CN117467022A - Chimeric antigen receptor and uses thereof - Google Patents
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Abstract
The invention provides chimeric antigen receptor capable of recognizing two antigen ligands simultaneously, corresponding nucleic acid construct, expression vector, transgenic immune cell, pharmaceutical composition and application thereof, wherein the chimeric antigen receptor comprises: an extracellular region comprising a first binding fragment and a second binding fragment, the first binding fragment and the second binding fragment being linked, the first binding fragment being an NKp30 extracellular segment or a fragment thereof, the second binding fragment being an NKG2D extracellular segment or a fragment thereof; the N end of the transmembrane region is connected with the C end of the extracellular region; and an intracellular region, wherein the N-terminal of the intracellular region is connected with the C-terminal of the transmembrane region. The chimeric antigen receptor of the invention simultaneously targets the NKp30 receptor ligand and the NKG2D receptor ligand, and the transgenic immune cells expressing the chimeric antigen receptor of the invention can effectively identify the tumor cells expressing the NKp30 receptor ligand and/or the NKG2D receptor ligand, namely can effectively kill the tumor cells which singly express the NKp30 receptor ligand or the NKG2D receptor ligand or simultaneously express the two receptor ligands, and especially can effectively identify the tumor cells losing one of the NKp30 or the NKG2D receptor ligand, thereby preventing the immune escape of tumors. Therefore, the transgenic immune cell provided by the invention has the advantages of high tumor cell killing activity, wide recognition spectrum, strong tumor inhibiting activity in vivo, low cell infusion dosage and good curative effect.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a chimeric antigen receptor and application thereof, and more particularly relates to a chimeric antigen receptor capable of simultaneously recognizing two antigen ligands, a corresponding nucleic acid construct, an expression vector, a transgenic immune cell, a pharmaceutical composition and application thereof.
Background
Chimeric antigen receptor T (chimeric antigen receptor T, CAR-T) cells have shown remarkable effects in hematological malignancy treatment. The antigen recognition region of a CAR molecule typically employs scFv sequences derived from antibodies, typically from murine, rabbit, alpaca, and the like, which are highly immunogenic. Even though the sequence is humanized, certain immunogenicity still exists, and anti-drug antibodies (ADA) are generated, so that the persistence of the CAR-T cells in the body is weakened. In recent years, there have been many new techniques applied to solve this problem, such as using a natural receptor protein sequence as a receptor, to reduce immunogenicity.
The NKp30 receptor is a natural NK cell activating receptor, is highly expressed on NK cells, and can recognize B7H6 molecules so as to mediate cytotoxicity. B7H6 is a member of the B7 family, and B7H6 molecules are typically highly expressed on a variety of primary tumor cells, including leukemia, lymphoma, colorectal, gastric, pancreatic, and the like, while being hardly expressed on normal tissue cells. Currently, the function of CAR-T cells constructed based on the extracellular segment of NKp30 as a receptor for B7H6 recognition has been verified in studies.
The NKG2D receptor is another natural NK cell surface receptor, mainly expressed on NK, CD8+T, γδ T, NKT and activated macrophage surface, recognizing stress proteins expressed by viral infection or malignant transformation of cells, such as MHC-class I-related proteins, MICA and MICB and cytomegalovirus UL-16 binding protein ULBP1-5. In the CN109400713A patent, NKG2D is taken as an intracellular signal transduction structural domain, and CART cells constructed based on the domain obviously strengthen the killing capacity of CAR T cells on tumor cells in a blood tumor and solid tumor killing test, and show good safety and anti-tumor activity in clinical application.
Based on the present situation of research and development, the chimeric conversion receptor with high killing activity and good curative effect is further researched so as to further improve the curative effect of the chimeric antigen receptor immune cell therapy.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art to at least some extent. Therefore, the invention provides a chimeric antigen receptor capable of simultaneously recognizing two antigen ligands, a corresponding nucleic acid construct, an expression vector, a transgenic immune cell, a pharmaceutical composition and application thereof, the chimeric antigen receptor of the invention simultaneously targets an NKp30 receptor ligand and an NKG2D receptor ligand, and the transgenic immune cell expressing the chimeric antigen receptor of the invention can effectively recognize tumor cells expressing the NKp30 receptor ligand and/or the NKG2D receptor ligand, namely can play an effective killing role on the tumor cells singly expressing the NKp30 receptor ligand or the NKG2D receptor ligand or simultaneously expressing the two receptor ligands, and particularly can effectively recognize the tumor cells losing one receptor ligand of the NKp30 or the NKG2D and prevent tumor immune escape. Therefore, the transgenic immune cell provided by the invention has the advantages of high tumor cell killing activity, wide recognition spectrum, strong tumor inhibiting activity in vivo, low cell infusion dosage and good curative effect.
In a first aspect of the invention, the invention provides a chimeric antigen receptor. The chimeric antigen receptor comprises:
an extracellular region comprising a first binding fragment and a second binding fragment, the first binding fragment and the second binding fragment being linked, the first binding fragment being an NKp30 extracellular segment or a fragment thereof, the second binding fragment being an NKG2D extracellular segment or a fragment thereof;
the N end of the transmembrane region is connected with the C end of the extracellular region;
and an intracellular region, wherein the N-terminal of the intracellular region is connected with the C-terminal of the transmembrane region.
The amino acid sequences according to the present invention are all shown from N-terminus to C-terminus.
According to the chimeric antigen receptor disclosed by the embodiment of the invention, the NKp30 receptor ligand and the NKG2D receptor ligand are simultaneously targeted, and the transgenic immune cells expressing the chimeric antigen receptor disclosed by the invention can effectively identify the tumor cells expressing the NKp30 receptor ligand and/or the NKG2D receptor ligand, namely, the chimeric antigen receptor can effectively kill the tumor cells which singly express the NKp30 receptor ligand or the NKG2D receptor ligand or simultaneously express both receptor ligands, and especially can effectively identify the tumor cells losing one of the NKG 30 or the NKG2D receptor ligand, so that the immune escape of tumors is prevented. Therefore, the transgenic immune cells expressing the chimeric antigen receptor of the invention have high tumor cell killing activity, wide tumor recognition spectrum, strong tumor inhibiting activity in vivo, low cell infusion dosage and good curative effect.
According to an embodiment of the invention, the first binding fragment has the sequence as set forth in SEQ ID NO:1 or a conservatively modified version thereof, said second binding fragment having the amino acid sequence set forth in SEQ ID NO:3 or a conservatively modified version thereof. Thus, the chimeric antigen receptor of the invention can simultaneously target NKp30 receptor ligand and NKG2D receptor ligand.
According to an embodiment of the invention, the first binding fragment has the sequence as set forth in SEQ ID NO:1, said second binding fragment having an amino acid sequence as set forth in SEQ ID NO:3, and a polypeptide having the amino acid sequence shown in 3. Thus, the chimeric antigen receptor of the invention targets both NKp30 receptor ligands and NKG2D receptor ligands.
In this context, the term "conservatively modified form of an amino acid sequence" refers to an amino acid modification that does not significantly affect or alter the ligand binding properties of a receptor comprising the amino acid sequence, including amino acid substitutions, additions and deletions. Modifications may be introduced into the receptors of the invention by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
According to an embodiment of the invention, the C-terminus of the first binding fragment is linked to the N-terminus of the second binding fragment, or the N-terminus of the first binding fragment is linked to the C-terminus of the second binding fragment.
According to an embodiment of the invention, the extracellular region further comprises a first linker peptide, and the first binding fragment and the second binding fragment are linked by the first linker peptide. Thereby, the binding ability of the chimeric antigen receptor of the present invention to the NKp30 receptor ligand and the NKG2D receptor ligand is further improved.
According to an embodiment of the invention, the C-terminus of the first binding fragment is linked to the N-terminus of the first linker peptide, which is linked to the N-terminus of the second binding fragment; or the C end of the second binding fragment is connected with the N end of the first connecting peptide, and the C end of the first connecting peptide is connected with the N end of the first binding fragment. Thus, the resulting chimeric antigen receptor of the invention targets both the NKp30 receptor ligand and the NKG2D receptor ligand.
According to an embodiment of the invention, the first connecting peptide is selected from (G 4 S) n Or the amino acid sequence is shown as SEQ ID NO: 5. 34 to 39, and n is an integer other than zero.
According to an embodiment of the invention, the first connecting peptide is (G 4 S) n N is any integer between 2 and 6.
According to an embodiment of the invention, the first connecting peptide is (G 4 S) 3 . Thus, the chimeric antigen receptor of the present invention having a good binding ability to the NKp30 receptor ligand and the NKG2D receptor ligand can be obtained.
According to an embodiment of the invention, the extracellular region further comprises a hinge region. Thereby, the binding ability of the chimeric antigen receptor of the present invention to the NKp30 receptor ligand and the NKG2D receptor ligand is further improved.
According to an embodiment of the invention, the C-terminal of the first binding fragment is linked to the N-terminal of the first linker peptide, the C-terminal of the first linker peptide is linked to the N-terminal of the second binding fragment, and the C-terminal of the second binding fragment is linked to the N-terminal of the hinge region; or the C end of the second binding fragment is connected with the N end of the first connecting peptide, the C end of the first connecting peptide is connected with the N end of the first binding fragment, and the C end of the first binding fragment is connected with the N end of the hinge region. Thus, the resulting chimeric antigen receptor of the invention targets both the NKp30 receptor ligand and the NKG2D receptor ligand.
According to an embodiment of the invention, the hinge region has the sequence as set forth in SEQ ID NO:9 or a conservatively modified version thereof.
According to an embodiment of the invention, the hinge region has the sequence as set forth in SEQ ID NO: 9.
According to an embodiment of the invention, the extracellular region further comprises a second connecting peptide.
According to an embodiment of the invention, the C-terminus of the first binding fragment is linked to the N-terminus of the first linker peptide, the C-terminus of the first linker peptide is linked to the N-terminus of the second binding fragment, the C-terminus of the second binding fragment is linked to the N-terminus of the second linker peptide, and the C-terminus of the second linker peptide is linked to the N-terminus of the hinge region; or (b)
The C-terminal of the second binding fragment is connected with the N-terminal of the first connecting peptide, the C-terminal of the first connecting peptide is connected with the N-terminal of the first binding fragment, the C-terminal of the first binding fragment is connected with the N-terminal of the second connecting peptide, and the C-terminal of the second connecting peptide is connected with the N-terminal of the hinge region.
Thereby, the binding ability of the chimeric antigen receptor of the present invention to the NKp30 receptor ligand and the NKG2D receptor ligand is further improved.
According to an embodiment of the invention, the second linking peptide is selected from (G 4 S) n Or the amino acid sequence is shown as SEQ ID NO: 5. 34 to 39, and n is an integer other than zero.
According to an embodiment of the present invention, the amino acid sequence of the second connecting peptide is as shown in SEQ ID NO: shown at 5. Thus, the chimeric antigen receptor of the present invention having a good binding ability to the NKp30 receptor ligand and the NKG2D receptor ligand can be obtained.
According to an embodiment of the invention, the transmembrane region comprises a CD8a molecule transmembrane segment or fragment thereof.
According to an embodiment of the invention, the CD8a molecule transmembrane segment has the sequence as set forth in SEQ ID NO:11, and a polypeptide comprising the amino acid sequence shown in seq id no.
According to an embodiment of the invention, the intracellular region comprises a co-stimulatory domain and an intracellular signaling domain.
According to an embodiment of the invention, the C-terminal of the co-stimulatory factor domain is connected to the N-terminal of the intracellular signaling domain.
According to an embodiment of the invention, the co-stimulatory domain is selected from at least one of a CD28 molecular intracellular segment or fragment thereof, a 4-1BB molecular intracellular segment or fragment thereof, a 2B4 molecular intracellular segment or fragment thereof, a DAP10 molecular intracellular segment or fragment thereof, a DAP12 molecular intracellular segment or fragment thereof, a CD27 molecular intracellular segment or fragment thereof, a CD40 molecular intracellular segment or fragment thereof, an OX40 molecular intracellular segment or fragment thereof, an ICOS molecular intracellular segment or fragment thereof.
According to an embodiment of the invention, the co-stimulatory domain is an intracellular segment of a 4-1BB molecule or a fragment thereof. Thus, the tumor killing activity of the immune cells modified by the chimeric antigen receptor gene of the present invention can be further enhanced.
According to an embodiment of the invention, the 4-1BB molecule intracellular segment or fragment thereof has the sequence as set forth in SEQ ID NO:13, and a nucleotide sequence shown in seq id no.
According to an embodiment of the invention, the intracellular signaling domain is the intracellular segment of a cd3ζ molecule or fragment thereof. Thus, the tumor killing activity of the immune cells modified by the chimeric antigen receptor gene of the present invention can be further enhanced.
According to an embodiment of the invention, the intracellular segment of the cd3ζ molecule has the sequence as set forth in SEQ ID NO:15, and a polypeptide having the amino acid sequence shown in seq id no.
According to an embodiment of the invention, the chimeric antigen receptor has the sequence as set forth in SEQ ID NO:19 or 21. Therefore, the immune cells modified by the chimeric antigen receptor gene have remarkably enhanced tumor cell killing activity and in-vivo tumor inhibiting activity.
In a second aspect of the invention, the invention provides a nucleic acid construct. The nucleic acid construct comprises: a first nucleic acid molecule encoding the chimeric antigen receptor described above.
Nucleic acid constructs according to embodiments of the invention may encode the aforementioned chimeric antigen receptor that targets both NKp30 receptor ligand and NKG2D receptor ligand.
According to an embodiment of the invention, the first nucleic acid molecule has the sequence as set forth in SEQ ID NO:20 or 22. Thereby, the expression level of the chimeric antigen receptor of the present invention on immune cells is further increased.
According to an embodiment of the invention, the nucleic acid construct further comprises a second nucleic acid molecule, the second nucleic acid molecule being linked to the first nucleic acid molecule, the second nucleic acid molecule encoding a first fusion protein or a second fusion protein, the first fusion protein comprising IL-15, a CD8a molecule hinge region and a CD8a molecule transmembrane region; the second fusion protein includes IL-15Rα and IL-15. Thus, the activation and proliferation of immune cells expressing the chimeric antigen receptor are further promoted, the quantity and activity of the immune cells in the local microenvironment of the tumor are maintained, and the killing activity and in-vivo tumor inhibiting activity of the tumor cells are further improved.
According to an embodiment of the invention, the C-terminus of the IL-15 of the first fusion protein is linked to the N-terminus of the CD8a molecular hinge region, and the C-terminus of the CD8a molecular hinge region is linked to the N-terminus of the CD8a molecular transmembrane region.
According to an embodiment of the invention, the CD8a molecular hinge region has the sequence set forth in SEQ ID NO: 27. Thereby promoting the formation of the preferred conformation of the first fusion protein on the surface of the immune cell.
According to an embodiment of the invention, the CD8a molecule transmembrane region has the sequence as set forth in SEQ ID NO:11, and a polypeptide comprising the amino acid sequence shown in seq id no. Thus, the first fusion protein is expressed in the immune cell membrane.
According to an embodiment of the invention, the first fusion protein has the sequence as set forth in SEQ ID NO:28, and a polypeptide comprising the amino acid sequence shown in seq id no. Thus, an immune cell expressing both the chimeric antigen receptor and the first fusion protein is obtained.
According to an embodiment of the invention, the second nucleic acid molecule has the sequence as set forth in SEQ ID NO: 29. Thereby, the expression level of the chimeric antigen receptor and the first fusion protein of the invention on immune cells is further increased.
According to an embodiment of the invention, the C-terminus of IL-15Rα of the second fusion protein is linked to the N-terminus of the IL-15, or the N-terminus of the IL-15Rα is linked to the C-terminus of the IL-15.
As used herein, the term "second fusion protein" is equivalent to "mbIL15RF", "super IL15", and refers to fusion proteins of IL-15Rα and IL-15.
According to an embodiment of the invention, the second fusion protein further comprises a third connecting peptide; the C end of the IL-15Rα is connected with the N end of the third connecting peptide, and the C end of the third connecting peptide is connected with the N end of the IL-15; or the C-terminal of the IL-15 is connected with the N-terminal of the third connecting peptide, and the C-terminal of the third connecting peptide is connected with the N-terminal of the IL-15 Ralpha. Thus, a second fusion protein expressed by the cell membrane was obtained.
According to an embodiment of the invention, the third connecting peptide is selected from (G 4 S) n or amino acid sequence shown in SEQ ID NO: 5. 34 to 39, and n is an integer other than zero.
According to an embodiment of the invention, the third connecting peptide is selected from (G 4 S) n, n is any integer between 2 and 6.
According to an embodiment of the invention, the third connecting peptide is (G 4 S) 3. Thus, the second fusion protein forms a preferred configuration on the surface of the immune cell.
According to an embodiment of the invention, the IL-15Rα has the amino acid sequence as set forth in SEQ ID NO:25, and a polypeptide comprising the amino acid sequence shown in seq id no.
According to an embodiment of the invention, the IL-15 has the sequence as set forth in SEQ ID NO:26, and a polypeptide comprising the amino acid sequence shown in seq id no.
According to an embodiment of the invention, the second fusion protein has the sequence as set forth in SEQ ID NO: 23. Thus, an immune cell expressing both the chimeric antigen receptor and the second fusion protein is obtained.
According to an embodiment of the invention, the second nucleic acid molecule has the sequence as set forth in SEQ ID NO:24, and a nucleotide sequence shown in seq id no. Thereby, the expression level of the chimeric antigen receptor of the invention and the second fusion protein on immune cells is further increased.
According to an embodiment of the invention, the nucleic acid construct further comprises a third nucleic acid molecule encoding a first linker, the first and second nucleic acid molecules being linked by the third nucleic acid molecule.
According to an embodiment of the invention, the first linker is selected from at least one of P2A, T2A, E a and F2A. Thus, immune cells can be obtained which more uniformly express the chimeric antigen receptor and the first fusion protein or the second fusion protein on the cell membrane.
According to an embodiment of the present invention, the first linker is P2A. Thus, the uniform expression of the chimeric antigen receptor and the first fusion protein or the second fusion protein on the cell membrane surface can be further promoted.
According to an embodiment of the invention, the first linker has the sequence as set forth in SEQ ID NO:30, and a nucleotide sequence shown in seq id no. Thus, an immune cell is obtained which uniformly expresses the chimeric antigen receptor and the first fusion protein or the second fusion protein on the cell membrane.
According to an embodiment of the invention, the third nucleic acid molecule has the sequence as set forth in SEQ ID NO: 31. Thereby, the expression level of the chimeric antigen receptor of the invention and the first fusion protein or the second fusion protein on immune cells is further increased.
According to an embodiment of the invention, the nucleic acid construct has the sequence as set forth in SEQ ID NO:32 or 33. Thereby, the expression level of the chimeric antigen receptor of the invention and the first fusion protein or the second fusion protein on immune cells is further increased.
In a third aspect of the invention, the invention provides an expression vector. The expression vector carries the aforementioned nucleic acid construct.
According to an embodiment of the invention, the expression vector is a non-pathogenic viral vector.
According to an embodiment of the invention, the non-pathogenic virus is optionally one of a retrovirus, a chronic virus and an adenovirus-associated virus.
According to an embodiment of the invention, the non-pathogenic virus is a lentivirus. Thus, the expression vector constructed may express the chimeric antigen receptor of the present invention and/or the aforementioned first fusion protein or second fusion protein in a host cell.
In the case of ligating the above-mentioned nucleic acid molecule to an expression vector, the nucleic acid molecule may be directly or indirectly linked to control elements on the expression vector, as long as these control elements are capable of controlling translation, expression, etc. of the nucleic acid molecule. Of course, these control elements may be directly from the carrier itself or may be exogenous, i.e. not from the carrier itself. Of course, the nucleic acid molecule may be operably linked to a control element. According to an embodiment of the invention, the expression vector is a non-pathogenic viral vector.
In this context, the term "vector" generally refers to a nucleic acid molecule capable of insertion into a suitable host for self-replication, which transfers the inserted nucleic acid molecule into and/or between host cells. The vector may include a vector mainly used for inserting DNA or RNA into a cell, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of transcription and/or translation of DNA or RNA. The carrier also includes a carrier having a plurality of functions as described above. The vector may be a polynucleotide capable of transcription and translation into a polypeptide when introduced into a suitable host cell. Typically, the vector will produce the desired expression product by culturing a suitable host cell comprising the vector.
According to an embodiment of the invention, it may be obtained by operably linking the nucleic acid with a commercially available vector, such as a plasmid or a viral vector. The vector of the present invention is not particularly limited, and commonly used plasmids such as pSeTag2, PEE14, pMH3, etc. can be used.
As used herein, the term "operably linked" refers to the linkage of a foreign gene to a vector such that control elements within the vector, such as transcription and translation control sequences, and the like, are capable of performing their intended functions of regulating transcription and translation of the foreign gene. The usual vectors may be, for example, viral vectors, plasmids, phages and the like. After the expression vector according to some embodiments of the present invention is introduced into a suitable immune cell, the expression of the nucleic acid molecule can be effectively achieved under the mediation of a regulatory system, so that the protein encoded by the nucleic acid molecule can be expressed in a large amount in a cell membrane, thereby obtaining a transgenic immune cell.
According to an embodiment of the invention, the expression vector is a eukaryotic vector or a prokaryotic vector.
According to an embodiment of the invention, the expression vector is a non-pathogenic viral vector.
According to an embodiment of the invention, the non-pathogenic virus is optionally one of a retrovirus, a lentivirus and an adenovirus-associated virus.
According to an embodiment of the invention, the virus is a lentivirus.
In a fourth aspect of the invention, the invention provides a transgenic immune cell. The transgenic immune cells express the chimeric antigen receptor; or carrying the aforementioned nucleic acid construct or the aforementioned expression vector.
The immune cells according to the embodiments of the present invention are obtained by transfecting or transforming the vector or transformant, and the cells can efficiently express the aforementioned chimeric antigen receptor or the aforementioned chimeric antigen receptor and the first fusion protein or the second fusion protein under appropriate conditions.
According to an embodiment of the invention, the transgenic immune cells are selected from at least one of T cells, NK cells, NKT cells, γδ T cells, macrophages, peripheral blood NK cells, umbilical cord blood NK cells, NK-92 cells and iPSC-derived immune cells of any of the above. The chimeric antigen receptor, the first fusion protein or the second fusion protein of the present invention can be expressed on the surface of immune cells such as T, NK, NKT, γδt, macrophage, iPSC, etc., by transduction of these immune cells with an expression vector (e.g., lentiviral vector).
According to an embodiment of the invention, the transgenic immune cells are NK cells.
The invention does not limit the source of NK cells strictly, and can be NK cells from peripheral blood NK cells, umbilical cord blood NK cells, iPSC or NK-92 and the like.
In a fourth aspect of the invention, the invention provides a pharmaceutical composition. The pharmaceutical composition comprises:
the chimeric antigen receptor described above, the nucleic acid construct described above, the expression vector described above or the transgenic immune cell described above.
According to an embodiment of the present invention, the pharmaceutical composition further comprises: pharmaceutically acceptable auxiliary materials.
The term "pharmaceutical composition" as used herein generally refers to unit dosage forms and may be prepared by any of the methods well known in the pharmaceutical arts. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. Generally, the compositions are prepared by uniformly and intimately bringing into association the active compound with liquid carriers, solid carriers, or both.
As used herein, the term "pharmaceutically acceptable excipients" may include any solvent, solid excipient, diluent or other liquid excipient, and the like, suitable for the particular dosage form of interest. In addition to the extent to which any conventional adjuvant is incompatible with the chimeric antigen receptor, nucleic acid, expression vector or transgenic immune cell of the invention, such as any adverse biological effects produced or interactions with any other component of the pharmaceutically acceptable composition in a deleterious manner, their use is also contemplated by the present invention.
Those skilled in the art will appreciate that the features and advantages described above for chimeric antigen receptors, nucleic acid constructs, expression vectors and transgenic immune cells are equally applicable to the pharmaceutical compositions and will not be described in detail herein.
In a fifth aspect of the invention, the invention provides the use of the aforementioned chimeric antigen receptor, the aforementioned nucleic acid construct, the aforementioned expression vector, the aforementioned transgenic immune cell or the aforementioned pharmaceutical composition for the preparation of a medicament for the prevention or treatment of a tumor or cancer.
According to an embodiment of the invention, the tumor is a solid tumor or a hematological tumor.
In some specific embodiments of the invention, the cancer is selected from one of leukemia, lymphoma, multiple myeloma, liver cancer, cholangiocarcinoma, esophageal cancer, lung cancer, head and neck cancer, thyroid cancer, glioma, cervical cancer, ovarian cancer, gastric sarcoma, osteosarcoma, breast cancer, pancreatic cancer, melanoma, colorectal cancer, renal cancer, and prostate cancer.
In a specific embodiment of the invention, the cancer is colorectal cancer.
Those skilled in the art will appreciate that the features and advantages described above for chimeric antigen receptors, nucleic acid constructs, expression vectors, and transgenic immune cells are equally applicable for this purpose and will not be described in detail herein.
In another aspect of the invention, the invention also provides a method of preventing and/or treating a tumor or cancer. According to an embodiment of the invention, the method comprises: administering to a subject a pharmaceutically acceptable amount of the transgenic immune cell described above or the pharmaceutical composition described above.
According to an embodiment of the invention, the tumor is a solid tumor or a hematological tumor.
In some specific embodiments of the invention, the cancer is selected from one of leukemia, lymphoma, multiple myeloma, liver cancer, cholangiocarcinoma, esophageal cancer, lung cancer, head and neck cancer, thyroid cancer, glioma, cervical cancer, ovarian cancer, gastric sarcoma, osteosarcoma, breast cancer, pancreatic cancer, melanoma, colorectal cancer, renal cancer, and prostate cancer.
In a specific embodiment of the invention, the cancer is colorectal cancer.
As used herein, the term "administering" refers to introducing a predetermined amount of a substance into a patient by some suitable means. The transgenic immune cell or pharmaceutical composition of the present invention may be administered by any common route as long as it can reach the intended tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the invention is not limited to these illustrated modes of administration. Preferably, the compositions of the present invention are administered intravenously.
In this context, the term "treatment" refers to the use to obtain a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or symptoms thereof, and/or may be therapeutic in terms of partially or completely curing the disease and/or adverse effects caused by the disease. As used herein, "treating" encompasses diseases in mammals, particularly humans, including: (a) Preventing the occurrence of a disease or disorder in an individual susceptible to the disease but not yet diagnosed with the disease; (b) inhibiting disease, e.g., arresting disease progression; or (c) alleviating a disease, e.g., alleviating symptoms associated with a disease. As used herein, "treating" encompasses any administration of a drug or transgenic immune cell to an individual to treat, cure, alleviate, ameliorate, reduce or inhibit a disease in the individual, including, but not limited to, administration of a drug comprising a chimeric antigen receptor-containing cell described herein to an individual in need thereof.
The effective amount of the transgenic immune cells and pharmaceutical compositions of the present invention may vary depending on the mode of administration, the severity of the disease to be treated, and the like. Preferably, the selection of an effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life etc.; the severity of the disease to be treated in the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. For example, separate doses may be administered several times per day, or the dose may be proportionally reduced, as dictated by the urgent need for the treatment of the condition.
As used herein, the term "effective amount" or "effective dose" refers to an amount that is functional or active in and acceptable to a human and/or animal.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a schematic diagram of the structure of a different chimeric antigen receptor according to example 1 of the present invention;
FIG. 2 is a graph showing the statistical results of killing efficiency of NCI-H716 cells by different CAR-NK cells of example 2 of the present invention;
FIG. 3 is a schematic representation of the CAR structure containing membrane-expressed IL15 (FIG. 3-A) or super IL15 (FIG. 3-B) according to example 3 of the present invention;
FIG. 4 is a graph showing fold change in NK-92 cell and CAR5-NK-92 cell of the present invention under different IL-2 culture concentrations, wherein the CAR5-NK-92 membrane in FIG. 4-A expresses IL15 and the CAR5-NK-92 membrane in FIG. 4-B expresses super IL15;
FIG. 5 is a graph showing the change in survival rate of NK-92 and CAR5-NK-92 cells of example 3 of the present invention at different IL-2 culture concentrations, wherein the CAR5-NK-92 membrane in FIG. 5-A expresses IL15 and the CAR5-NK-92 membrane in FIG. 5-B expresses super IL15;
FIG. 6A graph of the tumor-inhibiting effect of peripheral blood derived CAR5-NK cells of example 4 of the present invention on a mouse model with human colorectal cancer NCI-H716 cells tumor-bearing, wherein the CAR5-NK-92 membrane expressed IL15 in FIG. 6-A and the CAR5-NK-92 membrane expressed super IL15 in FIG. 6-B.
Detailed Description
Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and such range or value should be understood to encompass values approaching those range or value. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
Terms and definitions
In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but not to exclude other aspects.
Herein, the terms "according to embodiments of the present invention", "optional" or "optional" mean generally that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
Herein, the term "(G) 4 S) n "equivalent to" (Gly) 4 Ser) n ", which represents that 4 glycine and 1 serine are repeated n times, is a connecting peptide widely used at present, and can be positioned at V H C-terminal and V L Between N ends, also can be positioned at V L C-terminal and V H And N ends. At present, it is common to use (G 4 S) 3 Wherein glycine is the amino acid with the smallest molecular mass and the shortest side chain, which can increase the flexibility of the side chain, serine is the amino acid with the strongest hydrophilicity, and can increase the hydrophilicity of the connecting peptide. Thus, (G) 4 S) 3 Has good stability and activity. Connecting peptides with different lengths and sequences can be designed to construct scFv with different biological functions.
As used herein, the term "hinge region" refers to the attachment of CH in an antibody 1 And CH (CH) 2 (i.e., the heavy chain region linking Fab and Fc). Also sometimes referred to herein as an "Fc hinge region".
Herein, "carbon end" and "C end" are synonymous; "Nitrogen end" and "N end" are synonymous.
The details of the sequences involved in the present invention are shown in Table 1.
Table 1: nucleotide/amino acid sequence specification table
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The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The "plasmid" and "vector" described in the following examples have the same meaning and are used interchangeably.
Experimental example 1: preparation of NK cells expressing different CAR structures
1. Construction of different CAR structures
Different CAR sequences are synthesized through total genes, cloned to a lentiviral vector pCDH-EF1-MCS-TA 2-copGGFP through enzyme cutting sites XbaI and BamHI, and the plasmids pCDH-EF1a-CAR1, pCDH-EF1a-CAR2, pCDH-EF1a-CAR3 and pCDH-EF1a-CAR4 are obtained after sequencing verification to be correct.
The structural schematic of the genetic elements of the chimeric antigen receptor of this example is shown in FIG. 1, and the nucleotide/amino acid sequences are shown in Table 1. Wherein, the nucleotide sequences of CAR1, CAR2, CAR3 and CAR4 are set forth in SEQ ID NO:40 to 41, 20 and 22.
2. Packaging of lentiviruses
Taking 293T cells in logarithmic growth phase 5X 10 6 Inoculating into 10cm cell culture dish, adding 10mL DMEM medium, 37 deg.C, 5% CO 2 Culturing overnight in an incubator. When the cell density in the cell culture dish reaches 80-90%, 10mL of fresh DMEM medium is replaced, and the cell culture dish is placed in an incubator for standby. Preparing a slow virus packaging system, adding 6 mug of psPAX2 and 3 mug of pMD2.G of slow virus packaging auxiliary plasmid and 6 mug of target gene vector plasmid into 250 mug serum-free DMEM culture medium to prepare plasmid mixed solution, and uniformly mixing. Will be 15Added into 235 mu L of serum-free DMEM medium, and mixed uniformly. Will->The mixed solution is added into the plasmid mixed solution at one time, mixed evenly and incubated for 15min at room temperature. The mixture was added to 293T cell culture dishes. After 24h, the liquid was changed and the dish was returned to 37℃with 5% CO 2 In the incubator, the cell supernatant was collected after 48 hours, centrifuged at 400 Xg for 5 minutes, cell debris was removed, and the supernatant was filtered into a 50mL centrifuge tube with a 0.45 μm filter head. Adding 5 XPEG 8000 solution to make virus liquid concentration, The centrifuge tubes were turned upside down and mixed well and placed in a refrigerator at 4 ℃ overnight. Centrifuging at 4deg.C at 4000 Xg for 20min, discarding supernatant, adding appropriate amount of serum-free DMEM to resuspend virus precipitate, transferring into EP tube, and storing in refrigerator at-80deg.C.
3. Lentivirus infects human NK-92 cells
Sucking NK-92 cells in logarithmic phase, centrifuging at 100×g for 5min to obtain cells, adding appropriate amount of alpha-MEM culture medium to resuspend cells, and adjusting cell density to 5×10 5 And each mL. The respective 5X 10 holes are connected into 24 hole plates 5 NK-92 cells, 0.2mL virus concentrate, 0.8mL alpha-MEM medium and protamine (final concentration 8. Mu.g/mL) were mixed well. Placing at 37deg.C and 5% CO 2 Culturing in an incubator. After 24h, the cell status was observed, the liquid was changed, the infected cells were transferred into EP tube, centrifuged at 100 Xg for 5min, the cells were resuspended in a small amount of fresh alpha-MEM medium, transferred into cell culture flasks, and cultured for a further 48h with 10mL of fresh alpha-MEM medium and IL-2 (final concentration of 200 IU/mL). Cells were transferred into a inflow tube, 3mL of 1 XPBS solution was added, 100 XPS was centrifuged for 5min, the supernatant was discarded, the cell pellet was sprung, and washed once again with 1 XPBS solution. The expression rate of GFP was measured using a flow meter. And continuing to expand culture, and adjusting the state of NK-92 cells after infection to expand. The infected NK-92 cells were sorted by flow meter for GFP-positive CAR-NK92 cells for later experiments.
The inventor further tests and observes that the CAR3-NK92 cells and the CAR4-NK92 cells of the embodiment can play an effective killing role on tumor cells which singly express NKp30 receptor ligand or NKG2D receptor ligand or simultaneously express two receptor ligands, and especially can effectively identify tumor cells losing one receptor ligand of NKp30 or NKG2D and prevent immune escape of tumors.
Experimental example 2: comparison of killing efficiency of different CAR structures
In this example, the inventors examined the killing activity of the different CAR-NK cells obtained in example 1 in vitro, verifying the advantages of dual targeting over single targeting, and preferring CAR structures with optimal killing activity.
The inventor selects colorectal cancer cell line NCI-H716 cells which are highly expressed by NKG2D ligand MICA/B and NKG 30 ligand B7-H6 as target cells, and carries out in-vitro killing activity detection on different CAR-NK cells.
The specific method comprises the following steps: NCI-H716 cells were fluorescently labeled using CFSE according to 2X10 4 The individual cells/wells were accessed into 96-well plates and NK-92 or different CAR-NK-92 cells were plated separately and incubated with NCI-H716 cells for 4H. And then adding PI (polyimide) to dye and distinguish dead and living cells, and detecting the killing efficiency by a flow cytometer. The ratio of effector cells to target cells was examined to be 2:1.
The test results are shown in FIG. 2.
The investigation results of the killing activity of different CAR structures show that: compared with the single-targeted CAR1-NK-92 or CAR2-NK-92 cells, the killing efficiency of the double-targeted CAR3-NK-92 and CAR4-NK-92 cells on NCI-H716 cells is obviously enhanced.
Example 3: investigation of Membrane-expressed IL15 element to increase proliferation and survival of CAR-NK cells
1. CAR sequence construction containing Membrane-expressed IL15 elements
In this example, the inventors designed and constructed a CAR5 gene by adding a membrane-expressed IL15 element to the CAR3 sequence, taking CAR3 of example 1 as an example. The CAR5 gene is synthesized through the whole gene, cloned to a lentiviral vector pCDH-EF1-MCS-TA 2-copGGFP through enzyme cutting sites XbaI and BamHI, and the pCDH-EF1a-CAR5 plasmid is obtained after sequencing verification to be correct.
The structural schematic of the genetic elements of the chimeric antigen receptor of this example is shown in FIG. 3 and the nucleotide/amino acid sequence is shown in Table 1. Wherein, the nucleotide sequences of CAR5A and CAR5B are shown in SEQ ID NO: 32. 33.
2. CAR5 gene modification for promoting NK cell proliferation activity investigation
The inventors further validated the effect of membrane-expressed IL15 on NK-92 cells to promote proliferation in vitro.
The specific method comprises the following steps: respectively will be 3×10 6 Cell numbers NK-92 and CAR5-NK-92 cells were plated in T25 cell culture flasks and different IL-2 concentrations (200 IU/ml and 0 IU/ml) were set, and cell counts were performed every 2 days. And will be 3 x 10 6 Cell number transfer into novel T25 cell culture flaskAnd IL-2 is added with corresponding concentration, and the next round of cell culture and counting is started. If the total number of cells is less than 3X 10 6 Cells, all cells were transferred to a new T25 cell culture flask.
The results of NK cell proliferation fold investigation by day 10 of culture are shown in fig. 4. Wherein, in FIG. 4-A, the CAR5-NK-92 membrane expresses IL15, and in FIG. 4-B, the CAR5-NK-92 membrane expresses super IL15.
NK cell proliferation activity examination results showed that: at the concentration of 200IU/mL IL-2, the expansion times of the CAR5-NK-92 cells are obviously higher than that of the NK-92 cell group; at an IL-2 concentration of 0IU/mL, the expansion times of the CAR5-NK-92 cells are obviously higher than that of the NK-92 cell group, and the NK-92 cell group is basically not expanded.
The results show that the IL15 expressed by the membrane in the CAR5-NK-92 plays an important role in promoting the expansion of NK cells, can further improve the expansion capacity of NKR-NK cells in vivo, and can reduce the dependence of NK cell expansion on IL-2.
3. CAR5 gene modification facilitates NK cell survival rate investigation
The inventors further validated the effect of membrane-expressed IL15 on NK-92 cells to promote survival in vitro.
The specific method comprises the following steps: NK-92 and CAR5-NK-92 cell plates of the same cell number were plated in 24 well plates, respectively, and different IL-2 concentrations (200 IU/ml and 0 IU/ml) were set, and the apoptosis rate was examined by flow cytometry every 24 hours. Flow cytometry detection of apoptosis rate was performed according to the procedure of the kit instructions (biankia, cat No. AP 101), briefly: cells were collected in EP tubes, washed once by centrifugation with 1xPBS solution, and resuspended. mu.L of Annexin V-FITC and 10. Mu.L of PI were added to each tube. After gentle vortexing, incubation for 5min at room temperature in the dark, and flow detection of resuspended cells. Cell viability was the proportion of Annexin V-FITC and PI staining double negative.
The results of NK cell viability investigation by day 8 of culture are shown in fig. 5. Wherein, in FIG. 5-A, the CAR5-NK-92 membrane expresses IL15, and in FIG. 5-B, the CAR5-NK-92 membrane expresses super IL15.
NK cell viability investigation results show that: NK-92 group in the absence of IL-2 (IL-2 0 IU/ml), cell viability was significantly reduced from day 4 to day 8 when most of the cells had been apoptotic; and the CAR5-NK-92 group cells can still keep high cell viability even under the condition of completely removing IL-2, and few cells undergo apoptosis.
The results show that the IL15 expressed by the membrane in the CAR5-NK-92 plays an important role in promoting the survival of NK cells, can further improve the long-term survival of NKR-NK cells in vivo, and can reduce the dependence of NK cell survival on IL-2.
Example 4: in vivo tumor-inhibiting activity investigation of primary CAR5-NK cells
The inventor establishes a mouse subcutaneous tumor-bearing model by using human colorectal cancer NCI-H716 cells, prepares peripheral blood-derived CAR-NK cells by infecting human peripheral blood primary NK cells with CAR5 lentivirus, and observes the treatment effect of the primary CAR5-NK cells on the colorectal cancer tumor-bearing model.
The specific method comprises the following steps: NCG mice with 6 weeks of age were selected for underarm tumor loading until tumor volume reached 50mm 3 NK cell therapy was started at this time. Tumor volume size was measured prior to treatment and randomly divided into untreated, NK cell treated and CAR5-NK cell treated groups according to tumor volume size. In the NK cell treatment group, mice were treated 1 time every 2 days by tail vein reinfusion of NK cells 3 times, each tail vein reinfusion dose was 8×10 6 CD56 + NK cells and were maintained viable by intraperitoneal injection of IL-2 every 2 days; in the CAR5-NK cell treatment group, CAR5-NK cells were infused back through tail vein in single treatment at doses of 4×10, respectively 6 CAR5-NK cells/only, and no IL-2 injection. And continuously observing and measuring the size of the tumor volume, and drawing a tumor growth curve.
The test results are shown in FIG. 6. Wherein, in FIG. 6-A, the CAR5-NK-92 membrane expresses IL15, and in FIG. 6-B, the CAR5-NK-92 membrane expresses super IL15.
The in vivo tumor inhibition activity investigation result shows that: compared with a non-infected NK cell treatment group (a control group, NK cells do not carry any form of CAR or CSR), the peripheral blood source CAR5-NK cells prepared based on the CAR5 have the remarkably enhanced effect of inhibiting tumor growth, and the dose of reinfusion NK cells is lower.
The results show that the CAR5-NK cells have strong anti-tumor effect on colorectal cancer tumors, are lower in application dosage, and are expected to break through the bottleneck of poor treatment effect of immune cell therapy on solid tumors.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (12)
1. A chimeric antigen receptor comprising:
An extracellular region comprising a first binding fragment and a second binding fragment, the first binding fragment and the second binding fragment being linked, the first binding fragment being an NKp30 extracellular segment or a fragment thereof, the second binding fragment being an NKG2D extracellular segment or a fragment thereof;
the N end of the transmembrane region is connected with the C end of the extracellular region;
and an intracellular region, wherein the N-terminal of the intracellular region is connected with the C-terminal of the transmembrane region.
2. The chimeric antigen receptor according to claim 1, wherein the first binding fragment has the sequence set forth in SEQ ID NO:1 or a conservatively modified version thereof, said second binding fragment having the amino acid sequence set forth in SEQ ID NO:3 or a conservatively modified version thereof.
3. The chimeric antigen receptor according to claim 1, wherein the C-terminus of the first binding fragment is linked to the N-terminus of the second binding fragment or the N-terminus of the first binding fragment is linked to the C-terminus of the second binding fragment;
optionally, the extracellular region further comprises a first linker peptide, the first binding fragment and the second binding fragment being linked by the first linker peptide;
Optionally, the C-terminus of the first binding fragment is linked to the N-terminus of the first linker peptide, which is linked to the N-terminus of the second binding fragment; or (b)
The C end of the second binding fragment is connected with the N end of the first connecting peptide, and the C end of the first connecting peptide is connected with the N end of the first binding fragment;
optionally, the first connecting peptide is selected from (G 4 S) n Or the amino acid sequence is shown as SEQ ID NO: 5. 34 to 39, n is an integer other than zero;
optionally, the first connecting peptide is (G 4 S) n N is any integer between 2 and 6;
optionally, the first connecting peptide is (G 4 S) 3 ；
Optionally, the extracellular region further comprises a hinge region;
optionally, the C-terminus of the first binding fragment is linked to the N-terminus of the first linker peptide, the C-terminus of the first linker peptide is linked to the N-terminus of the second binding fragment, and the C-terminus of the second binding fragment is linked to the N-terminus of the hinge region; or (b)
The C end of the second binding fragment is connected with the N end of the first connecting peptide, the C end of the first connecting peptide is connected with the N end of the first binding fragment, and the C end of the first binding fragment is connected with the N end of the hinge region;
Optionally, the hinge region has the amino acid sequence as set forth in SEQ ID NO:9 or a conservatively modified version thereof;
optionally, the extracellular region further comprises a second connecting peptide;
optionally, the C-terminus of the first binding fragment is linked to the N-terminus of the first linker peptide, the C-terminus of the first linker peptide is linked to the N-terminus of the second binding fragment, the C-terminus of the second binding fragment is linked to the N-terminus of the second linker peptide, and the C-terminus of the second linker peptide is linked to the N-terminus of the hinge region; or (b)
The C end of the second binding fragment is connected with the N end of the first connecting peptide, the C end of the first connecting peptide is connected with the N end of the first binding fragment, the C end of the first binding fragment is connected with the N end of the second connecting peptide, and the C end of the second connecting peptide is connected with the N end of the hinge region;
optionally, the second connecting peptide is selected from (G 4 S) n Or the amino acid sequence is shown as SEQ ID NO: 5. 34 to 39, n is an integer other than zero;
optionally, the amino acid sequence of the second connecting peptide is as set forth in SEQ ID NO: shown at 5.
4. The chimeric antigen receptor according to claim 1, wherein the transmembrane region comprises a CD8a molecular transmembrane segment or fragment thereof;
Optionally, the CD8a molecule transmembrane segment has the amino acid sequence as set forth in SEQ ID NO:11, and a polypeptide comprising the amino acid sequence shown in seq id no;
optionally, the intracellular region comprises a costimulatory domain and an intracellular signaling domain;
optionally, the C-terminus of the co-stimulatory factor domain is linked to the N-terminus of the intracellular signaling domain;
optionally, the co-stimulatory domain is selected from at least one of a CD28 molecular intracellular segment or fragment thereof, a 4-1BB molecular intracellular segment or fragment thereof, a 2B4 molecular intracellular segment or fragment thereof, a DAP10 molecular intracellular segment or fragment thereof, a DAP12 molecular intracellular segment or fragment thereof, a CD27 molecular intracellular segment or fragment thereof, a CD40 molecular intracellular segment or fragment thereof, an OX40 molecular intracellular segment or fragment thereof, an ICOS molecular intracellular segment or fragment thereof;
optionally, the costimulatory domain is the intracellular segment of a 4-1BB molecule or a fragment thereof;
optionally, the 4-1BB molecule intracellular fragment or fragment thereof has the amino acid sequence as set forth in SEQ ID NO:13, an amino acid sequence shown in seq id no;
optionally, the intracellular signaling domain is an intracellular segment of a cd3ζ molecule or fragment thereof;
optionally, the cd3ζ molecule intracellular segment has an amino acid sequence as set forth in SEQ ID NO:15, and a polypeptide comprising the amino acid sequence shown in seq id no;
optionally, the chimeric antigen receptor has the amino acid sequence as set forth in SEQ ID NO:19 or 21.
5. A nucleic acid construct comprising a first nucleic acid molecule encoding the chimeric antigen receptor of any one of claims 1-4;
optionally, the first nucleic acid molecule has a sequence as set forth in SEQ ID NO:20 or 22.
6. The nucleic acid construct of claim 5, further comprising a second nucleic acid molecule, said second nucleic acid molecule being linked to the first nucleic acid molecule, said second nucleic acid molecule encoding a first fusion protein or a second fusion protein, said first fusion protein comprising IL-15, a CD8a molecule hinge region and a CD8a molecule transmembrane region; the second fusion protein comprises IL-15Rα and IL-15;
optionally, the C-terminus of the IL-15 of the first fusion protein is linked to the N-terminus of the CD8a molecular hinge region, the C-terminus of the CD8a molecular hinge region being linked to the N-terminus of the CD8a molecular transmembrane region;
optionally, the CD8a molecular hinge region has the amino acid sequence as set forth in SEQ ID NO:27, and a polypeptide sequence as set forth in seq id no;
optionally, the CD8a molecule transmembrane region has the amino acid sequence as set forth in SEQ ID NO:11, and a polypeptide comprising the amino acid sequence shown in seq id no;
optionally, the first fusion protein has the sequence as set forth in SEQ ID NO:28, and a polypeptide comprising the amino acid sequence shown in seq id no;
Optionally, the second nucleic acid molecule has a sequence as set forth in SEQ ID NO:29, a nucleotide sequence shown in seq id no;
optionally, the C-terminus of IL-15Rα of the second fusion protein is linked to the N-terminus of the IL-15, or the N-terminus of the IL-15Rα is linked to the C-terminus of the IL-15;
optionally, the second fusion protein further comprises a third connecting peptide; optionally, the C-terminus of the IL-15 ra is linked to the N-terminus of the third connecting peptide, which is linked to the N-terminus of the IL-15; or (b)
The C-terminal of the IL-15 is connected with the N-terminal of the third connecting peptide, and the C-terminal of the third connecting peptide is connected with the N-terminal of the IL-15 Ralpha;
optionally, the third connecting peptide is selected from (G 4 S) n or amino acid sequence shown in SEQ ID NO: 5. 34 to 39, n is an integer other than zero;
optionally, the third connecting peptide is selected from (G 4 S) n, n is any integer between 2 and 6;
optionally, the third connecting peptide is (G) 4 S)3；
Optionally, the IL-15 ra has the amino acid sequence as set forth in SEQ ID NO:25, an amino acid sequence shown in seq id no;
optionally, the IL-15 has the amino acid sequence as set forth in SEQ ID NO:26, and a polypeptide comprising the amino acid sequence shown in seq id no;
optionally, the second fusion protein has the sequence as set forth in SEQ ID NO:23, an amino acid sequence shown in seq id no;
Optionally, the second nucleic acid molecule has a sequence as set forth in SEQ ID NO:24, and a nucleotide sequence shown in seq id no.
7. The nucleic acid construct of claim 6, further comprising a third nucleic acid molecule encoding a first linker, the first and second nucleic acid molecules being linked by the third nucleic acid molecule;
optionally, the first linker is selected from at least one of P2A, T2A, E2A and F2A;
optionally, the first linker is P2A;
optionally, the first linker has the sequence set forth in SEQ ID NO:30, an amino acid sequence shown in seq id no;
optionally, the third nucleic acid molecule has a sequence as set forth in SEQ ID NO: 31.
8. The nucleic acid construct of claim 7, having the sequence set forth in SEQ ID NO:32 or 33.
9. An expression vector carrying the nucleic acid construct of any one of claims 5 to 8;
optionally, the expression vector is a non-pathogenic viral vector;
optionally, the non-pathogenic virus is optionally one of a retrovirus, a lentivirus virus, and an adenovirus-associated virus;
optionally, the non-pathogenic virus is a lentivirus.
10. A transgenic immune cell, the transgenic immune cell comprising:
expressing the chimeric antigen receptor of any one of claims 1 to 4; or alternatively
Carrying the nucleic acid construct of any one of claims 5 to 7, the expression vector of claim 9;
optionally, the transgenic immune cell is selected from at least one of T cells, NK cells, NKT cells, γδ T cells, macrophages, peripheral blood NK cells, umbilical cord blood NK cells, NK-92 cells, and iPSC-derived any one of the above immune cells;
optionally, the transgenic immune cell is an NK cell.
11. A pharmaceutical composition comprising:
the chimeric antigen receptor of any one of claims 1 to 4, the nucleic acid construct of any one of claims 5 to 7, the expression vector of claim 9, or the transgenic immune cell of claim 10;
optionally, further comprising: pharmaceutically acceptable auxiliary materials.
12. Use of the chimeric antigen receptor of any one of claims 1 to 4, the nucleic acid construct of any one of claims 5 to 7, the expression vector of claim 9, the transgenic immune cell of claim 10 or the pharmaceutical composition of claim 11 in the preparation of a medicament for the prevention and/or treatment of a tumor or cancer;
Optionally, the tumor is a solid tumor or a hematological tumor;
optionally, the cancer is colorectal cancer.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107982538A (en) * | 2017-12-26 | 2018-05-04 | 深圳市体内生物医药科技有限公司 | A kind of pharmaceutical composition and its application |
| CN108276493A (en) * | 2016-12-30 | 2018-07-13 | 南京传奇生物科技有限公司 | A kind of Novel chimeric antigen receptor and its application |
| CN109734813A (en) * | 2019-01-28 | 2019-05-10 | 广东昭泰体内生物医药科技有限公司 | A kind of Chimeric antigen receptor and its application |
| CN112154204A (en) * | 2018-05-15 | 2020-12-29 | 科济生物医药（上海）有限公司 | Genetically engineered cells and uses |
| CN112384534A (en) * | 2018-05-21 | 2021-02-19 | 指南针制药有限责任公司 | Compositions and methods for enhancing killing of target cells by NK cells |
| CN113698491A (en) * | 2020-05-22 | 2021-11-26 | 重庆精准生物技术有限公司 | Fusion protein, CD 19-targeted CAR and application thereof |
| WO2022048523A1 (en) * | 2020-09-02 | 2022-03-10 | 南京北恒生物科技有限公司 | Chimeric antigen receptor targeting nk activated receptor |
-
2023
- 2023-09-28 CN CN202311287169.5A patent/CN117467022A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108276493A (en) * | 2016-12-30 | 2018-07-13 | 南京传奇生物科技有限公司 | A kind of Novel chimeric antigen receptor and its application |
| CN107982538A (en) * | 2017-12-26 | 2018-05-04 | 深圳市体内生物医药科技有限公司 | A kind of pharmaceutical composition and its application |
| CN112154204A (en) * | 2018-05-15 | 2020-12-29 | 科济生物医药（上海）有限公司 | Genetically engineered cells and uses |
| CN112384534A (en) * | 2018-05-21 | 2021-02-19 | 指南针制药有限责任公司 | Compositions and methods for enhancing killing of target cells by NK cells |
| CN109734813A (en) * | 2019-01-28 | 2019-05-10 | 广东昭泰体内生物医药科技有限公司 | A kind of Chimeric antigen receptor and its application |
| CN113698491A (en) * | 2020-05-22 | 2021-11-26 | 重庆精准生物技术有限公司 | Fusion protein, CD 19-targeted CAR and application thereof |
| WO2022048523A1 (en) * | 2020-09-02 | 2022-03-10 | 南京北恒生物科技有限公司 | Chimeric antigen receptor targeting nk activated receptor |
| CN116063550A (en) * | 2020-09-02 | 2023-05-05 | 南京北恒生物科技有限公司 | Chimeric antigen receptor targeting NK-activating receptor |
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