Engineered immune cells and use thereof

Abstract

The present application relates to engineered immune cells (e.g., NK cells, NKT cells, and T cells), and methods of use thereof. The engineered immune cells show enhanced tumor eradication.

Description

ENGINEERED IMMUNE CELLS AND USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 492,994 filed on March 29, 2023, U.S. Provisional Application No. 63 / 492,992 filed on March 29, 2023, U.S. Provisional Application No. 63 / 492,989 filed on March 29, 2023, and U.S. Provisional Application No. 63 / 492,984 filed on March 29, 2023, each of which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] This application contains a computer readable Sequence Listing which has been submitted in XML file format with this application, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted with this application entitled “14767-002-228_SEQLISTING.xml” was created on March 20, 2024 and is 144,173 bytes in size.1. FIELD

[0003] The present application relates to engineered immune cells (e.g., NK cells, NKT cells, and T cells), and methods of use thereof. The engineered immune cells show enhanced tumor eradication.2. BACKGROUND

[0004] Immunotherapy has become a promising approach to treat cancer. T cells expressing chimeric antigen receptor (CAR) have demonstrated to be effective for liquid tumors, but with very limited effect against 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 essential part in innate immune system and constitute the first defense line against tumor (Campbell et al. (2013) Natural killer cell biology: an update and future directions. J Allergy Clin Immunol. 132(3):536-544). CAR engineered NK cells (CAR-NK) have become more attractive due to the unique biological property of NK cells. Firstly, CAR-NK has higher safety compared to CAR-T, including the lower risk of cytokine release syndrome (CRS) and neurotoxicity, lower risk of graft-versus-host disease (GVHD)(Liu et al. (2020) Use of CAR- Transduced Natural Killer Cells in CD19-Positive Lymphoid Tumors. N Engl 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). Secondly, multifunctionality of CAR-NK can kill target tumor cells inCAR-specific manner. In addition, NK cells exhibit potent cytotoxic activity against tumor cells through many constitutively expressed activating receptors such as NCRs, NKG2D, DNAM-1, activating KIRs (KIR2DS1, KIR2DS4, KIR2DL4), CD16-mediated antibody-dependent cellular cytotoxicity (ADCC), and TRAIL or FasL induced apoptosis processes (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 ADAM 17 as a regulatory checkpoint of CD16A in NK cells and as a potential target for cancer immunotherapy. J Leukoc Biol. 105(6): 1297-1303). Thirdly, CAR-NK cells can provide an off-the-shelf product, eliminating the need for a personalized and patient-specific product. The minimal risk for alloreactivity and GVHD potentially permits allogeneic CAR-NK cells to be procured from various sources, including NK cell line, peripheral blood mononuclear cell (PBMC), and pluripotent stem cell (iPSC)(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] The most of the current NK CARs, including the CAR-NKs investigated in the latest clinical trials, use T cell derived CARs, such as ScFv-CD8a hinge-CD28 TM and ICD-CD3zeta. It has been reported that NK specific CAR is better than T CAR 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] In one aspect, provided herein are engineered immune cells. In certain embodiments, provided herein is an engineered immune cell comprising(i) a chimeric antigen receptor (CAR), comprising:(a) an extracellular antigen binding domain;(b) a transmembrane domain; and(c) an intracellular signaling domain comprising an intracellular domain derived from a mutated CD84;(ii) a first nucleic acid encoding a mutated interleukin-2 (IL-2); and(iii) a second nucleic acid encoding an extracellular domain of transforming growth factor beta receptor II (TGFpRII).

[0008] In certain embodiments, provided herein is an engineered immune cell comprising a chimeric antigen receptor (CAR), comprising:(a) an extracellular antigen binding domain;(b) a transmembrane domain; and(c) an intracellular signaling domain comprising an intracellular domain derived from a mutated CD84, wherein the interaction between the intracellular signaling domain of the CAR and SH2 domaincontaining tyrosine phosphatase 2 (SHP2) is lower than the interaction between the intracellular domain of a second CAR and SHP2, and wherein the second CAR comprises an intracellular signaling domain comprising an intracellular domain derived from a wild-type CD84; or the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 7 and exon 8 are deleted.

[0009] In certain embodiments, provided herein is an engineered immune cell comprising(i) a chimeric antigen receptor (CAR), comprising:(a) an extracellular antigen binding domain;(b) a transmembrane domain; and(c) an intracellular signaling domain, and(ii) an exogenously introduced polynucleotide, wherein the exogenously introduced polynucleotide:(aa) encodes a mutated interleukin-2 (IL-2), wherein the mutated IL-2 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:63; or(bb) comprises a first nucleic acid encoding an extracellular domain of transforming growth factor beta receptor II (TGFpRII); and a second nucleic acid encoding a transmembrane domain derived from CD8a, CD4, CD7 or 2B4.

[0010] In certain embodiments, the mutated IL-2 is membrane-bound or linked to a transmembrane domain of a polypeptide directly, via a peptide linker, or via a hinge domain, optionally wherein the polypeptide is CD8. In certain embodiments, the mutated IL-2 comprises the amino acid sequence of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:63.

[0011] In certain embodiments, the engineered immune cell grows in a media that does not contain cytokine.

[0012] In certain embodiments, the first nucleic acid encoding the mutated IL-2 and the second nucleic acid encoding the extracellular domain of TGFpRII are on the same polynucleotide. In certain embodiments, the first nucleic acid encoding the mutated IL-2 or the second nucleic acid encoding the extracellular domain of TGFpRII is on a polynucleotide comprising a nucleic acid encoding the CAR. In certain embodiments, the first nucleic acid encoding the mutated IL-2 or the second nucleic acidencoding the extracellular domain of TGFpRII is on a polynucleotide comprising a nucleic acid encoding a transmembrane domain. In certain embodiments, the transmembrane domain comprises a transmembrane domain derived from CD8a, CD4, CD7 or 2B4. In certain embodiments, the transmembrane domain comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26. In certain embodiments, the second nucleic acid encoding the extracellular domain of TGFpRII is on a polynucleotide comprising a nucleic acid sequence encoding a signal peptide. In certain embodiments, the signal peptide comprises a CD8 signal peptide, a CD4 signal peptide, a CD7 signal peptide or a 2B4 signal peptide. In certain embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO : 1 , SEQ ID NO : 21 , SEQ ID NO : 23 , or SEQ ID NO : 25.

[0013] 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. In certain embodiments, the hinge domain of the CAR comprises a hinge domain derived from CD84.

[0014] In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain derived from CD84.

[0015] In certain embodiments, the intracellular signaling domain of the CAR further comprises an intracellular domain derived from DAP 10, DAP 12 or DNAM1. In certain embodiments, the intracellular signaling domain of the CAR further comprises an intracellular domain derived from CD3zeta. In certain embodiments, the intracellular signaling domain of the CAR further comprises an intracellular domain derived from SLAM-associated protein (SAP). In certain embodiments, the intracellular signaling domain of the CAR further comprises an intracellular domain derived from Ewing’s sarcoma-activated transcript-2 (EAT2). In certain embodiments, the intracellular domain derived from CD3zeta is linked to the intracellular domain derived from SAP or the intracellular domain derived from EAT2 directly or via a peptide linker. In certain embodiments, the intracellular domain derived from CD3zeta is linked to the intracellular domain derived from SAP or the intracellular domain derived from EAT2 via F2A, P2A, T2A, or E2A.

[0016] In certain embodiments, the engineered immune cell further comprises a second exogenously introduced polynucleotide encoding an intracellular domain derived from SLAM- associated protein (SAP) or Ewing’s sarcoma-activated transcript-2 (EAT2). In certain embodiments, the second exogenously introduced polynucleotide encodes the CAR, and wherein the intracellular signaling domain of the CAR comprises the intracellular domain derived from SAP or the intracellular domain derived from EAT2.

[0017] In certain embodiments, the extracellular antigen binding domain of the CAR comprises an scFv. In certain embodiments, the extracellular antigen binding domain of the CAR binds to a tumor antigen or a fragment thereof.

[0018] In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by one or more exons are deleted. In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 5 and exon 6 are deleted. In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 7 and exon 8 are deleted.

[0019] In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16. In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:6, SEQ ID NO: 106, or SEQ ID NO: 107. In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:69 or SEQ ID NO: 108. In certain embodiments, the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:8, SEQ ID NON, SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO:20. In certain embodiments, the extracellular domain of TGFpRII comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:28.

[0020] In certain embodiments, the engineered immune cell is an NK cell, an NKT cell, a T cell, or a Treg cell.

[0021] In certain embodiments, the CAR, from N-terminus to C-terminus, comprising:(i) an extracellular antigen binding domain;(ii) a transmembrane domain comprising an transmembrane domain derived from CD84;(iii) an intracellular signaling domain comprising(a) an intracellular domain derived from DAP 10;(b) an intracellular domain derived from a mutated CD84, wherein the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 7 and exon 8 are deleted; and(c) an intracellular domain derived from CD3zeta, and wherein the 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.

[0022] In one aspect, provided herein are polypeptides. In certain embodiments, provided herein is a polypeptide comprising(i) a chimeric antigen receptor (CAR), comprising:(a) an extracellular antigen binding domain;(b) a transmembrane domain; and(c) an intracellular signaling domain;(ii) a mutated interleukin-2 (IL-2) comprising an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:63.

[0023] In certain embodiments, provided herein is a polypeptide comprising(i) a chimeric antigen receptor (CAR), comprising:(a) an extracellular antigen binding domain;(b) a transmembrane domain; and(c) an intracellular signaling domain comprising an intracellular domain derived from a mutated CD84;(ii) a mutated interleukin-2 (IL-2); and(iii) an extracellular domain of transforming growth factor beta receptor II (TGFpRII).

[0024] In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16. In certain embodiments, the intracellular domain of the CAR comprises the amino acid sequence of SEQ ID NO: 16. In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:6, SEQ ID NO: 106, or SEQ ID NO: 107. In certain embodiments, the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO:20.

[0025] In certain embodiments, provided herein is a polypeptide comprising(i) an extracellular domain of growth factor beta receptor II (TGFpRII); and(ii) a transmembrane domain of CD8a, CD4, CD7 or 2B4.

[0026] In certain embodiments, the polypeptide comprises a CD8 signal peptide, a CD4 signal peptide, a CD7 signal peptide or a 2B4 signal peptide. In certain embodiments, the polypeptide further comprises a transmembrane domain of CD8a, CD4, CD7 or 2B4. In certain embodiments, the polypeptide further comprises P2A.

[0027] In certain embodiments, the extracellular domain of TGFpRII comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:28.

[0028] In certain embodiments, the polypeptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26. In certain embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO : 1 , SEQ ID NO : 21 , SEQ ID NO : 23 , or SEQ ID NO : 25. In certain embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:67, or SEQ ID NO:68.

[0029] In one aspect, provided herein are polynucleotides. In certain embodiments, provided herein is a polynucleotide encoding the polypeptide provided herein. In another aspect, provided herein are vectors. In certain embodiments, provided herein is a vector comprising the polynucleotide provided herein.

[0030] In one aspect, provided herein are pharmaceutical compositions. In certain embodiments, provided herein is a pharmaceutical composition comprising(i) the engineered immune cell provided herein, the polypeptide provided herein, the polynucleotide provided herein, or the vector provided herein; and(ii) a pharmaceutically acceptable excipient.

[0031] In another aspect, provided herein are methods of treating a disease or disorder in a subject. In certain embodiments, provided herein is a method of treating a disease or disorder in a subject, comprising administering to the subject an effective amount of the engineered immune cell provided herein or the pharmaceutical composition provided herein. In certain embodiments, the disease or disorder is cancer.

[0032] In another aspect, provided herein are methods of engineering an immune cell. In certain embodiments, provided herein is a method of engineering an immune cell, comprising introducing into the immune cell the polypeptide provided herein, the polynucleotide provided herein, or the vector provided herein.

[0033] In yet another aspect, provided herein are methods of culturing an immune cell. In certain embodiments, provided herein is a method of culturing an immune cell comprising culturing the immune cell in a media comprising a mutated interleukin-2 (IL-2), wherein the mutated IL-2 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:47, SEQ ID NO:52, SEQ ID NO:54, or SEQ ID NO:57.

[0034] In certain embodiments, the media does not contain cytokine. In certain embodiments, the mutated IL-2 is soluble.

[0035] In certain embodiments, the immune cell is an NK cell, an NKT cell, a T cell, or a Treg cell.

[0036] In certain embodiments, the mutated IL-2 comprises the amino acid sequence of SEQ ID NO:48, SEQ ID NO:53, SEQ ID NO:55, or SEQ ID NO:58.

[0037] In certain embodiments, the immune cell is an engineered immune cell. In certain embodiments, the engineered immune cell comprises a chimeric antigen receptor (CAR), wherein the CAR comprises:(i) an extracellular antigen binding domain;(ii) a transmembrane domain; and(iii) an intracellular signaling domain.

[0038] 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 transmembranedomain. In certain embodiments, the extracellular antigen binding domain of the CAR comprises an scFv. In certain embodiments, the extracellular antigen binding domain of the CAR binds to a tumor antigen or a fragment thereof.4. BRIEF DESCRIPTION OF THE FIGURES

[0039] FIGs. 1A-1G illustrate that mutated CD84 composed CAR-NK eradicated tumor cells efficiently. FIG. 1A: Designs of CD84-CAR and complete signaling CD84-CAR. CARO is empty vector control, CAR2 is the reported NKG2DTM CAR against CD 19, CAR17 is composed of CD84, and CAR18 is SAP enhanced CAR17, that is complete signaling CAR. FIG. IB: Complete signaling facilitated CD84-CAR-NK eradicating tumor cells efficiently. CD84 CAR-NK cells killed 293T- CD19 (left) and 293T-CD19-HLA-E cell (middle) for 4 h, and killed NALM6 cell (right) for 10 h at the indicated ratios. CD84-CAR (CAR17, CAR18) NK cells had better killing ability compared to NKG2D-CAR (CAR2), and the SAP strengthened complete signaling CD84-CAR (CAR18) was much better in killing against HLA-E expressing target cells. Experiments were performed three times. FIG. 1C: CD84 (SLAMF5) molecule structure and binding property. CD84 is the member of SLAMF 5. The ectodomain contains a tandem of the IgV-IgC2 motifs. In the CD84 cytoplasmic tail, there are two consensus immunoreceptor tyrosine-based switch motifs (ITSM), the first domain (TIY262TYI) recruits SAP and EAT2 via the Y262, the second domain (TVY299SEV) recruits EAT2 and SHP2 via Y299. In addition, Y279 and Y324 can also recruit signaling molecules. In the genome, Exon5 and Exon6 encode the part of first ITSM to the Y279, and the part of the second ITSM to the tail is encoded by Exon7 and Exon8. FIG. ID: Designs of mutated CD84-CAR with or without complete signaling. The designs of CARO, CAR2, CAR17, CAR18 are the same as in A. CAR116 uses CD84A56 ICD, which is deleted the part encoded by exon 5 and exon 6. CAR117 is SAP strengthened CAR116. CAR118 uses CD84A78 ICD, which is deleted the part encoded by exon 7 and exon 8. CAR119 is SAP strengthened CAR118. FIG. IE: With complete signaling dispensable of SAP, CD84A78-CAR overcame inhibitory signaling barrier efficiently. CAR-NK cells killed 293T- CD19 (left) and 293T-CD19-HLA-E cell (middle) for 4 h, and killed NALM6 cell (right) for 10 h at the indicated ratios. CD84-CAR (CAR17, CAR18) NK cells had better killing ability compared to NKG2D-CAR (CAR2), and the SAP strengthened complete signaling CD84-CAR (CAR18) was much better in killing against HLA-E expressing target cells. CD84A56-CAR (CAR116, CAR117) NK cells had impaired killing ability, and CD84A78-CAR (CAR118, CAR119) NK cells had excellent killing ability, and also, CAR118 was better than CAR119 in view of the fact that CD84A78-CAR did not need to express extra SAP to enhance the signaling. Experiments were performed three times. FIG. IF: CD84A78 CAR-NK had superior IFNg production and degranulation. 293T, 293T-CD19 and NALM6 stimulated degranulation and IFNg generation. NKlarget cells were used 1 : 1 , for CD 107a checking, 2pg / ml antibodies was co-cultured at 37°C for 2h, CD 107a+% was determined by FACS, and for IFNg checking, NK cells were stimulated by targets 2 h, then addedGolgiplus and continued to culture for 4 h, IFNg+% was determined by FACS. Experiments were performed three times. FIG. 1G: CD84A78 CAR-NK stimulated by 293T-CD19-HLA-E, SAP-CAR duolink and SHP2-CAR duolink. CARNK cells were stimulated by 293T-CD19-HLA-E for 30 min at 37°C . Duolink PLA assays were performed between SAP with CAR-GFP, and between SHP2 with CAR-GFP. Complete signaling SAP strengthened CAR18 NK cells had more interaction between SAP-CAR, and lower SHP2-CAR binding, compared to CAR17. CD84A56 CAR (CAR116) NK cells had lower SAP-CAR interaction, and normal SHP2-CAR binding. CD84A78 CAR (CAR118) NK cells had excellent SAP-CAR interaction, and also less SHP2-CAR binding, suggesting SHP2 prior to bind to the part encoded by exon7 and exon8. All experiments were performed three times with similar results. And *p<0.05, **p<0.01, ***p<0.001, and ns means no significance.

[0040] FIGs. 2A-2F illustrate that dominant negative TGFpRII without active receptor tail overcame the suppressiveness of TGF-p. FIG. 2A: NK cells expressed TbRI and TbRII on cell surface. Surface TGFpRI (TbRI) and TGFpRII (TbRII) expression on NK cells were analyzed by FACS. Iso, isotype. FIG. 2B: DNTbRII-GFP designs. DNTbRII DNA constructs were designed as shown on top, whereas the bottom table showed the different signal peptides (SP) that were used with the same transmembrane (TM) domain of raw TGFpRII (#1-5), and different TM were used with the same SP of CD8a (#2 and #6-9). FIG. 2C: DNTbRII-GFP expression on NK cells. Dominant negative TGFpRII (DNTbRII) expressed on NK cells were analyzed by FACS. DNTbRII-GFP is fusion protein, and the GFP is at C-terminal. FIG. 2D: DNTbRII-NK neutrolized TGFp-dose dependent immunosuppression of NK killing activity. NK cells were treated with different doses of TGFp for 5 days, NK cells were washed and co-incubated with K562 for 5 h, the killing assay was shown. FIG. 2E: DNTbRII-NK neutralized TGF-P and prevented TGF-P immunosuppression of NK killing activity. NK cells were treated with 5 ng / mL TGF-P or PBS for 5 days, NK cells were co-incubated with K562 for 5 h, and killing assay was shown. FIG. 2F: DNTbRII-NK neutralized TGF-P and prevented TGF-P immunosuppression of NK IFNg production. NK cells were stimulated with K562 (1: 1) for 2 h, after adding Golgiplus, 4 h more culture was performed, and IFNg generation was determined by FACS, the percentage of IFNg+% is shown. FIG.2G: DNTbRII-NK prevented phosphorylation of Smad2 and Smad3 in NK cells stimulated by TGF-p. Wild-type (WT) and DNTbRII NK cells were treated with or without 5ng / ml TGF-P for 5 days, cells were collected, lyzed and directly boiled with laemmli sample buffer. Western blot determination of pSMAD2, pSMAD3, SMAD2, SMAD3, and Actin as internal reference were shown. DNTbRII NK cells had no response to TGF-p. All experiments were performed three times with similar results. *p<0.05, **p<0.0I, ***p<0.00I.

[0041] FIGs. 3A-3H illustrate that membrane-bound mutated IL-2s (“safteins”) supported NK cells renewal. FIG. 3A: mbIL-2, mbIL-15 and mbIL-7, but not mbIL-18 nor mbIL-21 supported NK cell renewal. Membrane-bound cytokines IL-2, IL-15, IL-7, IL-18 or IL-21 were designed as CD8 signal peptide (SP)-mature cytokines-CD8 transmembrane (TM) (top). Membrane-bound cytokineexpressed NK cells were cultured and withdrew cytokines in media. 3 days later, FACS analysis was determined as shown. mbIL-7 NK cells survived and grew a few, mbIL-15 NK cells grew better, and mbIL-2 NK cells grew the best (bottom). FIG. 3B: mbIL-2 enhanced NK cell killing ability. mbIL-2 and mbIL-15 NK cells were co-incubated with K562 at different ratios for 6 h (left), or NK:K562 at 1:20 for 20h (right). The NK cell killing ability was shown. FIG. 3C: IL-2 mediated differential immune effects when binding to different IL-2 receptors The intermediate-affinity dimeric IL-2 receptor (IL-2R) comprised P chain and common y chain on effector T or NK cells, the IL-2 binding stimulated immune response; and the high-affinity trimeric IL-2R comprised a, and y chains on Treg cells and endothelial cells, IL-2 binding stimulated immune suppression and triggered vascular leak syndrome. FIG. 3D: Amino acids of IL-2 facing to IL-2Ra. IL-2’s interaction with receptor subsets of a, P and y were based on the structure of IL-2 and IL-2R. Amino acids of IL-2 facing to IL- 2Ra were with property of hydrophobic (F42, F44, Y45, P65, L72), and polar periphery (K35, R38, K43, E61, E62). FIG. 3E: Designs of saftein. To prevent binding of IL-2 to IL-2Ra, membrane-bound IL-2 mutants were designed. Saftein 0 is the raw IL-2, Saftein 1-14 are different IL-2 mutants as shown. The Saftein 6 and Saftein 7 were designed based on reports. FIG. 3F: Saftein supported NK growth. Membrane-bound saftein expressed NK cells were cultured without cytokine addition in the media. Live NK cell growth were calculated at day 1, day 3 and day 10 (top). And the day 10 NK cell growth showed in the bottom. Saftein 0, Saftein 2, Saftein 6, Saftein 7, Saftein 9, Saftein 11, Saftein 12 and Saftein 14 supported NK renewal and growth. FIG. 3G: Saftein supported the growth of T cells. Membrane-bound saftein expressed NK cells were co-cultured with T cells without cytokine, and CD4+ T cell number was calculated at day 0, day 5 and day 7. Saftein 0, Saftein 2, Saftein 6, Saftein 9, Saftein 11, Saftein 14 can support CD4+ T cell growth by bystander effect, and T cells plus rhIL-2 was used as positive control. FIG. 3H: Saftein supported the growth of Treg cells. Membranebound saftein expressed NK cells were co-cultured with T cells without cytokine, and Treg cell number was calculated at different time as shown in the left, and the Treg number fold change at day 10 was shown in the right. Saftein 0, Saftein 6, Saftein 9, Saftein 11, and Saftein 14 could enhance Treg growth, and Saftein 2, Saftein 6, Saftein 7, and Saftein 12 could not, T cell plus rhIL-2 was used as positive control. All experiments were performed three times with similar results. *p<0.05, **p<0.0I, ***p<0.00I.

[0042] FIGs. 4A-4E illustrate that selected soluble safteins supported NK, T, Treg cell growth. FIG. 4A: Soluble safteins expression in coomassie blue staining gel. Soluble safteins with 6xHIS were produced via 293T secretion, and purified by Ni-NTA beads. The purified saftein proteins were mixed with laemmli, boiled and dissolved in SDS-PAGE gel. Coomassie blue staining was performed as shown. FIG. 4B: pSTAT5 was induced by Saftein binding. NK cells were cultured without cytokines for 24 h, soluble safteins or rhIL-2 were added to the culture media, and 30 min later, pSTAT5 were treated and analyzed by phosflow method. pSTAT5 expression in different treated NK cells was shown. pSTAT5 was activated in Saftein 0, Saftein 2, Saftein 6, Saftein 9 treated NK cells, Saftein 7was not effective to activate pSTAT5 in NK cells, and rhIL-2 was used as positive control. FIG 4C: Soluble safteins supported NK growth. Soluble safteins were used to replace cytokines in NK cell culture media. Media only was used as negative control, and rhIL-2 was used as positive control. NK cell number was calculated at day 1, day 5 and day 10. All safteins and rhIL-2 supported NK cell growth, and soluble Saftein 2, and Saftein 9 were the best NK cell growth stimulators, which were more effective compared to Saftein 0 or rhIL-2. But Saftein 6 and Saftein 7 were not as good as Saftein 0 or rhIL-2, and had lower effects on NK cell growth. FIG. 4D: Soluble safteins supported T cell growth. Soluble safteins and rhIL-2 were used to culture T cells. Media only was used as negative control, and rhIL-2 were used as positive control to culture T cells. CD4+ T cell (left) or CD8+ T cell (right) number were calculated at day 1, day 5, and day 10. Similar as in FIG. 4C above to culture NK cells, Saftein 2 and Saftein 9 were the best T cell growth stimulators, and Saftein 6 and Saftein 7 had lower effects on T cell growth, compared to Saftein 0 or rhIL-2. FIG. 4E: Soluble safteins supported Treg cell growth. Soluble safteins and rhIL-2 were used to culture Treg cells. Media only was used as negative control, and rhIL-2 were used as positive control to culture Treg cells. At day 10, Treg cell number was calculated. Saftein 0, Saftein 9 and rhIL-2 effectively stimulated Treg growth, and Saftein 6 also stimulated Treg growth, but with a low level. All experiments were performed three times with similar results. **p<0.0I, ***p<0.00I.

[0043] FIGs. 5A-5I illustrate that Saftein and DNTbRII expressing complete signaling CAR-NK cells eliminated tumor effectively. FIG. 5 A: Design of sd-csCAR-reNK system: CD8aSP-CARl 18- P2A-CD8aSP-DNTbRII-(G3S)3 spacer-Saftein2-CD8aTM. Tumor cells produced TGF-P and suppressed NK cells. DNTbRII bound to TGF-p, but had no signaling to the cell, which means it neutralized the TGF-p. Saftein 2 supported the NK cell survival and growth. The complete signaling CAR bound to the tumor cells. The combination of the three parts expressed on NK cells eliminated tumor cell efficiently. FIG. 5B: sd-csCAR-reNK cell phenotypes. CAR expressed NK cells were analyzed by FACS. NK cell surface phenotypes such as NKG2D, NKG2A, NKp46, NKp44, 2B4 and CD94 were shown. There was no significant changes of phenotype between CAR0-NK, CAR118-NK, and sd-csCAR-reNK. FIG. 5C: sd-csCAR-reNK cells functioned better in short prime, and antagonized the suppressiveness of TGF-p. Sd-csCAR-reNK cells were cultured with or without 5ng / mL TGF-P for 5 days, and co-cultured with target NALM6 cells for 10 h. The killing ability of NK cells without TGF-P treated was shown in the top left, and the killing ability of NK cells treated with TGF-P was shown in the top right. For IFNg production assay (bottom), NK cells were primed by NALM6 for 2 h, and 4 h more culture after Golgiplus treatment. IFNg intracellular generation assay was performed by FACS. FIG. 5D: Tumor cells produced TGF-p. 293T-CD19-HLA-E, NALM6 and HUH7-HLA-E cells were stained with anti -TGF-P antibody, washed, fixed and intracellular stained again for anti-TGF-p. The representative FACS data were shown. FIG. 5E: sd- csCAR-reNK cells antagonized the suppressiveness of TGF-p. CAR-NK cells were co-incubated with target cells (1: 100). 3 days later, LDH assay was performed, the LDH OD value was shown. TheCAR-NK cells with Saftein2 killed target tumor effectively, and DNTbRII facilitated NK cell killing ability more effectively. FIG. 5F: sd-csCAR-reNK supported cell survival. CAR-NK cells as indicated were cultured without cytokine for 3 days, and the day 0 and day 3 NK cells were stained with CellEvent cleaved caspase3 / 7 green at 37 °C for 30 min, the FACS analysis were performed and the representative data were shown in the top, and accumulated data were shown in the bottom. Data showed that Saftein2 supported NK cell survival effectively. FIG. 5G: sd-csCAR-reNK supported cell renewal. CAR-NK cells as indicated were cultured and withdrew cytokine, cells were stained with CellTrace Violet (CTV) and analyzed by FACS at day 0, and day 3. The representative data were shown. FIGs. 5H and 51: NK cell number calculation cultured using no cytokine media. CAR-NK cells as indicated were cultured without cytokine and live cells were calculated at different days (FIG. 5H), and the day 7 NK cell number was shown in FIG. 51. Data showed that saftein2 could support NK cell survival effectively. All experiments were performed three times. *p<0.05, **p<0.0I, ***p<0.0015. DETAILED DESCRIPTION OF THE INVENTION

[0044] Provided herein are CARs, engineered immune cells (e.g., NK cells, NKT cells, and T cells), related polypeptides, related polynucleotides and vectors, related pharmaceutical compositions, related methods of treating a disease, and related methods of engineering an immune cells (e.g., NK cells, NKT cells, and T cells). In one aspect, provided herein is a CAR (see Section 5.2) comprising an extracellular antigen binding domain (see Section 5.2.3), an transmembrane domain (see Section5.2.4), an intracellular signaling domain (see Section 5.2.5), optionally a hinge domain (see Section5.2.5) and optionally one or more peptide linkers (see Section 5.2.7). In another aspect, the application relates to a mutated interleukin-2 (IL-2) (see Section 5.3). In certain embodiments, provided herein is an engineered immune cell comprising an exogenously introduced polynucleotide encoding a mutated IL-2 (see Section 5.3.2). In certain embodiments, provided herein is a polypeptide comprising an amino acid sequence of a mutated IL-2 (see Section 5.3.3). In certain embodiments, provided herein is a method of culturing an immune cell (see Section 5.3.4). In yet another aspect, the application relates to TGFpRII (see Section 5.4). In certain embodiments, provided herein is an engineered immune cell comprising an exogenously introduced polynucleotide comprises a first nucleic acid encoding an extracellular domain of TGFpRII and a second nucleic acid encoding a transmembrane domain (see Section 5.4.2). In certain embodiments, provided herein is a polypeptide comprising an extracellular domain of TGFpRII and a transmembrane domain (see Section 5.4.3). In yet another aspect, provided herein are engineered immune cells comprising a CAR, a first nucleic acid encoding a mutated IL-2, and a second nucleic acid encoding an extracellular domain of TGFpRII (see Section 5.5). In certain embodiments, provided herein is a polypeptide comprising a CAR, a mutated IL-2 and an extracellular domain of TGFpRII (see Section 5.5.3). In certain embodiments, provided herein is a polynucleotide (see Section 5.6). In certain embodiments, provided herein is a vector (see Section5.6). In certain embodiments, provided herein is a pharmaceutical composition comprising (i) the CAR provided in Section 5.2, the engineered immune cell provided herein, the polypeptide provided herein, the polynucleotide provided in Section 5.6, or the vector provided in Section 5.6; and (ii) a pharmaceutically acceptable excipient (see Section 5.7). In certain embodiments, provided herein is a therapeutic method or use related to the engineered immune cell (see Section 5.8). In certain embodiments, provided herein is a method of engineering an immune cell (see Section 5.9). In certain embodiments, provided herein are exemplary assays that can be used to make, use and measure the compositions provided herein (see Section 5.10).

[0045] When an engineered immune cell comprises (i) a CAR comprising an intracellular signaling domain comprising an intracellular domain derived from a mutated CD84 (e.g., CD84A7,8), (ii) a first nucleic acid encoding a mutated interleukin-2 (IL-2), and (iii) a second nucleic acid encoding an extracellular domain of transforming growth factor beta receptor II (TGF[3RII), the engineered immune cell showed superior CAR activation and cell sustenance and renewal. Moreover, the engineered immune cell does not need any cytokine to support the survival and growth, and the engineered immune cell effectively antagonizes the suppression by TGF-J3, resulting in enhanced tumor eradication activity. In other words, the combination of a CAR comprising an intracellular domain derived from a mutated CD84 (e.g., CD84A7,8), a mutated IL-2 (e.g., IL-2 A42-45) and DNTbRII in an engineered immune cell displayed unexpected and superior effect in, for example, CAR activation and cell sustenance and renewal, long-term-no-cytokine-supported cell survival and renewal, and antagonized inhibition by TGF-J3. These superior results suggested that the engineered immune cell can be used to treat cancer (e.g., solid tumor) efficiently.5.1 Definitions

[0046] Techniques and procedures described or referenced herein include those that are generally well understood and / or commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3d ed. 2001); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010); and Antibody Engineering Vols 1 and 2 (Kontermann and Diibel eds., 2d ed. 2010). Unless otherwise defined herein, technical and scientific terms used in the present description have the meanings that are commonly understood by those of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any description of a term set forth conflicts with any document incorporated herein by reference, the description of the term set forth below shall control.

[0047] The term “antibody,” “immunoglobulin,” or “Ig” is used interchangeably herein, and is used in the broadest sense and specifically covers, for example, monoclonal antibodies (includingagonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity), formed from at least two intact antibodies, single chain antibodies, and fragments thereof (e.g., domain antibodies), as described below. An antibody can be human, humanized, chimeric and / or affinity matured, as well as an antibody from other species, for example, mouse, rabbit, llama, etc. The term “antibody” is intended to include a polypeptide product of B cells within the immunoglobulin class of polypeptides that is able to bind to a specific molecular antigen and is composed of two identical pairs of polypeptide chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids, and each carboxy-terminal portion of each chain includes a constant region. See, e.g., Antibody Engineering (Borrebaeck ed., 2d ed. 1995); and Kuby, Immunology (3d ed. 1997). Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, single domain antibodies including from Camelidae species (e.g. , llama or alpaca) or their humanized variants, intrabodies, anti-idiotypic (anti- id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above, which refers to 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 was 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, diabody, triabody, tetrabody, and minibody. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, for example, antigen-binding domains or molecules that contain an antigen-binding site that binds to an antigen (e.g., one or more CDRs of an antibody). Such antibody fragments can be found in, for example, 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; Pltickthun and Skerra, 1989, Meth. Enzymol. 178:497-515; and Day, Advanced Immunochemistry (2d ed. 1990). The antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) of immunoglobulin molecule. Antibodies may be agonistic antibodies or antagonistic antibodies. Antibodies may be neither agonistic nor antagonistic.

[0048] An “antigen” is a structure to which an antibody can selectively bind. A target antigen may 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 in a cell.

[0049] An “intact” antibody is one comprising an antigen-binding site as well as a CL and at least heavy chain constant regions, CHI, CH2 and CH3. The constant regions may include humanconstant regions or amino acid sequence variants thereof. In certain embodiments, an intact antibody has one or more effector functions.

[0050] “Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the VH and VL antibody domains connected into 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 the sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0051] “Single domain antibody” or “sdAb” as used herein refers to a single monomeric variable antibody domain and which is capable of antigen binding (e.g., single domain antibodies that bind to CD 19). Single domain antibodies include VHH domains as described herein. Examples of single domain antibodies include, but are not limited to, antibodies naturally devoid of light chains such as those from Camelidae species (e.g., llama), single domain antibodies derived from conventional 4- chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit, and bovine. For example, a single domain antibody can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco, as described herein. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; VHHs derived from such other species are within the scope of the disclosure. In some embodiments, the single domain antibody (e g., VHH) provided herein has a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Single domain antibodies may be genetically fused or chemically conjugated to another molecule (e.g., an agent) as described herein. Single domain antibodies may be part of a bigger binding molecule (e.g., a multispecific antibody or a chimeric antigen receptor).

[0052] The terms “binds” or “binding” refer to an interaction between molecules including, for example, to form a complex. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non-covalent interactions between a single antigen-binding site on an antibody and a single epitope of a target molecule, such as an antigen, is the affinity of the antibody or functional fragment forthat epitope. The ratio of dissociation rate (koff) to association rate (kon) of a binding molecule (e.g., an antibody) to a monovalent antigen (koff / kon) is the dissociation constant KD, which is inversely related to affinity. The lower the KD value, the higher the affinity of the antibody. The value of KD varies for different complexes of antibody and antigen and depends on both konand kOff. The dissociation constant KDfor an antibody provided herein can be determined using any method provided herein or any other method well known to those skilled in the art. The affinity at one binding site does not always reflect the true strength of the interaction betweenan antibody and an antigen. When complex antigens containing multiple, repeating antigenic determinants, such as a polyvalent antigen, come in contact with antibodies containing multiple binding sites, the interaction of antibody with antigen at one site will increase the probability of a reaction at a second site. The strength of such multiple interactions between a multivalent antibody and antigen is called the avidity.

[0053] In connection with the binding molecules described herein terms such as “bind to,” “that specifically bind to,” and analogous terms are also used interchangeably herein and refer to binding molecules of antigen binding domains that specifically bind to an antigen, such as a polypeptide. A binding molecule or antigen binding domain that binds to or specifically binds to an antigen can be identified, for example, by immunoassays, Octet®, Biacore®, or other techniques known to those of skill in the art. In some embodiments, a binding molecule or antigen binding domain binds to or specifically binds to an antigen when it binds to an antigen with higher affinity than to any cross- reactive antigen as determined using experimental techniques, such as radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA). Typically, a specific or selective reaction is at least twice background signal or noise and may be more than 10 times background. See, e.g., Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989) for a discussion regarding binding specificity. In certain embodiments, the extent 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 particular target antigen, for example, as determined by fluorescence activated cell sorting (FACS) analysis or RIA. A binding molecule or antigen binding domain that binds to an antigen includes one that is capable of binding the antigen with sufficient affinity such that the binding molecule is useful, for example, as a therapeutic and / or diagnostic agent in targeting the antigen. In certain embodiments, a binding molecule or antigen binding domain that binds to an antigen has a dissociation constant (KD) of less than or equal to IpM, 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. In certain embodiments, a binding molecule or antigen binding domain binds to an epitope of an antigen that is conserved among the antigen from different species.

[0054] In certain embodiments, the binding molecules or antigen binding domains can comprise “chimeric” sequences in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit 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 include humanized sequences.

[0055] In certain embodiments, the binding molecules or antigen binding domains can comprise portions of “humanized” forms of nonhuman (e.g., camelid, murine, non-human primate) antibodiesthat include sequences from human immunoglobulins (e.g., recipient antibody) in which the native CDR residues are replaced by residues from the corresponding CDR of a nonhuman species (e.g., donor antibody) such as camelid, mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, one or more FR region residues of the human immunoglobulin sequences are replaced by corresponding nonhuman residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. A humanized antibody heavy or light chain can comprise substantially all of at least one or more variable regions, in which all or substantially all of the CDRs correspond to those of a nonhuman immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. In certain embodiments, the humanized antibody comprise at least a portion of an immunoglobulin constant region (Fc), typically that of 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.

[0056] In certain embodiments, the binding molecules or antigen binding domains can comprise portions of a “fully human antibody” or “human antibody,” wherein the terms are used interchangeably herein and refer to an antibody that comprises a human variable region and, for example, a human constant region. The binding molecules may comprise a single domain antibody sequence. In specific embodiments, the terms refer to an antibody that comprises a variable region and constant region of human origin. “Fully human” antibodies, in certain embodiments, can also encompass antibodies which bind polypeptides and are encoded by nucleic acid sequences which are naturally occurring somatic variants of human germline immunoglobulin nucleic acid sequence. The term “fully human antibody” includes 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, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). A “human antibody” is one that possesses an amino acid sequence which corresponds to that of an antibody produced by a human and / or has been made using any of the techniques for making human antibodies. This definition of a human antibody specifically excludes a humanized antibody 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)). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy 77 (1985); Boemer et a / ., J. Immunol. 147(l):86-95 (1991); and van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001). Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodiesin response to antigenic challenge, but whose endogenous loci have been disabled, e.g., mice (see, e.g., Jakobovits, Curr. Opin. Biotechnol. 6(5):561-66 (1995); Briiggemann and Taussing, Curr. Opin. Biotechnol. 8(4):455-58 (1997); and U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also, for example, Li et al., Proc. Natl. Acad. Sci. USA 103:3557- 62 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.

[0057] In certain embodiments, the binding molecules or antigen binding domains can comprise portions of a “recombinant human antibody,” wherein the phrase includes human antibodies that are prepared, expressed, created or isolated by recombinant means, 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., a mouse or cow) that is transgenic and / or transchromosomal for human immunoglobulin genes (see, e.g., Taylor, L. D. et al., Nucl. Acids Res. 20:6287-6295 (1992)) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies can have variable and constant regions derived from human germline immunoglobulin sequences (See Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0058] In certain embodiments, the binding molecules or antigen binding domains can comprise a portion of a “monoclonal antibody,” wherein the term as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts or well-known post-translational modifications such as amino acid iomerizatio or deamidation, methionine oxidation or asparagine or glutamine deamidation, each monoclonal antibody typically recognize a single epitope on the antigen. In specific embodiments, a “monoclonal antibody,” as used herein, is an antibody produced by a single hybridoma or other cell. The term “monoclonal” is not limited to any particular method for making the antibody. For example, the monoclonal antibodies useful in the present disclosure may be prepared by the hybridoma methodology first described by Kohler et al. , Nature 256:495 (1975), or may be made using recombinant DNA methods in bacterial or eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-28 (1991) and Marks et al., J. Mol. Biol. 222:581-97 (1991), for example. Other methods for the preparation of clonal cell lines and ofmonoclonal antibodies expressed thereby are well known in the art. See, e.g., Short Protocols in Molecular Biology (Ausubel et al. eds., 5th ed. 2002).

[0059] A typical 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the a and y chains and four CH domains for p and a isotypes. Each L chain has at the N- terminus, a variable domain (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain of the heavy chain (CHI). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, for example, Basic and Clinical Immunology 71 (Stites et al. eds., 8th ed. 1994); and Immunobiology (Janeway et al. eds., 5thed. 2001).

[0060] The term “Fab” or “Fab region” refers to an antibody region that binds to antigens. A conventional IgG usually comprises two Fab regions, each residing on one of the two arms of the Y- shaped IgG structure. Each Fab region is typically composed of one variable region and one constant region of each of the heavy and the light chain. More specifically, the variable region and the constant region of the heavy chain in a Fab region are VH and CHI regions, and the variable region and the constant region of the light chain in a Fab region are VL and CL regions. The VH, CHI, VL, and CL in a Fab region can be arranged in various ways to confer an antigen binding capability according to the present disclosure. For example, VH and CHI regions can be on one polypeptide, and VL and CL regions can be on a separate polypeptide, similarly to a Fab region of a conventional IgG. Alternatively, VH, CHI, VL and CL regions can all be on the same polypeptide and oriented in different orders as described in more detail the sections below.

[0061] The term “variable region,” “variable domain,” “V region,” or “V domain” refers to a portion of the light or heavy chains of an antibody that is generally located at the amino-terminal of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and are used in the binding and specificity of each particular antibody for its particular antigen. The variable region of the heavy chain may be referred to as “VH.” The variable region of the light chain may be referred to as “VL.” The term “variable” refers to the fact that certain segments of the variable regions differ extensively in sequence among antibodies. The V region mediates antigen binding and defines 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 regions. Instead, the V regions consist of less variable (e.g., relatively invariant) stretchescalled framework regions (FRs) of about 15-30 amino acids separated by shorter regions of greater variability (e.g., extreme variability) called “hypervariable regions” that are each about 9-12 amino acids long. The variable regions of heavy and light chains each comprise four FRs, largely adopting a P sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases form part of, the sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed. 1991)). The constant regions are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). The variable regions differ extensively in sequence between different antibodies. In specific embodiments, the variable region is a human variable region.

[0062] The term “variable region residue numbering according to Kabat” or “amino acid position numbering as in Kabat”, and variations thereof, refer to the numbering system used for heavy chain variable regions or light chain variable regions of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, an FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 and three inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence. The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., supra). The “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al. , supra). The “EU index as in Kabat” refers to the residue numbering of the human IgG 1 EU antibody. Other numbering systems have been described, for example, by AbM, Chothia, Contact, IMGT, and AHon.

[0063] The term “heavy chain” when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids, and a carboxy-terminal portion includes a constant region. The constant region can be one of five distinct types, (e.g., isotypes) referred to as alpha (a), delta (5), epsilon (a), gamma (y), and mu (p), based on the amino acid sequence of the heavy chain constant region. The distinct heavy chains differ in size: a, 5, and y contain approximately 450 amino acids, while p and 8 contain approximately 550 amino acids. When combined with a light chain, these distinct types of heavy chains give rise to five well known classes (e.g., isotypes) of antibodies, IgA, IgD, IgE, IgG, and IgM, respectively, including four subclasses of IgG, namely IgGl, IgG2, IgG3, and IgG4.

[0064] The term “light chain” when used in reference to an antibody refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids, and a carboxy-terminal portion includes a constant region. The approximate length of a light chain is 211 to 217 amino acids. There are two distinct types, referred to as kappa (K) or lambda (X) based on the amino acid sequence of the constant domains.

[0065] As used herein, the terms “hypervariable region,” “HVR,” “Complementarity Determining Region,” and “CDR” are used interchangeably. A “CDR” refers to one of three hypervariable regions (Hl, H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH [3-sheet framework, or one of three hypervariable regions (LI, L2 or L3) within the non-framework region of the antibody VL [3-sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences.

[0066] CDR regions are well known to those skilled in the art and have been defined by well- known numbering systems. For example, the Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (see, e.g., Kabat et al., supra). Chothia refers instead to the location of the structural loops (see, e.g., Chothia and Lesk, J. Mol. Biol. 196:901-17 (1987)). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the 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 regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software (see, e.g., Antibody Engineering Vol. 2 (Kontermann and Diibel eds., 2d ed. 2010)). The “contact” hypervariable regions are based on an analysis of the available complex crystal structures. Another universal numbering system that has been developed and widely adopted is ImMunoGeneTics (IMGT) Information System® (Lafranc et al., Dev. Comp. Immunol. 27(l):55-77 (2003)). IMGT is an integrated information system specializing in immunoglobulins (IG), T-cell receptors (TCR), and major histocompatibility complex (MHC) of human and other vertebrates. Herein, the CDRs are referred to in terms of both the amino acid sequence and the location within the light or heavy chain. As the “location” of the CDRs within the structure of the immunoglobulin variable domain is conserved between species and present in structures called loops, by using numbering systems that align variable domain sequences according to structural features, CDR and framework residues are readily identified. This information can be used in grafting and replacement of CDR residues from immunoglobulins of one species into an acceptor framework from, typically, a human antibody. An additional numbering system (AHon) has been developed by Honegger and Pltickthun, J. Mol. Biol. 309: 657-70 (2001). Correspondence between the numbering system, including, for example, the Kabat numbering and the IMGT unique numbering system, is well known to one skilled in the art (see, e.g., Kabat, supra,' Chothia and Lesk, supra,' Martin, supra,' Lefranc etal., supra). The residues from each of these hypervariable regions or CDRs are exemplified in Table 1 below.Table 1. Exemplary CDRs According to Various Numbering Systems

[0067] The boundaries of a given CDR may vary depending on the scheme used for identification. Thus, unless otherwise specified, the terms “CDR” and “complementary determining region” of a given antibody or region thereof, such as a variable region, as well as individual CDRs (e.g., CDR-H1, CDR-H2) of the antibody or region thereof, should be understood to encompass the complementary determining region as defined by any of the known schemes described herein above. In some instances, the scheme for identification of a particular CDR or CDRs is specified, such as the CDR as defined by the IMGT, Kabat, Chothia, or Contact method. In other cases, the particular amino acid sequence of a CDR is given. It should be noted CDR regions may also be defined by a combination of various numbering systems, e.g., a combination of Kabat and Chothia numbering systems, or a combination of Kabat and IMGT numbering systems. Therefore, the term such as “a CDR as set forth in a specific VH or VHH” includes any CDR1 as defined by the exemplary CDR numbering systems described above, but is not limited thereby. Once a variable region (e.g., a VHH, VH or VL) is given, those skilled in the art would understand that CDRs within the region can be defined by different numbering systems or combinations thereof.

[0068] Hypervariable regions may comprise “extended hypervariable regions” as follows: 24-36 or 24-34 (LI), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 or 26-35A (Hl), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in the VH.

[0069] The term “constant region” or “constant domain” refers to a carboxy terminal portion of the light and heavy chain which is not directly involved in binding of the antibody to antigen but exhibits various effector function, such as interaction with the Fc receptor. The term refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to theother portion of the immunoglobulin, the variable region, which contains the antigen binding site. The constant region may contain the CHI, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0070] The term “framework” or “FR” refers to those variable region residues flanking the CDRs. FR residues are present, for example, in chimeric, humanized, human, domain antibodies (e.g., single domain antibodies), diabodies, linear antibodies, and bispecific antibodies. FR residues are those variable domain residues other than the hypervariable region residues or CDR residues.

[0071] The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is often defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue. A “functional Fc region” possesses an “effector function” of a native sequence Fc region. Exemplary “effector functions” include Clq binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor), etc. Such effector functions generally require the Fc region to be combined with a binding region or binding domain (e.g., an antibody variable region or domain) and can be assessed using various assays known to those skilled in the art. A “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification (e.g., substituting, addition, or deletion). In certain embodiments, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, for example, from about one to about ten amino acid substitutions, or from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of a parent polypeptide. The variant Fc region herein can possess at least about 80% homology with a native sequence Fc region and / or with an Fc region of a parent polypeptide, or at least about 90% homology therewith, for example, at least about 95% homology therewith.

[0072] As used herein, an “epitope” is a term in the art and refers to a localized region of an antigen to which a binding molecule (e.g., an antibody comprising a single domain antibody sequence) can specifically bind. An epitope can be a linear epitope or a conformational, non-linear, or discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope can be contiguous amino acids of the polypeptide (a “linear” epitope) or an epitope can comprise amino acids from two or more non-contiguous regions of the polypeptide (a “conformational,” “non-linear” or“discontinuous” epitope). It will be appreciated by one of skill in the art that, in general, a linear epitope may or may not be dependent on secondary, tertiary, or quaternary structure. For example, in some embodiments, a binding molecule binds to a group of amino acids regardless of whether they are folded in a natural three dimensional protein structure. In other embodiments, a binding molecule requires amino acid residues making up the epitope to exhibit a particular conformation (e.g., bend, twist, turn or fold) in order to recognize and bind the epitope.

[0073] A “blocking” antibody or an “antagonist” antibody is one that inhibits or reduces a biological activity of the antigen it binds. In some embodiments, blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0074] An “agonist” or activating antibody is one that enhances or initiates signaling by the antigen to which it binds. In some embodiments, agonist antibodies cause or activate signaling without the presence of the natural ligand.

[0075] ‘Percent (%) amino acid sequence identity” and “homology” with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0076] ‘Chimeric antigen receptor” or “CAR” as used herein refers to genetically engineered receptors, which can be used to graft one or more antigen specificity onto immune effector cells, such as NK cells, NKT cells, and T cells. Some CARs are also known as “artificial T-cell receptors,” “chimeric T cell receptors,” or “chimeric immune receptors.” In some embodiments, the CAR comprises an extracellular antigen binding domain specific for one or more antigens (such as tumor antigens), a transmembrane domain, and an intracellular signaling domain of a T cell, NKT cell, or NK cell and / or other receptors. “CAR-T cell” refers to a T cell that expresses a CAR. “CAR-NKT cell” refers to an NK cell that expresses a CAR. “CAR-NK cell” refers to an NK cell that expresses a CAR.

[0077] The terms “polypeptide” and “peptide” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. Also included within the definition are, for example, polypeptidescontaining one or more analogs of an amino acid, including but not limited to, unnatural amino acids, as well as other modifications known in the art. It is understood that, because the polypeptides of this disclosure may be based upon antibodies or other members of the immunoglobulin superfamily, in certain embodiments, a “polypeptide” can occur as a single chain or as two or more associated chains.

[0078] “Polynucleotide” or “nucleic acid,” as used interchangeably herein, refers to polymers of nucleotides of any length and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs.“Oligonucleotide,” as used herein, refers to short, generally single -stranded, synthetic polynucleotides that are generally, but not necessarily, fewer than about 200 nucleotides in length. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides. A cell that produces a binding molecule of the present disclosure may include a parent hybridoma cell, as well as bacterial and eukaryotic host cells into which nucleic acids encoding the antibodies have been introduced. Unless specified otherwise, the left-hand end of any single -stranded polynucleotide sequence disclosed herein is the 5’ end; the left-hand direction of double -stranded polynucleotide sequences is referred to as the 5’ direction. The direction of 5’ to 3’ addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 5’ to the 5’ end of the RNA transcript are referred to as “upstream sequences”; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3 ’ to the 3’ end of the RNA transcript are referred to as “downstream sequences.”

[0079] An “isolated nucleic acid” is a nucleic acid, for example, an RNA, DNA, or a mixed nucleic acids, which is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence. An “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a specific embodiment, one or more nucleic acid molecules encoding a single domain antibody or an antibody as described herein are isolated or purified. The term embraces nucleic acid sequences that have been removed from their naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems. A substantially pure molecule may include isolated forms of the molecule. Specifically, an “isolated” nucleic acid molecule encoding a CAR or an sdAb described herein is a nucleic acid molecule that is identifiedand separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced.

[0080] The term “control sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0081] As used herein, the term “operatively linked,” and similar phrases (e.g., genetically fused), when used in reference to nucleic acids or amino acids, refer to the operational linkage of nucleic acid sequences or amino acid sequence, respectively, placed in functional relationships with each other. For example, an operatively linked promoter, enhancer elements, open reading frame, 5' and 3' UTR, and terminator sequences result in the accurate production of a nucleic acid molecule (e.g., RNA). In some embodiments, operatively linked nucleic acid elements result in the transcription of an open reading frame and ultimately the production of a polypeptide (z.e., expression of the open reading frame). As another example, an operatively linked peptide is one in which the functional domains are placed with appropriate distance from each other to impart the intended function of each domain.

[0082] The term “vector” refers to a substance that is used to carry or include a nucleic acid sequence, including for example, a nucleic acid sequence encoding a binding molecule (e.g., an antibody) as described herein, in order to introduce a nucleic acid sequence into a host cell. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. When two or more nucleic acid molecules are to be coexpressed (e.g., both an antibody heavy and light chain or an antibody VH and VL), both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The introduction of nucleic acid molecules into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the nucleic acid molecules are expressed in a sufficientamount to produce a desired product and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

[0083] The term “host” as used herein refers to an animal, such as a mammal (e.g., a human).

[0084] The term “host cell” as used herein refers to a particular subject cell that may be transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.

[0085] As used herein, the term “autologous” is meant to refer to any material derived from the same individual to whom it is later to be re-introduced into the individual.

[0086] “Allogeneic” refers to a graft derived from a different individual of the same species.

[0087] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous polynucleotide is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous polynucleotide. The cell includes the primary subject cell and its progeny. In certain embodiments, an exogenously introduced polynucleotide is not a polynucleotide that exits naturally in a cell, e.g., an engineered immune cell.

[0088] The term “pharmaceutically acceptable” as used herein means being approved by a regulatory agency of the Federal or a state government, or listed in United States Pharmacopeia, European Pharmacopeia, or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.

[0089] “Excipient” means 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, coloring agents, diluents, disintegrating agents, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, perfumes, preservatives, propellants, releasing agents, sterilizing agents, sweeteners, solubilizers, wetting agents and mixtures thereof. The term “excipient” can also refer to a diluent, adjuvant (e.g., Freunds’ adjuvant (complete or incomplete) or vehicle.

[0090] In some embodiments, excipients are pharmaceutically acceptable excipients. Examples of pharmaceutically acceptable excipients include buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (e.g., fewer than about 10 amino acid residues) polypeptide; 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™, polyethylene glycol (PEG), and PLURONICS™. Other examples of pharmaceutically acceptableexcipients are described in Remington and Gennaro, Remington’s Pharmaceutical Sciences (18th ed. 1990).

[0091] As used herein, the term “pharmaceutically acceptable” when used in reference to a carrier, is intended to mean that the carrier, diluent or excipient is not toxic or otherwise undesirable, (i.e., the material may be administered to a subject without causing any undesirable biological effects), and it is compatible with the other ingredients of the formulation. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as saline solutions. A saline solution can be a carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0092] In some embodiments, excipients are sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is an exemplary excipient when a composition (e.g., a pharmaceutical composition) is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, particularly for injectable solutions. An excipient can also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, 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, etc.

[0093] Compositions, including pharmaceutical compounds, may contain a binding molecule (e.g., an antibody), for example, in isolated or purified form, together with a suitable amount of excipients.

[0094] The term “effective amount” or “therapeutically effective amount” as used herein refers to the amount of a single domain antibody or a therapeutic molecule comprising an agent and the single domain antibody or pharmaceutical composition provided herein which is sufficient to result in the desired outcome.

[0095] The terms “subject” and “patient” may be used interchangeably. As used herein, in certain embodiments, a subject is a mammal, such as a non-primate or a primate (e.g., human). In specific embodiments, the subject is a human. In one embodiment, the subject is a mammal, e.g., a human, diagnosed with a disease or disorder. In another embodiment, the subject is a mammal, e.g., a human, at risk of developing a disease or disorder.

[0096] “Administer” or “administration” refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other method of physical delivery described herein or known in the art.

[0097] As used herein, the terms “treat,” “treatment” and “treating” refer to the reduction or amelioration of the progression, severity, and / or duration of a disease or condition resulting from the administration of one or more therapies. Treating may be determined by assessing whether there has been a decrease, alleviation and / or mitigation of one or more symptoms associated with the underlying disorder such that an improvement is observed with the patient, despite that the patient may still be afflicted with the underlying disorder. The term “treating” includes both managing and ameliorating the disease. The terms “manage,” “managing,” and “management” refer to the beneficial effects that a subject derives from a therapy which does not necessarily result in a cure of the disease.

[0098] The terms “prevent,” “preventing,” and “prevention” refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptom(s) (e.g., diabetes or a cancer).

[0099] As used herein, “delaying” the development of cancer means to defer, hinder, slow, retard, stabilize, and / or postpone development of the disease. This delay can be of varying lengths of time, depending on the history of the disease and / or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. A method that "delays" development of cancer is a method that reduces probability of disease development in a given time frame and / or reduces the extent of the disease in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a statistically significant number of individuals. Cancer development can be detectable using standard methods, including, but not limited to, computerized axial tomography (CAT Scan), Magnetic Resonance Imaging (MRI), abdominal ultrasound, clotting tests, arteriography, or biopsy. Development may also refer to cancer progression that may be initially undetectable and includes occurrence, recurrence, and onset.

[0100] The terms “about” and “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.

[0101] As used in the present disclosure and claims, the singular forms “a”, “an” and “the” include plural forms unless the context clearly dictates otherwise.

[0102] It is understood that wherever embodiments are described herein with the term “comprising” otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are also provided. It is also understood that wherever embodiments are described herein with the phrase “consisting essentially of’ otherwise analogous embodiments described in terms of “consisting of’ are also provided.

[0103] The term “between” as used in a phrase as such “between A and B” or “between A-B” refers to a range including both A and B.

[0104] The term “and / or” as used in a phrase such as “A and / or B” herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of 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).

[0105] As well-known in the art, polypeptide sequences and fragments thereof can be used in reversed format except for the signal peptide (SP). Various polypeptide sequences as provided herein, such as the amino acid sequences of hinge domain, transmembrane domain or intracellular domain of a polypeptide, can be used in reversed format. In other words, it is understood that whenever an amino acid sequence is provided herein, the reversed version of such is also provided in the application. As an illustration for clarity, the amino acid sequence of DAP 10 intracellular domain (ICD) is LCARPRRSPAQEDGKVYINMPGRG (SEQ ID NO:6), whereas a reversed version of the amino acid sequence of DAP 10 intracellular domain (ICD) is GRGPMNIYVKGDEDQAPSRRPRACL (SEQ ID NO: 104). Similarly, the amino acid sequence of SAP is SEQ ID NO:69, whereas the reversed version of such is SEQ ID NO: 105.5.2 Chimeric antigen receptor (CARs)

[0106] In one aspect, provided herein are chimeric antigen receptors (CARs). In certain embodiments, provided herein is a chimeric antigen receptor (CAR), comprising:(i) an extracellular antigen binding domain;(ii) a transmembrane domain; and(iii) an intracellular signaling domain comprising an intracellular domain derived from a mutated CD84, wherein the interaction between the intracellular signaling domain of the CAR and SH2 domaincontaining tyrosine phosphatase 2 (SHP2) is lower than the interaction between the intracellular domain of a second CAR and SHP2, and wherein the second CAR comprises an intracellular signaling domain comprising an intracellular domain derived from a wild-type CD84.

[0107] In certain embodiments, provided herein is a chimeric antigen receptor (CAR), comprising:(i) an extracellular antigen binding domain;(ii) a transmembrane domain; and(iii) an intracellular signaling domain comprising an intracellular domain derived from a mutated CD84, wherein the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 7 and exon 8 are deleted.

[0108] 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.

[0109] In certain embodiments, the hinge domain of the CAR comprises a hinge domain derived from CD84.

[0110] In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain derived from CD84.

[0111] In certain embodiments, the intracellular signaling domain further comprises an intracellular domain derived from DAP 10, DAP 12 or DNAM1. In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain derived from DAP 10.

[0112] In certain embodiments, the intracellular signaling domain of the CAR further comprises an intracellular domain derived from CD3zeta. In certain embodiments, the intracellular signaling domain of the CAR further comprises an intracellular domain derived from SLAM-associated protein (SAP). In certain embodiments, the intracellular signaling domain of the CAR further comprises an intracellular domain derived from Ewing’s sarcoma-activated transcript-2 (EAT2). In certain embodiments, the intracellular domain derived from CD3zeta is linked to the intracellular domain derived from SAP or the intracellular domain derived from EAT2 directly or via a peptide linker. In certain embodiments, the intracellular domain derived from CD3zeta is linked to the intracellular domain derived from SAP or the intracellular domain derived from EAT2 via P2A, F2A, T2A, or E2A.

[0113] In certain embodiments, the extracellular antigen binding domain of the CAR comprises an scFv. In certain embodiments, the extracellular antigen binding domain of the CAR binds to a tumor antigen or a fragment thereof.

[0114] In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by one or more exons are deleted. In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 5 and exon 6 are deleted. In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 7 and exon 8 are deleted.

[0115] In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16. In certain embodiments, the intracellular domain of the CAR comprises the amino acid sequence of SEQ ID NO: 16.

[0116] In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:6, SEQ ID NO: 106, or SEQ ID NO: 107. In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:69 or SEQ ID NO: 108.

[0117] In certain embodiments, the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO:20.

[0118] In certain embodiments, the CAR, from N-terminus to C-terminus, comprising:(i) an extracellular antigen binding domain;(ii) a transmembrane domain comprising an transmembrane domain derived from CD84;(iii) an intracellular signaling domain comprising(iii)-(a) an intracellular domain derived from DAP 10;(iii)-(b) an intracellular domain derived from mutated CD84, wherein the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 7 and exon 8 are deleted; and(iii)-(c) an intracellular domain derived from CD3zeta, and wherein the 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 Introduction

[0119] CD84 (SLAMF5) is a homophilic adhesion molecule, which is expressed on most immune cell surface. CD84 signaling can activate or inhibit cell functions including cytokine secretion, cytotoxicity depending on cell types, activation or differentiation status. In anti-CD3 activated human T cells, activation of CD84 increases T cell proliferation, IFNg generation, and TCR- mediated response (Agod et al. (2018) Signaling Lymphocyte Activation Molecule Family 5 Enhances Autophagy and Fine-Tunes Cytokine Response in Monocyte-Derived Dendritic Cells via Stabilization of Interferon Regulatory Factor 8. Front. Immunol. 9:62).

[0120] CD84 ectodomain contains two Ig-like domains, a tandem IgV-IgC2 motifs. The cytoplasmic tail of human CD84 contains 83 amino acids and 4 critical tyrosine residues. Y262 and Y299 are in two consensus immunoreceptor tyrosine-based switch motifs (ITSM), respectively. The phosphorylated Y262 and Y299 bind SLAM-associated protein (SAP), Ewing's sarcoma-associated transcript 2 (EAT-2), and SH2 enzymes such as SHP-2, SHP-1, Csk, and SHIP-1. Some study showed that Y262 is critical to recruit SAP, EAT2 can be recruited to both Y262 and Y299. SAP, SHP-2, and SHIP can also bind to non-phosphorylated Y262. (Tangye et al. (2003) Functional requirements for interactions between CD84 and Src homology 2 domain-containing proteins and their contribution to human T cell activation. J Immunol. 171(5) :2485-95). But recruitment of SAP is most efficient when CD84 is phophorylated (Cannons et al. (2010) Optimal germinal center responses require a multistage T cell:B cell adhesion process involving integrins, SLAM-associated protein, and CD84. Immunity.26;32(2):253-65.) (Sayos et al. (2001) Cell surface receptors Ly-9 and CD84 recruit the X-linked lymphoproliferative disease gene product SAP. Blood. 97(12):3867-74. )

[0121] However, other two sites Y279 and Y324 are not associated with SAP and EAT2 recruitment, which are essential for CD84 mediated FcsRI inhibitory signals in mast cells and CD84- mediated T cell activation and cytokine secretion. Y324 is in ITIM, Y279 and Y324 are supposed to recruit SH2 proteins SHP-2, SHP-1, Csk, and SHIP-1. (Wang et al. (2010) Cutting Edge: The Adapters EAT-2A and -2B Are Positive Regulators of CD244- and CD84-Dependent NK Cell Functions in the C57BL / 6 Mouse. J Immunol 185 (10): 5683-5687. ).

[0122] CD84 expression on T cells is elevated in systemic lupus erythematosus (SLE) patients, which contributes to SLE pathogenesis. In contrast, in lupus clinical remission, CD84 expression in T cells is significantly lower in lupus clinical remission compared to active lupus nephritis (Stratigou et al. (2017) Altered expression of signalling lymphocyte activation molecule receptors in T-cells from lupus nephritis patients-a potential biomarker of disease activity. Rheumatology (Oxford).56(7): 1206-1216.). In mouse NK cells, interactions of homophilic CD84 with target tumor cells enhance NK cytotoxicity. EAT-2A / B and SAP are positive regulators of CD84-mediated NK cell killing, and EAT2 can regulate the phosphorylation ofVav-1.

[0123] The CD84-encoding gene has eight exons. The exon 1 encodes the signal peptide, the exon 2 and exon 3 encode the two Ig-like domains, the exon 4 encodes transmembrane region, the exons 5-8 encode cytoplasmic tail, in which, exon 5 and exon 6 encode the first ITSM to the Y279, and the second ITSM to the tail is encoded by exon 7 and exon 8. CD84 lacking cytoplasmic tail (CD84A5,6) is overexpressed in T and NK cells in SLE patients (Cuenca et al. (2019) CD84 cell surface signaling molecule: An emerging biomarker and target for cancer and autoimmune disorders. Clin Immunol. 204:43-49; the disclosure of which is incorporated by reference in its entirety).

[0124] Here, we designed CD84-CAR using CD84 without (CAR17) or with (CAR18) SAP, and using mutant CD84 lacking cytoplasmic tail (CD84A5,6) or (CD84A7,8). CAR-expressing NK cell function assay showed that CAR18-NK killing ability was much higher than CAR17-NK; CD84A5,6 CAR-NK killing ability was impaired, but CD84A7,8 CAR-NK killing ability was enhanced dispensable of SAP.5.2.2 Exemplary CAR

[0125] In certain embodiments, the CAR provided herein, from N-terminus to C-terminus, comprises:(i) an extracellular antigen binding domain;(ii) optionally a hinge domain that can be, but is not limited to, a hinge domain derived from CD8 or CD84;(iii) a transmembrane domain optionally comprising an transmembrane domain derived from CD84;(iv) optionally a peptide linker that can be, but is not limited to, a peptide linker provided herein;(v) an intracellular signaling domain comprising:(v)-(a) an intracellular domain derived from DAP10, DNAM1, or DAP12;(v)-(b) an intracellular domain derived from mutated CD84, optionally wherein the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 7 and exon 8 are deleted; and(v)-(c) optionally an intracellular domain derived from CD3zeta;(v)-(d) optionally a peptide linker, that can be, but is not limited to, P2A, T2A, or E2A;(v)-(e) optionally an intracellular domain derived from SAP or an intracellular domain derived from EAT2.

[0126] In certain embodiments, the CAR comprises an amino acid sequence at least 90% (at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO:20. In certain embodiments, the CAR comprises the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO:20. In certain embodiments, the CAR comprises an amino acid sequence at least 90% (at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 19.

[0127] 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 the elements as illustrated in Table 2 while having one or more peptide linkers between certain elements, for example, between the hinge domain and the transmembrane domain, between the transmembrane domain and the intracellular signaling domain, and between various intracellular domains derived from different polypeptides within the intracellular signaling domain. In certain embodiments, the symbol in Table 2 represents linking directly or via a peptide linker, for example via a peptide linker as provided in Section 5.2.6.Table 2. Exemplary CAR constructs5.2.3 Extracellular antigen binding domain

[0128] In certain embodiments, the extracellular antigen binding domain of the CAR provided herein recognizes and binds to a specific binding element on the target of interest. Non-limitingexamples of the extracellular antigen binding domain include a single-chain variable fragment (scFv), nanobodies, ligands to cognate receptors, native receptors against targets, and small peptides. In certain embodiment, the extracellular antigen binding domain of the CAR comprises an scFv. In certain embodiments, the extracellular antigen binding domain of the CAR binds to a tumor antigen or a fragment thereof.

[0129] In certain embodiments, the extracellular antigen binding domain of the CAR binds to GPC3 or a fragment thereof. In certain embodiments, the extracellular antigen binding domain of the 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:97. In certain embodiments, the extracellular antigen binding domain of the CAR comprises the amino acid sequence of SEQ ID NO:97. In certain embodiments, the extracellular antigen binding domain of the CAR binds to GPC3 and comprises the VH CDR1-3 and VL CDR1-3 in an 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: 97. In certain embodiments, the extracellular antigen binding domain of the CAR binds to GPC3 and comprises the VH CDR1-3 and VL CDR1-3 in an scFv comprising the amino acid sequence of SEQ ID NO:97. In certain embodiments, the extracellular antigen binding domain of the CAR binds to GPC3 and comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO:98, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:99, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 100, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 101, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 102, and a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 103.

[0130] In certain embodiments, the extracellular antigen binding domain of the CAR binds to CD 19 or a fragment thereof. In certain embodiments, the extracellular antigen binding domain of the CAR binds to a polypeptide comprising the amino acid sequence of SEQ ID NO:70. In certain embodiments, the extracellular antigen binding domain of the 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 NOVO. In certain embodiments, the extracellular antigen binding domain of the CAR comprises the amino acid sequence of SEQ ID NOVO. In certain embodiments, the extracellular antigen binding domain of the CAR binds to CD 19 and comprises the VH CDR1-3 and VL CDR1-3 in an 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 NOVO. In certain embodiments, the extracellular antigen binding domain of the CAR binds to CD 19 and comprises the VH CDR1-3 and VL CDR1-3 in an scFv comprising the amino acid sequence of SEQ ID NOVO. In certain embodiments, the extracellular antigen binding domain of the CAR binds to CD 19 and comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO:91, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:92, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:93, a VL CDR1 comprising the amino acid sequence of SEQ ID NO:94, aVL CDR2 comprising the amino acid sequence of SEQ ID NO:95, and a VL CDR1 comprising the amino acid sequence of SEQ ID NO:96.

[0131] Various antigens suitable for targeting with a CAR are known in the art, and each are suitable for use with the present application. Tumor antigens are proteins that are produced by tumor cells that can elicit an immune response, particularly T-cell mediated immune responses. Exemplary tumor antigens include, but not limited to, a glioma-associated antigen, carcinoembryonic antigen (CEA), P-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2 / neu, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF- II, IGF-I receptor and mesothelin.

[0132] In some embodiments, the tumor antigen comprises one or more antigenic cancer epitopes associated with a malignant tumor. Malignant tumors express a number of proteins that can serve as target antigens for an immune attack. These molecules include, but are not limited to, tissuespecific antigens such as MART-1, tyrosinase and gplOO in melanoma and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules such as the oncogene HER2 / Neu / ErbB-2. Yet another group of target antigens are onco-fetal antigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma the tumor-specific idiotype immunoglobulin constitutes a truly tumor-specific immunoglobulin antigen that is unique to the individual tumor. In addition to CD 19, B-cell differentiation antigens such as CD20 and CD37 are other candidates for target antigens in B-cell lymphoma.

[0133] In some embodiments, the tumor antigen is a tumor-specific antigen (TSA) or a tumor- associated antigen (TAA). A TSA is unique to tumor cells and does not occur on other cells in the body. A TAA associated antigen is not unique to a tumor cell, and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen. The expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens that are expressed on normal cells during fetal development, when the immune system is immature, and unable to respond or they may be antigens that are normally present at extremely low levels on normal cells, but which are expressed at much higher levels on tumor cells.

[0134] Non-limiting examples of TSA or TAA antigens include: differentiation antigens such as MART-l / MelanA (MART-I), gp 100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; overexpressed embryonic antigens such as CEA and alpha-fetoprotein (AFP); overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER2 / neu; unique tumor antigens resulting fromchromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA, human immunodeficiency viruses (HIV) antigen CD32a, hepatitis B virus (HBV) antigen HBsAg, and the human papillomavirus (HPV) antigens E6 and E7.

[0135] 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, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alphafetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA- 50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, M0V18, 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 antigen(s) is selected from a group consisting of CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c- Met, EGFRvIII, GD-2, NY-ESO-1, MAGE A3, and glycolipid F77.5.2.4 Transmembrane domain

[0136] In certain embodiments, the CARs provided herein comprise a transmembrane domain that is linked to the extracellular antigen binding domain provided herein directly or via a hinge domain provided herein.

[0137] In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain derived from a signaling lymphocytic activation molecule family (SLAMF) receptor, CD2, CD28H, DNAM1, CD28, 4-1BB, 0X40, KIR-S, NKG2C, or NKG2E. In certain embodiments, the third polypeptide is a signaling lymphocytic activation molecule family (SLAMF) receptor, CD2, CD28H, DNAM1, CD28, 4-1BB, 0X40, 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.

[0138] In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain derived from SLAMF5 (CD84). In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain derived from CD8. In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain derived from SLAMF4 (CD244, 2B4). In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain derived from SLAMF3 (CD229, Ly9). In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain derived from SLAMF6 (CD352, NTB-A, SF2000). In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain derived from SLAMF7 (CD319, CRACC, CS1). In certainembodiments, the transmembrane domain of the CAR comprises a transmembrane domain derived from SLAMF1 (CD 150). In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain derived from SLAMF2 (CD48, FimH). In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain derived from SLAMF8 (CD353). In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain derived from SLAMF9 (CD84H1, SF2001). In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain derived from NKG2D. In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain derived from CD28H. In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain derived from DNAM1. In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain derived from CD2.

[0139] In some embodiments, the transmembrane domain of the CAR comprises the transmembrane domain (TD) of CD84, CD8, or NKG2D. In some embodiments, the transmembrane domain of the CAR comprises the transmembrane domain (TD) of CD84. In some embodiments, the transmembrane domain of the CAR comprises the transmembrane domain (TD) of SLAMF4 (CD244, 2B4), SLAMF3 (CD229, Ly9), SLAMF6 (CD352, NTB-A, SF2000), SLAMF7 (CD319, CRACC, CS1), SLAMF1 (CD150), SLAMF2 (CD48, FimH), SLAMF8 (CD353), SLAMF9 (CD84H1, SF2001), CD8, CD28, 4-1BB, 0X40, 2B4, CD2, CD28H, DNAM1, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, NKG2C, or NKG2E. In certain embodiments, the transmembrane domain of the 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:3, SEQ ID NO:4, or SEQ ID NO: 13. In certain embodiments, the transmembrane domain of the CAR comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO: 13. In certain embodiments, the transmembrane domain of the CAR comprises the amino acid sequence of SEQ ID NO: 13.

[0140] The transmembrane domain may be derived either from a natural or from a synthetic source. As used herein, a “transmembrane domain” refers to any protein structure that is thermodynamically stable in a cell membrane, preferably an eukaryotic cell membrane. Transmembrane domains compatible for use in the CARs described herein may be obtained from a naturally occurring protein. Alternatively, it can be a synthetic, non-naturally occurring protein segment, e.g. , a hydrophobic protein segment that is thermodynamically stable in a cell membrane.

[0141] Transmembrane domains are classified based on the three dimensional structure of the transmembrane domain. For example, transmembrane domains may form an alpha helix, a complex of more than one alpha helix, a beta-barrel, or any other stable structure capable of spanning the phospholipid bilayer of a cell. Furthermore, transmembrane domains may also or alternatively be classified based on the transmembrane domain topology, including the number of passes that the transmembrane domain makes across the membrane and the orientation of the protein. For example, single-pass membrane proteins cross the cell membrane once, and multi-pass membrane proteinscross the cell membrane at least twice (e.g., 2, 3, 4, 5, 6, 7 or more times). Membrane proteins may be defined as Type I, Type II or Type III depending upon the topology of their termini and membranepassing segment(s) relative to the inside and outside of the cell. Type I membrane proteins have a single membrane-spanning region and are oriented such that the N-terminus of the protein is present on the extracellular side of the lipid bilayer of the cell and the C-terminus of the protein is present on the cytoplasmic side. Type II membrane proteins also have a single membrane -spanning region but are oriented such that the C-terminus of the protein is present on the extracellular side of the lipid bilayer of the cell and the N-terminus of the protein is present on the cytoplasmic side. Type III membrane proteins have multiple membrane -spanning segments and may be further sub-classified based on the number of transmembrane segments and the location of N- and C-termini.

[0142] In some embodiments, the transmembrane domain of the CAR described herein is derived from a Type I single-pass membrane protein. In some embodiments, transmembrane domains from multi-pass membrane proteins may also be compatible for use in the CARs described herein. Multipass membrane proteins may comprise a complex (at least 2, 3, 4, 5, 6, 7 or more) alpha helices or a beta sheet structure. In some embodiments, the N-terminus and the C-terminus of a multi-pass membrane protein are present on opposing sides of the lipid bilayer, e.g., 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.

[0143] In some embodiments, the transmembrane domain of the CAR comprises a transmembrane domain chosen from the transmembrane domain of an alpha, beta or zeta chain of a T- cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CD1 la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD 160, CD 19, IL-2R beta, IL-2R gamma, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C, whereas the intracellular domain of the CAR comprises an intracellular domain derived from a first polypeptide that is different from the peptides listed in this paragraph.

[0144] Transmembrane domains for use in the CARs described herein can also comprise at least a portion of a synthetic, non-naturally occurring protein segment. In some embodiments, the transmembrane domain is a synthetic, non-naturally occurring alpha helix or beta sheet. In some embodiments, the protein segment is at least approximately 20 amino acids, e.g., at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids. Examples of synthetic transmembranedomains are known in the art, for example in U.S. Patent No.7,052,906 and PCT Publication No. WO 2000 / 032776, the relevant disclosures of which are incorporated by reference herein.5.2.5 Intracellular signaling domainMutated CD84

[0145] In certain embodiments, the intracellular signaling domain of the CAR provided herein comprises an intracellular domain derived from a mutated CD84. In certain embodiments, the interaction between the intracellular signaling domain of the CAR and SH2 domain-containing tyrosine phosphatase 2 (SHP2) is reduced. In certain embodiments, the interaction between the intracellular signaling domain of the CAR and SH2 domain-containing tyrosine phosphatase 2 (SHP2) is lower than the interaction between the intracellular domain of a second CAR and SHP2, and wherein the second CAR comprises an intracellular signaling domain comprising an intracellular domain derived from a wild-type CD84. In certain embodiments, the second CAR differs from the CAR provided herein only in that the second CAR comprises an intracellular signaling domain comprising an intracellular domain derived from a wild-type CD84 whereas the CAR provided herein comprises an intracellular signaling domain comprising an intracellular domain derived from a mutated CD84.

[0146] In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by one or more exons are deleted. In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 5 and exon 6 are deleted. In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 7 and exon 8 are deleted.

[0147] In certain embodiments, the intracellular domain of the 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: 15. In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the intracellular domain of the 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: 16. In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 16. In certain embodiments, the intracellular domain of the CAR comprises the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the intracellular domain of the CAR comprises the amino acid sequence of SEQ ID NO: 16.DAP10, DNAM1, or DAP12

[0148] In certain embodiments, the intracellular signaling domain of the CAR provided herein comprises an intracellular domain derived from DAP 10, DNAM1 or DAP 12. In certain embodiments, the intracellular signaling domain of the CAR provided herein comprises the intracellular domain ofDAP 10, DNAM1 or DAP 12. In certain embodiments, the first polypeptide is DAP 10. In certain embodiments, the intracellular signaling domain of the CAR provided herein comprises an intracellular domain derived from DAP 10. In certain embodiments, the intracellular signaling domain of the CAR provided herein comprises the intracellular domain of DAP 10 (DAP 10 ICD). In certain embodiments, the intracellular domain derived from the first polypeptide is linked to the C-terminus of the transmembrane domain directly or via a peptide linker provided herein.

[0149] In certain embodiments, the CAR provided herein comprises an intracellular domain derived from a polypeptide that is capable of activating PI3K at the site of membrane. In certain embodiments, the intracellular domain derived from the polypeptide is linked to the C-terminus of the transmembrane domain via a peptide linker. In certain embodiments, the peptide linker between the intracellular domain derived from the polypeptide and the C-terminus of the transmembrane domain is less than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids long so that the intracellular domain derived from the polypeptide is close to the C-terminus of the transmembrane domain. In certain embodiments, the peptide linker between the intracellular domain derived from the polypeptide and the C-terminus of the transmembrane domain is in a length that the intracellular domain derived from the first polypeptide is close to the C-terminus of the transmembrane domain and / or close to the membrane of the engineered immune cell. In certain embodiments, the peptide linker between the intracellular domain derived from the polypeptide and the C-terminus of the transmembrane domain is less than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids long so that the intracellular domain derived from the polypeptide is capable of activating PI3K at the site of membrane. In certain embodiments, the peptide linker between the intracellular domain derived from the polypeptide and the C-terminus of the transmembrane domain is in a length that the intracellular domain derived from the polypeptide is capable of activating PI3K at the site of membrane. In certain embodiments, the peptide linker between the intracellular domain derived from the polypeptide and the C-terminus of the transmembrane domain is GGGS (SEQ ID NO:80). In certain embodiments, the peptide linker between the intracellular domain derived from the 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, Notl, Bglll, BamhI, EcoRI, Nhel, Xhol, PacI, SacI, SacII, Xbal, Sall, and Clal.

[0150] In certain embodiments, the intracellular signaling domain of the 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:6, SEQ ID NO: 106, or SEQ ID NO: 107. In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:6, SEQ ID NO: 106, or SEQ ID NO: 107. In certain embodiments, the intracellular signaling domain of the CAR comprises the amino acid sequence of SEQ ID NO:6, SEQ ID NO: 106, or SEQ ID NO: 107. In certain embodiments, the intracellular signaling domain of the 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:6. In certain embodiments, the intracellular signaling domain of the CAR comprises the amino acid sequence of SEQ ID NO:6.CD3zeta, SAP, EAT2, and peptide linkers

[0151] In certain embodiments, the intracellular signaling domain of the CAR further comprises an intracellular domain derived from CD3zeta. In certain embodiments, the intracellular signaling domain of the CAR further comprises the intracellular domain of CD3zeta (CD3zeta ICD). In certain embodiments, the intracellular signaling domain of the 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:5. In certain embodiments, the intracellular signaling domain of the CAR comprises the amino acid sequence of SEQ ID NO:5.

[0152] In certain embodiments, when the intracellular signaling domain of the CAR comprises an intracellular domain derived a mutated CD84, the intracellular signaling domain of the CAR does not comprise an intracellular domain derived SLAM-associated protein (SAP), which nevertheless shows superior effect, for example superior effects in eradicating tumor cells.

[0153] In certain embodiments, the intracellular signaling domain of the CAR further comprises an intracellular domain derived from SLAM-associated protein (SAP). In certain embodiments, the intracellular signaling domain of the 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:69. In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:69. In certain embodiments, the intracellular signaling domain of the CAR comprises the amino acid sequence of SEQ ID NO: 69.

[0154] In certain embodiments, the intracellular signaling domain of the CAR further comprises an intracellular domain derived from EAT2. In certain embodiments, the intracellular signaling domain of the 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: 108. In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 108. In certain embodiments, the intracellular signaling domain of the CAR comprises the amino acid sequence of SEQ ID NO: 108.

[0155] In certain embodiments, the intracellular signaling domain of the CAR further comprises a peptide linker derived from P2A, F2A, T2A, or E2A. In certain embodiments, the intracellular signaling domain of the CAR further comprises P2A, F2A, T2A, or E2A. In certain embodiments, the intracellular signaling domain of the 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: 1, SEQ ID NO:2 or SEQ ID NO:3. In certain embodiments, the intracellular signaling domain of theCAR comprises the 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 the CAR further comprises a peptide linker derived from P2A. In certain embodiments, the intracellular signaling domain of the CAR further comprises P2A. In certain embodiments, the intracellular signaling domain of the 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: 1. In certain embodiments, the intracellular signaling domain of the CAR comprises the amino acid sequence of SEQ ID NO: 1.

[0156] In certain embodiments, the intracellular domain derived from CD3zeta is linked to the intracellular domain derived from SAP or the intracellular domain derived from EAT2 directly or via a peptide linker. In certain embodiments, the intracellular domain derived from CD3zeta is linked to the intracellular domain derived from SAP or the intracellular domain derived from EAT2 via P2A, F2A, T2A, or E2A.

[0157] In certain embodiments, without being bound by any particular theory, an intracellular domain derived from SAP or an intracellular domain derived from EAT2 can facilitate the CAR to trigger a complete activation signal and completely overcome the inhibitory receptor signaling.

[0158] In certain embodiments, the intracellular signaling domain of the CAR comprises a primary signaling domain and one or more co-stimulatory signaling domains. In certain embodiments, the primary signaling domain and the one or more co-stimulatory signaling domains are linked directly to each other or linked to each other via peptide linker(s).

[0159] In certain embodiments, the intracellular signaling domain of the CAR comprises a primary signaling domain comprising an intracellular domain derived from CD3zeta. In certain embodiments, the intracellular signaling domain of the CAR comprises a primary signaling domain comprising the intracellular domain of CD3zeta (CD3zeta ICD). In certain embodiments, the intracellular signaling domain of the CAR comprises a primary 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:5. In certain embodiments, the intracellular signaling domain of the CAR comprises a primary signaling domain comprising the amino acid sequence of SEQ ID NO:5.

[0160] In certain embodiments, the intracellular signaling domain of the CAR comprises a co- stimulatory signaling domain comprising an intracellular domain derived from DAP 10, DNAM1, or DAP 12. In certain embodiments, the intracellular signaling domain of the CAR comprises a co- stimulatory signaling domain comprising the intracellular domain of DAP 10, DNAM1, or DAP 12. In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory 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:6, SEQ ID NO: 106, or SEQ ID NO: 107. In certain embodiments, the intracellular signaling domain of the CAR comprises a co- stimulatory signaling domain comprising the amino acid sequence of SEQ ID NO:6, SEQ ID NO: 106,or SEQ ID NO: 107. In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling domain comprising an intracellular domain derived from DAP 10. In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling domain comprising the intracellular domain of DAP 10 (DAP 10 ICD). In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory 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: 6. In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling domain comprising the amino acid sequence of SEQ ID NO:6.

[0161] 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 that is different from a mutated CD84. In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by one or more exons are deleted. In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 5 and exon 6 are deleted. In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 7 and exon 8 are deleted.

[0162] In certain embodiments, the intracellular domain of the CAR comprises a co-stimulatory 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: 15. In certain embodiments, the intracellular domain of the CAR comprises a co-stimulatory signaling domain comprisingan amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the intracellular domain of the CAR comprises a co-stimulatory signaling domain comprisingan 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: 16. In certain embodiments, the intracellular domain of the CAR comprises a co-stimulatory signaling domain comprisingan amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 16. In certain embodiments, the intracellular domain of the CAR comprises a co-stimulatory signaling domain comprising the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the intracellular domain of the CAR comprises a co-stimulatory signaling domain comprisingthe amino acid sequence of SEQ ID NO: 16.

[0163] In certain embodiments, the intracellular signaling domain of the CAR comprises a co- stimulatory signaling domain comprising an intracellular domain derived from SAP or EAT2. In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling domain comprising SAP or EAT. In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory 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:69 or SEQ ID NO: 108. In certain embodiments, the intracellular signaling domain of theCAR comprises a co-stimulatory signaling domain comprising the amino acid sequence of SEQ ID NO:69 or SEQ ID NO: 108.5.2.6 Hinge domain

[0164] 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.

[0165] The CARs provided herein may comprise a hinge domain that is located between the extracellular antigen binding domain and the transmembrane domain. 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. In certain embodiments, the hinge domain of the CAR comprises a hinge domain derived from the second polypeptide provided herein.

[0166] In certain embodiments, the hinge domain of the CAR comprises a hinge domain derived from CD84. In certain embodiments, the hinge domain 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: 12. In certain embodiments, the hinge domain comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the hinge domain comprises the amino acid sequence of SEQ ID NO: 12.

[0167] In certain embodiments, the hinge domain of the CAR comprises a hinge domain derived from CD8. In certain embodiments, the hinge domain 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:2. In certain embodiments, the hinge domain comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:2. In certain embodiments, the hinge domain comprises the amino acid sequence of SEQ ID NO:2.

[0168] A hinge domain is an amino acid segment that is generally found between two domains of a protein and may allow for flexibility of the protein and movement of one or both of the domains relative to one another. Any amino acid sequence that provides such flexibility and movement of the extracellular antigen binding domain relative to the transmembrane domain of the effector molecule can be used.

[0169] The hinge domain may contain about 10-100 amino acids, e.g., about any one of 15-75 amino acids, 20-50 amino acids, or 30-60 amino acids. In some embodiments, the hinge domain may be at least about any one of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids in length.

[0170] In some embodiments, the hinge domain is a hinge domain of a naturally occurring protein. Hinge domains of any protein known in the art to comprise a hinge domain are compatible for use in the CARs provided herein. In some embodiments, the hinge domain is at least a portion of a hinge domain of a naturally occurring protein and confers flexibility to the chimeric receptor. In someembodiments, the hinge domain is a portion of the hinge domain of CD8a, e.g., a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8a.

[0171] Hinge domains of antibodies, such as an IgG, IgA, IgM, IgE, or IgD antibodies, are also compatible for use in the CAR described herein. In some embodiments, the hinge domain is the hinge domain that joins the constant domains CHI and CH2 of an antibody. In some embodiments, the hinge domain is of an antibody and comprises the hinge domain of the antibody and one or more constant regions of the antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the CH3 constant region of the antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the CH2 and CH3 constant regions of the 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 IgGl, IgG2, IgG3, or IgG4 antibody. In some embodiments, the hinge region comprises the hinge region and the CH2 and CH3 constant regions of an IgGl antibody. In some embodiments, the hinge region comprises the hinge region and the CH3 constant region of an IgGl antibody.

[0172] Non-naturally occurring peptides may also be used as hinge domains for the chimeric receptors described herein. In some embodiments, the hinge domain between the C-terminus of the extracellular ligand-binding domain of an Fc receptor and the N- terminus of the transmembrane domain is a peptide linker, such as a (GxS)n linker, wherein x and n, independently can be an integer between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more.5.2.7 Peptide linkers

[0173] In certain embodiments, provided herein are one or more peptide linkers. In certain embodiments, the CAR provided herein comprises one or more peptide linkers. In certain embodiments, the peptide linker is less than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids long. In certain embodiments, the peptide linker is GGGS (SEQ ID NO:80). 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, Notl, Bglll, BamhI, EcoRI, Nhel, Xhol, PacI, SacI, SacII, Xbal, Sall, and Clal.

[0174] In certain embodiments, the CAR provided herein comprises a peptide linker between the transmembrane domain and the intracellular signaling domain. In certain embodiments, the intracellular domain derived from DAP 10, DAP 12 or DNAM1 is linked to the C-terminus of the transmembrane domain via a peptide linker. In certain embodiments, the peptide linker between the intracellular domain derived from DAP 10, DAP 12 or DNAM1 and the C-terminus of the transmembrane domain is less than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids long so that the peptide linker between the intracellular domain derived from the first polypeptide is close to the C-terminus of the transmembrane domain. 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: 80). 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, Notl, Bglll, BamhI, EcoRI, Nhel, Xhol, PacI, SacI, SacII, Xbal, Sall, and Clal.

[0175] In certain embodiments, the CAR provided herein comprises one or more peptide linkers within the intracellular signaling domain. In certain embodiments, the one or more peptide linkers within the intracellular signaling domain is between intracellular domains derived from polypeptides. In certain embodiments, an intracellular domain derived from CD3zeta is linked to an intracellular domain derived from SAP or an intracellular domain derived from EAT2 via a peptide linker.

[0176] Each peptide linker in a CAR may have the same or different length and / or sequence depending on the structural and / or functional features of the single domain antibodies and / or the various domains. Each peptide linker may be selected and optimized independently. The length, the degree of flexibility and / or other properties of the peptide linker(s) used in the CARs may have some influence on properties, including but not limited to the affinity, specificity or avidity for one or more particular antigens or epitopes. For example, longer peptide linkers may be selected to ensure that two adjacent domains do not sterically interfere with one another. In some embodiments, a short peptide linker may be disposed between the transmembrane domain and the intracellular signaling domain of a CAR. In some embodiment, a peptide linker comprises flexible residues (such as glycine and serine) so that the adjacent domains are free to move relative to each other. For example, a glycine -serine doublet can be a suitable peptide linker.

[0177] The peptide linker can be of any suitable length. In some embodiments, the peptide linker is at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids long. In some embodiments, the peptide linker is no more than about any of 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or fewer amino acids long. In some embodiments, the length of the peptide linker is any of about 1 amino acid to about 10 amino acids, about 1 amino acids to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids long, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids.

[0178] The peptide linker may have a naturally occurring sequence, or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of heavy chain only antibodies may be used as the linker. See, for example, WO 1996 / 34103. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include but not limited to glycine polymers (G)n, glycineserine polymers (including, for example, (GS)n, (GSGGS)n, (GGGS)n, and (GGGGS)n, where n is an integer of at least one), glycine -alanine polymers, alanine -serine polymers, and other flexible linkers known in the art. Exemplary peptide linkers are listed in the table below.Table 3. Exemplary Peptide Linkers

[0179] Other linkers known in the art, for example, as described in WO2016014789, WO2015158671, WO2016102965, US20150299317, WO2018067992, US7741465, Colcher etal., J. Nat. Cancer Inst. 82: 1191-1197 (1990), and Bird et al., Science 242:423-426 (1988) may also be included in the CARs provided herein, the disclosure of each of which is incorporated herein by reference.5.3 Mutated interleukin-2 (IL-2)

[0180] In one aspect, provided herein are engineered immune cells. In certain embodiments, provided herein is an engineered immune cell comprising an exogenously introduced polynucleotide encoding a mutated interleukin-2 (IL-2), and the mutated IL-2 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:63.

[0181] In certain embodiments, the engineered immune cell grows in a media that does not contain cytokine.

[0182] In certain embodiments, the mutated IL-2 is membrane-bound. In certain embodiments, the mutated IL-2 is linked to a transmembrane domain of a polypeptide directly, via a peptide linker, or via a hinge domain. In certain embodiments, the polypeptide is CD8.

[0183] In certain embodiments, the engineered immune cell is an NK cell, an NKT cell, a T cell, or a Treg cell.

[0184] In certain embodiments, the engineered immune cell comprises a chimeric antigen receptor (CAR), wherein the CAR comprises:(i) an extracellular antigen binding domain;(ii) a transmembrane domain; and(iii) an intracellular signaling domain.

[0185] 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.

[0186] In certain embodiments, the extracellular antigen binding domain of the CAR comprises an scFv. In certain embodiments, the extracellular antigen binding domain of the CAR binds to a tumor antigen or a fragment thereof.

[0187] In certain embodiments, the intracellular signaling domain of the CAR comprises an intracellular domain derived from a mutated CD84. In certain embodiments, the interaction between the intracellular signaling domain of the CAR and SH2 domain-containing tyrosine phosphatase 2 (SHP2) is lower than the interaction between the intracellular domain of a second CAR and SHP2, and wherein the second CAR comprises an intracellular signaling domain comprising an intracellular domain derived from a wild-type CD84.

[0188] 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. In certain embodiments, the hinge domain of the CAR comprises a hinge domain derived from CD84.

[0189] In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain derived from CD84.

[0190] In certain embodiments, the intracellular signaling domain of the CAR further comprises an intracellular domain derived from DAP 10, DAP 12 or DNAM1. In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain derived from DAP 10. In certain embodiments, the intracellular signaling domain of the CAR further comprises an intracellular domain derived from CD3zeta. In certain embodiments, the intracellular signaling domain of the CAR further comprises an intracellular domain derived from SLAM-associated protein (SAP). In certain embodiments, the intracellular signaling domain of the CAR further comprises an intracellular domain derived from Ewing’s sarcoma-activated transcript-2 (EAT2). In certain embodiments, theintracellular domain derived from CD3zeta is linked to the intracellular domain derived from SAP or the intracellular domain derived from EAT2 directly or via a peptide linker.

[0191] In certain embodiments, the intracellular domain derived from CD3zeta is linked to the intracellular domain derived from SAP or the intracellular domain derived from EAT2 via P2A, F2A, T2A, or E2A.

[0192] In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by one or more exons are deleted. In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 5 and exon 6 are deleted. In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 7 and exon 8 are deleted.

[0193] In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16. In certain embodiments, the intracellular domain of the CAR comprises the amino acid sequence of SEQ ID NO: 16.

[0194] In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:6, SEQ ID NO: 106, or SEQ ID NO: 107. In certain embodiments, the intracellular domain of the CAR comprises the amino acid sequence of SEQ ID NO:6, SEQ ID NO: 106, or SEQ ID NO: 107.

[0195] In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:69 or SEQ ID NO: 108. In certain embodiments, the intracellular domain of the CAR comprises the amino acid sequence of SEQ ID NO:69 or SEQ ID NO: 108.

[0196] In certain embodiments, the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO:20. In certain embodiments, the CAR comprises the amino acid sequence of SEQ ID NO:8, SEQ ID NON, SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO:20.

[0197] In certain embodiments, the exogenously introduced polynucleotide encodes the CAR.

[0198] In certain embodiments, the exogenously introduced polynucleotide comprises a nucleic acid sequence encoding the CAR and a nucleic acid sequence encoding the mutated IL-2, and wherein the nucleic acid sequence encoding the CAR and the nucleic acid sequence encoding the mutated IL-2 are linked to each other directly or via a nucleic acid linker. In certain embodiments, the mutated IL-2 comprises the amino acid sequence of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:63.

[0199] In one aspect, provided herein are polypeptides. In certain embodiments, provided herein is a polypeptide comprising an amino acid sequence of a chimeric antigen receptor (CAR) and anamino acid sequence of a mutated IL-2, wherein the mutated IL-2 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:63, wherein the CAR comprises:(i) an extracellular antigen binding domain;(ii) a transmembrane domain; and(iii) an intracellular signaling domain.

[0200] In certain embodiments, the amino acid sequence of the CAR and the amino acid sequence of the mutated IL-2 is linked to each other directly or via a peptide linker.

[0201] In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16. In certain embodiments, the intracellular domain of the CAR comprises the amino acid sequence of SEQ ID NO: 16.

[0202] In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:6, SEQ ID NO: 106, or SEQ ID NO: 107. In certain embodiments, the intracellular domain of the CAR comprises the amino acid sequence of SEQ ID NO:6, SEQ ID NO: 106, or SEQ ID NO: 107.

[0203] In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:69 or SEQ ID NO: 108. In certain embodiments, the intracellular domain of the CAR comprises the amino acid sequence of SEQ ID NO:69 or SEQ ID NO: 108.

[0204] In certain embodiments, the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO:20. In certain embodiments, the CAR comprises the amino acid sequence of SEQ ID NO:8, SEQ ID NON, SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO:20.

[0205] In certain embodiments, the mutated IL-2 comprises the amino acid sequence of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:63.

[0206] In one aspect, provided herein are polynucleotides. In certain embodiments, provided herein is a polynucleotide encoding the polypeptide provided herein. In another aspect, provided herein are vectors. In certain embodiments, provided herein is a vector comprising the polynucleotide provided herein.

[0207] In one aspect, provided herein are pharmaceutical compositions. In certain embodiments, provided herein is a pharmaceutical composition comprising(ii) the engineered immune cell provided herein, the polypeptide provided herein, thepolynucleotide provided herein, or the vector provided herein; and(ii) a pharmaceutically acceptable excipient.

[0208] In another aspect, provided herein are methods of treating a disease or disorder in a subject. In certain embodiments, provided herein is a method of treating a disease or disorder in a subject, comprising administering to the subject an effective amount of the engineered immune cell provided herein or the pharmaceutical composition provided herein.

[0209] In certain embodiments, the disease or disorder is cancer.

[0210] In another aspect, provided herein are methods of engineering an immune cell. In certain embodiments, provided herein is a method of engineering an immune cell, comprising introducing into the immune cell the polypeptide provided herein, the polynucleotide provided herein, or the vector provided herein.

[0211] In certain embodiments, provided herein is a method of culturing an immune cell comprising culturing the immune cell in a media comprising a mutated interleukin-2 (IL-2), wherein the mutated IL-2 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:48, SEQ ID NO:53, SEQ ID NO:55, or SEQ ID NO:58.

[0212] In certain embodiments, the media does not contain cytokine. In certain embodiments, the mutated IL-2 is soluble.

[0213] In certain embodiments, the immune cell is an NK cell, an NKT cell, a T cell, or a Treg cell.

[0214] In certain embodiments, the mutated IL-2 comprises the amino acid sequence of SEQ ID NO:48, SEQ ID NO:53, SEQ ID NO:55, or SEQ ID NO:58.

[0215] In certain embodiments, the immune cell is an engineered immune cell. In certain embodiments, the engineered immune cell comprises a chimeric antigen receptor (CAR), wherein the CAR comprises:(i) an extracellular antigen binding domain;(ii) a transmembrane domain; and(iii) an intracellular signaling domain.

[0216] 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. In certain embodiments, the extracellular antigen binding domain of the CAR comprises an scFv. In certain embodiments, the extracellular antigen binding domain of the CAR binds to a tumor antigen or a fragment thereof.5.3.1 IntroductionMembrane-bound cytokines

[0217] The functional differentiation of secretory and membrane -bound cytokines has not been fully studied yet. Tumor cell vaccines expressing membrane -bound cytokines, such as mbGM-CSF,mbFK, mbTNFa, mbIL-2, or mbIL-4, showed comparable level of immune stimulatory effect as soluble form cytokine expressing vaccines. The toxicity and side effects of injected cytokines could be avoided by minimizing cytokines incorporation into blood circulation (Kim YS. (2009) Tumor Therapy Applying Membrane -bound Form of Cytokines. Immune Netw. 9(5): 158-68.).

[0218] Gene engineered artificial antigen-presenting cells (aAPCs) expressing membrane -bound IL-15 (mbIL-15) and membrane IL-21 (mbIL-21) have been used to expand clinical-grade NK cells for human immunotherapy clinical trials. (Denman et al. (2012) Membrane -bound IL-21 promotes sustained ex vivo proliferation of human natural killer cells. PLoS One. 7(l):e30264.). In addition, it has been shown that membrane -bound IL-15 (mbIL-15)-CAR T cells and mbIL-15-CARNK cells improved T and NK cell persistence independent of CAR signaling, and the long-term persistence was triggered by signaling through mbIL15 without exogenous cytokines (Hurton et al. (2016) Tethered IL- 15 augments antitumor activity and promotes a stem-cell memory subset in tumor-specific T cells. Proc Natl Acad Sci U S A. 113(48):E7788-E7797.)(Imamura et al. (2014) Autonomous growth and increased cytotoxicity of natural killer cells expressing membrane-bound interleukin-15. Blood. 124(7): 1081-8.).

[0219] We designed membrane-bound IL-2, IL-7, IL-15, IL-18, and IL-21 and the membranebound cytokine expressed NK cells’ survival and growth were screened. We found that mbIL-2 and mbIL-15 could efficiently support NK cell survival and growth, and mbIL-7 had a very low effect on supporting NK cell. Data further demonstrated that mbIL-2-NK enhanced killing ability, but mblL- 15-NK cell reduced the killing ability with unclear mechanism.IL-2 and IL-2R

[0220] Interleukin-2 (IL-2) is a pluripotent cytokine, which is produced by activated CD4+ T cells. IL-2 is first identified as T cell growth factor, which can promote T helper differentiation, Treg development, induce NK cell killing activation and activation-induced cell death (AICD), IL-2 also plays a critical role in promoting proliferation and growth of activated T cells, B cells, monocytes and NK cells.

[0221] Human IL-2 precursor has 153 amino acids, mature IL-2 contains 133 amino acids with four a-helical bundle type I cytokine. (Cheng et al. (2011) T-cell tolerance and the multi-functional role of IL-2R signaling in T-regulatory cells. Immunol Rev. 241( 1): 63-76; Liao et al. (2011) Modulation of cytokine receptors by IL-2 broadly regulates differentiation into helper T cell lineages. Nat Immunol. 12(6):551-9).

[0222] IL-2 receptor has three different subsets: interleukin 2 receptor alpha (IL-2Ra; CD25), beta (IL-2R[3; CD122), and gamma (IL-2Ry; CD132). IL-2Ra is low affinity IL-2 receptor, the binding of IL-2 with IL-2Ra has no biological activity. IL-2Rp / IL-2Ry is intermediate affinity receptor (Kd~10-9 M), which is expressed on CD8+ and CD4+ memory T cells and CD561ow NK cells. Trimeric IL-2R (IL-2Ra / IL-2Rp / IL-2Ry) is high affinity receptor (Kd~10-l 1 M), which isexpressed on activated lymphocytes such as CD8+ and CD4+ effector T cells, group 2 innate lymphoid cells (ILC2s), CD56bright NK cells, and CD4+ Treg cells (Waldmann TA. (2006) The biology of interleukin-2 and interleukin- 15: implications for cancer therapy and vaccine design. Nat Rev Immunol. 2006 Aug;6(8):595-601.).

[0223] IL-2 was used to activate patients’ effector T cell and NK cell populations, which was approved by US Food and Drug Administration (FDA) for the treatment of metastatic renal cell carcinoma (in 1992) and metastatic melanoma (in 1998) (Hernandez et al. (2022) Engineering IL-2 for immunotherapy of autoimmunity and cancer. Nat Rev Immunol. 22(10):614-628.), however, high- dose IL-2 therapy resulted in severe non-specific toxicities.

[0224] Single IL-2 as immunotherapy is insufficient to improve cancer patients’ survival due to its two direction properties on T cells and severe adverse effect in high dose. IL-2 has a short half-life (<15 min), so a high dose of IL-2 must be used to achieve optimal immune -stimulatory effect. Very high dose of intravenous injection of IL-2 (50-million unit, three time a day for 5 days) were used and had non-specific toxicity, such as vascular leak syndrome (VLS), pulmonary edema, hypotension and heart toxicity. Binding of IL-2 to CD25+ endothelial cells could induce vasodilation and VLS, in addition, high eNOS, angiopoietin 2, IL-2 fragment could also be designated as permeabilityenhancing peptide and resulting in VLS. (Jiang et al. (2016) Role of IL-2 in cancer immunotherapy. Oncoimmunology. 5(6):e 1163462); Epstein et al. (1988) A novel method for the detection of necrotic lesions in human cancers. Cancer Res. 48(20): 5842-8). IL-2 supports Treg cell function, also Treg increase in tumor TME is a prognostic factor for immunotherapy, which makes the tumor IL-2 immunotherapy more complicated, resulting in low complete response and off-target effects on Treg cells. (Hernandez et al. (2022) Engineering IL-2 for immunotherapy of autoimmunity and cancer. Nat Rev Immunol. 22(10): 614-628.).

[0225] Administering low dose IL-2 (about one million unit daily) for autoimmunity and other inflammatory diseases showed that Treg cells were selectively expanded due to the high expression of CD25 on Treg cells (Hernandez et al. (2022) Engineering IL-2 for immunotherapy of autoimmunity and cancer. Nat Rev Immunol. 22( 10):614-628). Low dose IL-2 has been tested in different kinds of patients such as with graft versus host disease (GVHD), hepatitis C virus (HCV), type 1 diabetes (T1D). Low dose IL-2 is safe to support endogenous Treg cells and adoptively transferred Treg cells in T1D patients, but does not support effector T cells. Furthermore, low-dose IL-2 administration requires repeat injection.

[0226] Even though drawbacks of IL-2 usage, IL-2 remains an attractive therapy method due to its effects on effector T cells, NK cells, and Treg cells. IL-2 analogues have been designed to target particular IL-2R to limit off-target immune responses, including muteins, PEGylated IL-2, IL-2- antibody complex, IL-2-CD25 complex (Hernandez et al. (2022) Engineering IL-2 for immunotherapy of autoimmunity and cancer. Nat Rev Immunol. 22(10): 614-628). The engineered IL-2 analogues were designed to bind to middle affinity receptor (IL-2Rp / IL-2Ry), but not high affinity receptor (IL-2Ra / IL-2Rp / IL-2Ry), leading to only activate effector cells, but not Treg cells.

[0227] For tumor immunotherapy, IL-2 may activate NK and effector T, but not Treg cells. IL-2 mutein was point mutated structurally to block the binding to IL-2Ra while with a similar affinity to that of IL-2 to the IL-2RPy complex. These point mutants could stimulate NK and T cells, without high level of pro-inflammatory cytokine (IFNy, IL- 1 P, TNF-a) and less VLS (Jiang et al. (2016) Role of IL-2 in cancer immunotherapy. Oncoimmunology. 5(6):el 163462; Merchant et al. (2022) Finetuned long-acting interleukin-2 superkine potentiates durable immune responses in mice and nonhuman primate. Journal for ImmunoTherapy of Cancer. 10:e003155.), also, IL-2 mutants could stimulate less expansion of Treg cells compared with wild-type IL-2. The structural alternation of IL- 2 to improve or reduce specific biological property is a milestone breakthrough in the cancer immunotherapy.

[0228] Crystal structure of IL-2 / IL-2R signaling complex showed that IL-2Ra / IL-2 interaction has high abundance of charge-charge interactions, and with rapid association rate and high-affinity interaction. In addition, IL-2Ra has no contacts with IL-2Rp / IL-2Ry, and only minor changes in the IL-2 protein interacting with receptor binding. Facing to IL-2Ra, amino acids of IL-2 play the principal role to form hydrophobic center and form charge-charge interaction, such as K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, K64, P65, E68, L72, and Y107. Among them, F42, F44, Y45, P65, and L72 combine to form hydrophobic center, and amino acids K35, R38, K43, E61, E62 to form polar periphery (Stauber et al. (2006) Crystal structure of the IL-2 signaling complex: paradigm for a heterotrimeric cytokine receptor. Proc Natl Acad Sci U S A. 103(8):2788-93).

[0229] We designed novel IL-2 mutants (saftein) to bind middle affinity receptor (IL-2RJ3 / IL- 2Ry), but not high affinity receptor (IL-2Ra / IL-2Rp / IL-2Ry), to activate effector immune response, but not immunosuppression and avoid toxicity such as VLS. We screened the membrane-bound form safteins and found that some formats of saftein are much better IL-2 analogues to bind IL-2R and support NK, T, and Treg cell growth, in addition, saftein 2 was found to be the best efficient effector NK and T cell growth factor, but does not support Treg survival and growth. Furthermore, soluble saftein 2 was confirmed to support NK, T cell survival and growth with significant higher efficiency than recombinant human IL-2, but did not support Treg cells.5.3.2 An engineered immune cell comprising an exogenously introduced polynucleotide encoding a mutated interleukin-2 (IL-2)

[0230] In certain embodiments, provided herein is an engineered immune cell comprising an exogenously introduced polynucleotide encoding a mutated interleukin-2 (IL-2), i.e., IL-2 mutants or saftein. In certain embodiments, the engineered immune cell expresses the mutated IL-2. In certain embodiments, the engineered immune cell expresses the mutated IL-2 on the cell surface. In certain embodiments, the engineered immune cell grows in a media that does not contain cytokine.

[0231] In certain embodiments, the mutated IL-2 binds a middle affinity receptor (IL-2RJ3 / IL- 2Ry), but not a high affinity receptor (IL-2Ra / IL-2Rp / IL-2Ry). In certain embodiments, the mutated IL-2 leads to activate effector immune response, but not immune suppressiveness and avoid toxicity such as VLS. In certain embodiments, the mutated IL-2 provided herein is designed based on the crystal structure of IL-2 / IL-2R complex, and the facing amino acids of IL-2 to IL-2R.

[0232] The mutated IL-2 are provided in FIG. 3E. In certain embodiments, the wild-type IL-2 comprises the amino acid of SEQ ID NO:39. In certain embodiments, the mutated IL-2 differs from wild-type IL-2 in that the amino acid residues at positions 35 to 38 and 61 to 62 are deleted (Saftein 1; IL-2 A35-38+A61-62). In certain embodiments, the mutated IL-2 differs from wild-type IL-2 in that the amino acid residues at positions 42 to 45 are deleted (Saftein 2; IL-2 A42-45). In certain embodiments, the mutated IL-2 differs from wild-type IL-2 in that the amino acid residues at positions 42 to 62 are deleted (Saftein 3; IL-2 A42-62). In certain embodiments, the mutated IL-2 differs from wild-type IL-2 in that the amino acid residues at positions 42 to 72 are deleted (Saftein 4; IL-2 A42-72). In certain embodiments, the mutated IL-2 differs from wild-type IL-2 in that the amino acid residues at positions 61 to 72 are deleted (Saftein 5; IL-2 A61-72). In certain embodiments, the mutated IL-2 differs from wild-type IL-2 by including mutations R38A, F42A, Y45A, and E62A (Saftein 6)(R. Vazquez-Lombardi, et al. (2017) Potent antitumour activity of interleukin-2 -Fc fusion proteins requires Fc-mediated depletion of regulatory T-cells. Nat. Commun. 8, p. 15373). In certain embodiments, the mutated IL-2 differs from wild-type IL-2 by including mutations F42A, Y45A, and L72G (Saftein 7)(Ahmed L. Alaofi. (2022) IL-2 Flexible Loops Might Play a Role in IL-2 Interaction with the High-Affinity IL-2 Receptor: A Molecular Dynamics (MD) Study. Journal of Chemistry, Article ID 3646375). In certain embodiments, the mutated IL-2 differs from wild-type IL-2 by including mutations F42A, K43A, F43A, and Y45A (Saftein 8). In certain embodiments, the mutated IL-2 differs from wild-type IL-2 in that the amino acid residues at positions 35 to 38 are deleted (Saftein 9; IL-2 A35-38). In certain embodiments, the mutated IL-2 differs from wild-type IL-2 in that the amino acid residues at positions 35 to 45 are deleted (Saftein 10; IL-2 A35-45). In certain embodiments, the mutated IL-2 differs from wild-type IL-2 in that the amino acid residues at positions 38 to 45 are deleted (Saftein 11; IL-2 A38-45). In certain embodiments, the mutated IL-2 differs from wild-type IL-2 in that the amino acid residues at positions 61 to 62 are deleted (Saftein 12; IL-2 A61-62). In certain embodiments, the mutated IL-2 differs from wild-type IL-2 in that the amino acid residues at positions 61 to 65 are deleted (Saftein 13; IL-2 A61-65). In certain embodiments, the mutated IL-2 differs from wild-type IL-2 in that the amino acid residues at positions 65 to 72 are deleted (Saftein 14; IL-2 A65-72).

[0233] In certain embodiments, the mutated IL-2 comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identical to the mutated IL-2 amino acid sequence as set forth in the amino acid sequence of 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, or SEQ ID NO: 122. In certain embodiments, the mutated IL-2 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the mutated IL-2 amino acid sequence as set forth in the amino acid sequence of 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, or SEQ ID NO: 122. In certain embodiments, the mutated IL-2 comprises the mutated IL-2 amino acid sequence as set forth in the amino acid sequence of 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, or SEQ ID NO: 122. In certain embodiments, the mutated IL-2 comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identical to the mutated IL-2 amino acid sequence as set forth in the amino acid sequence of SEQ ID NO: 110. In certain embodiments, the mutated IL-2 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the mutated IL-2 amino acid sequence as set forth in the amino acid sequence of SEQ ID NO: 110. In certain embodiments, the mutated IL-2 comprises the mutated IL-2 amino acid sequence as set forth in the amino acid sequence of SEQ ID NO: 110.

[0234] In certain embodiments, the mutated IL-2 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:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:63. In certain embodiments, the mutated IL-2 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:63. In certain embodiments, the mutated IL-2 comprises the amino acid sequence of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:63. In certain embodiments, the mutated IL-2 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: 47. In certain embodiments, the mutated IL-2 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:47. In certain embodiments, the mutated IL-2 comprises the amino acid sequence of SEQ ID NO:47.

[0235] In certain embodiments, the mutated IL-2 is membrane-bound. In certain embodiments, the mutated IL-2 is linked to a transmembrane domain of a polypeptide directly. In certain embodiments, the mutated IL-2 is linked to a transmembrane domain of a polypeptide via a peptide linker provided herein. In certain embodiments, the mutated IL-2 is linked to a transmembrane domain of a polypeptide via a hinge domain. In certain embodiments, the mutated IL-2 is linked to ahinge domain of a polypeptide, which is linked to a transmembrane domain of a polypeptide via a peptide linker provided herein. In certain embodiments, the hinge domain linked to the mutated IL-2 and the transmembrane domain linked to the hinge domain are derived from the same polypeptide. In certain embodiments, the hinge domain linked to the mutated IL-2 and the transmembrane domain linked to the hinge domain are derived from the same CD8. In certain embodiments, the hinge domain linked to the mutated IL-2 and the transmembrane domain linked to the hinge domain is derived from different polypeptides.

[0236] In certain embodiments, the hinge domain of a polypeptide can be any hinge domain of any polypeptide that can function in an engineered immune cell. A person of ordinary skill in the art can use known methods to test if a hinge domain of a polypeptide can function in an engineered immune cell, for example, methods as shown in the Example section herein. In certain embodiments, the hinge domain of a polypeptide is the hinge domain of CD8.

[0237] In certain embodiments, the transmembrane domain of a polypeptide can be any transmembrane domain of any polypeptide that can function in an engineered immune cell. A person of ordinary skill in the art can use known methods to test if a transmembrane domain of a polypeptide can function in an engineered immune cell, for example, methods as shown in the Example section herein. In certain embodiments, the transmembrane domain of a polypeptide is the transmembrane domain of CD 8.

[0238] In certain embodiments, the engineered immune cell is an NK cell. In certain embodiments, the engineered immune cell is an NKT cell. In certain embodiments, the engineered immune cell is a T cell, such as CAR-T, TIL, TCR-T, gamma-deltaT, and double negative DN-T. In certain embodiments, the engineered immune cell is a Treg cell.

[0239] In certain embodiments, the engineered immune cell further comprises a chimeric antigen receptor (CAR). In certain embodiments, the CAR is any CAR known in the art. In certain embodiments, the CAR is a CAR provided herein. In certain embodiments, the CAR is a CAR provided in U.S. Provisional Appl. No. 63 / 376,931, the entirety of which is incorporated by reference herein.5.3.3 A polypeptide comprising an amino acid sequence of a mutated IL-2

[0240] In one aspect, provided herein are polypeptides. In certain embodiments, provided herein is a polypeptide comprising an amino acid sequence of a mutated IL-2.

[0241] The mutated IL-2 are provided in FIG. 3E. In certain embodiments, the wild-type IL-2 comprises the amino acid of SEQ ID NO:39. In certain embodiments, the polypeptide comprises an amino acid sequence of an mutated IL-2 that differs from wild-type IL-2 in that the amino acid residues at positions 35 to 38 and 61 to 62 are deleted (Saftein 1; IL-2 A35-38+A61-62). In certain embodiments, the polypeptide comprises an amino acid sequence of an mutated IL-2 that differs from wild-type IL-2 in that the amino acid residues at positions 42 to 45 are deleted (Saftein 2; IL-2 A42-45). In certain embodiments, the polypeptide comprises an amino acid sequence of an mutated IL-2 that differs from wild-type IL-2 in that the amino acid residues at positions 42 to 62 are deleted (Saftein 3; IL-2 A42-62). In certain embodiments, the polypeptide comprises an amino acid sequence of an mutated IL-2 that differs from wild-type IL-2 in that the amino acid residues at positions 42 to 72 are deleted (Saftein 4; IL-2 A42-72). In certain embodiments, the polypeptide comprises an amino acid sequence of an mutated IL-2 that differs from wild-type IL-2 in that the amino acid residues at positions 61 to 72 are deleted (Saftein 5; IL-2 A61-72). In certain embodiments, the polypeptide comprises an amino acid sequence of an mutated IL-2 that differs from wild-type IL-2 by including mutations R38A, F42A, Y45A, and E62A (Saftein 6)(R. Vazquez-Lombardi, et al. (2017) Potent antitumour activity of interleukin-2 -Fc fusion proteins requires Fc-mediated depletion of regulatory T- cells. Nat. Commun. 8, p. 15373). In certain embodiments, the polypeptide comprises an amino acid sequence of an mutated IL-2 that differs from wild-type IL-2 by including mutations F42A, Y45A, and L72G (Saftein 7)(Ahmed L. Alaofi. (2022) IL-2 Flexible Loops Might Play a Role in IL-2 Interaction with the High-Affinity IL-2 Receptor: A Molecular Dynamics (MD) Study. Journal of Chemistry, Article ID 3646375). In certain embodiments, the polypeptide comprises an amino acid sequence of an mutated IL-2 that differs from wild-type IL-2 by including mutations F42A, K43A, F43A, and Y45A (Saftein 8). In certain embodiments, the polypeptide comprises an amino acid sequence of an mutated IL-2 that differs from wild-type IL-2 in that the amino acid residues at positions 35 to 38 are deleted (Saftein 9; IL-2 A35-38). In certain embodiments, the polypeptide comprises an amino acid sequence of an mutated IL-2 that differs from wild-type IL-2 in that the amino acid residues at positions 35 to 45 are deleted (Saftein 10; IL-2 A35-45). In certain embodiments, the polypeptide comprises an amino acid sequence of an mutated IL-2 that differs from wild-type IL-2 in that the amino acid residues at positions 38 to 45 are deleted (Saftein 11; IL-2 A38- 45). In certain embodiments, the polypeptide comprises an amino acid sequence of an mutated IL-2 that differs from wild-type IL-2 in that the amino acid residues at positions 61 to 62 are deleted (Saftein 12; IL-2 A61-62). In certain embodiments, the polypeptide comprises an amino acid sequence of an mutated IL-2 that differs from wild-type IL-2 in that the amino acid residues at positions 61 to 65 are deleted (Saftein 13; IL-2 A61-65). In certain embodiments, the polypeptide comprises an amino acid sequence of an mutated IL-2 that differs from wild-type IL-2 in that the amino acid residues at positions 65 to 72 are deleted (Saftein 14; IL-2 A65-72).

[0242] In certain embodiments, provided herein is a polypeptide comprising an amino acid sequence of a chimeric antigen receptor (CAR) provided herein and an amino acid sequence of a mutated IL-2 provided herein. In certain embodiments, the mutated IL-2 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO: 63.

[0243] In certain embodiments, provided herein is a polypeptide comprising an amino acid sequence of a mutated IL-2 provided herein, an amino acid sequence of a hinge domain, and an amino acid sequence of a transmembrane domain.

[0244] In certain embodiments, the polypeptide 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: 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, or SEQ ID NO: 122. In certain embodiments, the polypeptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of 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, or SEQ ID NO: 122. In certain embodiments, the polypeptide comprises the amino acid sequence of 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, or SEQ ID NO: 122. In certain embodiments, the polypeptide 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: 110. In certain embodiments, the polypeptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 110. In certain embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 110.5.3.4 Method of culturing an immune cell

[0245] In one aspect, provided herein are methods of culturing an immune cell. In certain embodiments, provided herein is a method of culturing an immune cell comprising culturing the immune cell in a media comprising a mutated interleukin-2 (IL-2) provided herein. In certain embodiments, the mutated IL-2 supports the growth of immune cells, such as T cells and NK cells. In certain embodiments, the mutated IL-2 does not support the grows of Regulatory T cells (Tregs). In certain embodiments, the mutated IL-2 show lower level of support of the growth of Regulatory T cells (Tregs) than that of the growth of other immune cells.

[0246] In certain embodiments, the mutated IL-2 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:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:63. In certain embodiments, the mutated IL-2 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:63. In certain embodiments, the mutated IL-2comprises the amino acid sequence of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:63. In certain embodiments, the mutated IL-2 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: 47. In certain embodiments, the mutated IL-2 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:47. In certain embodiments, the mutated IL-2 comprises the amino acid sequence of SEQ ID NO:47.

[0247] In certain embodiments, the mutated IL-2 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:48, SEQ ID NO:53, SEQ ID NO:55, or SEQ ID NO:58. In certain embodiments, the mutated IL-2 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:48, SEQ ID NO:53, SEQ ID NO:55, or SEQ ID NO:58. In certain embodiments, the mutated IL-2 comprises the amino acid sequence of SEQ ID NO:48, SEQ ID NO:53, SEQ ID NO:55, or SEQ ID NO:58. In certain embodiments, the mutated IL-2 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:48. In certain embodiments, the mutated IL-2 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:48. In certain embodiments, the mutated IL-2 comprises the amino acid sequence of SEQ ID NO:48.

[0248] In certain embodiments, the media does not contain cytokine other than a mutated IL-2. In certain embodiments, the mutated IL-2 is soluble.

[0249] In certain embodiments, the immune cell is an NK cell. In certain embodiments, the immune cell is an NKT cell. In certain embodiments, the engineered immune cell is a T cell, such as CAR-T, TIL, TCR-T, gamma-deltaT, and double negative DN-T. In certain embodiments, the immune cell is a Treg cell.

[0250] In certain embodiments, the immune cell is an engineered immune cell. In certain embodiments, the engineered immune cell comprises a CAR. In certain embodiments, the CAR is any CAR known in the art. In certain embodiments, the CAR is a CAR provided herein. In certain embodiments, the CAR is a CAR provided in U.S. Provisional Appl. No. 63 / 376,931, the entirety of which is incorporated by reference herein. In certain embodiments, the engineered immune cell is an engineered immune cell provided in Section 5.4.2.5.4 Transforming growth factor beta receptor II (TGF RII)

[0251] In one aspect, provided herein are engineered immune cells. In certain embodiments, provided herein is an engineered immune cell comprising an exogenously introduced polynucleotide, wherein the exogenously introduced polynucleotide comprises:(i) a first nucleic acid encoding an extracellular domain of transforming growth factor beta receptor II (TGF[3RII); and(ii) a second nucleic acid encoding a transmembrane domain derived from CD8a, CD4, CD7 or 2B4.

[0252] In certain embodiments, the exogenously introduced polynucleotide further comprises a nucleic acid encoding a CD8 signal peptide, a CD4 signal peptide, a CD7 signal peptide or a 2B4 signal peptide.

[0253] In certain embodiments, the engineered immune cell is an NK cell, an NKT cell, a T cell, or a Treg cell.

[0254] In certain embodiments, the engineered immune cell comprises a chimeric antigen receptor (CAR), wherein the CAR comprises:(i) an extracellular antigen binding domain;(ii) a transmembrane domain; and(iii) an intracellular signaling domain.

[0255] 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. In certain embodiments, the extracellular antigen binding domain of the CAR comprises an scFv. In certain embodiments, the extracellular antigen binding domain of the CAR binds to a tumor antigen or a fragment thereof.

[0256] In certain embodiments, the intracellular signaling domain of the CAR comprises an intracellular domain derived from a mutated CD84.

[0257] In certain embodiments, the interaction between the intracellular signaling domain of the CAR and SH2 domain-containing tyrosine phosphatase 2 (SHP2) is lower than the interaction between the intracellular domain of a second CAR and SHP2, and wherein the second CAR comprises an intracellular signaling domain comprising an intracellular domain derived from a wildtype CD84.

[0258] 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. In certain embodiments, the hinge domain of the CAR comprises a hinge domain derived from CD84.

[0259] In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain derived from CD84.

[0260] In certain embodiments, the intracellular signaling domain of the CAR further comprises an intracellular domain derived from DAP 10, DAP 12 or DNAM1. In certain embodiments, the intracellular domain of the CAR comprises an intracellular domain derived from DAP 10.

[0261] In certain embodiments, the intracellular signaling domain of the CAR further comprises an intracellular domain derived from CD3zeta. In certain embodiments, the intracellular signalingdomain of the CAR further comprises an intracellular domain derived from SLAM-associated protein (SAP). In certain embodiments, the intracellular signaling domain of the CAR further comprises an intracellular domain derived from Ewing’s sarcoma-activated transcript-2 (EAT2). In certain embodiments, the intracellular domain derived from CD3zeta is linked to the intracellular domain derived from SAP or the intracellular domain derived from EAT2 directly or via a peptide linker. In certain embodiments, the intracellular domain derived from CD3zeta is linked to the intracellular domain derived from SAP or the intracellular domain derived from EAT2 via P2A, F2A, T2A, or E2A.

[0262] In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by one or more exons are deleted. In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 5 and exon 6 are deleted. In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 7 and exon 8 are deleted.

[0263] In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16. In certain embodiments, the intracellular domain of the CAR comprises the amino acid sequence of SEQ ID NO: 16.

[0264] In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:6, SEQ ID NO: 106, or SEQ ID NO: 107. In certain embodiments, the intracellular domain of the CAR comprises the amino acid sequence of SEQ ID NO:6, SEQ ID NO: 106, or SEQ ID NO: 107.

[0265] In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:69 or SEQ ID NO: 108. In certain embodiments, the intracellular domain of the CAR comprises the amino acid sequence of SEQ ID NO:69 or SEQ ID NO: 108.

[0266] In certain embodiments, the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO:20. In certain embodiments, the CAR comprises the amino acid sequence of SEQ ID NO:8, SEQ ID NON, SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO:20.

[0267] In certain embodiments, the exogenously introduced polynucleotide further comprises a nucleic acid sequence encoding the CAR.

[0268] In certain embodiments, the extracellular domain of TGFpRII comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:28. In certain embodiments, the extracellular domain of TGFpRII comprises the amino acid sequence of SEQ ID NO:28.

[0269] In certain embodiments, the transmembrane domain derived from CD8a comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:3. In certain embodiments, the transmembrane domain derived from CD4 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:22. In certain embodiments, the transmembrane domain derived from CD7 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:24. In certain embodiments, the transmembrane domain derived from 2B4 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:26.

[0270] In certain embodiments, the CD8 signal peptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the CD4 signal peptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:21. In certain embodiments, the signal peptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:23. In certain embodiments, the 2B4 signal peptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:25.

[0271] In certain embodiments, the exogenously introduced polynucleotide comprises a nucleic acid sequence encoding a transmembrane domain of CD8a. In certain embodiments, the transmembrane domain of CD8a comprises the amino acid sequence of SEQ ID NO:3.

[0272] In certain embodiments, the exogenously introduced polynucleotide further comprises a nucleic acid sequence encoding a CD8 signal peptide. In certain embodiments, the CD8 signal peptide comprises the amino acid sequence of SEQ ID NO: 1.

[0273] In one aspect, provided herein are polypeptides. In certain embodiments, provided herein is a polypeptide comprising an extracellular domain of growth factor beta receptor II (TGF[3RII) and a transmembrane domain of CD8a, CD4, CD7 or 2B4.

[0274] In certain embodiments, the extracellular domain of TGFpRII and the transmembrane domain of CD8a, CD4, CD7 or 2B4 are linked to each other directly or via a linker.

[0275] In certain embodiments, the polypeptide further comprises a CD4 signal peptide, a CD7 signal peptide or a 2B4 signal peptide.

[0276] In certain embodiments, the transmembrane domain derived from CD8a comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:3. In certain embodiments, the transmembrane domain derived from CD4 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:22. In certain embodiments, the transmembrane domain derived from CD7 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:24. In certain embodiments, the transmembrane domain derived from 2B4 comprises an aminoacid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:26.

[0277] . In certain embodiments, the CD8 signal peptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the CD4 signal peptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:21. In certain embodiments, the CD7 signal peptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:23. In certain embodiments, the 2B4 signal peptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:25.

[0278] In certain embodiments, the polypeptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38.

[0279] In certain embodiments, the exogenously introduced polynucleotide comprises a nucleic acid sequence encoding a transmembrane domain of CD8a. In certain embodiments, the transmembrane domain of CD8a comprises the amino acid sequence of SEQ ID NO:3.

[0280] In certain embodiments, the exogenously introduced polynucleotide further comprises a nucleic acid sequence encoding a CD8 signal peptide. In certain embodiments, the CD8 signal peptide comprises the amino acid sequence of SEQ ID NO: 1.

[0281] In one aspect, provided herein are polynucleotides. In certain embodiments, provided herein is a polynucleotide encoding the polypeptide provided herein. In another aspect, provided herein are vectors. In certain embodiments, provided herein is a vector comprising the polynucleotide provided herein.

[0282] In one aspect, provided herein are pharmaceutical compositions. In certain embodiments, provided herein is a pharmaceutical composition comprising(iii) the engineered immune cell provided herein, the polypeptide provided herein, the polynucleotide provided herein, or the vector provided herein; and(ii) a pharmaceutically acceptable excipient.

[0283] In another aspect, provided herein are methods of treating a disease or disorder in a subject. In certain embodiments, provided herein is a method of treating a disease or disorder in a subject, comprising administering to the subject an effective amount of the engineered immune cell provided herein or the pharmaceutical composition provided herein.

[0284] In certain embodiments, the disease or disorder is cancer.

[0285] In another aspect, provided herein are methods of engineering an immune cell. In certain embodiments, provided herein is a method of engineering an immune cell, comprising introducing into the immune cell the polypeptide provided herein, the polynucleotide provided herein, or the vector provided herein.5.4.1 IntroductionTumor microenvironment and TGF-

[0286] Solid tumor microenvironment (TME) is heterogeneous, which is complex and continuously evolving entity, with the property of low nutrient, toxic, hypoxic, and immunosuppressive. The establishment of tumor means the solid tumor adapts to the unfavorable environment and overcomes glucose deprivation, oxidative stress, hypoxia, and immune response. Tumor TME is composed of immune cells, stromal cells, blood vessels, and extracellular matrix (ECM). Tumor TME is an active promoter of cancer progression, which forms a barrier to protect the tumor from immune systems and finally leads to tumor metastasis (Anderson et al. (2020) The tumor microenvironment. Curr Biol. 30(16):R921-R925).

[0287] In tumor TME, TGF-P can be produced by many kinds of cells, such as fibroblasts, mesenchymal stem cells, epithelial tumor cells, adipose tissue derived stem cells, and endothelial cells. TGF-P suppresses early tumorigenesis by inhibiting cell growth and apoptosis, and promotes advanced tumor progression and metastasis by promoting epithelial mesenchymal transition (ETM), establishment of fibrotic TME, promoting angiogensis, producing extracellular matrix (ECM), and inhibiting immune response. TGF-P also impairs the immune response of T cell, NK cell, macrophage, and dendritic cell (DC). (Morikawa et al. (2016) TGF-P and the TGF-P Family: Context- Dependent Roles in Cell and Tissue Physiology. Cold Spring Harb Perspect Biol. 8(5):a021873.).

[0288] As a potent immune suppressor, TGF-P suppresses tumor adaptive immunity. TGF-P inhibits CD4+T cell differentiation into effector subtypes, but it can induce T cell differentiation into regulatory T cells (Treg) and Treg proliferation. TGF-P also inhibits the processing of tumor antigens and presentation by DC, resulting in the inhibition of cytotoxic CD8+T cell activation, maturation and expression of IFNg and IL-2. Additionally, TGF-P promotes programmed cell death protein 1 (PD-1) in CD8 + T cells leading to T cell exhaustion.

[0289] TGF-P impairs NK cell function through direct or indirect ways. On one hand, TGF-P directly inhibits the T-bet transcription factor, downregulates the expression of activation receptor such as NKp30, NKG2D, leading to inhibition of NK cell cytotoxicity and IFN-y production (Melaiu et al. (2020) Influence of the Tumor Microenvironment on NK Cell Function in Solid Tumors. Front Immunol. 10:3038.). The expression of DNAX activating protein 12 kDa (DAP12), an essential NKG2D adaptor in human NK cells, were downregulated by TGF-p-induced miR-183. TGF-P can inhibit NK cell function by repressing the IL- 15 induced activation by targeting the serine and threonine kinase mTOR (Bou-Tayeh et al. (2021) Chronic IL-15 Stimulation and Impaired mTOR Signaling and Metabolism in Natural Killer Cells During Acute Myeloid Leukemia. Front Immunol. 12:730970.). TGF-P can also differentiate NK cells into ILC type 1 (ILC1), missing of cytotoxic functions. On the other hand, TGF-P indirectly inhibits NK cells. First, TGF-P can downregulate NK cell activation receptor ligands in the TME, such as the MICA (NKG2D ligand) in cancer patients.Second, TGF-P was shown to bind and enhance suppressive cells such as MDSCs and Treg cells in liver cancer model (Zalfa et al. (2021) Natural Killer Cell Interactions With Myeloid Derived Suppressor Cells in the Tumor Microenvironment and Implications for Cancer Immunotherapy. Front Immunol. 12:633205.). Third, TGF-P can impair CD56dim NK recruitment and favor that of CD56bright NK cells by reducing the expression of chemokines (CXCL2, CX3CL1, CXCL1, and CXCL8), and increasing chemokines (CXCL9, CXCL10, CXCL19, and CCL5) (Melaiu et al. (2020) Influence of the Tumor Microenvironment on NK Cell Function in Solid Tumors. Front Immunol. 10:3038.).

[0290] The TGF-p-induced cell suppression responses lead to cancer cell escape, that is one of major reasons of cancer patients’ recurrence after chemotherapy, radiation and other targeted therapy. But by far, single therapy targeting TGF-P results in very limited response. And clinical trials combining PD-L1 antibody and TGF-P antibody to treat tumor was more efficient than single treatment. (Gulley et al. (2022) Dual inhibition of TGF-P and PD-L1: a novel approach to cancer treatment. Mol Oncol. 16(11):2117-2134.).Dominant negative-TGFpRII (DNTbRII)

[0291] TGF-pi, 2 and 3 are large, secreted protein precursors with 70-80% homology. They are synthesized in a latent form and must be activated to bind to tetrameric receptor complex of TGF-P receptors I and II. TGF-P first binds to receptor TGFpRII, then the complex is recognized and bound by TGFpRI, and then the constitutively active TGFpRII phosphorylates TGFpRI at glycine and serine residues (GS domain), Phosphorylation of the GS domain triggers the substrates SMAD2 / SMAD3 and cascade. (Heldin et al. (2016) Signaling Receptors for TGF-P Family Members. Cold Spring Harb Perspect Biol. 8(8):a022053.).

[0292] Blocking TGF-P signaling is beneficial to improve the killing ability of NK cells against breast cancer cells. Dominant negative TGFpRII (DNTbRII) favored NK cell products resistant to the suppressive effects of TGF-P (Kennedy et al. (2022) Challenges to the broad application of allogeneic natural killer cell immunotherapy of cancer. Stem Cell Res Ther. 13(1): 165.). In T cells, truncated TGFpRII, but not absence of TGFpRII mediated signaling, induces memory CD8+T cell expansion (Ishigame et al. (2013) Truncated form of TGFpRII, but not its absence, induces memory CD8+ T cell expansion and lymphoproliferative disorder in mice. J Immunol. 190( 12): 6340-50). DNTbRII-T cells targeted to tumor through natural receptors or CARs showed elevated adoptive immunotherapy in several preclinical studies (Andrea et al. (2022) Advances in CAR-T Cell Genetic Engineering Strategies to Overcome Hurdles in Solid Tumors Treatment. Front Immunol. 13:830292).

[0293] We designed the dominant negative TGFpRII (DNTbRII) using the extracellular part of mature TGFpRII using different signal peptide and different transmembrane, and without intracellular tail, we found good expression of the newly designed DNTbRII using CD8, CD4 or 2B4 signal peptide and the raw transmembrane domain of TGFpRII or the transmembrane domain of CD8.Importantly, the NK cells expressing DNTbRII could efficiently neutralize the suppressive role of TGF-P on NK cells and lead to significantly enhanced tumor eradication.5.4.2 An engineered immune cell comprising an exogenously introduced polynucleotide comprises a first nucleic acid encoding an extracellular domain of TGF RII and a second nucleic acid encoding a transmembrane domain

[0294] In one aspect, provided herein are engineered immune cells. In certain embodiments, provided herein is an engineered immune cell comprising an exogenously introduced polynucleotide, wherein the exogenously introduced polynucleotide comprises:(i) a first nucleic acid encoding an extracellular domain of transforming growth factor beta receptor II (TGF[3RII); and(ii) a second nucleic acid encoding a transmembrane domain derived from CD8a, CD4, CD7 or 2B4.

[0295] In certain embodiments, provided herein is an engineered immune cell comprising an exogenously introduced polynucleotide, wherein the exogenously introduced polynucleotide comprises a first nucleic acid encoding an extracellular domain of TGF[3RII and a second nucleic acid encoding a transmembrane domain derived from CD8a. In certain embodiments, provided herein is an engineered immune cell comprising an exogenously introduced polynucleotide, wherein the exogenously introduced polynucleotide comprises a first nucleic acid encoding an extracellular domain of TGF[3RII and a second nucleic acid encoding a transmembrane domain derived from CD4. In certain embodiments, provided herein is an engineered immune cell comprising an exogenously introduced polynucleotide, wherein the exogenously introduced polynucleotide comprises a first nucleic acid encoding an extracellular domain of TGF[3RII and a second nucleic acid encoding a transmembrane domain derived from CD7. In certain embodiments, provided herein is an engineered immune cell comprising an exogenously introduced polynucleotide, wherein the exogenously introduced polynucleotide comprises a first nucleic acid encoding an extracellular domain of TGF[3RII and a second nucleic acid encoding a transmembrane domain derived from 2B4.

[0296] In certain embodiments, the exogenously introduced polynucleotide further comprises a nucleic acid encoding a signal peptide. In certain embodiments, the exogenously introduced polynucleotide further comprises a nucleic acid encoding a CD8 signal peptide. In certain embodiments, the exogenously introduced polynucleotide further comprises a nucleic acid encoding a CD4 signal peptide. In certain embodiments, the exogenously introduced polynucleotide further comprises a nucleic acid encoding a CD7 signal peptide. In certain embodiments, the exogenously introduced polynucleotide further comprises a nucleic acid encoding a 2B4 signal peptide.

[0297] In certain embodiments, the engineered immune cell is an NK cell. In certain embodiments, the engineered immune cell is an NKT cell. In certain embodiments, the engineered immune cell is a T cell, such as CAR-T, TIL, TCR-T, gamma-deltaT, and double negative DN-T. In certain embodiments, the engineered immune cell is a Treg cell.

[0298] In certain embodiments, the engineered immune cell further comprises a chimeric antigen receptor (CAR). In certain embodiments, the CAR is any CAR known in the art. In certain embodiments, the CAR is a CAR provided herein. In certain embodiments, the CAR is a CAR provided in U.S. Provisional Appl. No. 63 / 376,931, the entirety of which is incorporated by reference herein. In certain embodiments, the exogenously introduced polynucleotide further comprises a nucleic acid sequence encoding the CAR.

[0299] In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by one or more exons are deleted. In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 5 is deleted. In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 6 is deleted. In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 7 is deleted. In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 8 is deleted. In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 5 and exon 6 are deleted. In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 7 and exon 8 are deleted.

[0300] In certain embodiments, the extracellular domain of TGFpRII 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:28. In certain embodiments, the extracellular domain of TGFpRII comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:28. In certain embodiments, the extracellular domain of TGFpRII comprises the amino acid sequence of SEQ ID NO:28.

[0301] In certain embodiments, the transmembrane domain derived from CD8a 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:3. In certain embodiments, the transmembrane domain derived from CD8a comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:3. In certain embodiments, the transmembrane domain derived from CD4 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:22. In certain embodiments, the transmembrane domain derived from CD4 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:22. In certain embodiments, the transmembrane domain derived from CD7 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:24. In certain embodiments, the transmembrane domain derived from CD7 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:24. In certain embodiments, the transmembrane domain derived from 2B4 comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identical to theamino acid sequence of SEQ ID NO:26. In certain embodiments, the transmembrane domain derived from 2B4 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:26.

[0302] In certain embodiments, the CD8 signal peptide 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: 1. In certain embodiments, the CD8 signal peptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the CD4 signal peptide 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:21. In certain embodiments, the CD4 signal peptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:21. In certain embodiments, the signal peptide 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:23. In certain embodiments, the signal peptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:23. In certain embodiments, the 2B4 signal peptide 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:25. In certain embodiments, the 2B4 signal peptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:25.

[0303] In certain embodiments, the exogenously introduced polynucleotide comprises a nucleic acid sequence encoding a transmembrane domain of CD8a. In certain embodiments, the transmembrane domain of CD8a comprises the amino acid sequence of SEQ ID NO:3. In certain embodiments, the exogenously introduced polynucleotide further comprises a nucleic acid sequence encoding a CD8 signal peptide. In certain embodiments, the CD8 signal peptide comprises the amino acid sequence of SEQ ID NO: 1.5.4.3 A polypeptide comprising an extracellular domain of TGF RII and a transmembrane domain

[0304] In one aspect, provided herein are polypeptides. In certain embodiments, provided herein is a polypeptide comprising an extracellular domain of growth factor beta receptor II (TGF[3RII) and a transmembrane domain of CD8a, CD4, CD7 or 2B4.

[0305] In certain embodiments, the extracellular domain of TGF[3RII and the transmembrane domain of CD8a, CD4, CD7 or 2B4 are linked to each other directly or via a linker.

[0306] In certain embodiments, the polypeptide further comprises a CD4 signal peptide, a CD7 signal peptide or a 2B4 signal peptide.

[0307] In certain embodiments, the transmembrane domain derived from CD8a 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:3. In certain embodiments, the transmembrane domainderived from CD8a comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:3. In certain embodiments, the transmembrane domain derived from CD4 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:22. In certain embodiments, the transmembrane domain derived from CD4 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:22. In certain embodiments, the transmembrane domain derived from CD7 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:24. In certain embodiments, the transmembrane domain derived from CD7 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:24. In certain embodiments, the transmembrane domain derived from 2B4 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:26. In certain embodiments, the transmembrane domain derived from 2B4 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:26.

[0308] In certain embodiments, the CD8 signal peptide 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: 1. In certain embodiments, the CD8 signal peptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the CD4 signal peptide 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:21. In certain embodiments, the CD4 signal peptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:21. In certain embodiments, the signal peptide 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:23. In certain embodiments, the signal peptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:23. In certain embodiments, the 2B4 signal peptide 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:25. In certain embodiments, the 2B4 signal peptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:25.

[0309] In certain embodiments, the polypeptide 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:35, SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38. In certain embodiments, the polypeptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, or SEQ ID NO:38.

[0310] In certain embodiments, the exogenously introduced polynucleotide comprises a nucleic acid sequence encoding a transmembrane domain of CD8a. In certain embodiments, the transmembrane domain of CD8a comprises the amino acid sequence of SEQ ID NO:3. In certain embodiments, the exogenously introduced polynucleotide further comprises a nucleic acid sequence encoding a CD8 signal peptide. In certain embodiments, the CD8 signal peptide comprises the amino acid sequence of SEQ ID NO: 1.5.5 Engineered immune cells comprising a CAR, a first nucleic acid encoding a mutated IL- 2, and a second nucleic acid encoding an extracellular domain of TGFpRII

[0311] In one aspect, provided herein are engineered immune cells. In certain embodiments, provided herein is an engineered immune cell comprising(i) a chimeric antigen receptor (CAR), comprising:(a) an extracellular antigen binding domain;(b) a transmembrane domain; and(c) an intracellular signaling domain comprising an intracellular domain derived from a mutated CD84;(ii) a first nucleic acid encoding a mutated interleukin-2 (IL-2); and(iii) a second nucleic acid encoding an extracellular domain of transforming growth factor beta receptor II (TGFpRII).

[0312] In certain embodiments, the interaction between the intracellular signaling domain of the CAR and SH2 domain-containing tyrosine phosphatase 2 (SHP2) is lower than the interaction between the intracellular domain of a second CAR and SHP2, and wherein the second CAR comprises an intracellular signaling domain comprising an intracellular domain derived from a wildtype CD84.

[0313] In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 7 and exon 8 are deleted.

[0314] In certain embodiments, the mutated IL-2 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:63. In certain embodiments, the mutated IL-2 comprises the amino acid sequence of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:63.

[0315] In certain embodiments, the engineered immune cell grows in a media that does not contain cytokine.

[0316] In certain embodiments, the first nucleic acid and the second nucleic acid are on the same polynucleotide. In certain embodiments, the first nucleic acid or the second nucleic acid is on a polynucleotide comprising a nucleic acid encoding the CAR.

[0317] In certain embodiments, the nucleic acid encoding the CAR, the second nucleic acid encoding the extracellular domain of TGF[3RII, and the first nucleic acid encoding the mutated IL-2 are each linked to each other directly or via a nucleic acid encoding a linker.

[0318] 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. In certain embodiments, the hinge domain of the CAR comprises a hinge domain derived from CD84. In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain derived from CD84.

[0319] In certain embodiments, the intracellular signaling domain of the CAR further comprises an intracellular domain derived from DAP 10, DAP 12 or DNAM1. In certain embodiments, the intracellular signaling domain of the CAR further comprises an intracellular domain derived from CD3zeta.

[0320] In certain embodiments, the extracellular antigen binding domain of the CAR comprises an scFv. In certain embodiments, the extracellular antigen binding domain of the CAR binds to a tumor antigen or a fragment thereof.

[0321] In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by one or more exons are deleted. In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 5 and exon 6 are deleted. In certain embodiments, the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 7 and exon 8 are deleted.

[0322] In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

[0323] In certain embodiments, the intracellular domain of the CAR comprises the amino acid sequence of SEQ ID NO: 16.

[0324] In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:6, SEQ ID NO: 106, or SEQ ID NO: 107.

[0325] In certain embodiments, the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO:20.

[0326] In certain embodiments, the extracellular domain of TGF[3RII comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:28.

[0327] In certain embodiments, the first nucleic acid or the second nucleic acid is on a polynucleotide comprising a nucleic acid encoding a transmembrane domain.

[0328] In certain embodiments, the transmembrane domain comprises a transmembrane domain derived from CD8a, CD4, CD7 or 2B4.

[0329] In certain embodiments, the transmembrane domain comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26. In certain embodiments, the transmembrane domain comprises a transmembrane domain derived from CD8a.

[0330] In certain embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO:3.

[0331] In certain embodiments, the second nucleic acid is on a polynucleotide comprising a nucleic acid sequence encoding a signal peptide.

[0332] In certain embodiments, the signal peptide comprises a CD8 signal peptide, a CD4 signal peptide, a CD7 signal peptide or a 2B4 signal peptide. In certain embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:25. In certain embodiments, the signal peptide comprises a CD8 signal peptide. In certain embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 1.

[0333] In certain embodiments, the engineered immune cell is an NK cell, an NKT cell, a T cell, or a Treg cell.

[0334] In certain embodiments, the CAR, from N-terminus to C-terminus, comprising:(i) an extracellular antigen binding domain;(ii) a transmembrane domain comprising an transmembrane domain derived from CD84;(iii) an intracellular signaling domain comprising(a) an intracellular domain derived from DAP 10;(b) an intracellular domain derived from a mutated CD84, wherein the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 7 and exon 8 are deleted; and(c) an intracellular domain derived from CD3zeta, and wherein the 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.

[0335] In one aspect, provided herein are polypeptides. In certain embodiments, provided herein is a polypeptide polypeptide comprising(i) a chimeric antigen receptor (CAR), comprising:(a) an extracellular antigen binding domain;(b) a transmembrane domain; and(c) an intracellular signaling domain comprising an intracellular domain derived from a mutated CD84;(ii) a mutated interleukin-2 (IL-2); and(iii) an extracellular domain of transforming growth factor beta receptor II (TGFpRII).

[0336] In certain embodiments, the polypeptide further comprises a CD8 signal peptide, a CD4 signal peptide, a CD7 signal peptide or a 2B4 signal peptide.

[0337] In certain embodiments, the polypeptide further comprises a CD8 signal peptide. In certain embodiments, the polypeptide further comprises a transmembrane domain of CD8a, CD4, CD7 or 2B4. In certain embodiments, the polypeptide further comprises a transmembrane domain of CD8a. In certain embodiments, the polypeptide further comprises P2A. In certain embodiments, the polypeptide further comprises one or more spacers.

[0338] In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16. In certain embodiments, the intracellular domain of the CAR comprises the amino acid sequence of SEQ ID NO: 16.

[0339] In certain embodiments, the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:6, SEQ ID NO: 106, or SEQ ID NO: 107.

[0340] In certain embodiments, the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO:20.

[0341] In certain embodiments, the extracellular domain of TGF RII comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:28.

[0342] In certain embodiments, the polypeptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26. In certain embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:3. In certain embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO : 1 , SEQ ID NO : 21 , SEQ ID NO : 23 , or SEQ ID NO : 25.

[0343] In certain embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:67, or SEQ ID NO:68.

[0344] In one aspect, provided herein are polynucleotides. In certain embodiments, provided herein is a polynucleotide encoding the polypeptide provided herein. In another aspect, provided herein are vectors. In certain embodiments, provided herein is a vector comprising the polynucleotide provided herein.

[0345] In one aspect, provided herein are pharmaceutical compositions. In certain embodiments, provided herein is a pharmaceutical composition comprising(iv) the engineered immune cell provided herein, the polypeptide provided herein, thepolynucleotide provided herein, or the vector provided herein; and(ii) a pharmaceutically acceptable excipient.

[0346] In another aspect, provided herein are methods of treating a disease or disorder in a subject. In certain embodiments, provided herein is a method of treating a disease or disorder in a subject, comprising administering to the subject an effective amount of the engineered immune cell provided herein or the pharmaceutical composition provided herein.

[0347] In certain embodiments, the disease or disorder is cancer.

[0348] In another aspect, provided herein are methods of engineering an immune cell. In certain embodiments, provided herein is a method of engineering an immune cell, comprising introducing into the immune cell the polypeptide provided herein, the polynucleotide provided herein, or the vector provided herein.5.5.1 Introduction

[0349] CD84, also named SLAM family member 5 (SLAMF5), contains four critical tyrosines on the cytoplasmic tail, two of which lie in consensus immunoreceptor tyrosine-based switch motifs (ITSM), and recruit SLAM-associated protein (SAP), Ewing's sarcoma-associated transcript 2 (EAT- 2), and SH2 enzymes including SHP-2, SHP-1, Csk, and SHIP-1. Here, we designed CD84 chimeric antigen receptors (CARs) and expressed them on natural killer (NK) cells and found that SAP facilitates CD84 CAR to trigger the full activation ofNK cells. CD84A5,6 CAR has impaired activation signaling, while CD84A7,8 CAR, with complete signaling, overcomes the barrier of inhibitory signaling in NK cells, dispensible of SAP.

[0350] Solid tumor microenvironment (TME) is immunosuppressive. TGF-J3, a potent immune suppressor in the TME, impairs the immune response of T cells, NK cells, macrophages, and dendritic cells. Blocking TGF-J3 signaling is beneficial to the killing ability of NK cells towards tumor cells.Overexpressing dominant negative TGF[3RII (DNTbRII) renders NK cells and T cells to escape from the suppressive effects of TGF-J3. We thus designed DNTbRII composed of extracellular TGF-J3 receptor II with CD8a transmembrane and found that the DNTbRII -NK cells efficiently antagonized the suppression of NK cells by TGF-J3, resulting in significantly enhanced tumor eradication.

[0351] Membrane-bound cytokines play the similar role to the soluble form of cytokines. In addition, the toxicity and side-effects induced by cytokines can be prevented by minimizing cytokine releasing into circulation. Accordingly, We designed and screened various membrane -bound cytokines including IL-2, IL-7, IL- 15, IL- 18 and IL-21 and tested their effects on NK cell survival and growth. We found that mbIL-2 and mbIL-15 efficiently supported NK cell survival and growth. More importantly, mbIL-2 enhanced NK cell killing ability significantly.

[0352] IL-2 as a pluripotent cytokine, is produced by activated CD4+T cells, and supports T cell growth andl differentiation, Treg development, and NK cell activation, survival and growth. In patient, treatment with IL-2 not only supports effector T or NK cell functions, but also triggers Tregcell proliferation. We thus designed different types of IL-2 mutants (“Saftein”) and screened their binding affinities to effector T or NK cells, but not to Treg cells. We selected Saftein 2 as the best candidate. Either of its membrane-bound and soluble forms, supported the survival and growth of T and NK cells, but not those of Treg cells.

[0353] CD84A7,8 CAR combining saftein 2 and DNTbRII was expressed on NK cells and showed excellent complete signaling CAR activation and NK cell sustenance and renewal, and the renewable NK (reNK) cells do not need cytokines to support their survival and growth, and the reNK cells effectively antagonize the suppression by TGF-p, resulting in enhanced tumor eradication activity.

[0354] In certain embodiments, we combined the saftein 2 and DNTbRII to arm CD84A78- csCARNK cells (sd-csCAR-reNK), and found that the CAR-NK cells could antagonize TGF-p, gain the property of renewal, and could be completely activated by overcoming the barrier of inhibitory receptor signaling, resulting in significantly higher efficiency in elimination of tumor cells.5.5.2 Exemplary engineered immune cells

[0355] In certain embodiments, provided herein is an engineered immune cell comprising(i) a chimeric antigen receptor (CAR);(ii) a first nucleic acid encoding a mutated interleukin-2 (IL-2); and(iii) a second nucleic acid encoding an extracellular domain of transforming growth factor beta receptor II (TGFpRII).

[0356] In certain embodiments, the CAR is any CAR known in the art. In certain embodiments, the CAR is a CAR provided herein. In certain embodiments, the CAR is a CAR provided in U.S. Provisional Appl. No. 63 / 376,931, the entirety of which is incorporated by reference herein.

[0357] In certain embodiments, the mutated IL-2 is any mutated IL-2 known in the art. In certain embodiments, the mutated IL-2 is a mutated IL-2 provided herein.

[0358] In certain embodiments, provided herein is an engineered immune cell comprising(i) a chimeric antigen receptor (CAR), comprising:(a) an extracellular antigen binding domain;(b) a transmembrane domain; and(c) an intracellular signaling domain comprising an intracellular domain derived from a mutated CD84;(ii) a first nucleic acid encoding a mutated interleukin-2 (IL-2); and(iii) a second nucleic acid encoding an extracellular domain of transforming growth factor beta receptor II (TGFpRII).

[0359] In certain embodiments, the engineered immune cell grows in a media that does not contain cytokine.

[0360] In certain embodiments, the first nucleic acid and the second nucleic acid are on the same polynucleotide. In certain embodiments, the first nucleic acid or the second nucleic acid is on a polynucleotide comprising a nucleic acid encoding the CAR.

[0361] In certain embodiments, the nucleic acid encoding the CAR, the second nucleic acid encoding the extracellular domain of TGF[3RII, and the first nucleic acid encoding the mutated IL-2 are each linked to each other directly or via a nucleic acid encoding a linker.

[0362] In certain embodiments, the first nucleic acid or the second nucleic acid is on a polynucleotide comprising a nucleic acid encoding a transmembrane domain, certain embodiments, the transmembrane domain comprises a transmembrane domain derived from CD 8a. In certain embodiments, the transmembrane domain comprises a transmembrane domain derived from CD4. In certain embodiments, the transmembrane domain comprises a transmembrane domain derived from CD7. In certain embodiments, the transmembrane domain comprises a transmembrane domain derived from B2A.

[0363] In certain embodiments, the transmembrane domain derived from CD8a 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:3. In certain embodiments, the transmembrane domain derived from CD8a comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:3. In certain embodiments, the transmembrane domain derived from CD4 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:22. In certain embodiments, the transmembrane domain derived from CD4 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:22. In certain embodiments, the transmembrane domain derived from CD7 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:24. In certain embodiments, the transmembrane domain derived from CD7 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:24. In certain embodiments, the transmembrane domain derived from 2B4 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:26. In certain embodiments, the transmembrane domain derived from 2B4 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:26. In certain embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO:3.

[0364] In certain embodiments, the second nucleic acid is on a polynucleotide comprising a nucleic acid sequence encoding a signal peptide. In certain embodiments, the CD8 signal peptide 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: 1. In certain embodiments, the CD8 signal peptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the aminoacid sequence of SEQ ID NO: 1. In certain embodiments, the CD4 signal peptide 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:21. In certain embodiments, the CD4 signal peptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:21. In certain embodiments, the signal peptide 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:23. In certain embodiments, the signal peptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:23. In certain embodiments, the 2B4 signal peptide 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:25. In certain embodiments, the 2B4 signal peptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:25. In certain embodiments, the signal peptide comprises a CD8 signal peptide. In certain embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 1.

[0365] In certain embodiments, the engineered immune cell is an NK cell. In certain embodiments, the engineered immune cell is an NKT cell. In certain embodiments, the engineered immune cell is a T cell, such as CAR-T, TIL, TCR-T, gamma-deltaT, and double negative DN-T. In certain embodiments, the engineered immune cell is a Treg cell.

[0366] In certain embodiments, provided herein is an engineered immune cell comprising(i) a chimeric antigen receptor (CAR), comprising:(a) an extracellular antigen binding domain;(b) a transmembrane domain; and(c) an intracellular signaling domain; wherein the interaction between the intracellular signaling domain of the CAR and SH2 domain-containing tyrosine phosphatase 2 (SHP2) is reduced;(ii) a first nucleic acid encoding a mutated interleukin-2 (IL-2); and(iii) a second nucleic acid encoding an extracellular domain of transforming growth factor beta receptor II (TGFpRII).

[0367] In certain embodiments, provided herein is an engineered immune cell comprising(i) a chimeric antigen receptor (CAR), comprising:(a) an extracellular antigen binding domain;(b) a transmembrane domain; and(c) an intracellular signaling domain comprising an intracellular domain derived from a mutated CD84 provided herein;(ii) a first nucleic acid encoding a mutated interleukin-2 (IL-2); and(iii) a second nucleic acid encoding an extracellular domain of transforming growth factor beta receptor II (TGFpRII).

[0368] In certain embodiments, provided herein is an engineered immune cell comprising(i) a chimeric antigen receptor (CAR), comprising:(a) an extracellular antigen binding domain;(b) a transmembrane domain; and(c) an intracellular signaling domain comprising an intracellular domain derived from a mutated CD84, wherein the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 7 and exon 8 are deleted;(ii) a first nucleic acid encoding a mutated interleukin-2 (IL-2); and(iii) a second nucleic acid encoding an extracellular domain of transforming growth factor beta receptor II (TGF[3RII).

[0369] In certain embodiments, provided herein is an engineered immune cell comprising(i) a chimeric antigen receptor (CAR), comprising:(a) an extracellular antigen binding domain;(b) a transmembrane domain; and(c) an intracellular signaling domain; wherein the interaction between the intracellular signaling domain of the CAR and SH2 domain-containing tyrosine phosphatase 2 (SHP2) is reduced;(ii) a first nucleic acid encoding a mutated interleukin-2 (IL-2), wherein the mutated IL-2 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:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO: 60, SEQ ID NO:61, SEQ ID NO: 62, or SEQ ID NO: 63; and(iii) a second nucleic acid encoding an extracellular domain of transforming growth factor beta receptor II (TGFpRII).

[0370] In certain embodiments, provided herein is an engineered immune cell comprising(i) a chimeric antigen receptor (CAR), comprising:(a) an extracellular antigen binding domain;(b) a transmembrane domain; and(c) an intracellular signaling domain comprising an intracellular domain derived from a mutated CD84, wherein the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 7 and exon 8 are deleted;(ii) a first nucleic acid encoding a mutated interleukin-2 (IL-2), wherein the mutated IL-2 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:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO: 60, SEQ ID NO:61, SEQ ID NO: 62, or SEQ ID NO: 63; and(iii) a second nucleic acid encoding an extracellular domain of transforming growth factor betareceptor II (TGFpRII).

[0371] In certain embodiments, provided herein is an engineered immune cell comprising(i) a chimeric antigen receptor (CAR), comprising:(a) an extracellular antigen binding domain;(b) a transmembrane domain; and(c) an intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 16;(ii) a first nucleic acid encoding a mutated interleukin-2 (IL-2), wherein the mutated IL-2 comprises the amino acid sequence of SEQ ID NO:47; and(iii) a second nucleic acid encoding an extracellular domain of transforming growth factor beta receptor II (TGFpRII).

[0372] In certain embodiments, provided herein is an engineered immune cell comprising(i) a chimeric antigen receptor (CAR), comprising:(a) an extracellular antigen binding domain;(b) a transmembrane domain; and(c) an intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 16;(ii) a first nucleic acid encoding a mutated interleukin-2 (IL-2), wherein the mutated IL-2 comprises the amino acid sequence of SEQ ID NO:47; and(iii) a second nucleic acid encoding an extracellular domain of transforming growth factor beta receptor II (TGFpRII), wherein the extracellular domain of TGFpRII comprises the amino acid sequence of SEQ ID NO:28.5.5.3 A polypeptide comprising a CAR, a mutated IL-2 and an extracellular domain of TGFPRII

[0373] In one aspect, provided herein are polypeptides. In certain embodiments, provided herein is a polypeptide comprising(i) a chimeric antigen receptor (CAR), comprising:(a) an extracellular antigen binding domain;(b) a transmembrane domain; and(c) an intracellular signaling domain comprising an intracellular domain derived from a mutated CD84;(ii) a mutated interleukin-2 (IL-2); and(iii) an extracellular domain of transforming growth factor beta receptor II (TGFpRII).

[0374] In certain embodiments, the polypeptide further comprises a CD8 signal peptide, a CD4 signal peptide, a CD7 signal peptide or a 2B4 signal peptide.

[0375] In certain embodiments, the polypeptide further comprises a CD8 signal peptide. In certain embodiments, the polypeptide further comprises a transmembrane domain of CD8a, CD4,CD7 or 2B4. In certain embodiments, the polypeptide further comprises a transmembrane domain of CD8a. In certain embodiments, the polypeptide further comprises P2A, F2A, E2A or T2A. In certain embodiments, the polypeptide further comprises one or more spacers.

[0376] In certain embodiments, the polypeptide 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:3, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26. In certain embodiments, the polypeptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26.

[0377] In certain embodiments, the polypeptide 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: 1, SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:25. In certain embodiments, the polypeptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:25. In certain embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:3. In certain embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:25.

[0378] In certain embodiments, the polypeptide 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:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:67, or SEQ ID NO:68. In certain embodiments, the polypeptide comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:67, or SEQ ID NO:68. In certain embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO:67, or SEQ ID NO:68.5.6 Polynucleotides and vectors

[0379] In yet another aspect, provided herein are polynucleotide. In certain embodiments, provided herein is a polynucleotide encoding the CAR provided herein. In certain embodiments, provided herein is a polynucleotide encoding the polypeptide provided herein.

[0380] The polynucleotides of the disclosure can be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double -stranded or singlestranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand. In some embodiments, the polynucleotide is in the form of cDNA. In some embodiments, the polynucleotide is a synthetic polynucleotide.

[0381] The present application further relates to variants of the polynucleotides described herein, wherein the variant encodes, for example, fragments, analogs, and / or derivatives of the CAR provided herein or the polypeptide provided herein. In certain embodiments, the present application provides apolynucleotide comprising a polynucleotide having a nucleotide sequence 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 to a polynucleotide encoding the CAR provided herein or the polypeptide provided herein. As used herein, the phrase “a polynucleotide having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence” is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence can include up to five point mutations per each 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 in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence. These mutations of the reference sequence can occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

[0382] The polynucleotide variants can contain alterations in the coding regions, non-coding regions, or both. In some embodiments, a polynucleotide variant contains alterations which produce silent substitutions, additions, or deletions, but does not alter the properties or activities of the encoded polypeptide. In some embodiments, a polynucleotide variant comprises silent substitutions that results in no change to the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code). Polynucleotide variants can be produced for a variety of reasons, for example, to optimize codon expression for a particular host (i. e. , change codons in the human mRNA to those preferred by a bacterial host such as E. coli). In some embodiments, a polynucleotide variant comprises at least one silent mutation in a non-coding or a coding region of the sequence.

[0383] In some embodiments, a polynucleotide variant is produced to modulate or alter expression (or expression levels) of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to increase expression of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to decrease expression of the encoded polypeptide. In some embodiments, a polynucleotide variant has increased expression of the encoded polypeptide as compared to a parental polynucleotide sequence. In some embodiments, a polynucleotide variant has decreased expression of the encoded polypeptide as compared to a parental polynucleotide sequence.

[0384] In yet another aspect, provided herein are vectors. In certain embodiments, provided herein is a vector comprising the polynucleotide provided herein.

[0385] In an embodiment, the polynucleotide can be incorporated into a recombinant expression vector. The present application provides recombinant expression vectors comprising any of the polynucleotide provided herein. As used herein, the term “recombinant expression vector” means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of anmRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell. The vectors described herein are not naturally-occurring as a whole; however, parts of the vectors can be naturally-occurring. The described recombinant expression vectors can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides. The recombinant expression vectors can comprise naturally- occurring or non-naturally-occurring intemucleotide linkages, or both types of linkages. The non- naturally occurring or altered nucleotides or intemucleotide linkages do not hinder the transcription or replication of the vector.5.7 Pharmaceutical composition

[0386] In yet another aspect, provided herein are pharmaceutical compositions. In certain embodiments, provided herein is a pharmaceutical composition comprising(i) the CAR provided herein, the engineered immune cell provided herein, the polypeptide provided herein, the polynucleotide provided herein, or the vector provided herein; and(ii) a pharmaceutically acceptable excipient.

[0387] In certain embodiments, provided herein is a pharmaceutical composition comprising(i) the CAR provided herein, and(ii) a pharmaceutically acceptable excipient.

[0388] In certain embodiments, provided herein is a pharmaceutical composition comprising(i) the engineered immune cell provided herein; and(ii) a pharmaceutically acceptable excipient.

[0389] In certain embodiments, provided herein is a pharmaceutical composition comprising(i) the polypeptide provided herein; and(ii) a pharmaceutically acceptable excipient.

[0390] In certain embodiments, provided herein is a pharmaceutical composition comprising(i) the polynucleotide provided herein; and(ii) a pharmaceutically acceptable excipient.

[0391] In certain embodiments, provided herein is a pharmaceutical composition comprising(i) the vector provided herein; and(ii) a pharmaceutically acceptable excipient.

[0392] In certain embodiments, provided herein is a pharmaceutical composition comprising the CAR provided herein, the engineered immune cell provided herein, the polypeptide provided herein, the polynucleotide provided herein, or the vector provided herein. In certain embodiments, provided herein is a pharmaceutical composition comprising the CAR provided herein. In certain embodiments,provided herein is a pharmaceutical composition comprising the engineered immune cell provided herein. In certain embodiments, provided herein is a pharmaceutical composition comprising the polypeptide provided herein. In certain embodiments, provided herein is a pharmaceutical composition comprising the polynucleotide provided herein. In certain embodiments, provided herein is a pharmaceutical composition comprising the vector provided herein.

[0393] In certain embodiments, provided herein is a pharmaceutical composition comprising an effective amount of the CAR provided herein, an effective amount of the engineered immune cell provided herein, an effective amount of the polypeptide provided herein, an effective amount of the polynucleotide provided herein, or an effective amount of the vector provided herein. In certain embodiments, provided herein is a pharmaceutical composition comprising an effective amount of the CAR provided herein. In certain embodiments, provided herein is a pharmaceutical composition comprising an effective amount of the engineered immune cell provided herein. In certain embodiments, provided herein is a pharmaceutical composition comprising an effective amount of the polypeptide provided herein. In certain embodiments, provided herein is a pharmaceutical composition comprising an effective amount of the polynucleotide provided herein. In certain embodiments, provided herein is a pharmaceutical composition comprising an effective amount of the vector provided herein.

[0394] In one embodiment, each component is “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio. See, e.g., Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, pharmaceutically acceptable excipients are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. In some embodiments, a pharmaceutically acceptable excipient is an aqueous pH buffered solution.

[0395] In a specific embodiment, the term “excipient” can also refer to a diluent, adjuvant (e.g., Freunds’ adjuvant (complete or incomplete), carrier or vehicle. Pharmaceutical excipients can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, 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. Such compositions contain a prophylactically or therapeutically effective amount of the active ingredient provided herein, such as in purified form, together with a suitable amount of excipient so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0396] In some embodiments, the choice of excipient is determined in part by the particular cell, binding molecule, and / or antibody, and / or by the method of administration. Accordingly, there are a variety of suitable formulations.

[0397] Typically, acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants including ascorbic acid, methionine, Vitamin E, sodium metabisulfite; preservatives, isotonicifiers, stabilizers, metal complexes (e.g. Zn-protein complexes); chelating agents such as EDTA and / or non-ionic surfactants.

[0398] Buffers may be used to control the pH in a range which optimizes the therapeutic effectiveness, especially if stability is pH dependent. Suitable buffering agents for use with the present disclosure include both organic and inorganic acids and salts thereof. For example, citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. Additionally, buffers may comprise histidine and trimethylamine salts such as Tris.

[0399] Preservatives may be added to retard microbial growth. Suitable preservatives for use with the present disclosure include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3 -pentanol, and m-cresol.

[0400] Tonicity agents, sometimes known as “stabilizers” can be present to adjust or maintain the tonicity of liquid in a composition. When used with large, charged biomolecules such as proteins and antibodies, they are often termed “stabilizers” because they can interact with the charged groups of the amino acid side chains, thereby lessening the potential for inter and intra-molecular interactions. Exemplary tonicity agents include polyhydric sugar alcohols, trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.

[0401] Additional exemplary excipients include: (1) bulking agents, (2) solubility enhancers, (3) stabilizers and (4) agents preventing denaturation or adherence to the container wall. Such excipients include: polyhydric sugar alcohols (enumerated above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol; sulfur containing reducing agents, such as urea,glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglycerol and sodium thio sulfate; 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.

[0402] Non-ionic surfactants or detergents (also known as “wetting agents”) may be present to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. Suitable non-ionic surfactants include, e.g., polysorbates (20, 40, 60, 65, 80, etc.), polyoxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid ester, methyl celluose and carboxymethyl cellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyle sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0403] In order for the pharmaceutical compositions to be used for in vivo administration, they are preferably sterile. The pharmaceutical composition may be rendered sterile by filtration through sterile filtration membranes. The pharmaceutical compositions herein generally can be placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0404] The route of administration is in accordance with known and accepted methods, such as by single or multiple bolus or infusion over a long period of time in a suitable manner, e.g., injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, topical administration, inhalation or by sustained release or extended-release means.

[0405] In another embodiment, a pharmaceutical composition can be provided as a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release (see, e.g., Sefton, Crit. Ref. Biomed. Eng. 14:201-40 (1987); Buchwald et al., Surgery 88:507-16 (1980); and Saudek et al., N. Engl. I. 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, e.g., 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, I. 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., I. 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. WO 99 / 15154 and WO 99 / 20253). Examples of polymers used in sustained releaseformulations include, but are not limited to, poly(2 -hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, polyethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In one embodiment, the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In yet another embodiment, a controlled or sustained release system can be placed in proximity of a particular target tissue, for example, the nasal passages or lungs, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release Vol. 2, 115-38 (1984)). Controlled release systems are discussed, for example, by Langer, Science 249: 1527-33 (1990). Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more agents as described herein (see, e.g., U.S. Pat. No. 4,526,938, PCT publication Nos. WO 91 / 05548 and WO 96 / 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)).

[0406] The active ingredients may also be entrapped in microcapsules prepared, for example, by coascervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methyhnethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 18th edition.

[0407] Various compositions and delivery systems are known and can be used with the therapeutic agents provided herein, including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the single domain antibody or therapeutic molecule provided herein, construction of a nucleic acid as part of a retroviral or other vector, etc.

[0408] In some embodiments, the pharmaceutical composition provided herein contains the binding molecules and / or cells in amounts effective to treat or prevent the disease or disorder, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined.5.8 Therapeutic Methods and Uses

[0409] In yet another aspect, provided herein are methods of treating a disease or disorder in a subject. In certain embodiments, provided herein is a method of treating a disease or disorder in asubject, comprising administering to the subject an effective amount of the engineered immune cell provided herein or the pharmaceutical composition provided herein.

[0410] In certain embodiments, the method of treating a disease or disorder in a subject, comprises administering to the subject an effective amount of the engineered immune cell provided herein. In certain embodiments, the method of treating a disease or disorder in a subject, comprises administering to the subject an effective amount of the pharmaceutical composition provided herein.

[0411] As provided herein, the engineered immune cells provided herein are suitable for use as a cell-based therapy or therapies, such as in a method of treating cancer. In certain embodiments, treatment with the engineered immune cell provided herein or the pharmaceutical composition provided herein results in one, two, or more, or all of the following: (1) a reduction in severity, progression, spread, and / or frequency of one or more symptoms, (2) elimination of one or more symptoms and / or underlying cause, (3) prevention of the occurrence of one or more symptoms and / or their underlying cause, and (4) improvement or remediation of damage. In specific embodiments, treatment includes therapeutic treatment as well as prophylactic, or suppressive measures for the condition, disease or disorder.

[0412] Such methods and uses include therapeutic methods and uses, for example, involving administration of the molecules, cells, or compositions containing the same, to a subject having a disease, condition, or disorder. In some embodiments, the molecule, cell, and / or composition is administered in an effective amount to effect treatment of the disease or disorder. Uses include uses of the engineered immune cell provided herein or the pharmaceutical composition provided herein in such methods and treatments, and in the preparation of a medicament in order to carry out such therapeutic methods. In some embodiments, the methods are carried out by administering the engineered immune cell provided herein or the pharmaceutical composition, to the subject having or suspected of having the disease or condition. In some embodiments, the methods thereby treat the disease or disorder in the subject.

[0413] In some embodiments, the treatment provided herein cause complete or partial amelioration or reduction of a disease or disorder, or a symptom, adverse effect or outcome, or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. The terms include, but do not imply, complete curing of a disease or complete elimination of any symptom or effect(s) on all symptoms or outcomes.

[0414] As used herein, in some embodiments, the treatment provided herein delay development of a disease or disorder, e.g., defer, hinder, slow, retard, stabilize, suppress and / or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and / or individual being treated. As is evident to one skilled in the art, a sufficient orsignificant delay can, in effect, encompass prevention, in that the individual does not develop the disease or disorder. For example, a late stage cancer, such as development of metastasis, may be delayed.

[0415] In some embodiments, the methods include administration of the engineered immune cell provided herein or the pharmaceutical composition provided herein to a subject, tissue, or cell, such as one having, at risk for, or suspected of having the disease or disorder.

[0416] In some embodiments, the subject, to whom the engineered immune cell provided herein or the pharmaceutical composition provided herein are administered is a primate, such as a human. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some examples, the subject is a validated animal model for disease, adoptive cell therapy, and / or for assessing toxic outcomes.

[0417] The engineered immune cell provided herein or the pharmaceutical composition provided herein, can be administered by any suitable means, for example, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon's injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, they are administered by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

[0418] The amount of a prophylactic or therapeutic agent provided herein that is effective in the prevention and / or treatment of a disease or condition can be determined by standard clinical techniques. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. For the prevention or treatment of disease, the appropriate dosage of the binding molecule or cell may depend on the type of disease or disorder to be treated, the type of binding molecule, the severity and course of the disease or disorder, whether the therapeutic agent is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the agent, and the discretion of the attending physician. The compositions, molecules and cells are in some embodiments suitably administered to the patient at one time or over a series of treatments.

[0419] In the context of engineered immune cells, in some embodiments, a subject may be administered the range of about one million to about 100 billion cells and / or that amount of cells per kilogram of body weight. In some embodiments, wherein the pharmaceutical composition comprises any one of the engineered immune cells described herein, the pharmaceutical composition is administered at a dosage of at least about any of 104, 105, 106, 107, 108, or 109cells / kg of body weight of the individual. Dosages may vary depending on attributes particular to the disease or disorder and / or patient and / or other treatments.

[0420] In some embodiments, the pharmaceutical composition is administered for a single time. In some embodiments, the pharmaceutical composition is administered for multiple times (such as 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. A dosing cycle can be, e.g., 1, 2, 3, 4, 5 or more week(s), or 1, 2, 3, 4, 5, or more month(s). The optimal dosage and treatment regime for a particular patient can be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0421] In some embodiments, the engineered immune cell provided herein or the pharmaceutical composition provided herein are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as another antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent.

[0422] In some embodiments, the engineered immune cell provided herein or the pharmaceutical composition provided herein are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells or antibodies are administered prior to the one or more additional therapeutic agents. In some embodiments, the cells or antibodies are administered after to the one or more additional therapeutic agents.5.9 Methods of engineering an immune cell

[0423] In yet another aspect, provided herein are methods of engineering an immune cell. In certain embodiments, provided herein is a method of engineering an immune cell, comprising introducing into the immune cell the polynucleotide provided herein, the vector provided herein, or the polypeptide provided herein.

[0424] In certain embodiments, provided herein is a method of engineering an immune cell, comprising introducing into the immune cell the polynucleotide provided herein. In certain embodiments, provided herein is a method of engineering an immune cell, comprising introducing into the immune cell the vector provided herein. In certain embodiments, provided herein is a method of engineering an immune cell, comprising introducing into the immune cell the polypeptide provided herein.

[0425] In certain embodiments, the engineered immune cell is an NK cell. In certain embodiments, the engineered immune cell is an NKT cell. In certain embodiments, the engineered immune cell is a T cell, such as CAR-T, TIL, TCR-T, gamma-deltaT, and double negative DN-T. In certain embodiments, the engineered immune cell is a Treg cell.

[0426] In some embodiments, provided herein is a method of engineering an immune cell, comprising introducing into the immune cell a polynucleotide provided herein. In some embodiments,the method further comprises, selecting a immune cell for expression of the CAR encoded by the polynucleotide or the vector provided herein.5.10 Assays

[0427] The practice of the embodiments provided herein can employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, and immunology, which are within the skill of those working in the art. Such techniques are explained fully in the literature. Examples of particularly suitable texts for consultation include the following: Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., 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, N.Y., 2d ed. 1987); and / or Janeway, et al. Immunobiology (Garland Science, 7th ed. 2008).

[0428] 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 populations is measured by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered immune cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of engineered immune cells also can be measured by assaying expression and / or secretion of certain cytokines, such as CD107a, IFNy, IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.5.10.1 Cells, cell culture, and cloning

[0429] 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 healthy human donors’ peripheral blood. Any known cloning technology can be used herein.5.10.2 In vitro killing, degranulation assays

[0430] The killing activity of an engineered immune cell can be evaluated using various known methods in the art. For example, 293T, 293T-CD19, NALM6, and Huh7 target cells can be genetically modified and stably expressed Luc2-GFP as described previously (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(61 l):eabg4328). To assess killing activities, CAR-NK cellscan be co-incubated with target cells with different ratios for different time as indicated in figure legends. D-Luciferin can be used to measure luminescence emission spectra. Target cells without NK cells can be used as 100% live cell control. The killing ability can be determined based on the normalized spectrum and background subtraction and calculated using the equation killing%=( 1 - (exp / 100%live 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(61 l):eabg 4328).

[0431] The degranulation activity of an engineered immune cell can be evaluated using various known methods in the art. For example, to assess degranulation, fluorescently conjugated antiCD 107a antibody can be added to the NK-target cocultures and incubated, cells can then be washed with FACS buffer, and stained with anti-CD56 antibodies. CD56 stained CD107a+ NK cells can be quantified by flow cytometry analysis (Beckman Coulter CytoFLEX LX).5.10.3 Flow cytometry analysis

[0432] The IFN production can be evaluated using various known methods in the art. For example, to assess IFN production, live NK cells can be co-incubated with target cells in U-bottom 96-well plates after spinning. Brefeldin A can be added to the cells and incubated. Cell surface markers can be stained, washed, and fixed with IC Fixation Buffer (eBioscience), and washed and stained with cytokine antibodies using permeabilization buffer (eBioscience), and then analyzed by flow cytometry.

[0433] To check CD107a, 2pg / ml antibodies were co-cultured at 37°C for 2h, cells were stained for the surface molecule firstly, washed, fixed, and stained the CD 107a as performed for IFNg staining. Cells were analyzed by flow cytometry (Beckman Coulter CytoFLEX LX).5.10.4 Duolink PLA

[0434] As an example, molecule-molecule interaction assays can be determined using the in situ Duolink proximity ligation assay (PLA) red kit (Sigma- Aldrich) following the manufacturer’s instructions. The NK cells can be attached to the poly-1 -lysine glass, target cells can then be added and incubated. Unattached cells can be removed by washing with PBS. slides can be fixed and stained with anti-GFP (Rabbit IgG) and anti -DAP 10 (Mouse IgG), washed, and stained with anti -rabbit and anti-mouse PLA probes, followed by probe DNA ligation, amplification, Fluorescent staining can be then performed and observed using confocal microscopy.5.10.5 Retroviral transduction

[0435] Any known method for NK cell transduction can be used herein. For example, for NK cell transduction, Phoenix A cells can be transfected with expression vectors together with the packaging vector GagPol using the calcium method. Filtered viral supernatant, polybrene and IL-2can be mixed with NK line cells or 3-day expanded primary NK cells in 6-well plates. The plates cam be centrifuged. The transduced NK line cells can be cultured with respective media for in vitro experiments, the transduced primary NK cells can be co-cultured with inactivated K562-CD137L and recombinant IL-2 and used for in vitro validation and adoptive cell therapy experiment in mice in the next step.5.10.6 Soluble protein production and purification

[0436] Any known method for soluble protein production and purification can be used herein. For example, for soluble protein production, vectors can be used to transfect suspension cultured 293T using PEI MAX (25K), according to the protocol reported (Longo et al. (2013) Transient mammalian cell transfection with polyethylenimine (PEI). Methods Enzymol. 2013;529:227-40.). Cells can be cultured 72 h at 37 °C and 8% CO2. Cell culture supernatant can be collected, and 6HIS protein can be purified using Ni-NTA HISTrap column (Cytiva) and Superdex 200 10 / 300 GL chromatography column using AKTA (Cytiva). Protein can be dialyzed by PBS, and stored in -20 °C. Each protein sample (Ipg) can be dissolved using SDS-PAGE, and coomassie blue staining can be performed.5.10.7 WB assay

[0437] Any known method for measuring pSMAD2 and pSMAD3 levels can be used herein. For example, NK cells can be cultured with 5 pg / mL TGF-J3 or not for 5 days. NK cells can be lyzed using lysis buffer (50 mM Tris, 150 mM NaCl, 10 mM Sodium Pyrophosphate, 1 mM Phenylmethylsulfonyl Fluoride, 1 mM Sodium Metavanadate, 2 mM Sodium Fluoride, 0.5% Triton X-100, protease inhibitor cocktail (Roche) and lOOuM Sodium orthovanadate), Laemmili sample buffer can be added and boiled directly at 70 °C for 10 min, samples can be dissolved using SDS- PAGE and transferred to nitrogen membrane, the membrane can be blocked with 5% BSA, and stained with anti-pSMAD2, anti-pSMAD3, anti-SMAD2, anti-SMAD3, and internal control [3-actin antibodies. Fluorescent second antibodies can be used to stain the membrane, and the blot can be observed using iBright FL 1500 (ThermoFisher Scientific).5.10.8 Apoptosis assay, and proliferation assay

[0438] To quantify apoptosis, NK cells were cultured without cytokine for 3 days, the day 0 and day 3 NK cells were incubated 500 nM CellEvent Caspase 3 / 7 detection reagent (Thermo Fisher Scientific), and antibodies for cell surface markers BV605-CD56 for 30 minutes at 37 °C before the experiment end. To determine NK cell renewal, proliferation assay was performed using CellTrace Violet (CTV) cell proliferation kit (Thermofisher Scientific). NK cells was stained with CTV, and cultured without cytokine. CTV fluorescence was determined by flow cytometry (Beckman Coulter CytoFLEX LX) at day 0 and day 3 (Zhang et al. (2021) Ectopic activation of the miR-200c-EpCAMaxis enhances antitumor T cell responses in models of adoptive cell therapy. Sci Transl Med. 13(61 l):eabg4328.).6. EXAMPLES6.1 EXAMPLE 1: Exemplary materials and methods6.1.1 Cells

[0439] All cells were cultured at 37°C and 5% CO2. NK92 cells were cultured in RPMI-1640 supplemented with 2mM L-glutamine (Sigma), 1.5 g / L sodium bicarbonate (Sigma), 0.2 mM inositol (Sigma), 0.1 mM 2-Mercaptoethanol (Sigma), 0.02 mM Folic acid (Sigma), 100 lU / 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 10 mM HEPES, 2 mM L-glutamine, 1 x non- essential amino acids, ImM sodium pyruvate, 50 pM 2-mercaptoethanol and 10% FBS. 293T and HUH7 cells were cultured in complete Dulbecco’s Modified Eagle Medium DMEM with 10% FBS.

[0440] Human primary NK cells were separated from healthy donors’ peripheral blood by NK cell negative selection kit (STEMCELL Technoloties Inc.) following manufacturer’s instruction. The purified NK cells were mixed with Paraformaldehyde (PF A) fixed K562-CD137L cells (1: 1) with lOOIU / mL IL-2 and cultured at 37°C, 5% CO2 in serum -free OpTmizer T Cell Expansion medium (Gibco) supplemented with 10% of human IL-2 preparation (Hemagen Diagnostics). NK cells were gene modified at day 3, and the cultured NK cells were applied 2 weeks after continued expansion.6.1.2 Vector constructs

[0441] Primers and long DNA fragments were synthesized by ThermoFisher Scientific and GeneScript. CAR, mb-saftein, and DNTbRII-CD8aTM constructs were cloned into Bglll / Xhol of MSCV retrovirus vectors. In the combined CAR, the saftein and DNTbRII was led by P2A and follow the CAR sequence. For soluble protein expression, saftein DNA was optimized, synthesized and cloned into vector pcDNA3.1-HIS by GeneScript.Table 4. Gene Sequences Used in experiments6.1.3 In vitro killing, degranulation assays

[0442] 293T, 293T-CD19, 293T-CD19-HLA-E, K562, NALM6, HUH7 and Huh7-HLA-E target cells were genetically modified and stably expressed Luc2-GFP as described previously (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(61 l):eabg4328). To determine NK cell killing ability, CAR-NK cells were co-incubated with target cells at 37 °C with different ratios as indicated in figure. D-Luciferin (0.75 mg / mL) was used to measure luminescence. Target cells only were used as live cell control. The killing ability was determined based on the normalized luminescence spectrum and background subtraction. Killing assay was calculated via the equation killing%=(l-(exp / 100%live 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(61 l):eabg4328.). To observe the effect of TGF-P on NK killing ability, NK cells were incubated with TGF-P (5ng / mL or the dose indicated in graph) for 5 days before killing assay performed.

[0443] For the killing assay using LDH approach, NK cells were co-incubated with target cells (1 : 100) for 72 h, the LDH OD value was determined and minus that of the background with target cell only without NK cell.

[0444] To determine degranulation, NK-target cells were co-cultured with 2 pg / ml Allophycocyanin (APC) conjugated anti-CD107a antibody at 37 °C for 2 hours, cells were harvested, washed with PBS buffer with ImM EDTA, and stained with anti-CD56 antibodies. CD107a+ percentage in CD56+ NK cells was determined by flow cytometry (Beckman Coulter CytoFLEX LX). 6.1.4 Flow cytometry analysis of Treg, intracellular IFNg, and TGF-P on cell surface and intracell.

[0445] To check Treg, cells were stained for anti-CD4 / anti-CD25 on surface, washed, fixed and foxp3 was stained with Foxp3 / transcription factor staining kit according to manufacturer’s instruction (eBioscience).

[0446] To analyze IFNg production in NK cells, NK cells were stimulated by target cells (1: 1) at 37°C for 2 hours, Brefeldin A was added to media at final 5pg / ml, and continued to culture for 4 morehours. Cells were stained with surface marker, washed, fixed using IC Fixation Buffer (eBioscience), washed again and stained with cytokines in permeabilization buffer (eBioscience), and then analyzed by flow cytometry (Beckman Coulter CytoFLEX LX).

[0447] For TGF-J3 staining, tumor cells were stained for the surface molecule firstly, washed, fixed, and stained the intracellular TGF-P molecule as performed for IFNg staining. Cells were analyzed by flow cytometry (Beckman Coulter CytoFLEX LX).6.1.5 Phosflow analysis

[0448] For pSTAT5 determination, NK cells were starved 24 h using media without serum and cytokine. These NK cells were then cultured in media with soluble safteins for 30 min at 37°C. NK cells were collected and stained with surface marker on ice, then washed and fixed using 4% paraformaldehyde (PFA) for 10 min, after washing using PBS, methanol was used to fix cells again overnight at -20°C. Cells were washed twice using lx permeabilization buffer (eBioscience), and stained with pSTAT5. Cells were analyzed using flow cytometry (Beckman Coulter CytoFLEX LX).6.1.6 Retroviral transduction

[0449] For NK cell gene modification, phoenix A cells were transfected with MSCV and packaging vectors using CaPO4 method. Virus in the supernatant, mixed with 8 pg / ml polybrene and IL-2 (lOOIU / mL), was added to NK cell lines or 3 days primary cells in 6-well plates. Plates were centrifuged at 1400xg for 2 hours at 35 °C . The transduced NK cell lines were cultured with the same media as before and the transduced primary NK cells were expanded for 14 days and used for experiments.6.1.7 Soluble protein production and purification

[0450] For soluble protein production, saftein-pcDNA3.1-HIS vectors was used to transfect suspension cultured 293T using PEI MAX (25K), according to the protocol reported (Longo et al. (2013) Transient mammalian cell transfection with polyethylenimine (PEI). Methods Enzymol. 2013;529:227-40.). Cells were cultured 72 h at 37 °C and 8% CO2. Cell culture supernatant was collected, and 6HIS protein was purified using Ni-NTA HISTrap column (Cytiva) and Superdex 200 10 / 300 GL chromatography column using AKTA (Cytiva). Protein was dialyzed by PBS, and stored in -20 °C. Each protein sample (Ipg) was dissolved using SDS-PAGE, and coomassie blue staining was performed.6.1.8 WB assay

[0451] For checking pSMAD2 and pSMAD3 levels, NK cells was cultured with 5 pg / mL TGF-J3 or not for 5 days. NK cells were lyszed using lysis buffer (50 mM Tris, 150 mM NaCl, 10 mM Sodium Pyrophosphate, 1 mM Phenylmethylsulfonyl Fluoride, 1 mM Sodium Metavanadate, 2 mM Sodium Fluoride, 0.5% Triton X-100, protease inhibitor cocktail (Roche) and lOOuM Sodium ortho vanadate), Laemmili sample buffer was added and boiled directly at 70 °C for 10 min, sampleswere dissolved using SDS-PAGE and transferred to nitrogen membrane, the membrane was blocked with 5% BSA, and stained with anti-pSMAD2, anti-pSMAD3, anti-SMAD2, anti-SMAD3, and internal control [3-actin antibodies. Fluorescent second antibodies were used to stain the membrane, and the blot was observed using iBright FL1500 (ThermoFisher Scientific).6.1.9 Apoptosis assay, and proliferation assay

[0452] To quantify apoptosis, NK cells were cultured without cytokine for 3 days, the day 0 and day 3 NK cells were incubated 500 nM CellEvent Caspase 3 / 7 detection reagent (Thermo Fisher Scientific), and antibodies for cell surface markers BV605-CD56 for 30 minutes at 37 °C before the experiment end.

[0453] To determine NK cell renewal, proliferation assay was performed using CellTrace Violet (CTV) cell proliferation kit (Thermofisher Scientific). NK cells was stained with CTV, and cultured without cytokine. CTV fluorescence was determined by flow cytometry (Beckman Coulter CytoFLEX LX) at day 0 and day 3 (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(61 l):eabg4328.).6.1.10 Duolink PLA assay

[0454] Molecule-molecule interaction assays were determined using the in situ Duolink proximity ligation assay (PLA) red kit (Sigma-Aldrich) following the manufacturer’s instructions. CARO is an empty vector with GFP expression as control, or CAR-GFP fusion expressing NK cells were plated to the poly-L-lysine treated glass, target 293T-CD19-HLA-E cells were then co-incubated at 37°C for 30 minutes. Free cells were washed with PBS. Slides were fixed and stained with anti- GFP (Rabbit IgG) and anti-SAP (Mouse IgG), or anti-GFP (Rabbit IgG) and anti-SHP2 (Mouse IgG), washed, and stained with the second antibodies with DNA probes, probes DNA was ligated, amplified, fluorescent substrate stained, and then slides were observed using confocal microscopy,6.2 EXAMPLE 2: Mutated CD84 composed CAR-NK eradicated tumor cells efficiently.

[0455] Chimeric antigen receptor (CAR) engineered natural killer (NK) cells (CAR-NK) have been shown potential for adaptive anti-tumor immunotherapy. SLAM family molecules such as 2B4, CD84 are used to design CAR backbone for NK cells. However, the cytoplasmic immunoreceptor tyrosine-based switch motif (ITSM) motif can bind to SLAM-associated protein (SAP), and also competitively bind inhibitory molecules such as SHP (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 ofNK cell activity by 2B4, NTB-A and CRACC. Front Biosci. 13:956-65). We designed complete signaling csCAR for fully activation of NK cells to enhance activation signaling and overcome the barrier of inhibitory signaling (FIG. 1A) (U.S. Provisional Appl. No. 63 / 376,931). Here the CARs’ scFv is CD19, CARO is the empty vector asnegative control, CAR2 is the reported NKG2D-TM CAR as a positive control...

Claims

WHAT IS CLAIMED:

1. An engineered immune cell comprising(i) a chimeric antigen receptor (CAR), comprising:(a) an extracellular antigen binding domain;(b) a transmembrane domain; and(c) an intracellular signaling domain comprising an intracellular domain derived from a mutated CD84;(ii) a first nucleic acid encoding a mutated interleukin-2 (IL-2); and(iii) a second nucleic acid encoding an extracellular domain of transforming growth factor beta receptor II (TGFpRII).

2. An engineered immune cell comprising a chimeric antigen receptor (CAR), comprising:(a) an extracellular antigen binding domain;(b) a transmembrane domain; and(c) an intracellular signaling domain comprising an intracellular domain derived from a mutated CD84, wherein the interaction between the intracellular signaling domain of the CAR and SH2 domain-containing tyrosine phosphatase 2 (SHP2) is lower than the interaction between the intracellular domain of a second CAR and SHP2, and wherein the second CAR comprises an intracellular signaling domain comprising an intracellular domain derived from a wild-type CD84; or the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 7 and exon 8 are deleted.

3. An engineered immune cell comprising(i) a chimeric antigen receptor (CAR), comprising:(a) an extracellular antigen binding domain;(b) a transmembrane domain; and(c) an intracellular signaling domain, and(ii) an exogenously introduced polynucleotide, wherein the exogenously introduced polynucleotide:(aa) encodes a mutated interleukin-2 (IL-2), wherein the mutated IL-2 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:63; or(bb) comprises a first nucleic acid encoding an extracellular domain of transforming growth factor beta receptor II (TGFpRII); and a second nucleic acid encoding a transmembrane domain derived from CD8a, CD4, CD7 or 2B4.

4. The engineered immune cell of claim 1 or 3, wherein the mutated IL-2 is membrane -bound or linked to a transmembrane domain of a polypeptide directly, via a peptide linker, or via a hinge domain, optionally wherein the polypeptide is CD8.

5. The engineered immune cell of any one of claims 1, 3, or 4, wherein the mutated IL-2 comprises the amino acid sequence of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:63.

6. The engineered immune cell of any one of claims 1 to 5, wherein the engineered immune cell grows in a media that does not contain cytokine.

7. The engineered immune cell of claim 1, wherein the first nucleic acid encoding the mutated IL-2 and the second nucleic acid encoding the extracellular domain of TGFpRII are on the same polynucleotide.

8. The engineered immune cell of claim 1 or 7, wherein the first nucleic acid encoding the mutated IL-2 or the second nucleic acid encoding the extracellular domain of TGFpRII is on a polynucleotide comprising a nucleic acid encoding the CAR.

9. The engineered immune cell of claim 1, wherein the first nucleic acid encoding the mutated IL-2 or the second nucleic acid encoding the extracellular domain of TGFpRII is on a polynucleotide comprising a nucleic acid encoding a transmembrane domain.

10. The engineered immune cell of claim 9, wherein the transmembrane domain comprises a transmembrane domain derived from CD8a, CD4, CD7 or 2B4.

11. The engineered immune cell of claim 10, wherein the transmembrane domain comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26.

12. The engineered immune cell of any one of claims 1 and 7 to 11, wherein the second nucleic acid encoding the extracellular domain of TGFpRII is on a polynucleotide comprising a nucleic acid sequence encoding a signal peptide.

13. The engineered immune cell of claim 12, wherein the signal peptide comprises a CD8 signalpeptide, a CD4 signal peptide, a CD7 signal peptide or a 2B4 signal peptide.

14. The engineered immune cell of claim 13, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO : 1 , SEQ ID NO : 21 , SEQ ID NO : 23 , or SEQ ID NO : 25.

15. The engineered immune cell of any one of claims 1 to 14, wherein 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.

16. The engineered immune cell of claim 15, wherein the hinge domain of the CAR comprises a hinge domain derived from CD84.

17. The engineered immune cell of any one of claims 1 to 16, wherein the transmembrane domain of the CAR comprises a transmembrane domain derived from CD84.

18. The engineered immune cell of any one of claims 1 to 43, wherein the intracellular signaling domain of the CAR further comprises an intracellular domain derived from DAP 10, DAP 12 or DNAM1.

19. The engineered immune cell of any one of claims 1 to 18, wherein the intracellular signaling domain of the CAR further comprises an intracellular domain derived from CD3zeta.

20. The engineered immune cell of any one of claims 1 to 19, wherein the intracellular signaling domain of the CAR further comprises an intracellular domain derived from SLAM-associated protein (SAP).

21. The engineered immune cell of any one of claims 1 to 20, wherein the intracellular signaling domain of the CAR further comprises an intracellular domain derived from Ewing’s sarcoma- activated transcript-2 (EAT2).

22. The engineered immune cell of claim 20 or 21, wherein the intracellular domain derived from CD3zeta is linked to the intracellular domain derived from SAP or the intracellular domain derived from EAT2 directly or via a peptide linker.

23. The engineered immune cell of claim 22, wherein the intracellular domain derived from CD3zeta is linked to the intracellular domain derived from SAP or the intracellular domain derived from EAT2 via F2A, P2A, T2A, or E2A.

24. The engineered immune cell of any one of claims 1 to 19, wherein the engineered immunecell further comprises a second exogenously introduced polynucleotide encoding an intracellular domain derived from SLAM-associated protein (SAP) or Ewing’s sarcoma-activated transcript-2 (EAT2).

25. The engineered immune cell of claim 24, wherein the second exogenously introduced polynucleotide encodes the CAR, and wherein the intracellular signaling domain of the CAR comprises the intracellular domain derived from SAP or the intracellular domain derived from EAT2.

26. The engineered immune cell of any one of claims 1 to 25, wherein the extracellular antigen binding domain of the CAR comprises an scFv.

27. The engineered immune cell of any one of claims 1 to 26, wherein the extracellular antigen binding domain of the CAR binds to a tumor antigen or a fragment thereof.

28. The engineered immune cell of any one of claims 1, 2 and 4 to 27, wherein the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by one or more exons are deleted.

29. The engineered immune cell of any one of claims 1, 2 and 4 to 28, wherein the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 5 and exon 6 are deleted.

30. The engineered immune cell of any one of claims 1, 2 and 4 to 28, wherein the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 7 and exon 8 are deleted.

31. The engineered immune cell of any one of claims 1 to 30, wherein the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

32. The engineered immune cell of any one of claims 1 to 31, wherein the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 106, or SEQ ID NO: 107.

33. The engineered immune cell of any one of claims 1 to 32, wherein the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:69 or SEQ ID NO: 108.

34. The engineered immune cell of any one of claims 1 to 33, wherein the CAR comprises anamino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO:20.

35. The engineered immune cell of any one of claims 1, 3 to 34, wherein the extracellular domain of TGFPRII comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:28.

36. The engineered immune cell of any one of claims 1 to 35, wherein the engineered immune cell is an NK cell, an NKT cell, a T cell, or a Treg cell.

37. The engineered immune cell of claim 1, wherein the CAR, from N-terminus to C-terminus, comprising:(i) an extracellular antigen binding domain;(ii) a transmembrane domain comprising an transmembrane domain derived from CD84;(iii) an intracellular signaling domain comprising(a) an intracellular domain derived from DAP 10;(b) an intracellular domain derived from a mutated CD84, wherein the mutated CD84 differs from wild-type CD84 in that the amino acid residues encoded by exon 7 and exon 8 are deleted; and(c) an intracellular domain derived from CD3zeta, and wherein the 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.

38. A polypeptide comprising(i) a chimeric antigen receptor (CAR), comprising:(a) an extracellular antigen binding domain;(b) a transmembrane domain; and(c) an intracellular signaling domain;(ii) a mutated interleukin-2 (IL-2) comprising an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:63.

39. A polypeptide comprising(i) a chimeric antigen receptor (CAR), comprising:(a) an extracellular antigen binding domain;(b) a transmembrane domain; and(c) an intracellular signaling domain comprising an intracellular domain derived from a mutated CD84;(ii) a mutated interleukin-2 (IL-2); and(iii) an extracellular domain of transforming growth factor beta receptor II (TGFpRII).

40. The polypeptide of claim 38 or 39, wherein the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

41. The polypeptide of any one of claims 38 to 40, wherein the intracellular domain of the CAR comprises the amino acid sequence of SEQ ID NO: 16.

42. The polypeptide of any one of claims 38 to 41, wherein the intracellular domain of the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 106, or SEQ ID NO: 107.

43. The polypeptide of any one of claims 38 to 42, wherein the CAR comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO:20.

44. A polypeptide comprising(i) an extracellular domain of growth factor beta receptor II (TGFpRII); and(ii) a transmembrane domain of CD8a, CD4, CD7 or 2B4.

45. The polypeptide of any one of claims 39 to 44 further comprising a CD8 signal peptide, a CD4 signal peptide, a CD7 signal peptide or a 2B4 signal peptide.

46. The polypeptide of claim 39 to 45 further comprising a transmembrane domain of CD8a, CD4, CD7 or 2B4.

47. The polypeptide of any one of claims 39 to 46 further comprising P2A.

48. The polypeptide of any one of claims 39 to 47, wherein the extracellular domain of TGFpRII comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 28.

49. The polypeptide of any one of claims 39 to 48, wherein the polypeptide comprises an aminoacid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26.

50. The polypeptide of any one of claims 39 to 49, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO : 1 , SEQ ID NO : 21 , SEQ ID NO : 23 , or SEQ ID NO : 25.

51. The polypeptide of any one of claims 38 to 50, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 67, or SEQ ID NO:68.

52. A polynucleotide encoding the polypeptide of any one of claims 38 to 51.

53. A vector comprising the polynucleotide of claim 52.

54. A pharmaceutical composition comprising(i) the engineered immune cell of any one of claims 1 to 37, the polypeptide of any one of claims 38 to 51, the polynucleotide of claim 52, or the vector of claim 53; and(ii) a pharmaceutically acceptable excipient.

55. A method of treating a disease or disorder in a subject, comprising administering to the subject an effective amount of the engineered immune cell of any one of claims 1 to 37 or the pharmaceutical composition of claim 54.

56. The method of claim 55, wherein the disease or disorder is cancer.

57. A method of engineering an immune cell, comprising introducing into the immune cell the polynucleotide of claim 52 or the vector of claim 53.

58. A method of culturing an immune cell comprising culturing the immune cell in a media comprising a mutated interleukin-2 (IL-2), wherein the mutated IL-2 comprises an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO:47, SEQ ID NO:52, SEQ ID NO:54, or SEQ ID NO:57.

59. The method claim 58, wherein the media does not contain cytokine.

60. The method of claim 58 or 59, wherein the mutated IL-2 is soluble.

61. The method of any one of claims 58 to 60, wherein the immune cell is an NK cell, an NKT cell, a T cell, or a Treg cell.

62. The method of any one of claims 58 to 61, wherein the mutated IL-2 comprises the amino acid sequence of SEQ ID NO:48, SEQ ID NO:53, SEQ ID NO:55, or SEQ ID NO:58.

63. The method of any one of claims 58 to 61, wherein the immune cell is an engineered immune cell.

64. The method of claim 63, wherein the engineered immune cell comprises a chimeric antigen receptor (CAR), wherein the CAR comprises:(i) an extracellular antigen binding domain;(ii) a transmembrane domain; and(iii) an intracellular signaling domain.

65. The method of claim 64, wherein 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.

66. The method of claim 64 or 65, wherein the extracellular antigen binding domain of the CAR comprises an scFv.

67. The method of any one of claims 64 to 66, wherein the extracellular antigen binding domain of the CAR binds to a tumor antigen or a fragment thereof.

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