CN116925236B - Chimeric transition receptors and uses thereof - Google Patents
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- CN116925236B CN116925236B CN202310541428.6A CN202310541428A CN116925236B CN 116925236 B CN116925236 B CN 116925236B CN 202310541428 A CN202310541428 A CN 202310541428A CN 116925236 B CN116925236 B CN 116925236B
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Abstract
The invention provides a chimeric conversion receptor capable of simultaneously recognizing ligand antigens of two immune receptors and application thereof. The chimeric transition receptor comprises: an extracellular region comprising a first binding fragment and a second binding fragment; 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 conversion receptor can simultaneously recognize ligand antigens of two immune receptors, and immune cells expressing the chimeric conversion receptor can be activated after recognizing any one of the ligand antigens of the two immune receptors, so that the logical gating effect of immune cells on tumor antigen recognition is realized, and the curative effect of CSR-immune cells is improved.
Description
Technical Field
The invention relates to the field of bio-pharmaceuticals, in particular to a chimeric conversion receptor and application thereof, in particular to a CSR-immune cell and application thereof in the field of tumor treatment, and more particularly to a chimeric conversion receptor capable of simultaneously recognizing two antigen ligands, a corresponding nucleic acid molecule, an expression vector, a lentiviral vector, a transgenic immune cell, a pharmaceutical composition and application thereof.
Background
The chimeric transducer Receptor (CSR, chimeric Switch Receptor) is an artificially constructed recombinant Receptor comprising an extracellular antigen recognition domain, a transmembrane region and an intracellular signaling domain. Unlike the extracellular antigen recognition domain of a chimeric antigen receptor, which is composed of single chain antibodies, the extracellular antigen recognition domain of a chimeric switch receptor is composed mainly of inhibitory surface receptors of immune cells. The chimeric transducer receptor converts the original immunosuppressive signal into an immunocompetent signal by recognizing the ligand of the inhibitory receptor, activates immune cells, and triggers an immune response against target cells expressing the ligand of the inhibitory receptor. Immune cell therapies based on chimeric switching receptors are an emerging direction in the field of tumor therapy.
The immune cell therapy of the chimeric conversion receptor has better application prospect in tumor treatment, but the curative effect still needs to be further improved.
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 chimeric conversion receptor can simultaneously recognize ligand antigens of two immune receptors, and immune cells expressing the chimeric conversion receptor can be activated after recognizing any one of the ligand antigens of the two immune receptors, so that the logical gating effect of immune cells on tumor antigen recognition is realized, the problems of tumor immune escape and tumor antigen heterogeneity are solved, the inhibition of inhibitory signals from tumor microenvironment is more effectively reversed, immune cells such as NK cells are prevented from being subjected to immune exhaustion, and the therapeutic effect of immune cells based on the chimeric conversion receptor is effectively improved.
The present invention has been completed based on the following work of the inventors:
Tumors are heterogeneous, and although the ligand of the inhibitory receptor TIGIT and the ligand of the inhibitory receptor PD-1 are highly expressed in tumor cells, they are often not expressed simultaneously even in the same tumor cell, such as occurs when only a portion of the tumor cells express one ligand and another portion of the tumor cells express another ligand; at the same time, the tumor escape mechanism also causes the ligand loss of tumor cells TIGIT or PD-1. Therefore, conventional single chimeric antigen receptors or chimeric conversion receptors cannot be recognized effectively and have limited therapeutic effects.
In order to solve the above problems, the inventors have largely proposed the following idea: two chimeric conversion receptors are simultaneously expressed in one immune cell (such as NK cells or T cells), and the obtained immune cell is expected to effectively identify tumor cells under the two conditions, so that the anti-tumor effect is triggered and the curative effect is improved. Further, the inventors selected the extracellular domains of the human inhibitory receptor TIGIT and the human inhibitory receptor PD-1 as extracellular antigen recognition domains of chimeric switching receptors. Through a large number of bioinformatics simulation and screening tests, two TIGIT and PD-1 extracellular truncations with good binding activity with PD-L1 and CD155 are obtained.
To further enhance the therapeutic effect of immune cells, the inventors skillfully linked TIGIT and the extracellular truncations of PD-1 and used as extracellular antigen recognition domains to obtain chimeric transition receptors containing both antigen recognition domains.
Further test results show that NK cells modified by the chimeric conversion receptor gene containing the TIGIT and PD-1 antigen recognition domains can simultaneously recognize the TIGIT ligand and the PD-1 ligand, and can reverse any inhibitory signals transmitted by the TIGIT or PD-1 recognition corresponding ligand into activation signals, so that NK cells in tumor microenvironment are prevented from losing functions or being exhausted. Further animal level test results show that the genetically modified NK cells can effectively identify tumor cells which partially express TIGIT ligand or partially express PD-1 ligand and also can effectively identify tumor cells which lose one receptor ligand of PD-1 or TIGIT. Thus, the problems of tumor immune escape and tumor antigen heterogeneity are solved, and the therapeutic effect of immune cells based on chimeric conversion receptors is improved.
Thus, in a first aspect of the invention, the invention provides a chimeric transducer receptor. The chimeric transition receptor comprises: an extracellular region comprising a first binding fragment and a second binding fragment, the first binding fragment having a sequence as set forth in SEQ ID NO:10, said second binding fragment having an amino acid sequence as set forth in SEQ ID NO:11, and a polypeptide comprising the amino acid sequence shown in seq id no; 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 conversion receptor of the invention has the binding activity of PD-L1 and CD155, and immune cells expressing the chimeric conversion receptor of the invention can effectively identify tumor cells partially expressing TIGIT ligand or partially expressing PD-1 ligand and also can effectively identify tumor cells losing one receptor ligand of PD-1 or TIGIT.
In a second aspect of the invention, the invention provides a nucleic acid molecule encoding the chimeric transducer receptor described above. The chimeric conversion receptor of the first aspect of the invention can be expressed in immune cells by carrying the nucleic acid molecules, and can activate the immune cells after recognizing any one of TIGIT ligand and PD-1 ligand, so as to realize the logical gate effect of the immune cells on tumor antigen recognition.
In a third aspect of the invention, the invention provides an expression vector carrying a nucleic acid molecule of the second aspect of the invention. Thus, the chimeric conversion receptor of the first aspect of the present invention can be expressed efficiently in cells by using the expression vector thus constructed.
In a fourth aspect of the invention, the invention provides a lentiviral vector carrying a nucleic acid sequence having the amino acid sequence as set forth in SEQ ID NO:1 or SEQ ID NO:15, and a nucleotide sequence shown in seq id no. After introducing the lentiviral vector of the fourth aspect of the invention into a recipient cell, the recipient cell is capable of effecting expression of the chimeric switch receptor of the first aspect of the invention.
In a fifth aspect of the invention, the invention provides a transgenic immune cell expressing the chimeric switching receptor of the first aspect of the invention, or carrying the nucleic acid molecule of the second aspect of the invention, the expression vector of the third aspect of the invention or the lentiviral vector of the fourth aspect of the invention. Thus, the obtained transgenic immune cells can be further used for tumor treatment, and have significantly improved tumor killing activity and effectively reduced immune cell depletion state. Clinically, the drug resistance of tumor patients to therapeutic immune cells, which is generated by immune escape and/or tumor antigen heterogeneity, can be effectively eliminated only by applying the immune cells of the invention. Meanwhile, compared with a treatment scheme of sequentially or simultaneously applying a plurality of therapeutic immune cells to identify different tumor cell ligands, the immune cell provided by the invention is safer and more convenient in clinical application, and is beneficial to clinical application of cell therapy.
In a sixth aspect of the invention, the invention provides a pharmaceutical composition comprising the chimeric switching receptor of the first aspect of the invention, the nucleic acid molecule of the second aspect of the invention, the expression vector of the third aspect of the invention, the lentiviral vector of the fourth aspect of the invention or the transgenic immune cell of the fifth aspect of the invention. The obtained pharmaceutical composition can be further used for preventing or treating tumor diseases.
In a seventh aspect of the invention, the invention provides the use of a chimeric switch receptor of the first aspect of the invention, a nucleic acid molecule of the second aspect of the invention, an expression vector of the third aspect of the invention, a lentiviral vector of the fourth aspect of the invention, a transgenic immune cell of the fifth aspect of the invention or a pharmaceutical composition of the sixth aspect of the invention in the manufacture of a medicament for the prevention or treatment of a tumor. The chimeric conversion receptor and the corresponding nucleic acid molecules, expression vectors, lentiviral vectors, transgenic immune cells or pharmaceutical compositions can be further prepared into medicaments which can be clinically used for preventing or treating diseases.
Those skilled in the art will appreciate that the features and advantages described above for chimeric transducer receptors, nucleic acid molecules, expression vectors, lentiviral vectors, transgenic immune cells and pharmaceutical compositions are equally applicable for this use and will not be described in detail herein.
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 a chimeric double (or gate) receptor according to example 1 of the present invention;
FIG. 2 is a graph showing the results of in vitro cytotoxicity test of NK cells for the double (or gate) chimeric receptor according to example 2 of the present invention;
FIG. 3 is a graph showing the results of in vitro degranulation level investigation of NK cells of the double (or gate) chimeric receptor of example 3 of the present invention;
FIG. 4 is a graph showing the results of in vitro interferon gamma production level investigation of NK cells of the double (or gate) chimeric receptor of example 3 of the present invention;
FIG. 5 is a graph showing the results of examining ligands expressed by PD-1 and TIGIT in the ovarian cancer cell line HEY of example 4;
FIG. 6 is a graph showing the results of examining the killing efficiency of NK cells of the double (or gate) chimeric receptor against HEY cells of ovarian cancer in example 4 of the present invention;
FIG. 7 is a graph showing the in vivo anti-tumor effect of NK cells of the double (or gate) chimeric receptor of example 4 on ovarian cancer.
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 are understood to encompass values approaching those ranges or values. 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.
In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.
In this context, the term "Logic Gate" is equivalent to "Logic Gate", AND is based on chimeric antigen receptor, by designing two OR more antigen recognition domains, AND applying the principles of Logic Gate "AND Gate", "OR Gate" AND the like, the precise recognition of immune cells on target cells is achieved.
As used herein, the term "chimeric transition Receptor", which is equivalent to "CSR", which is equivalent to "CHIMERIC SWITCH Receptor", is an artificially constructed recombinant Receptor comprising an extracellular antigen-recognition domain, a transmembrane region, and an intracellular signaling domain. Unlike the extracellular antigen recognition domain of a chimeric antigen receptor, which is composed of single chain antibodies, the extracellular antigen recognition domain of a chimeric switch receptor is composed mainly of inhibitory surface receptors of immune cells.
As used herein, the term "chimeric receptor-expressing immune cell", which is equivalent to "CSR-immune cell", is a transgenic immune cell that expresses a particular chimeric receptor and is further useful in the prevention or treatment of disease. In some specific cases, including but not limited to, can be CSR-T cells, but also can be CSR-NK cells, CSR-NKT cells, CSR-gamma delta T cells, CSR-macrophages, CSR-peripheral blood NK cells, CSR-umbilical cord blood NK cells or any one of the immune cells of CSR-iPSC origin, and any one of the CSR-NK cell lines. In some cases, the binding context is equivalent to "chimeric receptor gene modified immune cell technology" and is equivalent to "chimeric receptor gene modified immune cell".
In this context, the term "dual chimeric transducer receptor" is equivalent to "or a door chimeric transducer receptor", in some cases, in combination with the context, to "chimeric transducer receptor described in the present invention", to "chimeric transducer receptor described above", to "chimeric transducer receptor of the first aspect of the present invention"; the term "double chimeric transceptor immune cell" is equivalent to "or" portal chimeric transceptor immune cell ", in some cases, in combination with the context, to" transgenic immune cell according to the invention ", to" transgenic immune cell described above ", to" transgenic immune cell according to the fifth aspect of the invention ". In this context, the double (or gate) chimeric receptor is a novel chimeric receptor proposed by the present invention, which can recognize ligands of two inhibitory receptors expressed on the surface of solid tumor or blood tumor cells at the same time, and can activate immune cells when recognizing any one of the ligands.
As used herein, the term "finger domain", which is equivalent to "Spacer domain", is a small polypeptide sequence that is present in the structure of a single chain antibody or derivative thereof and serves to separate the scFv domain from an immune cell (e.g., T cell). The length of the Spacer sequence or whether it is used is dependent on the number of sequences including but not limited to: the actual conditions such as the triggering of downstream signal intensity after the specific receptor binds to the ligand are determined, and the efficacy is further determined through experiments and cannot be predicted in advance.
As used herein, the term "single chain antibody", equivalent to "SINGLE CHAIN FV", equivalent to "scFv", is a small molecule antibody made up of an immunoglobulin heavy chain variable region (V H) and a light chain variable region (V L) joined by a linker peptide that is flexible enough to allow the free folding of V H and V L to give the correct configuration of the antibody binding region.
In this context, the term "(G 4S)n" is identical to "(Gly 4 Ser") N ", meaning that 4 glycine residues and 1 serine residues are repeated N times, is a type of connecting peptide which is widely used at present, and can be located between the C terminal of V H and the N terminal of V L, and also between the C terminal of V L and the N terminal of V H.
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.
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, etc., suitable for the particular dosage form of interest. In addition to the extent to which any conventional adjuvant is incompatible with the chimeric switch receptor, nucleic acid molecule, expression vector, lentiviral vector or transgenic immune cell of the invention, such as any adverse biological effect produced or interaction with any other component of the pharmaceutically acceptable composition in a deleterious manner, their use is also contemplated by the present invention.
As used herein, the term "administering" refers to introducing a predetermined amount of a substance into a patient by some suitable means. The chimeric switch receptor, nucleic acid molecule, expression vector, lentiviral vector or transgenic immune cell or pharmaceutical composition of the invention may be administered by any common route, provided that it reaches the desired 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" is intended to mean obtaining 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 transition receptor-containing cell described herein to an individual in need thereof.
Herein, "carbon end" and "C end" are synonymous; "Nitrogen end" and "N end" are synonymous.
The invention provides a chimeric conversion receptor capable of simultaneously recognizing ligand antigens of two immune receptors, and corresponding nucleic acid molecules, expression vectors, lentiviral vectors, transgenic immune cells, pharmaceutical compositions and uses thereof, and the chimeric conversion receptor and the corresponding nucleic acid molecules, the expression vectors, the lentiviral vectors, the transgenic immune cells, the pharmaceutical compositions and the uses thereof are respectively described in detail below.
Chimeric transition receptors
The present invention provides a chimeric transition receptor. The chimeric transition receptor comprises: an extracellular region comprising a first binding fragment and a second binding fragment, the first binding fragment having a sequence as set forth in SEQ ID NO:10, said second binding fragment having an amino acid sequence as set forth in SEQ ID NO:11, and a polypeptide comprising the amino acid sequence shown in seq id no; 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.
According to the embodiment of the invention, the TIGIT extracellular truncations with specific amino acid sequences (with the amino acid sequence shown as SEQ ID NO: 10) and the PD-1 extracellular truncations (with the amino acid sequence shown as SEQ ID NO: 11) have good binding activity to PD-L1 and CD155, and can simultaneously exert the binding activity in the chimeric conversion receptor. The immune cells expressing the chimeric conversion receptor of the invention can effectively identify tumor cells expressing TIGIT ligand or PD-1 ligand and also can effectively identify tumor cells losing one receptor ligand of PD-1 or TIGIT.
According to the embodiment of the invention, the extracellular antigen recognition domain TIGIT extracellular truncations (recognizing CD155 ligand) and the PD-1 extracellular truncations (recognizing PD-L1 ligand) selected by the invention show synergistic binding activity in chimeric conversion receptors, and thus the constructed immune cells show stronger tumor cell killing activity. Compared with immune cells only expressing TIGIT or PD1 chimeric converting receptor, the immune cells expressing the chimeric converting receptor of the invention have stronger killing effect on tumor cells simultaneously expressing TIGIT and PD1 ligand, and the expression level of anti-tumor effector IFN-gamma is also obviously improved.
The inventors further observed that, according to the examples of the present invention, CD107a expression levels of immune cells transduced with the chimeric transducer of the present invention were significantly increased, indicating significantly increased NK cell degranulation levels, as well as demonstrating that NK cells now have a greater tumor killing capacity, compared to immune cells transduced with either TIGIT or PD1 chimeric transducer alone.
According to an embodiment of the present invention, the chimeric conversion receptor may further include at least one of the following technical features:
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 connecting peptide. In specific embodiments, the TIGIT extracellular truncations, PD-1 extracellular truncations are linked by a linker peptide, thereby better exerting CD155, PD-L1 ligand binding activity.
According to an embodiment of the invention, the C-terminus of the first binding fragment is linked to the N-terminus of the linker peptide, and the C-terminus of the linker peptide is linked to the N-terminus of the second binding fragment; or the C-terminal of the second binding fragment is linked to the N-terminal of the connecting peptide, and the C-terminal of the connecting peptide is linked to the N-terminal of the first binding fragment.
According to an embodiment of the invention, the linking peptide is selected from at least one of (G4S)n、ESGRS GGGGSGGGGS、EGKSSGSGSESKST、EGKSSGSGSESKSTQ、GSTSGSGKSSEGKG、KESGS VSSEQ LAQFR SLD、ESGSVSSEELAFRSLD, n is an integer other than zero.
According to an embodiment of the invention, the linking peptide is selected from (G 4S)n, n is any integer between 2 and 6).
According to an embodiment of the invention, the connecting peptide is (G 4S)4.
According to an embodiment of the invention, the extracellular region does not comprise a range domain. The inventors have further studied and found that, unlike the general chimeric receptor, the chimeric receptor of the present invention does not include a hinge region between the extracellular domain and the transmembrane region of other receptors such as a CD8a hinge region, thereby facilitating the binding between the chimeric receptor and the ligand and exerting the activating effect of the chimeric receptor on immune cells.
According to an embodiment of the invention, the transmembrane region comprises a CD8a molecular transmembrane segment.
In some alternative embodiments of the invention, the CD8a molecule transmembrane segment has a sequence as set forth in SEQ ID NO:12, and a polypeptide having the amino acid sequence shown in FIG. 12.
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 factor domain is selected from at least one of the CD28, 4-1BB, 2B4, DAP10, DAP12, CD27, CD40, OX40, ICOS intracellular domains.
According to an embodiment of the invention, the co-stimulatory domain is the intracellular segment of a 4-1BB molecule.
In some alternative embodiments of the invention, the 4-1BB co-stimulatory factor domain has a 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 the cd3ζ molecule.
In some alternative embodiments of the invention, the cd3ζ molecule intracellular segment has an amino acid sequence as set forth in SEQ ID NO:14, and a polypeptide having the amino acid sequence shown in seq id no.
In one embodiment of the invention, the chimeric transition receptor has the amino acid sequence as set forth in SEQ ID NO:16, and a polypeptide having the amino acid sequence shown in seq id no.
Nucleic acid molecules
The present invention provides a nucleic acid molecule. The nucleic acid molecules encode the chimeric transition receptors described above. The immune cells carrying the nucleic acid molecules can express the chimeric conversion receptor, and the chimeric conversion receptor can activate the immune cells after recognizing any one of TIGIT ligand and PD-1 ligand, so that the logical gating effect of the immune cells on tumor antigen recognition is realized.
It is noted that, for the nucleic acid molecules mentioned herein, one skilled in the art will understand that either one or both of the complementary double strands are actually included. For convenience, although only one strand is shown in most cases herein, the other strand complementary thereto is actually disclosed. In addition, the molecular sequence in the present invention includes a DNA form or an RNA form, and disclosure of one of them means that the other is also disclosed.
Expression vector
The invention provides an expression vector. The expression vector carries the nucleic acid molecule described above. Thus, the expression vector constructed can express the chimeric transducer receptor of the present invention in a receptor 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.
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 recipient cell, the expression of the nucleic acid molecule described above can be effectively achieved under the mediation of a regulatory system, thereby achieving in vitro mass-production of the protein encoded by the nucleic acid molecule.
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.
Lentiviral vector
The invention provides a lentiviral vector. The lentiviral vector carries a nucleic acid sequence having the sequence shown in SEQ ID NO:1 or SEQ ID NO:15, and a nucleotide sequence shown in seq id no. Thus, expression of the chimeric switch receptor of the invention in immune cells can be achieved after introduction of the lentiviral vector into the recipient cell.
Cells
The present invention provides a transgenic immune cell. The transgenic immune cells express the chimeric conversion receptor; or carrying the nucleic acid molecule described above, the expression vector described above or the lentiviral vector described above. Therefore, the tumor cell killing activity of the obtained transgenic immune cells is obviously improved, and the loss rate of the transgenic immune cells in the tumor treatment process is effectively reduced.
According to an embodiment of the invention, the transgenic immune cells are any at least one selected from the group consisting of T cells, NK cells, NKT cells, γδ T cells, macrophages, peripheral blood NK cells, umbilical cord blood NK cells, and iPSC-derived immune cells of any of the above.
In some specific embodiments, the transgenic immune cell is any selected from NK cells.
In some more specific embodiments, the transgenic immune cell is optionally derived from any one of the NK cell lines.
The chimeric transducer of the present invention can be expressed on the surface of immune cells such as T, NK, NKT, γδt, and macrophages by transduction of these immune cells with an expression vector (lentiviral vector).
According to embodiments of the invention, the transgenic immune cells of the invention have better clinical efficacy and safety than immune cells modified by chimeric switching receptor genes containing only TIGIT or PD-1 antigen recognition domain administered sequentially or simultaneously at comparable doses and in the same manner of administration.
Pharmaceutical composition
The invention provides a pharmaceutical composition. The pharmaceutical composition comprises the chimeric transition receptor, the nucleic acid molecule, the expression vector, the lentiviral vector or the transgenic immune cell. The pharmaceutical composition thus obtained is further used for the treatment of tumors.
According to an embodiment of the present invention, the pharmaceutical composition further comprises: pharmaceutically acceptable auxiliary materials.
Those skilled in the art will appreciate that the features and advantages described above for chimeric transducer receptors, nucleic acid molecules, expression vectors, lentiviral vectors, transgenic immune cells are equally applicable to the pharmaceutical compositions and will not be described in detail herein.
Use of the same
The invention provides the use of the chimeric transition receptor, the nucleic acid molecule, the expression vector, the lentiviral vector, the transgenic immune cell or the pharmaceutical composition in the preparation of medicaments for preventing or treating tumors.
According to an embodiment of the invention, the tumor is a solid tumor or a hematological tumor.
According to an embodiment of the present invention, the solid tumor includes at least one selected from the group consisting of tangible tumors occurring in organs, including pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, cholangiocarcinoma, gastric cancer, esophageal cancer, colorectal cancer, lung cancer, head and neck cancer, cervical cancer, glioma, renal cancer, breast cancer, prostate cancer, melanoma, and the like.
According to an embodiment of the invention, the hematological neoplasm comprises at least one selected from acute myeloid leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, multiple myeloma, etc. within the blood cell and hematopoietic system.
Method for preventing or treating tumor
The present invention provides a method of treating and/or preventing an immune system disorder. 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.
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. The selection of the preferred 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.
The details of the sequences involved in the present invention are shown in Table 1.
Table 1: nucleotide/amino acid sequence specification table
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.
In the following examples, lentivirus production and human NK cell transduction methods were as follows:
Replication-defective lentiviral vectors were prepared and collected by centrifugation for transduction of human NK cells. The experimental procedure for lentiviral vector preparation, collection and concentration is briefly described below: 293T cells were plated in cell culture dishes with a bottom area of 150 square centimeters and plasmid transfection of 293T cells was performed using Lipofectamine 3000 (available from Thermo Fisher, waltham, USA) according to the instructions. 47.37 micrograms of lentiviral transgene plasmid, 30.8 micrograms of psPAX plasmid, 16.58 micrograms of pMD2.G plasmid, 189.48 microliters of P3000, and 118.43 microliters of Lipofectamine 3000 were added per dish of cells. The supernatant was collected at 24 hours, centrifuged for 250g for 5 minutes (centrifuge was Hunan Kecheng L4-5K) to remove the precipitate, and the supernatant was mixed with 1/4 volume of PEG-IT (available from Systems Biosciences, palo Alto, USA), placed overnight at 4℃and centrifuged 1500g for 30 minutes on the next day. Finally, the viral vector pellet was resuspended in 0.3ml DMEM serum free medium.
Peripheral blood from unnamed healthy volunteer donors was isolated by density gradient centrifugation of human peripheral blood lymphocyte isolates (from Daidae, shenzhen, china) and enriched by human NK cell enrichment kit (from Miltenyi, bergischGladbach, germany) to obtain human primary human NK cells. Human NK cells were cultured in RPMI-1640 complete medium and stimulated with human IL-2 at a final concentration of 1000U/mL and human IL-21 at a final concentration of 20ng/mL, and after 48 hours of activation of human NK cells, 0.5X10 6 NK cells were plated per well in 24 well plates, with a culture volume of 0.3mL of 1640 complete medium containing 100U/mL. To each well of cells was added 0.3ml of the above resuspended virus supernatant and Polybrene (concentration 8 micrograms/ml). After 12 hours, 0.45mL of the culture supernatant was aspirated, and 0.85mL of 1640 complete medium containing 100U/mL was added. After 3 days of continuous culture, the culture can be used for doing functional analysis and subsequent experiments.
The flow cytometer used in the examples of the present invention was Beckman Cytoflex (available from Beckman Coulter) and the flow cytometric analysis data was analyzed using Cytexpert software (Beckman Coulter).
In the following examples, the in vitro CFSE-7AAD cytotoxicity assay was performed as follows:
Cytotoxic activity of the or gate chimeric transduction receptor NK cells was assessed using a 4 hour CFSE-7AAD assay. The method comprises the following specific steps: target test cells were labeled with PBS containing a final concentration of 5mM CFSE for 15 minutes at 37 degrees Celsius. After labelling, the cells were rinsed with RPMI medium containing 10% Foetal Calf Serum (FCS). After rinsing, the cells were resuspended in the same medium at a concentration of 1X 10 5/ml. NK cells were added to the target test cell suspension at various target cell ratios (E: T) after transduction, and cells were inoculated into 96-well round bottom plates, with a total volume per well of 200. Mu.l. Cells were cultured in a 37 degree celsius incubator for 4 hours. After 4 hours, the whole cell suspension was aspirated from each well, 3 μl of 7AAD solution was added to each well, and after 1 minute of standing in the dark, the proportion of 7AAD positive cells in CFSE positive cells, i.e. the proportion of target cell death, was detected by flow cytometry Beckman Cytoflex (purchased from Beckman Coulter) to reflect the cytotoxic level of NK cells.
In the following examples, NK cell degranulation and interferon gamma production levels were detected as follows:
In the examples, the ability of the or gate chimeric switching receptor NK cells to degranulate and produce interferon gamma was assessed by co-culturing NK cells with tumor cells. The method comprises the following specific steps: target test cells and transduced NK cells were expressed at 1:3 (E: T) was inoculated into a 96-well round bottom plate, the total volume per well was 200. Mu.l, and 2. Mu.l of a fluorescent antibody against human CD107a per well was added. Cells were cultured in a 37 degree celsius incubator for 3 hours. After 3 hours, the whole cell suspension was aspirated from each well, labeled with anti-human CD56 fluorescent antibody, incubated for 15 minutes in the absence of light, washed, cells were sequentially fixed, membrane-pierced with a fixing solution, a membrane-piercing solution (purchased from Biolegend, san Diego, USA) according to the instructions, and labeled with anti-human interferon gamma fluorescent antibody, and after washing, the proportion of CD107a and interferon gamma positive cells in CD56 positive NK cells was detected by a flow cytometer Beckman Cytoflex (purchased from Beckman Coulter), wherein the former represents the level of NK cell degranulation.
In the following examples, there are 4 target cells for performance testing, respectively: (1) WT K562 cells, hereinafter referred to as "WT K562"; (2) The K562 cells overexpressing CD155, hereinafter referred to as "155tg K562"; (3) K562 cells from which CD112 and CD155 were knocked out and over-expressed PDL1, hereinafter referred to as "112&155KO PDL1tg K562"; (4) The K562 cells that overexpress PDL1 and CD155 are hereinafter referred to as "155& PDL1tg K562".
The "plasmid" and "vector" described in the following examples have the same meaning and are used interchangeably.
Example 1
In this example, vectors expressing the double (or gate) chimeric transducer receptor were constructed as follows:
The coding human CD8 alpha signal peptide (the amino acid sequence is shown as SEQ ID NO:9, the nucleotide sequence is shown as SEQ ID NO: 2) and the nucleotide sequence of the chimeric conversion receptor (the amino acid sequence is shown as SEQ ID NO:16, the nucleotide sequence is shown as SEQ ID NO: 15) are cloned on a lentiviral vector (LENTIVIRAL VECTOR) containing EF-1 alpha promoter, and the lentiviral vector for expressing the chimeric conversion receptor of double (or gate) is obtained through enzyme digestion, connection, screening and amplification of target plasmids.
Wherein the chimeric converting receptor comprises, in order from the N-terminal to the C-terminal, a sequence of a human TIGIT extracellular region, a sequence of a human PD-1 extracellular region, a sequence of a human CD8 alpha transmembrane region, a sequence of a human 4-1BB intracellular region and a sequence of a CD3ζ molecule intracellular segment.
The structure of the chimeric receptor of the double (or gate) of this example is shown in FIG. 1, and specific sequence information is shown in reference to Table 1.
Example 2
In this example, the inventors further examined the killing effect of NK cells expressing the bis (or gate) chimeric transducer receptor of example 1 on tumor cells expressing TIGIT and PD-1 ligand according to the following method:
Replication-defective lentiviral vectors were prepared and collected by centrifugation for transduction of human NK cells: 293T cells were plated in cell culture dishes with a bottom area of 150 square centimeters and plasmid transfection of 293T cells was performed using Lipofectamine 3000 (available from Thermo Fisher, waltham, USA) according to the instructions. A total of 4 groups, each group differing only in "lentiviral transgene plasmid": 47.37 micrograms of lentiviral transgene plasmid (Ctrl, TIGIT-NKR, PD1-NKR, TIGIT-PD1-NKR, PD1-TIGIT-NKR, respectively), 30.8 micrograms of psPAX plasmid, 16.58 micrograms of pmd2.g plasmid, 189.48 microliters of P3000, and 118.43 microliters of Lipofectamine 3000 were added per dish of cells. The supernatant was collected at 24 hours, centrifuged for 250g for 5 minutes (centrifuge was Hunan Kecheng L4-5K) to remove the precipitate, and the supernatant was mixed with 1/4 volume of PEG-IT (available from Systems Biosciences, palo Alto, USA), placed overnight at 4℃and centrifuged 1500g for 30 minutes on the next day. Finally, the viral vector pellet was resuspended in 0.3ml DMEM serum free medium. Peripheral blood from unnamed healthy volunteer donors was isolated by density gradient centrifugation of human peripheral blood lymphocyte isolates (from Daidae, shenzhen, china) and enriched by human NK cell enrichment kit (from Miltenyi, bergischGladbach, germany) to obtain human primary human NK cells. Human NK cells were cultured in RPMI-1640 complete medium and stimulated with human IL-2 at a final concentration of 1000U/mL and human IL-21 at a final concentration of 20ng/mL, and after 48 hours of activation of human NK cells, 0.5X10 6 NK cells were plated per well in 24 well plates, with a culture volume of 0.3mL of 1640 complete medium containing 100U/mL. To each well of cells was added 0.3ml of the 4 resuspended viral supernatants and Polybrene (8 microgram/ml concentration). After 12 hours, 0.45mL of the culture supernatant was aspirated, and 0.85mL of 1640 complete medium containing 100U/mL was added. Transduced NK cells were harvested 3-7 days after lentiviral vector transduction for CFSE-7AAD cytotoxicity experiments in vitro.
Target cells from 4 assays, WT K562, 155tg K562, 112&155KO PDL1tg K562, 155& PDL1tg K562, were individually labeled with PBS containing a final concentration of 5mM CFSE for 15 minutes at 37 ℃. After labelling, the cells were rinsed with RPMI medium containing 10% Foetal Calf Serum (FCS). After rinsing, the cells were resuspended in the same medium at a concentration of 1X 10 5/ml. NK cells were transduced at 1:1 effective target cell ratio (E: T) was added to the target test cell suspension and the cells were seeded into 96-well round bottom plates in a total volume of 200. Mu.l per well. Cells were cultured in a 37 degree celsius incubator for 4 hours. After 4 hours, the whole cell suspension was aspirated from each well, 3 μl of 7AAD solution was added to each well, and after 1 minute of standing in the dark, the proportion of 7AAD positive cells in CFSE positive cells, i.e. the proportion of target cell death, was detected by flow cytometry Beckman Cytoflex (purchased from Beckman Coulter) to reflect the cytotoxic level of NK cells.
The examination results are shown in fig. 2: NK cells transduced with TIGIT chimeric switch receptor ("TIGIT") kill TIGIT ligand-positive tumor cells ("155 tg K562" or "PDL1&155tg K562") at a higher killing rate than control NK cells ("Ctrl"), but kill TIGIT ligand-negative tumor cells ("WT K562" or "112&155KO PDL1tg K562") at a rate that is not significantly different from control NK cells; NK cells transduced with the PD1 chimeric transduction receptor ("PD 1") kill PD1 ligand-positive tumor cells ("112&155KO PDL1tg K562", "PDL1&155tg K562") at a higher kill rate than control NK cells ("Ctrl"), but kill PD1 ligand-negative tumor cells ("WT K562" or "155tg K562") at no significant difference from control NK cells; while NK cells transduced with the double chimeric transducer (or gate chimeric transducer) ("TIGIT-PD 1" or "PD 1-TIGIT") killed either TIGIT ligand single positive ("155tg K562"), PD1 ligand single positive ("112&155KO PDL1tg K562") or TIGIT and PD1 ligand double positive ("PDL 1&155tg K562") tumor cells were killed at a higher rate than the control, while NK cells transduced with both TIGIT and PD1 ligand double negative ("WT K562") were not significantly different from the control, and NK cells transduced with the double (or gate) chimeric transducer) ("TIGIT-PD 1" or "PD 1-TIGIT") killed TIGIT and PD1 ligand double positive ("PDL 1&155tg K562") tumor cells were killed at a higher rate than either TIGIT or PD1 chimeric transducer alone.
The results indicate that the double (or gate) chimeric conversion receptor can identify tumor types with heterogeneity expressed by TIGIT/PD1 ligand, compared with TIGIT or PD1 single chimeric conversion receptor, or gate can simultaneously identify tumors with TIGIT ligand single positive, PD1 ligand single positive, TIGIT and PD1 ligand double positive, and can cope with the situation that the ligand expression of one receptor is lost, and the ligand of the other receptor is still expressed, and killing is carried out.
Example 3
In this example, the inventors further examined the degranulation and production of interferon gamma levels of NK cells expressing the bis (or gate) chimeric transduction receptor of example 1 when stimulated by tumor cells expressing TIGIT and PD-1 ligand, according to the following method:
Replication-defective lentiviral vectors were first prepared and collected by centrifugation for transduction of human cells: 293T cells were plated in cell culture dishes with a bottom area of 150 square centimeters and plasmid transfection of 293T cells was performed using Lipofectamine 3000 (available from Thermo Fisher, waltham, USA) according to the instructions. A total of 4 groups, each group differing only in "lentiviral transgene plasmid": 47.37 micrograms of lentiviral transgene plasmid (Ctrl, TIGIT-NKR, PD1-NKR, TIGIT-PD1-NKR, PD1-TIGIT-NKR, respectively), 30.8 micrograms of psPAX plasmid, 16.58 micrograms of pmd2.g plasmid, 189.48 microliters of P3000, and 118.43 microliters of Lipofectamine 3000 were added per dish of cells. The supernatant was collected at 24 hours, centrifuged for 250g for 5 minutes (centrifuge was Hunan Kecheng L4-5K) to remove the precipitate, and the supernatant was mixed with 1/4 volume of PEG-IT (available from Systems Biosciences, palo Alto, USA), placed overnight at 4℃and centrifuged 1500g for 30 minutes on the next day. Finally, the viral vector pellet was resuspended in 0.3ml DMEM serum free medium. Peripheral blood from unnamed healthy volunteer donors was isolated by density gradient centrifugation of human peripheral blood lymphocyte isolates (from Daidae, shenzhen, china) and enriched by human NK cell enrichment kit (from Miltenyi, bergischGladbach, germany) to obtain human primary human NK cells. Human NK cells were cultured in RPMI-1640 complete medium and stimulated with human IL-2 at a final concentration of 1000U/mL and human IL-21 at a final concentration of 20ng/mL, and after 48 hours of activation of human NK cells, 0.5X10 6 NK cells were plated per well in 24 well plates, with a culture volume of 0.3mL of 1640 complete medium containing 100U/mL. To each well of cells was added 0.3ml of the above resuspended virus supernatant and Polybrene (concentration 8 micrograms/ml). After 12 hours, 0.45mL of the culture supernatant was aspirated, and 0.85mL of 1640 complete medium containing 100U/mL was added. The transduced NK cells were harvested 3-7 days after lentiviral vector transduction for in vitro tumor cell stimulated degranulation and detection experiments to produce interferon gamma levels.
Experiment settings WT K562, 155tg K562, 112&155KO PDL1tg K562, 155& pdl1tg K562, and "no target cell" group ("NT") for a total of 5 groups.
The target cells to be tested and transduced NK cells were mixed at a ratio of 1:4 (E: T) was inoculated into a 96-well round bottom plate, the total volume per well was 200. Mu.l, and 2. Mu.l of a fluorescent antibody against human CD107a per well was added. Cells were cultured in a 37 degree celsius incubator for 3 hours. After 3 hours, the whole cell suspension was aspirated from each well, labeled with anti-human CD56 fluorescent antibody, incubated for 15 minutes in the absence of light, washed, cells were sequentially fixed and membrane-pierced with a fixing solution, a membrane-piercing solution (purchased from Biolegend, san Diego, USA) according to the instructions, and labeled with anti-human interferon gamma fluorescent antibody, and after washing, the proportion of CD107a and interferon gamma positive cells in CD56 positive NK cells was detected by a flow cytometer Beckman Cytoflex (purchased from Beckman Coulter), wherein the former is the NK cell degranulation level.
The examination results are shown in fig. 3 and 4: NK cells transduced with TIGIT chimeric switch receptor ("TIGIT") produced IFN-gamma and expressed CD107a levels higher than control NK cells ("Ctrl") when co-cultured with TIGIT ligand positive tumor cells ("155 tg K562" or "PDL1&155tg K562"), but produced IFN-gamma and expressed CD107a levels not significantly different from control NK cells when co-cultured with TIGIT ligand negative tumor cells ("WT K562" or "112&155KO PDL1tg K562"); NK cells transduced with the PD1 chimeric transduction receptor ("PD 1") produced IFN-gamma and expressed CD107a levels higher than control NK cells ("Ctrl") when co-cultured with PD1 ligand positive tumor cells ("112&155KO PDL1tg K562", "PDL1&155tg K562"), but produced IFN-gamma and expressed CD107a levels that were not significantly different from control NK cells when co-cultured with PD1 ligand negative tumor cells ("WT K562" or "155tg K562"); while NK cells transduced with the double (or gate) chimeric transduction receptor (TIGIT-PD 1' or "PD 1-TIGIT") produced IFN-gamma and expressed CD107a levels higher than those of the control group when co-cultured with TIGIT ligand single positive ("155 tg K562"), PD1 ligand single positive ("112&155KO PDL1tg K562") or TIGIT and PD1 ligand double positive ("PDL 1&155tg K562") tumor cells, and produced IFN-gamma and expressed CD107a levels not significantly different from those of the control group when co-cultured with TIGIT and PD1 ligand double negative ("WT K562") tumor cells, and NK cells transduced with the double (or gate) chimeric transduction receptor produced IFN-gamma and expressed CD107a levels significantly higher than that of NK cells transduced with TIGIT alone or PD1 chimeric transduction receptor alone.
Example 4
10 5 Human ovarian cancer cells HEY were incubated in 100uL PBS suspension with 0.5 μg of the following flow-through fluorescent antibodies, respectively, for 10 minutes in the dark: PE-anti-human PDL1, APC-anti-human PDL2, PE-anti-human CD155, APC-anti-human CD112. The flow-through assay was then performed by washing once with PBS. The results show that the ligand of human ovarian cancer cells HEY that simultaneously highly expressed TIGIT and PD1 (as shown in fig. 5) should be recognized by the double (or gate) chimeric receptor, and thus the inventors first selected HEY cells for the identification of the anti-tumor ability of NK cells of the double (or gate) chimeric receptor.
In this example, the inventors examined the killing efficiency of NK cells expressing the bis (or gate) chimeric transduction receptor of example 1 against human ovarian cancer cells HEY according to the method of example 3.
The examination results are shown in fig. 6: NK cells transduced with TIGIT chimeric transducer ("TIGIT") or PD1 chimeric transducer ("PD 1") kill HEY cells at a higher rate than control NK cells ("Ctrl"), whereas NK cells transduced with double (or gate) chimeric transducer ("TIGIT-PD 1" or "PD 1-TIGIT") kill HEY cells at a higher rate than control NK cells transduced with TIGIT alone or PD1 chimeric transducer alone. The experimental results show that TIGIT-PD1 or PD1-TIGIT or a chimeric transfer receptor of the door can identify the tumor type (such as ovarian cancer) expressing the TIGIT/PD1 ligand, and the tumor type has higher killing efficiency compared with the single TIGIT or PD1 chimeric transfer receptor.
Further, based on the above-described results of effectiveness, the inventors further identified the ability of NK cells expressing the bis (or gate) chimeric transduction receptor of example 1 to resist tumors in mice. The specific method comprises the following steps:
The human ovarian cancer HEY cells are subcutaneously injected under armpit to establish an ovarian cancer model. Immunodeficient non-obese diabetic (NOD) -SCID gamma mice (NSG) (NOD-SCID mice background and with IL-2 receptor gamma chain defects) were selected for subcutaneous tumor-bearing, 5 x 10 6 HEY cells per mouse subcutaneous injection. Starting on the third day after tumor bearing, tail vein injection of 10 7 NK92 cells once a week until the end of the experiment; starting on the day of the first injection of NK92 cells, 50000 units of Interleukin 2 were intraperitoneally injected twice a week for increasing the survival level of NK92 cells in vivo. The long diameter and the short diameter of the tumor are measured every week, the tumor volume is calculated, and the tumor growth curve is drawn.
The examination results are shown in fig. 7: NK92 cells transduced with TIGIT chimeric transducer receptor ("TIGIT") or PD1 chimeric transducer receptor ("PD 1") had a therapeutic effect of inhibiting tumor growth compared to NK92 Wild Type (WT), whereas NK92 cells transduced with double (or gate) chimeric transducer receptor ("TIGIT-PD 1" or "PD 1-TIGIT") had a better therapeutic effect and could inhibit tumor growth to a greater extent compared to NK92 cells. Demonstrating that the double (or gate) chimeric transition receptor of the invention confers a greater ability to NK92 cells to recognize tumors and activate anti-tumor effector functions.
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 (20)
1. A chimeric transition receptor comprising:
An extracellular region comprising a first binding fragment and a second binding fragment, the amino acid sequence of the first binding fragment being as set forth in SEQ ID NO:10, the amino acid sequence of the second binding fragment is shown in SEQ ID NO: 11;
A transmembrane region, wherein the N-terminal of the transmembrane region is connected with the C-terminal of the extracellular region, and the transmembrane region comprises a CD8a molecular transmembrane segment; the amino acid sequence of the CD8a molecule transmembrane segment is shown in SEQ ID NO: shown at 12;
an intracellular region, wherein the N end of the intracellular region is connected with the C end of the transmembrane region, the intracellular region comprises a costimulatory domain and an intracellular signaling domain, the costimulatory domain is a 4-1BB molecular intracellular segment, the intracellular signaling domain is a CD3 zeta molecular intracellular segment, and the amino acid sequence of the 4-1BB costimulatory factor domain is shown as SEQ ID NO:13, the amino acid sequence of the intracellular segment of the CD3 zeta molecule is shown as SEQ ID NO: 14;
the extracellular region further comprises a connecting peptide;
the C end of the first binding fragment is connected with the N end of the connecting peptide, and the C end of the connecting peptide is connected with the N end of the second binding fragment; or (b)
The C-terminal of the second binding fragment is linked to the N-terminal of the linker peptide, and the C-terminal of the linker peptide is linked to the N-terminal of the first binding fragment.
2. The chimeric transition receptor according to claim 1, wherein the linking peptide is selected from (G 4S)n, n is any integer between 2 and 6.
3. The chimeric transition receptor according to claim 2, wherein the linking peptide is (G 4S)4.
4. The chimeric transition receptor according to claim 1, wherein the extracellular region does not include a finger domain.
5. The chimeric transition receptor according to claim 1, wherein the chimeric transition receptor has the amino acid sequence set forth in SEQ ID NO:16, and a polypeptide having the amino acid sequence shown in seq id no.
6. A nucleic acid molecule encoding the chimeric switch receptor of any one of claims 1 to 5.
7. An expression vector carrying the nucleic acid molecule of claim 6.
8. The expression vector of claim 7, wherein the expression vector is a non-pathogenic viral vector.
9. The expression vector of claim 8, wherein the non-pathogenic viral vector is optionally one of a retroviral vector, a lentiviral vector, and an adenoviral vector.
10. The expression vector of claim 8, wherein the non-pathogenic viral vector is a lentiviral vector.
11. A lentiviral vector comprising a nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:15, and a nucleic acid molecule as shown in seq id no.
12. A transgenic immune cell, the transgenic immune cell comprising:
expressing the chimeric transduction receptor according to any one of claims 1 to 5; or alternatively
Carrying the nucleic acid molecule of claim 6, the expression vector of any one of claims 7 to 10 or the lentiviral vector of claim 11.
13. The transgenic immune cell of claim 12, wherein the transgenic immune cell is selected from at least one of T cells, NK cells, macrophages.
14. The transgenic immune cell of claim 13, wherein the T cell is selected from at least one of NKT cells, γδ T cells, and the NK cell is selected from at least one of peripheral blood NK cells, umbilical cord blood NK cells.
15. The transgenic immune cell of any one of claims 13 or 14, wherein the T cell, NK cell or macrophage is iPSC-derived.
16. The transgenic immune cell of claim 12, wherein the transgenic immune cell is selected from NK cells.
17. A pharmaceutical composition comprising:
The chimeric switching receptor of any one of claims 1 to 5, the nucleic acid molecule of claim 6, the expression vector of any one of claims 7 to 10, the lentiviral vector of claim 11, or the transgenic immune cell of any one of claims 12 to 16.
18. The pharmaceutical composition of claim 17, further comprising: pharmaceutically acceptable auxiliary materials.
19. Use of the chimeric switch receptor of any one of claims 1 to 5, the nucleic acid molecule of claim 6, the expression vector of any one of claims 7 to 10, the lentiviral vector of claim 11, the transgenic immune cell of any one of claims 12 to 16, or the pharmaceutical composition of any one of claims 17 to 18 in the manufacture of a medicament for treating a tumor, the tumor being a solid tumor or a hematological tumor, the hematological tumor being hodgkin's lymphoma.
20. The use according to claim 19, wherein the solid tumor comprises at least one selected from the group consisting of a tangible tumor occurring in an organ, including pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, cholangiocarcinoma, gastric cancer, esophageal cancer, colorectal cancer, lung cancer, head and neck cancer, cervical cancer, brain glioma, renal cancer, breast cancer, prostate cancer, melanoma.
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