaling in Immune Cells of the Tumor Microenvironment. Front Immunol. 9: 2809), which belongs to the signaling lymphocyte activation molecule family (SLAMF4). Without being limited by theory, the natural ligand of 2B4 has been identified as CD48. Without being limited by theory, the cytoplasmic tail of 2B4 contains four immunoreceptor tyrosine-based switch motifs (ITSM). Without being limited by theory, ITSM can recruit signaling lymphocyte activation molecule (SLAM)-associated protein (SAP), which competitively binds to ITSM and inhibits SHP recruitment, thereby being associated with activation signaling. Without being limited by theory, ITSM can also recruit Ewing's sarcoma activated transcript-2 (EAT2), which is associated with activation and inhibition of signaling. Without being limited by theory, SAP can bind to both non-phosphorylated and phosphorylated ITSM, but EAT2 can only bind to phosphorylated SLAMF ITSM (Morra et al. (2001) Structural basis for the interaction of the freeSH2 domain EAT-2 with SLAM receptors in hematopoietic cells. EMBO J. 20(21): 5840-52), thereby limiting the effect of EAT2 in 2B4 signaling by only partially inhibiting SHP binding to ITSM. Without being limited by theory, SAP and EAT2 have two functions: 1) help activate Fyn in SLAMF signaling, 2) block SHP binding to pTyr in 2B4's ITSM, thereby eliminating the inhibition caused by the SHP molecule.

[0315] We designed Figure 2AThe novel CAR shown in . 2B4 hinge TM, DAP10ICD and CD3ζICD are used to produce this novel and effective NK CAR. Not limited to theory, since 2B4 exhibits both inhibition and activation functions, according to the CAR construct, NK CAR is designed to have a P2A-guided SAP signal or EAT2 signal. After translation, P2A is cleaved to release CAR and SAP or EAT2 protein (Liu et al. (2017) Systematic comparison of 2A peptides forcloning multi-genes in a polycistronic vector. Sci Rep. 7 (1): 2193). Not limited to theory, SAP or EAT2 binds to the ITSM at the tail of 2B4 and competes with the inhibitory signal molecule SHP. Therefore, full activation signal transduction can be achieved by NK-CAR. Unexpectedly, the data showed that compared with the empty vector (CAR0) and the control CAR1 without intracellular signal transduction DAP10 and CD3ζ, all of these designs successfully triggered activation signal transduction ( FIG. 2C to FIG. 2E ).

[0316] The hinge is believed to play a key role in the effective expression and function of CAR. We compared the 2B4 hinge and CD8 hinge, both of which can trigger the effective activation of CAR in NK cells, indicating that the 2B4 hinge can be used in NK-CAR to provide effective activation signals for NK cells ( FIG. 2C to FIG. 2E ).

[0317] Inhibitory receptors (e.g., NKG2A) can bind to the HLA-I molecule HLA-E and be triggered by it, leading to the triggering of the inhibitory molecule SHP. 2B4 can bind to SHP, thereby inhibiting receptor signaling, and SAP or EAT2 can bind to the tail of 2B4 to compete with SHP, resulting in net activation signaling. Unexpectedly, the data show that SAP (in CAR8) and EAT2 (in CAR10) can be used as effective signaling enhancers to generate a complete signal to activate CAR-NK ( Figure 2C To E). Among them, SAP is more effective than EAT2.

[0318] In addition, CAR4 and CAR6, namely CD8 hinge-2B4-TM / ICD CAR and 2B4-hinge / TM / ICD CAR, are both effective in triggering activation signals, but are still limited because they do not have the fourth SAP signal ( Figure 2C to E), which is subsequently discussed in FIG. 6 .

[0319] FIG. 2A to FIG. 2EThe following results were confirmed: 1. 2B4 can be used in TM and ICD to prepare effective NK CAR. 2. 2B4 hinge can be used as an effective hinge in CAR constructs. 3. Unexpectedly, DAP10 used to replace NKG2D performed well in CAR constructs. 4. SAP and EAT2 unexpectedly enhanced NK activation signal transduction, which constituted a complete signal transduction NK-CAR (NK-csCAR), in which SAP may be better than EAT2. 5. CAR4 and CAR6, i.e. CD8 hinge-2B4-TM / ICD CAR and 2B4-hinge / TM / ICD CAR, are not as effective as NKG2D-CAR (Figure 6).

[0320] The above data indicate that our novel design performs unexpectedly well and that the complete signaling CAR can effectively trigger NK cell activation to an unexpected degree. 6.5 Example 5: Complete Signaling Enhanced by SAP

[0321] As shown in Figure 2, 2B4 CAR can successfully trigger NK cell activation (CAR6), while SAP, as a complete signal enhancer, can effectively block the inhibitory receptor signaling barrier mediated by SHP (in CAR8).

[0322] To study the effect of using a fourth signal SAP and using the CD8 hinge instead of 2B4 in CAR4, SAP signal enhancers were added to CAR2 and CAR4 to design novel constructs CAR3 and CAR5 ( Figure 3A ). Similar to CAR6 to CAR8 with the addition of SAP (Figure 2), CAR3 can be prepared from CAR2 and CAR5 can be prepared from CAR4 by adding SAP, and the same effect can be achieved. The signal enhancer SAP effectively enhances the signal transduction of CAR3 and CAR5. Compared with CAR2-NK cells, the killing ability, degranulation and IFNg production of CAR3-NK cells were significantly increased, and compared with CAR4-NK cells, the killing ability, degranulation and IFNg production of CAR5-NK cells were also improved ( Figure 3B and 3C ). Although the function of CAR5-NK cells is similar to that of CAR2-NK cells, the function of CAR4-NK cells is still limited, the same phenomenon as shown in Figure 2, which will be discussed later in Figure 6.

[0323] Figures 3A to 3C The following results were confirmed: 1. Unexpectedly, SAP can enhance the killing ability of CAR-NK cells. 2. Unexpectedly, SAP can provide complete signaling to CAR-NK cells to stimulate degranulation and IFNg production. 3. Similarly, 2B4 CAR is not as effective as NKG2D CAR, which will be explained later in Figure 6.

[0324] The above data indicate that SAP-enhanced CAR can provide complete signal transduction of NK cells and effectively improve the function of NK cells. 6.6 Example 6: 2B4-CAR-NK cells have normal natural NKG2D function, and the complete signaling CAR enhances the natural 2B4 signaling

[0325] As shown in Figure 1, we demonstrated that NKG2D CAR (CAR2) NK cells have low expression of constitutive natural NKG2D and have dysfunctional NKG2D. To determine whether the same phenomenon exists in our novel designed CAR NK cells, we compared the NKG2D function of CAR2, CAR4, CAR6 and CAR8 ( Figure 4A Among CAR-expressing NK cells, NKG2D expression was reduced in CAR2-expressing NK cells but normal in other CAR-NK cells. Similarly, 2B4 expression was similar between these CAR-NK cells ( Figure 4B ).

[0326] Duolink PLA analysis between NKG2D and DAP10 showed that, unlike CAR2-NK cells, several novel designed CAR-NK cells had normal recruitment of DAP10 to NKG2D receptors ( Figure 4C ), which indicates that the NKG2D receptor has normal signal transduction in these novel designed CAR-NK cells. Consistently, the NKG2D-mediated 293T-MICA cell killing experiment showed that CAR2 reduced the killing effect, while CAR4, CAR6, and CAR8 improved the killing process. In the 293T-CD48 cell killing experiment, although there was synergy between NKG2D and 2B4 receptors, the killing percentage did not increase, with the exception of CAR8 NK cells; this indicates similar 2B4 function, and only in CAR8 did signal transduction significantly enhanced ( Figure 4D ), which may be because 2B4 receptor signaling has two directions, and inhibitory polarized signaling limits NK cell activation.

[0327] 4A to 4DThe following results were confirmed: 1. NKG2D CAR with natural NKG2D is dysfunctional and has lower NKG2D-DAP10 binding. 2. 2B4-DAP10-CD3Z CAR-NK cells have normal NKG2D function. 3. 2B4 expression in CAR-NK cells is normal, and 2B4 has two guiding functions. 4. Unexpectedly, SAP complete signaling CAR has enhanced activation and killing capabilities.

[0328] In conclusion, our novel designed 2B4 CAR possesses normal and effective natural NKG2D receptor function, and the novel designed 2B4 CAR, especially CAR8 with SAP-enhanced complete signaling NK-CAR, can maintain the multifunctional properties of NK cells during CAR-NK cell therapy. 6.7 Example 7: Complete signaling CAR (csCAR) can effectively overcome the HLA-I inhibitory barrier

[0329] Without being limited to any particular theory, HLA class I (HLA-I) causes NK cells to have an inhibitory response and promotes the "permission" or "education" process, but in tumor immunotherapy, adoptively transferred NK cells must overcome the inhibitory signaling barrier from HLA-I expressed on tumor cells (Long et al. (2013) Controlling natural killer cell responses: integration of signals for activation and inhibition. Annu Rev Immunol. 31: 227-58). Without being limited to any particular theory, the inhibitory receptors of NK cells usually use SHP to inhibit vav1 function and phosphorylate CSK to affect cytoskeletal organization. Without being limited to any particular theory, when binding to pTry at the ITSM tail of SLAMF, SAP and SHP compete, which blocks the binding of SHP, thereby eliminating the inhibitory potential of HLA-I. In the complete signaling CAR (csCAR) construct ( Figure 5A ), SAP and CAR (in CAR3, CAR5, and CAR8) were expressed together, and we then determined whether CAR could overcome inhibitory signals when triggered by antigens on tumor cells.

[0330] Without being limited to any particular theory, HLA-E (major histocompatibility complex, class I, E) belongs to the HLA class I heavy chain paralog, and is the major ligand for the natural killer cell inhibitory receptor CD94 / NKG2A. Without being limited to any particular theory, NKG2A is an inhibitory receptor expressed by NK cells that binds to HLA-E and triggers inhibitory signaling through recruitment of SHP. We established stable expression of HLA-E on 293T-CD19 cells, while NALM6 cells constitutively expressed HLA-E ( Figure 5B Although NKG2D expression is reduced in CAR2 NK cells ( Figure 4B ), but there was no significant difference in NKG2A expression between different CAR-NK cells ( Figure 5C ). However, the interaction between HLA-E and NKG2A provides inhibitory signaling, thereby reducing the killing ability of NK cells, especially the killing ability of NK cells with CAR2, CAR4 or CAR6 ( Figure 5D ).

[0331] Surprisingly, SAP-enhanced CAR provided complete signaling and effectively overcame the HLA-E inhibitory barrier in CAR3, CAR5, and CAR8 NK cells ( Figure 5D ). Here, CAR3 was derived from CAR2 (NKG2D CAR) with the addition of P2A-SAP, CAR5 was derived from CAR4 with the addition of P2A-SAP, and CAR8 was derived from CAR6 with the addition of P2A-SAP. The data showed that SAP-enhanced CARs can enhance killing ability and overcome the inhibitory signaling barrier from inhibitory receptors triggered by HLA-I.

[0332] Unexpectedly, CAR and SAP gene-modified NK cells maintained extremely high function after several weeks of culture using feeder cells ( Figure 5D ), indicating that NK cell inhibition is eliminated only when triggered by tumor antigens, while in the absence of tumor antigens, normal licensing processes can be maintained when in contact with HLA-I on healthy cells. It can be seen that the culture enhancement process, survival and licensing process after in vitro culture and intravenous injection, as well as the very efficient function of NK cells when encountering tumor cells expressing antigens, indicate that our novel NK csCAR design is a very effective solution for cancer immunotherapy.

[0333] FIG. 5A to FIG. 5D The following results were confirmed: 1. The expression of NKG2A was the same in different CAR-NK cells. 2. HLA-E and NKG2A interacted to provide inhibitory signal transduction. 3. SAP-enhanced CAR complete signaling can effectively overcome the HLA-E barrier. 6.8 Example 8: The signaling domains (DAP10 and CD3ζ) of NK-CAR in NK signaling are essential, and DAP10 anchored at the endomembrane is a key factor in enhancing the activation and killing activity of NK cells

[0334] As shown in Figures 2 and 3, although CAR4 and CAR6 can effectively trigger activation signal transduction, they do not have the fourth signal SAP, and the effect is limited. In theory, even in the absence of SAP, the signal is sufficient, at least the effect should reach the level of CAR2. We instruct that NKG2D does not have a signal tail, but recruits DAP10 ligands to deliver signals, and DAP10 is a transmembrane protein with a YxxM motif in its tail. Through YxxM, DAP10 can activate phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathways, resulting in cell survival and proliferation, and PI3K is a plasma membrane-associated lipid kinase group that works on the membrane region (Yang et al. (2019) Targeting PI3K in cancer: mechanisms and advances in clinicaltrials. Mol Cancer. 18 (1): 26; Chen et al. (2020) Research Progress on NK Cell Receptors and Their Signaling Pathways. Mediators Inflamm. 2020: 6437057). In CAR4 and CAR6, DAP10 follows the 2B4 ICD away from the plasma membrane, thereby limiting its function.

[0335] Using CAR4 as a model, we designed CARs with or without CD3ζ or DAP10, and also varied the location of DAP10 so that it was anchored or not in the endoplasmic membrane ( Fig. 6A When comparing NK cells expressing these CARs, we found that CAR activation required CD3ζ and DAP10 ( FIG. 6B to FIG. 6D ). Again, DAP10 is surprisingly position-sensitive. DAP10 anchored at the endoplasmic membrane is a key factor in strongly enhancing activation and killing abilities ( Figure 6Bto D). Essentially, DAP10 is recruited to NKG2D by the NKG2D transmembrane domain to trigger GRB2 and vav1 function. DAP10 anchored at the cell membrane uses YxxM to activate PI3K. Without being limited to any particular theory, when DAP10 is located behind the tail of the 2B4 ICD, it is too far from the cell membrane to function, and therefore, its effect in activating PI3K is reduced. Because PI3K uses ATP and PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) to generate phosphatidylinositol 3,4,5-triphosphate (PIP3) at sites on the intracellular membrane, we adjust the position of DAP10 so that it is anchored at the intracellular membrane, thereby bringing it closer to the substrate and playing a correct role.

[0336] After discovering the critical DAP10 position in CAR4, we compared the DAP10 position effect in CAR4, CAR5, CAR6, and CAR8 ( Fig. 6E ), similar to CAR4, for other CARs, DAP10 anchored at the inner membrane also showed better activation signal transduction ( FIG. 6F to FIG. 6H ). All data confirm that full CAR activation requires DAP10 and / or CD3ζ domains, and when DAP10 is anchored at the endoplasmic membrane following the TM domain, activation signaling in CAR NK cells is significantly improved. Accordingly, the most favorable main chain of csCAR may be ScFv-SLAMF hinge-SLAMF TM-DAP10-SLAMF ICD-CD3Z-P2A-SAP.

[0337] 6A to 6H The following results were confirmed: 1. CD3Z and DAP10 are critical for CAR activation. 2. DAP10 is position sensitive, and DAP10 anchored at the inner membrane is unexpectedly critical for enhancing the good effects of activation and killing. 3. DAP10 at the inner membrane improves CAR signaling, whether it uses or does not use the SAP signal enhancer. 6.9 Example 9: Broader Screening of SLAMF and Non-SLAMF CARs in NK Cell Activation and Killing

[0338] Several SLMAF family genes are expressed on NK cells, but not SLAMF2 (CD48), which does not have an intracellular signaling domain ( Figure 8). A novel NK CAR using the hinge, TM and ICD of other SLAMF family genes was designed as 2B4 CAR (CAR 6 and CAR8), including CD229 (CAR11-12), NTB-A (CAR13-14), CD84 (CAR17-18) and CRACC (CAR15-16), and DAP10 was located in front of the SLAMF ICD, that is, DAP10 was anchored on the inner membrane ( Fig. 7A ).

[0339] When CAR used 2B4, CAR using CD229, NTB-A, CD84 and CRACC was effective in activating NK signaling ( Figure 7B and 7C ), and CAR can be enhanced by SAP, with the exception of CRACC CAR, indicating that these genes can serve as extremely excellent CAR triggers, especially CAR12, 14, and 18 that use SAP as an enhancer to overcome inhibitory signaling barriers.

[0340] However, in CRACC CAR (CAR15-16), the HLA-E-stimulated signal greatly inhibited the function of CAR NK, and even used SAP as a signal enhancer ( Figure 7B and 7C ). Reports show that EAT2 can be recruited to pTyr304 within the ITSM of CRACC, and EAT2 recruits and activates PLCγ. SAP can also be recruited to pTyr304, but with much lower affinity. If EAT2 is missing, SHIP-1 can be recruited to pTyr and mediate inhibitory signal transduction (Campbell et al. (2018) Mechanisms of NK Cell Activation and Clinical Activity of the Therapeutic SLAMF7 Antibody, Elotuzumab in Multiple Myeloma. Front Immunol. 9: 2551). This should be the reason why SAP cannot serve as an effective signal enhancer for CRACC CAR. Therefore, we designed CAR16B, using EAT2 instead of SAP in CAR16. The data show that EAT2 is the correct signal enhancer for CRACC CAR (CAR16B), which can improve NK cell activation and completely overcome the HLA-I inhibitory signal transduction barrier ( Fig.7D and 7E ).

[0341] Without being limited to any particular theory, according to the nature of NK cell signaling, CAR can be fully activated as long as there are three synergistic signals. Therefore, we also used non-SLAMF genes to design CARs, such as CD2 (CAR19-20), CD28H (CAR21-22) and DNAM1 (CAR23-24). In these designs, we still use the natural hinge, TM and ICD of CD2, CD28H or DNAM1 and DAP10ICD with CD3ζ, and DAP10 is also anchored at the cell inner membrane ( Fig. 7A ).

[0342] Surprisingly, we found that CD2 CAR and CH28H CAR designs (CAR21, 23) worked well, but did not require SAP signaling (CAR22, 24) ( Figure 7B and Figure 7C ). Without being limited to any particular theory, considering that CD2 and CD28H do not have ITSM in their tails within the cell, they cannot recruit SAP, and therefore, SAP cannot help them improve activation signal transduction. However, CD2 CAR (CAR19) and CD28H CAR (CAR21) can effectively overcome the inhibitory signal transduction barrier induced by HLA-I.

[0343] DNAM1 CAR is quite special. We designed DNAM1 CAR as CAR23 and CAR24. DNAM1CAR (CAR23) is effective, but the effect is still limited. However, when it is combined with the fourth signal SAP enhancer, the effect is excellent, although it does not belong to SLAMF ( Figure 7B and 7C ). Without being bound by any particular theory, DNAM1 does not belong to the SLAMF family, and its tail, like 2B4, does not have an ITSM, so SAP should not bind to it. Without being bound by any particular theory, although DNAM1 works with LFA-1 (de Andrade et al. (2014) DNAM-1 control of natural killer cells functions through nectin and nectin-like proteins. Immunol Cell Biol. 92(3): 237-44), SAP can help LFA-1 activate Fyn, and Fyn is recruited to the tail of DNAM-1, thereby enhancing the activation signal and fully activating it.

[0344] 7A to 7EThe following results were confirmed: 1. SLAMF family CD229, NTB-A, CD84 CARs are effective and can be enhanced by SAP, while CRACC CAR can be enhanced by EAT2. 2. Non-SLAMF CD2 and CD28H CARs are effective and do not require SAP signaling. 3. DNAM1 CAR is effective and can be enhanced by SAP, although it is not a SLAMF.

[0345] In summary, in addition to 2B4 CAR, we performed a broader screening of SLAMF and non-SLAMF csCARs in NK cells and confirmed a series of excellent CARs, such as CD229 CAR (CAR12), NTB-ACAR (CAR13, CAR14), CRACC CAR (CAR16B), CD84 CAR (CAR18), CD2 CAR (CAR19), CD28H CAR (CAR 21, CAR22) and DNAM-1CAR (CAR24), which can effectively improve NK cell activation and successfully overcome the inhibitory signal barrier induced by HLA-I, thereby promoting the realization of successful anti-tumor immunotherapy. 7. Equivalents

[0346] The polypeptides, polynucleotides, cells, methods and compositions disclosed herein are not limited by the specific embodiments described herein. In fact, in addition to the polypeptides, polynucleotides, cells, methods and compositions described herein, those skilled in the art can implement various changes according to this article and the accompanying drawings. These changes also fall within the scope of protection of the present application.

[0347] Various publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. 8. Sequence

Claims

1. A chimeric antigen receptor, characterized in that Include: (i) an extracellular antigen-binding domain; (ii) a transmembrane domain; and (iii) comprising an intracellular signaling domain derived from the intracellular domain of the first polypeptide, wherein the intracellular domain derived from the first polypeptide is linked to the C-terminus of the transmembrane domain directly or via a peptide linker, and The first polypeptide is DAP10 or DAP12.

2. A chimeric antigen receptor, characterized in that Include: (i) an extracellular antigen-binding domain; (ii) comprises a transmembrane domain derived from the transmembrane domain of a second polypeptide; and (iii) comprising an intracellular signaling domain derived from the intracellular domain of the first polypeptide, wherein the second polypeptide is different from the first polypeptide, and wherein the intracellular domain derived from the first polypeptide is linked directly or via a peptide linker to the C-terminus of the transmembrane domain derived from the second polypeptide, and The first polypeptide is DNAM1.

3. The chimeric antigen receptor of claim 1, wherein the transmembrane domain of the chimeric antigen receptor comprises a transmembrane domain derived from a second polypeptide, wherein the second polypeptide is different from the first polypeptide, and wherein the intracellular domain derived from the first polypeptide is linked directly or via a peptide linker to the C-terminus of the transmembrane domain derived from the second polypeptide.

4. The chimeric antigen receptor of claim 3, wherein the intracellular signaling domain of the chimeric antigen receptor further comprises an intracellular domain derived from a third polypeptide, wherein the third polypeptide is different from the first polypeptide. The chimeric antigen receptor of claim 4 , wherein the second polypeptide and the third polypeptide are the same polypeptide.

6. The chimeric antigen receptor according to any one of claims 1 to 5, wherein the chimeric antigen receptor further comprises a hinge domain between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain.

7. The chimeric antigen receptor of claim 6, wherein the hinge domain of the chimeric antigen receptor comprises a hinge domain derived from the second polypeptide.

8. The chimeric antigen receptor of any one of claims 3 to 7, wherein the second polypeptide or the third polypeptide is a signaling lymphocyte activation molecule family (SLAMF) receptor, CD2, CD28H, DNAM1, CD28, 4-1BB, OX40, KIR-S, NKG2C or NKG2E.

9. The chimeric antigen receptor of claim 8, wherein the SLAMF receptor is SLAMF4 (CD244, 2B4), SLAMF3 (CD229, Ly9), SLAMF6 (CD352, NTB-A, SF2000), SLAMF5 (CD84), SLAMF7 (CD319, CRACC, CS1), SLAMF1 (CD150), SLAMF2 (CD48, FimH), SLAMF8 (CD353) or SLAMF9 (CD84H1, SF2001).

10. The chimeric antigen receptor of claim 8, wherein the KIR-S is KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, or KIR3DS1.

11. The chimeric antigen receptor according to any one of claims 1 to 10, wherein the intracellular signaling domain of the chimeric antigen receptor further comprises an intracellular domain derived from CD3ζ.

12. The chimeric antigen receptor according to any one of claims 1 to 11, wherein the intracellular signaling domain of the chimeric antigen receptor further comprises an intracellular domain derived from a SLAM-associated protein (SAP).

13. The chimeric antigen receptor according to any one of claims 1 to 11, wherein the intracellular signaling domain of the chimeric antigen receptor further comprises an intracellular domain derived from Ewing's sarcoma activated transcript-2 (EAT2).

14. The chimeric antigen receptor according to claim 12 or 13, wherein the intracellular domain derived from CD3ζ is linked directly or via a peptide linker to the intracellular domain derived from SAP or the intracellular domain derived from EAT2.

15. The chimeric antigen receptor according to claim 14, wherein the intracellular domain derived from CD3ζ is linked to the intracellular domain derived from SAP or the intracellular domain derived from EAT2 via P2A, T2A or E2A.

16. The chimeric antigen receptor of any one of claims 1 to 15, wherein the extracellular antigen binding domain of the chimeric antigen receptor comprises a scFv.

17. The chimeric antigen receptor according to any one of claims 1 to 16, wherein the extracellular antigen binding domain of the chimeric antigen receptor binds to a tumor antigen or a fragment thereof.

18. The chimeric antigen receptor of any one of claims 1 and 3 to 17, wherein the first polypeptide is DAP10.

19. The chimeric antigen receptor according to any one of claims 1 and 3 to 18, comprising an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:

20.

20. The chimeric antigen receptor of any one of claims 1 to 19, comprising an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:

86.

21. The chimeric antigen receptor of any one of claims 1 to 19, comprising an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:

87.

22. The chimeric antigen receptor of any one of claims 1 to 19, comprising a chimeric antigen receptor having a residue identical to SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144 or SEQ ID NO:145, or an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144 or SEQ ID NO:

145.

23. The chimeric antigen receptor according to any one of claims 3 to 19, comprising from N-terminus to C-terminus: (i) an extracellular antigen-binding domain; (ii) comprising a transmembrane domain derived from the transmembrane domain of the second polypeptide; (iii) an intracellular signaling domain comprising (iii)-(a) derived from the intracellular domain of DAP10; (iii)-(b) derived from the intracellular domain of CD3ζ; and (iii)-(c) derived from the intracellular domain of SAP or derived from the intracellular domain of EAT2, in, The intracellular signaling domain comprises intracellular domains connected to each other directly or via one or more peptide linkers, and The extracellular antigen-binding domain, the transmembrane domain and the intracellular domain are connected to each other directly or via one or more peptide linkers.

24. An engineered immune cell, characterized in that: Comprising a chimeric antigen receptor according to any one of claims 1 to 23, wherein the engineered immune cell is a NK cell, a NKT cell or a T cell.

25. An engineered immune cell, characterized in that: comprising a chimeric antigen receptor, wherein the chimeric antigen receptor comprises: (i) an extracellular antigen-binding domain; (ii) a transmembrane domain; and (iii) an intracellular signaling domain, and wherein the engineered immune cell further comprises an exogenously introduced polynucleotide encoding an intracellular domain derived from a SLAM-associated protein (SAP), and The engineered immune cells are NK cells, NKT cells or T cells.

26. An engineered immune cell, characterized in that: comprising a chimeric antigen receptor, wherein the chimeric antigen receptor comprises: (i) an extracellular antigen-binding domain; (ii) a transmembrane domain; and (iii) an intracellular signaling domain, and wherein the engineered immune cell further comprises an exogenously introduced polynucleotide encoding an intracellular domain derived from Ewing's sarcoma activated transcript-2 (EAT2), and The engineered immune cells are NK cells, NKT cells or T cells.

27. The engineered immune cell according to claim 25 or 26, wherein the exogenously introduced polynucleotide encodes the chimeric antigen receptor, and wherein the intracellular signaling domain of the chimeric antigen receptor comprises the intracellular domain derived from SAP or the intracellular domain derived from EAT2.

28. The engineered immune cell of any one of claims 25 to 27, wherein the intracellular signaling domain of the chimeric antigen receptor comprises an intracellular domain derived from a first polypeptide, and wherein the first polypeptide is DAP10, DNAM1 or DAP12.

29. The engineered immune cell of claim 28, wherein the intracellular domain of the first polypeptide is linked to the C-terminus of the transmembrane domain directly or via a peptide linker.

30. The engineered immune cell of any one of claims 25 to 29, wherein the transmembrane domain of the chimeric antigen receptor comprises a transmembrane domain derived from a second polypeptide.

31. The engineered immune cell of any one of claims 25 to 30, wherein the intracellular signaling domain of the chimeric antigen receptor comprises an intracellular domain derived from a third polypeptide.

32. The engineered immune cell of any one of claims 25 to 31, wherein the second polypeptide or the third polypeptide is a signaling lymphocyte activation molecule family (SLAMF) receptor, CD2, CD28H, DNAM1, CD28, 4-1BB, OX40, KIR-S, NKG2C or NKG2E.

33. The engineered immune cell of claim 32, wherein the SLAMF receptor is SLAMF4 (CD244, 2B4), SLAMF3 (CD229, Ly9), SLAMF6 (CD352, NTB-A, SF2000), SLAMF5 (CD84), SLAMF7 (CD319, CRACC, CS1), SLAMF1 (CD150), SLAMF2 (CD48, FimH), SLAMF8 (CD353) or SLAMF9 (CD84H1, SF2001).

34. The engineered immune cell of claim 32, wherein the KIR-S is KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, or KIR3DS1.

35. The engineered immune cell of any one of claims 25 to 34, wherein the intracellular signaling domain of the chimeric antigen receptor comprises an intracellular domain derived from CD3ζ.

36. An engineered immune cell according to claim 35, wherein the intracellular signaling domain of the chimeric antigen receptor further comprises an intracellular domain derived from a SLAM-associated protein (SAP) or an intracellular domain derived from EAT2, and wherein the intracellular domain derived from CD3ζ is connected directly or via a peptide linker to the intracellular domain derived from SAP or the intracellular domain derived from EAT2.

37. The engineered immune cell of claim 36, wherein the intracellular domain derived from CD3ζ is linked to the intracellular domain derived from SAP or the intracellular domain derived from EAT2 via P2A, T2A or E2A.

38. The engineered immune cell of any one of claims 25 to 37, wherein the extracellular antigen binding domain of the chimeric antigen receptor comprises a scFv.

39. The engineered immune cell of any one of claims 25 to 38, wherein the extracellular antigen binding domain of the chimeric antigen receptor binds to a tumor antigen or a fragment thereof.

40. The engineered immune cell of any one of claims 25 to 39, wherein the chimeric antigen receptor comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:

20.

41. The engineered immune cell of any one of claims 25 to 40, wherein the chimeric antigen receptor comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:

86.

42. The engineered immune cell of any one of claims 25 to 40, wherein the chimeric antigen receptor comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:

87.

43. The engineered immune cell of any one of claims 25 to 39, wherein the chimeric antigen receptor comprises a chimeric antigen receptor that is SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144 or SEQ ID NO:145 are amino acid sequences that are at least 90%, at least 95% or 100% identical to the amino acid sequences of SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, 44. A polypeptide, characterized in that A chimeric antigen receptor comprising any one of claims 1 to 23.

45. A polypeptide, characterized in that The invention relates to a chimeric antigen receptor and an intracellular domain derived from a SLAM-associated protein (SAP), wherein the chimeric antigen receptor comprises: (i) an extracellular antigen-binding domain; (ii) a transmembrane domain; and (iii) an intracellular signaling domain, and wherein the chimeric antigen receptor is linked to the intracellular domain derived from SAP via a peptide linker.

46. ​​A polypeptide, characterized in that The invention relates to a chimeric antigen receptor and an intracellular domain derived from Ewing's sarcoma activated transcript-2 (EAT2), wherein the chimeric antigen receptor comprises: (i) an extracellular antigen-binding domain; (ii) a transmembrane domain; and (iii) an intracellular signaling domain, and wherein the chimeric antigen receptor is linked to the intracellular domain derived from EAT2 via a peptide linker.

47. The polypeptide of claim 45 or 46, wherein the chimeric antigen receptor is linked to the intracellular domain derived from SAP or the intracellular domain derived from EAT2 via P2A, T2A or E2A.

48. The polypeptide of any one of claims 45 to 47, wherein the polypeptide comprises an amino acid sequence that is at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO: 86 or SEQ ID NO:

87.

49. The polypeptide of any one of claims 44 to 47, wherein the polypeptide comprises a residue identical to SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: : 144, or SEQ ID NO:

145.

50. A polynucleotide, characterized in that It encodes the chimeric antigen receptor according to any one of claims 1 to 23, or it encodes the polypeptide according to any one of claims 44 to 49.

51. A carrier, characterized in that Comprising the polynucleotide of claim 50.

52. A pharmaceutical composition, characterized in that Include: (i) the chimeric antigen receptor of any one of claims 1 to 23, the engineered immune cell of any one of claims 24 to 43, the polypeptide of any one of claims 44 to 49, the polynucleotide of claim 50, or the vector of claim 51; and (ii) a pharmaceutically acceptable excipient.

53. A method of treating a disease or condition in an individual, characterized in that The method comprises administering to the individual an effective amount of the engineered immune cell of any one of claims 24 to 43 or the pharmaceutical composition of claim 52.

54. The method of claim 53, wherein the disease or condition is cancer.

55. A method for engineering immune cells, characterized in that: The method comprises introducing the polynucleotide of claim 50, the vector of claim 51, or the polypeptide of any one of claims 44 to 49 into the immune cell.

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