Protease-responsive gpcr receptors and uses thereof

Abstract

This invention discloses a protease-responsive GPCR receptor and its applications. The protease-responsive GPCR receptor of this invention comprises an extracellular protease recognition domain, a transmembrane domain, and an intracellular signaling domain; the extracellular protease recognition domain contains a peptide hydrolyzed by the protease; the intracellular signaling domain contains an HCV enzyme hydrolyzed peptide, a nuclear localization sequence (NLS), a Cas protease, and a coupled nuclear transcription factor. This protease-responsive GPCR receptor can induce endogenous dCas9 assembly into the nucleus and activate the chimeric antigen receptor signaling pathway in natural killer cells in response to extracellular proteases, providing a new solution for developing novel precision immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of immunocellular therapy technology, and in particular to protease-responsive GPCR receptors and their applications. Background Technology

[0002] Cells respond to their environment by sensing surrounding signals and translating them into changes in gene expression. In recent years, synthetic networks have been designed in both prokaryotic and eukaryotic systems to create new functions for specific applications. Among these, redesigning cellular sensors to trigger unnatural responses has become a fundamental engineering aspect of cell therapy. In related technologies, research on enhancing the function of chimeric antigen receptor immune cells has primarily focused on improving artificially synthesized receptors; however, the design of engineered cells still lacks efficient and multifunctional molecular devices.

[0003] Advances in CRISPR / Cas technology, particularly the use of clustered, regularly spaced short palindromic repeats, have revolutionized the functional study of gene expression. Related research has shown that dCas9-coupled transcription factors (e.g., VP64-P65) inactivated by restriction enzymes can be used to control the expression of specific genes, making the dCas9 activation system a powerful tool for precisely controlling cellular signaling pathways. Researchers have used CRISPR / dCas9-coupled G protein-coupled receptors (GPCRs) to design Tango and ChaCha artificial signaling systems to respond to extracellular soluble small molecules. However, these designs have only utilized artificial or natural ligands of GPCRs, and there are few reports on the design of artificially modified GPCR receptors that respond to artificial proteases.

[0004] Therefore, further research on cell signaling pathways is needed to improve immunotherapy. This invention aims to further expand the application of artificial signaling pathways by designing artificially modified GPCR receptors that respond to artificial proteases, and to provide new ideas for tumor immunotherapy. Summary of the Invention

[0005] This invention aims to address at least one of the technical problems existing in the prior art. To this end, this invention proposes a protease-responsive GPCR receptor that, by sensing and responding to extracellular proteases, induces endogenous dCas9 assembly into the nucleus to activate and regulate the GPCR receptor signaling pathway within immune cells, providing an innovative solution for developing novel, precision-guided immunotherapy.

[0006] The present invention also provides a protease-responsive GPCR receptor composition.

[0007] The present invention also provides nucleic acid molecules encoding GPCR receptors or GPCR receptor compositions that encode the above-mentioned protease responses.

[0008] The present invention also provides lentiviral particles of nucleic acid molecules encoding GPCR receptors or GPCR receptor compositions that encode the above-mentioned protease responses.

[0009] The present invention also provides recombinant proteins, including the above-described protease-responsive GPCR receptors or GPCR receptor compositions.

[0010] The present invention also provides the use of the above-mentioned protease-responsive GPCR receptor, protease-responsive GPCR receptor composition, nucleic acid molecule encoding the above-mentioned protease-responsive GPCR receptor or GPCR receptor composition, lentiviral particle encoding the above-mentioned protease-responsive GPCR receptor or GPCR receptor composition, or recombinant cell containing the above-mentioned protease-responsive GPCR receptor or GPCR receptor composition in the preparation of drugs for treating or preventing tumors.

[0011] According to a first aspect of the present invention, a protease-responsive GPCR receptor includes an extracellular protease recognition domain, a transmembrane domain, and an intracellular signaling domain.

[0012] The extracellular protease recognition domain contains peptides that are hydrolyzed by proteases;

[0013] The intracellular signaling domain contains a protease-hydrolyzed peptide of hepatitis C virus (HCV).

[0014] The amino acid sequence of the HCV enzyme hydrolyzed peptide is shown in SEQ ID NO.3.

[0015] In some embodiments of the present invention, at least the following beneficial effects are achieved: the protease-responsive GPCR receptor can regulate the activation of the GPCR receptor signaling pathway in immune cells by inducing the assembly of endogenous dCas9 into the nucleus in response to extracellular protease signals, which can provide an innovative solution for developing new precision-guided immune cell therapies.

[0016] According to some embodiments of the present invention, the intracellular signaling domain further comprises a nuclear localization sequence (NLS);

[0017] Preferably, the amino acid sequence of the nuclear localization sequence NLS is shown in SEQ ID NO.5.

[0018] According to some embodiments of the present invention, the intracellular signaling domain further comprises a Cas protease.

[0019] According to some embodiments of the present invention, the intracellular signaling domain further comprises coupled nuclear transcription factors.

[0020] According to some embodiments of the present invention, the protease-hydrolyzed peptides include at least one selected from the following: Tobacco Etch Virus protease (TEV protease) hydrolyzed peptides, thrombin hydrolyzed peptides, MMP1 hydrolyzed peptides, PLAU urokinase plasminogen activator (PLAU) hydrolyzed peptides, MMP13 hydrolyzed peptides, integrin metalloproteinase 10 hydrolyzed peptides, integrin metalloproteinase 17 hydrolyzed peptides, enterokinase hydrolyzed peptides, coagulation factor Xa hydrolyzed peptides, furin protease hydrolyzed peptides, hepatitis C virus protease hydrolyzed peptides, human rhinovirus 3C protease hydrolyzed peptides, plumpox virus protease hydrolyzed peptides, and sunflower mild mosaic virus protease hydrolyzed peptides.

[0021] Preferably, the amino acid sequence of the TEV protease hydrolysate peptide is: ENLYFQG (SEQ ID NO.2).

[0022] Preferably, the amino acid sequence of the thrombin hydrolysate peptide is: ENLYFQG (SEQ ID NO.20);

[0023] The amino acid sequence of the MMP1 hydrolyzed peptide is: GTAGLIGQ (SEQ ID NO.25).

[0024] The amino acid sequence of the PLAU hydrolyzed peptide is: GGGRR (SEQ ID NO.27).

[0025] The amino acid sequence of the MMP13 hydrolyzed peptide is: GPAGLYEK (SEQ ID NO.30).

[0026] The amino acid sequence of the integrin metalloproteinase 10 hydrolysate is: PRAEALKGG (SEQ ID NO. 31).

[0027] The amino acid sequence of the deintegrin metalloproteinase 17 hydrolysate peptide is: PRAAAVKSP (SEQ ID NO. 32).

[0028] The amino acid sequence of the enterokinase hydrolysate peptide is: DDDDK (SEQ ID NO.33);

[0029] The amino acid sequence of the hydrolyzed peptide of coagulation factor Xa is: IEGD (SEQ ID NO.34) or INGD (SEQ ID NO.35).

[0030] The amino acid sequence of the furin protease hydrolysate peptide is: RXRR (SEQ ID NO.36) or RXKR (SEQ ID NO.37).

[0031] The amino acid sequence of the HCV protease hydrolysate peptide is: DEMEECSQHL (SEQ ID NO. 3).

[0032] The amino acid sequence of the human rhinovirus 3C protease hydrolysate peptide is: LEVLFQP (SEQ ID NO.38) or LEVLFGP (SEQ ID NO.39).

[0033] The amino acid sequence of the protease hydrolysate peptide of the poxvirus is: NVVVHQA (SEQ ID NO.40).

[0034] The amino acid sequence of the sunflower mild mosaic virus protease hydrolysate peptide is: EEIHLQS (SEQ ID NO. 41) or EEIHLQG (SEQ ID NO. 42).

[0035] According to some embodiments of the present invention, the transmembrane domain includes at least one of the GPR56 transmembrane peptide, the ADGRL3 transmembrane peptide, the CD97 transmembrane peptide, and the EMR1 transmembrane peptide.

[0036] Preferably, the transmembrane domain includes the GPR56 transmembrane peptide.

[0037] According to some embodiments of the present invention, when the peptide hydrolyzed by the protease is a peptide hydrolyzed by TEV protease, the amino acid sequence of the extracellular protease recognition domain-transmembrane domain peptide (TEV-GPCR peptide) is as shown in SEQ ID NO.1.

[0038] The underlined portion of the amino acid sequence SEQ ID NO.1 is the TEV protease hydrolysate peptide.

[0039] Preferably, when the peptide hydrolyzed by the protease is a thrombin hydrolyzed peptide, the amino acid sequence of the extracellular protease recognition domain-transmembrane domain peptide (thrombin-GPCR peptide) is as shown in SEQ ID NO.19.

[0040] The underlined portion of the amino acid sequence SEQ ID NO.19 is a thrombin hydrolysate peptide.

[0041] Preferably, when the peptide hydrolyzed by the protease is an MMP1 hydrolyzed peptide, the amino acid sequence of the extracellular protease recognition domain-transmembrane domain peptide (MMP1-GPCR peptide) is as shown in SEQ ID NO.24.

[0042] The underlined portion of the amino acid sequence SEQ ID NO.24 is the MMP1 hydrolysate peptide.

[0043] Preferably, when the peptide hydrolyzed by the protease is a PLAU hydrolyzed peptide, the amino acid sequence of the extracellular protease recognition domain-transmembrane domain peptide (PLAU-GPCR peptide) is as shown in SEQ ID NO.26.

[0044] The underlined portion of the amino acid sequence SEQ ID NO.26 is the PLAU hydrolyzed peptide.

[0045] According to some embodiments of the present invention, the Cas protease includes dCas9 protease or dCas12 protease;

[0046] Preferably, the Cas protease is dCas9 protease;

[0047] Preferably, the amino acid sequence of the dCas9 protease is shown in SEQ ID NO.6.

[0048] According to some embodiments of the present invention, the coupled nuclear transcription factor includes at least one of VP64, MCP, P65, T2A and Puromycin.

[0049] According to some embodiments of the present invention, the amino acid sequence of VP64 is shown in SEQ ID NO.7.

[0050] According to some embodiments of the present invention, the amino acid sequence of the MCP is shown in SEQ ID NO.8.

[0051] According to some embodiments of the present invention, the amino acid sequence of P65 is shown in SEQ ID NO.9.

[0052] According to some embodiments of the present invention, the amino acid sequence of T2A is shown in SEQ ID NO.17.

[0053] According to some embodiments of the present invention, the amino acid sequence of the Puromycin is shown in SEQ ID NO.18.

[0054] Preferably, the coupled nuclear transcription factor is the "VP64-MCP-P65" sequence;

[0055] Preferably, the amino acid sequence of the “VP64-MCP-P65” sequence is shown in SEQ ID NO.23.

[0056] According to some embodiments of the present invention, the sgRNA sequence of the dCas9 protease is gagcactgtcctccgaacgt (SEQ ID NO.29).

[0057] According to some embodiments of the present invention, the method for obtaining the protease-responsive GPCR receptor includes the following steps:

[0058] Step S1: Integrate the extracellular recognition sequence encoding the peptide containing protease hydrolysis and the gene encoding the transmembrane domain into the lentiviral expression vector to obtain an expression vector containing the target gene.

[0059] Step S2: Transfect the host cell with the expression vector containing the target gene to obtain a protease-responsive GPCR receptor.

[0060] A protease-responsive GPCR receptor composition according to a second aspect of the present invention comprises a first protease-responsive GPCR receptor and a second protease-responsive GPCR receptor; wherein,

[0061] The first protease-responsive GPCR receptor is the protease-responsive GPCR receptor described above, wherein the peptide of the Cas protease is the N-terminal peptide of the Cas protease.

[0062] The second protease-responsive GPCR receptor is the protease-responsive GPCR receptor described above, wherein the peptide of the Cas protease is the C-terminal peptide of the Cas protease.

[0063] Preferably, the Cas protease is dCas9 protease, and the amino acid sequence of the N-terminal peptide of the Cas protease is shown in SEQ ID NO.16; the amino acid sequence of the C-terminal peptide of the Cas protease is shown in SEQ ID NO.22.

[0064] According to some embodiments of the present invention, the protease-responsive GPCR receptor composition comprises a first protease-responsive GPCR receptor and a second protease-responsive GPCR receptor.

[0065] The first protease-responsive GPCR receptor is the protease-responsive GPCR receptor described above, wherein the peptide of the Cas protease is the N-terminal peptide of the dCas9 protease; the amino acid sequence of the N-terminal peptide of the dCas9 protease is shown in SEQ ID NO. 16.

[0066] The second protease-responsive GPCR receptor is the protease-responsive GPCR receptor described above, wherein the peptide of the Cas protease is the C-terminal peptide of the dCas9 protease; the amino acid sequence of the C-terminal peptide of the dCas9 protease is shown in SEQ ID NO. 22.

[0067] According to some embodiments of the present invention, the nuclear transcription factors coupled to the first protease-responsive GPCR receptor include T2A and Puromycin.

[0068] According to some embodiments of the present invention, the coupled nuclear transcription factors of the second protease-responsive GPCR receptor include VP64, MCP and P65.

[0069] According to some embodiments of the present invention, the peptides hydrolyzed by the protease in the extracellular protease recognition domain of the first protease-responsive GPCR receptor and the second protease-responsive GPCR receptor may be the same or different.

[0070] Preferably, the extracellular protease recognition domains of the first protease-responsive GPCR receptor and the second protease-responsive GPCR receptor contain different peptides that are hydrolyzed by the protease.

[0071] According to some embodiments of the present invention, the protease in the first protease-responsive GPCR receptor and the protease in the second protease-responsive GPCR receptor may be the same or different.

[0072] According to some embodiments of the present invention, the protease in the first protease-responsive GPCR receptor is different from the protease in the second protease-responsive GPCR receptor.

[0073] A nucleic acid molecule according to a third aspect of the present invention, which encodes the above-described protease-responsive GPCR receptor or a composition of GPCR receptors that encode the above-described protease-responsive GPCR receptor.

[0074] Since the nucleic acid molecule encodes the protease-responsive GPCR receptor or protease-responsive GPCR receptor composition of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments.

[0075] A lentiviral particle according to a fourth aspect of the present invention comprises a nucleotide sequence encoding a GPCR receptor for the protease response or a nucleotide sequence encoding a GPCR receptor composition for the protease response.

[0076] Preferably, the lentiviral particle further comprises a nucleotide sequence encoding an adaptor protein.

[0077] Preferably, the lentiviral particle further contains a nucleotide sequence encoding an HCV enzyme.

[0078] According to some embodiments of the present invention, the amino acid sequence of the adaptor protein is shown in SEQ ID NO.12.

[0079] According to some embodiments of the present invention, the amino acid sequence of the HCV enzyme is shown in SEQ ID NO.13.

[0080] According to some embodiments of the present invention, the method for obtaining the lentiviral particles includes the following steps:

[0081] Step S1: Construct a lentiviral vector expressing protease-hydrolyzed peptides and transmembrane peptides;

[0082] Step S2: Construct a lentiviral vector expressing adaptor protein coupled with HCV enzyme;

[0083] Step S3: The lentiviral vector obtained in steps S1 and S2 is packaged and then transfected into host cells to obtain the lentiviral particles.

[0084] Preferably, the lentiviral vector is a pHR lentiviral vector.

[0085] Preferably, the pHR lentiviral vector contains a CMV or EFS promoter.

[0086] Preferably, the transmembrane peptide is selected from at least one of GPR56 transmembrane peptide, ADDRL3 transmembrane peptide, CD97 transmembrane peptide, and EMR1 transmembrane peptide.

[0087] A recombinant cell according to a fifth aspect of the present invention expresses the above-described protease-responsive GPCR receptor and a protease-responsive GPCR receptor composition.

[0088] Preferably, the recombinant cells also express adaptor protein-coupled HCV enzyme.

[0089] Preferably, the amino acid sequence of the adaptor protein coupled with the HCV enzyme is shown in SEQ ID NO.10.

[0090] According to some embodiments of the present invention, the recombinant cells include natural killer cells, natural killer T cells, macrophages, regulatory T cells, and γδT cells.

[0091] Preferably, the recombinant cells are NK-92 natural killer cells.

[0092] The use of the above-described protease-responsive GPCR receptor according to a sixth aspect of the present invention in the preparation of a drug for treating or preventing tumors.

[0093] The use of the above-described protease-responsive GPCR receptor composition according to a seventh aspect of the present invention in the preparation of a drug for treating or preventing tumors.

[0094] The use of a nucleic acid molecule encoding a GPCR receptor or a composition of a GPCR receptor encoding the above-described protease response according to an eighth aspect embodiment of the present invention in the preparation of a drug for treating or preventing tumors.

[0095] The use of the above-described lentiviral particles according to a ninth aspect of the present invention in the preparation of a drug for treating or preventing tumors.

[0096] The use of the above-described recombinant cells in the preparation of a drug for treating or preventing tumors, according to a tenth aspect embodiment of the present invention.

[0097] According to some embodiments of the present invention, at least the following beneficial effects are achieved:

[0098] 1. This invention constructs modularly modified GPCR receptors that respond to artificial or natural proteases and logically designs to regulate gene expression at the posttranscriptional level, thereby prompting mammalian cells to respond to extracellular input in a predictable manner, overcoming the challenges of applications such as cancer immunotherapy and smart cell implantation.

[0099] 2. This invention utilizes a modified adhesive GPCR receptor to sense and respond to different concentrations of extracellular proteases, enabling the design of a chimeric antigen receptor signaling pathway that induces endogenous dCas9 assembly into the nucleus and regulates this pathway within natural killer cells. This protease-responsive chimeric antigen receptor signaling pathway will provide a useful tool for synthetic biology and innovative solutions for developing novel, precision-guided immunotherapy.

[0100] Furthermore, the chimeric antigen receptor signal transduction method based on protease response of the present invention will greatly expand the antigen recognition spectrum of immunocellular therapy and provide a new mode for the activation of chimeric antigen receptor expression by the overexpression of proteases in the tumor microenvironment.

[0101] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0102] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0103] Figure 1 This is a schematic diagram of the artificially modified GPCR receptor vector for TEV protease response constructed according to embodiments of the present invention, and a schematic diagram of the vector for the Arrestin-HCV protease complex recruited after receptor activation.

[0104] Figure 2This is a schematic diagram of the TEV protease-responsive artificially modified GPCR receptor-mediated dCas9 nuclear translocation to activate chimeric antigen receptor gene expression in an embodiment of the present invention, wherein PCS is a TEV protease cleavage peptide and HCS is an HCV protease cleavage peptide.

[0105] Figure 3 This is a schematic diagram of the artificially modified GPCR receptor vector that responds to both TEV protease and thrombin constructed according to embodiments of the present invention, and the vector for the Arrestin-HCV protease complex recruited after receptor activation.

[0106] Figure 4 This is a schematic diagram of the artificially modified GPCR receptor mediated by TEV protease and thrombin co-response in an embodiment of the present invention to activate the expression of chimeric antigen receptor gene via dCas9 nuclear translocation, wherein PCS1 is a TEV protease cleavage peptide; PCS2 is a thrombin cleavage peptide; and HCS is an HCV protease cleavage peptide.

[0107] Figure 5 This is a schematic diagram of the artificially modified GPCR receptor vector co-responsive to MMP1 and PLAU proteases constructed in this embodiment of the invention, the Arrestin-HCV protease complex vector recruited after receptor activation, and the chimeric antigen receptor vector activated by dCas9, wherein CAR is the chimeric antigen receptor gene.

[0108] Figure 6 This is a schematic diagram illustrating the expression of chimeric antigen receptor mediated by dCas9 nuclear translocation mediated by the co-response of MMP1 and PLAU proteases in the present invention. PCS1 is the MMP1 protease cleavage peptide; PCS2 is the PLAU protease cleavage peptide; and HCS is the HCV protease cleavage peptide.

[0109] Figure 7 This is a fluorescence image showing the expression level of the chimeric antigen receptor after TEV protease response in HEK293T cells according to the present invention.

[0110] Figure 8 This is a comparison chart of the killing rates of transfected and untransfected cells in an embodiment of the present invention. Detailed Implementation

[0111] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0112] Where specific techniques or conditions are not specified in the implementation methods, they shall be performed in accordance with the techniques and conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0113] Unless otherwise specified, all cell lines were derived from the American Type Culture Collection (ATCC), and all unmodified vectors were derived from Addgene, including the packaging plasmid psPAX2 (Addgene, #12260), the envelope plasmid pMD2.G (Addgene, #12259), and the pHR plasmid (Addgene, #79121). The modified pHR lentiviral vector was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and all reagents used for cell culture were derived from Thermo Fisher Scientific (China) Co., Ltd. All chemical reagents used in the examples were derived from Merk / Sigma.

[0114] Example 1: An artificially engineered GPCR receptor responsive to TEV protease

[0115] This embodiment provides an artificially modified GPCR receptor that responds to the TEV protease, specifically obtained through the following method:

[0116] 1. Construct a lentiviral vector containing the TEV-GPR56 sequence.

[0117] The nucleotide sequence “TEV-GPR56”, containing an extracellular protease recognition domain-transmembrane domain peptide (TEV-GPCR peptide), was introduced into a pHR lentiviral vector using genetic engineering techniques. In this embodiment, the TEV-GPCR sequence in the lentiviral vector containing the TEV-GPR56 sequence was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and its amino acid information is shown below:

[0118] MTPQSLLQTTLFLLSLLFLVQGAHGRGHREDFRFCSQRNQTHRSSLHYKPTPDLRISIENSEEALTVHAPFPAAHPASRSFPDPRGLYHFCLYWNRHAGRLHLLYGKRDFLLSDKASSLLCFQHQEESLAQGPPLLATSVTSWWSPQNISLPSAASFTFSFHSPPHTAAHNASVDMCELKRDLQLLSQFL KHPQKASRRPSAAPASQQLQSLESKLTSVRFMGDMVSFEEDRINATVWKLQPTAGLQDLHIHSRQEEEQSEIMEYSVLLPRTLFQRTKGRSGEAEKRLLLVDFSSQALFQDKNSSQVLGEKVLGIVVQNTKVANLTEPVVLTFQHQLQPKNVTLQCVFWVEDPTLSSPGHWSSAGCETVRRETQTSCFCNH ENLYFQG TYFAVLMVSSVEVDAVHKHYLSLLSYVGCVVSALACLVTIAAYLCSRRKPRDYTIKVHMNLLLAVFLLDTSFLLSEPVALTGSEAGCRASAIFLHFSLLTCLSWMGLEGYNLYRLVVEVFGTYVPGYLLKLSAMGWGFPIFLVTLVALVDVDNY GPIILAVHRTPEGVIYPSMCWIRDSLVSYITNLGLFSLVFLFNMAMLATMVVQILRLRPHTQKWSHVLTLLGLSLLVLGLPWALIFFSFASGTFQLVVLYLFSIITSFQGFLIFIWYWSMRLQARGGPSPLKSNSDSARLPISSGSTSSSRI (SEQ ID NO.1).

[0119] The underlined portion of the amino acid sequence SEQ ID NO.1 is the TEV protease hydrolysate peptide (SEQ ID NO.2), with the first part being the N-terminal domain of GPR56 and the second part being the C-terminal domain of GPR56.

[0120] After the target fragment "TEV-GPR56" was inserted into the multiple cloning site of the pHR lentiviral vector, its nucleotide sequence is as follows:

[0121] ATGACTCCCCAGTCGCTGCTGCAGACGACACTGTTCCTGCTGAGTCTGCTCTTCCT GGTCCAAGGTGCCCACGGCAGGGGCCACAGGGAAGACTTTCGCTTCTGCAGCCAGCGGAACCAGACACACAGGAGC AGCCTCCACTACAAACCCACACCAGACCTGCGCATCTCCATCGAGAACTCCGAAGAGGCCCTCACAGTCCATGCCC CTTTCCCTGCAGCCCACCCTGCTTCCCGATCCTTCCCTGACCCCAGGGGCCTCTACCACTTCTGCCTCTACTGGAA CCGACATGCTGGGAGATTACATCTTCTCTATGGCAAGCGTGACTTCTTGCTGAGTGACAAAGCCTCTAGCCTCCTC TGCTTCCAGCACCAGGAGGAGAGCCTGGCTCAGGGCCCCCCGCTGTTAGCCACTTCTGTCACCTCCTGGTGGAGCC CTCAGAACATCAGCCTGCCCAGTGCCGCCAGCTTCACCTTCTCCTTCCACAGTCCTCCCCACACGGCCGCTCACAA TGCCTCGGTGGACATGTGCGAGCTCAAAAGGGACCTCCAGCTGCTCAGCCAGTTCCTGAAGCATCCCCAGAAGGCC TCAAGGAGGCCCTCGGCTGCCCCCGCCAGCCAGCAGTTGCAGAGCCTGGAGTCGAAACTGACCTCTGTGAGATTCA TGGGGGACATGGTGTCCTTCGAGGAGGACCGGATCAACGCCACGGTGTGGAAGCTCCAGCCCACAGCCGGCCTCCA GGACCTGCACATCCACTCCCGGCAGGAGGAGGAGCAGAGCGAGATCATGGAGTACTCGGTGCTGCTGCCTCGAACA CTCTTCCAGAGGACGAAAGGCCGGAGCGGGGAGGCTGAGAAGAGACTCCTCCTGGTGGACTTCAGCAGCCAAGCCC TGTTCCAGGACAAGAATTCCAGCCAAGTCCTGGGTGAGAAGGTCTTGGGGATTGTGGTACAGAACACCAAAGTAGC CAACCTCACGGAGCCCGTGGTGCTCACTTTCCAGCACCAGCTACAGCCGAAGAATGTGACTCTGCAATGTGTGTTC TGGGTTGAAGACCCCACATTGAGCAGCCCGGGGCATTGGAGCAGTGCTGGGTGTGAGACCGTCAGGAGAGAAACCC AAACATCCTGCTTCTGCAACCACGAAAACCTGTATTTTCAGGGCACCTACTTTGCAGTGCTGATGGTCTCCTCGGT GGAGGTGGACGCCGTGCACAAGCACTACCTGAGCCTCCTCTCCTACGTGGGCTGTGTCGTCTCTGCCCTGGCCTGC CTTGTCACCATTGCCGCCTACCTCTGCTCCAGGAGGAAACCTCGGGACTACACCATCAAGGTGCACATGAACCTGC TGCTGGCCGTCTTCCTGCTGGACACGAGCTTCCTGCTCAGCGAGCCGGTGGCCCTGACAGGCTCTGAGGCTGGCTG CCGAGCCAGTGCCATCTTCCTGCACTTCTCCCTGCTCACCTGCCTTTCCTGGATGGGCCTCGAGGGGTACAACCTC TACCGACTCGTGGTGGAGGTCTTTGGCACCTATGTCCCTGGCTACCTACTCAAGCTGAGCGCCATGGGCTGGGGCT TCCCCATCTTTCTGGTGACGCTGGTGGCCCTGGTGGATGTGGACAACTATGGCCCCATCATCTTGGCTGTGCATAG GACTCCAGAGGGCGTCATCTACCCTTCCATGTGCTGGATCCGGGACTCCCTGGTCAGCTACATCACCAACCTGGGC CTCTTCAGCCTGGTGTTTCTGTTCAACATGGCCATGCTAGCCACCATGGTGGTGCAGATCCTGCGGCTGCGCCCCC ACACCCAAAAGTGGTCACATGTGCTGACACTGCTGGGCCTCAGCCTGGTCCTTGGCCTGCCCTGGGCCTTGATCTT CTTCTCCTTTGCTTCTGGCACCTTCCAGCTTGTCGTCCTCTACCTTTTCAGCATCATCACCTCCTTCCAAGGCTTC CTCATCTTCATCTGGTACTGGTCCATGCGGCTGCAGGCCCGGGGTGGCCCCTCCCCTCTGAAGAGCAACTCAGACA GCGCCAGGCTCCCCATCAGCTCGGGCAGCACCTCGTCCAGCCGCATC

[0122] The underlined portion in SEQ ID NO.43 is the nucleotide sequence of the inserted target sequence “TEV-GPCR sequence” (SEQ ID NO.44).

[0123] A schematic diagram of the lentiviral vector containing the TEV-GPCR sequence is shown below. Figure 1 As shown in 'a'.

[0124] HCS is the HCV restriction site, and its amino acid sequence is DEMEECSQHL (SEQ ID NO.3).

[0125] The specific amino acid sequence information for “NLS-dCas9-VP64-MCP-P65” is as follows:

[0126] PKKKRKVPKKKRKV EASGRA DALDDFDLDMLGSDALDDFDLDML GSDALDDFDLDMLGSDALDDFDLDML INGTASGSGEGRGSLLTCGDVEENPGPVSKLM ASNFTQFVLVDNGGTGDV TVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPI FATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY SAGGGGSGGGGSGGGGSGPKKKRKVAAAGS PSGQISNQALA LAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTPGPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGN STDPGVFTDLASVDNSEFQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED FSSIADMDFSALLSQISS SGQGGGGSGFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPRPPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAEDPTISLLTGSEPPKAKDPTVS (SEQ ID NO. 4).

[0127] In the amino acid sequence SEQ ID NO.4, the underlined part is the NLS nuclear localization sequence: PKKKRKV (as shown in SEQ ID NO.5); the bold black part is the dCas9 sequence (as shown in SEQ ID NO.6); the italicized bold part is the VP64 sequence (as shown in SEQ ID NO.7); the bold underlined part is the MCP sequence (as shown in SEQ ID NO.8); and the italicized underlined part is P65 (as shown in SEQ ID NO.9).

[0128] 2. Construct a lentiviral vector containing an adaptor protein coupled with an HCV enzyme sequence.

[0129] Using genetic engineering techniques, the adaptor protein was linked to the HCV protease via a linker fragment to obtain the Arrestin-HCV enzyme fragment. The amino acid sequence of the Arrestin-HCV enzyme fragment is as follows:

[0130] MGEKPGTRVFKKSSPNCKLTVYLGKRDFVDHLDKVDPVDGVVLVDPDYLKDRKVFVTLTCAFRYGREDLDVLGLSFRKDLFIATYQAFPPVPNPPRPPTRLQDRLLRKLGQHAHPFFFTIPQNLPCSVTLQPGPEDTGKACGVDFEIRAFCAKSLEEKSHKRNSVRLVIRKVQFAPEKPGPQPSAETTRHFLMSDRSLHLEASL DKELYYHGEPLNVNVHVTNNSTKTVKKIKVSVRQYADICLFSTAQYKCPVAQLEQDDQVSPSSTFCKVYTITPLLSDNREKRGLALDGKLKHEDTNLASSTI VKEGANKEVLGILVSYRVKVKLVVSRGGDVSVELPFVLMHPKPHDHIPLPRPQSAAPETDVPVDTNLIEFDTNYATDDDIVFEDFARLRLKGMKDDDYDDQLC GSGGGGSGGGGSGGGGS DYKDDDDKGSSGTGSGSGTSAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTFLATCINGVCWAVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGS SDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVTLTHPITKIDTKYIMTCMSADLEVVT (SEQ IDNO.10).

[0131] The underlined portion of the amino acid sequence SEQ ID NO.10 is the linker fragment (as shown in SEQ ID NO.11);

[0132] The sequence preceding the linker fragment in SEQ ID NO.10 is the amino acid sequence of the adaptor protein (as shown in SEQ ID NO.12).

[0133] The sequence following the linker fragment in SEQ ID NO.10 is the HCV enzyme amino acid sequence (as shown in SEQ ID NO.13).

[0134] Subsequently, NES exonuclear transport signal fragments were added to both ends of the Arrestin-HCV enzyme fragment. After synthesis by Sangon Biotech (Shanghai) Co., Ltd., the fragment was introduced into the multiple cloning sites BamHI and NotI of the pHR lentiviral vector to obtain the lentiviral vector containing the adaptor protein-coupled HCV enzyme sequence. Specific information on the lentiviral vector containing the adaptor protein-coupled HCV enzyme sequence is as follows: Figure 1 As shown in Figure b, the amino acid sequence of the NES exotropic signal fragment is: LPPLELTL (SEQ ID NO.14).

[0135] 3. Transfecting cells

[0136] First, the lentiviral vectors described above were cloned into DH5α competent cells (purchased from Thermo Fisher Scientific, catalog number 18258012). Single clones were selected, and endotoxin-free lentiviral plasmids were extracted. The lentiviral vectors were then used to transfect HEK293T cells to prepare a TEV protease-responsive artificially modified GPCR receptor. The specific preparation process is as follows:

[0137] (1) One day in advance, HEK293T cells were prepared at a concentration of 2.5 × 10⁻⁶. 5 The cells were seeded at a rate of 100 cells / mL in 6-well plates and cultured in DMEM high-glucose complete medium with 10% fetal bovine serum until the confluence reached 70%.

[0138] (2) Day 1 of packaging: Before the experiment, the old culture medium of HEK293T cells was aspirated and washed once with PBS. Culture medium was added, and then two pHR viral vectors containing 0.75 μg of the target gene were mixed. 1 μg of packaging plasmid psPAX2 (Addgene, #12260) and 1 μg of envelope plasmid pMD2.G (Addgene, #12259) were mixed in 250 μL of Opti-MEM culture medium and 7.5 μL of Mirus TransIT-LT1 transfection reagent to obtain the transfection complex. The transfection complex was cultured at room temperature (37℃±1℃) for 30 minutes, and then added dropwise to HEK293T cell culture medium evenly. After being gently mixed, it was placed in a 37℃, 5% CO2 incubator for culture.

[0139] (3) On the second day after packaging: remove the old culture medium of HEK293T cells, add 8 ml of fresh DMEM high glucose complete culture medium, and place in an incubator to continue culturing.

[0140] (4) On the third day of packaging: lentivirus was extracted from the supernatant using a sterile syringe and filtered through a 0.45 μm polyvinylidene fluoride filter to obtain lentivirus particles expressing the target gene. Then, the particles were immediately transduced into target cells or frozen at -80°C for later use.

[0141] During the preparation process, when 5-20 U / mL TEV protease is added to the culture medium of transformed HEK-293T cells, dCas9 responds to the protease entering the nucleus and activating the expression of the chimeric antigen receptor gene containing fluorescent protein. The specific flowchart is as follows: Figure 2 As shown in the example (refer to the detection example for specific activation and expression process verification). The expression level of fluorescent protein can be used to quantitatively analyze the artificial signaling pathway activated by the protease, and then the optimized vector design can be transplanted into immune cells.

[0142] Example 2: TEV-thrombin-responsive artificially modified GPCR receptor composition

[0143] This embodiment provides two protease-responsive artificially modified GPCR receptor compositions, which are obtained through the following methods:

[0144] 1. Construct a lentiviral vector containing the TEV-aGPCR-dCas9 (N segment) sequence.

[0145] The "TEV-GPR56" sequence, which serves as an extracellular recognition element, was introduced into a pHR lentiviral vector using genetic engineering techniques. In this embodiment, the TEV-GPR56 sequence in the lentiviral vector containing the TEV-GPR56 sequence was synthesized by Sangon Biotech (Shanghai) Co., Ltd. The amino acid information of the TEV-GPR56 sequence is shown in SEQ ID NO.1, where the underlined part in the amino acid sequence SEQ ID NO.1 is the TEV protease hydrolysis peptide (SEQ ID NO.2).

[0146] A schematic diagram of the lentiviral vector containing the TEV-GPR56 sequence prepared by inserting the target fragment "TEV-GPR56" into the multiple cloning sites BamHI and NotI of the pHR lentiviral vector is shown below. Figure 3 As shown in 'a'.

[0147] HCS is the HCV restriction site, and its amino acid sequence is shown in SEQ ID NO.3;

[0148] The amino acid sequence of “NLS-dCas9(N-terminus)-T2A-Puromycin” is: PKKKRKV DKKYSIGLAIGTNSVG WAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKV DDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFL IEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASM IKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMREGRGSLLTCGDVEENPGPMTEEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIG PRMAELSGSRLAAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA (SEQ ID NO.15)

[0149] Among them, the underlined part in the amino acid sequence SEQ ID NO.15 is the amino acid sequence of dCas9 (N-terminus) (as shown in SEQ ID NO.16).

[0150] The amino acid sequence in SEQ ID NO.15 with the bolded portion being T2A (as shown in SEQ ID NO.17); the amino acid sequence in SEQ ID NO.15 with no underscore and the bolded portion being Puromycin (as shown in SEQ ID NO.18).

[0151] 2. Construct a lentiviral vector containing the thrombin-aGPCR-dCas9 (C-terminus) sequence.

[0152] Using genetic engineering techniques, the "thrombin-GPR56" sequence, which serves as an extracellular recognition element, was introduced into the multiple cloning sites BamHI and NotI of the pHR lentiviral vector. In this embodiment, the thrombin-GPR56 sequence in the lentiviral vector containing the thrombin-GPR56 sequence was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and its amino acid information is shown below:

[0153] MTPQSLLQTTLFLLSLLFLVQGAHGRGHREDFRFCSQRNQTHRSSLHYKPTPDLRISIENSEEALTVHAPFPAAHPASRSFPDPRGLYHFCLYWNRHAGRLHLLYGKRDFLLSDKASSLLCFQHQEESLAQGPPLLATSVTSWWSPQNISLPSAASFTFSFHSPPHTAAHNASVDMCELKRDLQLLSQFL KHPQKASRRPSAAPASQQLQSLESKLTSVRFMGDMVSFEEDRINATVWKLQPTAGLQDLHIHSRQEEEQSEIMEYSVLLPRTLFQRTKGRSGEAEKRLLLVDFSSQALFQDKNSSQVLGEKVLGIVVQNTKVANLTEPVVLTFQHQLQPKNVTLQCVFWVEDPTLSSPGHWSSAGCETVRRETQTSCFCNH LVPRGS TYFAVLMVSSVEVDAVHKHYLSLLSYVGCVVSALACLVTIAAYLCSRRKPRDYTIKVHMNLLLAVFLLDTSFLLSEPVALTGSEAGCRASAIFLHFSLLTCLSWMGLEGYNLYRLVVEVFGTYVPGYLLKLSAMGWGFPIFLVTLVALVDVDNY GPIILAVHRTPEGVIYPSMCWIRDSLVSYITNLGLFSLVFLFNMAMLATMVVQILRLRPHTQKWSHVLTLLGLSLLVLGLPWALIFFSFASGTFQLVVLYLFSIITSFQGFLIFIWYWSMRLQARGGPSPLKSNSDSARLPISSGSTSSSRI (SEQ ID NO.19).

[0154] The underlined portion of the amino acid sequence SEQ ID NO.19 is the thrombin hydrolysate peptide (SEQ ID NO.20).

[0155] A schematic diagram of the lentiviral vector containing the thrombin-GPR56 sequence prepared by inserting the target fragment "thrombin-GPR56" into the multiple cloning site of the pHR lentiviral vector is shown below. Figure 3 As shown in b in the figure.

[0156] HCS is the HCV restriction site, and its amino acid sequence is shown in SEQ ID NO.3;

[0157] The amino acid sequence of “NLS-dCas9 (C-terminus)-VP64-MCP-P65” is: PKKKRKV KPAFLSGEQKKAIVDLL FKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDELEDIVLTLTLFED REMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRER MKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV LTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVA QILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEF VYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVR KVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSV KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLKPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFL YLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHL FTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO.21)

[0158] Among them, the amino acid sequence SEQ ID NO.21 contains an underlined portion of the dCas9 (C-terminus) amino acid sequence (as shown in SEQ ID NO.22); and an ununderlined portion of the VP64-MCP-P65 amino acid sequence (as shown in SEQ ID NO.23).

[0159] 3. Construct a lentiviral vector containing an adaptor protein coupled with an HCV enzyme sequence.

[0160] Using genetic engineering techniques, the adaptor protein was linked to the HCV protease via a linker fragment to obtain the Arrestin-HCV enzyme fragment. The amino acid sequence of the Arrestin-HCV enzyme fragment is shown in SEQ ID NO.10.

[0161] Subsequently, after adding the NES exotropic signal fragment to both ends of the Arrestin-HCV enzyme fragment, it was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and introduced into the multiple cloning site of the pHR lentiviral vector to obtain the product. The amino acid sequence of the linker fragment is shown in SEQ ID NO.11, and the amino acid sequence of the NES exotropic signal fragment is shown in SEQ ID NO.14.

[0162] 4. Transfecting cells

[0163] First, the lentiviral vectors described above were cloned into DH5α competent cells (purchased from Thermo Fisher Scientific, catalog number 18258012). Single clones were selected, and endotoxin-free lentiviral plasmids were extracted. The TEV protease-responsive artificially modified GPCR receptor was prepared by transfecting HEK293T cells using this lentiviral vector. The specific preparation process is as follows:

[0164] (1) One day in advance, HEK293T cells were prepared at a concentration of 2.5 × 10⁻⁶. 5 The cells were seeded at a rate of 10 cells / mL in 6-well plates and cultured until confluence reached 70%.

[0165] (2) Day 1 of packaging: Before the experiment, the old culture medium of HEK293T cells was aspirated and washed once with PBS. 8 ml of DMEM high glucose complete culture medium was added. Then, 3 pHR lentiviral vectors containing 0.75 μg of the target sequence were mixed. 1 μg of packaging plasmid psPAX2 and 1 μg of envelope plasmid pMD2.G were mixed in 250 μL of Opti-MEM culture medium and 7.5 μL of Mirus TransIT-LT1 transfection reagent to obtain the transfection complex. The transfection complex was incubated at room temperature for 30 minutes. Then, it was added dropwise to HEK293T cell culture medium evenly and gently mixed. The cells were then incubated in a 37±1℃, 5% CO2 incubator.

[0166] (3) On the second day after packaging: remove the old culture medium of HEK293T cells, add 8 ml of fresh DMEM high glucose complete culture medium, and place in an incubator to continue culturing.

[0167] (4) On the third day of packaging: lentivirus was extracted from the supernatant using a sterile syringe and filtered through a 0.45 μm polyvinylidene fluoride filter to obtain lentivirus particles expressing the target gene, which were then frozen at -80℃ for later use.

[0168] Using the above methods, lentiviral particles containing the Arrestin-HCV enzyme fragment, lentiviral particles containing the TEV-aGPR56-dCas9 (N-terminal) fragment, and lentiviral particles containing the thrombin-aGPR56-dCas9 (C-terminal) fragment were obtained, respectively. These were then transfected into HEK-293T cells, and positive clones were screened. Upon protease response, the dCas9 protease enters the nucleus and activates the expression of the chimeric antigen receptor gene containing fluorescent protein. The specific flowchart is shown below. Figure 4 As shown.

[0169] Example 3: Artificially Modified GPCR Receptor Composition Responding to MMP1-PLAU Protease

[0170] 1. Construct a lentiviral vector containing the MMP1-GPCR-dCas9 sequence.

[0171] Using genetic engineering techniques, the extracellular target molecule binding domain "MMP1-GPR56" sequence, serving as an extracellular recognition element, was introduced into the multiple cloning sites BamHI and NotI of the pHR lentiviral vector. In this embodiment, the MMP1-GPR56 sequence in the lentiviral vector containing the MMP1-GPR56 sequence was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and its amino acid information is shown below:

[0172] MTPQSLLQTTLFLLSLLFLVQGAHGRGHREDFRFCSQRNQTHRSSLHYKPTPDLRISIENSEEALTVHAPFPAAHPASRSFPDPRGLYHFCLYWNRHAGRLHLLYGKRDFLLSDKASSLLCFQHQEESLAQGPPLLATSVTSWWSPQNISLPSAASFTFSFHSPPHTAAHNASVDMCELKRDLQLLSQFL KHPQKASRRPSAAPASQQLQSLESKLTSVRFMGDMVSFEEDRINATVWKLQPTAGLQDLHIHSRQEEEQSEIMEYSVLLPRTLFQRTKGRSGEAEKRLLLVDFSSQALFQDKNSSQVLGEKVLGIVVQNTKVANLTEPVVLTFQHQLQPKNVTLQCVFWVEDPTLSSPGHWSSAGCETVRRETQTSCFCNH GTAGLIGQTYFAVLMVSSVEVDAVHKHYLSLLSYVGCVVSALACLVTIAAYLCSRRKPRDYTIKVHMNLLLAVFLLDTSFLLSEPVALTGSEAGCRASAIFLHFSLLTCLSWMGLEGYNLYRLVVEVFGTYVPGYLLKLSAMGWGFPIFLVTLVALVDVDNY GPIILAVHRTPEGVIYPSMCWIRDSLVSYITNLGLFSLVFLFNMAMLATMVVQILRLRPHTQKWSHVLTLLGLSLLVLGLPWALIFFSFASGTFQLVVLYLFSIITSFQGFLIFIWYWSMRLQARGGPSPLKSNSDSARLPISSGSTSSSRI (SEQ ID NO.24).

[0173] The underlined portion of the amino acid sequence SEQ ID NO.24 is the MMP1 hydrolyzed peptide (SEQ ID NO.25).

[0174] A schematic diagram of the lentiviral vector containing the MMP1-GPR56 sequence obtained by inserting the target fragment "MMP1-GPR56" into the multiple cloning site of the pHR lentiviral vector is shown below. Figure 5 As shown in 'a'.

[0175] HCS is the HCV restriction site, and its amino acid sequence is shown in SEQ ID NO.3; the amino acid sequence of “NLS-dCas9(N-terminus)-T2A-Puromycin” is shown in SEQ ID NO.15.

[0176] 2. Construct a lentiviral vector containing the PLAU-aGPCR-dCas9 sequence.

[0177] Using genetic engineering techniques, the extracellular target molecule binding domain "PLAU-GPR56" sequence, serving as an extracellular recognition element, was introduced into the multiple cloning sites BamHI and NotI of the pHR lentiviral vector. In this embodiment, the PLAU-GPR56 sequence in the lentiviral vector containing the GPR56-T PLAU sequence was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and its amino acid information is shown below:

[0178] MTPQSLLQTTLFLLSLLFLVQGAHGRGHREDFRFCSQRNQTHRSSLHYKPTPDLRISIENSEEALTVHAPFPAAHPASRSFPDPRGLYHFCLYWNRHAGRLHLLYGKRDFLLSDKASSLLCFQHQEESLAQGPPLLATSVTSWWSPQNISLPSAASFTFSFHSPPHTAAHNASVDMCELKRDLQLLSQFL KHPQKASRRPSAAPASQQLQSLESKLTSVRFMGDMVSFEEDRINATVWKLQPTAGLQDLHIHSRQEEEQSEIMEYSVLLPRTLFQRTKGRSGEAEKRLLLVDFSSQALFQDKNSSQVLGEKVLGIVVQNTKVANLTEPVVLTFQHQLQPKNVTLQCVFWVEDPTLSSPGHWSSAGCETVRRETQTSCFCNH GGGRR TYFAVLMVSSVEVDAVHKHYLSLLSYVGCVVSALACLVTIAAYLCSRRKPRDYTIKVHMNLLLAVFLLDTSFLLSEPVALTGSEAGCRASAIFLHFSLLTCLSWMGLEGYNLYRLVVEVFGTYVPGYLLKLSAMGWGFPIFLVTLVALVDVDNY GPIILAVHRTPEGVIYPSMCWIRDSLVSYITNLGLFSLVFLFNMAMLATMVVQILRLRPHTQKWSHVLTLLGLSLLVLGLPWALIFFSFASGTFQLVVLYLFSIITSFQGFLIFIWYWSMRLQARGGPSPLKSNSDSARLPISSGSTSSSRI (SEQ ID NO.26).

[0179] The underlined portion of the amino acid sequence SEQ ID NO.26 is the PLAU hydrolyzed peptide (SEQ ID NO.27).

[0180] A schematic diagram of the lentiviral vector containing the PLAU-GPR56 sequence obtained by inserting the target fragment "PLAU-GPR56" into the multiple cloning site of the pHR lentiviral vector is shown below. Figure 5 As shown in b in the figure.

[0181] HCS is the HCV restriction site, and its amino acid sequence is shown in SEQ ID NO.3;

[0182] The amino acid sequence of “NLS-dCas9(C-terminus)-VP64-MCP-P65” is shown in SEQ ID NO.21.

[0183] 3. Construct a lentiviral vector containing an adaptor protein coupled with an HCV enzyme sequence.

[0184] Using genetic engineering techniques, the adaptor protein was linked to the HCV protease via a linker fragment to obtain the Arrestin-HCV enzyme fragment. The amino acid sequence of the Arrestin-HCV enzyme fragment is shown in SEQ ID NO.10.

[0185] Subsequently, after adding NES exonuclear transport signal fragments to both ends of the Arrestin-HCV enzyme fragment, the fragment was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and then introduced into the multiple cloning site of the pHR lentiviral vector to obtain the final product. For detailed vector information, please refer to [link to vector information]. Figure 5 As shown in c, the amino acid sequence of the linker fragment is shown in SEQ ID NO.11, and the amino acid sequence of the NES exonuclear transport signal fragment is shown in SEQ ID NO.14.

[0186] 4. Transfecting cells

[0187] First, the lentiviral vectors described above were cloned into DH5α competent cells (purchased from Thermo Fisher Scientific, catalog number 18258012). Single clones were selected, and endotoxin-free lentiviral plasmids were extracted. The TEV protease-responsive artificially modified GPCR receptor was prepared by transfecting HEK293T cells using this lentiviral vector. The specific preparation process is as follows:

[0188] (1) One day in advance, HEK293T cells were prepared at a concentration of 2.5 × 10⁻⁶. 5 The cells were seeded at a rate of 10 cells / mL in 6-well plates and cultured until confluence reached 70%.

[0189] (2) Day 1 of packaging: Before the experiment, the old culture medium of HEK293T cells was aspirated and washed once with PBS. The cells were then added to DMEM high glucose complete culture medium. Three pHR lentiviral vectors containing 0.75 μg of the target sequence were mixed. 1 μg of packaging plasmid psPAX2 and 1 μg of envelope plasmid pMD2.G were mixed in 250 μL of Opti-MEM culture medium and 7.5 μL of Mirus TransIT-LT1 transfection reagent to obtain the transfection complex. The transfection complex was cultured at 37℃±1℃ for 30 min at room temperature. Then, it was added dropwise to HEK293T cell culture medium and gently mixed. The cells were then incubated at 37℃±1℃ in a 5% CO2 incubator.

[0190] (3) On the second day after packaging: remove the old culture medium of HEK293T cells, add 8 ml of fresh DMEM high glucose complete culture medium, and place in an incubator to continue culturing.

[0191] (4) On the third day of packaging: lentivirus was extracted from the supernatant using a sterile syringe and filtered through a 0.45 μm polyvinylidene fluoride filter to obtain lentivirus particles expressing the target gene, which were then frozen at -80℃ for later use.

[0192] In this embodiment, the target fragment is introduced into a lentiviral pHR vector containing a CMV or EFS promoter, and then transfected into HEK-293T cells or immune cells. Positive clones expressing the fragment are selected. During cell culture, when MMP1 and PLAU proteases are present simultaneously, dCas9 assembles into the nucleus and binds to the VP64-GAL4 orthogonal transcription factor, promoting the expression of the chimeric antigen receptor on natural killer cells. For detailed procedures, please refer to [link to documentation]. Figure 6 As shown. Furthermore, this embodiment detects the expression of dCas9-activated chimeric antigen receptors by inserting a chimeric antigen receptor gene into the vector (Addgene##85427). The specific nucleotide sequence of its GAL4 upstream activation sequence is: GGAGCACTGTCCTCCGAACG (SEQ ID NO.28).

[0193] Detection example

[0194] To verify the activation of artificial signaling pathways by the artificially modified GPCR receptor in response to protease, this embodiment used the lentiviral vector particles obtained in Example 1 above and transfected them into HEK293T cells to verify the activation of chimeric antigen receptor expression by the artificially modified GPCR receptor. In this embodiment, the nucleotide sequence of the dCas9 sgRNA is GAGCACTGTCCTCCGAACGT (SEQ ID NO. 29), and the sgRNA sequence was cloned into the lenti U6-sgRNA / EF1a-mCherry vector (Addgene, #114199).

[0195] 1. Detection of expression levels of chimeric antigen receptors in protease response

[0196] In this example, HEK293T cells expressing a chimeric antigen receptor carrying the mEGFP tag were created by replacing mCherry with the mEGFP-containing chimeric antigen receptor gene in the vector (Addgene#79123) (see reference). Figure 5 (d) was obtained by screening BFP-positive cells using flow cytometry.

[0197] In this embodiment, fluorescence microscopy was used to detect the mEGFP fluorescent protein to characterize the expression level of the chimeric antigen receptor. The specific detection method is as follows:

[0198] (1) 48 hours before the test, the old culture medium of HEK293T cells was aspirated and washed once with PBS. The cells were then added to DMEM high glucose complete culture medium. 1.5 μg of lenti U6-sgRNA / EF1a-mCherry vector containing sgRNA sequence, 1 μg of packaging plasmid psPAX2 and 1 μg of envelope plasmid pMD2.G were mixed in 250 μL of Opti-MEM culture medium and 7.5 μL of Lirus TransIT-LT1 transfection reagent to obtain the transfection complex. The transfection complex was cultured at room temperature (28℃±2℃) for 30 minutes. Then, it was added dropwise to HEK293T cell culture medium and gently mixed. The cells were then incubated at 37℃ in a 5% CO2 incubator.

[0199] (2) 12 hours before the test, add 10 U / mL of TEV protease to the cell culture medium, continue to incubate at 37℃±1℃ for 12 hours, and then observe under a fluorescence microscope.

[0200] Test results as follows Figure 7 As shown, from Figure 7 As can be seen from the figure, in the control group without the addition of protease (left figure), the expression level of fluorescent protein is very low. After the addition of TEV protease, the expression level of chimeric antigen receptor with green fluorescent protein mEGFP tag is significantly increased (right figure).

[0201] 2. Verification of anti-tumor effects

[0202] First, untransfected and transfected NK-92 cells (2×10⁶ cells per cell line) were separately divided into two groups. 5 NK-92 cells and target cells (MDA-MB-231 cells) were co-cultured in 96-well plates for 8 h, with effector cell (NK-92 cells) to target cell (MDA-MB-231 cells) ratios of 3:1, 5:1, and 10:1. MDA-MB-231 cells were cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin; NK-92 cells were cultured in α-MEM medium supplemented with 200 U / ml human type II interferon, 10% horse serum, and 10% fetal bovine serum. All cell cultures were incubated in a humidified incubator at 37℃±1℃ with 5% CO2.

[0203] Cells from the co-culture system were collected, and the expression levels of CD107a degranulation, interferon-gamma (IFNγ), and TNFα were detected by flow cytometry. The specific detection method for CD107a expression was as follows:

[0204] (1) Add 20 μL of antibody CD107a-FITC to 80 μL of NK-92 cell co-culture suspension (2 × 10⁻⁶ cells / mL). 6Cells / ml ~ 3 × 10 6 (cells / ml) to obtain an antibody cell suspension.

[0205] (2) The antibody-cell suspension was added to a flat-bottomed 96-well plate, and 2 μL of CD28 / CD49d co-stimulatory antibody was injected into each well. Additionally, 100 μL of CEF peptide at a concentration of 64 μg / ml was added to the solution as a CEF treatment control group, and the same amount of cell culture medium was used as a negative control group. The plates were incubated at 37°C in a 5% CO2 incubator for 60 min, and then 0.5 μL of BD-GolgiStop containing monensin was added, followed by incubation for another 120 min.

[0206] (3) After washing, the cells were co-incubated with CD3-APC and CD8 PreCP antibodies (1:200) for 30 min. After centrifugation, the supernatant was discarded and the cells were resuspended in 130 μL of cell culture medium. The cells were then analyzed by flow cytometry, and at least 50,000 events were collected and recorded.

[0207] The specific detection method for the expression levels of IFNγ and TNFα was as follows: Cells were fixed with 100 μL of BD-Cytofix / Cytoperm solution and incubated in the dark at 4°C for 20 min. After washing and centrifugation, the cells were resuspended in Perm / Wash buffer, and 20 μL of IFN-γ-PE and 20 μL of TNF-α-PE antibody (1:200) were added. The cells were then incubated in the dark at 4°C for 30 min, followed by flow cytometry analysis, collecting and recording at least 50,000 events.

[0208] like Figure 8 As shown, the results indicate that, compared with untransfected NK-92 cells, transfected NK-92 cells have significant tumor cell killing efficiency at target cell to tumor cell ratios of 1:3, 1:5, and 1:10, and the killing efficiency increases with the increase of the NK-92 to tumor cell ratio.

[0209] In summary, this invention overcomes the challenges of applications such as cancer immunotherapy and smart cell implantation by constructing modularly modified GPCR receptors that respond to artificial or natural proteases and logically designing to regulate gene expression at the posttranscriptional level, thereby prompting mammalian cells to respond to extracellular input in a predictable manner.

[0210] Furthermore, this invention establishes the expression of chimeric antigen receptors in immune cells that respond to proteases in the extracellular microenvironment. By using a modified adhesive GPCR receptor to sense and respond to different concentrations of extracellular proteases, it is used to design and induce endogenous dCas9 assembly into the nucleus to activate and regulate the chimeric antigen receptor signaling pathway within natural killer cells. This protease response platform will provide a useful tool for synthetic biology and an innovative solution for developing new, precision-guided immunotherapy.

[0211] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A protease-responsive GPCR receptor, characterized in that, The GPCR receptor is composed of an extracellular protease recognition domain, a transmembrane domain, and an intracellular signaling domain. The extracellular protease recognition domain is an amino acid sequence of the TEV protease hydrolysis peptide as shown in SEQ IN NO.2; The transmembrane domain is composed of the N-terminal domain of GPR56 as shown in the amino acid sequence of SEQ ID NO.1 (positions 1-381) and the C-terminal domain of GPR56 as shown in the amino acid sequence of SEQ ID NO.1 (positions 389-693), wherein the extracellular protease recognition domain connects the N-terminal domain of GPR56 and the C-terminal domain of GPR56. The amino acid sequence of the extracellular protease recognition domain linked to the transmembrane domain is shown in SEQ ID NO.1; The intracellular signaling domain consists of the HCV enzyme hydrolysis peptide with the amino acid sequence shown in SEQ ID NO. 3 and the NLS with the amino acid sequence shown in SEQ ID NO.

4. dCas9 VP64 MCP P65 composition.

2. A nucleic acid molecule, characterized in that, Encodes a GPCR receptor that responds to the protease as described in claim 1.

3. A lentiviral particle, characterized in that, It contains a nucleotide sequence encoding a GPCR receptor that responds to the protease as described in claim 1.

4. The lentiviral particle according to claim 3, characterized in that, The lentiviral particle also contains a nucleotide sequence encoding an adaptor protein, the amino acid sequence of which is shown in SEQ ID NO.

12.

5. The lentiviral particle according to claim 3 or 4, characterized in that, The lentiviral particle also contains a nucleotide sequence encoding an HCV enzyme, the amino acid sequence of which is shown in SEQ ID NO.

23.

6. A recombinant cell, characterized in that, Expressing the protease-responsive GPCR receptor as described in claim 1.

7. The recombinant cell according to claim 6, characterized in that, The recombinant cells also express adaptor protein-coupled HCV enzyme.

8. The recombinant cell according to claim 6 or 7, characterized in that, The recombinant cells include natural killer cells.

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