CN112029001A

CN112029001A - Chimeric antigen receptors targeting NK activating receptors

Info

Publication number: CN112029001A
Application number: CN202010907250.9A
Authority: CN
Inventors: 周亚丽; 陈功; 姜小燕; 任江涛; 贺小宏; 王延宾; 韩露
Original assignee: Nanjing Bioheng Biotech Co Ltd
Current assignee: Nanjing Bioheng Biotech Co Ltd
Priority date: 2020-09-02
Filing date: 2020-09-02
Publication date: 2020-12-04
Anticipated expiration: 2040-09-02
Also published as: CN112029001B; WO2022048523A1; CN116063550A

Abstract

The invention relates to a novel chimeric antigen receptor targeting an NK activating receptor, which can treat malignant tumors caused by NK cell lesions and can avoid killing of introduced therapeutic CAR cells by NK cells in a patient in the background of universal CAR cells. The invention also relates to engineered immune cells expressing this novel chimeric antigen receptor, compositions comprising the same and their use in treating diseases.

Description

Chimeric antigen receptors targeting NK activating receptors

Technical Field

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

Background

Chimeric Antigen Receptor (CAR) cell therapy, as a new type of precise targeted therapy targeting tumors, has demonstrated in recent years good results in tumor therapy, especially in hematological tumor therapy. The basic principle is that exogenous chimeric antigen receptor is loaded in immune cells (such as commonly used T cells, NK cells and the like) through genetic engineering, and after in vitro amplification, the modified immune cells are returned to a human body, so that targeted tumor cells are killed. The chimeric antigen receptor generally comprises an antigen binding region, a transmembrane region, and an intracellular signaling region, and by binding of the antigen binding region to a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA) expressed on the surface of a tumor cell, a signal is transmitted into an immune cell via the intracellular signaling region, thereby activating the immune cell to exert its effector function.

NK cells are cytotoxic lymphocytes, can kill target cells through a variety of mechanisms, such as release of perforin and granzyme to cause cytolysis, activation of apoptosis pathway to cause apoptosis, release of cytokines directly on target cells, cell-mediated cytotoxicity, and the like, and play important roles 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 killing of self-healthy cells by NK cells. However, when the expression of MHC-I molecules on the cell surface is reduced or lost, or tumor cells, virus-infected cells, and the like are bound to an activating receptor via a surface antigen, the activating signal exceeds the inhibiting signal, and NK cells are activated to kill target cells.

In general CAR-T cells, it is often desirable to inhibit or knock out MHC class I molecules, such as HLA and B2M, to reduce host immune rejection of a foreign introduced CAR-T cell. However, such inhibition or knockout can activate NK cells in the host, leading to killing of CAR-T cells by host NK cells, seriously affecting proliferation and survival of CAR-T cells in vivo, and further affecting the efficacy of CAR-T cells.

Therefore, there is a need to develop a novel chimeric antigen receptor to address the problem of NK cell killing of therapeutic CAR cells.

Disclosure of Invention

In a first aspect, 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.

In one embodiment, the NK activating receptor is selected from the NKG2 family, preferably from NKG2C, NKG2D, NKG2E, NKG2F and NKG2H, more preferably NKG 2D. In one embodiment, the NK activating receptor is selected from the Natural Cytotoxicity Receptors (NCRs) family, preferably from NKp30, NKp44, NKp46 and NKp80, more preferably from NKp30 and NKp 46. In one embodiment, the NK activating receptor is selected from the KIR-S family, preferably from KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5 and KIR3DS1, more preferably KIR2DS 4. In one embodiment, the NK activating receptor is selected from the group consisting of co-receptors, preferably selected from the group consisting of 2B4, DNAM-1, CD2 and LFA-1.

In one embodiment, the novel 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, TnAg, 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, ERBB 20 (Her 20/neu), MUC 20, EGFR, CS 20, CD138, NCAM, Claudin18.2, Prostase, BCPAP, Nyhrf 2, Nyhrin 20, Epsilon 20, EPTC-20, EPTC-72, EPTC-20, EPTC-20, EPTC-20, EPTC-20, EPTC, LY6, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPV E6, E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostate specific protein, survivin and telomerase, PCTA-l/Galectin 8, MelanA/MARTl, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2ETS fusion gene), NA17, PAX3, BlAR, Cyclin, MYCN, RhoC, TRP-2, TRP 1B, RIS, SART3, PART 6342, OY 1, AKK-AP 28, LACK-869, LRRU-IRU 867, RAKE 8679, CD a, RARE-LRRU-7, RAKE 8479, CD 867, CD a, CD 8679, CD a, CD-LRRU 2, RAG-LRRU 2, and RNA, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGF β, APRIL, NKG2D, and any combination thereof. Preferably, the target is selected from the group consisting of CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, MUC1, 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 an immunoglobulin molecule, Fab ', F (ab')2, Fv fragment, scFv, disulfide-linked Fv (sdfv), heavy chain variable region (VH) or light chain variable region (VL) of an antibody, Fd fragment consisting of VH and CH1 domains, a linear antibody, a single domain antibody, a nanobody, a natural 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: TCR α chain, TCR β chain, TCR γ chain, TCR chain, CD3 ζ subunit, CD3 subunit, CD3 γ subunit, CD3 subunit, CD45, CD4, CD5, CD8 α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, and CD 154. Preferably, the transmembrane domain is selected from the transmembrane domains of CD8 α, CD4, CD28 and CD 278.

In one embodiment, the intracellular signaling domain is selected from the signaling domains of the following proteins: FcR γ, FcR β, CD3 γ, CD3, CD3, CD3 ζ, CD22, CD79a, CD79b, and CD66 d. Preferably, the intracellular signaling domain is a signaling domain comprising CD3 ζ.

In one embodiment, the chimeric antigen receptor further comprises a co-stimulatory domain comprising one or more intracellular regions of a protein 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, zaslp 76, PD-1, LIGHT, TRIM, CD94, LTB, p70, and combinations thereof. In a preferred embodiment, the co-stimulatory domain comprises one or more intracellular regions of a protein selected from the group consisting of: DAP10, DAP12, CD27, CD28, CD134, 4-1BB, or CD 278. For example, in one embodiment, the co-stimulatory domain comprises the intracellular region of 4-1 BB. In one embodiment, the co-stimulatory domain comprises the intracellular region of CD 28. In one embodiment, the costimulatory domain comprises the intracellular region of DAP 10. In one embodiment, the costimulatory domain comprises the intracellular region of DAP 12. More preferably, the co-stimulatory domain comprises two intracellular domains of a protein selected from the group consisting of: DAP10, DAP12, CD27, CD28, CD134, 4-1BB, or CD 278. In one embodiment, the co-stimulatory domain comprises the intracellular region of 4-1BB and the intracellular region of CD 28. In one embodiment, the co-stimulatory domain comprises the 4-1BB intracellular region and the intracellular region of DAP 10. In one embodiment, the co-stimulatory domain comprises the intracellular region of 4-1BB and the intracellular region of DAP 12. In one embodiment, the co-stimulatory domain comprises the intracellular region of CD28 and the intracellular region of DAP 10. In one embodiment, the co-stimulatory domain comprises the intracellular region of CD28 and the intracellular region of DAP 12.

The 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 invention also provides engineered immune cells expressing the novel chimeric antigen receptors described above.

In one embodiment, the engineered immune cell expresses two chimeric antigen receptors, wherein a first chimeric antigen receptor comprises a first antigen-binding region that targets an NK-activating receptor and a second chimeric antigen receptor comprises a second antigen-binding region that targets a tumor antigen.

In one embodiment, the engineered immune cell further comprises at least one gene whose expression is inhibited or silenced selected from the group consisting of: CD, GR, dCK, TCR/CD genes (e.g., TRAC, TRBC, CD γ, CD ζ), MHC associated genes (HLA- - -2, HLA-DPA, HLA-DQ, HLA-DRA, TAP, LMP, RFX, RFXAP, RFXANK, CIITA) and immune checkpoint genes, such as PD, LAG, TIM, CTLA, PPP2, PTPN, PDCD, HAVCR, BTLA, CD160, TIGIT, CD, CRTAM, TNFRSF10, CASP, FADD, FAS, TGFBRII, TGFRBRI, SMAD, SKI, SKIL, TGTGTGT, IL10, HMIF, IL6 AK, CSK, PAG, PRBAT, SIXP, FOCY, GUCY1A, GUDM 1B, GUAB 1, GU, and GU B. Preferably, the engineered immune cell further comprises at least one gene whose expression is inhibited or silenced selected from the group consisting of: TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA, PD1, LAG3, TIM3, CTLA 4.

In one embodiment, to reduce mutual killing between engineered immune cells, 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 NKp 46.

In one embodiment, the engineered immune cell is selected from a T cell, a macrophage, a dendritic cell, a monocyte, an NK cell, or an NKT cell. Preferably, the T cell is a CD4+/CD8+ T cell, CD4+ helper T cell, CD8+ T cell, tumor infiltrating cell, memory T cell, naive T cell, gamma-T cell, or alpha beta-T cell. In one embodiment, the immune cell is derived from a stem cell, such as an adult stem cell, an embryonic stem cell, a cord blood stem cell, a progenitor cell, a bone marrow stem cell, an induced pluripotent stem cell, a totipotent stem cell, or a hematopoietic stem cell, and the like.

In one embodiment, the invention also provides a pharmaceutical composition comprising an engineered immune cell, nucleic acid molecule or vector of the invention, and one or more pharmaceutically acceptable excipients.

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

In one embodiment, the invention also provides the use of a novel nucleic acid and antigen receptor, nucleic acid molecule, vector, engineered immune cell or pharmaceutical composition according to the invention in the manufacture of a medicament for the treatment of cancer, infection or autoimmune disease.

The novel chimeric antigen receptor has the advantages that the specific targeting NK fine activating receptor is used for starting killing of NK cells, on one hand, malignant tumors caused by NK cell pathological changes can be treated, on the other hand, the killing of the introduced therapeutic CAR cells by the NK cells in a patient body can be avoided under the background of general CAR cells (especially under the condition of inhibiting or knocking out MHC (major histocompatibility complex)) so that the survival of the CAR cells is enhanced, and the treatment effect is improved.

Detailed Description

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

Chimeric antigen receptors

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 (e.g., an antibody or antigen-binding portion thereof), a transmembrane domain, a costimulatory domain, and an intracellular signaling domain, each linked by a linker. CARs are able to redirect the specificity and reactivity of T cells and other immune cells to selected targets in a non-MHC-restricted manner using the antigen-binding properties of monoclonal antibodies. non-MHC-restricted antigen recognition gives CAR-expressing T cells the ability to recognize antigen independent of antigen processing, thus bypassing the major mechanism of tumor escape. Furthermore, when expressed in T cells, the CAR advantageously does 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 to an antigen. The antigen binding region may be an antibody, including but not limited to monoclonal, polyclonal, recombinant, human, humanized, murine, chimeric, and functional fragments thereof, or a natural ligand for the antigen or a functional fragment thereof. For example, antigen binding regions include, but are not limited to, immunoglobulin molecules, Fab ', F (ab')2, Fv fragments, scFv, disulfide-linked Fv (sdfv), the heavy chain variable region (VH) or light chain variable region (VL) of an antibody, Fd fragments consisting of the VH and CH1 domains, linear antibodies, single domain antibodies, nanobodies, natural ligands of the antigen or functional fragments thereof, and the like, 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 the two identical fragments produced by papain cleavage of an immunoglobulin molecule, consisting of the entire light and heavy chain N-terminal portions linked by disulfide bonds, wherein the heavy chain N-terminal portion includes the heavy chain variable region and CH 1. Compared to intact IgG, Fab has no Fc fragment, higher mobility and tissue penetration, and binds antigen monovalent without mediating antibody effects.

"Single-chain antibody" or "scFv" is an antibody in which an antibody variable region (VH) and a light chain variable region (VL) are linked via a linker. The optimal length and/or amino acid composition of the linker may be selected. The length of the linker will significantly affect the variable region folding and interaction profiles of the scFv. In fact, if shorter linkers are used (e.g., between 5-10 amino acids), intra-strand folding may be prevented. For the choice of linker size and composition, see, e.g., Hollinger et al, 1993Proc Natl Acad. Sci. U.S.A.90: 6444-; U.S. patent application publication nos. 2005/0100543, 2005/0175606, 2007/0014794; and PCT publication nos. WO2006/020258 and WO2007/024715, which are incorporated herein by reference in their entirety. The scFv may comprise a VH and a VL connected in any order, for example a VH-linker-VL or a VL-linker-VH.

"Single domain antibody" or "sdAb" refers to an antibody that naturally lacks a light chain, which comprises only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, also referred to as "heavy chain antibodies".

"Nanobody" or "Nb" refers to a VHH structure that is cloned and expressed individually, has structural stability comparable to that of an original heavy chain antibody and binding activity to an antigen, and is the smallest unit currently known to bind to a target antigen.

As used herein, the term "NK activating receptor" refers to an NK cell surface receptor having or binding to an Immunoreceptor tyrosine-based activation motif (ITAM). Most NK-activating receptors initiate intracellular signaling by binding of arginine or lysine residues of their transmembrane regions to the corresponding adaptor proteins (e.g., DAP12, FcRI γ, CD3 ζ, etc.). The NK activating receptor is combined with the ligand to enable ITAM contained in the adaptor protein to generate tyrosine phosphorylation, thereby recruiting tyrosine kinase such as Syk, Zap70 and the like, transmitting downstream signals and inducing NK cell activation.

In one embodiment, the NK activating receptor is selected from the NKG2 family, preferably from NKG2C, NKG2D, NKG2E, NKG2F and NKG2H, more preferably NKG 2D. These NKG2 family active receptors share a high degree of homology in their extracellular domains, mostly containing a C-type lectin-like domain. Of these, NKG2E and NKG2H are encoded by the same gene, and the latter is a truncated sequence of the former. Expression of NKG2C and NKG2E/H requires heterodimerization with CD94, allowing rapid phosphorylation of two tyrosine residues in ITAMs by binding to DAP12 molecules containing ITAMs, which in turn recruit ZAP-70 and Syk, leading to NK cell activation. The amino acid sequence of NKG2F is similar to that of NKG2C, having lysine residues in the transmembrane region, and also transmits an activation signal by binding to DAP 12. NKG2D is expressed very widely, not only on all NK cells in humans, but also on most gamma T cells and activated CD8+ α β T cells, and on all NK cells, a fraction of gamma T cells and a fraction of macrophages in mice. NKG2D is expressed as a homodimer, which binds to two different adaptor proteins DAP10 and DAP12, mediating different functions via two signaling pathways, respectively. On the one hand, NKG2D can bind to DAP10 via positively charged arginine residues of the transmembrane region, phosphorylate DAP10, and activate NK cells to exert cytotoxic effects via PI 3K-dependent Ras-independent signaling pathway. On the other hand, NKG2D may also bind to ITAM-containing DAP12, recruiting tyrosine kinases such as Syk and ZAP-70 by phosphorylation of tyrosine residues, leading to the transduction of downstream signals, inducing the release of cytokines and chemokines.

In a preferred embodiment, the chimeric antigen receptor of the invention comprises an antigen binding region which is an antibody or a functional fragment thereof targeting NKG2D, preferably Fab, scFv, sdAb and nanobodies targeting NKG2D, more preferably an scFv targeting NKG 2D. In a preferred embodiment, the chimeric antigen receptor of the invention comprises an anti-NKG 2D antibody comprising:

(i) respectively shown in SEQ ID NO: 61. 62 and 63, CDR-L1, CDR-L2 and CDR-L3, and the amino acid sequences as set forth in SEQ ID NOs: 64. 65 and 66, CDR-H1, CDR-H2 and CDR-H3;

(ii) respectively shown in SEQ ID NO: 67. 68 and 69, CDR-L1, CDR-L2 and CDR-L3, and the amino acid sequences as set forth in SEQ ID NOs: 70. 71 and 72, CDR-H1, CDR-H2 and CDR-H3;

(iii) respectively shown in SEQ ID NO: 67. 68 and 73, and CDR-L1, CDR-L2, and CDR-L3 as set forth in SEQ ID NOs: 74. CDR-H1, CDR-H2 and CDR-H3 shown in 75 and 76; or

(iv) Respectively shown in SEQ ID NO: 77. 78 and 79, and CDR-L1, CDR-L2 and CDR-L3 as set forth in SEQ ID NOs: 80. 81 and 82, CDR-H1, CDR-H2 and CDR-H3.

Preferably, the chimeric antigen receptor of the invention comprises an anti-NKG 2D antibody comprising an amino acid sequence identical to SEQ ID NO: 27, 1-121, SEQ ID NO: 29, 1-109, SEQ ID NO: 31, 1-109 or SEQ ID NO: 33, 1-108 and a light chain variable region sequence 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 set forth in SEQ ID NO: 27, position 137-246, SEQ ID NO: position 122-243 of 29, SEQ ID NO: 31 position 122-236 or SEQ ID NO: 33, 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 depicted in position 121-243. More preferably, the chimeric antigen receptor of the invention comprises an anti-NKG 2D antibody comprising the amino acid sequence as set forth in SEQ ID NO: 27. 29, 31 or 33.

In one embodiment, instead of the NKG2D antibody, the chimeric antigen receptor of the invention may also comprise a natural ligand for NKG 2D. NKG2D ligands in humans are MIC genes (including MICA and MICB) and ULBP genes (including ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6), and in mice are Rae-1, H60, and MULT 1. MICA and MICB are located on one side of the HLA-B locus in the MHC gene complex and have homology as high as 91%. The ULBP gene is similar in structure to MHC class I molecules, and contains both alpha 1 and alpha 2 domains, but no alpha 3 domain and beta 2 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 MULT 1.

In one embodiment, the NK activating receptor is selected from the Natural Cytotoxicity Receptors (NCRs) family, preferably from NKp30, NKp44, NKp46 and NKp80, more preferably from NKp30 and NKp 46. NKp30 is one of the key factors of NK cells to exert killing activity, and the expression level is disordered when various tumors and viruses are infected, and the NKp30 may be involved in immune escape of the tumors and the viruses. The extracellular domain of NKp30 is an immunoglobulin-like domain of type V linked to the arginine-rich transmembrane domain by a hydrophobic amino acid sequence. NKp30 binds to CD3 ζ and transmits activation signals intracellularly via ITAMs of the latter. The extracellular domain of NKp44 contains a V-type domain, and the transmembrane domain has a charged lysine that binds KAPAP/DAP 12. Although the intracellular domain of NKp44 contains ITIMs, it lacks inhibitory functions and does not attenuate the activation signal delivered by DAP 12. Research shows that NKp44 can bind to virus hemagglutinin and exert the antiviral effect of NK cells. The extracellular domain of NKp46 has two Ig-like domains of type C2, the transmembrane domain contains a positively charged arginine residue, and the intracellular domain does not contain the ITAM motif, so that it is also required to co-deliver activation signals intracellularly, similar to NKp30, by forming complexes with molecules such as CD3 ζ and/or FcRI γ, whose intracellular segments contain ITAM. NKp46 is expressed on the surface of all mature NK cells and is a key receptor for initiating NK cell killing function. NKp46 also binds to viral hemagglutinin and exerts the antiviral effect of NK cells. NKp80 is expressed on the surface of almost all NK cells in the form of homodimer, and can rapidly activate NK cells after being combined with activation-induced C-type lectin (AICL) induced by ligand activation, thereby improving the cytotoxicity of the NK cells and the capability of secreting inflammatory cytokines.

In a preferred embodiment, the chimeric antigen receptor of the invention comprises an antigen binding region which is an antibody or a functional fragment thereof targeting NKp46, preferably Fab, scFv, sdAb and nanobody targeting NKp46, more preferably scFv targeting NKp 46. In a preferred embodiment, the chimeric antigen receptor of the invention comprises an anti-NKp 46 antibody comprising

(i) Respectively shown in SEQ ID NO: 83. 84 and 85, and CDR-L1, CDR-L2, and CDR-L3 as set forth in SEQ ID NOs: 86. 87 and 88, CDR-H1, CDR-H2 and CDR-H3;

(ii) respectively shown in SEQ ID NO: 89. 90 and 91, CDR-L1, CDR-L2 and CDR-L3, and the amino acid sequence as set forth in SEQ ID NOs: 92. 93 and 94, CDR-H1, CDR-H2 and CDR-H3;

(iii) respectively shown in SEQ ID NO: 95. 96 and 97, and CDR-L1, CDR-L2, and CDR-L3 as set forth in SEQ ID NOs: 98. 99 and 100, CDR-H1, CDR-H2 and CDR-H3;

(iv) respectively shown in SEQ ID NO: 101. 84 and 102, and CDR-L1, CDR-L2, and CDR-L3 as set forth in SEQ ID NOs: 103. 87 and 104, CDR-H1, CDR-H2 and CDR-H3; or

(v) Respectively shown in SEQ ID NO: 105. 106 and 107, and CDR-L1, CDR-L2 and CDR-L3 as set forth in SEQ ID NOs: 108. 109 and 110, CDR-H1, CDR-H2 and CDR-H3.

Preferably, the chimeric antigen receptor of the invention comprises an anti-NKp 46 antibody comprising an amino acid sequence identical to SEQ ID NO: 35, positions 1-107, SEQ ID NO: 37, 1-107, SEQ ID NO: 39, 1-107, SEQ ID NO: 41, 1-107 or SEQ ID NO:43, and a light chain variable region sequence 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 set forth in positions 1-107 of SEQ ID NO: position 123-244 of 35, SEQ ID NO: position 123-238 of 37, SEQ ID NO: position 123-238 of SEQ ID NO: position 123-242 of 41 or SEQ ID NO:43, 123-237, having 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 invention comprises an anti-NKp 46 antibody comprising the amino acid sequence as set forth in SEQ ID NO: 35. 37, 39, 41 or 43.

In one embodiment, instead of an NKp46 antibody, the chimeric antigen receptor of the invention may comprise a natural ligand for NKp 46. It is found that NK cells recognize and agglutinate virus Hemagglutinin (HA) through NKp46, and further kill cells infected by influenza virus. In addition, NKp46 also binds to a soluble glycoprotein, complement factor p (cfp). Patients lacking CFP are reported to be more susceptible to infection by neisseria meningitidis, and treatment of this infection with CFP relies on the action of NKp 46. Thus, in this embodiment, the chimeric antigen receptor of the invention may also comprise HA or CFP or a functional fragment thereof.

In one embodiment, the NK activating receptor is selected from the KIR-S family, preferably from KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5 and KIR3DS1, more preferably KIR2DS 4. Killer cell Ig-like receptors (KIRs) are type I transmembrane proteins belonging to the immunoglobulin superfamily, the structure of which includes an extracellular domain, a transmembrane domain, and a cytoplasmic domain. KIRs can also be divided into KIR2D and KIR3D subfamilies, depending on the number of Ig-like domains comprised by the extracellular region. Furthermore, KIRs can be classified into a long type (KIR-L) and a short type (KIR-S) depending on the size of the cytoplasmic region. The cytoplasmic region of KIR-S contains no ITIM, and similarly to NCR, binds to the adaptor protein DAP12 or FcRI γ, again through charged transmembrane residues, recruiting tyrosine kinases and mediating downstream activation signals.

In one embodiment, the NK activating receptor is selected from the group consisting of co-receptors, preferably selected from the group consisting of 2B4, DNAM-1, CD2 and LFA-1. 2B4, also known as CD244, is a membrane protein with an extracellular region with a V-type immunoglobulin domain and a C2-type immunoglobulin-like domain, a transmembrane region without any charged amino acids, and an intracellular region containing an Immunoreceptor Tyrosine Switch Motif (ITSM) that is recognized by the cytoplasmic SH2 region of the adaptor proteins SAP, EAT-2, DRT, etc. DNAM-1, also known as CD226, is the primary helper activation receptor that initiates NK cell function. DNAM-1 contains 2 immunoglobulin V-like domains of the extracellular domain, 1 transmembrane region, and contains tyrosine and serine residues of potential phosphorylation sites of the cytoplasmic region. CD2, also known as LFA-2, is a single-chain glycoprotein consisting of 327 amino acids expressed on the surface of mature T cells, most thymocytes and some NK cells. LFA-1 is formed by the non-covalent linkage of two polypeptide chains: the alpha subunit (CD11a) and the beta subunit (CD 18). These synergistic receptors cannot activate NK cells alone, but rely on other NK activating receptors such as NCR to initiate activation signals, which then participate together in amplifying the activation signals, more effectively promoting NK cell activation. Antibodies known in the art that target 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 antigen binding region of the chimeric antigen receptor of the present invention.

In one embodiment, in addition to the antigen binding region targeting the NK activating receptor, the novel chimeric antigen receptor of the present invention comprises a second antigen binding region which 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, TnAg, 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, ERBB 20 (Her 20/neu), MUC 20, EGFR, CS 20, CD138, NCAM, Claudin18.2, Prostase, BCPAP, Nyhrf 2, Nyhrin 20, Epsilon 20, EPTC-20, EPTC-72, EPTC-20, EPTC-20, EPTC-20, EPTC-20, EPTC, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPV E6, E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutants, prostate specific protein, survivin and telomerase, PCTA-l/Galectin 8, MelanA/MARTl, Ras mutants, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2ETS fusion gene), NA17, PAX3, BlAR, Cyclin, MYCN, RhoC, TRP-2, TRP 1B, RIS, SART3, PATES 5, OY-TES1, AK 464-SSK 464, SSAK 584, LAR 5819, RAKE 24-RAG 9, RAKE 5, CD 5979, CD 599, RGE 5, CD 5979, RGE 5, CD 599, RGE 5, RG 5, CD 599, RG 5, RGE 5, and its complement 5, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGF β, APRIL, NKG2D, and any combination thereof. Preferably, the tumor antigen is selected from the group consisting of CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, MUC1, 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 to target the above-mentioned 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) respectively shown in SEQ ID NO: 49. 50 and 51, and CDR-L1, CDR-L2 and CDR-L3, and the amino acid sequences set forth in SEQ ID NOs: 52. 53 and 54, CDR-H1, CDR-H2 and CDR-H3; or

(ii) Respectively shown in SEQ ID NO: 55. 56 and 57, and CDR-L1, CDR-L2 and CDR-L3 as set forth in SEQ ID NOs: 58. 59 and 60, CDR-H1, CDR-H2 and CDR-H3.

Preferably, the chimeric antigen receptor of the invention comprises an antibody targeting the NK activating receptor and an antibody targeting CD19, said antibody targeting CD19 comprising an amino acid sequence identical to SEQ ID NO:1, 1-107 or SEQ ID NO: 25, position 1-107, and a light chain variable region sequence 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 set forth in SEQ ID NO:1 position 123-242 or SEQ ID NO: 25, 123-238, having 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 invention comprises an antibody targeting the NK activating receptor and an antibody targeting CD19, said antibody targeting CD19 comprising the amino acid sequence as set forth 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 (i.e., substitution, deletion, or insertion) as compared to the parent amino acid sequence, provided that the variant retains 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 an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing the amino acid sequence. Such 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 those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β -branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Conservative modifications may 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" has at least 75%, preferably at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a parent amino acid sequence and retains the biological activity, e.g., binding activity, of the parent amino acid.

As used herein, the term "sequence identity" refers to the degree to which two (nucleotide or amino acid) sequences have the same residue at the same position in an alignment, and is typically expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies of an identical sequence have 100% identity. One 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 structure that enables a chimeric antigen receptor to be expressed on the surface of an immune cell (e.g., a lymphocyte, NK cell, or NKT cell) and to direct the cellular response of the immune cell against a target cell. The transmembrane domain may be natural or synthetic, and may be derived from any membrane-bound or transmembrane protein. The transmembrane domain is capable of signaling when the chimeric antigen receptor binds to a target antigen. Transmembrane domains particularly suitable for use in the present invention may be derived from, for example, the TCR α chain, the TCR β chain, the TCR γ chain, the TCR chain, the CD3 ζ subunit, the CD3 subunit, the CD3 γ subunit, the CD3 subunit, CD45, CD4, CD5, CD8 α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and functional fragments thereof. 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 the CD8 a chain or CD28, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID No. 3 or5, or whose coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the nucleotide sequence shown in SEQ ID No. 4 or 6.

In one embodiment, the chimeric antigen receptor of the present invention may further comprise a hinge region located 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 connect a transmembrane domain to a ligand binding domain. In particular, 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 may be derived in whole or in part from a naturally occurring molecule, such as 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 portion of the hinge region of the CD8 a chain, CD28, Fc γ RIII a receptor, IgG4 or IgG1, more preferably a hinge from CD8 a, CD28 or IgG4, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID No. 19, 21 or 23, or whose coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the nucleotide sequence shown in SEQ ID No. 20, 22 or 24.

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

In one embodiment, the intracellular signaling domain comprised by the chimeric antigen receptor of the present invention may be the cytoplasmic sequences of the T cell receptor and co-receptor that work together to trigger primary signaling upon antigen receptor binding, as well as any derivative or variant of these sequences and any synthetic sequence with the same or similar function. The intracellular signaling domain may contain a number of Immunoreceptor Tyrosine-based Activation Motifs (ITAMs). Non-limiting examples of intracellular signaling domains of the invention include, but are not limited to, those derived from FcR γ, FcR β, CD3 γ, CD3, CD3, CD3 ζ, CD22, CD79a, CD79b, and CD66 d. In a preferred embodiment, the signalling domain of a CAR of the invention may comprise a CD3 zeta signalling domain which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID No. 11 or 13 or whose coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID No. 12 or 14.

In one embodiment, the chimeric antigen receptor of the present invention further comprises one or more co-stimulatory domains. The co-stimulatory domain may be an intracellular functional signaling domain from a co-stimulatory molecule, which comprises the entire intracellular portion of the co-stimulatory molecule, or a functional fragment thereof. "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 co-stimulatory domains of the invention include, but are not limited to, co-stimulatory signaling domains derived from: 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 co-stimulatory domain comprises one or more intracellular regions of a protein selected from the group consisting of: DAP10, DAP12, CD27, CD28, CD134, 4-1BB, or CD 278. For example, in one embodiment, the co-stimulatory domain comprises the intracellular region of 4-1 BB. In one embodiment, the co-stimulatory domain comprises the intracellular region of CD 28. In one embodiment, the costimulatory domain comprises the intracellular region of DAP 10. In one embodiment, the costimulatory domain comprises the intracellular region of DAP 12. More preferably, the co-stimulatory domain comprises two intracellular domains of a protein selected from the group consisting of: DAP10, DAP12, CD27, CD28, CD134, 4-1BB, or CD 278. In one embodiment, the co-stimulatory domain comprises the intracellular region of 4-1BB and the intracellular region of CD 28. In one embodiment, the co-stimulatory domain comprises the intracellular region of 4-1BB and the intracellular region of DAP 10. In one embodiment, the co-stimulatory domain comprises the intracellular region of 4-1BB and the intracellular region of DAP 12. In one embodiment, the co-stimulatory domain comprises the intracellular region of CD28 and the intracellular region of DAP 10. In one embodiment, the co-stimulatory domain comprises the intracellular region of CD28 and the intracellular region of DAP 12.

In one embodiment, the intracellular region of 4-1BB has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence depicted in SEQ ID NO. 9, 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 to the nucleotide sequence depicted in SEQ ID NO. 10. 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% sequence identity to the amino acid sequence shown in SEQ ID NO. 7 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 to the nucleotide sequence shown in SEQ ID NO. 8. 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% sequence identity to the amino acid sequence shown in SEQ ID No. 45 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 to the nucleotide sequence shown in SEQ ID No. 46. 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% sequence identity to the amino acid sequence shown in SEQ ID No. 47 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 to the nucleotide sequence shown in SEQ ID No. 48.

In one embodiment, the CAR of the invention may further comprise a signal peptide such that when it is expressed in a cell, for example 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 hydrophobic amino acid segment that has a tendency to form a single alpha-helix. At the end of the signal peptide there is usually a stretch of amino acids which is recognized and cleaved by the signal peptidase. The signal peptidase may cleave during translocation or after completion to produce a free signal peptide and a mature protein. The free signal peptide is then digested by a specific protease. Signal peptides useful in the present invention are well known to those skilled in the art, such as those derived from CD8 α, IgG1, GM-CSFR α, B2M, and the like. In one embodiment, the signal peptide useful in the present invention has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO. 15 or 17, or the coding sequence of the signal peptide has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO. 16 or 18.

In one embodiment, the CAR of the invention may further comprise a switch structure to regulate the time of expression of the CAR. For example, the switch structure may be in the form of a dimerization domain that causes a conformational change upon binding to its corresponding ligand, exposing the extracellular binding domain to allow binding to the targeted antigen, thereby activating the signaling pathway. Alternatively, a switch domain may be used to connect the binding domain and the signaling domain, respectively, such that the binding domain and the signaling domain are connected together via a dimer only when the switch domains are bound to each other (e.g., in the presence of an inducing compound) to activate the signaling pathway. The switch structure may also be in the form of a masking peptide. The masking peptide can mask the extracellular binding domain, preventing its binding to the antigen to be targeted, and when the masking peptide is cleaved, for example by a protease, the extracellular binding domain can be exposed, making it a "normal" CAR structure. Various switch configurations known to those skilled in the art may be used with the present invention.

In one embodiment, the CAR of the invention may also comprise a suicide gene, i.e. such that it expresses a cell death signal that can be induced by foreign substances, to eliminate CAR cells when needed (e.g. when severe toxic side effects are produced). For example, the suicide gene may be in the form of an inserted epitope, such as the CD20 epitope, RQR8, etc., and when desired, the CAR cells can be eliminated by the addition of antibodies or agents that target these epitopes. The suicide gene may also be herpes simplex virus thymidine kinase (HSV-TK), which causes cell death induced by treatment with ganciclovir. The suicide gene can also be iCaspase-9, and the iCaspase-9 can be induced to dimerize by chemical induction drugs such as AP1903, AP20187 and the like, so that downstream Caspase3 molecules are activated, and apoptosis is caused. 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, or mixtures thereof (including hybrid molecules). Thus, nucleic acids according to the invention include DNA (such as dsDNA, ssDNA, cDNA), RNA (such as dsRNA, ssRNA, mRNA, ivtRNA), combinations or derivatives thereof (such as PNA). Preferably, the nucleic acid is DNA or RNA, more preferably mRNA.

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

The nucleic acid sequences of the invention may be codon optimized for optimal expression in a desired host cell (e.g., an immune cell); 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, with the codons before and after the replacement encoding the same amino acid. Thus, the choice of optimal codons depends on the codon usage bias of the host genome.

As used herein, the term "vector" is a vector nucleic acid molecule used as a vehicle for transferring (foreign) genetic material into a host cell where it can, for example, be replicated and/or expressed.

Vectors generally include targeting vectors and expression vectors. A "targeting vector" is a medium for delivering an isolated nucleic acid to the interior of a cell, for example, by homologous recombination or by using a hybrid recombinase that targets sequences at a site specifically. An "expression vector" is a vector for the transcription of heterologous nucleic acid sequences (such as those encoding the chimeric antigen receptor polypeptides of the invention) in a suitable host cell and the translation of their mRNA. 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 invention include, but are not limited to, plasmids, viruses (e.g., retroviruses, lentiviruses, adenoviruses, vaccinia viruses, rous sarcoma viruses (RSV, polyoma viruses and adeno-associated viruses (AAV), etc.), bacteriophages, phagemids, cosmids, and artificial chromosomes (including BACs and YACs). the vectors themselves are typically nucleotide sequences, typically DNA sequences comprising an insert (transgene) and a larger sequence that serves as a "backbone" for the vector. The vector is an in vitro transcription 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 (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC). For example, the immune cell may be a T cell, macrophage, dendritic cell, monocyte, NK cell, and/or NKT cell. In one embodiment, the immune cell is derived from a stem cell, such as an adult stem cell, an embryonic stem cell, a cord blood stem cell, a progenitor cell, a bone marrow stem cell, an induced pluripotent stem cell, a totipotent stem cell, or a hematopoietic stem cell, and the like. Preferably, the immune cell is a T cell. The T cell may be any T cell, such as an in vitro cultured T cell, e.g., a primary T cell, or a T cell from an in vitro cultured T cell line, e.g., Jurkat, SupT1, etc., or a T cell 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 the site of infection, ascites, pleural effusion, spleen tissue, and tumors. T cells may also be concentrated or purified. The T cells may be at any developmental stage, including, but not limited to, CD4+/CD8+ T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, gamma-T cells, alpha beta-T cells, and the like. In a preferred embodiment, the immune cell is a human T cell. T cells can be obtained from the blood of a subject using a variety of techniques known to those skilled in the art, such as Ficoll isolation.

In one embodiment, the engineered immune cell expresses 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 binding region is defined as described above.

The nucleic acid sequence encoding the chimeric antigen receptor and the exogenous gene can be introduced into the immune cell using conventional methods known in the art (e.g., by transduction, transfection, transformation, etc.). "transfection" is the process of introducing a nucleic acid molecule or polynucleotide (including vectors) into a target cell. One example is RNA transfection, the process of introducing RNA (e.g., in vitro transcribed RNA, ivtRNA) into a host cell. The term is used primarily for non-viral methods in eukaryotic cells. The term "transduction" is generally used to describe virus-mediated transfer of a nucleic acid molecule or polynucleotide. Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane to allow uptake of the material. Transfection may be performed using calcium phosphate, by electroporation, by cell extrusion, or by mixing cationic lipids with the material to create liposomes that fuse with the cell membrane and deposit their cargo into the interior. 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. The term "transformation" is used to describe the non-viral transfer of a nucleic acid molecule or polynucleotide (including vectors) into bacteria, but also into non-animal eukaryotic cells (including plant cells). Thus, transformation is a genetic alteration of a bacterial or non-animal eukaryotic cell, which is produced by direct uptake of the cell membrane from its surroundings and subsequent incorporation of foreign genetic material (nucleic acid molecules). The transformation may be achieved by artificial means. In order for transformation to occur, the cell or bacteria must be in a competent state. For prokaryotic transformation, techniques may include heat shock mediated uptake, bacterial protoplast fusion with intact cells, microinjection, and electroporation. After introducing the nucleic acid or vector into the immune cells, the resulting immune cells can be expanded and activated by one skilled in the art by conventional techniques.

In one embodiment, to reduce the risk of graft versus host disease, the engineered immune cell further comprises at least one gene whose expression is suppressed or silenced selected from the group consisting of: CD, GR, dCK, TCR/CD genes (e.g., TRAC, TRBC, CD γ, CD ζ), MHC associated genes (HLA- - -2, HLA-DPA, HLA-DQ, HLA-DRA, TAP, LMP, RFX, RFXAP, RFXANK, CIITA) and immune checkpoint genes, such as PD, LAG, TIM, CTLA, PPP2, PTPN, PDCD, HAVCR, BTLA, CD160, TIGIT, CD, CRTAM, TNFRSF10, CASP, FADD, FAS, TGFBRII, TGFRBRI, SMAD, SKI, SKIL, TGTGTGT, IL10, HMIF, IL6 AK, CSK, PAG, PRBAT, SIXP, FOCY, GUCY1A, GUDM 1B, GUAB 1, GU, and GU B. Preferably, the engineered immune cell further comprises at least one gene whose expression is inhibited or silenced 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, expression of the corresponding endogenous NK-activating receptor targeted by the chimeric antigen receptor in the engineered immune cells is inhibited or silenced. For example, in engineered immune cells that target NKG2D (e.g., CAR-T or CAR-NK cells), expression of its endogenous NKG2D is inhibited or silenced; expression of endogenous NKp46 is inhibited or silenced in engineered immune cells (e.g., CAR-T or CAR-NK cells) targeted to NKp 46.

Methods for 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, Transdominant Negative Protein (TNP), chimeric/fusion proteins, chemokine ligands, anti-infective cellular proteins, intracellular antibodies (sFv), nucleoside analogs (NRTI), non-nucleoside analogs (NNRTI), integrase inhibitors (oligonucleotides, dinucleotides, and chemical agents), and protease inhibitors can be used to inhibit expression of a gene. In addition, DNA fragmentation can also be mediated by, for example, meganucleases, zinc finger nucleases, TALE nucleases or Cas enzymes in CRISPR systems to silence the gene.

Pharmaceutical composition

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

As used herein, the term "pharmaceutically acceptable excipient" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient (i.e., capable of eliciting a desired therapeutic effect without causing any undesirable local or systemic effects), which are well known in the art (see, e.g., Remington's Pharmaceutical sciences. edited by genomic AR,19th ed. pennsylvania: mach Publishing Company, 1995). Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coatings, adsorbents, anti-adherents, 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 adjusting agents. The selection of suitable excipients to prepare the desired pharmaceutical compositions of the present invention is known to those skilled in the art. Exemplary excipients for use in the pharmaceutical compositions of the present invention include saline, buffered saline, dextrose, and water. In general, the choice of suitable excipients depends, inter alia, on the active agent used, the disease to be treated and the desired dosage form of the pharmaceutical composition.

The pharmaceutical composition according to the present invention may be suitable for administration by various routes. Typically, administration is accomplished parenterally. Methods of parenteral delivery 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 in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures or extracts of fluid extracts, or in a form which is particularly suitable for the desired method of administration. Processes known in the art for the manufacture of medicaments may comprise, for example, conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions comprising immune cells such as described herein are typically provided in solution form and preferably comprise a pharmaceutically acceptable buffer.

The pharmaceutical compositions according to the invention may also be administered in combination with one or more other agents suitable for the treatment and/or prevention of the diseases to be treated. Preferred examples of the pharmaceutical agents suitable for combination include known anticancer drugs such as cisplatin, maytansine derivatives, rebeccin (rachelmycin), calicheamicin (calicheamicin), docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer porphyrin sodium ii (sorfimer Sodiumtofrin ii), temozolomide, topotecan, glucuronide (trimetrenate glucoside), oritavastin e (auristatin E), vincristine, and adriamycin; peptide cytotoxins such as ricin, diphtheria toxin, pseudomonas bacterial exotoxin A, DNA enzyme, 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, and the like; antibodies or fragments thereof, such as anti-CD 3 antibodies or fragments thereof, complement activators, heterologous protein domains, homologous protein domains, viral/bacterial protein domains, and viral/bacterial peptides. In addition, the pharmaceutical compositions of the present invention may also be used in combination with one or more other therapeutic methods, such as chemotherapy, radiation therapy.

Therapeutic applications

The invention also provides a method of treating a subject having cancer, an infection or an autoimmune disease, comprising administering to the subject an effective amount of an immune cell or a pharmaceutical composition according to the invention. Thus, the invention also encompasses the use of said chimeric antigen receptor, nucleic acid molecule, vector, engineered immune cell and pharmaceutical composition for the preparation of a medicament for the treatment of cancer, infection or autoimmune disease.

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 treatment method of the invention is ex vivo treatment. Specifically, the method comprises the following steps: (a) providing a sample comprising immune cells; (b) introducing in vitro the chimeric antigen receptor of the invention and an exogenous gene (if present) into said immune cell and optionally inhibiting or silencing expression of a specific gene (if desired) in said immune cell, obtaining a modified immune cell, (c) administering said modified immune cell 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; and the immune cells can be obtained from a sample (particularly a blood sample) of a subject by conventional methods known in the art. However, other immune cells capable of expressing the chimeric antigen receptor 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 immune system of the subject, i.e. preferably the immune cells do not elicit an immunogenic response. For example, "universal recipient cells," i.e., universally compatible lymphocytes that can be grown and expanded in vitro to function as desired biological effects, can be used. The use of such cells would not require the obtaining and/or provision of subject autologous lymphocytes. The ex vivo introduction of step (c) may be carried out by introducing the nucleic acid or vector described herein into an immune cell via electroporation or by infecting an immune cell with a viral vector, which is a lentiviral, adenoviral, adeno-associated viral vector or retroviral vector as described previously. Other conceivable methods include the use of transfection reagents (such as liposomes) or transient RNA transfection.

In one embodiment, the immune cell is an autologous or allogeneic cell, preferably a T cell, macrophage, dendritic cell, monocyte, NK cell and/or NKT cell, more preferably a T cell, NK cell or NKT cell.

As used herein, the term "autologous" means that any material derived from an individual will be reintroduced into the same individual at a later time.

As used herein, the term "allogeneic" refers to any material derived from a different animal or patient of the same species as the individual into which the material is introduced. When the genes at one or more loci are different, two or more individuals are considered allogeneic to each other. In some cases, genetic differences in allogenic material from individuals of the same species may be sufficient for antigen interactions to occur.

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

In one embodiment, the cancer is selected from: brain glioma, blastoma, sarcoma, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancers, 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, gastric cancer (including gastrointestinal cancer), Glioblastoma (GBM), liver cancer, hepatoma, intraepithelial tumors, kidney cancer, larynx cancer, liver tumor, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenoid lung cancer, and squamous lung cancer), melanoma, myeloma, neuroblastoma, oral cancer (e.g., lip, 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 cancer, gastric cancer, colon cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, malignant tumors of the urinary system, vulvar cancer, Waldenstrom's macroglobulinemia, lymphomas (including hodgkin's lymphoma and non-hodgkin's lymphoma, such as B-cell lymphoma (including low-grade/follicular non-hodgkin's lymphoma (NHL), Small Lymphocytic (SL) NHL, intermediate-grade/follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cracked cellular NHL, large lump disease NHL), mantle cell lymphoma, AIDS-related lymphoma, burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, MALT lymphoma, marginal zone lymphoma, plasmablastinoma, plasmacytoid dendritic cell lymphoma, etc.), NK cell lymphoma, aggressive NK cell leukemia, leukemia (including acute leukemia, for example acute lymphocytic leukemia, acute myelogenous leukemia, acute non-lymphocytic leukemia such as acute myelogenous leukemia (including undifferentiated and partially differentiated), acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, erythroleukemia, acute megakaryocytic leukemia; chronic leukemias, e.g., chronic myelogenous leukemia, chronic lymphocytic leukemia, chronic monocytic leukemia; and other specific 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 disease, myelodysplasia, multiple myeloma, myelodysplasia, and post-transplant lymphoproliferative disorder (PTLD). Preferably, the diseases that can be treated with the engineered immune cells or the pharmaceutical compositions of the 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 I diabetes, celiac disease, graves 'disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, addison's disease, sjogren's syndrome, hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, and systemic lupus erythematosus, among others.

The invention will be described in detail below with reference to the accompanying drawings and examples. It should be noted that the drawings and their embodiments of the present invention are for illustrative purposes only and are not to be construed as limiting the invention. The embodiments and features of the embodiments in the present application may be combined with each other without contradiction.

Drawings

FIG. 1: CAR structure schematic constructed in example 1.

FIG. 2: scFv expression levels in CAR-T cells comprising different NKG2D scFv.

FIG. 3: killing effect of CAR-T cells comprising different NKG2D scFv on NK92 cells.

FIG. 4: scFv expression levels in NKG 2D-targeted CAR-T cells.

FIG. 5: killing effect of NKG 2D-targeted CAR-T cells on NK92 cells.

FIG. 6: CD107 expression levels after co-culture of NKG 2D-targeted CAR-T cells with NK92 cells.

FIG. 7: cytokine release levels of CAR-T cells targeted to NKG 2D.

FIG. 8: CD8+ T cell proportion in a population of CAR-T cells targeted to NKG2D in which endogenous NKG2D is knocked out.

FIG. 9: level of scFv expression in NKG2D-CD19 dual target CAR-T cells.

FIG. 10: killing effect of NKG2D-CD19 double-target CAR-T cells on NK92 cells and Nalm6 cells.

FIG. 11: CD107 expression levels after co-culture of NKG2D-CD19 dual-target CAR-T cells with Nalm6 cells.

FIG. 12: CD107 expression levels after co-culture of NKG2D-CD19 dual-target CAR-T cells with NK92 cells.

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

FIG. 14: scFv expression levels in CAR-T cells comprising different NKp46 scFv.

FIG. 15: killing effect of CAR-T cells comprising different NKp46scFv on NK92 cells.

FIG. 16: level of scFv expression in NKp46-CD19 dual target CAR-T cells.

FIG. 17: killing effect of NKp46-CD19 double-target CAR-T cells on NK92 cells and Nalm6 cells.

FIG. 18: killing effect of NKp46-CD19 double-target CAR-NK cells on NK92 cells and Nalm6 cells.

FIG. 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 invention were primary human CD4+ CD8+ T cells isolated from healthy donors by leukapheresis using Ficoll-Paque (TM) PREMIUM (GE Healthcare, cat. No. 17-5442-02).

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

TABLE 1

Example 1 preparation of CAR T cells comprising different scFv and validation of function

1.1 CAR T cell preparation

Sequences encoding the following proteins were synthesized and cloned into a pLVX vector (Public Protein/Plasmid Library (PPL), cat # PPL00157-4 a): CD8 a signal peptide (SEQ ID NO: 17), anti-NKG 2D scFv, CD8 a hinge region (SEQ ID NO: 19), CD8 a transmembrane region (SEQ ID NO: 3), 4-1BB intracellular region (SEQ ID NO: 9), CD3 ζ intracellular signaling domain (SEQ ID NO: 11), and correct insertion of the target sequence was confirmed by sequencing. Wherein the NKG2D-1CAR comprises the anti-NKG 2D scFv with the amino acid sequence shown in SEQ ID NO: 27 is shown; the amino acid sequence of the anti-NKG 2D scFv comprised by the NKG2D-2CAR is as shown in SEQ ID NO: 29 is shown; the amino acid sequence of the anti-NKG 2D scFv comprised by the NKG2D-3CAR is as shown in SEQ ID NO: 31, shown in the figure; the amino acid sequence of the anti-NKG 2D scFv comprised by the NKG2D-4CAR is as shown in SEQ ID NO: shown at 33.

After diluting the above plasmid by adding 3ml of Opti-MEM (Gibco, cat # 31985-: the packaging vector psPAX2(Addgene, cat # 12260) and the envelope vector pmd2.g (Addgene, cat # 12259) were added at a ratio of 4:2:1 for the viral envelope vector. Then, 120ul of X-treme GENE HP DNA transfection reagent (Roche, cat # 06366236001) was added, mixed immediately, incubated at room temperature for 15min, and the plasmid/vector/transfection reagent mixture was added dropwise to the 293T cell culture flask. The viruses were collected at 24 hours and 48 hours, and after combining them, concentrated lentiviruses were obtained by ultracentrifugation (25000g, 4 ℃, 2.5 hours).

CTS with DynaBeads CD3/CD28^TM(Gibco, cat. No. 40203D) activated T cells and cultured at 37 ℃ and 5% CO2 for 1 day. Then, after adding concentrated lentivirus and continuing the culture for 3 days, CAR T cells expressing different scFv were obtained. Unmodified wild-type T cells were used as negative control (NT). The results are shown in FIG. 2

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

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

To test the longer killing ability of CAR-T cells in vitro, first 5X10⁵NK92 target cells were plated into 24-well plates and then plated at 2:1 ratio of effective targets (i.e. ratio of effector T cells to target cells) CAR T cells expressing different scfvs prepared as above were plated into 24-well plates for co-culture, and fluorescence was measured 16-18 hours later using a microplate reader. According to the calculation formula: (mean value of fluorescence of target cells-mean value of fluorescence of sample)/mean value of fluorescence of target cells x 100%, and the killing efficiency was calculated, and the results are shown in FIG. 3.

It can be seen that CAR T cells comprising 4 different scfvs targeting NKG2D all had significant killing of NK92 cells, with NKG2D-1CAR T cells having the strongest killing ability, were used in subsequent experiments and renamed to 2Dbbz-CAR T cells.

Example 2: CAR T cell preparation and killing effect and cytokine release on NK92 target cells

2.1 CAR T cell preparation

Sequences encoding the following proteins were synthesized and cloned into a pLVX vector (Public Protein/Plasmid Library (PPL), cat # PPL00157-4 a): CD8 α signal peptide (SEQ ID NO: 17), anti-NKG 2D scFv (SEQ ID NO: 27), CD8 α hinge region (SEQ ID NO: 19), CD8 α transmembrane region (SEQ ID NO: 3), costimulatory domain, CD3 ζ intracellular signaling domain (SEQ ID NO: 11), wherein the costimulatory domain comprises the CD28 intracellular region (SEQ ID NO: 7), or the 4-1BB intracellular region (SEQ ID NO: 9) + DAP10 intracellular region (SEQ ID NO: 45), 2D28z-CAR and 2Dbb10z-CAR were obtained and correct insertion of the target sequence was confirmed by sequencing. FIG. 1 shows the CAR structure constructed in this example.

The preparation of CAR T cells was performed according to the method described in example 1, and the expression levels on 2 Dbbz-scFv, 2D28z-CAR T cells, 2Dbb10z-CAR T cells were examined by flow cytometry using Biotin-SP (long spacer) AffiniP Goat Anti-Mouse IgG, F (ab') Fragment specificity (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, cat # 115-065-072) as a primary antibody, APC Streptavidin (BD Pharmingen, cat # 554067) or PE Streptavidin (BD Pharmingen, cat # 554061) as secondary antibodies, and the results are shown in FIG. 4.

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

To test the longer killing ability of CAR-T cells in vitro, first 5X10⁵NK92 target cells were plated into 24-well plates and then plated at 2:1 (i.e. effector T cell to target cell ratio) CAR-T cells expressing 2Dbbz-CAR, 2D28z-CAR and 2Dbb10z-CAR were plated into 24-well plates for co-culture (D0) and co-cultured according to 2: effective target ratio of 1The killing rate of CAR T cells was calculated by continuously adding target cells and then detecting the residue of NK92 cells by flow cytometry using PE-mouse anti-human CD56 at D2 and D10, and the results are shown in fig. 5.

It can be seen that all three CAR T cells had significant specific killing of NK92 target cells compared to NT. And as the culture time is prolonged, the killing activity of the 2Dbbz-CAR T cells is gradually weakened, while the 2Dbb10z-CAR T cells can still maintain higher killing capability, and the killing activity of the D10 and the D10 are obviously different. This indicates that the addition of the intracellular region of DAP10 as a costimulatory domain significantly improved the persistence of the killing activity of CAR T cells.

2.3 detection of expression of CD107a

Cytotoxic T lymphocytes (CTL cells) contain high concentrations of cytotoxic particles in the form of vesicles in the cytoplasm, and lysosomal associated membrane protein I (CD107a) is the major component of the vesicle membrane protein. When the CTL cells kill the target cells, the toxic particles will reach and fuse with the cell membrane (at which time the CD107a molecule is transported to the cell membrane surface), causing the release of the particle contents, eventually leading to the death of the target cells. Thus, the CD107a molecule is a sensitive marker of CTL cell degranulation, and can respond to cell killing activity.

At 1x10⁵Concentration of Individual cells/well target cells (NK92 cells) were plated in 96-well plates, and then CAR-T and NT cells of the invention were added to each well at a 1:1 ratio, together with 10. mu.l PE-anti-human CD107a (BD Pharmingen, cat 555801) at 37 ℃ with 5% CO₂Co-culturing under culture conditions. After 1h, Goigstop (BD Pharmingen, cat # 51-2092KZ) was added and incubation continued for 2.5 h. Then, 5. mu.l of APC-anti human CD8(BD Pharmingen, cat # 555369) and 5. mu.l of FITC-anti human CD4(BD Pharmingen, cat # 561005) were added to each well, and after incubation at 37 ℃ for 30 minutes, the expression of CD107a was detected by flow cytometry, as shown in FIG. 6.

It can be seen that the expression of CD107a was significantly up-regulated after co-culturing the 2Dbbz-CAR T cells, 2D28z-CAR T cells, and 2Dbb10z-CAR T cells prepared by the present invention with target cells, indicating that the CAR-T cells of the present invention can significantly kill NK cells. Furthermore, CD107a expression of 2Dbb10z-CAR T cells was significantly higher in CD8+ T cells than 2Dbbz-CAR T cells, indicating that 2Dbb10z-CAR T cells had a stronger killing power than 2Dbbz-CAR T cells.

2.4 cytokine release by CAR-T cells

When the T cells kill target cells, the target cells decrease in number and release cytokines IL-2, IFN-gamma, and the like. The levels of release of cytokines IL-2 and IFN- γ upon killing of target cells by CAR T cells were determined using enzyme-linked immunosorbent assay (ELISA) according to the following procedure.

(1) Collecting cell co-culture supernatant

At 1x10⁵Concentration per well target cells NK92 were plated in 96-well plates, CAR T cells and NT cells of the invention were then co-cultured with target cells at a ratio of 1:1, respectively, and cell co-culture supernatants were collected 18-24 hours later.

(2) ELISA detection of IL-2 and IFN-gamma secretion in supernatants

The 96-well plate was coated with capture Antibody Purified anti-human IL2 Antibody (Biolegend, cat. No. 500302) or Purified anti-human IFN-. gamma.antibody (Biolegend, cat. No. 506502) and incubated overnight at 4 deg.C, then the Antibody solution was removed, 250. mu.L of PBST (1 XPBS with 0.1% Tween) solution containing 2% BSA (sigma, cat. No. V900933-1kg) was added and incubated for 2 hours at 37 deg.C. The plates were then washed 3 times with 250 μ L of PBST (1 XPBS with 0.1% Tween). mu.L of cell co-culture supernatant or standard was added to each well and incubated at 37 ℃ for 1 hour, after which the plates were washed 3 times with 250. mu.L of PBST (1 XPBS with 0.1% Tween). Then 50. mu.L of the Anti-Interferon gamma antibody [ MD-1] (Biotin) (abcam, cat # ab25017) was added to each well, and after 1 hour of incubation at 37 ℃ the plates were washed 3 times with 250. mu.L of PBST (1 XPBS containing 0.1% Tween). HRP Streptavidin (Biolegend, cat # 405210) was added, and after incubation at 37 ℃ for 30 minutes, the supernatant was discarded, 250. mu.L of PBST (1 XPBS containing 0.1% Tween) was added, and washed 5 times. To each well 50 μ L of TMB substrate solution was added. The reaction was allowed to occur at room temperature in the dark for 30 minutes, after which 50. mu.L of 1mol/L H SO was added to each well to stop the reaction. Within 30 minutes of stopping the reaction, absorbance at 450nm was measured using a microplate reader, and the content of cytokine was calculated from a standard curve (plotted according to the reading and concentration of the standard), and the result is shown in FIG. 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 invention was significantly higher than the control NT cells. Furthermore, the level of IL-2 and IFN- γ release from 2Dbb10z-CAR T cells was significantly lower than for 2Dbbz-CAR T cells, suggesting that the former is safer than the latter, since too high cytokine release may lead to serious side effects such as cytokine storm.

The above results indicate that CAR-T cells targeting NK activating receptors are able to continuously and efficiently kill NK cells. Furthermore, further inclusion of the intracellular region of DAP10 may increase the sustained killing activity of CAR-T cells, while secreting less cytokines, increasing safety compared to traditional CARs containing only the 4-1BB co-stimulatory domain.

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

Endogenous NKG2D in NT, 2Dbbz-CAR T cells, 2D28z-CAR T and 2Dbb10z-CAR T cells were knocked out by the CRISP/Cas9 system and NKG2D expression levels in CAR T cells after knockdown were measured by flow cytometry with PE mouse anti-human NKG2D (biolegend cat No. 302806) (table 1) and the proportion of CD8+ T cells in CAR-T cells was measured with APC-anti human CD8(BD Pharmingen, 555369) (fig. 8). Non-knockdown NT cells served as controls.

TABLE 1 NKG2D expression levels in CAR-T cells

Name of Gene	2DbbzKO-CAR	2Dbb10zKO-CAR	2D28zKO-CAR	NT
					NKG2D	5.00％	6.20％	6.50％	39.40％

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

As can be seen from figure 8, when a CAR targeting NKG2D was expressed, further knock-out of endogenous NKG2D could significantly increase the proportion of CD8+ T cells in the CAR T cell population, probably due to reduced mutual killing between CAR-T cells. Since CD8+ T cells are cytotoxic lymphocytes, this elevated proportion of CD8+ T cells can enhance the killing effect of CAR T cells in vitro and in vivo overall.

Example 4 preparation of Dual target CAR-T cells and verification of their function

19CAR T cells and 2D19CAR T cells were prepared according to the method described in example 1. 19CAR T cells differ from 2Dbbz-CAR T cells only by replacing the anti-NKG 2D scFv with anti-CD 19scFv (SEQ ID NO: 1). The 2D19CAR T cells differed from the 2Dbbz-CAR T cells only in that the CAR further comprised an anti-CD 19 scFv.

In addition, universal CAR-tKO T cells in which TCR and MHC-related genes (e.g., HLA-class I molecules such as B2M and HLA-class II molecules such as RFX5) were knocked out were also prepared by the CRISP/Cas9 system, and the expression efficiencies of TCR/CD3(TCR α), HLA-I (CAR 2M), HLA-II (RFX5) in the knocked out T cells were measured by flow cytometry using FITC Mouse Anti-Human CD3(BD Pharmingen, cat No. 555916) antibody, PE Mouse Anti-Human HLA-I (R & D cat No. FAB7098P), and APC Anti-Human DR, DP, DQ (cat No. 361714) antibodies, as shown in table 2 below. Non-knockdown NT cells served as controls.

TABLE 2 expression levels of TCR and MHC associated genes in CAR-T cells

Name of knockout gene	TCR/CD3	HLA-I(B2M)	HLA-II(RFX5)
				19CAR-tKO	5.60％	14％	11％
2D19CAR-tKO	3.90％	16.30％	10.40％
				NT	97％	98％	82％

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

In addition, the expression level of scFv on CAR-T cells was measured by flow cytometry using Biotin-SP (long spacer) affinity Goat Anti-Mouse IgG, F (ab') Fragment specificity (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, cat # 115-065-072) as a secondary antibody and APC Streptavidin (BD Pharmingen, cat # 554067) or PE Streptavidin (BD Pharmingen, cat # 554061) as a secondary antibody, as shown in FIG. 9.

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

The killing effect of the above cells on NK92 target cells was tested according to the method described in 1.2 in example 1, while the killing ability of CAR T cells on target cells Nalm6 was tested as follows: first at 1x10⁴Nalm6 target cells carrying a fluorescein gene were plated in 96-well plates, CAR T cells and untransfected T cells (NT) were plated in 96-well plates at an effective-to-target ratio (i.e., ratio of effector T cells to target cells) of 4:1 for co-culture, and fluorescence was measured using a microplate reader after 16-18 hours. According to the calculation formula: (mean value of fluorescence of target cells-mean value of fluorescence of sample)/mean value of fluorescence of target cells x 100%, and 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 have significant killing of Nalm 6. Two CAR-T cells containing only anti-CD 19scFv did not kill NK92 as they could not be recognized; while both 2D19CAR T cells and 2D19CAR-tKO T cells comprising anti-NKG 2D scFv were able to kill NK92 cells efficiently. Furthermore, TCR/MHC-associated gene knockout 2D19CAR-tKO T cells have reduced killing of target cells compared to 2D19CAR T cells, probably because at the same time CAR-T cells kill NK cells, the knockout of these genes also allows NK cells to produce some degree of killing of CAR-T cells.

The expression level of CD107a after the above cells were co-cultured with target cells Nalm6 (expressing CD19) and NK92 (expressing NKG2D), respectively, was examined according to the method described in 2.3 in example 2, and the results are shown in fig. 11 and 12. It can be seen that 19CAR T cells and 19CAR-tKO T cells only detected significantly elevated levels of CD107a after cocultivation with Nalm6 compared to NT cells; significantly elevated CD107a levels were detected after co-culture of both 2D19CAR T cells and 2D19CAR-tKO T cells with Nalm6 and NK92 cells.

The IFN-. gamma.release levels of the above cells after coculture with the target cells Nalm6 (expressing CD19) and NK92 (expressing NKG2D), respectively, were examined according to the method described in 2.4 of example 2, and the results are shown in FIG. 13. It can be seen that CAR-T cells containing only anti-CD 19scFv detected significantly elevated IFN- γ levels only after co-culture with Nalm6, whereas little release of IFN- γ was detected after co-culture with NK92 cells that did not express CD 19. However, CAR-T cells containing both anti-CD 19scFv and anti-NKG 2D scFv could detect IFN- γ release upon co-culture with both target cells.

The above results indicate that the CAR-T cells recognizing double targets of CD19 and NKG2D and the universal CAR-T cells in which MHC-related genes are knocked out prepared by the present invention can effectively kill target cells expressing CD19 or NKG 2D. This dual-targeted CAR design that targets both NK-activating receptors and tumor antigens allows on the one hand killing of NK cells and thus prolonging survival of CAR-T cells, while on the other hand killing of target cells expressing tumor antigens with high efficiency.

Example 5 preparation of CAR cells comprising different scFv sequences targeting NKp46 and validation of their function

CAR T cells were prepared according to the method of example 1, which differed from 2Dbbz-CAR T cells only in the replacement of anti-NKG 2D scFv with anti-NKp 46 scFv. Wherein the NKp46-1CAR comprises the anti-NKp 46scFv with the amino acid sequence shown in SEQ ID NO: 35 is shown in the figure; the amino acid sequence of the anti-NKp 46scFv comprised by NKp46-2CAR is as set forth in SEQ ID NO: 37 is shown in the figure; the amino acid sequence of the anti-NKp 46scFv comprised by NKp46-3CAR is as set forth in SEQ ID NO: 39; the amino acid sequence of the anti-NKp 46scFv comprised by the NKp46-4CAR is as shown in SEQ ID NO: 41 is shown; the amino acid sequence of the anti-NKp 46scFv comprised by the NKp46-5CAR is set forth in SEQ ID NO: shown at 43.

Expression levels of scFv on CAR-T cells were detected by flow cytometry using Biotin-SP (long spacer) AffiniP Goat Anti-Mouse IgG, F (ab') Fragment specificity (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, cat # 115-065-072) as a primary antibody, APC Streptavidin (BD Pharmingen, cat # 554067) or PE Streptavidin (BD Pharmingen, cat # 554061) as a secondary antibody, and the results are shown in FIG. 14.

It can be seen that CAR-T cells containing different NKp46 scfvs all efficiently expressed scfvs.

The killing effect of the CAR T cells on NK92 cells was tested 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 comprising 5 different scfvs targeting NKp46 all had significant killing of NK92 cells, with NKp46-5-CAR T cells having the strongest killing ability, were used in subsequent experiments and renamed to 46CAR-T cells.

The inventors also generated 4619CAR-T cells targeting both NKp46 and CD19 targets, which differed from 2D19bbz-CAR T cells only by replacing the anti-NKG 2D scFv with anti-NKp 46scFv (SEQ ID NO: 43). scFv expression was detected by flow cytometry in 19CAR-T cells, 46CAR-T cells and 4619CAR-T cells, and the results are shown in FIG. 16. The killing effect of the above three CAR T cells on NK92 cells and Nalm6 was tested according to the method described in example 1, and the results are shown in fig. 17.

It can be seen that CARs in 19CAR-T cells, 46CAR-T cells and 4619CAR-T cells were all efficiently expressed, and 46CAR T cells killed NK92 target cells only, 19CAR T cells killed Nalm6 target cells only, while 4619CAR T cells were able to kill both target cells effectively.

Example 6 preparation of NKp 46-Targeted CAR-NK cells and verification of their function

Preparing CAR-NK cells targeting NKp46, CD19, or both, and KO-CAR NK cells in which NKp46 is knocked out, as follows: endogenous NKp46 in NK92 cells was first knocked out by CRISPR/Cas9 system and NKp46 in NK92 cells was confirmed to be efficiently knocked out by flow cytometry (table 3).

TABLE 3 NKp46 expression levels in NK92 cells before and after knockdown

Name of Gene	NK92KO	NK92
			NKp46	23.00％	92.00％

Then, after packaging 19CAR comprising CD19scFv (SEQ ID NO:1), 46CAR comprising NKp46scFv (SEQ ID NO:43) and 4619CAR vector comprising CD19scFv (SEQ ID NO:1) and NKp46scFv (SEQ ID NO:43) with the virus according to the virus packaging method in example 1, the virus was centrifuged to co-infect NK92 cells or NK92KO cells, respectively (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 CAR-NK cells on NK92 cells and Nalm6 cells was examined according to the methods described in example 2 and example 3, and the results are shown in fig. 18 and fig. 19. It can be seen that 46CAR NK cells and 46KO-CAR NK cells kill only NK92 target cells, 19CAR NK and 19KO-CAR NK cells kill only Nalm6 target cells, while 4619CAR NK and 4619KO-CAR NK cells kill effectively on both target cells.

It should be noted that the above-mentioned embodiments are merely preferred examples of the present invention, and the present invention is not limited thereto. It will be understood by those skilled in the art that any modification, equivalent replacement, or improvement made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Sequence listing

<110> Nanjing Beijing Heng Biotechnology Ltd

<120> chimeric antigen receptor targeting NK activating receptor

<130> BHCN25

<160> 110

<170> SIPOSequenceListing 1.0

<210> 1

<211> 242

<212> PRT

<213> Artificial Sequence

<220>

<223> CD19 scFv-1

<400> 1

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys

130 135 140

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

145 150 155 160

Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser

165 170 175

Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile

180 185 190

Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln

195 200 205

Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

210 215 220

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val

225 230 235 240

Ser Ser

<210> 2

<211> 726

<212> DNA

<213> Artificial Sequence

<220>

<223> CD19 scFv-1

<400> 2

gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120

gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240

gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300

gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360

ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 420

tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 480

cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 540

tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 600

ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 660

tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc 720

tcctca 726

<210> 3

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> CD8 alpha transmembrane domain

<400> 3

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys Lys

20 25

<210> 4

<211> 75

<212> DNA

<213> Artificial Sequence

<220>

<223> CD8 alpha transmembrane domain

<400> 4

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gcaaa 75

<210> 5

<211> 27

<212> PRT

<213> Artificial Sequence

<220>

<223> transmembrane domain of CD28

<400> 5

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 6

<211> 81

<212> DNA

<213> Artificial Sequence

<220>

<223> transmembrane domain of CD28

<400> 6

ttttgggtcc tcgtcgtagt tggaggggta cttgcctgtt atagcctcct ggttaccgta 60

gcatttatta tattctgggt g 81

<210> 7

<211> 41

<212> PRT

<213> Artificial Sequence

<220>

<223> CD28 costimulatory domain

<400> 7

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 8

<211> 123

<212> DNA

<213> Artificial Sequence

<220>

<223> CD28 costimulatory domain

<400> 8

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120

tcc 123

<210> 9

<211> 40

<212> PRT

<213> Artificial Sequence

<220>

<223> 4-1BB Co-stimulatory Domain

<400> 9

Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg

1 5 10 15

Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro

20 25 30

Glu Glu Glu Glu Gly Gly Cys Glu

35 40

<210> 10

<211> 120

<212> DNA

<213> Artificial Sequence

<220>

<223> 4-1BB Co-stimulatory Domain

<400> 10

cggggcagaa agaaactcct gtatatattc aaacaaccat ttatgagacc agtacaaact 60

actcaagagg aagatggctg tagctgccga tttccagaag aagaagaagg aggatgtgaa 120

<210> 11

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> CD3 zeta signaling domain

<400> 11

Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

1 5 10 15

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

20 25 30

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

35 40 45

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

50 55 60

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

65 70 75 80

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

85 90 95

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

100 105 110

Arg

<210> 12

<211> 339

<212> DNA

<213> Artificial Sequence

<220>

<223> CD3 zeta signaling domain

<400> 12

ctgagagtga agttcagcag gagcgcagac gcccccgcgt accagcaggg ccagaaccag 60

ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caagagacgt 120

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 180

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 240

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 300

acctacgacg cccttcacat gcaggccctg ccccctcgc 339

<210> 13

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> CD3 zeta signaling domain

<400> 13

Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

1 5 10 15

Gly Gln Asn Gln Leu Phe Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

20 25 30

Phe Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

35 40 45

Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu

50 55 60

Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly

65 70 75 80

Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Phe Gln Gly Leu Ser

85 90 95

Thr Ala Thr Lys Asp Thr Phe Asp Ala Leu His Met Gln Ala Leu Pro

100 105 110

Pro Arg

<210> 14

<211> 342

<212> DNA

<213> Artificial Sequence

<220>

<223> CD3 zeta signaling domain

<400> 14

ctgagagtga agttcagcag gagcgcagac gcccccgcgt accagcaggg ccagaaccag 60

ctctttaacg agctcaatct aggacgaaga gaggagttcg atgttttgga caagagacgt 120

ggccgggacc ctgagatggg gggaaagccg cagagaagga agaaccctca ggaaggcctg 180

tacaatgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg gatgaaaggc 240

gagcgccgga ggggcaaggg gcacgatggc cttttccagg gtctcagtac agccaccaag 300

gacacctttg acgcccttca catgcaggcc ctgccccctc gc 342

<210> 15

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> B2M Signal peptide

<400> 15

Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser

1 5 10 15

Gly Leu Glu Ala

20

<210> 16

<211> 60

<212> DNA

<213> Artificial Sequence

<220>

<223> B2M Signal peptide

<400> 16

atgtcccgct ctgttgcttt ggctgtgctg gcccttttgt cccttagcgg actggaggcc 60

<210> 17

<211> 21

<212> PRT

<213> Artificial Sequence

<220>

<223> CD8 alpha Signal peptide

<400> 17

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 18

<211> 63

<212> DNA

<213> Artificial Sequence

<220>

<223> CD8 alpha Signal peptide

<400> 18

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccg 63

<210> 19

<211> 45

<212> PRT

<213> Artificial Sequence

<220>

<223> CD8 alpha hinge region

<400> 19

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 20

<211> 135

<212> DNA

<213> Artificial Sequence

<220>

<223> CD8 alpha hinge region

<400> 20

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 21

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> CD28 hinge region

<400> 21

Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn

1 5 10 15

Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu

20 25 30

Phe Pro Gly Pro Ser Lys Pro

35

<210> 22

<211> 117

<212> DNA

<213> Artificial Sequence

<220>

<223> CD28 hinge region

<400> 22

attgaagtta tgtatcctcc tccttaccta gacaatgaga agagcaatgg aaccattatc 60

catgtgaaag ggaaacacct ttgtccaagt cccctatttc ccggaccttc taagccc 117

<210> 23

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> IgG4 hinge region

<400> 23

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

1 5 10

<210> 24

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223> IgG4 hinge region

<400> 24

gaaagcaaat acgggccgcc gtgtccaccc tgtccg 36

<210> 25

<211> 238

<212> PRT

<213> Artificial Sequence

<220>

<223> CD19 scFv-2

<400> 25

Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Thr Ser Leu Gly

1 5 10 15

Glu Thr Val Thr Ile Gln Cys Gln Ala Ser Glu Asp Ile Tyr Ser Gly

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Asp Leu Gln Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Thr Ser Met Gln Thr

65 70 75 80

Glu Asp Glu Gly Val Tyr Phe Cys Gln Gln Gly Leu Thr Tyr Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln

115 120 125

Ser Gly Ala Glu Leu Val Arg Pro Gly Thr Ser Val Lys Leu Ser Cys

130 135 140

Lys Val Ser Gly Asp Thr Ile Thr Phe Tyr Tyr Met His Phe Val Lys

145 150 155 160

Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Glu

165 170 175

Asp Glu Ser Thr Lys Tyr Ser Glu Lys Phe Lys Asn Lys Ala Thr Leu

180 185 190

Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr Leu Lys Leu Ser Ser Leu

195 200 205

Thr Ser Glu Asp Thr Ala Thr Tyr Phe Cys Ile Tyr Gly Gly Tyr Tyr

210 215 220

Phe Asp Tyr Trp Gly Gln Gly Val Met Val Thr Val Ser Ser

225 230 235

<210> 26

<211> 714

<212> DNA

<213> Artificial Sequence

<220>

<223> CD19 scFv-2

<400> 26

gacatccaga tgacccagag ccctgccagc ctgtctacca gcctgggcga gacagtgacc 60

atccagtgtc aggccagcga ggacatctac tctggcctgg cttggtatca gcagaagccc 120

ggcaagagcc ctcagctgct gatctacggc gccagcgacc tgcaggacgg cgtgcctagc 180

agattcagcg gcagcggctc cggaacccag tacagcctga agatcaccag catgcagacc 240

gaggacgagg gcgtgtactt ctgccagcaa ggcctgacct accctagaac cttcggagga 300

ggcaccaagc tggaactgaa gggcggaggc ggaagtggag gcggaggatc tggcggcgga 360

ggctctgaag tgcagctgca gcagtctggc gctgaactgg tccggcctgg cactagcgtg 420

aagctgtcct gcaaggtgtc cggcgacacc atcaccttct actacatgca cttcgtgaag 480

cagaggccag gacagggcct ggaatggatc ggcagaatcg accctgagga cgagagcacc 540

aagtacagcg agaagttcaa gaacaaggcc accctgaccg ccgacaccag cagcaacacc 600

gcctacctga agctgtctag cctgacctcc gaggacaccg ccacctactt ttgcatctac 660

ggcggctact acttcgacta ctggggccag ggcgtgatgg tcaccgtgtc cagc 714

<210> 27

<211> 246

<212> PRT

<213> Artificial Sequence

<220>

<223> NKGD scFv-1

<400> 27

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Gly Leu Gly Asp Gly Thr Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Ala Leu Thr Gln Pro Ala

130 135 140

Ser Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys Ser Gly

145 150 155 160

Ser Ser Ser Asn Ile Gly Asn Asn Ala Val Asn Trp Tyr Gln Gln Leu

165 170 175

Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Asp Asp Leu Leu Pro

180 185 190

Ser Gly Val Ser Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala

195 200 205

Phe Leu Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr

210 215 220

Cys Ala Ala Trp Asp Asp Ser Leu Asn Gly Pro Val Phe Gly Gly Gly

225 230 235 240

Thr Lys Leu Thr Val Leu

245

<210> 28

<211> 738

<212> DNA

<213> Artificial Sequence

<220>

<223> NKGD scFv-1

<400> 28

caggtccagt tggttgaaag tggaggaggg ttggtgaaac ccggaggatc cttgcgactg 60

tcttgcgcag ctagtggctt cacgttcagt tcctatggca tgcattgggt gagacaagct 120

cctggtaaag gcttggaatg ggtggccttc ataaggtatg acggttcaaa taaatattac 180

gccgactccg tcaagggtcg atttaccatt tccagggaca attcaaaaaa tacattgtac 240

ttgcagatga atagccttag agcggaggac acggccgtct actactgcgc gaaggatcga 300

gggttgggcg acggcactta ctttgactac tggggacagg gaacgacggt cacagtctcc 360

tcaggcggtg gtgggagcgg tggtggagga agtggaggtg gtggttcaca gtccgcactc 420

actcagccag cgtccgttag tggttcccct ggccagagta tcaccattag ttgcagtggc 480

agttcctcaa atattgggaa taacgcagtg aattggtatc agcaattgcc tggcaaagcg 540

cccaaattgc ttatttatta cgacgatctc cttccctctg gagtttccga tcggttctca 600

ggctctaaga gcggtacctc tgcgtttctc gccatatctg gtcttcaatc agaagatgaa 660

gccgactatt actgcgctgc atgggatgac agccttaatg gtccggtttt tgggggcgga 720

accaaactta cagtcctg 738

<210> 29

<211> 243

<212> PRT

<213> Artificial Sequence

<220>

<223> NKGD scFv-2

<400> 29

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Gly Gly Gly

100 105 110

Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser

115 120 125

Gly Gly Gly Val Val Arg Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala

130 135 140

Ala Ser Gly Phe Thr Phe Asp Asp Tyr Gly Met Thr Trp Val Arg Gln

145 150 155 160

Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly Ile Asn Trp Asn Gly

165 170 175

Gly Ser Thr Gly Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser

180 185 190

Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg

195 200 205

Ala Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Arg Glu Arg Glu Leu Tyr

210 215 220

Tyr Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gln Gly Thr Thr Val Thr

225 230 235 240

Val Ser Ser

<210> 30

<211> 729

<212> DNA

<213> Artificial Sequence

<220>

<223> NKGD scFv-2

<400> 30

gaaatcgttc ttacacaaag ccctggtact ttgtctctgt cccctggaga gcgagcgact 60

ctgagctgta gagcatccca atctgttagt agcagttacc tggcttggta ccaacagaaa 120

ccaggccagg cgccgcgctt gctcatttac ggtgctagta gtagagcaac aggaatcccc 180

gaccgcttta gtgggtcagg aagtggtact gactttacgc tgacgatcag cagactggaa 240

ccagaggatt tcgccgttta ttactgccaa cagtatggca gttcaccctt caccttcgga 300

cctggtacaa aagtggacat taaacgaggt gggggaagcg gaggagggtc aggaggtggt 360

tcagaggttc agctcgtgga gagtgggggt ggcgtagtac gaccaggtgg ttccctccga 420

ctgagctgcg ccgctagtgg ttttactttc gacgattacg gaatgacctg ggtcaggcaa 480

gcaccaggca agggtttgga gtgggtttct gggattaact ggaatggggg ttctacggga 540

tacgcagact ctgttaaggg gcggtttaca atatcacgag ataacgctaa aaactccttg 600

tatttgcaga tgaacagttt gcgggctgaa gacactgctc tttactactg cgcacgggaa 660

cgagaattgt actattatta ttacgggctc gatgtttggg gccaagggac aacagtgact 720

gtttccagc 729

<210> 31

<211> 236

<212> PRT

<213> Artificial Sequence

<220>

<223> NKGD scFv-3

<400> 31

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly

100 105 110

Ser Gly Gly Gly Ser Gly Gly Gly Ser Gln Val His Leu Gln Glu Ser

115 120 125

Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr

130 135 140

Val Ser Asp Asp Ser Ile Ser Ser Tyr Tyr Trp Ser Trp Ile Arg Gln

145 150 155 160

Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly His Ile Ser Tyr Ser Gly

165 170 175

Ser Ala Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val

180 185 190

Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala

195 200 205

Ala Asp Thr Ala Val Tyr Tyr Cys Ala Asn Trp Asp Asp Ala Phe Asn

210 215 220

Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

225 230 235

<210> 32

<211> 708

<212> DNA

<213> Artificial Sequence

<220>

<223> NKGD scFv-3

<400> 32

gaaattgtgc ttacccagtc ccctggcaca ctcagcctca gtccagggga acgcgcgacg 60

ctgtcatgta gggcctcaca atctgtgagt tccagctatc tggcgtggta tcagcaaaag 120

ccgggtcaag cccctcgctt gcttatttac ggagcaagca gtagagccac tggtattcct 180

gatcgcttca gcggcagcgg aagcggcacc gacttcacgt tgaccattag ccggcttgaa 240

ccggaagatt ttgcagttta ctattgccaa caatatgggt cctctccctg gactttcgga 300

caggggacga aggtggaaat aaagcgaggg ggcggcagtg gcggaggcag tggaggagga 360

agccaagtac acctgcagga atctggcccg ggtctcgtca aaccttccga aacattgagt 420

cttacttgta cagtttcaga tgatagcatt tccagctact attggtcttg gattaggcaa 480

ccccccggaa aaggtttgga atggattggt cacattagtt attcagggtc agctaattat 540

aatccttctt tgaaatcaag ggtaacaata tcagtggaca cgagtaaaaa ccagtttagt 600

cttaaactta gcagcgtaac agcagcggat actgccgtat attactgtgc aaattgggac 660

gacgccttta atatctgggg acaggggacg atggttacgg tatctagc 708

<210> 33

<211> 243

<212> PRT

<213> Artificial Sequence

<220>

<223> NKGD scFv-4

<400> 33

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Ser

100 105 110

Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Gln Ser Gly

115 120 125

Ala Glu Val Lys Glu Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Asn

130 135 140

Ser Gly Tyr Ser Phe Thr Asn Tyr Trp Val Gly Trp Val Arg Gln Met

145 150 155 160

Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser

165 170 175

Asp Thr Arg Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala

180 185 190

Asp Lys Ser Ile Asn Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala

195 200 205

Ser Asp Thr Ala Met Tyr Tyr Cys Gly Arg Leu Thr Met Phe Arg Gly

210 215 220

Ile Ile Ile Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

225 230 235 240

Val Ser Ser

<210> 34

<211> 729

<212> DNA

<213> Artificial Sequence

<220>

<223> NKGD scFv-4

<400> 34

gaaatagtcc ttacgcaatc cccagctaca ttgagcttga gtccaggaga gcgagcgacc 60

ctcagttgta gagctagtca gtcagtaagc tcctatctcg cttggtatca gcaaaagcca 120

ggacaagccc ccagactcct catttacgac gccagtaaca gggcaactgg tatccctgcg 180

cgctttagtg gcagcggttc aggcacagat ttcactttga ccatatcatc attggagccc 240

gaagatttcg cagtgtacta ctgccaacaa cgctcaaact ggccctggac tttcggtcag 300

gggaccaagg ttgaaatcaa gaggggaggc ggttccggcg gaggatcagg aggaggctca 360

gaagttcagt tggtgcaatc tggagcggag gtaaaagaac cgggggaatc attgaaaata 420

agctgtaaaa atagtggata tagtttcaca aactactggg tcgggtgggt tcgccagatg 480

ccgggtaagg gactggagtg gatggggata atctatccag gcgactcaga cactcgctat 540

agtccttctt ttcaagggca agtcacaata agtgccgaca agagtattaa caccgcatat 600

cttcagtggt cctccttgaa agcgagtgat acggcgatgt attactgcgg acgacttact 660

atgttccgag gtataatcat tggatacttc gactattggg gccaagggac cctggtgacg 720

gtttcaagc 729

<210> 35

<211> 244

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-1

<400> 35

Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Val Ser Glu Asn Ile Tyr Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val

35 40 45

Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys

130 135 140

Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp Tyr Ala Trp Asn Trp Ile

145 150 155 160

Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp Met Gly Tyr Ile Thr Tyr

165 170 175

Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Glu Ser Arg Ile Ser Ile

180 185 190

Thr Arg Asp Thr Ser Thr Asn Gln Phe Phe Leu Gln Leu Asn Ser Val

195 200 205

Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Arg Gly Gly Tyr Tyr

210 215 220

Gly Ser Ser Trp Gly Val Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

225 230 235 240

Thr Val Ser Ala

<210> 36

<211> 732

<212> DNA

<213> Artificial Sequence

<220>

<223> NKp46 scFv-1

<400> 36

gatattcaga tgacacagtc tcctgcctct ctgtctgcaa gcgttggtga aacggtcact 60

ataacatgtc gggtgagtga aaatatctat tcatatctcg cctggtacca gcagaaacaa 120

ggcaagagtc cccaattgct tgtttacaat gccaaaacgc tggctgaagg tgttccatct 180

cggtttagcg gatcaggaag cggaacgcag ttttccctta agatcaattc ccttcaacct 240

gaggactttg gatcttatta ttgccaacat cattatggga caccttggac gtttggagga 300

gggacaaaac tggagataaa aggaggtggt ggcagtggtg gaggaggaag tgggggtggg 360

ggatcagaag tacagctgca agagagcggg ccgggtctcg ttaaaccatc ccaatccctt 420

agcctcactt gtactgttac gggatacagc ataacctcag attatgcctg gaactggatc 480

agacagttcc ccggcaacaa actcgaatgg atgggctaca taacttacag tggtagcact 540

tcctacaacc ccagtttgga gtcacgcatt agcatcacga gggatactag cactaatcag 600

ttctttcttc agctgaatag tgttaccacg gaggatacgg cgacgtatta ctgcgccagg 660

ggcggttatt atgggtcatc ctggggagta tttgcttact gggggcaagg aacccttgtc 720

actgtctcag cg 732

<210> 37

<211> 238

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-2

<400> 37

Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly

1 5 10 15

Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln

115 120 125

Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys

130 135 140

Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr Thr Met His Trp Val Lys

145 150 155 160

Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly Gly Ile Ser Pro Asn

165 170 175

Ile Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu

180 185 190

Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu

195 200 205

Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Arg Gly Gly Ser

210 215 220

Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

225 230 235

<210> 38

<211> 714

<212> DNA

<213> Artificial Sequence

<220>

<223> NKp46 scFv-2

<400> 38

gatattgtga tgactcaaag tcccgctact ctttctgtta caccgggaga tcgcgtatca 60

ctgagctgcc gcgcctcaca gtccatctct gattatcttc actggtatca acaaaagtca 120

catgagtctc caaggctttt gataaaatat gcgagccagt ccatctctgg cattcctagt 180

cgctttagcg ggagtggctc aggaagcgac ttcaccctca gcatcaattc agttgaacct 240

gaggacgtcg gggtttatta ttgccagaat gggcactcct tccccctgac atttggtgcc 300

ggcaccaaac ttgagcttaa aggcggagga ggttctggtg gtggtggatc cgggggcggc 360

ggatccgagg tgcagcttca acagagcggt cccgaattgg tcaagccggg agcttccgtt 420

aaaattagct gtaaaacctc tggatatact tttacggaat atactatgca ttgggttaag 480

cagtcacacg gaaagagtct tgaatggata ggcggtatct ctcccaatat cgggggcacc 540

tcttacaacc agaagttcaa gggtaaagct acgttgacgg tggacaagtc ttcttcaacc 600

gcttatatgg aattgagatc actcacttca gaagactctg cagtatatta ctgtgctcga 660

cggggtggtt cctttgatta ttggggtcag ggaactacgc tgactgttag tagc 714

<210> 39

<211> 238

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-3

<400> 39

Asp Ile Gln Met Ile Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn

20 25 30

Leu Ala Trp Phe Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val

35 40 45

Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Gly Ile Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln

115 120 125

Ser Ala Val Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys

130 135 140

Lys Ala Ser Gly Tyr Thr Phe Thr Ser Phe Thr Met His Trp Val Lys

145 150 155 160

Gln Arg Pro Gly Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Ser

165 170 175

Gly Tyr Thr Glu Tyr Asn Gln Lys Phe Lys Asp Lys Thr Thr Leu Thr

180 185 190

Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Asp Ser Leu Thr

195 200 205

Ser Asp Asp Ser Ala Val Tyr Tyr Cys Val Arg Gly Ser Ser Arg Gly

210 215 220

Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala

225 230 235

<210> 40

<211> 714

<212> DNA

<213> Artificial Sequence

<220>

<223> NKp46 scFv-3

<400> 40

gatatacaaa tgatccagag ccctgcatca ctttcagttt ctgtggggga gactgtcaca 60

attacttgta gggcgagtga aaacatctat tcaaacctcg cgtggtttca acagaagcag 120

ggcaagtctc ctcaactgct cgtatatgcg gcaaccaatc tggcggacgg cgtcccctcc 180

aggttttccg gtagcggatc tggtactcag tactccttga agattaattc cctgcaaagt 240

gaagatttcg gaatctacta ttgccaacat ttttggggta cgccgagaac gtttggaggc 300

gggacaaagc tcgaaattaa aggaggggga ggcagtgggg gcggtggatc aggtggtggt 360

gggagccaag tgcaacttca gcaatctgcc gtagaacttg ctaggcccgg ggcgtcagtt 420

aaaatgtctt gtaaggcatc tgggtataca ttcacgtcat tcactatgca ctgggttaaa 480

caacgcccgg gaggattgga gtggataggg tatataaacc ccagtagtgg gtacactgag 540

tacaatcaaa agtttaaaga taagactacg ttgactgccg ataaatcctc aagcacagcg 600

tatatgcaac tcgattctct tacttctgat gattccgcag tgtattactg tgtacgcggg 660

tcatcacgcg gttttgacta ttggggccaa ggaactcttg taactgtaag tgcg 714

<210> 41

<211> 242

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-4

<400> 41

Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Ile Tyr Ser Tyr

20 25 30

Leu Ala Trp Cys Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val

35 40 45

Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr His Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Gly Ile Tyr Tyr Cys Gln His His Tyr Asp Thr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Gln Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys

130 135 140

Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp Tyr Ala Trp Asn Trp Ile

145 150 155 160

Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp Met Gly Tyr Ile Thr Tyr

165 170 175

Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Ile Ser Ile

180 185 190

Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu Gln Leu Asn Ser Val

195 200 205

Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Arg Cys Trp Asp Tyr

210 215 220

Ala Leu Tyr Ala Met Asp Cys Trp Gly Gln Gly Thr Ser Val Thr Val

225 230 235 240

Ser Ser

<210> 42

<211> 726

<212> DNA

<213> Artificial Sequence

<220>

<223> NKp46 scFv-4

<400> 42

gatattcaaa tgacacagtc cccagccagt cttagcgcta gtgtgggtga gaccgtgact 60

ataacttgca gaacgtctga gaatatatat tcctatctgg cttggtgcca acaaaaacag 120

gggaagagtc cgcaactgct cgtctacaac gcgaaaaccc tcgccgaagg tgtccccagt 180

cgatttagtg gatctggcag tggcacccat ttcagcctca aaatcaactc actccagcca 240

gaggattttg ggatatatta ctgtcagcat cattacgata cgcctctgac ttttggggca 300

ggcacaaagc tcgaattgaa gggaggaggt ggatctggcg gaggtggcag tggcgggggt 360

gggagcgatg tccagcttca ggaatctggc ccgggccttg ttaagccatc acagagtctg 420

agcctgacat gcactgtgac cgggtattcc atcacaagcg attatgcctg gaattggata 480

cggcaatttc ccggcaacaa gttggagtgg atggggtaca tcacttattc agggtcaacg 540

aattataatc ccagtttgaa gtcccgcatc agtataacgc gagatacctc caagaaccag 600

ttttttctcc agcttaatag tgttacgaca gaggataccg ctacctatta ctgtgctcgg 660

tgttgggatt atgctttgta tgcaatggat tgctgggggc agggcacctc cgtcacagtg 720

tcaagc 726

<210> 43

<211> 237

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-5

<400> 43

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser

20 25 30

Leu Asn Trp Leu Gln Gln Glu Pro Asp Gly Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Thr Ser Arg Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

65 70 75 80

Glu Asp Phe Val Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Ser Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln

115 120 125

Pro Gly Ser Val Leu Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys

130 135 140

Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser Trp Met His Trp Ala Lys

145 150 155 160

Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly His Ile His Pro Asn

165 170 175

Ser Gly Ile Ser Asn Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu

180 185 190

Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr Val Asp Leu Ser Ser Leu

195 200 205

Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ser Arg Gly Gly Arg Phe

210 215 220

Asp Asp Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser

225 230 235

<210> 44

<211> 711

<212> DNA

<213> Artificial Sequence

<220>

<223> NKp46 scFv-5

<400> 44

gatatacaga tgactcagag cccatcctca ttgagtgcat ccctgggaga gagggtatca 60

ctgacttgtc gcgcctcaca agatattggg tcttcactta attggctcca acaggagcca 120

gatggtacaa tcaaaaggct tatttatgct acatcacgct tggatagcgg cgttcctaaa 180

cgcttcagcg gtagccgaag tggtagcgat tatagtctga ccatcagcag cctcgaatca 240

gaagacttcg tggactatta ctgccttcaa tacgcttcat ccccctggac gtttggcggt 300

ggaaccaagc ttgaaattaa aggagggggt ggttcaggtg gtggcggaag tggtggggga 360

ggttcacaag tccaactcca gcaacccggg agcgttctcg taaggccggg tgcttctgtt 420

aagttgtcat gcaaagcttc tggttacact ttcaccagtt catggatgca ctgggccaag 480

cagcgccctg gtcaaggact cgaatggatc ggacatattc atcccaacag cgggatttcc 540

aattacaatg agaagttcaa agggaaagca actcttaccg tggacactag ctcatcaact 600

gcctacgtgg atctgagttc actgacttca gaggattcag cggtttacta ctgctcacgg 660

ggtggtagat tcgatgattg gggagcaggg accacagtaa cggtgtccag c 711

<210> 45

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> intracellular region of DAP10

<400> 45

Pro Arg Arg Ser Pro Ala Gln Glu Asp Gly Lys Val Tyr Ile Asn Met

1 5 10 15

Pro Gly Arg Gly

20

<210> 46

<211> 60

<212> DNA

<213> Artificial Sequence

<220>

<223> intracellular region of DAP10

<400> 46

ccacgccgca gccccgccca agaagatggc aaagtctaca tcaacatgcc aggcaggggc 60

<210> 47

<211> 49

<212> PRT

<213> Artificial Sequence

<220>

<223> intracellular region of DAP12

<400> 47

Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg

1 5 10 15

Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly

20 25 30

Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr

35 40 45

Lys

<210> 48

<211> 147

<212> DNA

<213> Artificial Sequence

<220>

<223> intracellular region of DAP12

<400> 48

ggccgcctgg tgccgcgcgg ccgcggcgcg gcggaagcgg cgacccgcaa acagcgcatt 60

accgaaaccg aaagcccgta tcaggaactg cagggccagc gcagcgatgt gtatagcgat 120

ctgaacaccc agcgcccgta ttataaa 147

<210> 49

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> CD19 scFv-1 CDR-L1

<400> 49

Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn

1 5 10

<210> 50

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> CD19 scFv-1 CDR-L2

<400> 50

His Thr Ser Arg Leu His Ser

1 5

<210> 51

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CD19 scFv-1 CDR-L3

<400> 51

Gln Gln Gly Asn Thr Leu Pro Tyr Thr

1 5

<210> 52

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> CD19 scFv-1 CDR-H1

<400> 52

Gly Val Ser Leu Pro Asp Tyr

1 5

<210> 53

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> CD19 scFv-1 CDR-H2

<400> 53

Trp Gly Ser Glu Thr

1 5

<210> 54

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> CD19 scFv-1 CDR-H3

<400> 54

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

1 5 10

<210> 55

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> CD19 scFv-2 CDR-L1

<400> 55

Gln Ala Ser Glu Asp Ile Tyr Ser Gly Leu Ala

1 5 10

<210> 56

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> CD19 scFv-2 CDR-L2

<400> 56

Gly Ala Ser Asp Leu Gln Asp

1 5

<210> 57

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CD19 scFv-2 CDR-L3

<400> 57

Gln Gln Gly Leu Thr Tyr Pro Arg Thr

1 5

<210> 58

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> CD19 scFv-2 CDR-H1

<400> 58

Gly Asp Thr Ile Thr Phe Tyr

1 5

<210> 59

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> CD19 scFv-2 CDR-H2

<400> 59

Asp Pro Glu Asp Glu Ser

1 5

<210> 60

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> CD19 scFv-1 CDR-H3

<400> 60

Gly Gly Tyr Tyr Phe Asp Tyr

1 5

<210> 61

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> NKG2D scFv-1 CDR-L1

<400> 61

Gly Phe Thr Phe Ser Ser Tyr

1 5

<210> 62

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> NKG2D scFv-1 CDR-L2

<400> 62

Arg Tyr Asp Gly Ser Asn

1 5

<210> 63

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> NKG2D scFv-1 CDR-L3

<400> 63

Asp Arg Gly Leu Gly Asp Gly Thr Tyr Phe Asp Tyr

1 5 10

<210> 64

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> NKG2D scFv-1 CDR-H1

<400> 64

Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Ala Val Asn

1 5 10

<210> 65

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> NKG2D scFv-1 CDR-H2

<400> 65

Tyr Asp Asp Leu Leu Pro Ser

1 5

<210> 66

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> NKG2D scFv-1 CDR-H3

<400> 66

Ala Ala Trp Asp Asp Ser Leu Asn Gly Pro Val

1 5 10

<210> 67

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> NKG2D scFv-2 CDR-L1

<400> 67

Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala

1 5 10

<210> 68

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> NKG2D scFv-2 CDR-L2

<400> 68

Gly Ala Ser Ser Arg Ala Thr

1 5

<210> 69

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> NKG2D scFv-2 CDR-L3

<400> 69

Gln Gln Tyr Gly Ser Ser Pro Phe Thr

1 5

<210> 70

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> NKG2D scFv-2 CDR-H1

<400> 70

Gly Phe Thr Phe Asp Asp Tyr

1 5

<210> 71

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> NKG2D scFv-2 CDR-H2

<400> 71

Asn Trp Asn Gly Gly Ser

1 5

<210> 72

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> NKG2D scFv-2 CDR-H3

<400> 72

Glu Arg Glu Leu Tyr Tyr Tyr Tyr Tyr Gly Leu Asp Val

1 5 10

<210> 73

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> NKG2D scFv-3 CDR-L3

<400> 73

Gln Gln Tyr Gly Ser Ser Pro Trp Thr

1 5

<210> 74

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> NKG2D scFv-3 CDR-H1

<400> 74

Asp Asp Ser Ile Ser Ser Tyr

1 5

<210> 75

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> NKG2D scFv-3 CDR-H2

<400> 75

Ser Tyr Ser Gly Ser

1 5

<210> 76

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> NKG2D scFv-2 CDR-H3

<400> 76

Trp Asp Asp Ala Phe Asn

1 5

<210> 77

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> NKG2D scFv-4 CDR-L1

<400> 77

Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala

1 5 10

<210> 78

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> NKG2D scFv-4 CDR-L2

<400> 78

Asp Ala Ser Asn Arg Ala Thr

1 5

<210> 79

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> NKG2D scFv-4 CDR-L3

<400> 79

Gln Gln Arg Ser Asn Trp Pro Trp Thr

1 5

<210> 80

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> NKG2D scFv-4 CDR-H1

<400> 80

Gly Tyr Ser Phe Thr Asn Tyr

1 5

<210> 81

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> NKG2D scFv-4 CDR-H2

<400> 81

Tyr Pro Gly Asp Ser Asp

1 5

<210> 82

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> NKG2D scFv-4 CDR-H3

<400> 82

Leu Thr Met Phe Arg Gly Ile Ile Ile Gly Tyr Phe Asp Tyr

1 5 10

<210> 83

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-1 CDR-L1

<400> 83

Arg Val Ser Glu Asn Ile Tyr Ser Tyr Leu Ala

1 5 10

<210> 84

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-1 CDR-L2

<400> 84

Asn Ala Lys Thr Leu Ala Glu

1 5

<210> 85

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-1 CDR-L3

<400> 85

Gln His His Tyr Gly Thr Pro Trp Thr

1 5

<210> 86

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-1 CDR-H1

<400> 86

Gly Tyr Ser Ile Thr Ser Asp Tyr

1 5

<210> 87

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-1 CDR-H2

<400> 87

Thr Tyr Ser Gly Ser

1 5

<210> 88

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-1 CDR-H3

<400> 88

Gly Gly Tyr Tyr Gly Ser Ser Trp Gly Val Phe Ala Tyr

1 5 10

<210> 89

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-2 CDR-L1

<400> 89

Arg Ala Ser Gln Ser Ile Ser Asp Tyr Leu His

1 5 10

<210> 90

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-2 CDR-L2

<400> 90

Tyr Ala Ser Gln Ser Ile Ser

1 5

<210> 91

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-2 CDR-L3

<400> 91

Gln Asn Gly His Ser Phe Pro Leu Thr

1 5

<210> 92

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-2 CDR-H1

<400> 92

Gly Tyr Thr Phe Thr Glu Tyr

1 5

<210> 93

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-2 CDR-H2

<400> 93

Ser Pro Asn Ile Gly Gly

1 5

<210> 94

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-2 CDR-H3

<400> 94

Arg Gly Gly Ser Phe Asp Tyr

1 5

<210> 95

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-3 CDR-L1

<400> 95

Arg Ala Ser Glu Asn Ile Tyr Ser Asn Leu Ala

1 5 10

<210> 96

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-3 CDR-L2

<400> 96

Ala Ala Thr Asn Leu Ala Asp

1 5

<210> 97

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-3 CDR-L3

<400> 97

Gln His Phe Trp Gly Thr Pro Arg Thr

1 5

<210> 98

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-3 CDR-H1

<400> 98

Gly Tyr Thr Phe Thr Ser Phe

1 5

<210> 99

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-3 CDR-H2

<400> 99

Asn Pro Ser Ser Gly Tyr

1 5

<210> 100

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-3 CDR-H3

<400> 100

Gly Ser Ser Arg Gly Phe Asp Tyr

1 5

<210> 101

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-4 CDR-L1

<400> 101

Arg Thr Ser Glu Asn Ile Tyr Ser Tyr Leu Ala

1 5 10

<210> 102

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-4 CDR-L3

<400> 102

Gln His His Tyr Asp Thr Pro Leu Thr

1 5

<210> 103

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-4 CDR-H1

<400> 103

Gly Tyr Ser Ile Thr Ser Asp Tyr

1 5

<210> 104

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-4 CDR-H3

<400> 104

Cys Trp Asp Tyr Ala Leu Tyr Ala Met Asp Cys

1 5 10

<210> 105

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-5 CDR-L1

<400> 105

Arg Ala Ser Gln Asp Ile Gly Ser Ser Leu Asn

1 5 10

<210> 106

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-5 CDR-L2

<400> 106

Ala Thr Ser Arg Leu Asp Ser

1 5

<210> 107

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-5 CDR-L3

<400> 107

Gln His Phe Trp Gly Thr Pro Arg Thr

1 5

<210> 108

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-5 CDR-H1

<400> 108

Gly Tyr Thr Phe Thr Ser Ser

1 5

<210> 109

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-5 CDR-H2

<400> 109

His Pro Asn Ser Gly Ile

1 5

<210> 110

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> NKp46 scFv-5 CDR-H3

<400> 110

Gly Gly Arg Phe Asp Asp

1 5

Claims

1. 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.

2. The chimeric antigen receptor of claim 1, wherein the NK-activating receptor is selected from the NKG2 family, NCR family, KIR-S family, 2B4, DNAM-1, CD2, and LFA-1.

3. 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 NKp 80.

4. The chimeric antigen receptor of claim 3, wherein the NK activating receptor is selected from NKG2D and NKp 46.

5. The chimeric antigen receptor of claim 1, wherein the chimeric antigen receptor comprises an antibody targeting NKG2D or NKp46, the NKG 2D-targeting antibody comprising:

(iv) Respectively shown in SEQ ID NO: 77. 78 and 79, and CDR-L1, CDR-L2 and CDR-L3 as set forth in SEQ ID NOs: 80. CDR-H1, CDR-H2 and CDR-H3 shown in 81 and 82;

the NKp 46-targeting antibody comprises:

6. The chimeric antigen receptor of claim 5, wherein the antibody targeting NKG2D comprises an amino acid sequence identical to SEQ ID NO: 27, 1-121, SEQ ID NO: 29, 1-109, SEQ ID NO: 31, 1-109 or SEQ ID NO: 33, 1-108 and a light chain variable region sequence 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 set forth in SEQ ID NO: 27, position 137-246, SEQ ID NO: position 122-243 of 29, SEQ ID NO: 31 position 122-236 or SEQ ID NO: 33, 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 depicted in position 121-243;

the NKp 46-targeting antibody comprises an amino acid sequence identical to SEQ ID NO: 35, positions 1-107, SEQ ID NO: 37, 1-107, SEQ ID NO: 39, 1-107, SEQ ID NO: 41, 1-107 or SEQ ID NO:43, and a light chain variable region sequence 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 set forth in positions 1-107 of SEQ ID NO: position 123-244 of 35, SEQ ID NO: position 123-238 of 37, SEQ ID NO: position 123-238 of SEQ ID NO: position 123-242 of 41 or SEQ ID NO:43, 123-237, having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity.

7. The chimeric antigen receptor of any one of claims 1-6, wherein the antigen binding region is selected from the group consisting of an immunoglobulin molecule, Fab ', F (ab')2, Fv fragment, scFv, disulfide-linked Fv (sdFv), heavy chain variable region (VH) or light chain variable region (VL) of an antibody, Fd fragment consisting of VH and CH1 domains, a linear antibody, a single domain antibody, a nanobody, and a natural ligand for the antigen or a functional fragment thereof.

8. 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: TCR α chain, TCR β chain, TCR γ chain, TCR chain, CD3 ζ subunit, CD3 subunit, CD3 γ subunit, CD3 subunit, CD45, CD4, CD5, CD8 α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, and CD 154.

9. 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: FcR γ, FcR β, CD3 γ, CD3, CD3, CD3 ζ, CD22, CD79a, CD79b, and CD66 d. Preferably, the intracellular signaling domain is a signaling domain comprising CD3 ζ.

10. 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 regions of a protein 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, zaslp 76, PD-1, LIGHT, TRIM, CD94, LTB, p70, and combinations thereof.

11. The chimeric antigen receptor of claim 10, wherein the co-stimulatory domain comprises one or more intracellular regions of a protein selected from the group consisting of: DAP10, DAP12, CD27, CD28, CD134, 4-1BB, or CD 278.

12. 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, TnAg, 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, ERBB 20 (Her 20/neu), MUC 20, EGFR, CS 20, CD138, NCAM, Claudin18.2, Prostase, BCPAP, Nyhrf 2, Nyhrin 20, Epsilon 20, EPTC-20, EPTC-72, EPTC-20, EPTC-20, EPTC-20, EPTC-20, EPTC, LY6, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPV E6, E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostate specific protein, survivin and telomerase, PCTA-l/Galectin 8, MelanA/MARTl, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2ETS fusion gene), NA17, PAX3, Blandrogen receptor, Cyclin, MYCN, RhoC, TRP-2, BO 1B 5, RIS, SART3, PAX5, OY-TES1, AKAP 1, LAAK 24-RAKE 639, LACK 1, LAR-IRV-599, RAKE 6379, CD 599, RU-IRU 2, RAKE 639, CD 599, RU-LR 2, RAKE 639, CD 599, LR2, and LR2, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGF β, APRIL, NKG2D, and any combination thereof.

13. A nucleic acid molecule encoding the chimeric antigen receptor of any one of claims 1-12.

14. A vector comprising the nucleic acid molecule of claim 13.

15. 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 that targets a tumor antigen.

17. The engineered immune cell of any one of claims 15-16, further comprising at least one gene whose expression is inhibited or silenced selected from the group consisting of: TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA, PD1, LAG3, TIM3, CTLA 4.

18. The engineered immune cell of any one of claims 14-16, wherein expression of a corresponding endogenous NK-activating receptor targeted by the chimeric antigen receptor is inhibited or silenced.

19. The engineered immune cell of claim 18, wherein the NK-activating receptor is NKG2D or NKp 46.

20. The engineered immune cell of any one of claims 15-19, wherein the engineered immune cell is selected from a T cell, a macrophage, a dendritic cell, a monocyte, an NK cell, or an NKT cell.

21. The engineered immune cell of any one of claims 15-20, wherein the immune cell is derived from a stem cell.

22. A pharmaceutical composition comprising the chimeric antigen receptor of any one of claims 1-12, the nucleic acid molecule of claim 13, the vector of claim 14, or the engineered immune cell of any one of claims 15-21, and one or more pharmaceutically acceptable excipients.

23. Use of 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 the pharmaceutical composition of claim 22 in the preparation of a medicament for treating cancer, an infection, or an autoimmune disease.