WO2022048523A1

WO2022048523A1 - Chimeric antigen receptor targeting nk activated receptor

Info

Publication number: WO2022048523A1
Application number: PCT/CN2021/115495
Authority: WO
Inventors: 周亚丽; 陈功; 姜小燕; 任江涛; 贺小宏; 王延宾; 韩露
Original assignee: 南京北恒生物科技有限公司
Priority date: 2020-09-02
Filing date: 2021-08-31
Publication date: 2022-03-10
Also published as: CN112029001B; CN116063550A; CN112029001A

Abstract

The present invention relates to a novel chimeric antigen receptor targeting an NK activated receptor. The chimeric antigen receptor can be used to treat malignant tumors caused by NK cytopathy, and can also prevent NK cells in a patient from killing introduced therapeutic CAR cells under the background of universal CAR cells. The present invention also relates to an engineered immune cell expressing the novel chimeric antigen receptor, a composition containing same, and the use thereof in the treatment of diseases.

Description

Chimeric antigen receptor targeting NK activating receptor

technical field

The present invention belongs to the field of immunotherapy. More specifically, the present invention relates to chimeric antigen receptors targeting NK activating receptors, engineered immune cells expressing such chimeric antigen receptors, and uses thereof.

Background technique

Chimeric antigen receptor (CAR) cell therapy, as a new type of precise targeted therapy targeting tumors, has demonstrated its good effect in tumor treatment, especially in the treatment of hematological tumors in recent years. Its basic principle is to load exogenous chimeric antigen receptors in immune cells (such as commonly used T cells, NK cells, etc.) through genetic engineering, and after in vitro expansion, the modified immune cells are then infused back into human body, thereby killing targeted tumor cells. Chimeric antigen receptors are generally composed of an antigen-binding region, a transmembrane region and an intracellular signal transduction region. Through the binding of the antigen-binding region to tumor-associated antigen (TAA) or tumor-specific antigen (TSA) expressed on the surface of tumor cells, The signal is transmitted to the interior of immune cells through the intracellular signal transduction region, thereby activating the immune cells to exert their effector functions.

NK cells are a type of cytotoxic lymphocytes that can kill target cells through a variety of mechanisms, such as releasing perforin and granzyme to cause cytolysis, activating apoptotic pathways to cause apoptosis, releasing cytokines to directly act on target cells, cells It plays an important role in tumor immunity and antiviral infection. The activation state of NK cells is determined by the balance between activating and inhibitory receptors. Normally, inhibitory receptors that recognize MHC class I molecules dominate the balance to prevent NK cells from killing their own healthy cells. However, when the expression of MHC-I molecules on the cell surface is reduced or absent, or when tumor cells, virus-infected cells, etc. bind to activating receptors through surface antigens, the activation signal will exceed the inhibitory signal, thereby activating NK cells to kill target cells.

In general-purpose CAR-T cells, it is usually necessary to inhibit or knock out MHC-I molecules, such as HLA and B2M, to reduce the host's immune rejection of exogenously introduced CAR-T cells. However, this inhibition or knockout will activate NK cells in the host, resulting in the killing of CAR-T cells by host NK cells, which seriously affects the proliferation and survival of CAR-T cells in the body, thereby affecting the efficacy of CAR-T cells.

Therefore, it is necessary to develop a novel chimeric antigen receptor to address the killing of therapeutic CAR cells by NK cells.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a novel chimeric antigen receptor comprising an antigen binding domain, a transmembrane domain and an intracellular signaling domain, wherein the antigen binding domain targets an NK activating receptor .

In one embodiment, the NK activating receptor is selected from the NKG2 family, preferably NKG2C, NKG2D, NKG2E, NKG2F and NKG2H, more preferably NKG2D. In one embodiment, the NK activating receptor is selected from the natural cytotoxicity receptor (NCR) family, preferably selected from NKp30, NKp44, NKp46 and NKp80, more preferably selected from NKp30 and NKp46. In one embodiment, the NK activating receptor is selected from the KIR-S family, preferably KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5 and KIR3DS1, more preferably KIR2DS4. In one embodiment, the NK activating receptor is selected from co-receptors, preferably selected from 2B4, DNAM-1, CD2 and LFA-1.

In one embodiment, the novel chimeric antigen receptor comprises a second antigen binding region that binds a tumor antigen selected from the group consisting of: TSHR, CD19, CD123, CD22, BAFF-R, CD30, CD171, CS-1 , CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-l lRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, AFP, Folate receptor α, ERBB2(Her2/neu), MUC1, EGFR, CS1, CD138, NCAM, Claudin18 .2, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gploo, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl -GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD 179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, Podin, HPV E6, E7, MAGE A1, ETV6-AML, spermatin 17, XAGE1, Tie 2, MAD- CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutant, prostate specific protein, survivin and telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, Sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2ETS fusion gene), NA17, PAX3, androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES 1 , LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF , CLEC12A, BST2, EMR2, LY75, GP C3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGF[beta], APRIL, NKG2D, and any combination thereof. Preferably, the target is selected from CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, MUCl, AFP, Folate receptor alpha, CEA, PSCA, PSMA, Her2, EGFR, IL13Ra2, GD2, NKG2D , EGFRvIII, CS1, BCMA, mesothelin, and any combination thereof.

In one embodiment, the antigen binding region is an antibody or functional fragment thereof, including but not limited to immunoglobulin molecules, Fab, Fab', F(ab')2, Fv fragments, scFv, disulfide-linked Fv (sdFv), heavy chain variable region (VH) or light chain variable region (VL) of antibodies, Fd fragments composed of VH and CH1 domains, linear antibodies, single domain antibodies, Nanobodies, the The native ligand of the antigen or a functional fragment thereof.

In one embodiment, the transmembrane domain is selected from the transmembrane domains of the following proteins: TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. Preferably, the transmembrane domain is selected from the transmembrane domains of CD8α, CD4, CD28 and CD278.

In one embodiment, the intracellular signaling domain is selected from the signaling domains of the following proteins: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. Preferably, the intracellular signaling domain is a CD3ζ-comprising signaling domain.

In one embodiment, the chimeric antigen receptor further comprises a costimulatory domain comprising one or more intracellular regions selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18(LFA-1), CD27, CD28, CD30, CD40, CD54(ICAM), CD83, CD134(OX40), CD137(4-1BB) ), CD270(HVEM), CD272(BTLA), CD276(B7-H3), CD278(ICOS), CD357(GITR), DAP10, DAP12, LAT, NKG2C, NKG2D, SLP76, PD-1, LIGHT, TRIM, CD94 , LTB, ZAP70 and their combinations. In a preferred embodiment, the costimulatory domain comprises one or more intracellular domains selected from the group consisting of DAP10, DAP12, CD27, CD28, CD134, 4-1BB or CD278. For example, in one embodiment, the costimulatory domain comprises the intracellular region of 4-1BB. In one embodiment, the costimulatory domain comprises the intracellular region of CD28. In one embodiment, the costimulatory domain comprises the intracellular region of DAP10. In one embodiment, the costimulatory domain comprises the intracellular region of DAP12. More preferably, the costimulatory domain comprises two intracellular domains selected from the group consisting of DAP10, DAP12, CD27, CD28, CD134, 4-1BB or CD278. In one embodiment, the costimulatory domain comprises the intracellular domain of 4-1BB and the intracellular domain of CD28. In one embodiment, the costimulatory domain comprises the 4-1BB intracellular domain and the intracellular domain of DAP10. In one embodiment, the costimulatory domain comprises the intracellular domain of 4-1BB and the intracellular domain of DAP12. In one embodiment, the costimulatory domain comprises the intracellular domain of CD28 and the intracellular domain of DAP10. In one embodiment, the costimulatory domain comprises the intracellular domain of CD28 and the intracellular domain of DAP12.

The present invention also provides nucleic acid molecules encoding the novel chimeric antigen receptors described above and vectors comprising the nucleic acid molecules.

In a second aspect, the present invention also provides engineered immune cells expressing the novel chimeric antigen receptors described above.

In one embodiment, the engineered immune cells express two chimeric antigen receptors, wherein the first chimeric antigen receptor comprises a first antigen binding region targeting an NK activating receptor and the second chimeric antigen receptor is The antibody contains a second antigen binding region that targets the tumor antigen.

In one embodiment, the engineered immune cells further comprise suppressed or silenced expression of at least one gene selected from the group consisting of CD52, GR, dCK, TCR/CD3 genes (eg, TRAC, TRBC, CD3γ, CD3δ, CD3ε , CD3ζ), MHC-related genes (HLA-A, HLA-B, HLA-C, B2M, HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA) and immune checkpoint genes such as PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7 , FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3 , GUCY1B2 and GUCY1B3. Preferably, the engineered immune cells further comprise suppressed or silenced expression of at least one gene selected from the group consisting of TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA, PD1, LAG3, TIM3, CTLA4.

In one embodiment, to reduce mutual killing between the engineered immune cells, the expression of the corresponding endogenous NK activating receptor targeted by the chimeric antigen receptor in the engineered immune cells is inhibited or silenced. In a preferred embodiment, the NK activating receptor is NKG2D or NKp46.

In one embodiment, the engineered immune cells are selected from T cells, macrophages, dendritic cells, monocytes, NK cells or NKT cells. Preferably, the T cells are CD4+/CD8+ T cells, CD4+ helper T cells, CD8+ T cells, tumor infiltrating cells, memory T cells, naive T cells, γδ-T cells or αβ-T cells. In one embodiment, the immune cells are derived from stem cells, such as adult stem cells, embryonic stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells, and the like.

In one embodiment, the present invention also provides a pharmaceutical composition comprising the engineered immune cells, nucleic acid molecules or vectors of the present invention, and a plurality of pharmaceutically acceptable excipients.

In a third aspect, the present invention also provides a method of treating a subject suffering from cancer, infection or autoimmune disease, comprising administering to the subject an effective amount of an immune cell according to the present invention or pharmaceutical composition.

In one embodiment, the present invention also provides the use of novel money and antigen receptors, nucleic acid molecules, vectors, engineered immune cells or pharmaceutical compositions according to the present invention in the manufacture of a medicament for the treatment of cancer, infection or autoimmune disease .

The advantage of the novel chimeric antigen receptor of the present invention lies in that by specifically targeting the NK cell activating receptor to initiate the killing of NK cells, on the one hand, it can treat malignant tumors caused by NK cell lesions, and on the other hand, it can also In the context of universal CAR cells (especially in the case of inhibiting or knocking out MHC-related genes), the killing of the introduced therapeutic CAR cells by NK cells in the patient is avoided, thereby enhancing the survival of CAR cells and improving the therapeutic effect.

Detailed description of the invention

Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Chimeric Antigen Receptor

The present invention provides a novel chimeric antigen receptor comprising an antigen binding region, a transmembrane domain and an intracellular signaling domain, wherein the antigen binding region targets an NK activating receptor.

As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide that generally includes an antigen-binding region (eg, an antibody or antigen-binding portion thereof), a transmembrane domain, a co- The stimulatory domain and the intracellular signaling domain are linked by linkers. CARs can exploit the antigen-binding properties of monoclonal antibodies to redirect the specificity and reactivity of T cells and other immune cells to selected targets in an MHC-non-restricted manner. Non-MHC-restricted antigen recognition confers CAR-expressing T cells the ability to recognize antigen independent of antigen processing, thus bypassing the primary mechanism of tumor escape. Furthermore, when expressed within T cells, CARs advantageously do not dimerize with the alpha and beta chains of the endogenous T cell receptor (TCR).

As used herein, "antigen binding region" refers to any structure or functional variant thereof that can bind an antigen. The antigen binding region can be an antibody, including but not limited to monoclonal, polyclonal, recombinant, human, humanized, murine, chimeric, and functional fragments thereof, or natural ligands of the antigen. body or a functional fragment thereof. For example, antigen binding regions include, but are not limited to, immunoglobulin molecules, Fab, Fab', F(ab')2, Fv fragments, scFv, disulfide-linked Fv (sdFv), heavy chain variable regions of antibodies ( VH) or light chain variable region (VL), Fd fragments composed of VH and CH1 domains, linear antibodies, single domain antibodies, Nanobodies, natural ligands of the antigen or functional fragments thereof, etc., preferably selected From Fab, scFv, sdAb and Nanobodies. In the present invention, the antigen binding region may be monovalent or bivalent, and may be a monospecific, bispecific or multispecific antibody.

"Fab" refers to either of two identical fragments produced upon cleavage of an immunoglobulin molecule by papain, consisting of an intact light chain and an N-terminal portion of a heavy chain linked by disulfide bonds, wherein the N-terminal portion of the heavy chain includes Heavy chain variable region and CH1. Compared to intact IgG, Fab has no Fc fragment, has higher mobility and tissue penetration, and can bind antigen monovalently without mediating antibody effects.

A "single chain antibody" or "scFv" is an antibody composed of an antibody heavy chain variable region (VH) and light chain variable region (VL) linked by a linker. The optimal length and/or amino acid composition of the linker can be selected. The length of the linker significantly affects the variable region folding and interaction of scFv. In fact, intrachain folding can be prevented if shorter linkers are used (eg between 5-10 amino acids). For selection of linker size and composition, see, eg, Hollinger et al., 1993 Proc Natl Acad. Sci. USA 90:6444-6448; US Patent Application Publication Nos. 2005/0100543, 2005/0175606, 2007/0014794; and PCT Publication Nos. WO2006/020258 and WO2007/024715, the entire contents of which are incorporated herein by reference. The scFv can comprise VH and VL linked in any order, eg, VH-linker-VL or VL-linker-VH.

"Single-domain antibody" or "sdAb" refers to an antibody that is naturally deficient in its light chain, and which contains only one variable heavy chain region (VHH) and two conventional CH2 and CH3 regions, also referred to as a "heavy chain" antibody".

"Nanobody" or "Nb" refers to a single cloned and expressed VHH structure, which has the same structural stability and antigen-binding activity as the original heavy chain antibody, and is the smallest known unit that can bind to the target antigen. .

As used herein, the term "NK activating receptor" refers to an NK cell surface receptor that has or binds to an immunoreceptor tyrosine-based activation motif (ITAM). Most NK-activating receptors bind to corresponding adaptor proteins (eg, DAP12, FcεRIγ, CD3ζ, etc.) through arginine or lysine residues in their transmembrane regions, thereby initiating intracellular signal transduction. The binding of NK activating receptors to ligands causes tyrosine phosphorylation of ITAM contained in adaptor proteins, and then recruits tyrosine kinases such as Syk, Zap70, etc., and transmits downstream signals to induce NK cell activation.

In one embodiment, the NK activating receptor is selected from the NKG2 family, preferably NKG2C, NKG2D, NKG2E, NKG2F and NKG2H, more preferably NKG2D. The extracellular segments of these NKG2 family active receptors are highly homologous and most contain a C-type lectin-like domain. Among them, NKG2E and NKG2H are encoded by the same gene, and the latter is a truncated sequence of the former. The expression of NKG2C and NKG2E/H requires the formation of heterodimers with CD94. By binding to the DAP12 molecule containing ITAM, the two tyrosine residues in ITAM are rapidly phosphorylated, which in turn recruits ZAP-70 and Syk, resulting in NK cell activation. The amino acid sequence of NKG2F is similar to that of NKG2C, with lysine residues in the transmembrane region, and it also transmits an activation signal by binding to DAP12. NKG2D is widely expressed, not only on all NK cells in humans, but also on the vast majority of γδT cells and activated CD8+αβT cells, and in mice on all NK cells, some γδT cells, and some macrophages . NKG2D is expressed as a homodimer, which can bind to two different adaptor proteins, DAP10 and DAP12, and mediate different functions via two signaling pathways. On the one hand, NKG2D can bind to DAP10 through positively charged arginine residues in the transmembrane region, phosphorylate DAP10, and then activate NK cells to exert cytotoxic effects through a PI3K-dependent Ras-independent signaling pathway. On the other hand, NKG2D can also bind to DAP12, which contains ITAM, and recruit tyrosine kinases such as Syk and ZAP-70 through phosphorylation of tyrosine residues, leading to the transduction of downstream signals, inducing the release of cytokines and chemokines.

In a preferred embodiment, the antigen binding region comprised by the chimeric antigen receptor of the present invention is an antibody or functional fragment thereof targeting NKG2D, preferably a Fab, scFv, sdAb and Nanobody targeting NKG2D, more preferably scFv targeting NKG2D. In a preferred embodiment, the chimeric antigen receptor of the invention comprises an anti-NKG2D antibody comprising:

(i) CDR-L1, CDR-L2 and CDR-L3 as shown in SEQ ID NOs: 61, 62 and 63, and CDR-H1, CDR-H2 as shown in SEQ ID NOs: 64, 65 and 66, respectively and CDR-H3;

(ii) CDR-L1, CDR-L2 and CDR-L3 as shown in SEQ ID NOs: 67, 68 and 69, and CDR-H1, CDR-H2 as shown in SEQ ID NOs: 70, 71 and 72, respectively and CDR-H3;

(iii) CDR-L1, CDR-L2 and CDR-L3 as set forth in SEQ ID NOs: 67, 68 and 73, and CDR-Hl, CDR-H2 as set forth in SEQ ID NOs: 74, 75 and 76, respectively and CDR-H3; or

(iv) CDR-L1, CDR-L2 and CDR-L3 as set forth in SEQ ID NOs: 77, 78 and 79, and CDR-Hl, CDR-H2 as set forth in SEQ ID NOs: 80, 81 and 82, respectively and CDR-H3.

Preferably, the chimeric antigen receptor of the present invention comprises an anti-NKG2D antibody comprising SEQ ID NO: 27 positions 1-121, SEQ ID NO: 29 positions 1-109, SEQ ID NO: 31 positions 1-109 A light chain may have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity to the amino acid sequence shown in positions 1-108 of SEQ ID NO: 33. The variable region sequence sum is shown in SEQ ID NO: 27, 137-246, SEQ ID NO: 29, 122-243, SEQ ID NO: 31, 122-236, or SEQ ID NO: 33, 121-243 The amino acid sequence of the heavy chain variable region sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity. More preferably, the chimeric antigen receptor of the present invention comprises an anti-NKG2D antibody comprising the amino acid sequence shown in SEQ ID NO: 27, 29, 31 or 33.

In one embodiment, instead of NKG2D antibodies, the chimeric antigen receptors of the invention may also comprise a natural ligand for NKG2D. The ligands of NKG2D in humans are the MIC genes (including MICA and MICB) and the ULBP genes (including ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 and ULBP6), and the ligands in mice are Rae-1, H60 and MULT1. MICA and MICB are located on the side of the HLA-B locus in the MHC gene complex, and their homology is as high as 91%. ULBP gene is similar in structure to MHC-I molecules, both containing α1 and α2 domains, but not α3 domain and β2 microglobulin. Thus, in this embodiment, the chimeric antigen receptor of the invention may further comprise the following proteins or functional fragments thereof: MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, Rae-1, H60 and MULT1 .

In one embodiment, the NK activating receptor is selected from the natural cytotoxicity receptor (NCR) family, preferably selected from NKp30, NKp44, NKp46 and NKp80, more preferably selected from NKp30 and NKp46. NKp30 is one of the key factors for the killing activity of NK cells, and its expression level is deregulated in the infection of various tumors and viruses, which may be involved in the immune escape of tumors and viruses. The extracellular segment of NKp30 is a V-type immunoglobulin-like domain linked by a hydrophobic amino acid sequence to an arginine-rich transmembrane region. NKp30 binds to CD3ζ and transmits an activation signal intracellularly through the ITAM of the latter. The extracellular region of NKp44 contains a V-type domain and the transmembrane region has charged lysines that bind to KAPAP/DAP12. Although the intracellular domain of NKp44 contains ITIM, its lack of inhibitory function does not attenuate the activation signal transmitted by DAP12. Studies have shown that NKp44 can combine with viral hemagglutinin to exert the antiviral effect of NK cells. The extracellular region of NKp46 has two C2-type Ig-like domains, the transmembrane region contains a positively charged arginine residue, and the intracellular region does not contain an ITAM motif, so similar to NKp30, it also needs to interact with the cell. Molecules such as CD3ζ and/or FcεRIγ containing ITAM in the inner segment form complexes, which together transmit activation signals into cells. NKp46 is expressed on the surface of all mature NK cells and is a key receptor for initiating NK cell killing. NKp46 can also bind to viral hemagglutinin to exert the antiviral effect of NK cells. NKp80 is expressed on the surface of almost all NK cells in the form of homodimers, and after binding to its ligand activation-induced C-type lectin (AICL), it can rapidly activate NK cells and improve NK cells. Cytotoxicity and ability to secrete inflammatory cytokines.

In a preferred embodiment, the antigen binding region comprised by the chimeric antigen receptor of the present invention is an antibody or functional fragment thereof targeting NKp46, preferably Fab, scFv, sdAb and Nanobody targeting NKp46, more preferably scFv targeting NKp46. In a preferred embodiment, the chimeric antigen receptor of the invention comprises an anti-NKp46 antibody comprising

(i) CDR-L1, CDR-L2 and CDR-L3 as shown in SEQ ID NOs: 83, 84 and 85, respectively, and CDR-H1, CDR-H2 as shown in SEQ ID NOs: 86, 87 and 88 and CDR-H3;

(ii) CDR-L1, CDR-L2 and CDR-L3 as shown in SEQ ID NOs: 89, 90 and 91, and CDR-H1, CDR-H2 as shown in SEQ ID NOs: 92, 93 and 94, respectively and CDR-H3;

(iii) CDR-L1, CDR-L2 and CDR-L3 as shown in SEQ ID NOs: 95, 96 and 97, and CDR-H1, CDR-H2 as shown in SEQ ID NOs: 98, 99 and 100, respectively and CDR-H3;

(iv) CDR-L1, CDR-L2 and CDR-L3 as set forth in SEQ ID NOs: 101, 84 and 102, and CDR-Hl, CDR-H2 as set forth in SEQ ID NOs: 103, 87 and 104, respectively and CDR-H3; or

(v) CDR-L1, CDR-L2 and CDR-L3 as set forth in SEQ ID NOs: 105, 106 and 107, and CDR-Hl, CDR-H2 as set forth in SEQ ID NOs: 108, 109 and 110, respectively and CDR-H3.

Preferably, the chimeric antigen receptor of the present invention comprises an anti-NKp46 antibody comprising an position, SEQ ID NO: 41 1-107 or SEQ ID NO: 43 1-107 of the amino acid sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, A light chain variable region sequence of 99% or 100% sequence identity and the The amino acid sequence shown in positions 123-242 of SEQ ID NO: 41 or positions 123-237 of SEQ ID NO: 43 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or heavy chain variable region sequences with 100% sequence identity. More preferably, the chimeric antigen receptor of the present invention comprises an anti-NKp46 antibody comprising the amino acid sequence shown in SEQ ID NO: 35, 37, 39, 41 or 43.

In one embodiment, instead of NKp46 antibodies, the chimeric antigen receptors of the invention may comprise a natural ligand for NKp46. Studies have found that NK cells recognize viral hemagglutinin (HA) through NKp46, and agglutinate with it, thereby killing influenza virus-infected cells. In addition, NKp46 also binds a soluble glycoprotein, complement factor P (CFP). It has been reported that patients lacking CFP are more susceptible to N. meningitidis infection, and treatment of this infection with CFP relies on the action of NKp46. Thus, in this embodiment, the chimeric antigen receptor of the present invention may also comprise HA or CFP or functional fragments thereof.

In one embodiment, the NK activating receptor is selected from the KIR-S family, preferably KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5 and KIR3DS1, more preferably KIR2DS4. Killer Ig-like receptors (KIRs) are type I transmembrane proteins belonging to the immunoglobulin superfamily whose structures include extramembrane, transmembrane and cytoplasmic domains. According to the number of Ig-like domains contained in the extramembrane region, KIRs can also be divided into KIR2D and KIR3D subfamilies. In addition, according to the length of the cytoplasmic region, KIR can be divided into long (KIR-L) and short (KIR-S). The cytoplasmic region of KIR-S does not contain ITIM, and similar to NCR, it also binds to the adaptor protein DAP12 or FcεRIγ through charged transmembrane residues, thereby recruiting tyrosine kinases and mediating downstream activation signals.

In one embodiment, the NK activating receptor is selected from co-receptors, preferably selected from 2B4, DNAM-1, CD2 and LFA-1. 2B4, also known as CD244, is a membrane protein whose extracellular domain It has a V-type immunoglobulin domain and a C2-type immunoglobulin-like domain, the transmembrane region does not contain any charged amino acids, and the intracellular region contains an immunoreceptor tyrosine-based inhibitory motif. switch motif, ITSM), this motif can be recognized by the cytoplasmic SH2 region of adaptor proteins SAP, EAT-2, DRT, etc. DNAM-1, also known as CD226, is the major co-activating receptor that initiates NK cell function. DNAM-1 contains the extracellular domain of two immunoglobulin V-like domains, a transmembrane domain, and a cytoplasmic domain containing potential phosphorylation sites for tyrosine and serine residues. CD2, also known as LFA-2, is a single-chain glycoprotein composed of 327 amino acids that is expressed on the surface of mature T cells, most thymocytes, and some NK cells. LFA-1 consists of two polypeptide chains linked by non-covalent bonds: an alpha subunit (CD11a) and a beta subunit (CD18). These co-receptors cannot activate NK cells alone, but rely on other NK activating receptors such as NCR to initiate activation signals, and then jointly participate in amplifying the activation signals to more effectively promote NK cell activation. Antibodies known in the art targeting 2B4, DNAM-1, CD2 or LFA-1, or natural ligands of 2B4, DNAM-1, CD2 or LFA-1 and functional fragments thereof can be used as the chimeras of the present invention the antigen-binding region of the antigen receptor.

In one embodiment, the novel chimeric antigen receptors of the invention comprise, in addition to the antigen binding region targeting the NK activating receptor, a second antigen binding region that binds to a tumor antigen selected from the group consisting of TSHR, CD19 , CD123, CD22, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, AFP, Folate receptor α, ERBB2 ( Her2/neu), MUC1, EGFR, CS1, CD138, NCAM, Claudin18.2, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gploo, bcr-abl, tyrosinase, EphA2 , Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, Podlin, HPV E6, E7, MAGE A1 , ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutant, prostate specific protein, survivin and telomerase, PCTA -l/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2ETS fusion gene), NA17, PAX3, androgen receptor, Cyclin Bl, MYCN, RhoC, TRP -2, CYP1B 1, BORIS, SART3, PAX5, OY-TES 1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70 -2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLE C12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGFβ, APRIL, NKG2D, and any combination thereof. Preferably, the tumor antigen is selected from CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, MUCl, AFP, Folate receptor alpha, CEA, PSCA, PSMA, Her2, EGFR, IL13Ra2, GD2, NKG2D, EGFRvIII, CS1, BCMA, mesothelin and any combination thereof. Antibodies known in the art targeting the above tumor antigens can be used as the second antigen binding region in the present invention.

In a preferred embodiment, the second antigen binding region comprises an antibody targeting CD19 comprising:

(i) CDR-L1, CDR-L2 and CDR-L3 as shown in SEQ ID NOs: 49, 50 and 51, respectively, and CDR-H1, CDR-H2 as shown in SEQ ID NOs: 52, 53 and 54, respectively and CDR-H3; or

(ii) CDR-L1, CDR-L2 and CDR-L3 as shown in SEQ ID NOs: 55, 56 and 57, and CDR-H1, CDR-H2 as shown in SEQ ID NOs: 58, 59 and 60, respectively and CDR-H3.

Preferably, the chimeric antigen receptor of the present invention comprises an antibody targeting the NK activating receptor and an antibody targeting CD19, the antibody targeting CD19 comprising positions 1-107 of SEQ ID NO: 1 or SEQ ID The amino acid sequence shown at positions 1-107 of NO:25 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity to the light chain variable region sequence and have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, Heavy chain variable region sequences of 99% or 100% sequence identity. More preferably, the chimeric antigen receptor of the present invention comprises an antibody targeting NK activating receptor and an antibody targeting CD19, the antibody targeting CD19 comprising the amino acid sequence shown in SEQ ID NO: 1 or 25 .

The term "functional variant" or "functional fragment" refers to a variant that substantially comprises the amino acid sequence of a parent but contains at least one amino acid modification (ie, substitution, deletion or insertion) compared to the parent amino acid sequence, provided that all The variants retain the biological activity of the parent amino acid sequence. In one embodiment, the amino acid modification is preferably a conservative modification.

As used herein, the term "conservative modification" refers to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing the amino acid sequence. These conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the chimeric antigen receptors of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which amino acid residues are replaced by amino acid residues having similar side chains. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid) ), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine) acid, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). Conservative modifications can be selected, for example, based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.

Thus, a "functional variant" or "functional fragment" is at least 75%, preferably at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84% of the parent amino acid sequence %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, And retain the biological activity of the parent amino acid, such as binding activity.

As used herein, the term "sequence identity" refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same positions in an alignment, and is usually expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Therefore, two copies with the exact same sequence are 100% identical. Those skilled in the art will recognize that several algorithms can be used to determine sequence identity using standard parameters, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215:403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147:195-197) and ClustalW.

As used herein, the term "transmembrane domain" refers to a polypeptide that enables expression of a chimeric antigen receptor on the surface of immune cells (eg, lymphocytes, NK cells, or NKT cells) and directs the cellular response of the immune cells against target cells structure. The transmembrane domain can be natural or synthetic, and can be derived from any membrane-bound or transmembrane protein. The transmembrane domain is capable of signaling when the chimeric antigen receptor binds to the target antigen. Transmembrane domains particularly useful in the present invention may be derived from, for example, TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α , CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and their functional fragments. Alternatively, the transmembrane domain may be synthetic and may contain predominantly hydrophobic residues such as leucine and valine. Preferably, the transmembrane domain is derived from CD8 alpha chain or CD28, which is at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, and at least 70% of the amino acid sequence shown in SEQ ID NO: 3 or 5 % or 99% or 100% sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.

In one embodiment, the chimeric antigen receptors of the present invention may further comprise a hinge region between the ligand binding domain and the transmembrane domain. As used herein, the term "hinge region" generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the ligand binding domain. Specifically, the hinge region serves to provide greater flexibility and accessibility to the ligand binding domain. The hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. The hinge region can be derived in whole or in part from a native molecule, such as in whole or in part from the extracellular region of CD8, CD4 or CD28, or in whole or in part from an antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a fully synthetic hinge sequence. In a preferred embodiment, the hinge region comprises a hinge region portion of a CD8α chain, CD28, FcγRIIIα receptor, IgG4 or IgG1, more preferably a hinge from CD8α, CD28 or IgG4, which is the same as SEQ ID NO: 19, 21 or The amino acid sequence shown in 23 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity, or its coding sequence and SEQ ID NO: 20, 22 The nucleotide sequence shown in or 24 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.

As used herein, the term "intracellular signaling domain" refers to the portion of a protein that transduces effector function signals and directs cells to perform specified functions. The intracellular signaling domain is responsible for primary intracellular signaling following binding of the ligand binding domain to an antigen, resulting in the activation of immune cells and immune responses. In other words, the intracellular signaling domain is responsible for activating at least one of the normal effector functions of the immune cells in which the CAR is expressed. For example, the effector function of T cells can be cytolytic activity or helper activity, including secretion of cytokines.

In one embodiment, the chimeric antigen receptors of the invention comprise intracellular signaling domains that may be cytoplasmic sequences of T cell receptors and co-receptors that act together upon antigen receptor binding to initiate primary signaling conduction, as well as any derivatives or variants of these sequences and any synthetic sequences that have the same or similar function. The intracellular signaling domain can contain a number of immunoreceptor tyrosine-based activation motifs (ITAM). Non-limiting examples of intracellular signaling domains of the invention include, but are not limited to, those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In a preferred embodiment, the signaling domain of the CAR of the present invention may comprise a CD3ζ signaling domain having at least 70%, preferably at least 80%, with the amino acid sequence shown in SEQ ID NO: 11 or 13 , more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity, or its coding sequence has at least 70%, preferably at least 80%, with the nucleotide sequence shown in SEQ ID NO: 12 or 14 %, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.

In one embodiment, the chimeric antigen receptors of the invention further comprise one or more costimulatory domains. A costimulatory domain may be an intracellular functional signaling domain from a costimulatory molecule, comprising the entire intracellular portion of the costimulatory molecule, or a functional fragment thereof. A "costimulatory molecule" refers to a cognate binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (e.g., proliferation) of the T cell. Costimulatory molecules include, but are not limited to, MHC class 1 molecules, BTLA and Toll ligand receptors. Non-limiting examples of costimulatory domains of the invention include, but are not limited to, costimulatory signaling domains derived from the following proteins: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11 , CD2, CD7, CD8, CD18(LFA-1), CD27, CD28, CD30, CD40, CD54(ICAM1), CD83, CD134(OX40), CD137(4-1BB), CD270(HVEM), CD272(BTLA) , CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, CD94, LTB and ZAP70 and combinations thereof.

In a preferred embodiment, the costimulatory domain comprises one or more intracellular domains selected from the group consisting of DAP10, DAP12, CD27, CD28, CD134, 4-1BB or CD278. For example, in one embodiment, the costimulatory domain comprises the intracellular region of 4-1BB. In one embodiment, the costimulatory domain comprises the intracellular region of CD28. In one embodiment, the costimulatory domain comprises the intracellular region of DAP10. In one embodiment, the costimulatory domain comprises the intracellular region of DAP12. More preferably, the costimulatory domain comprises two intracellular domains selected from the group consisting of DAP10, DAP12, CD27, CD28, CD134, 4-1BB or CD278. In one embodiment, the costimulatory domain comprises the intracellular domain of 4-1BB and the intracellular domain of CD28. In one embodiment, the costimulatory domain comprises the intracellular domain of 4-1BB and the intracellular domain of DAP10. In one embodiment, the costimulatory domain comprises the intracellular domain of 4-1BB and the intracellular domain of DAP12. In one embodiment, the costimulatory domain comprises the intracellular domain of CD28 and the intracellular domain of DAP10. In one embodiment, the costimulatory domain comprises the intracellular domain of CD28 and the intracellular domain of DAP12.

In one embodiment, the intracellular region of 4-1BB is at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the amino acid sequence set forth in SEQ ID NO:9 % sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% with the nucleotide sequence shown in SEQ ID NO: 10 sequence identity. In one embodiment, the intracellular region of CD28 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the amino acid sequence set forth in SEQ ID NO:7 The sequence identity, or its coding sequence, has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence with the nucleotide sequence shown in SEQ ID NO:8 identity. In one embodiment, the intracellular region of DAP10 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the amino acid sequence set forth in SEQ ID NO:45 The sequence identity, or its coding sequence, has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence with the nucleotide sequence shown in SEQ ID NO:46 identity. In one embodiment, the intracellular region of DAP12 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the amino acid sequence set forth in SEQ ID NO:47 The sequence identity, or its coding sequence, has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence with the nucleotide sequence shown in SEQ ID NO:48 identity.

In one embodiment, the CAR of the present invention may further comprise a signal peptide such that when it is expressed in a cell such as a T cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface. The core of the signal peptide may contain a long segment of hydrophobic amino acids that has a tendency to form a single alpha-helix. At the end of the signal peptide, there is usually a segment of amino acids that is recognized and cleaved by signal peptidases. The signal peptidase can cleave during or after translocation to generate the free signal peptide and mature protein. Then, the free signal peptide is digested by specific proteases. Signal peptides useful in the present invention are well known to those skilled in the art, eg, signal peptides derived from CD8α, IgG1, GM-CSFRα, B2M, and the like. In one embodiment, the signal peptide useful in the present invention is at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% of the amino acid sequence set forth in SEQ ID NO: 15 or 17 or 100% sequence identity, or the coding sequence of the signal peptide and the amino acid sequence shown in SEQ ID NO: 16 or 18 have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.

In one embodiment, the CAR of the present invention may further comprise a switch structure to regulate the expression time of the CAR. For example, the switch structure can be in the form of a dimerization domain that induces a conformational change upon binding to its corresponding ligand, exposing the extracellular binding domain for binding to the targeted antigen, thereby activating a signaling pathway. Alternatively, switch domains can be used to connect the binding and signaling domains, respectively, which can dimerize only when the switch domains bind to each other (eg, in the presence of an inducing compound) linked together, thereby activating signaling pathways. The switch structure can also be in the form of a masked peptide. The masking peptide can mask the extracellular binding domain, preventing it from binding to the targeted antigen, and when the masking peptide is cleaved by, for example, a protease, the extracellular binding domain can be exposed, making it a "normal" CAR structure. Various switch structures known to those skilled in the art can be used in the present invention.

In one embodiment, the CAR of the present invention may further comprise a suicide gene, i.e., causing it to express a cell death signal that can be induced by a foreign substance, to clear the CAR cells when needed (eg, when severe toxic side effects occur). For example, suicide genes can be in the form of inserted epitopes, such as CD20 epitopes, RQR8, etc., and when desired, CAR cells can be eliminated by adding antibodies or reagents targeting these epitopes. The suicide gene can also be herpes simplex virus thymidine kinase (HSV-TK), which causes cell death induced by ganciclovir treatment. The suicide gene can also be iCaspase-9, which can be induced to dimerize by chemical inducing drugs such as AP1903, AP20187, etc., thereby activating the downstream Caspase3 molecule, leading to apoptosis. Various suicide genes known to those skilled in the art can be used in the present invention.

Nucleic Acids and Vectors

As used herein, the term "nucleic acid molecule" includes sequences of ribonucleotides and deoxyribonucleotides, such as modified or unmodified RNA or DNA, each in linear or circular form in single- and/or double-stranded form form, or their mixtures (including hybrid molecules). Thus, nucleic acids according to the present invention include DNA (eg dsDNA, ssDNA, cDNA), RNA (eg dsRNA, ssRNA, mRNA, ivtRNA), combinations or derivatives thereof (eg PNA). Preferably, the nucleic acid is DNA or RNA, more preferably mRNA.

Nucleic acids may contain conventional phosphodiester bonds or unconventional bonds (eg, amide bonds, such as found in peptide nucleic acids (PNA)). Nucleic acids of the invention may also contain one or more modified bases, such as, for example, tritylated bases and uncommon bases such as inosine. Other modifications are also contemplated, including chemical, enzymatic, or metabolic modifications, so long as the multi-chain CARs of the invention can be expressed from polynucleotides. Nucleic acids can be provided in isolated form. In one embodiment, nucleic acids may also include regulatory sequences, such as transcriptional control elements (including promoters, enhancers, operators, repressors, and transcription termination signals), ribosome binding sites, introns, and the like.

The nucleic acid sequences of the invention can be codon optimized for optimal expression in desired host cells (eg, immune cells); or for expression in bacterial, yeast, or insect cells. Codon optimization refers to the replacement of codons present in the target sequence that are generally rare in highly expressed genes of a given species with codons that are generally common in highly expressed genes of such species, and the codons before and after the replacement code for the same amino acid. Therefore, the choice of optimal codons depends on the codon usage preferences of the host genome.

As used herein, the term "vector" is a nucleic acid molecule used as a vehicle for the transfer of (exogenous) genetic material into a host cell, in which the nucleic acid molecule can eg be replicated and/or expressed.

Vectors generally include targeting vectors and expression vectors. A "targeting vector" is a medium that delivers an isolated nucleic acid to the interior of a cell, for example, by homologous recombination or using a hybrid recombinase that specifically targets the sequence at the site. An "expression vector" is a vector used for the transcription of heterologous nucleic acid sequences, such as those encoding the chimeric antigen receptor polypeptides of the invention, in suitable host cells and the translation of their mRNAs. Suitable carriers for use in the present invention are known in the art and many are commercially available. In one embodiment, the vectors of the present invention include, but are not limited to, plasmids, viruses (eg, retroviruses, lentiviruses, adenoviruses, vaccinia virus, Rous sarcoma virus (RSV, polyoma virus, and adeno-associated virus (AAV)), and the like ), phages, phagemids, cosmids, and artificial chromosomes (including BAC and YAC). The vector itself is usually a nucleotide sequence, usually a DNA sequence containing the insert (transgene) and a larger sequence that serves as the "backbone" of the vector .Engineered vectors typically also contain an origin of autonomous replication in the host cell (if stable expression of the polynucleotide is desired), a selectable marker, and a restriction enzyme cleavage site (eg, a multiple cloning site, MCS). The vector may additionally contain a promoter , polyadenylation tail (polyA), 3'UTR, enhancer, terminator, insulator, operon, selectable marker, reporter gene, targeting sequence and/or protein purification tag and other elements. In a specific embodiment , the vector is an in vitro transcribed vector.

engineered immune cells

As used herein, the term "immune cell" refers to any cell of the immune system that has one or more effector functions (eg, cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC). For example, the immune cells can be T cells, macrophages, dendritic cells, monocytes, NK cells, and/or NKT cells. In one embodiment, the immune cells are derived from stem cells, such as adult stem cells, embryonic stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells, and the like. Preferably, the immune cells are T cells. The T cells can be any T cells, such as T cells cultured in vitro, such as primary T cells, or T cells from T cell lines cultured in vitro such as Jurkat, SupT1, etc., or T cells obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. T cells can also be concentrated or purified. T cells can be at any stage of development, including, but not limited to, CD4+/CD8+ T cells, CD4+ helper T cells (eg, Th1 and Th2 cells), CD8+ T cells (eg, cytotoxic T cells), tumor-infiltrating cells, memory T cells, naive T cells, γδ-T cells, αβ-T cells, etc. In a preferred embodiment, the immune cells are human T cells. T cells can be obtained from the blood of a subject using a variety of techniques known to those of skill in the art, such as Ficoll separation.

In one embodiment, the engineered immune cells express a second chimeric antigen receptor comprising a second antigen binding region in addition to the chimeric antigen receptor targeting the NK activating receptor, wherein the second antigen binds Zones are defined as above.

Nucleic acid sequences encoding chimeric antigen receptors and exogenous genes can be introduced into immune cells using conventional methods known in the art (eg, by transduction, transfection, transformation, etc.). "Transfection" is the process of introducing a nucleic acid molecule or polynucleotide, including a vector, into a target cell. An example is RNA transfection, the process of introducing RNA (eg, in vitro transcribed RNA, ivtRNA) into a host cell. The term is mainly used for non-viral methods in eukaryotic cells. The term "transduction" is generally used to describe virus-mediated transfer of nucleic acid molecules or polynucleotides. Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane to allow uptake of material. Transfection can be performed using calcium phosphate, by electroporation, by cell extrusion, or by mixing cationic lipids with materials to create liposomes that fuse with cell membranes and deposit their cargo inside. Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate/DNA co-precipitation), microinjection, and electroporation. perforation. The term "transformation" is used to describe the non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria, but also into non-animal eukaryotic cells (including plant cells). Thus, transformation is the genetic alteration of a bacterial or non-animal eukaryotic cell, which is produced by the direct uptake of the cell membrane from its surroundings and subsequent incorporation of exogenous genetic material (nucleic acid molecules). Conversion can be achieved by manual means. For transformation to occur, the cells or bacteria must be in a competent state. For prokaryotic transformation, techniques can include heat shock-mediated uptake, fusion of bacterial protoplasts with intact cells, microinjection, and electroporation. After the nucleic acid or vector is introduced into immune cells, those skilled in the art can expand and activate the resulting immune cells by conventional techniques.

In one embodiment, to reduce the risk of graft-versus-host disease, the engineered immune cells further comprise suppressed or silenced expression of at least one gene selected from the group consisting of CD52, GR, dCK, TCR/CD3 genes ( such as TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ), MHC-related genes (HLA-A, HLA-B, HLA-C, B2M, HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA) and immune checkpoint genes such as PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A , CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT , FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, and GUCY1B3. Preferably, the engineered immune cells further comprise suppressed or silenced expression of at least one gene selected from the group consisting of TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA, PD1, LAG3, TIM3, CTLA4, more preferably TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA.

In one embodiment, to reduce mutual killing between engineered immune cells, the expression of the corresponding endogenous NK activating receptors targeted by chimeric antigen receptors in the engineered immune cells is inhibited or silenced. For example, in engineered immune cells targeting NKG2D (e.g. CAR-T or CAR-NK cells), the expression of endogenous NKG2D is inhibited or silenced; engineered immune cells targeting NKp46 (e.g. CAR-T or CAR-NK cells) CAR-NK cells), its endogenous NKp46 expression was inhibited or silenced.

Methods of inhibiting gene expression or silencing genes are well known to those skilled in the art. For example, antisense RNA, RNA decoys, RNA aptamers, siRNA, shRNA/miRNA, trans dominant negative protein (TNP), chimeric/fusion proteins, chemokine ligands, anti-infective cellular proteins, cellular Intrabodies (sFv), nucleoside analogs (NRTI), non-nucleoside analogs (NNRTI), integrase inhibitors (oligonucleotides, dinucleotides and chemicals) and protease inhibitors to inhibit gene expression. In addition, gene silencing can also be achieved by mediating DNA fragmentation, eg, by meganucleases, zinc finger nucleases, TALE nucleases, or Cas enzymes in the CRISPR system.

pharmaceutical composition

The present invention also provides a pharmaceutical composition comprising the chimeric antigen receptor, nucleic acid molecule, carrier or engineered immune cell of the present invention as an active agent, and a variety of pharmaceutically acceptable excipients.

As used herein, the term "pharmaceutically acceptable excipient" means pharmacologically and/or physiologically compatible with the subject and the active ingredient (ie, capable of eliciting the desired therapeutic effect without causing any inconvenience desired local or systemic effect) carriers and/or excipients, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coatings, adsorbents, antiadherents, glidants, antioxidants, flavoring agents, colorants, Sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delaying agents, stabilizers and tonicity modifiers . It is known to those skilled in the art to select suitable excipients to prepare the desired pharmaceutical compositions of the present invention. Exemplary excipients for use in the pharmaceutical compositions of the present invention include saline, buffered saline, dextrose and water. In general, the selection of suitable excipients depends, among other things, on the active agent used, the disease to be treated and the desired dosage form of the pharmaceutical composition.

The pharmaceutical compositions according to the present invention may be suitable for administration by various routes. Typically, administration is accomplished parenterally. Parenteral delivery methods include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual or intranasal administration.

The pharmaceutical compositions according to the invention can also be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, in particular ointments, creams, transdermal patches, gels, powders, tablets, solutions, gaseous In the form of aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures or liquid extracts, or in a form particularly suitable for the desired method of administration. Processes known in the present invention for the manufacture of pharmaceuticals may include, for example, conventional mixing, dissolving, granulating, dragee-making, milling, emulsifying, encapsulating, entrapping, or lyophilizing processes. Pharmaceutical compositions comprising immune cells such as those described herein are typically provided in solution and preferably comprise a pharmaceutically acceptable buffer.

The pharmaceutical compositions according to the present invention may also be administered in combination with one or more other agents suitable for the treatment and/or prevention of the disease to be treated. Preferred examples of agents suitable for combination include known anticancer drugs such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide , gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetate glucuronate, auristatin E, vincristine and doxorubicin; peptide cytotoxins such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNase and RNase; radionuclides such as iodine 131, rhenium 186, indium 111, iridium 90, bismuth 210 and 213, actinium 225 and astatine 213; prodrugs, such as antibody-directed enzyme prodrugs; immunostimulants, such as platelet factor 4, melanoma growth-stimulating protein, etc.; antibodies or fragments thereof, such as anti-CD3 antibodies or fragments thereof, complement activators, heterologous protein domains, homologous protein domains, viral/bacterial protein domains and viral/bacterial peptides. In addition, the pharmaceutical composition of the present invention can also be used in combination with one or more other treatment methods, such as chemotherapy and radiotherapy.

therapeutic application

The present invention also provides a method of treating a subject suffering from cancer, infection or autoimmune disease, comprising administering to the subject an effective amount of an immune cell or a pharmaceutical composition according to the present invention. Accordingly, the present invention also encompasses the use of the chimeric antigen receptors, nucleic acid molecules, vectors, engineered immune cells and pharmaceutical compositions in the manufacture of medicaments for the treatment of cancer, infection or autoimmune diseases.

In one embodiment, an effective amount of an immune cell and/or pharmaceutical composition of the invention is administered directly to a subject.

In another embodiment, the method of treatment of the present invention is an ex vivo treatment. Specifically, the method comprises the steps of: (a) providing a sample comprising immune cells; (b) introducing the chimeric antigen receptor of the present invention and, if present, an exogenous gene into the immune cells in vitro and optionally inhibiting or silencing the expression of specific genes in the immune cells (if desired), obtaining modified immune cells, (c) administering the modified immune cells to a subject in need thereof. Preferably, the immune cells provided in step (a) are selected from macrophages, dendritic cells, monocytes, T cells, NK cells and/or NKT cells; Conventional methods are obtained from a subject's sample, particularly a blood sample. However, other immune cells capable of expressing the chimeric antigen receptors of the invention and performing the desired biological effector functions as described herein may also be used. Furthermore, the immune cells are typically selected to be compatible with the subject's immune system, ie preferably the immune cells do not elicit an immunogenic response. For example, "universal recipient cells," ie, universally compatible lymphocytes that can be grown and expanded in vitro, can be used that perform the desired biological effector function. The use of such cells would not require obtaining and/or providing the subject's own lymphocytes. The ex vivo introduction of step (c) can be carried out by introducing a nucleic acid or vector as described herein into immune cells via electroporation or by infecting immune cells with a viral vector such as a lentiviral vector, adenovirus Viral vector, adeno-associated viral vector or retroviral vector. Other conceivable methods include the use of transfection reagents (such as liposomes) or transient RNA transfection.

In one embodiment, the immune cells are autologous or allogeneic cells, preferably T cells, macrophages, dendritic cells, monocytes, NK cells and/or NKT cells, more preferably T cells, NK cells cells or NKT cells.

As used herein, the term "autologous" refers to any material derived from an individual to be later reintroduced into that same individual.

As used herein, the term "allogeneic" refers to any material derived from a different animal or different patient of the same species as the individual into which the material is introduced. Two or more individuals are considered allogeneic to each other when the genes at one or more loci are different. In some cases, allogeneic material from individuals of the same species may be genetically different enough for antigenic interactions to occur.

As used herein, the term "subject" is a mammal. The mammal can be a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow, but is not limited to these examples. Mammals other than humans can advantageously be used as subjects representing animal models of cancer. Preferably, the subject is a human.

In one embodiment, the cancer is selected from the group consisting of: brain glioma, blastoma, sarcoma, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer, peritoneal cancer, cervical cancer , choriocarcinoma, colon and rectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer (including gastrointestinal cancer), glioblastoma (GBM), Liver cancer, hepatocellular tumor, intraepithelial tumor, kidney cancer, laryngeal cancer, liver tumor, lung cancer (eg, small cell lung cancer, non-small cell lung cancer, adenocarcinoma, and squamous lung cancer), melanoma, myeloma, neuroblastoma , oral cancer (e.g. lips, tongue, mouth and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, mesothelioma, retinoblastoma, rhabdomyosarcoma, rectal cancer, cancer of the respiratory system, salivary gland cancer, skin cancer, squamous cell carcinoma squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, malignancies of the urinary system, vulvar cancer, Waldenstrom macroglobulinemia, lymphomas (including Hodgkin lymphoma and non-Hodgkin lymphoma) Neoplasms, such as B-cell lymphomas (including low-grade/follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade/follicular NHL, intermediate-grade diffuse NHL, high-grade immunogenic cellular NHL, high-grade lymphoblastic NHL, high-grade small non-cleaving cell NHL, bulky NHL), mantle cell lymphoma, AIDS-related lymphoma, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, MALT lymphoma, marginal zone lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell tumor, etc.), NK cell lymphoma, aggressive NK cell leukemia, leukemia (including acute leukemia, For example acute lymphoblastic leukemia, acute myeloid leukemia, acute non-lymphocytic leukemia such as acute myeloid leukemia (including undifferentiated and partially differentiated), acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia Cellular leukemia, erythroleukemia, acute megakaryocytic leukemia; chronic leukemia, such as chronic myeloid leukemia, chronic lymphocytic leukemia, chronic monocytic leukemia; and other special types of leukemia such as hairy cell leukemia, prolymphocytic leukemia, plasma cell leukemia , adult T-cell leukemia, eosinophilic leukemia, basophilic leukemia, etc.), blastic plasmacytoid dendritic cell tumor, malignant lymphoproliferative disorders, myelodysplasia, multiple myeloma, myeloproliferation Abnormalities, and Post-Transplant Lymphoproliferative Disorder (PTLD). Preferably, the diseases that can be treated with the engineered immune cells or pharmaceutical compositions of the present invention are selected from: leukemia, lymphoma, multiple myeloma, brain glioma, pancreatic cancer, gastric cancer, and the like.

In one embodiment, the infection includes, but is not limited to, infections caused by viruses, bacteria, fungi, and parasites.

In one embodiment, the autoimmune disease includes, but is not limited to, type 1 diabetes, celiac disease, Graves disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, Addison Illness, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia and systemic lupus erythematosus, etc.

The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with examples. It should be noted that those skilled in the art should understand that the accompanying drawings and the embodiments of the present invention are only for the purpose of illustration and do not constitute any limitation to the present invention. The embodiments in the present application and the features in the embodiments may be combined with each other where there is no contradiction.

Description of drawings

Figure 1: Schematic diagram of the CAR structure constructed in Example 1.

Figure 2: scFv expression levels in CAR-T cells containing different NKG2D scFvs.

Figure 3: Killing effect of CAR-T cells containing different NKG2D scFvs on NK92 cells.

Figure 4: scFv expression levels in CAR-T cells targeting NKG2D.

Figure 5: Killing effect of CAR-T cells targeting NKG2D on NK92 cells.

Figure 6: CD107 expression levels after co-culture of NKG2D-targeted CAR-T cells with NK92 cells.

Figure 7: Cytokine release levels from CAR-T cells targeting NKG2D.

Figure 8: Proportion of CD8+ T cells in NKG2D-targeting CAR-T cell population in which endogenous NKG2D is knocked out.

Figure 9: scFv expression levels in NKG2D-CD19 dual-target CAR-T cells.

Figure 10: The killing effect of NKG2D-CD19 dual-target CAR-T cells on NK92 cells and Nalm6 cells.

Figure 11: CD107 expression levels of NKG2D-CD19 dual-target CAR-T cells co-cultured with Nalm6 cells.

Figure 12: CD107 expression levels of NKG2D-CD19 dual-target CAR-T cells co-cultured with NK92 cells.

Figure 13: Cytokine release levels after co-culture of NKG2D-CD19 dual-target CAR-T cells with Nalm6 cells and NK92 cells.

Figure 14: scFv expression levels in CAR-T cells containing different NKp46 scFvs.

Figure 15: Killing effect of CAR-T cells containing different NKp46scFv on NK92 cells.

Figure 16: scFv expression levels in NKp46-CD19 dual-target CAR-T cells.

Figure 17: Killing effect of NKp46-CD19 dual-target CAR-T cells on NK92 cells and Nalm6 cells.

Figure 18: Killing effect of NKp46-CD19 dual-target CAR-NK cells on NK92 cells and Nalm6 cells.

Figure 19: Killing effect of NKp46-CD19 dual-target CAR-NK cells in which endogenous NKp46 is knocked out on NK92 cells and Nalm6 cells.

detailed description

The T cells used in all examples of the present invention were primary human CD4+CD8+ T cells isolated from healthy donors using leukapheresis by Ficoll-Paque™ PREMIUM (GE Healthcare, Cat. No. 17-5442-02).

The scFv sequences used in the following examples, the positions of the heavy chain variable region (VH) and light chain variable region (VL) contained therein in the corresponding scFv, and the CDR-L1, CDR-L2, CDR-L1, CDR-L2, CDR- The sequences of L3, CDR-H1, CDR-H2, and CDR-H3 are shown in Table 1 below.

Table 1

Example 1. Preparation of CAR T cells containing different scFvs and verification of function

1.1 Preparation of CAR T cells

Sequences encoding the following proteins were synthesized and cloned into the pLVX vector (Public Protein/Plasmid Library (PPL), Cat. No.: PPL00157-4a): CD8α signal peptide (SEQ ID NO: 17), anti-NKG2D scFv, CD8α hinge region ( SEQ ID NO: 19), CD8α transmembrane region (SEQ ID NO: 3), 4-1BB intracellular region (SEQ ID NO: 9), CD3ζ intracellular signaling domain (SEQ ID NO: 11), and through Sequencing confirms the correct insertion of the target sequence. Wherein, the amino acid sequence of the anti-NKG2D scFv contained in the NKG2D-1 CAR is shown in SEQ ID NO: 27; the amino acid sequence of the anti-NKG2D scFv contained in the NKG2D-2 CAR is shown in SEQ ID NO: 29; The amino acid sequence of the NKG2D scFv is shown in SEQ ID NO: 31; the amino acid sequence of the anti-NKG2D scFv contained in the NKG2D-4 CAR is shown in SEQ ID NO: 33.

Add 3ml Opti-MEM (Gibco, Item No. 31985-070) to a sterile tube to dilute the above plasmid, and then add the packaging vector psPAX2 (Addgene, Cat. No. 12260) and the envelope vector pMD2.G (Addgene, Cat. No. 12259). Then, add 120ul of X-treme GENE HP DNA transfection reagent (Roche, Cat. No. 06366236001), mix immediately, incubate at room temperature for 15 min, and then add the plasmid/vector/transfection reagent mixture dropwise to the culture flask of 293T cells . Viruses were collected at 24 hours and 48 hours, pooled, and ultracentrifuged (25000 g, 4°C, 2.5 hours) to obtain concentrated lentiviruses.

T cells were activated with DynaBeads CD3/CD28 CTS ^™ (Gibco, Cat. No. 40203D) and cultured for 1 day at 37°C and 5% CO2. Then, the concentrated lentivirus was added and cultured for 3 days to obtain CAR T cells expressing different scFvs. Unmodified wild-type T cells were used as negative controls (NT). The result is shown in Figure 2

It can be seen that the scFv in the CAR T cells prepared by the present invention can be effectively expressed.

1.2 Killing effect of CAR-T cells on NK92 target cells

In order to test the long-term killing ability of CAR-T cells in vitro, NK92 target cells were firstly plated into 24-well plates at 5× ^{10 5} /well, and then the effector-target ratio (ie the ratio of effector T cells to target cells) was 2:1 respectively. ) The CAR T cells expressing different scFvs prepared as above were plated into 24-well plates for co-culture, and the fluorescence value was measured by a microplate reader after 16-18 hours. According to the calculation formula: (mean fluorescence of target cells - mean fluorescence of samples)/mean fluorescence of target cells×100%, the killing efficiency was calculated, and the results are shown in FIG. 3 .

It can be seen that CAR T cells containing 4 different scFvs targeting NKG2D have significant killing ability to NK92 cells, among which NKG2D-1 CAR T cells have the strongest killing ability, which was used for subsequent experiments and renamed 2Dbbz- CAR T cells.

Example 2: Preparation of CAR T cells and their killing effect on NK92 target cells and cytokine release

2.1 Preparation of CAR T cells

Sequences encoding the following proteins were synthesized and cloned into the pLVX vector (Public Protein/Plasmid Library (PPL), Cat. No.: PPL00157-4a): CD8α signal peptide (SEQ ID NO: 17), anti-NKG2D scFv (SEQ ID NO: 27), CD8α hinge region (SEQ ID NO: 19), CD8α transmembrane region (SEQ ID NO: 3), costimulatory domain, CD3ζ intracellular signaling domain (SEQ ID NO: 11), wherein the costimulatory structure domain comprising CD28 intracellular region (SEQ ID NO: 7), or 4-1BB intracellular region (SEQ ID NO: 9)+DAP10 intracellular region (SEQ ID NO: 45) to obtain 2D28z-CAR and 2Dbb10z-CAR, And confirm the correct insertion of the target sequence by sequencing. Figure 1 shows the CAR structure constructed in this example.

The preparation of CAR T cells was completed according to the method described in Example 1, and Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG, F(ab') ₂ Fragment Specific (min X Hu, Bov, Hrs Sr Prot) was used (jackson immunoresearch, Cat. No. 115-065-072) as primary antibody, APC Streptavidin (BD Pharmingen, Cat. No. 554067) or PE Streptavidin (BD Pharmingen, Cat. No. 554061) as secondary antibodies, 2Dbbz-CAR T cells were detected by flow cytometry, The expression levels of scFv on 2D28z-CAR T cells and 2Dbb10z-CAR T cells, the results are shown in Figure 4.

2.2 Killing effect of CAR-T cells on NK92 target cells

In order to test the long-term killing ability of CAR-T cells in vitro, NK92 target cells were firstly plated into 24-well plates at 5× ^{10 5} /well, and then the effector-target ratio (ie the ratio of effector T cells to target cells) was 2:1 respectively. ) CAR-T cells expressing 2Dbbz-CAR, 2D28z-CAR and 2Dbb10z-CAR were plated into 24-well plates for co-culture (D0), and the target was continuously added at a 2:1 effector-target ratio on D2, D4 and D8, respectively. Cells, and then on D2 and D10, PE-mouse anti-human CD56 was used to detect the residual NK92 cells by flow cytometry, and the killing rate of CAR T cells was calculated. The results are shown in Figure 5.

It can be seen that, compared with NT, all three CAR T cells have significant specific killing on NK92 target cells. And with the prolongation of culture time, the killing activity of 2Dbbz-CAR T cells gradually weakened, while 2Dbb10z-CAR T cells could still maintain a high killing ability, and there was a significant difference in the killing activity of the two at D10. This indicates that the addition of the intracellular domain of DAP10 as a costimulatory domain can significantly improve the persistence of CAR T cell killing activity.

2.3 Detection of CD107a expression

Cytotoxic T lymphocytes (CTL cells) contain high concentrations of cytotoxic granules in the form of vesicles in the cytoplasm, and lysosome-associated membrane protein I (CD107a) is the main component of vesicle membrane proteins. When CTL cells kill target cells, the toxic particles will reach the cell membrane and fuse with the cell membrane (the CD107a molecule is transported to the cell membrane surface at this time), resulting in the release of the particle contents and eventually the death of the target cells. Therefore, CD107a molecule is a sensitive marker of CTL cell degranulation, which can reflect cell killing activity.

The target cells (NK92 cells) were plated in a 96-well plate at a concentration of 1x10 ⁵ cells/well, and then the CAR-T and NT cells of the present invention were added to each well at a ratio of 1:1, and 10 μl PE-anti- human CD107a (BD Pharmingen, Cat. No. 555801), co-cultured at 37°C, 5% CO ₂ . After 1 h, Goigstop (BD Pharmingen, Cat. No. 51-2092KZ) was added and incubated for 2.5 hours. Then, 5 μl APC-anti human CD8 (BD Pharmingen, Cat. No.: 555369) and 5 μl FITC-anti human CD4 (BD Pharmingen, Cat. No.: 561005) were added to each well, and after incubation at 37°C for 30 minutes, CD107a was detected by flow cytometry expression, and the results are shown in Figure 6.

It can be seen that after the 2Dbbz-CAR T cells, 2D28z-CAR T and 2Dbb10z-CAR T cells prepared by the present invention are co-cultured with the target cells, the expression of CD107a is significantly up-regulated, indicating that the CAR-T cells of the present invention can significantly kill NK cells . In addition, the expression of CD107a in CD8+ T cells of 2Dbb10z-CAR T cells was significantly higher than that of 2Dbbz-CAR T cells, indicating that 2Dbb10z-CAR T cells have stronger killing ability than 2Dbbz-CAR T cells.

2.4 Cytokine release from CAR-T cells

When T cells kill target cells, the number of target cells decreases and the cytokines IL-2 and IFN-γ are also released. According to the following procedure, enzyme-linked immunosorbent assay (ELISA) was used to measure the release levels of cytokines IL-2 and IFN-γ when CAR T cells killed target cells.

(1) Collect cell co-culture supernatant

The target cells NK92 were plated in a 96-well plate at a concentration of 1×10 ⁵ /well, and then the CAR T cells and NT cells of the present invention were co-cultured with the target cells at a ratio of 1:1, and the cells were collected after 18-24 hours. Culture supernatant.

(2) ELISA to detect the secretion of IL-2 and IFN-γ in the supernatant

Coat a 96-well plate with capture antibody Purified anti-human IL2 Antibody (Biolegend, Cat. No. 500302) or Purified anti-human IFN-γ Antibody (Biolegend, Cat. No. 506502) and incubate at 4°C overnight, then remove the antibody solution and add 250 μL containing 2% A solution of BSA (sigma, Cat. No. V900933-1 kg) in PBST (1XPBS with 0.1% Tween) was incubated at 37°C for 2 hours. Plates were then washed 3 times with 250 [mu]L of PBST (1XPBS with 0.1% Tween). 50 μL of cell co-culture supernatant or standards were added to each well and incubated at 37° C. for 1 hour, then the plate was washed 3 times with 250 μL of PBST (1XPBS with 0.1% Tween). Then, 50 μL of detection antibody Anti-Interferon gamma antibody [MD-1] (Biotin) (abcam, Cat. No. ab25017) was added to each well, incubated at 37°C for 1 hour, and washed with 250 μL of PBST (1XPBS containing 0.1% Tween) plate 3 times. Then, HRP Streptavidin (Biolegend, product number 405210) was added, and after incubation at 37° C. for 30 minutes, the supernatant was discarded, 250 μL of PBST (1XPBS containing 0.1% Tween) was added, and the mixture was washed 5 times. 50 [mu]L of TMB substrate solution was added to each well. The reaction was allowed to occur at room temperature for 30 minutes in the dark, after which 50 μL of ₁ mol/L _H2SO4 was added to each well to stop the reaction. Within 30 minutes of stopping the reaction, use a microplate reader to detect the absorbance at 450 nm, and calculate the content of cytokines according to the standard curve (drawn according to the reading value and concentration of the standard). The results are shown in Figure 7.

It can be seen that the cytokine release water of the 2Dbbz-CAR T cells, 2D28z-CAR T and 2Dbb10z-CAR T cells of the present invention is significantly higher than that of the control NT cells. In addition, both IL-2 and IFN-γ release levels of 2Dbb10z-CAR T cells were significantly lower than those of 2Dbbz-CAR T cells, suggesting that the former is safer than the latter, since excessive cytokine release may lead to severe Side effects, such as cytokine storm.

The above results show that CAR-T cells targeting NK activating receptors can continuously and effectively kill NK cells. In addition, compared with traditional CARs containing only the 4-1BB co-stimulatory domain, further inclusion of the DAP10 intracellular domain can improve the sustained killing activity of CAR-T cells while secreting fewer cytokines and improving safety.

Example 3. Preparation of CAR-T cells in which the targeted NK activating receptor is knocked out

Knockout of endogenous NKG2D in NT, 2Dbbz-CAR T cells, 2D28z-CAR T and 2Dbb10z-CAR T cells by CRISP/Cas9 system and PE mouse anti-human NKG2D by flow cytometry (biolegend Cat. No. 302806) The expression level of NKG2D in CAR T cells after knockout was detected (Table 1), and APC-anti human CD8 (BD Pharmingen, 555369) was used to detect the proportion of CD8+ T cells in CAR-T cells (Figure 8). Non-knockout NT cells served as controls.

Table 1. NKG2D expression levels in CAR-T cells

基因名称gene name	2DbbzKO-CAR2DbbzKO-CAR	2Dbb10zKO-CAR2Dbb10zKO-CAR	2D28zKO-CAR2D28zKO-CAR	NTNT
NKG2DNKG2D	5.00％5.00%	6.20％6.20%	6.50％6.50%	39.40％39.40%

The results in Table 1 showed that NKG2D was effectively knocked out in each CAR-T cell.

As can be seen from Figure 8, when a CAR targeting NKG2D is expressed, further knockdown of endogenous NKG2D can significantly increase the proportion of CD8+ T cells in the CAR T cell population, which may be due to reduced CAR-T cell interaction kill each other. Since CD8+ T cells are cytotoxic lymphocytes, this elevated ratio of CD8+ T cells can generally enhance the killing effect of CAR T cells in vitro and in vivo.

Example 4. Preparation of dual-target CAR-T cells and verification of their functions

19CAR T cells and 2D19CAR T cells were prepared according to the method described in Example 1. 19CAR T cells differed from 2Dbbz-CAR T cells only by replacing the anti-NKG2D scFv with an anti-CD19 scFv (SEQ ID NO: 1). 2D19CAR T cells differ from 2Dbbz-CAR T cells only in that the CAR further contains an anti-CD19 scFv.

In addition, general-purpose CAR-tKO T cells in which TCR and MHC-related genes (for example, HLA-class I molecules such as B2M and HLA-class II molecules such as RFX5) were knocked out were prepared by the CRISP/Cas9 system and treated with FITC Mouse Anti-Human CD3 (BD Pharmingen, Cat. No. 555916), PE mouse anti-human HLA-I (R&D Cat. No. FAB7098P) and APC anti-human DR, DP, DQ (biolegend, Cat. No. 361714) antibodies by flow cytometry The expression efficiency of TCR/CD3 (TCRα), HLA-I (B2M), and HLA-II (RFX5) in knockout CAR T cells was detected, and the results are shown in Table 2 below. Non-knockout NT cells served as controls.

Table 2. Expression levels of TCR and MHC-related genes in CAR-T cells

敲除基因名称Knockout gene name	TCR/CD3TCR/CD3	HLA-I(B2M)HLA-I (B2M)	HLA-II(RFX5)HLA-II (RFX5)
19CAR-tKO19CAR-tKO	5.60％5.60%	14％14%	11％11%
2D19CAR-tKO2D19CAR-tKO	3.90％3.90%	16.30％16.30%	10.40％10.40%
NTNT	97％97%	98％98%	82％82%

The results in Table 2 showed that TCR/CD3, HLA-I and HLA-II genes were effectively knocked out in each CAR-T cell.

In addition, Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG, F(ab') ₂ Fragment Specific (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, Cat. No. 115-065-072) was used as a Antibodies, APC Streptavidin (BD Pharmingen, Cat. No. 554067) or PE Streptavidin (BD Pharmingen, Cat. No. 554061) were used as secondary antibodies to detect the expression level of scFv on CAR-T cells by flow cytometry. The results are shown in Figure 9.

It can be seen that the scFv in the CAR T cells prepared in this example can be effectively expressed, and knockout of TCR and MHC-related genes will not affect the expression of CAR.

The killing effect of the above cells on the NK92 target cells was detected according to the method described in 1.2 in Example 1, and the killing ability of the CAR T cells on the target cell Nalm6 was detected according to the following method: First, the Nalm6 carrying the fluorescein gene was mixed with 1×10 ⁴ /well. Target cells were plated into 96-well plates, and then CAR T cells and untransfected T cells (NT) were plated into 96-well plates at a 4:1 effector-to-target ratio (ie, the ratio of effector T cells to target cells). After 16-18 hours of incubation, the fluorescence value was measured by a microplate reader. According to the calculation formula: (mean fluorescence of target cells - mean fluorescence of samples)/mean fluorescence of target cells×100%, the killing efficiency was calculated, and the results are shown in FIG. 10 .

It can be seen that both 19CAR T cells and 2D19CAR T cells can significantly kill Nalm6. The two CAR-T cells containing only anti-CD19 scFv did not kill NK92 because they could not recognize it; while both 2D19CAR T cells and 2D19CAR-tKO T cells containing anti-NKG2D scFv could effectively kill NK92 cells. In addition, compared with 2D19CAR T cells, TCR/MHC-related gene knockout 2D19CAR-tKO T cells had decreased killing of target cells, probably because the knockout of these genes was at the same time that CAR-T cells killed NK cells. It also makes NK cells kill CAR-T cells to a certain extent.

According to the method described in 2.3 in Example 2, the expression levels of CD107a after the above cells were co-cultured with target cells Nalm6 (expressing CD19) and NK92 (expressing NKG2D) were detected, and the results are shown in Figure 11 and Figure 12 . It can be seen that 19CAR T cells and 19CAR-tKO T cells detected significantly elevated CD107a levels only after co-culture with Nalm6 compared to NT cells; 2D19CAR T cells and 2D19CAR-tKO T cells were co-cultured with Nalm6 and NK92 cells. Significantly elevated levels of CD107a could be detected after culture.

According to the method described in 2.4 in Example 2, the IFN-γ release levels after the above cells were co-cultured with target cells Nalm6 (expressing CD19) and NK92 (expressing NKG2D), respectively, were detected. The results are shown in Figure 13 . It can be seen that CAR-T cells containing only anti-CD19 scFv detected significantly elevated levels of IFN-γ only after co-culture with Nalm6, while almost no IFN-γ was detected after co-culture with NK92 cells that did not express CD19. gamma release. However, CAR-T cells containing both anti-CD19 scFv and anti-NKG2D scFv had detectable IFN-γ release after co-culture with both target cells.

The above results show that the CAR-T cells that recognize the dual targets of CD19 and NKG2D prepared by the present invention and the universal CAR-T cells in which MHC-related genes are knocked out can effectively kill target cells expressing CD19 or NKG2D. This dual-target CAR design, which simultaneously targets NK activating receptors and tumor antigens, can kill NK cells to prolong the survival of CAR-T cells on the one hand, and efficiently kill target cells expressing tumor antigens on the other hand. .

Example 5. Preparation of CAR cells containing different scFv sequences targeting NKp46 and verification of their function

CAR T cells were prepared according to the method of Example 1, which differed from 2Dbbz-CAR T cells only by substituting anti-NKp46 scFv for anti-NKG2D scFv. Wherein, the amino acid sequence of the anti-NKp46 scFv contained in the NKp46-1 CAR is shown in SEQ ID NO: 35; the amino acid sequence of the anti-NKp46 scFv contained in the NKp46-2 CAR is shown in SEQ ID NO: 37; The amino acid sequence of the anti-NKp46 scFv is shown in SEQ ID NO: 39; the amino acid sequence of the anti-NKp46 scFv contained in the NKp46-4 CAR is shown in SEQ ID NO: 41; the amino acid sequence of the anti-NKp46 scFv contained in the NKp46-5 CAR is shown in SEQ ID NO: 41 ID NO: 43.

Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG, F(ab') ₂ Fragment Specific (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, Cat. No. 115-065-072) was used as primary antibody, APC Streptavidin (BD Pharmingen, Cat. No. 554067) or PE Streptavidin (BD Pharmingen, Cat. No. 554061) were used as secondary antibodies to detect the expression level of scFv on CAR-T cells by flow cytometry. The results are shown in Figure 14.

It can be seen that CAR-T cells containing different NKp46 scFv can effectively express scFv.

The killing effect of the above-mentioned CAR T cells on NK92 cells was detected according to the method described in 1.2 in Example 1, and the results are shown in Figure 15. It can be seen that CAR T cells containing 5 different scFvs targeting NKp46 have significant killing ability to NK92 cells, among which NKp46-5-CAR T cells have the strongest killing ability, which was used for subsequent experiments and renamed 46CAR -T cells.

The inventors also prepared 4619 CAR-T cells targeting dual targets of NKp46 and CD19, which differ from 2D19bbz-CAR T cells only by replacing the anti-NKG2D scFv with an anti-NKp46 scFv (SEQ ID NO: 43). The scFv expression in 19CAR-T cells, 46CAR-T cells and 4619CAR-T cells was detected by flow cytometry, and the results are shown in Figure 16. The killing effect of the above three CAR T cells on NK92 cells and Nalm6 was detected according to the method described in Example 1, and the results are shown in Figure 17.

It can be seen that CAR in 19CAR-T cells, 46CAR-T cells and 4619CAR-T cells can be effectively expressed, and 46CAR T cells only kill NK92 target cells, 19CAR T cells only kill Nalm6 target cells, and 4619CAR T can effectively kill both target cells.

Example 6. Preparation of CAR-NK cells targeting NKp46 and verification of their function

CAR-NK cells targeting NKp46, CD19, or both, and KO-CAR NK cells in which NKp46 is knocked out, were prepared by first knocking out endogenous NKp46 in NK92 cells by CRISPR/Cas9 Flow cytometry confirmed that NKp46 was efficiently knocked out in NK92 cells (Table 3).

Table 3. NKp46 expression levels in NK92 cells before and after knockdown

基因名称gene name	NK92KONK92KO	NK92NK92
NKp46NKp46	23.00％23.00%	92.00％92.00%

Then, 19CAR comprising CD19 scFv (SEQ ID NO: 1), 46 CAR comprising NKp46 scFv (SEQ ID NO: 43), and 19 CAR comprising CD19 scFv (SEQ ID NO: 1) and NKp46 according to the viral packaging method in Example 1 After the 4619CAR vector of scFv (SEQ ID NO:43) was packaged with virus, the virus was co-infected with NK92 cells or NK92KO cells by centrifugation (1000g, 90min) to obtain 19CAR NK, 46CAR NK, 4619CAR NK and 19KO-CAR NK, 46KO-CAR NK, 4619KO-CAR NK cells.

The killing effect of the above-mentioned CAR-NK cells on NK92 cells and Nalm6 cells was detected according to the methods described in Example 2 and Example 3, and the results are shown in Figure 18 and Figure 19 . It can be seen that 46CAR NK cells and 46KO-CAR NK cells kill only NK92 target cells, 19CAR NK cells and 19KO-CAR NK cells only kill Nalm6 target cells, and 4619CAR NK and 4619KO-CAR NK cells kill both target cells. Can effectively kill.

It should be noted that the above are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. It is understood by those skilled in the art that any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

A novel chimeric antigen receptor comprising an antigen binding region, a transmembrane domain and an intracellular signaling domain, wherein the antigen binding region targets a NK activating receptor.
The chimeric antigen receptor of claim 1, wherein the NK activating receptor is selected from the group consisting of NKG2 family, NCR family, KIR-S family, 2B4, DNAM-1, CD2 and LFA-1.
2. The chimeric antigen receptor of claim 2, wherein the NK activating receptor is selected from the group consisting of NKG2C, NKG2D, NKG2E, NKG2F, NKG2H, NKp30, NKp44, NKp46 and NKp80.
3. The chimeric antigen receptor of claim 3, wherein the NK-activating receptor is selected from the group consisting of NKG2D and NKp46.
The chimeric antigen receptor of claim 1, wherein the chimeric antigen receptor comprises an antibody targeting NKG2D or NKp46, the antibody targeting NKG2D comprising:

(i) CDR-L1, CDR-L2 and CDR-L3 as shown in SEQ ID NOs: 61, 62 and 63, and CDR-H1, CDR-H2 as shown in SEQ ID NOs: 64, 65 and 66, respectively and CDR-H3;

(ii) CDR-L1, CDR-L2 and CDR-L3 as shown in SEQ ID NOs: 67, 68 and 69, and CDR-H1, CDR-H2 as shown in SEQ ID NOs: 70, 71 and 72, respectively and CDR-H3;

(iii) CDR-L1, CDR-L2 and CDR-L3 as set forth in SEQ ID NOs: 67, 68 and 73, and CDR-Hl, CDR-H2 as set forth in SEQ ID NOs: 74, 75 and 76, respectively and CDR-H3; or

(iv) CDR-L1, CDR-L2 and CDR-L3 as set forth in SEQ ID NOs: 77, 78 and 79, and CDR-Hl, CDR-H2 as set forth in SEQ ID NOs: 80, 81 and 82, respectively and CDR-H3;

The antibody targeting NKp46 comprises:

(i) CDR-L1, CDR-L2 and CDR-L3 as shown in SEQ ID NOs: 83, 84 and 85, and CDR-H1, CDR-H2 as shown in SEQ ID NOs: 86, 87 and 88, respectively and CDR-H3;

(ii) CDR-L1, CDR-L2 and CDR-L3 as shown in SEQ ID NOs: 89, 90 and 91, and CDR-H1, CDR-H2 as shown in SEQ ID NOs: 92, 93 and 94, respectively and CDR-H3;

(iii) CDR-L1, CDR-L2 and CDR-L3 as shown in SEQ ID NOs: 95, 96 and 97, and CDR-H1, CDR-H2 as shown in SEQ ID NOs: 98, 99 and 100, respectively and CDR-H3;

(iv) CDR-L1, CDR-L2 and CDR-L3 as set forth in SEQ ID NOs: 101, 84 and 102, and CDR-Hl, CDR-H2 as set forth in SEQ ID NOs: 103, 87 and 104, respectively and CDR-H3; or

(v) CDR-L1, CDR-L2 and CDR-L3 as set forth in SEQ ID NOs: 105, 106 and 107, and CDR-Hl, CDR-H2 as set forth in SEQ ID NOs: 108, 109 and 110, respectively and CDR-H3.
The chimeric antigen receptor of claim 5, wherein the antibody targeting NKG2D comprises the The amino acid sequence shown in positions 1-109 or SEQ ID NO: 33, 1-108 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity Light chain variable region sequences and SEQ ID NO: 27, positions 137-246, SEQ ID NO: 29, positions 122-243, SEQ ID NO: 31, positions 122-236, or SEQ ID NO: 33, positions 121-243 The amino acid sequence shown at position has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity to the heavy chain variable region sequence;

The antibody targeting NKp46 comprises positions 1-107 of SEQ ID NO:35, positions 1-107 of SEQ ID NO:37, positions 1-107 of SEQ ID NO:39, positions 1-107 of SEQ ID NO:41 The amino acid sequence shown at position 107 or SEQ ID NO: 43 at positions 1-107 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity. Chain variable region sequences and SEQ ID NO: 35 positions 123-244, SEQ ID NO: 37 positions 123-238, SEQ ID NO: 39 positions 123-238, SEQ ID NO: 41 positions 123-242 Or a heavy chain having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity to the amino acid sequence shown in positions 123-237 of SEQ ID NO: 43 can be variable region sequence.
The chimeric antigen receptor of any one of claims 1-6, wherein the antigen binding region is selected from the group consisting of immunoglobulin molecules, Fab, Fab', F(ab')2, Fv fragments, scFv, disulfide bonds - linked Fv (sdFv), variable heavy (VH) or variable light (VL) domains of antibodies, Fd fragments consisting of VH and CH1 domains, linear antibodies, single domain antibodies, Nanobodies and the native ligand of the antigen or a functional fragment thereof.
The chimeric antigen receptor of any one of claims 1-7, wherein the transmembrane domain is selected from the transmembrane domains of the following proteins: TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
The chimeric antigen receptor of any one of claims 1-8, wherein the intracellular signaling domain is selected from the signaling domains of the following proteins: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b and CD66d. Preferably, the intracellular signaling domain is a CD3ζ-comprising signaling domain.
The chimeric antigen receptor of any one of claims 1-9, wherein the chimeric antigen receptor further comprises a costimulatory domain comprising one or more intracellular proteins selected from the group consisting of Regions: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18(LFA-1), CD27, CD28, CD30, CD40, CD54(ICAM), CD83, CD134(OX40), CD137(4-1BB), CD270(HVEM), CD272(BTLA), CD276(B7-H3), CD278(ICOS), CD357(GITR), DAP10, DAP12, LAT, NKG2C, NKG2D , SLP76, PD-1, LIGHT, TRIM, CD94, LTB, ZAP70, and combinations thereof.
10. The chimeric antigen receptor of claim 10, wherein the costimulatory domain comprises one or more intracellular domains selected from the group consisting of DAP10, DAP12, CD27, CD28, CD134, 4-1BB or CD278.
The chimeric antigen receptor of any one of claims 1-11, wherein the chimeric antigen receptor comprises a second antigen binding region that binds to a tumor antigen selected from the group consisting of TSHR, CD19, CD123, CD22, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3 , KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, AFP, Folate receptor α, ERBB2(Her2/neu), MUC1, EGFR, CS1, CD138, NCAM, Claudin18.2, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gploo, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe , GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD 179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR -1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, bean protein, HPV E6, E7, MAGE A1, ETV6-AML, Sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutant, prostate specific protein, survivin and telomerase, PCTA-l/Galectin 8 , MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B 1. BORIS, SART3, PAX5, OY-TES 1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mu hsp70-2, CD79a , CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2 , EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGFβ, APRIL, NKG2D, and any combination thereof.
A nucleic acid molecule encoding the chimeric antigen receptor of any one of claims 1-12.
A vector comprising the nucleic acid molecule of claim 13.
An engineered immune cell comprising the chimeric antigen receptor of any one of claims 1-12, the nucleic acid molecule of claim 13 or the vector of claim 14.
16. The engineered immune cell of claim 15, further comprising a second chimeric antigen receptor targeting a tumor antigen.
The engineered immune cell of any one of claims 15-16, further comprising suppressed or silenced expression of at least one gene selected from the group consisting of TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA, PD1, LAG3, TIM3, CTLA4.
16. The engineered immune cell of any one of claims 14-16, wherein expression of the corresponding endogenous NK activating receptor targeted by the chimeric antigen receptor is inhibited or silenced.
The engineered immune cell of claim 18, wherein the NK activating receptor is NKG2D or NKp46.
The engineered immune cell of any one of claims 15-19, wherein the engineered immune cell is selected from T cells, macrophages, dendritic cells, monocytes, NK cells, or NKT cells.
20. The engineered immune cell of any one of claims 15-20, wherein the immune cell is derived from a stem cell.
A pharmaceutical composition comprising the chimeric antigen receptor of any one of claims 1-12, the nucleic acid molecule of claim 13, the carrier of claim 14 or any one of claims 15-21 The engineered immune cells, and a variety of pharmaceutically acceptable excipients.
The chimeric antigen receptor of any one of claims 1-12, the nucleic acid molecule of claim 13, the vector of claim 14, the engineered immune cell of any one of claims 15-21, or Use of the pharmaceutical composition of claim 22 in the preparation of a medicament for treating cancer, infection or autoimmune disease.