Chimeric receptor complex based on transmembrane structure of nkg2d

Abstract

Provided are a chimeric receptor complex, a nucleic acid molecule encoding and capable of expressing the complex, a vector comprising the nucleic acid molecule, engineered T cells and engineered natural killer cells expressing the chimeric receptor complex, a method for producing such engineered cells, a cell population obtained by the method comprising the engineered cells, and a pharmaceutical composition or kit comprising the chimeric receptor complex, the nucleic acid molecule, the vector, the engineered cells, and the cell population, as well as a method for using the pharmaceutical composition or kit for treating diseases or disorders.

Description

Chimeric receptor complex based on NKG2D transmembrane structure Technical Field

[0001] This invention provides a chimeric receptor complex based on an NKG2D transmembrane structure, a nucleic acid molecule encoding and capable of expressing the complex, a vector containing the nucleic acid molecule, engineered T cells and engineered natural killer cells expressing the chimeric receptor complex, a method for producing such engineered cells, a cell population containing the engineered cells obtained by the method, a pharmaceutical composition or kit containing the chimeric receptor complex based on the NKG2D transmembrane structure, the nucleic acid molecule, the vector, the engineered cells, and the cell population, and a method for using the pharmaceutical composition or kit to treat a disease or condition. Background Technology

[0002] Adoptive cell immunotherapy, represented by chimeric antigen receptor (CAR) T cells, has become one of the most promising therapeutic technologies for treating malignant tumors. CAR design involves sequentially tandemly linking extracellular antigen recognition and binding domains, transmembrane domains, and intracellular signal transduction domains. After binding to the target antigen, these receptors continuously activate T cells, potentially leading to T cell exhaustion and triggering a severe cytokine storm.

[0003] NKG2D is a type II transmembrane-anchored activating / co-stimulatory immune receptor belonging to the CD94 / NKG2 family of type C lectin-like receptors (Houchins et al., 1991). It recognizes and binds to a wide range of structurally diverse ligands, including MHC chain-associated molecules A and B (MICA and MICB) and six cytomegalovirus UL16-binding protein families (ULBP1-6). NKG2D ligands are widely expressed in virus-infected and malignantly transformed cells, but are either absent or poorly expressed in normal healthy cells (Eagle and Trowsdale, 2007; El Gazzar et al., 2013). Therefore, NKG2D has attracted widespread attention as a broad-spectrum tumor target. To date, reported NKG2D receptor-based CAR structures utilize the extracellular domain of the NKG2D receptor directly tandem with the transmembrane structure and intracellular activation domain of the CAR, serving as targeting elements capable of targeting various tumor cells.

[0004] Because the natural NKG2D molecule exists with its N-terminus located on the cytoplasmic side, opposite to the orientation of currently used CAR molecule platforms, using the NKG2D transmembrane domain in existing CAR molecules would alter the conformation and function of other components. Furthermore, the cytoplasmic domain of NKG2D is known to lack signal transduction motifs, relying solely on polymerization with transmembrane signal transduction adaptor proteins DAP10 or DAP12 to transmit signals. These factors, along with the inherent instability of the NKG2D protein, pose challenges when using the NKG2D transmembrane domain in CARs to mediate the activation of immune cells, particularly T cells. For example, Yuxiao Wang and his team reported constructing a chimeric antigen receptor using HER2 scFv and the NKG2D transmembrane region, but failed to detect the expression of the corresponding receptor molecule on the surface of transfected NK cells. WO2015142661A1 reported a modulated chimeric antigen receptor containing a component of natural killer cell receptor (RNKR-CAR), but it required the use of a chemically dependent dimerization switch to achieve the polymerization of RNKR-CAR with a co-stimulatory signaling element, and ultimately did not construct or express any CAR molecule containing the NKG2D transmembrane region. Summary of the Invention

[0005] Through in-depth research, the inventors of this application have surprisingly discovered that, compared to traditional CARs (scFv-TM-co-stimulatory domain-CD3ζ CARs), novel chimeric receptor complexes designed based on the NKG2D transmembrane structure and adapter conduction structure, when expressed on T cells, enable engineered T cells to exhibit higher sensitivity and stronger cytotoxicity against tumor cells. Therefore, this type of novel chimeric receptor complex can be applied to cell therapy to directly treat a variety of diseases and conditions, including tumors.

[0006] Therefore, a first objective of the present invention is to provide a novel chimeric receptor complex (CRC) comprising a chimeric receptor molecule and a chimeric adapter molecule, wherein the chimeric receptor molecule comprises an extracellular binding element and a transmembrane element based on an NKG2D transmembrane domain, the extracellular binding element comprising at least one antibody, receptor, ligand, or antigen-binding fragment thereof that specifically recognizes and binds to a target antigen; and the chimeric adapter molecule comprises a DAP10 transmembrane structure or a DAP12 transmembrane structure and comprises at least one intracellular signal transduction element.

[0007] In some embodiments, the chimeric receptor molecule does not contain an intracellular signaling element and a co-stimulatory element. In some embodiments, the chimeric receptor molecule contains at least one intracellular signaling element and / or at least one co-stimulatory element.

[0008] In some embodiments, the chimeric receptor molecule disclosed herein does not contain some or all of the extracellular domain of the NKG2D receptor. In some embodiments, the chimeric receptor molecule does not contain amino acid residues 99 to 216 of the human NKG2D receptor (SEQ ID NO:23). In some embodiments, the chimeric NKG2D receptor of this application does not contain amino acid residues 115 to 232 of the mouse NKG2D receptor (SEQ ID NO:28).

[0009] In some embodiments, the extracellular binding element of the chimeric receptor molecule disclosed herein is attached to the C-terminus of the transmembrane element. In some embodiments, the extracellular binding element of the chimeric receptor molecule disclosed herein is attached to the N-terminus of the transmembrane element. In some embodiments, the extracellular binding element of the chimeric receptor molecule disclosed herein is directly linked to the C-terminus of the transmembrane element via a covalent bond. In some embodiments, the extracellular binding element of the chimeric receptor molecule disclosed herein is indirectly linked to the C-terminus of the transmembrane element via a linker or spacer sequence. In some embodiments, the extracellular binding element of the chimeric receptor molecule disclosed herein is directly linked to the N-terminus of the transmembrane element via a covalent bond. In some embodiments, the extracellular binding element of the chimeric receptor molecule disclosed herein is indirectly linked to the N-terminus of the transmembrane element via a hinge region. In a preferred embodiment, the hinge region has the amino acid sequence shown in SEQ ID NO:26, 30, 91, 92, or 94. In a more preferred embodiment, the hinge region has the amino acid sequence shown in SEQ ID NO:26. In some embodiments, the hinge region has an amino acid sequence that is at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence shown in SEQ ID NO:26, 30, 91, 92, or 94, or has an amino acid sequence that has one or more (e.g., 1, 2, 3, 4, or 5) amino acid deletions, substitutions, or insertions compared to the amino acid sequence shown in SEQ ID NO:26, 30, 91, 92, or 94.

[0010] In some embodiments, the antigen-binding fragment as an extracellular binding element is selected from any one or any combination of diabody, Fab, Fab', F(ab')2, Fd, Fv, disulfide-stabilized Fv (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody, single-chain antibody (scFv), scFv dimer (divalent diabody), multispecific antibody, heavy-chain-only antibody, single-domain antibody (such as camel-derived single-domain antibody), or nanobody. In preferred embodiments, the antigen-binding fragment as an extracellular binding element is selected from any one or any combination of single-chain antibody (scFv), single-domain antibody (such as camel-derived single-domain antibody), or nanobody. In some preferred embodiments, the antigen-binding fragment as an extracellular binding element is a single-chain antibody (scFv). In some preferred embodiments, the antigen-binding fragment as an extracellular binding element is a single-domain antibody or nanobody. In some preferred embodiments, the ligand serving as the extracellular binding element is a natural ligand. In some preferred embodiments, the ligand may be selected from any one or a combination of CD256, BAFF, IL5, or TIGIT. In some embodiments, the receptor serving as the extracellular binding element is the extracellular binding domain of a cell surface receptor. In some embodiments, the receptor serving as the extracellular binding element is not the extracellular domain of NKG2D.

[0011] In some preferred embodiments, each chimeric receptor molecule may contain one, two, three, or four antibodies, receptors, ligands, or their antigen-binding fragments (e.g., scFv, single-domain antibodies, nanobodies, extracellular binding domains, receptor affinity domains) in its extracellular binding element, wherein each antibody, receptor, ligand, or their antigen-binding fragment may bind to a target antigen. In some embodiments, two or more antibodies, receptors, ligands, or their antigen-binding fragments may bind to different antigens. In some embodiments, two or more antibodies, receptors, ligands, or their antigen-binding fragments may bind to the same antigen, such as the same or different epitopes on the same antigen. In some embodiments, two or more antibodies, receptors, ligands, or their antigen-binding fragments may optionally be linked by a linker or spacer sequence. In a preferred embodiment, the linker or spacer sequence is a flexible linker or flexible spacer sequence. In a more preferred embodiment, the linker or spacer sequence is a GS-type linker, such as a (Gly4Ser)3 linker or a (Gly3Ser2)3 linker.

[0012] In some embodiments, the target antigens disclosed herein are tumor-associated antigens. In preferred embodiments, the tumor-associated antigens are selected from CD19, CD20, CD22, BCMA, BAFF-R, ROR1, GPRC5D, CD38, CD40, CD33, CD123, CD125, CLL1, CD70, CD137, CTLA-4, PD-1, TIGIT, OX40, α4β7, CD5, CD7, CD90, CD276, CD22, CD23, CD25, CD134, LEY, CD47, CD79b, IL-4R, CD96, CD44v6, CD56, B7H3, B7H4, B7H6, and GPC. 3. Any one or any combination thereof from the group consisting of GPC2, CEA, AFP, FORL1, MSLN, CD133, Trop2, SIRPα, Epcam, EphA2, PSMA, uPAR, 5T4, HLA-G, HLA-E, MUC1, FAPα, CAIX, TAG72, OR2H1, Claudin6, Claudin18.2, GD2, HER2, HER3, L13Rα2, EGFR, EGFRvIII, VEGFR-1, VEGFR-2, αvβ3, αvβ6, DLL2, DLL3, and ROBO1. In a more preferred embodiment, the tumor-associated antigen is CD125.

[0013] In some embodiments, the extracellular antigen-binding element in the chimeric receptor molecule disclosed herein is a single-chain antibody (scFv) that specifically recognizes and binds to CD125. In a preferred embodiment, the scFv has the amino acid sequence shown in SEQ ID NO:9, or has an amino acid sequence having at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with the amino acid sequence shown in SEQ ID NO:9, or has an amino acid sequence with one or more amino acid deletions, substitutions, or insertions compared to the amino acid sequence shown in SEQ ID NO:9. In a preferred embodiment, the substitutions are conservative substitutions. In a preferred embodiment, the scFv has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid deletions, substitutions, or insertions compared to the amino acid sequence shown in SEQ ID NO:9.

[0014] In some embodiments, the transmembrane element of the chimeric receptor molecule disclosed herein comprises an NKG2D transmembrane domain rich in acidic amino acids. In some embodiments, the transmembrane element comprises an NKG2D transmembrane domain rich in lysine and / or aspartic acid. In a preferred embodiment, the NKG2D transmembrane domain is a human NKG2D transmembrane domain. In another preferred embodiment, the NKG2D transmembrane domain is a mouse NKG2D transmembrane domain. In a more preferred embodiment, the human NKG2D transmembrane domain has the amino acid sequence shown in SEQ ID NO:19. In other embodiments, the human NKG2D transmembrane domain has an amino acid sequence having at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with the amino acid sequence shown in SEQ ID NO:19. In other embodiments, the human NKG2D transmembrane domain has an amino acid sequence with one or more amino acid deletions, substitutions, or insertions compared to the amino acid sequence shown in SEQ ID NO:19. In some embodiments, the human NKG2D transmembrane domain has an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids deleted, substituted, or inserted compared to the amino acid sequence shown in SEQ ID NO: 19. In another preferred embodiment, the mouse NKG2D transmembrane domain has the amino acid sequence shown in SEQ ID NO: 21. In other embodiments, the mouse NKG2D transmembrane domain has an amino acid sequence that is at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence shown in SEQ ID NO: 21. In other embodiments, the mouse NKG2D transmembrane domain has an amino acid sequence with one or more amino acids deleted, substituted, or inserted compared to the amino acid sequence shown in SEQ ID NO: 21. In some embodiments, the mouse NKG2D transmembrane domain has an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids missing, substituted or inserted compared to the amino acid sequence shown in SEQ ID NO:21.

[0015] The complexes disclosed herein are formed by the polymerization of chimeric receptor molecules and chimeric adapter molecules. In some embodiments, the polymerization of the chimeric receptor and chimeric adapter molecules is achieved through non-covalent interactions between transmembrane elements and DAP10 or DAP12 transmembrane structures. In some embodiments, the polymerization of the chimeric receptor and chimeric adapter molecules is achieved through electrostatic interactions between transmembrane elements and DAP10 or DAP12 transmembrane structures. In some embodiments, the non-covalent or electrostatic interactions primarily occur between the NKG2D transmembrane domain and the DAP10 or DAP12 transmembrane structure. In some embodiments, the polymerization of the transmembrane element with the DAP10 or DAP12 transmembrane structure results in the generation and / or increase of intracellular signal transduction by the intracellular signal transduction elements of the complex disclosed herein. In some embodiments, the signal generated by the intracellular signal transduction elements is an activation signal.

[0016] In some embodiments, the chimeric acceptor molecule of this application does not contain elements outside the transmembrane element that can form complexes (such as polymerization) with the chimeric adapter molecule or its contained elements. In some embodiments, the chimeric adapter molecule of this application does not contain elements outside the DAP10 transmembrane structure or the DAP12 transmembrane structure that can form complexes (such as polymerization) with the chimeric acceptor molecule or its contained elements. For example, the first switching domain, the second switching domain, and the dimerization switch described in WO2015142661A1.

[0017] In some embodiments, the DAP10 transmembrane structure has an amino acid sequence as shown in SEQ ID NO:32 or 40, or has an amino acid sequence that is at least about 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the amino acid sequence shown in SEQ ID NO:32 or 40, or has an amino acid sequence that has one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid deletions, substitutions or insertions compared to the amino acid sequence shown in SEQ ID NO:32 or 40. In other embodiments, the DAP12 transmembrane structure has an amino acid sequence as shown in SEQ ID NO:36 or 44, or has an amino acid sequence that is at least about 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the amino acid sequence shown in SEQ ID NO:36 or 44, or has an amino acid sequence that has one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid deletions, substitutions or insertions compared to the amino acid sequence shown in SEQ ID NO:36 or 44.

[0018] In some embodiments, the chimeric adapter molecule includes DAP10 and at least one intracellular signaling element. In some embodiments, the chimeric adapter molecule includes DAP12 and at least one intracellular signaling element. In some embodiments, DAP10 or DAP12 is human DAP10 or human DAP12.

[0019] In a preferred embodiment, the chimeric adapter molecule has an amino acid sequence selected from that shown in SEQ ID NO:56, or has an amino acid sequence that is at least about 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the amino acid sequence shown in SEQ ID NO:56, or has an amino acid sequence that has one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid deletions, substitutions or insertions compared to the amino acid sequence shown in SEQ ID NO:56.

[0020] In some embodiments, the chimeric receptor complexes disclosed herein comprise a chimeric receptor molecule based on the human NKG2D transmembrane domain and a chimeric adapter molecule based on the human DAP10 transmembrane structure. In other embodiments, the chimeric receptor complexes disclosed herein comprise a chimeric receptor molecule based on the mouse NKG2D transmembrane domain and a chimeric adapter molecule based on the mouse DAP10 transmembrane structure. In still other embodiments, the chimeric receptor complexes disclosed herein comprise a chimeric receptor molecule based on the human NKG2D transmembrane domain and a chimeric adapter molecule based on the mouse DAP10 transmembrane structure. In yet another embodiment, the chimeric receptor complexes disclosed herein comprise a chimeric receptor molecule based on the mouse NKG2D transmembrane domain and a chimeric adapter molecule based on the human DAP10 transmembrane structure. In some embodiments, the chimeric receptor complexes disclosed herein comprise a chimeric receptor molecule based on the human NKG2D transmembrane domain and a chimeric adapter molecule based on the human DAP12 transmembrane structure. In other embodiments, the chimeric receptor complex disclosed herein comprises a chimeric receptor molecule based on the mouse NKG2D transmembrane domain and a chimeric adapter molecule based on the mouse DAP12 transmembrane structure. In other embodiments, the chimeric receptor complex disclosed herein comprises a chimeric receptor molecule based on the human NKG2D transmembrane domain and a chimeric adapter molecule based on the mouse DAP12 transmembrane structure. In other embodiments, the chimeric receptor complex disclosed herein comprises a chimeric receptor molecule based on the mouse NKG2D transmembrane domain and a chimeric adapter molecule based on the human DAP12 transmembrane structure.

[0021] In a preferred embodiment, the chimeric adapter molecule disclosed herein comprises one intracellular signaling element, such as an intracellular signaling domain selected from TCRζ (CD3ζ). In a more preferred embodiment, the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:48, or comprises an amino acid sequence having at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with the amino acid sequence shown in SEQ ID NO:48, or comprises an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid deletions, substitutions, or insertions compared to the amino acid sequence shown in SEQ ID NO:48. In some embodiments, the intracellular signaling element comprises two or more identical or different intracellular signaling domains, wherein at least one intracellular signaling domain comprises an ITAM motif. In some embodiments, the chimeric adapter molecules disclosed herein simultaneously include an intracellular signal transduction domain of DAP10 or DAP12 (e.g., the ITAM motif) and at least one other intracellular signal transduction domain derived from CD27, ICOS, CD134, Lck, ZAP-70, PKC, LAT, SLP-76, TCRζ (CD3ζ), FcRγ (FCER1G), FcRβ (FCER1B), CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS"), FcεRI, CD32 (FcγRIIa), CD66d, CD28, 4-1BB, or OX40, preferably an intracellular signal transduction domain of TCRζ (CD3ζ).

[0022] In some embodiments, the chimeric receptor molecules disclosed herein do not contain a co-stimulatory element. In some embodiments, the chimeric receptor molecules disclosed herein contain at least one co-stimulatory element.

[0023] In one embodiment, the chimeric adapter molecule disclosed herein includes a DAP10 transmembrane domain and at least one intracellular signal transduction element. In one embodiment, the chimeric adapter molecule disclosed herein includes a DAP12 transmembrane domain and at least one intracellular signal transduction element. In one embodiment, the chimeric adapter molecule disclosed herein includes a DAP10 transmembrane domain, at least one intracellular signal transduction element, and at least one co-stimulatory element. In one embodiment, the chimeric adapter molecule disclosed herein includes a DAP12 transmembrane domain, at least one intracellular signal transduction element, and at least one co-stimulatory element.

[0024] In some embodiments, the intracellular signaling elements disclosed herein comprise the ITAM motif. In a preferred embodiment, the intracellular signaling element comprises an intracellular signaling domain selected from CD27, ICOS, CD134, Lck, ZAP-70, PKC, LAT, SLP-76, TCRζ (CD3ζ), FcRγ (FCER1G), FcRβ (FCER1B), CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS"), FcεRI, CD32 (FcγRIIa), CD66d, CD28, 4-1BB, OX40, DAP10, or DAP12, or a combination thereof.

[0025] In some embodiments, the co-stimulatory element comprises a selection from CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, CD278 (ICOS), ICAM-1, LFA1 (CD11a / CD18), CD2, CD7LIGHT, NKG2C, B7-H3, GITR, BAFFR, HVEM (LIGHTR), SLAMf7, NKP80 (KLRF1), CD160 (BY35), CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, C49f, ITGAD, CD11d, ITGAE, and CD103. Co-stimulatory domains of LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGR2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (C244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A), SLAM (CD150), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, PAG / Cbp, SLP-76, NKP44, NKP30, and NKP46. In a preferred embodiment, the co-stimulatory element comprises an amino acid sequence selected from those shown in SEQ ID NO:87 or 89, or an amino acid sequence having at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with the amino acid sequence shown in SEQ ID NO:87 or 89, or an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid deletions, substitutions, or insertions compared to the amino acid sequence shown in SEQ ID NO:87 or 89.

[0026] In some embodiments, the chimeric adapter molecule disclosed herein simultaneously comprises a TCRζ (CD3ζ) intracellular signal transduction domain and a 4-1BB co-stimulatory domain (SEQ ID NO: 89). In other embodiments, the chimeric adapter molecule disclosed herein simultaneously comprises a CD28 domain (SEQ ID NO: 87) and a 4-1BB domain. In some embodiments, the adaptor molecule of this application simultaneously comprises a CD28 domain, a 4-1BB domain, and a CD3ζ domain.

[0027] A second objective of this invention is to provide a nucleic acid molecule encoding the chimeric receptor complex of this application.

[0028] In some embodiments, the nucleic acid molecules disclosed herein comprise (i) sequences encoding a) a chimeric receptor molecule and b) a chimeric adapter molecule, said sequences being located on a single nucleic acid molecule; or (ii) a sequence encoding a) a chimeric receptor molecule being located on a first nucleic acid molecule and a sequence encoding b) a chimeric adapter molecule being located on a second nucleic acid molecule.

[0029] In some embodiments, the nucleic acid molecules disclosed herein also include a sequence encoding the protective element c). In optional embodiments, the sequences encoding a), b), and c) are all located on a single nucleic acid molecule. In other embodiments, the sequences encoding two of a), b), and c) (e.g., a) and b)) are located on a first nucleic acid molecule, while the sequence encoding the third (e.g., c)) is located on a second nucleic acid molecule. In still other embodiments, the sequence encoding a) is located on a first nucleic acid molecule, the sequence encoding b) is located on a second nucleic acid molecule, and the sequence encoding c) is located on a third nucleic acid molecule.

[0030] In some embodiments, the sequence encoding the chimeric receptor molecule disclosed herein is operatively linked to a first regulatory element, and the sequence encoding the chimeric adapter molecule disclosed herein is operatively linked to a second regulatory element. In some embodiments, the sequence encoding a protective element is operatively linked to a third regulatory element. In other embodiments, the sequences encoding the chimeric receptor molecule and the chimeric adapter molecule are operatively linked to the same regulatory element, with a nucleic acid sequence encoding a peptide cleavage site selected from T2A, P2A, E2A, and F2A as a spacer between them.

[0031] In some embodiments, the nucleic acid molecules provided herein are deoxyribonucleic acid (DNA), ribonucleic acid (RNA) (e.g., mRNA, circular RNA, ccRNA), threonucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA having a β-D-ribose configuration, α-LNA having an α-L-ribose configuration (diastereomers of LNA), 2'-amino-LNA with 2'-amino functionalization and 2'-amino-α-LNA with 2'-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA) and / or chimeras and / or combinations thereof.

[0032] A third objective of this application is to provide a vector system comprising one or more vectors encoding a nucleic acid molecule described herein and a regulatory element operatively linked to said nucleic acid molecule.

[0033] In some embodiments, the vectors described herein encode all elements of the chimeric receptor complexes disclosed herein. For example, the chimeric receptor molecule and the chimeric adapter molecule are encoded on a single vector. In other embodiments, the chimeric receptor molecule is encoded on a first vector of a vector system, and the chimeric adapter molecule is encoded on a second vector of the vector system.

[0034] In a preferred embodiment, the vector described herein further encodes a cell-protective element. For example, a chimeric receptor molecule, a chimeric adapter molecule, and a cell-protective element are encoded on a single vector. In other embodiments, the chimeric receptor molecule and the chimeric adapter molecule are encoded on a first vector of the vector system, and the cell-protective element is encoded on a second vector of the vector system. In still other embodiments, the chimeric receptor molecule is encoded on a first vector of the vector system, the chimeric adapter molecule is encoded on a second vector of the vector system, and the cell-protective element is encoded on a third vector of the vector system.

[0035] In some embodiments, the vectors described herein comprise vectors selected from plasmids, nanoplasmids, granules, viral vectors, small circular vectors, RNA vectors, or linear or circular DNA (e.g., transposon DNA) or RNA molecules. In some embodiments, the viral vector is selected from lentiviral vectors, adenoviral vectors, retroviral vectors, adeno-associated virus vectors, or oncolytic virus vectors. In some embodiments, the viral vector is a bicistronic lentiviral vector. In other embodiments, the viral vector is a tricistronic lentiviral vector.

[0036] A fourth objective of this application is to provide liposomes that contain the nucleic acid molecules or carriers disclosed herein. In some embodiments, the nucleic acid molecules contained in the liposomes disclosed herein are mRNA.

[0037] A fifth objective of this application is to provide engineered cells that express the chimeric receptor complex, the nucleic acid molecule, or the vector provided herein.

[0038] In some embodiments, the engineered cells of this application are immune cells, which may be selected from any of T cells, natural killer (NK) cells, B cells, macrophages, monocytes, dendritic cells, neutrophils, or γδT cells. In a preferred embodiment, the engineered cells of this application endogenously express NKG2D. In a preferred embodiment, the engineered cells of this application are selected from T cells and NK cells.

[0039] In a more preferred embodiment, the T cells are selected from CD8. + T cells, CD4 +T cells, cytotoxic T cells, terminal effector T cells, memory T cells, naive T cells, regulatory T cells, natural killer T cells, γ-δ T cells, cytokine-induced killer (CIK) T cells, and tumor-infiltrating lymphocytes. In the most preferred embodiment, the engineered cells of this application are engineered CD8 cells. + T cells.

[0040] In some embodiments, the engineered cells of this application are autologous cells, such as autologous T cells. In some embodiments, the engineered cells of this application are allogeneic cells, such as allogeneic T cells.

[0041] In some embodiments, the engineered cells of this application further comprise a cell protection element capable of specifically reducing the mRNA or protein levels of the NKG2D ligand disclosed herein in the engineered cells. In some embodiments, the cell protection element is capable of specifically knocking down the endogenous NKG2D ligand gene or mRNA transcription in the engineered cells. In some embodiments, the cell protection element is capable of specifically degrading the endogenous NKG2D ligand molecule in the engineered cells. In some embodiments, the cell protection element is a gene editor capable of specifically knocking out or knocking down the endogenous NKG2D ligand gene. In some embodiments, the gene editor is selected from CRISPR, a base editor, a prime editor, TALEN, or a complex of zinc finger proteins. In some embodiments, the cell protection element is an antisense nucleic acid capable of specifically knocking out or knocking down the mRNA transcription of the endogenous NKG2D ligand. In some embodiments, the antisense nucleic acid may be selected from antisense DNA, antisense RNA, or a ribozyme, preferably siRNA or shRNA that specifically knocks out or knocks down the endogenous NKG2D transcription. In some embodiments, the cell protection element is a targeted degradation element that specifically degrades the endogenous NKG2D ligand. In some embodiments, the targeted degradation element may be selected from, for example, PROTAC, molecular glue, AUTOTAC, or targeted degradation molecules based on endoplasmic reticulum-associated degradation mechanisms. In a preferred embodiment, the cytoprotective element is a targeted degradation element comprising an endoplasmic reticulum-associated degradation mechanism protein-binding domain and an NKG2D ligand-targeting domain. For example, the chimeric protein construct for targeted protein degradation disclosed in PCT International Application No. PCT / CN2022 / 075302 is incorporated herein by reference in its entirety. In some embodiments, the cytoprotective element is incorporated into the genome of engineered cells or is stably present in engineered cells. In other embodiments, the cytoprotective element is not incorporated into the genome of engineered cells, but is temporarily expressed in engineered cells, for example, through transient transfection.

[0042] The sixth objective of this application is to provide a method for generating engineered cells as described herein, comprising introducing the nucleic acid molecules disclosed herein into starting cells under in vitro or in vivo conditions.

[0043] In some embodiments, the starting cells are selected from any of T cells, NK cells, B cells, macrophages, monocytes, dendritic cells, or neutrophils. In a preferred embodiment, the T cells may be selected from CD8 cells. + T cells, CD4 + T cells, cytotoxic T cells, terminal effector T cells, memory T cells, naive T cells, regulatory T cells, natural killer T cells, γδ T cells, cytokine-induced killer (CIK) T cells, and tumor-infiltrating lymphocytes. In a more preferred embodiment, the T cells are CD8+ T cells. + T cells.

[0044] The seventh objective of this application is to provide a population of cells that is generated in vitro or in vivo by the methods described herein.

[0045] In some embodiments, the cell populations disclosed herein comprise at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% engineered cells. In preferred embodiments, the cell populations disclosed herein comprise about 50% to about 100% of the engineered cells described herein. In some embodiments, the cell populations disclosed herein comprise substantially pure engineered cells described herein.

[0046] The eighth objective of this application is to provide a pharmaceutical composition or kit comprising (i) the chimeric receptor complex described herein, a nucleic acid molecule, a carrier, engineered cells or cell populations, and (ii) a pharmaceutically acceptable medium.

[0047] In some embodiments, pharmaceutically acceptable media include, but are not limited to, buffers, excipients, stabilizers, or preservatives. In some embodiments, pharmaceutically acceptable media also include cryoprotectants.

[0048] In some embodiments, the pharmaceutical composition or kit may also contain other active ingredients that can be used for cell therapy of a specific disease or condition, said other active ingredients having activities complementary to said cells. In some embodiments, the other active ingredients do not adversely affect the respective activities of the engineered cells described herein. In some embodiments, the other active ingredients may be selected from, for example, chemotherapy agents. In some embodiments, chemotherapy agents may be selected from, for example, asparaginase, busulfan, carboplatin, cisplatin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vincristine, and / or vinblastine.

[0049] A ninth object of the present invention is to provide a method for treating a symptom or disease, comprising administering to the subject a therapeutically effective amount of the engineered cells, cell populations, pharmaceutical compositions or kits provided herein.

[0050] In some implementations, the subjects are humans.

[0051] In some embodiments, the disease is selected from the group consisting of solid tumors, hematologic malignancies, viral infectious diseases, autoimmune diseases, degenerative diseases, and metabolic diseases. In a preferred embodiment, the solid tumor is selected from the group consisting of nervous system tumors, head and neck tumors, thoracic tumors, digestive system tumors, genitourinary system tumors, soft tissue and skin tumors, and bone tumors. In a preferred embodiment, the nervous system tumor is selected from the group consisting of diffuse glioma, diffuse astrocytoma and anaplastic astrocytoma, glioblastoma, oligodendroglioma, oligodendroastrocytoma, pediatric diffuse glioma, other astrocytomas, ependymoma, neuronal and mixed neuronal-glial tumors, medulloblastoma, other embryonal tumors, schwannomas, meningiomas, solitary fibrous tumors, and hemangiopericytoma. In a preferred embodiment, the head and neck tumor is selected from the group consisting of malignant tumors of the nasal cavity and sinuses, nasopharyngeal carcinoma, oral cancer, laryngeal cancer, salivary gland tumors, intracranial tumors, thyroid cancer, and tongue cancer. In a preferred embodiment, the thoracic tumor is selected from any one of the group consisting of lung cancer, esophageal cancer, gastric cardia cancer, breast cancer, and mediastinal tumors. In a preferred embodiment, the digestive system tumor is selected from any one of the group consisting of gastric cancer, colorectal cancer and sigmoid colon and rectal cancer, liver cancer, pancreatic cancer and periampullary cancer, biliary tract cancer, and small intestinal malignant tumors. In a preferred embodiment, the genitourinary system tumor is selected from any one of the group consisting of kidney cancer, prostate cancer, bladder cancer, testicular malignant tumors, penile cancer, cervical cancer, endometrial cancer, and ovarian cancer. In a preferred embodiment, the soft tissue and skin tumor is selected from any one of the group consisting of malignant fibrous histiocytoma, rhabdomyosarcoma, synovial sarcoma, and malignant melanoma of the skin. In a preferred embodiment, the bone tumor is selected from osteosarcoma or Ewing's sarcoma. In a preferred embodiment, the colon cancer is a colonic adenoma. In a preferred embodiment, the breast cancer is triple-negative breast cancer cells. In a preferred embodiment, the liver cancer is hepatocellular carcinoma. In a preferred embodiment, the hematologic malignancy is selected from any one of the group consisting of leukemia, lymphoma (HL), multiple myeloma (MM), and myelodysplastic syndrome (MDS). In a preferred embodiment, the leukemia is selected from any one of the group consisting of B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, and acute myeloid leukemia.

[0052] In other embodiments, the viral infectious disease is selected from the group consisting of respiratory viral diseases, gastrointestinal viral diseases, hepatic viral diseases, skin and mucous membrane viral diseases, ocular viral diseases, central nervous system viral diseases, lymphocytic viral diseases, worm-borne viral diseases, and lentiviral infections. In a preferred embodiment, the respiratory viral disease is selected from the group consisting of infections such as rhinovirus, adenovirus, respiratory syncytial virus, parainfluenza virus, and coronavirus, influenza, and mumps. In a preferred embodiment, the gastrointestinal viral disease is selected from the group consisting of poliomyelitis, Cooksackie virus infection, ECHO virus infection, and viral gastroenteritis. In a preferred embodiment, viral gastroenteritis is selected from the group consisting of rotavirus gastroenteritis, norovirus gastroenteritis, adenovirus gastroenteritis, astrovirus gastroenteritis, coronavirus gastroenteritis, and calicivirus gastroenteritis. In a preferred embodiment, the viral liver disease is selected from the group consisting of hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, Epstein-Barr virus (EBV) hepatitis, and cytomegalovirus (CMV) hepatitis. In a preferred embodiment, the viral skin and mucous membrane disease is selected from the group consisting of measles, rubella, roseola infantum, varicella and herpes zoster, smallpox, herpes simplex virus infection, rabies, and foot-and-mouth disease. In a preferred embodiment, the viral eye disease is selected from the group consisting of epidemic keratoconjunctivitis, follicular conjunctivitis, and herpetic keratoconjunctivitis. In a preferred embodiment, the viral central nervous system disease is selected from the group consisting of epidemic encephalitis B, western equine encephalitis, eastern equine encephalitis, St. Louis encephalitis, Venezuelan equine encephalitis, Murray Valley encephalitis, California encephalitis, tick-borne encephalitis, and lymphocytic choroid plexus meningitis. In a preferred embodiment, the lymphocytic viral disease is selected from any one of the group consisting of infectious mononucleosis, cytomegalovirus infection, and acquired immunodeficiency syndrome. In a preferred embodiment, the worm-borne viral disease is selected from any one of the group consisting of viral hemorrhagic fever, epidemic hemorrhagic fever, yellow fever, Crimean-Congo hemorrhagic fever, Rift Valley fever, Argentine hemorrhagic fever, Bolivian hemorrhagic fever, Lassa fever, Omsk hemorrhagic fever, Marburg disease and Ebola hemorrhagic fever, dengue fever and dengue hemorrhagic fever, West Nile fever, Colorado tick-borne fever, and sandfly fever. In a preferred embodiment, the slow viral infection disease is selected from any one of the group consisting of subacute sclerosing panencephalitis, kuru, progressive multifocal leukoencephalopathy, and subacute spongiform encephalopathy (corticostriatal degeneration).

[0053] In other embodiments, the autoimmune disease is selected from the group consisting of organ-specific autoimmune diseases and systemic autoimmune diseases. In a preferred embodiment, the organ-specific autoimmune disease is selected from the group consisting of chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cirrhosis, multiple cerebral sclerosis, and acute idiopathic polyneuritis. In a preferred embodiment, the systemic autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, thyroid autoimmune disease, and ulcerative colitis.

[0054] In other embodiments, the neurological diseases are selected from any one of the following groups: peripheral nerve diseases, spinal cord diseases, cerebrovascular diseases, infectious diseases of the central nervous system, demyelinating diseases of the central nervous system, movement disorders, epilepsy, headache, neurodegenerative diseases, hereditary diseases of the nervous system, developmental disorders of the nervous system, neuromuscular junction and muscle diseases, autonomic nervous system diseases, nervous system tumors, and paraneoplastic syndromes of the nervous system. In a preferred embodiment, the peripheral nerve diseases are selected from any one of the following groups: trigeminal neuralgia, facial paralysis, hemifacial spasm, vestibular neuronitis, glossopharyngeal neuralgia, mononeuropathy, brachial plexus neuralgia, multiple mononeuropathy, polyneuropathy, acute inflammatory demyelinating polyneuropathy, and chronic inflammatory demyelinating polyneuropathy. In a preferred embodiment, the spinal cord diseases are selected from any one of the following groups: myelitis, compressive myelopathy, subacute combined degeneration of the spinal cord, syringomyelia, spinal vascular disease, and spinal arachnoiditis. In a preferred embodiment, the cerebrovascular disease is selected from any one of the following groups: transient ischemic attack, cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, and intracranial venous thrombosis. In a preferred embodiment, the central nervous system infectious disease is selected from any one of the following groups: meningitis or encephalitis caused by viral, bacterial, fungal, or parasitic infections, and slow viral encephalitis caused by slow viral infection. In a preferred embodiment, the central nervous system demyelinating disease is selected from any one of the following groups: multiple sclerosis, neuromyelitis optica, acute infectious encephalomyelitis, and leukodystrophy. In a preferred embodiment, the movement disorder is selected from any one of the following groups: Parkinson's disease, chorea, Wilson's disease, dystonia, essential tremor, and tardive dyskinesia. In a preferred embodiment, the headache is selected from any one of the following groups: migraine, tension headache, and cluster headache. In a preferred embodiment, the neurodegenerative disease is selected from any one of the following groups: motor neuron disease, Alzheimer's disease, Lewy body dementia, frontotemporal dementia, and multiple system atrophy. In a preferred embodiment, the hereditary neurological disease is selected from any one of the following groups: hereditary ataxia, hereditary spastic paraplegia, peroneal muscular atrophy, neurofibromatosis, tuberous sclerosis, and cerebral facial angiomatosis. In a preferred embodiment, the developmental neurological disorder is selected from any one of the following groups: congenital hydrocephalus, cerebral palsy, basilar impression, and cerebellar tonsillar disease. In a preferred embodiment, the neuromuscular junction and muscle disease are selected from any one of the following groups: myasthenia gravis, periodic paralysis, polymyositis, progressive muscular dystrophy, myotonic myopathy (myotrophic lateral sclerosis, congenital myotonia), and metabolic myopathy (mitochondrial myopathy, lipid storage myopathy, glycogen storage disease).In a preferred embodiment, the autonomic nervous system disease is selected from any one of the following groups: Raynaud's disease, erythromelalgia, hemifacial atrophy, generalized autonomic dysfunction, spontaneous hyperhidrosis, and progressive lipodystrophy. In a preferred embodiment, the nervous system tumor is selected from any one of the following groups: glioma, lymphoma, and meningioma. In a preferred embodiment, the paraneoplastic syndrome is selected from any one of the following groups: paraneoplastic cerebellar degeneration, paraneoplastic encephalomyelitis, subacute necrotizing myelopathy, subacute motor neuron disease, and paraneoplastic sensory neuron disease.

[0055] In other embodiments, metabolic diseases are selected from any one of the following groups: glucose metabolism-related diseases such as diabetes and tumor glucose metabolism; lipid metabolism-related diseases such as tumor lipid metabolism, hyperlipidemia, and non-alcoholic fatty liver disease; gout and atherosclerosis. Attached Figure Description

[0056] Figure 1 shows schematic diagrams of the natural NKG2D / adapter complex (Figure 1A) and the chimeric receptor complex designed based on the extracellular domain and / or transmembrane structure of NKG2D (Figures 1B to 1D).

[0057] Figure 2 is a schematic diagram showing exemplary chimeric receptor complexes with different extracellular binding elements;

[0058] Figure 3 illustrates the tumor-killing activity of an exemplary chimeric receptor complex and a conventional chimeric antigen receptor against multiple myeloma cells (Figure 3A) or B-cell lymphoma cells (Figure 3B), respectively.

[0059] Figure 4 illustrates the tumor-killing activities of an exemplary chimeric receptor complex and a conventional chimeric antigen receptor against chronic eosinophilic leukemia cells and IgE plasmacytoma cells, respectively.

[0060] Figure 5 shows a schematic diagram of a chimeric antigen receptor constructed based on the extracellular domain of the NK cell receptor (Figure 5A) and a chimeric receptor complex constructed based on the transmembrane structure of the NK cell receptor (Figure 5B).

[0061] Figure 6 shows the use of nanobody V HH A schematic diagram of a chimeric receptor complex constructed as an extracellular binding element. Detailed Implementation

[0062] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0063] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. For example, see Sam Brook et al., *Molecular Cloning: A Laboratory Manual*, 4th Edition, Cold Spring Harbor Laboratory Press, 2012; and Ausubel et al., *Current Protocols in Molecular Biology*, Wiley Online Press, updated periodically. Unless otherwise specified, the materials, reagents, and instruments used in the following examples are commercially available. Unless otherwise specified, the quantitative experiments in the following examples are the average values ​​of three replicate experiments. Unless otherwise specified, in the following examples, nucleotide sequences are written in order from the 5' to the 3' end, and amino acid sequences are written from left to right in order from the amino terminus to the carboxyl terminus. In case of discrepancies between the sequences in the instruction manual and the sequence listing, the sequences described in the instruction manual shall prevail.

[0064] definition

[0065] As used in the specification of this invention, the following words and phrases are generally considered to have the meanings set forth below, unless otherwise specified in the context in which they are used.

[0066] As used herein, the terms “comprising” or “including” mean that a sequence, composition, or method comprises the described components or steps, but does not exclude other components or steps. “Mainly composed of”, when used to define a sequence, composition, or method, should exclude any other components or steps that are obviously essential to the composition. Therefore, compositions defined herein as mainly composed of these components will not exclude trace contamination from separation and purification methods and pharmaceutically acceptable carriers such as phosphate-buffered saline, preservatives, etc. “Composed of” should exclude other ingredients used in the application of the compositions of the invention and trace components of the substantial methods. Examples defined by these provisional terms are within the scope of this invention.

[0067] Unless otherwise expressly stated, the singular forms “a / an” and “the” include plural referents. Thus, for example, a reference to “a cell” includes a combination of two or more cells, or an entire culture of cells. Unless expressly stated or obvious from the context, the term “or” is understood to be inclusive as used herein.

[0068] Unless explicitly stated or obvious from the context, as used herein, the terms “about” or “substantially” should be understood as falling within the normal tolerance range in the field, such as within 2 standard deviations of the mean. “About” or “substantially” can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% of the stated value. Unless otherwise obvious from the context, all numerical values ​​provided herein are modified by the term “about”.

[0069] As used herein, the terms “first” and “second” are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Unless otherwise expressly stated, a feature defined as “first” or “second” may explicitly or implicitly include at least one of that feature.

[0070] As used herein, the terms “treatment” and “improvement” are used interchangeably to refer to a method of obtaining a beneficial or desired outcome, including but not limited to therapeutic benefits and / or preventive benefits. A therapeutic benefit means the eradication or reduction of an underlying disease being treated. A therapeutic benefit can be achieved by eradicating or reducing one or more physiological symptoms associated with an underlying disease, thereby resulting in an improvement observed in the patient. For the purposes of this invention, therapeutic benefits include, but are not limited to: symptom reduction, disease severity reduction, delay or slowing of disease progression, improvement or mitigation of a disease state, and remission (whether partial or complete), whether detectable or undetectable. For preventive benefits, the pharmaceutical compositions disclosed herein may also be administered to patients at risk of developing a specific disease or to patients reporting one or more physiological symptoms of a disease, even if the disease has not yet been diagnosed. As used herein, the term “prevention” means a reduction in the frequency of the disease or condition in a treated sample relative to an untreated control sample, or a delay in the onset and / or reduction in the severity of one or more symptoms of a disease or condition relative to an untreated control sample.

[0071] In some respects, the term "treatment" includes both preventative and therapeutic treatment. Generally, treatment is preventative (i.e., protecting the subject from developing the unwanted condition) if administered before the clinical manifestation of an unwanted condition (e.g., a hallmark behavior such as tremor, or the observation of typical pathological molecular markers); and therapeutic (i.e., aiming to reduce, improve, or stabilize the existing unwanted condition or its side effects) if administered after the manifestation of the unwanted condition.

[0072] As used herein, a “therapeutic effective amount” refers to an amount of a specific substance sufficient to enable a subject receiving treatment to achieve the desired effect. For example, this could be an amount necessary to stop or inhibit tumor growth. In one embodiment, a therapeutic effective amount is an amount necessary to eliminate, reduce the size of a tumor, or prevent tumor metastasis, such as an amount that reduces the size and / or volume of a tumor by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even 100% compared to its pre-treatment size / volume / number, and / or reduces the number and / or size / volume of metastases by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even 100%. When administered to a subject, the commonly used dose will reach a concentration in the target tissue (e.g., in a tumor) that has been shown to achieve the desired in vitro effect.

[0073] The term "vector" herein refers to a nucleic acid molecule that can be introduced into a host cell to produce a transformed host cell. A vector may include a nucleic acid sequence capable of replicating within the host cell, such as an origin of replication. A vector may also include one or more optional marker genes and other genetic elements known in the art. In some embodiments, the vector is a viral vector, such as a lentiviral vector.

[0074] As used herein, the terms “individual,” “subject,” “patient,” “host,” “object of need,” or similar expressions refer to any mammal or non-mammal. Mammals include, but are not limited to, cats, other vertebrates such as rodents, humans, and non-human primates such as cattle, horses, dogs, pigs, sheep, goats, giraffes, deer, camels, sheep, rats, mice, hares, and rabbits.

[0075] As used herein, the term "administration" means the provision or administration of a drug, such as the monoclonal antibody, CAR, or CAR-expressing cell of the present invention, to a subject via any effective route. Exemplary routes of administration include, but are not limited to, oral, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, intraprostatic, and intratumoral), sublingual, rectal, percutaneous, intranasal, vaginal, and inhalation.

[0076] As used herein, "operably ligated" means that the first nucleic acid sequence and the second nucleic acid sequence are operably ligated when they have a functional relationship. For example, if a promoter affects the transcription or expression of a coding sequence, then the promoter is operably ligated to the coding sequence. Typically, operably ligated DNA sequences are contiguous and, when it is necessary to ligate two protein-coding regions, are within the same reading frame.

[0077] As used herein, the term "pharmaceutically acceptable medium" refers to various excipients routinely used in the preparation of pharmaceutical compositions, see, for example, Remington: The Science and practice of Pharmacy, edited by The University of the Sciences in Philadelphia, Lippincott, Williams, & Wilkins, 21st Edition. Philadelphia, PA. (2005). These are generally safe and non-toxic, and do not possess biologically or otherwise undesirable properties. In general, the nature of the medium will depend on the specific route of administration employed. For example, parenteral formulations typically contain injectable fluids, including pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, glucose solutions, glycerol, etc., as mediums. For solid compositions in the form of powders, pills, tablets, or capsules, routinely used non-toxic solid excipients may include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral excipients, the pharmaceutical composition to be administered may contain small amounts of non-toxic excipients, such as wetting agents or emulsifiers, preservatives, and pH buffers, such as sodium acetate or sorbitan monolaurate.

[0078] As used herein, the term “substantially identical” means that two or more objects being compared have approximately the same quantity or level of the characteristics being compared, for example, when taking into account the error range of the measurement methods used.

[0079] As used herein, the term "conservative substitution" means that the substitution substantially does not affect or reduce the affinity of the protein (e.g., an anti-CD125 antibody). For example, a monoclonal antibody that specifically binds to CD125 may include up to about one, up to two, up to five, up to ten, or up to fifteen conserved substitutions that specifically bind to the CD125 polypeptide. The term "conservative substitution" also includes the use of substituted amino acids in place of unsubstituted parental amino acids, provided that the antibody specifically binds to CD125. Correspondingly, "non-conservative substitution" is a substitution that reduces the activity or binding ability of a protein to a target antigen.

[0080] Conservative amino acid substitutions are well known to those skilled in the art. The following six groups are examples of amino acids considered to be conserved substitutes for each other:

[0081] 1) Alanine (A), Serine (S), Threonine (T);

[0082] 2) Aspartic acid (D), glutamic acid (E);

[0083] 3) Asparagine (N), glutamine (Q);

[0084] 4) Arginine (R), Lysine (K);

[0085] 5) Isoleucine (I), leucine (L), methionine (M), valine (V); and

[0086] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0087] The term "cell population" generally refers to a group of cells. A cell population can consist of cells with a common phenotype (e.g., co-expression of chimeric receptor complexes and protective elements), or may contain at least a subset of cells with a common phenotype. Cells are considered to have a common phenotype when they are substantially similar or identical in one or more provable characteristics, including but not limited to morphological appearance, the presence, absence, or level of expression of specific cellular components or products (e.g., RNA, proteins, or other markers), activity of certain biochemical pathways, proliferative capacity and / or kinetics, differentiation potential and / or response to differentiation signals, or behavior during in vitro culture (e.g., adhesion, non-adhesion, monolayer growth, proliferation kinetics, etc.). Thus, such provable characteristics can define a cell population or its fractions.

[0088] NKG2D

[0089] NKG2D is a type II transmembrane-anchored activating / co-stimulatory immune receptor belonging to the CD94 / NKG2 family of type C lectin-like receptors (Houchins et al., 1991), which can modulate innate and adoptive immune responses. In humans, NKG2D is normally detected on all NK cells, natural killer T cells (NKT), and CD8+ cells. + It is expressed in T cells and γδT cell subsets (Zhang et al., 2015).

[0090] NKG2D can recognize and bind to a wide range of structurally diverse ligands. For example, human NKG2D recognizes MHC I chain-associated molecules A and B families (MICA and MICB, commonly referred to as MIC) and six cytomegalovirus UL16-binding protein families (ULBP1-6), which are widely expressed in virus-infected and malignantly transformed cells, but are either absent or poorly expressed in normal healthy cells (Eagle and Trousdale, 2007; El Gazzar et al., 2013). NKG2D itself lacks signal transduction motifs in its cytoplasmic domains; therefore, upon binding to its ligands, NKG2D in natural killer (NK) cells activates downstream signal transduction pathways by recruiting other molecules to its vicinity. The 10 kDa DNAX activator protein (DAP10) was the first adapter molecule identified as binding to NKG2D (Wu et al., 1999). Activated NKG2D in the cell membrane consists of a hexamer structure formed by the assembly of one NKG2D homodimer and two DAP10 homodimers (Garrity et al., 2005). NKG2D is also associated with another DNAX activating protein, DAP12 (Lanier et al., 1998).

[0091] The binding adapter molecule depends on the cell type and the NKG2D isoform. In resting mouse NK cells, NKG2D binds only to DAP10; while in activated NK cells, NKG2D can bind to either DAP10 or DAP12 (Diefenbach et al., 2002; Lanier et al., 1998). In human T cells, due to the difference in the transmembrane domain between human and mouse NKG2D and the lack of endogenous DAP12 expression in human T cells, human NKG2D binds only to DAP10 (Rosen et al., 2004). The DAP10 molecule contains a YXXM tyrosine-based motif that recruits the p85 subunit of phosphatidylinositol kinase-3 (P13K) and growth factor receptor-binding protein 2 (Grb2) (Wu et al., 1999). This then facilitates the transduction of intracellular signals in a manner similar to the signaling cascade of T cell co-stimulatory molecules CD28 and ICOS. During NK activation, DAP10 can recruit downstream signaling molecules and enhance TCR-mediated signaling events (Wallin et al., 2001). The DAP12 molecule contains an immune receptor tyrosine-based activation motif (ITAM) that recruits ZAP70 (ζ-chain-associated protein kinase 70) and Syk (spleen tyrosine kinase), directly mediating NK activation, cytokine production, and cytotoxicity (Diefenbach et al., 2002).

[0092] The amino acid sequence of the human NKG2D receptor polypeptide can be obtained from the NCBI database, see accession number NP_031386.2; GI:169234653, NP_001186734.1, etc.

[0093] The extracellular domain of NKG2D, as described herein, refers to the segment located at the C-terminus of the stalk domain in the amino acid sequence of naturally occurring NKG2D, such as amino acid residues 99 to 216 of human NKG2D, as shown in SEQ ID NO:25, or amino acid residues 115 to 232 of mouse NKG2D (SEQ ID NO:28). In some embodiments, the extracellular domain of NKG2D has at least 70%, 80%, 85%, 90%, 95%, or 99% homology to a control sequence (e.g., the extracellular domain of naturally occurring NKG2D). In some embodiments, the residue difference between the extracellular domain of NKG2D and the control sequence (e.g., the extracellular domain of naturally occurring NKG2D) is no more than 15%, 10%, 5%, 2%, or 1%. In some embodiments, the extracellular domain of NKG2D differs from the control sequence (e.g., the extracellular domain of naturally occurring NKG2D) by no more than 5, 4, 3, 2, or 1 residue. In some implementations, the NKG2D extracellular domain is indistinguishable from or 100% homologous to a control sequence (e.g., the naturally occurring NKG2D extracellular domain).

[0094] The NKG2D transmembrane domain herein refers to positions 52 to 72 of the amino acid sequence of naturally occurring human NKG2D, as shown in SEQ ID NO:19, or positions 67 to 89 of the amino acid sequence of mouse NKG2D, as shown in SEQ ID NO:21. In some embodiments, the NKG2D transmembrane domain has at least 70%, 80%, 85%, 90%, 95%, or 99% homology to a control sequence (e.g., a naturally occurring NKG2D transmembrane domain). In some embodiments, the residue difference between the NKG2D transmembrane domain and the control sequence (e.g., a naturally occurring NKG2D transmembrane domain) is no more than 15%, 10%, 5%, 2%, or 1%. In some embodiments, the NKG2D transmembrane domain differs from the control sequence (e.g., a naturally occurring NKG2D transmembrane domain) by no more than 5, 4, 3, 2, or 1 residue. In some implementations, the NKG2D transmembrane domain is indistinguishable from or 100% homologous to a control sequence (e.g., a naturally occurring NKG2D transmembrane domain).

[0095] The intracellular domain of NKG2D herein refers to positions 1 to 51 of the amino acid sequence of naturally occurring human NKG2D, as shown in SEQ ID NO:27, or positions 1 to 66 of the amino acid sequence of mouse NKG2D, as shown in SEQ ID NO:31. In some embodiments, the intracellular domain of NKG2D has at least 70%, 80%, 85%, 90%, 95%, or 99% homology to a control sequence (e.g., the intracellular domain of naturally occurring NKG2D). In some embodiments, the residue difference between the intracellular domain of NKG2D and the control sequence (e.g., the intracellular domain of naturally occurring NKG2D) is no more than 15%, 10%, 5%, 2%, or 1%. In some embodiments, the intracellular domain of NKG2D differs from the control sequence (e.g., the intracellular domain of naturally occurring NKG2D) by no more than 5, 4, 3, 2, or 1 residue. In some implementations, the NKG2D intracellular domain is indistinguishable from or 100% homologous to a control sequence (e.g., the naturally occurring NKG2D intracellular domain).

[0096] Extracellular antigen binding element

[0097] As used herein, the term "extracellular antigen-binding element" refers to a molecule that has an affinity for a target antigen, typically an antigen on target cells such as cancer cells. Exemplary antigen-binding elements comprise polypeptides, such as antibody molecules (which include full-length antibodies and their antigen-binding fragments, such as single-domain antibodies (sdAbs) or single-chain antibodies (scFvs),) or non-antibody scaffolds, such as fibronectin, etc. In some embodiments, the antigen-binding element is a single polypeptide. In other embodiments, the antigen-binding element comprises one, two, or more polypeptides.

[0098] The choice of antigen-binding element can depend on the type and number of ligands or receptors that define the target cell surface. Examples of targets that can serve as ligands or receptors include cell surface markers associated with specific disease states, such as viral diseases, bacterial diseases, parasitic infections, autoimmune diseases, and conditions associated with unwanted cell proliferation, such as cancer. "Tumor antigen" refers to an antigen common to a specific proliferative disease. In some embodiments, tumor antigens include, but are not limited to, CD19, CD20, CD22, BCMA, ROR1, CD38, CD33, CD123, CD125, CLL1, CD70, OX40, α4β7, CD5, CD90, CD276, CD22, CD23, CD25, CD134, LeY, CD47, CD96, CD44v6, CD56, B7H3, B7H6, GPC3, GPC2, CEA, MSLN, CD133, Epcam, EphA2, PSMA, uPAR, 5T4, MUC1, FAPα, CAIX, TAG72, OR2H1, Claudin18.2, GD2, HER2, HER3, L13Rα2, EGFR, EGFRvIII, VEGFR-1, VEGFR-2, αvβ3, αvβ6, DLL2, DLL3, and ROBO1.

[0099] Antigen-binding fragments derived from antibodies

[0100] The term "antibody" in this document refers to a polypeptide ligand containing at least one variable region that specifically recognizes and binds to an epitope of an antigen. Mammalian antibody molecules (also known as immunoglobulins) are typically composed of heavy chains (H) and light chains (L). Based on the heavy chain class, mammalian immunoglobulins are classified into five major types (or isotypes): IgG, IgM, IgA, IgD, and IgE. Other antibody isotypes have been found in animals other than mammals, including IgX, IgY, IgW, and IgNAR. IgX antibodies are present in amphibians. IgY antibodies are primary antibodies produced by birds and reptiles, functionally similar to mammalian IgG and IgE. IgW and IgNAR antibodies are present in cartilaginous fish.

[0101] The amino-terminal domains of the heavy or light chain of an antibody constitute variable regions, referred to as the heavy chain variable region (VH) and the light chain variable region (VL), respectively. Variable regions are typically the most variable parts of an antibody (relative to other antibodies of the same class), containing antigen-binding sites. For conventional four-chain antibodies, both the VH and VL are responsible for binding to the antigen recognized by the antibody. Cameloidea antibodies possess only a single heavy chain variable region called a "VHH." Therefore, the VHH is a special type of VH.

[0102] However, variability is not uniformly distributed across the entire variable region. In fact, within the variable regions of both the light and heavy chains, variability is concentrated in three segments known as hypervariable regions (HVRs), also referred to as complementarity-determining regions (CDRs). The residue sequences in the variable regions outside the HVRs are more conserved, and these regions are called frame regions (FRs). The CDRs are the regions within the variable region primarily responsible for binding to epitopes of the antigen, and are generally considered to define the binding affinity and specificity of the antibody; while the frame regions are mainly used for locating and aligning CDRs in three-dimensional space. Using any of many well-known numbering schemes, including those described below, the amino acid sequence boundaries of a given CDR can be readily determined: the Kabat numbering rule (Kabat et al., Sequences of Proteins of Immunological Interest, USDapartment of Health and Human Services, 1991), the Chothia numbering rule (Chothia and Lesk, J Mol Biol 196:901-917, 1987; Chothia et al., Nature 342:877, 1989; and Al-Lazikani et al., JMB 273,927-948, 1997), and the Paratome CDR (Kunik et al., PLoS Comput Biol 8:el002388, 2012; Kunik et al., Nucleic Acids Res 40 (Web Server)). (Issue: W521-524, 2012) and IMGT numbering rules (ImMunoGeneTics (IMGT) database, see Lefranc, Nucleic Acids Res 29:207-9, 2001). Kabat, Paratome, and IMGT databases are all available online. Among them, Kabat numbering is based on sequence variability and is the most commonly used. However, unless otherwise specified, the numbering of antibody heavy chain residues in this application is determined according to the IMGT numbering rules.

[0103] Mammalian immunoglobulins each possess three CDRs in their light and heavy chains, named L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. Single-domain antibodies contain three CDRs, referred to in this paper as CDR1, CDR2, and CDR3. Taking camel VHH domain antibodies as an example, it is generally believed, based on Kabat numbering, that VHH FR1 contains amino acid residues 1-30, CDR1 contains amino acid residues 31-35, FR2 contains amino acid residues 36-49, CDR2 contains amino acid residues 50-65, FR3 contains amino acid residues 66-94, CDR3 contains amino acid residues 95-102, and FR4 contains amino acid residues 103-113. It should be noted that, as known in the art regarding VH and VHH domains, the total number of amino acid residues in each CDR can vary and may not correspond to the total number of amino acid residues indicated by the Kabat number (i.e., one or more positions according to the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat number). These variations correspond to the shortening or insertion of FR or HVR in the variable region. For example, the heavy chain variable region may include a single amino acid insertion after residue 52 (residue 52a according to Kabat) and an insertion after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). For a given antibody, those skilled in the art can determine the Kabat number of the residue by aligning it to a “standard” Kabat numbered sequence in the sequence homologous region. Similarly, the IMGT number of the residue can also be determined by alignment.

[0104] As used herein, the term "single-domain antibody (abbreviated as sdAb)" refers to an antibody having a single domain (variable domain) capable of specifically binding to an antigen or epitope in the absence of other antibody domains. As non-limiting examples, single-domain antibodies include, for instance, VH domain antibodies, VNAR antibodies, (camelidae) VHH antibodies, and VL domain antibodies. VNAR antibodies are produced by cartilaginous fish such as nurse sharks, wobbegon sharks, spiny dogfish, and bamboo sharks. VHH antibodies are produced by various species of camels, including camels, llamas, alpacas, dromedary camels, and guanacos, which are capable of producing naturally occurring heavy chain antibodies lacking the light chain. A classic VHH antibody, from N-terminus to C-terminus, comprises frame region 1 (FR1), complementarity-determining region 1 (CDR1), frame region 2 (FR2), complementarity-determining region 2 (CDR2), frame region 3 (FR3), complementarity-determining region 3 (CDR3), and frame region 4 (FR4). The overall size of a single “single-domain antibody” is on the order of nanometers, so it can be used interchangeably with “nanobody” in this paper.

[0105] As used herein, the terms "specific binding," "specific recognition," or "specific to" refer to a measurable and reproducible interaction, such as the binding between a target and an antigen-binding protein (e.g., an antibody, receptor, ligand, or its antigen-binding fragment), in the presence of a heterogeneous group of molecules (including biomolecules), where the binding is a decisive factor in the presence of the target. For example, an antigen-binding protein that specifically binds to a target molecule (or epitope) is one that, compared to binding to a non-target molecule, exhibits greater affinity, stronger binding, and / or longer binding duration to the target molecule. In some embodiments, as determined by, for example, radioimmunoassay (RIA), the degree of binding of the antigen-binding protein to a non-target molecule is about 10% less than its binding to the target molecule. In some embodiments, the antigen-binding protein of the present invention binds to the target antigen with a binding affinity of at least about 1 × 10⁻⁶. -6 M, at least approximately 0.5 × 10 -6 M, at least approximately 1×10 -7 M, at least approximately 0.5 × 10 -7 M, at least approximately 1×10 -8 M, at least approximately 0.5 × 10 -8 M, at least approximately 1×10 -9 M, at least approximately 0.5 × 10 -9 M, or at least about 0.1 × 10 -9 M. In some embodiments, the antigen-binding protein of the present invention (e.g., anti-CD125 scFv) specifically binds to the target molecule (e.g., CD125), and the binding constant is at least 10³ M⁻¹, 10⁴ M⁻¹, or 10⁵ M⁻¹ greater than the binding constant of other molecules in the sample or subject. In some embodiments, the dissociation constant (Kd) of the complex formed after the antigen-binding protein of the present invention specifically binds to the target is ≤1000 nM, ≤750 nM, ≤500 nM, ≤250 nM, ≤100 nM, ≤50 nM, ≤25 nM, ≤10 nM, ≤5 nM, ≤2.5 nM, ≤1 nM, ≤0.5 nM, ≤0.25 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 nM or less, e.g., from 10). -5 M to 10 -10 M, for example, 10 -6 M to 10 -9Methods for determining binding affinity are known in the art, and any of them may be used for the purposes of this application. In some embodiments, instruments capable of measuring protein-protein interactions (such as the Octet RED384 protein interaction instrument) are used to determine parameters characterizing the strength of binding and dissociation. In some embodiments, binding affinity is measured using the Octet system (Creative Biolabs) based on biolayer interferometry (BLI) technology. In some embodiments, Kd is measured using surface plasmon resonance assays with a BIACORES-2000 or BIACORES-3000 (BIAcore, Inc., Piscataway, NJ). In some embodiments, specific binding may include, but is not required to be, exclusive binding.

[0106] As used herein, the term "valence" refers to the number of binding sites in an antigen-binding protein. For example, a natural tetrachain antibody has two binding sites and is divalent. Therefore, the terms "trivalent," "tetravalent," "pentavalent," and "hexavalent" indicate that an antigen-binding protein has two, three, four, five, and six binding sites, respectively.

[0107] A "humanized" antibody is an immunoglobulin comprising a human framework region and one or more core receptor (CDRs) of a non-human (e.g., mouse, rabbit, rat, shark, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDR is called the "donor," and the human immunoglobulin providing the framework is called the "receptor." In one embodiment, all CDRs are derived from the donor immunoglobulin. Constant regions are not necessarily present, but if present, they must be substantially identical to the constant regions of human immunoglobulins, i.e., at least about 85–90% identical, for example, about 95% or higher. Therefore, all portions of the humanized immunoglobulin, except for the CDRs, are substantially identical to the corresponding portions of the native human immunoglobulin sequence. The humanized antibody binds to the same determinants as the donor antibody that provides the CDR. Furthermore, the humanized antibody may contain residues not present in the receptor antibody or the donor antibody. These modifications (e.g., substitution of one or more FR residues) can further improve antibody properties, such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FR is typically no more than six in the H chain. In some cases, the number of amino acid substitutions in the FR of the H chain is 6, 5, 4, 3, 2, or 1.

[0108] As used in this article, the term "sequence identity" measures the degree of similarity between amino acid or nucleic acid sequences, usually expressed as a percentage of identity. The higher the percentage, the more similar the two sequences are. "Identity" is the percentage of identical matching positions between two or more sequences in a gap (if any) alignment processed by a specific mathematical model or computer program (such as an algorithm). The methods and computer programs used for alignment are well known in the field, such as the common BLAST suite (Stephen F. Altschul, et al. (1997), Nucleic Acids Res. 25:3389-3402), local alignment methods based on the Smith-Waterman algorithm (Smith, TF & Waterman, MS (1981) J. Mol. Biol. 147:195-197), and the Needleman-Wunsch algorithm (Needleman, SB & Wunsch, CD (1970) J. Mol. Biol. 48:443-453), which is a general global alignment method based on dynamic programming. It is understood that identity depends on the calculation of the identity percentage, but this value may vary due to gaps and penalties introduced in the calculation.

[0109] The NCBI Basic Local Alignment Search Tool (BLAST) is a commonly used tool for sequence alignment and identity calculation, available from multiple sources, such as the National Center for Biotechnology Information (NCBI, Bethesda, Md.). Depending on the sequence type and intended use, matching analysis programs such as blastp, blastn, blastx, tblastn, and tblastx can be selected. Instructions for using this program to determine sequence identity are available free of charge from the NCBI website. For comparisons of amino acid sequences longer than approximately 30 amino acids, the Blast2 sequence function can be used with the default BLOSUM62 matrix (vacancy presence cost of 11, cost per residue vacancy of 1) set to default parameters. When aligning short peptides (less than approximately 30 amino acids), the Blast2 sequence function is used with the PAM30 matrix set to default parameters (generating a vacancy penalty of 9, an extension vacancy penalty of 1). When evaluated using this method, proteins with higher similarity to a reference sequence will show an increased percentage of identity, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When comparing sequence identity less than the entire sequence, homologs and variants typically have at least 80% sequence identity within a short window of 10–20 amino acids, and may have at least 85%, at least 90%, or at least 95% sequence identity, depending on their similarity to the reference sequence. Methods for determining sequence identity within a short window are available on the NCBI website. Those skilled in the art will understand that these sequence identity ranges are provided for guidance only; it is entirely possible to obtain highly significant homologs beyond the provided ranges.

[0110] As used herein, the term "scFv" refers to a fusion protein comprising an antibody VL chain and a VH chain, wherein the VH and VL are linked, for example, by a short, flexible peptide linker (e.g., a short peptide linker). scFv can be expressed as a single-chain polypeptide and retains the specificity of the complete antibody from which it originates. The VL and VH variable chains can be linked in any order; for example, scFv may contain VL-linker-VH or VH-linker-VL. Methods for preparing scFv are known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. 1998, 1997. 85:5879-5883).

[0111] In some embodiments, the antigen-binding element is a single-domain antigen-binding (SDAB) molecule. An SDAB molecule includes a molecule whose complementarity-determining region is part of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain variable domains, binding molecules naturally lacking light chains, single-domain scaffolds derived from conventional four-chain antibodies, engineered domains, and single-domain scaffolds other than those derived from antibodies. SDABs can be derived from any species, including but not limited to mice, humans, camels, llamas, fish, sharks, goats, rabbits, and cattle. As non-limiting examples of single-domain antibodies, one could cite, for instance, variable regions (VHs) derived from antibody heavy chains, which typically consist of about 120 amino acids, have a molecular weight of about 12–15 kDa, and are about 4 x 2.5 nM in size, much smaller than conventional four-chain antibodies; a large number of natural IgG (HCAb) containing only heavy chains have been found in the serum of camels, which, due to the natural absence of the light chain found in conventional four-chain antibodies, consist of two heavy chains, each containing a heavy chain variable region (VHH), a hinge region, and two CH2 and CH3 domains; VNAR found in the serum of cartilaginous fish (Feng et al., Antib Ther, 2, 1–11, 2019); and heavy chain-only antibodies found in some human heavy chain diseases (Prelli and Frangione, J Immunol, 148, 949–952, 1992).

[0112] Interlocking adapter molecules

[0113] The chimeric adapter molecule of the present invention polymerizes with the chimeric acceptor molecule of the present invention to form a complex. The polymerization may be due to electrostatic forces, such as electrostatic interactions generated by amino acids with opposite charges. Alternatively, it may be due to polymerization facilitated by hydrogen bonding or hydrophobic interactions.

[0114] Natural NKG2D has been shown to form hexameric complexes in mammals. Wild-type human NKG2D homodimers can assemble with two DNAX activator protein 10 (DAP10) homodimers to form hexamers. Wild-type mouse NKG2D homodimers can form hexamers with DAP10 and / or DAP12. It should be understood that in cells capable of expressing endogenous DAP10 or endogenous DAP12 (such as T cells), the chimeric receptor molecule of the present invention can form hexameric complexes with endogenous DAP10 or DAP12, and correspondingly, the chimeric adapter molecule of the present invention can also form hexameric complexes with endogenous NKG2D. Therefore, various hexameric complex formations can be observed on the surface of immune-responding cells engineered using the chimeric receptor complexes disclosed herein.

[0115] An example of the chimeric adapter of the present invention is a fusion polypeptide formed by DNAX activator protein 10 (DAP10) and an intracellular signal transduction element. The DAP10 polypeptide can be derived from humans or non-human mammals, such as rodents (e.g., mice or rats). In one embodiment, the DAP10 polypeptide is human. Wild-type human DAP10 has the amino acid sequence shown in SEQ ID NO:34. The polypeptide is 93 amino acids in length, with the first 18 amino acids at the N-terminus generally considered to be the leader sequence, positions 19 to 48 being the extracellular domain, positions 49 to 69 being the transmembrane domain, and positions 70 to 93 being the cytoplasmic / intracellular domain.

[0116] Therefore, in one embodiment, the chimeric adapter of the present invention may comprise the DAP10 polypeptide shown in SEQ ID NO:34. In other embodiments, the chimeric adapter of the present invention may also comprise various truncated forms of the DAP10 polypeptide, as long as the transmembrane structure is retained. For example, the chimeric adapter may comprise only the truncated form of DAP10 shown in SEQ ID NO:34, consisting of amino acid residues 49 to 69 (i.e., consisting only of the transmembrane structure). Other truncated forms may comprise amino acid residues 19 to 93 of SEQ ID NO:34 (i.e., lacking the leader sequence), amino acid residues 19 to 69 of SEQ ID NO:34 (i.e., the extracellular domain and the transmembrane domain), amino acid residues 1 to 69 of SEQ ID NO:34 (i.e., the leader sequence, the extracellular domain, and the transmembrane domain), and amino acid residues 49 to 93 of SEQ ID NO:34 (i.e., the transmembrane domain and the cytoplasmic / intracellular domain).

[0117] In another embodiment, the DAP10 polypeptide or a variant thereof is derived from rodents, preferably mice. Wild-type mouse DAP10 has the amino acid sequence shown in SEQ ID NO:42. The polypeptide is 79 amino acids in length, and it is generally believed that the first 17 amino acids at the N-terminus are the leader sequence, positions 18 to 35 are the extracellular domain, positions 36 to 56 are the transmembrane domain, and positions 57 to 79 are the cytoplasmic / intracellular domain.

[0118] Therefore, in one embodiment, the chimeric adapter of the present invention may comprise the DAP10 polypeptide shown in SEQ ID NO:42. In other embodiments, the chimeric adapter of the present invention may also comprise various truncated forms of the mouse DAP10 polypeptide that retain the transmembrane structure. For example, it may comprise only the truncated form of DAP10 shown in SEQ ID NO:42, containing only amino acid residues 36 to 56 (i.e., the transmembrane structure), containing amino acid residues 18 to 79 of SEQ ID NO:42 (i.e., lacking the leader sequence), containing amino acid residues 18 to 56 of SEQ ID NO:42 (i.e., containing only the extracellular and transmembrane domains of DAP10), containing amino acid residues 1 to 56 of SEQ ID NO:42 (i.e., the leader sequence, the extracellular domain, and the transmembrane domain), and containing amino acid residues 36 to 79 of SEQ ID NO:42 (i.e., the transmembrane domain and the cytoplasmic / intracellular domain).

[0119] Wild-type DAP10 assembles into a hexameric complex in the form of a homodimer. Therefore, in one embodiment, the engineered immune cells disclosed herein comprise a homodimer of a chimeric adapter, said homodimer comprising two chimeric adapter molecules disclosed herein. In one embodiment, the engineered immune cells comprise a chimeric adapter heterodimer, each peptide of which comprises a different chimeric adapter disclosed herein.

[0120] signal peptide

[0121] Any chimeric molecule disclosed herein may also include a signal peptide (also referred to as a leader sequence). Specifically, chimeric receptor molecules and / or chimeric adapter molecules may further include a signal peptide. The signal peptide may optionally be fused to the N-terminus of the chimeric polypeptide. A variety of signal peptides are known and may be used in the chimeric molecules disclosed herein, such as the CD8α signal peptide sequence (amino acid residues 1 to 21 of UniProt: P01732, SEQ ID NO: 82), and the signal peptide sequences of DAP10 and DAP12 (SEQ ID NO: 83–86).

[0122] Variants of the signal peptide may also be used. It should be understood that said variant is a functional variant that substantially retains the functional activity of the signal peptide, or even improves that functional activity. For example, the signal peptide variant may have at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% sequence identity with any of SEQ ID NO:82 to 86, or have one or more (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) point mutations relative to any of SEQ ID NO:82 to 86.

[0123] Activating motifs (ITAMs) and co-stimulatory elements of tyrosine

[0124] Any chimeric molecule disclosed herein may also contain the ITAM motif. ITAM is a conserved sequence of four amino acids repeated twice in the cytoplasmic tail of a non-catalytic tyrosine phosphorylation receptor.

[0125] The chimeric receptor molecules and / or chimeric adapter molecules disclosed herein can be directly fused to ITAM. Alternatively, they can be linked via a linker. ITAM can be fused to the N- or C-terminus of the chimeric peptide. Optionally, ITAM is fused to the C-terminus of the chimeric adapter molecule.

[0126] It is known that ITAM exists in a variety of receptors, such as the zeta chain of the T cell receptor, the eta chain of the T cell receptor, the δ chain of the T cell receptor, the γ chain of the T cell receptor, or the ε chain (i.e., the CD3 chain) of the T cell receptor or the γ subunit of the FcRl receptor.

[0127] Various co-stimulatory elements can also be added to the fusion peptides disclosed herein. These are examples commonly used in conventional chimeric antigen receptors. As examples, the following domains can serve as co-stimulatory elements.

[0128] 4-1BB(CD137) intracellular domain: SEQ ID NO:89;

[0129] CD28 intracellular domain: SEQ ID NO:87;

[0130] CD3ζ intracellular domain: SEQ ID NO:48.

[0131] Hinge area

[0132] The hinge region is responsible for connecting extracellular binding elements to the transmembrane domain. It should be understood that hinge regions used in chimeric antigen receptors may be used, including but not limited to the CD8α hinge, human IgG1 hinge, or human IgG4 hinge.

[0133] Human IgG1 hinge: SEQ ID NO:90;

[0134] CD8α hinge: SEQ ID NO:91;

[0135] Human IgG4 hinge: SEQ ID NO:94.

[0136] Connector or spacer sequence

[0137] In the chimeric molecules disclosed herein, one or more antibodies, receptors, ligands, or their antigen-binding fragments and / or other structural units may be directly linked and / or linked via one or more linkers or spacer sequences. Suitable linkers are preferably peptide linkers. Peptide linkers are used to construct fusion peptides, and methods for their selection and design are known in the art (see, for example, Chen X et al., 2013, Adv. Drug Deliv. Rev. 65(10):135701369 and Wriggers W et al., 2005, Biopolymers 80:736-746).

[0138] Preferred linker or spacer sequences are those suitable for use with pharmaceutical proteins or peptides, and more preferably for linking antibody fragments or antibody domains. For example, linkers for constructing bispecific antibodies or ScFv fragments. When using affinity domains of nanobodies, receptors, or ligands, since the affinity domains themselves form complete antigen-binding sites, there is no need to consider the limitation of spatially close proximity of the spaced affinity domains to form complete antigen-binding sites. Therefore, the length or flexibility of the linkers used herein is only required not to prevent the affinity domains in the multivalent construct from binding to the corresponding target epitopes or target antigens. As a non-limiting example, the linkers herein may be, for example, amino acid sequences having 1 to 50, preferably 1 to 30, such as 1 to 15 amino acid residues. Those skilled in the art can determine suitable linkers for the antibodies of the present invention based on the disclosure herein, optionally after limited conventional experiments. In some instances, the linker includes (Gly x Ser y XAA z ) n A connector of type LE, where x is any integer from 1 to 4, y is any integer from 1 to 3, z is 0 or 1, and n is any integer from 1 to 8, such as 1, 2, 3, 4, 5, 6, 7, or 8, like (Gly4Ser)3 or (Gly3Ser2)3. In a preferred embodiment, the connector sequence is GGGGS (SEQ ID NO: 58). Other exemplary connectors include, but are not limited to, those having the sequence LE, (GGGGS). n (n is 2, 3 or 4) (SEQ ID NO: 60~62), (Gly) n (n is 6, 7 or 8) (SEQ ID NO: 63~65), (EAAAK) n (n is 1, 2 or 3) (SEQ ID NO: 66~68), A(EAAAK) nA (n is 2, 3, 4 or 5) (SEQ ID NO: 69-72), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 73), PAPAP (SEQ ID NO: 74), KESGSVSSEQLAQFRSLD (SEQ ID NO: 75), EGKSSGSGSESKST (SEQ ID NO: 76), GSAGSAAGSGEF (SEQ ID NO: 77). In some embodiments, the linker is, for example, one or more of GGGSE (SEQ ID NO: 78), GSESG (SEQ ID NO: 79), GSEGS (SEQ ID NO: 80), GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGGSEGGS (SEQ ID NO: 81), or linkers with G, S and E randomly placed at 4 amino acid intervals. Suitable adapters or spacer sequences are found, for example, in WO99 / 42077, WO06 / 040153, WO06 / 122825, and WO94 / 04678. Other suitable adapters may include organic compounds or polymers, such as polyethylene glycol components for linking antibody domains (see, for example, WO04 / 081026). When two or more adapters are used for the peptides disclosed herein, these adapters may be the same or different.

[0139] The preferred embodiments for carrying out the present invention will now be described. It should be noted that the embodiments described below are examples illustrating representative embodiments of the present invention, but the present invention is not limited to these embodiments.

[0140] Two or more of the methods described below can be combined, and such combinations are also included in this invention.

[0141] Example

[0142] Experimental materials, reagents, instruments and experimental methods

[0143] Cell lines:

[0144] Multiple myeloma RPMI8226 cells were purchased from the Cell Bank of the Chinese Academy of Sciences;

[0145] Raji cells for B-cell lymphoma were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0146] EOL-1 cells for chronic eosinophilic leukemia were purchased from Yaji Biotechnology.

[0147] IgE plasmacytoma U266 cells were purchased from Yaji Biotechnology.

[0148] Example 1: Design of a chimeric receptor complex (CRC) based on the NKG2D transmembrane structure

[0149] This embodiment illustrates the structural differences between the natural NKG2D receptor complex, the CNK receptor complex, and the chimeric receptor complex based on the NKG2D transmembrane structure.

[0150] In the natural NKG2D receptor complex, two NKG2D molecules form a homodimer, and each NKG2D molecule also polymerizes with two adapter molecules, DAP10, ultimately forming a hexamer structure (Figure 1A). In the CNK receptor complex, NKG2D molecules are covalently linked to costimulatory elements, and adapter DAP10 molecules are covalently linked to intracellular signal transduction domains (such as the CD3ζ intracellular signal transduction domain), ultimately forming a hexamer (Figure 1B). In the type I chimeric receptor complex based on the NKG2D transmembrane structure, the main chimeric receptor molecule retains only the transmembrane structure of NKG2D, connecting the affinity domain of the functional receptor or ligand on the cell to one end of the NKG2D transmembrane structure through a hinge region, and also connecting the costimulatory element to the other end of the NKG2D transmembrane structure; simultaneously, each chimeric receptor molecule can polymerize with two adapter molecules, DAP10, that are chimeric with intracellular signal transduction domains (such as the CD3ζ intracellular signal transduction domain), ultimately forming a hexamer (Figure 1C). In type II chimeric receptor complexes based on the NKG2D transmembrane structure, the main chimeric receptor molecule is not attached to the affinity domain of a functional receptor or ligand at one end of the NKG2D transmembrane structure, but rather to an antibody or its antigen-binding fragment that can bind to the target antigen (Figure 1D).

[0151] Example 2: Design of CRC-T elements expressing chimeric receptor complexes

[0152] This embodiment designs three types of CRC-T elements that express chimeric receptor complexes.

[0153] 2.1 CRC-T complex 1 (CD256 CRC)

[0154] The CRC-T complex 1 encodes a CD256 chimeric receptor complex that targets BCMA and TACI. Complex 1 comprises a chimeric adapter molecule (DAP10-CD3ζ, SEQ ID NO:56) linked by a self-cleaving peptide 2A and a CD256 chimeric receptor molecule (NKG2D ICD-TM-CD256 ECD-G4S linker-CD256 ECD-G4S linker-CD256 ECD, SEQ ID NO:50). The CD256 chimeric receptor molecule consists of a transmembrane element based on the NKG2D transmembrane structure and three repeating CD256 affinity domains (CD256 BDs) tandemly, with each affinity domain separated by a G4S linker. Complex 1 can be described as DAP10-CD3ζ-T2A-NKG2D ICD-TM-CD256 ECD-G4S linker-CD256 ECD-G4S linker-CD256 ECD (Figure 2A). Complex 1 is expressed as a single nucleic acid molecule and constitutes the two core elements of the chimeric receptor complex.

[0155] 2.2 CRC-T complex 2 (IL5 CRC)

[0156] IL5 is a cytokine secreted by activated T cells and is the natural ligand of the interleukin-5 receptor (CD125). The CRC-T complex 2 encodes a chimeric receptor complex containing an IL5-based extracellular binding element. Complex 2 comprises a chimeric adapter molecule (DAP10-CD3ζ) linked by a self-cleaving polypeptide 2A and an IL5 chimeric receptor molecule (NKG2DICD-TM--IL5-G4S linker-IL5, SEQ ID NO:52). The IL5 chimeric receptor molecule consists of a transmembrane element based on the NKG2D transmembrane structure and two repeating IL5 molecules tandemly, with G4S linkers separating the affinity domains. Complex 2 can be described as DAP10-CD3ζ-T2A-NKG2D ICD-TM-IL5-G4S linker-IL5 (Figure 2B).

[0157] 2.3 CRC-T Complex 3 (Anti-CD125 CRC)

[0158] The CRC-T complex 3 encodes a chimeric receptor complex that targets CD125. Complex 3 comprises a chimeric adapter molecule (DAP10-CD3ζ) linked by a self-cleaving peptide 2A and an anti-CD125 chimeric receptor molecule (DAP10-CD3ζ-T2A-NKG2D ICD-TM-anti-CD125 scFV, SEQ ID NO:54). The anti-CD125 chimeric receptor molecule is composed of a transmembrane element based on the NKG2D transmembrane structure and an anti-CD125 single-chain antibody (scFv) tandemly. Complex 3 can be described as DAP10-CD3ζ-T2A-NKG2D ICD-TM-anti-CD125 scFV (Figure 2C).

[0159] Example 3: Preparation of expression plasmids for chimeric receptor complexes

[0160] The nucleic acid sequence encoding the CRC-T complex was synthesized and ligated into the lentiviral vector pCDH-CMV-MCS-EF1α-Puro (Ubibio) or pLVX-EF1α-AcGFP1-C1 Vector (Takara) via molecular cloning. This ligation was operatively linked to the EF1α promoter to construct the CRC-T lentiviral vector. The correctly sequenced vector was transformed into DH5α competent cells (Thermo Fisher), and single colonies were picked for large-scale culture. Then, the plasmid extraction was performed using the PureLink plasmid extraction kit. TM The plasmid was purified using the HiPure Plasmid Maxiprep Kit (Thermo Fisher) to obtain the CRC-T lentiviral plasmid.

[0161] The nucleotide sequence encoding CRC-T complex 1 is shown in SEQ ID NO: 96, and the protein it encodes is shown in SEQ ID NO: 95;

[0162] The nucleotide sequence encoding CRC-T complex 2 is shown in SEQ ID NO: 98, and the protein it encodes is shown in SEQ ID NO: 97;

[0163] The nucleotide sequence encoding CRC-T complex 3 is shown in SEQ ID NO: 100, and the protein it encodes is shown in SEQ ID NO: 99.

[0164] Example 3.1: Preparation of co-expression plasmids for chimeric receptor complexes

[0165] The two core elements of the chimeric receptor complex of this application can also be placed in two separate plasmids and co-expressed to generate the chimeric receptor complex by co-transfection of host cells. For example, the nucleic acid sequence encoding DAP10-CD3ζ and the nucleic acid sequence encoding NKG2D ICD-TM-CD256 ECD-G4S linker-CD256 ECD-G4S linker-CD256 ECD are respectively placed after the EF1α promoter in the lentiviral vector pCDH-CMV-MCS-EF1α-Puro (Yobo Biotechnology) or pLVX-EF1α-AcGFP1-C1 Vector (Takara), and the lentiviral vectors of the chimeric receptor molecule and the chimeric adapter molecule are prepared respectively according to the method described in Example 3.

[0166] Example 4: Preparation of CRC-T Lentiviral Virus

[0167] The CRC-T lentiviral plasmid obtained in Example 3, along with packaging plasmids psPAX2 and pMD2.G (Addgene, catalog numbers: 12259 & 12260), were co-transfected into 293T cells (ATCC, CRL3216TM) using polyethyleneimine transfection reagent (408727, Sigma) (DNA: μg PEI = 1:3) at a ratio of 1.64 pmol: 1.3 pmol: 0.72 pmol. The packaging plasmid was prepared according to PureLink's method. TM The HiPure Plasmid Maxiprep Kit (K210006, Thermo) was used, and transfection was performed according to the Sigma transfection reagent instructions. Sixteen hours after transfection, the medium was replaced with complete culture medium (Life Technologies, 11995-065). After 24, 48, and 72 hours of incubation, the supernatant containing lentivirus was collected, combined, and centrifuged at -80°C and 3000 rpm for 10–15 minutes. Then, it was filtered through a 0.45 μm filter membrane. The CRC-T lentivirus concentrate was concentrated by ultracentrifugation at 4°C and 25000 rpm for 2–3 hours, and stored at -80°C.

[0168] Example 5: Preparation of CRC-T cells

[0169] Fresh peripheral blood mononuclear cells were obtained from fresh peripheral blood of healthy donors. CD3 sorting magnetic beads, MS separation column, and MiniMACS were then used for separation. TM The separation device (Miltenyi Biotec) positively sorts out CD3. +T cells were then stimulated and cultured using magnetic beads conjugated with anti-CD3 and anti-CD28 antibodies (Human T-Activator CD3 / CD28, Invitrogen, 11161D). The specific steps were as follows: peripheral blood mononuclear cells were diluted to a concentration of 1×10⁻⁶. 6 Single cells / ml were seeded in 24-well plates, and magnetic beads were added at a 1:1 ratio and mixed well. The cells were then resuspended in culture medium (OpTmizer™ T-Cell Expansion SFM, A1048503, Life Technologies). IL 250 U / ml and IL 15 5 ng / ml were added, and the plates were incubated at 37°C in a 5% CO2 incubator for 1 day. Lentiviral virus loaded with CRC-T elements, prepared in Example 4, was then added for transfection. The specific steps are as follows: the virus solution was added to the culture medium at an MOI of 3–5, and 10 μg / ml of Polybrene was added. The plates were centrifuged at low speed (500g–1000g / min) for 30–60 minutes using a flat-angle centrifuge, and then incubated at 37°C for 48 hours to obtain CRC-T expressing cells (CRC-T cells). Cell phenotype could be detected by flow cytometry.

[0170] Example 6: Assay of tumor-killing activity of CRC-T cells

[0171] 6.1 For multiple myeloma and B-cell lymphoma

[0172] This embodiment investigated the cytotoxic activity of CD256 CRC-T cells prepared by the method described in Example 5 against CD256-positive tumors. Multiple myeloma RPMI8226 cells and B-cell lymphoma Raji cells were used. Stable GFP-expressing cell lines RPMI8226-FFluc-GFP and Raji-FFluc-GFP were constructed by transfecting FFluc-GFP with lentivirus. Two BCMACAR-T cells (expressing chimeric antigen receptors anti-BCMA-CD8HT-41BB-Z or anti-BCMA-CD8HT-CD28Z, respectively) were used as controls to evaluate the cytotoxic effect of CD256 CRC-T cells against the two tumors.

[0173] The cell lines RPMI8226 and Raji cells were mixed at a ratio of 0.5 × 10⁻⁶. 610 cells / well were seeded into 24-well plates. CD256 CRC-T cells (DAP10-CD3ζ-T2A-NKG2D ICD-TM-3XCD256 ECD) or BCMACAR-T cells were added at an effector-to-target ratio of 1:1 or 1:2, respectively. Untransfected tumor cells were used as a blank control. Cells were harvested after 48 h and stained with CD3-APC and BCMA-PE (purchased from Biolegend). The composition of the co-cultured cells was analyzed by flow cytometry. Because the tumor cells express GFP, the number of remaining FITC+ tumor cells could be counted by flow cytometry, and the results are shown in Figures 3A and 3B.

[0174] It is evident that CD256 CRC-T cells exhibit good killing activity against both RPMI8226 cells and Raji cells, and their tumor-killing effect is superior to that of BCMACAR-T cells expressing chimeric antigen receptors (using CD28 and 41BB as co-stimulatory molecules).

[0175] 6.2 For chronic eosinophilic leukemia and IgE plasmacytoma

[0176] This embodiment investigates the cytotoxic activity of CD125(IL5Ra) CRC-T cells prepared by the method described in Example 5 against CD125-positive tumors. Chronic eosinophilic leukemia EOL-1 cells and IgE plasmacytoma U266 cells were used. Similarly, stable GFP-expressing cell lines EOL-1-FFluc-GFP and U266-FFluc-GFP were constructed. Two CD125 CAR-T cells (expressing chimeric antigen receptors anti-CD125-CD8HT-41BB-Z or anti-CD125-CD28-Z, respectively) were used as controls to evaluate the cytotoxic effects of CD125 CRC-T (DAP10-CD3ζ-T2A-NKG2DICD-TM-anti-CD125 scFv) cells and IL5 CRC-T (DAP10-CD3ζ-T2A-NKG2DICD-TM-IL5-G4S linker-IL5) cells against the two tumors.

[0177] As described in Section 6.1, T cells and tumor cells were co-cultured. The results are shown in Figure 4. It can be seen that CD125 CRC-T cells and IL5 CRC-T cells have good killing activity against EOL-1 cells and U266 cells, and their tumor-killing effect is significantly better than that of the two conventional chimeric antigen receptors expressing CD125.

[0178] This embodiment demonstrates that the chimeric receptor complex disclosed herein can activate T cells more effectively than chimeric antigen receptor molecules, transforming them into tumor-killing T cells.

[0179] In addition, compared with the chimeric receptor complex constructed using the transmembrane structure of NK cell receptors (such as NKP44) (Figure 5), the engineered T cells constructed using the chimeric receptor complex of this application also showed significantly higher tumor killing effect (data not shown).

[0180] Example 7

[0181] This embodiment describes the preparation of a chimeric receptor complex based on nanobodies. Specifically, elements expressing the chimeric receptor complex are prepared according to the method described in Example 2, except that the receptor, ligand, or antigen-binding fragment in any of the elements in 2.1 to 2.3 is replaced with a V anti-target protein. HH Antibodies, from which antibodies can be obtained that express 1 to 3 V groups. HH Antibodies serve as chimeric receptor complex expression elements for extracellular binding (Figures 6A to 6C). Furthermore, by describing the methods in Examples 3-5, expression cassettes, expression plasmids, recombinant lentiviruses, and engineered T cells expressing the corresponding chimeric receptor complexes can be obtained.

[0182] V contained in the same complex HH The antibodies can be the same, for example, DAP10-CD3ζ-T2A-NKG2DICD-TM-3Xanti-CD125-V HH It can also be not exactly the same or completely different, for example, DAP10-CD3ζ-T2A-NKG2D ICD-TM-anti-CD125-V HH -2xanti-BCMA-V HH Or DAP10-CD3ζ-T2A-NKG2D ICD-TM-anti-CD125-V HH -anti-BCMA-V HH -anti-CD19-V HH .

Claims

1. A chimeric receptor complex, characterized in that, The complex comprises a chimeric receptor molecule and a chimeric adapter molecule. The chimeric receptor molecule includes an extracellular binding element and a transmembrane element based on the NKG2D transmembrane domain. The extracellular binding element is at least one antibody, receptor, ligand, or antigen-binding fragment thereof that specifically recognizes and binds to the target antigen, and optionally also includes at least one intracellular signaling element. The chimeric adapter molecule includes a DAP10 transmembrane structure or a DAP12 transmembrane structure and includes at least one intracellular signal transduction element, optionally also including at least one co-stimulatory element.

2. The complex of claim 1, wherein, The extracellular binding element is an antigen-binding fragment, preferably selected from any one or any combination of the following: diabody, Fab, Fab', F(ab')2, Fd, Fv, disulfide-stabilized Fv(dsFv), (dsFv)2, bispecific dsFv(dsFv-dsFv'), disulfide-stabilized diabody, single-chain antibody (scFv), scFv dimer (divalent diabody), multispecific antibody, heavy chain only antibody, single-domain antibody (such as camel-derived single-domain antibody), or nanobody. Preferably, the extracellular binding element is selected from any one or any combination of single-chain antibodies (scFv), single-domain antibodies (such as camel-derived single-domain antibodies) or nanobodies; More preferably, the extracellular binding element is a single-chain antibody (scFv); Optionally, the extracellular binding element is at least one single-chain antibody (scFv) that specifically recognizes and binds to CD125. Preferably, the single-chain antibody comprises HCDR1 having the amino acid sequence shown in SEQ ID NO:1, HCDR2 having the amino acid sequence shown in SEQ ID NO:2, HCDR3 having the amino acid sequence shown in SEQ ID NO:3, LCDR1 having the amino acid sequence shown in SEQ ID NO:4, LCDR2 having the amino acid sequence shown in SEQ ID NO:5, and LCDR3 having the amino acid sequence shown in SEQ ID NO:

6. More preferably, the single-chain antibody comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:7 and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:8; More preferably, the single-chain antibody has an amino acid sequence as shown in SEQ ID NO:9, or has an amino acid sequence that is at least about 90% identical to the amino acid sequence shown in SEQ ID NO:9, or has an amino acid sequence with one or more amino acid deletions, substitutions or insertions compared to the amino acid sequence shown in SEQ ID NO:

9. Optionally, the extracellular binding element is a natural ligand or a derivative thereof; Preferably, the natural ligand is selected from any one or a combination of CD256, BAFF, IL5, or TIGIT. Optionally, the extracellular binding element is a natural ligand or a fragment thereof; Preferably, the natural ligand or fragment thereof has an amino acid sequence as shown in any of SEQ ID NO:13 to 16, or has an amino acid sequence that is at least about 90% identical to the amino acid sequence shown in any of SEQ ID NO:13 to 16, or has an amino acid sequence with one or more amino acid deletions, substitutions or insertions compared to the amino acid sequence shown in any of SEQ ID NO:13 to 16.

3. The complex of claim 1 or 2, wherein the target antigen is a tumor-associated antigen; Preferably, the tumor-associated antigen is selected from CD19, CD20, CD22, BCMA, BAFF-R, ROR1, GPRC5D, CD38, CD40, CD33, CD123, CD125, CD137, CTLA-4, PD-1, TIGIT, CLL1, CD70, OX40, α4β7, CD5, CD7, CD90, CD276, CD22, CD23, CD25, CD134, LEY, CD47, CD79b, IL-4R, CD96, CD44v6, CD56, B7H3, B7H4, B7H6, GPC3, Any one or any combination thereof from the group consisting of GPC2, CEA, AFP, FORR1, MSLN, CD133, Trop2, SIRPα, Epcam, EphA2, PSMA, uPAR, 5T4, HLA-G, HLA-E, MUC1, FAPα, CAIX, TAG72, OR2H1, Claudin6, Claudin18.2, GD2, HER2, HER3, L13Rα2, EGFR, EGFRvIII, VEGFR-1, VEGFR-2, αvβ3, αvβ6, DLL2, DLL3, and ROBO1.

4. The complex according to any one of claims 1 to 3, wherein, The NKG2D transmembrane domain is either a human NKG2D transmembrane domain or a mouse NKG2D transmembrane domain. Preferably, the human NKG2D transmembrane domain has an amino acid sequence as shown in SEQ ID NO:19, or has an amino acid sequence that is at least about 90% identical to the amino acid sequence shown in SEQ ID NO:19, or has an amino acid sequence with one or more amino acid deletions, substitutions or insertions compared to the amino acid sequence shown in SEQ ID NO:

19. Preferably, the mouse NKG2D transmembrane structure has an amino acid sequence as shown in SEQ ID NO:21, or has an amino acid sequence that is at least about 90% identical to the amino acid sequence shown in SEQ ID NO:21, or has an amino acid sequence with one or more amino acid deletions, substitutions or insertions compared to the amino acid sequence shown in SEQ ID NO:

21.

5. The complex according to any one of claims 1 to 4, wherein, The DAP10 and DAP12 transmembrane structures are derived from human or non-human mammalian DAP10 or DAP12, preferably from human or mouse DAP10 or DAP12. Preferably, the DAP10 transmembrane structure has an amino acid sequence as shown in SEQ ID NO:32 or 40, or has an amino acid sequence that is at least about 90% identical to the amino acid sequence shown in SEQ ID NO:32 or 40, or has an amino acid sequence with one or more amino acid deletions, substitutions or insertions compared to the amino acid sequence shown in SEQ ID NO:32 or 40. The DAP12 transmembrane structure has an amino acid sequence as shown in SEQ ID NO:36 or 44, or has an amino acid sequence that is at least about 90% identical to the amino acid sequence shown in SEQ ID NO:36 or 44, or has an amino acid sequence with one or more amino acid deletions, substitutions or insertions compared to the amino acid sequence shown in SEQ ID NO:36 or 44.

6. The complex according to any one of claims 1 to 5, wherein, The intracellular signal transduction element contains the ITAM motif; Preferably, the intracellular signal transduction element may be selected from intracellular signal transduction domains of CD27, ICOS, CD134, Lck, ZAP-70, PKC, LAT, SLP-76, TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI, CD32 (FcγRIIa), CD66d, CD28, 4-1BB, OX40, DAP10 or DAP12 or combinations thereof; More preferably, the intracellular signal transduction element has an amino acid sequence selected from the amino acid sequence shown in SEQ ID NO:48, 87 or 89, or has an amino acid sequence that is at least about 90% identical to the amino acid sequence shown in SEQ ID NO:48, 87 or 89, or has an amino acid sequence that has one or more amino acid deletions, substitutions or insertions compared to the amino acid sequence shown in SEQ ID NO:48, 87 or 89.

7. The complex according to any one of claims 1 to 6, wherein, The chimeric acceptor molecule has an amino acid sequence selected from SEQ ID NO:50, 52 or 54, or has an amino acid sequence that is at least about 90% identical to the amino acid sequence shown in SEQ ID NO:50, 52 or 54, or has an amino acid sequence that has one or more amino acid deletions, substitutions or insertions compared to the amino acid sequence shown in SEQ ID NO:50, 52 or 54. Optionally, the chimeric adapter molecule has an amino acid sequence selected from that shown in SEQ ID NO:56, or has an amino acid sequence that is at least about 90% identical to the amino acid sequence shown in SEQ ID NO:56, or has an amino acid sequence with one or more amino acid deletions, substitutions or insertions compared to the amino acid sequence shown in SEQ ID NO:

56.

8. A nucleic acid molecule encoding a complex as described in any one of claims 1 to 7; Optionally, the sequence encoding the chimeric receptor molecule and the sequence encoding the chimeric adapter molecule are located in the same or different reading frames, respectively; Optionally, the nucleic acid molecule is deoxyribonucleic acid (DNA), ribonucleic acid (RNA) (e.g., mRNA, circular RNA, ccRNA), threonucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA with β-D-ribose conformation, α-LNA with α-L-ribose conformation (diastereomers of LNA), 2'-amino-LNA with 2'-amino functionalization and 2'-amino-α-LNA with 2'-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA) and / or chimeras and / or combinations thereof.

9. A carrier comprising a nucleic acid molecule as described in claim 8 and a regulatory element operatively linked to said nucleic acid molecule; Optionally, the vector is selected from plasmids, nanoparticles, granules, viral vectors, small circular vectors, RNA vectors, or linear or circular DNA or RNA molecules; Optionally, the viral vector is selected from retroviral vectors, adenovirus vectors, lentiviral vectors, and adeno-associated virus vectors.

10. A liposome comprising the nucleic acid molecule as described in claim 8.

11. An engineered cell expressing the complex as described in any one of claims 1 to 7, or comprising the nucleic acid molecule as described in claim 8, or comprising the vector as described in claim 9; Optionally, the engineered cells are immune cells, which may be selected from any one of T cells, natural killer (NK) cells, B cells, macrophages, monocytes, dendritic cells, neutrophils or γδT cells. Optionally, the T cells are selected from CD8. + T cells, CD4 + T cells, cytotoxic T cells, terminal effector T cells, memory T cells, naive T cells, regulatory T cells, natural killer T cells, γ-δ T cells, cytokine-induced killer (CIK) T cells, and tumor-infiltrating lymphocytes; Optionally, the engineered cells further comprise a cell protection element capable of specifically reducing the mRNA or protein levels of the NKG2D ligand in the engineered cells; Preferably, the cell protection element is capable of specifically knocking down the endogenous NKG2D ligand gene or specifically degrading the endogenous NKG2D ligand molecule; Optionally, the cell protection element is selected from gene editors, targeted degradation molecules, or antisense nucleic acid molecules; Optionally, the gene editor is selected from a complex of CRISPR, base editor, leader editor, TALEN, or zinc finger protein that specifically knocks out or knocks down the endogenous NKG2D ligand gene. Optionally, the targeted degradation molecule is selected from PROTAC, molecular glue, AUTOTAC, and targeted degradation molecules based on endoplasmic reticulum-related degradation mechanisms that specifically degrade endogenous NKG2D in cells. Optionally, the antisense nucleic acid molecule is selected from antisense DNA, antisense RNA or ribozyme transcribed from mRNA that specifically knocks out or knocks down the endogenous NKG2D ligand in the cell, preferably siRNA or shRNA; Preferably, the cell protection element comprises a targeted degradation element consisting of an endoplasmic reticulum-associated degradation mechanism protein-binding domain and an NKG2D ligand-targeting domain.

12. A method for producing engineered cells as claimed in claim 11, comprising introducing a nucleic acid molecule as claimed in claim 8 into a starting cell under in vitro or in vivo conditions; Optionally, the starting cells are selected from any one of T cells, natural killer (NK) cells, B cells, macrophages, monocytes, dendritic cells, neutrophils or γδT cells; Preferably, the T cells are selected from CD8+ T cells, CD4+ T cells, cytotoxic T cells, terminal effector T cells, memory T cells, naive T cells, regulatory T cells, natural killer T cells, γ-δ T cells, cytokine-induced killer (CIK) T cells, and tumor-infiltrating lymphocytes.

13. A cell population, which is generated in vitro by the method of claim 12.

14. A pharmaceutical composition or kit comprising (i) a complex as described in any one of claims 1 to 7, or a nucleic acid molecule as described in claim 8, or a carrier as described in claim 9, or a liposome as described in claim 10, or an engineered cell as described in claim 11, or a cell population as described in claim 13, and (ii) a pharmaceutically acceptable medium.

15. A method of treating a symptom or disease, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition or kit as described in claim 14; Optionally, the disease is selected from any one of the group consisting of solid tumors, hematologic malignancies, viral infectious diseases, autoimmune diseases, degenerative diseases, and metabolic diseases; Preferably, the solid tumor is selected from any one of the group consisting of nervous system tumors, head and neck tumors, chest tumors, digestive system tumors, genitourinary system tumors, soft tissue and skin tumors, and bone tumors; Preferably, the nervous system tumor is selected from any one of the following groups: diffuse glioma, diffuse astrocytoma and anaplastic astrocytoma, glioblastoma, oligodendroglioma, oligodendroastrocytoma, pediatric diffuse glioma, other astrocytomas, ependymoma, neuronal and mixed neuronal-glial tumors, medulloblastoma, other embryonal tumors, schwannoma, meningioma, solitary fibrous tumor, and angiopericytoma; Preferably, the head and neck tumor is selected from any one of the following groups: malignant tumors of the nasal cavity and sinuses, nasopharyngeal carcinoma, oral cancer, laryngeal cancer, salivary gland tumors, intracranial tumors, thyroid cancer, and tongue cancer. Preferably, the thoracic tumor is selected from any one of the group consisting of lung cancer, esophageal cancer, gastric cardia cancer, breast cancer, and mediastinal tumors; Preferably, the digestive system tumor is selected from any one of the following groups: gastric cancer, colorectal cancer and sigmoid colon and rectal cancer, liver cancer, pancreatic cancer and periampullary cancer, biliary tract cancer, and small intestinal malignant tumors. Preferably, the urogenital system tumor is selected from any one of the group consisting of kidney cancer, prostate cancer, bladder cancer, testicular malignant tumor, penile cancer, cervical cancer, endometrial cancer, and ovarian cancer; Preferably, the soft tissue and skin tumors are selected from any one of the group consisting of malignant fibrous histiocytoma, rhabdomyosarcoma, synovial sarcoma, and malignant melanoma of the skin; Preferably, the bone tumor is selected from osteosarcoma or Ewing's sarcoma; Preferably, the colon cancer is a colonic adenoma; Preferably, the breast cancer is triple-negative breast cancer cells; Preferably, the liver cancer is hepatocellular carcinoma; Preferably, the hematologic malignancy is selected from any one of the group consisting of leukemia, lymphoma (HL), multiple myeloma (MM), and myelodysplastic syndrome (MDS); Preferably, the leukemia is selected from any one of the group consisting of B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, and acute myeloid leukemia; Preferably, the viral infectious disease is selected from any one of the group consisting of respiratory viral diseases, gastrointestinal viral diseases, liver viral diseases, skin and mucous membrane viral diseases, eye viral diseases, central nervous system viral diseases, lymphocytic viral diseases, worm-borne viral diseases, and lentiviral infectious diseases. Preferably, the respiratory viral disease is selected from any one of the group consisting of rhinovirus, adenovirus, respiratory syncytial virus, parainfluenza virus and coronavirus, influenza, and mumps. Preferably, the gastrointestinal viral disease is selected from any one of the group consisting of polio, Cooksackie virus infection, ECHO virus infection, and viral gastroenteritis; Preferably, the viral gastroenteritis is selected from any one of the group consisting of rotavirus gastroenteritis, norovirus gastroenteritis, adenovirus gastroenteritis, astrovirus gastroenteritis, coronavirus gastroenteritis, and calicivirus gastroenteritis; Preferably, the viral liver disease is selected from any one of the group consisting of hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, EB virus hepatitis, and cytomegalovirus hepatitis. Preferably, the viral diseases of the skin and mucous membranes are selected from any one of the group consisting of measles, rubella, roseola infantum, chickenpox and herpes zoster, smallpox, herpes simplex virus infection, rabies and foot-and-mouth disease; Preferably, the ocular viral disease is selected from any one of the group consisting of epidemic keratoconjunctivitis, follicular conjunctivitis and herpetic keratoconjunctivitis; Preferably, the viral disease of the central nervous system is selected from any one of the group consisting of Japanese encephalitis, Western equine encephalitis, Eastern equine encephalitis, St. Louis encephalitis, Venezuelan equine encephalitis, Murray Valley encephalitis, California encephalitis, tick-borne encephalitis, and lymphocytic choroid plexus meningitis. Preferably, the lymphocytic viral disease is selected from any one of the group consisting of infectious mononucleosis, cytomegalovirus infection, and acquired immunodeficiency syndrome; Preferably, the worm-borne viral disease is selected from any one of the following groups: viral hemorrhagic fever, epidemic hemorrhagic fever, yellow fever, Crimean-Congo hemorrhagic fever, Rift Valley fever, Argentine hemorrhagic fever, Bolivian hemorrhagic fever, Lassa fever, Omsk hemorrhagic fever, Marburg disease and Ebola hemorrhagic fever, dengue fever and dengue hemorrhagic fever, West Nile fever, Colorado tick-borne fever, and sandfly fever. Preferably, the lentiviral infection is selected from any one of the group consisting of subacute sclerosing panencephalitis, kuru, progressive multifocal leukoencephalopathy, and subacute spongiform encephalopathy (corticostriatal spinal cord degeneration). Preferably, the autoimmune disease is selected from the group consisting of organ-specific autoimmune diseases and systemic autoimmune diseases; Preferably, the organ-specific autoimmune disease is selected from any one of the following groups: acute and chronic GVHD after stem cell transplantation, organ transplant rejection, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cirrhosis, multiple cerebral sclerosis, and acute idiopathic polyneuritis. Preferably, the systemic autoimmune disease is selected from any one of the group consisting of systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, thyroid autoimmune disease, and ulcerative colitis. Preferably, the neurological diseases are selected from any one of the following groups: peripheral nerve diseases, spinal cord diseases, cerebrovascular diseases, infectious diseases of the central nervous system, demyelinating diseases of the central nervous system, movement disorders, epilepsy, headache, neurodegenerative diseases, hereditary diseases of the nervous system, developmental disorders of the nervous system, neuromuscular junction and muscle diseases, autonomic nervous system diseases, nervous system tumors, and paraneoplastic syndromes of the nervous system. Preferably, the peripheral nerve disease is selected from any one of the following groups: trigeminal neuralgia, facial paralysis, hemifacial spasm, vestibular neuronitis, glossopharyngeal neuralgia, mononeuropathy, brachial plexus neuralgia, multiple mononeuropathy, polyneuropathy, acute inflammatory demyelinating polyneuropathy, and chronic inflammatory demyelinating polyneuropathy. Preferably, the spinal cord disease is selected from any one of the group consisting of myelitis, compressive myelopathy, subacute combined degeneration of the spinal cord, syringomyelia, spinal vascular disease, and spinal arachnoiditis. Preferably, the cerebrovascular disease is selected from any one of the group consisting of transient ischemic attack, cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, and intracranial venous system thrombosis. Preferably, the central nervous system infectious disease is selected from any one of the group consisting of meningitis and encephalitis caused by viral, bacterial, fungal or parasitic infections, and slow virus encephalitis caused by slow virus infection; Preferably, the central nervous system demyelinating disease is selected from any one of the group consisting of multiple sclerosis, neuromyelitis optica, acute infectious encephalomyelitis, and leukodystrophy. Preferably, the movement disorder is selected from any one of the group consisting of Parkinson's disease, chorea, Wilson's disease, dystonia, essential tremor, and tardive dyskinesia. Preferably, the headache is selected from any one of the group consisting of migraine, tension headache, and cluster headache; Preferably, the neurodegenerative disease is selected from any one of the following groups: free motor neuron disease, Alzheimer's disease, Lewy body dementia, frontotemporal dementia, and multiple system atrophy. Preferably, the hereditary neurological disease is selected from any one of the group consisting of hereditary ataxia, hereditary spastic paraplegia, peroneal muscular atrophy, neurofibromatosis, tuberous sclerosis, and cerebral facial angiomatosis. Preferably, the neurodevelopmental disorder is selected from any one of the group consisting of congenital hydrocephalus, cerebral palsy, basilar impression, and cerebellar hypotonsillar lesion; Preferably, the neuromuscular junction and muscle disease are selected from any one of the group consisting of myasthenia gravis, periodic paralysis, polymyositis, progressive muscular dystrophy, myotonic myopathy (myotrophic lateral sclerosis, congenital myotonia), and metabolic myopathy (mitochondrial myopathy, lipid storage myopathy, glycogen storage disease). Preferably, the autonomic nervous system disease is selected from any one of the following groups: Raynaud's disease, erythromelalgia, hemifacial atrophy, generalized autonomic dysfunction, spontaneous hyperhidrosis, and progressive lipodystrophy. Preferably, the nervous system tumor is selected from any one of the group consisting of glioma, lymphoma, and meningioma: Preferably, the paraneoplastic syndrome of the nervous system is selected from any one of the group consisting of paraneoplastic cerebellar degeneration, paraneoplastic encephalomyelitis, subacute necrotizing myelopathy, subacute motor neuron disease, and paraneoplastic sensory neuron disease. Preferably, the metabolic diseases are selected from any one of the following groups: glucose metabolism-related diseases such as diabetes and tumor glucose metabolism; lipid metabolism-related diseases such as tumor lipid metabolism, hyperlipidemia, and non-alcoholic fatty liver disease; gout and atherosclerosis.

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