SynNotch-based engineered macrophage and application thereof

Abstract

The invention provides a synNotch receptor which comprises an extracellular domain, a transmembrane domain and an intracellular domain, and the extracellular domain comprises a single-chain variable fragment of a targeted tumor specific antigen. The invention also provides an engineered macrophage based on the synNotch receptor, and a preparation method of the engineered macrophage. The synNotch receptor disclosed by the invention can be used for specifically recognizing a TRP1 antigen on the surface of a tumor cell. The engineered macrophage provided by the invention has a good specific targeting effect on subcutaneous tumor and metastatic tumor, can effectively inhibit tumor growth, and has no side effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to an engineered macrophage based on synNotch and an application thereof. Background Art

[0002] Macrophages are a type of mononuclear leukocytes distributed throughout the body, which participate in many life processes of the body. Macrophages have a tropism for a variety of diseased tissues and participate in the occurrence and development of diseases. Tumors usually contain a large number of macrophages. For example, in breast cancer, the mass proportion of macrophages can reach 70%. Most opinions believe that most macrophages in tumor sites are derived from bone marrow and peripheral blood monocytes, which are recruited to the tumor site and transformed into tumor-associated macrophages under the action of chemokines and cytokines secreted by tumor-associated stromal cells and tumor cells. In addition to primary tumors, macrophages are also one of the first immune cells recruited to metastatic niches, providing favorable conditions for the metastasis and colonization of tumor cells.

[0003] Synthetic Notch (synNotch) receptors are modified from natural Notch proteins. Researchers have not only modified the extracellular part of the Notch protein to enable it to recognize specific antigens, but also modified the intracellular part of the Notch protein so that the downstream response of synNotch after recognizing specific antigens can also be artificially controlled. The synNotch receptor usually consists of three parts: the extracellular antigen recognition domain, which is responsible for recognizing antigens; the transmembrane Notch core, which will be cleaved by the γ-secretase on the cell membrane after the extracellular part recognizes the antigen and releases the intracellular part of synNotch into the cytoplasm; and the intracellular artificial transcription factor, which will enter the cell nucleus after the Notch core is cleaved to trigger the expression of specific genes. Therefore, cells engineered with synNotch can not only recognize specific tumor cells, but also make personalized responses after recognizing specific tumor cells. However, at present, synNotch receptors are mainly used in cells that are not easy to infiltrate tumors, such as T cells, which limits the role of synNotch receptors in tumors.

[0004] Immune checkpoint blockade therapies, such as anti-programmed death receptor 1 antibodies, have achieved clinical success in the treatment of certain cancer types. However, immune checkpoint blockade therapies are not effective in immunologically "cold" tumors due to the lack of pre-existing anti-tumor immunity. This limitation has prompted the development of therapies in combination with inflammatory cytokines. Among them, interleukin-12 (IL-12) promotes the polarization of T helper type 1 (Th1) cells and the secretion of interferon-γ (IFN-γ), thereby bridging the gap between innate and adaptive immunity. Despite promising results in preclinical studies, systemic administration of IL-12 has not been ideal in clinical trials due to limited accumulation of the drug within tumors and stimulation of lymphocytes in healthy tissues, which can produce adverse reactions.

[0005] Immune checkpoint blockers used clinically are mainly in the form of antibodies. However, due to their large molecular weight and complex post-translational modifications, antibodies are difficult to express simply through recombinant vectors and are not easy to form fusion proteins with other proteins. Nanobodies are usually only 15KDa in size and are easily expressed through recombinant vectors and are easy to form fusion proteins with other proteins. Summary of the invention

[0006] In view of the problems existing in the above-mentioned prior art, the inventors have provided a therapy based on synNotch engineered macrophages after long-term technical and practical exploration. The synNotch engineered macrophages of the present invention can recognize specific tumor antigens and then secrete a fusion protein of IL-12 and anti-PD-L1 nanoantibody (PD-L1 Nb). This therapy is not only suitable for the treatment of primary tumors, but also for metastatic tumors. It does not produce obvious toxic side effects while inducing a strong anti-tumor immune response.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a synNotch receptor comprising an extracellular domain, a transmembrane domain and an intracellular domain, wherein the extracellular domain comprises a single-chain variable fragment targeting a tumor-specific antigen.

[0009] According to some embodiments of the present invention, the tumor-specific antigen is selected from one or more of melanoma-specific antigens tyrosinase-related protein 1 (TRP1), CD19, CD20, CD133, HER2, EGFR, mesothelin, IL13RA2, c-MET and EphA2.

[0010] Preferably, the tumor-specific antigen is the melanoma-specific antigen tyrosinase-related protein 1 (TRP1).

[0011] According to some embodiments of the invention, the single chain variable fragment is a TA99 single chain antibody (TA99 scFv).

[0012] Preferably, the amino acid sequence of the TA99 single-chain antibody comprises the amino acid sequence shown in SEQ ID NO: 1, or the amino acid sequence of the TA99 single-chain antibody is as shown in SEQ ID NO: 1.

[0013] SEQ ID NO: 1:

[0014] MDFQVQIFSFLLISASVIMSRQFQVKLEESGAELVRPGALVKLSCKTSGFNIKDYFLHWVRQRPDQGLEWIGWINPDNGNTVYDPKLQGTASLTADTSSNTVYLQLSGLTSEDTAVYFCTRRDYTYEKAALDYWGQGASVIVSSAKTTAPSVYPLAPVCGDTTG SSVTLGCLVKRSGGGGSGGGGSGGGGSGARHCAHTDSKMSMSVGERVTLTCKASENVVTYVSWYQQKPEQSPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQGYSYPYTFGGGTKLEIKRADAAPTYPHHHHHHHLQISSTVAAARV

[0015] According to some embodiments of the invention, the extracellular domain further comprises a membrane targeting signal peptide and a peptide tag.

[0016] Preferably, the membrane targeting signal peptide is selected from one or more of a CD8α signal peptide, an immunoglobulin heavy chain signal peptide, an immunoglobulin light chain signal peptide and a GM-CSF signal peptide. More preferably, the membrane targeting signal peptide is a CD8α signal peptide.

[0017] Further preferably, the amino acid sequence of the CD8α signal peptide is as shown in SEQ ID NO: 2.

[0018] SEQ ID NO: 2:MALPVTALLLPLALLLHAARP

[0019] Preferably, the peptide tag is selected from one or more of a MYC-tag, a 6×His-tag and a Flag-tag. More preferably, the peptide tag is a MYC-tag.

[0020] Further preferably, the amino acid sequence of the MYC-tag is as shown in SEQ ID NO: 3.

[0021] SEQ ID NO: 3: EQKLISEEDL

[0022] According to some preferred embodiments of the present invention, the extracellular domain comprises a CD8α signal peptide, a MYC-Tag and a TA99 scFv which are operably linked in sequence.

[0023] More preferably, the amino acid sequence of the extracellular domain is as shown in SEQ ID NO: 4.

[0024] SEQ ID NO: 4:

[0025] MALPVTALLLPLALLLHAARPEQKLISEEDLMDFQVQIFSFLLISASVIMSRQFQVKLEESGAELVRPGALVKLSCKTSGFNIKDYFLHWVRQRPDQGLEWIGWINPDNGNTVYDPKLQGTASLTADTSSNTVYLQLSGLTSEDTAVYFCTRRDYTYEKAALDYWGQGASVIVSSAKTT APSVYPLAPVCGDTTGSSVTLGCLVKRSGGGGSGGGGSGGGGSGARHCAHTDSKMSMSVGERVTLTCKASENVVTYVSWYQQEQSPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQGYSYPYTFGGGTKLEIKRADAAPTYPHHHHHHHHLQISSTVAAARV

[0026] According to some embodiments of the present invention, the transmembrane domain comprises a Notch core, wherein the N-terminus of the Notch core is connected to the C-terminus of the extracellular domain, and the C-terminus of the Notch core is connected to the N-terminus of the intracellular domain.

[0027] Preferably, the amino acid sequence of the Notch core comprises the amino acid sequence shown in SEQ ID NO: 5, or the amino acid sequence of the Notch core is as shown in SEQ ID NO: 5.

[0028] SEQ ID NO: 5:

[0029] PCVGSNPCYNQGTCEPTSENPFYRCLCPAKFNGLLCHILDYSFTGGAGRDIPPPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLA AGTLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALASLGSLNIPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCGVLLSRKRRR

[0030] According to some embodiments of the invention, the intracellular domain comprises a transcription factor.

[0031] Preferably, the transcription factor is selected from one or more of GAL4-VP64, GAL4-p65-GR, GAL4-VPR and GAL4-MiniVPR. More preferably, the transcription factor is GAL4-VP64.

[0032] Further preferably, the amino acid sequence of the GAL4-VP64 comprises the amino acid sequence shown in SEQ ID NO: 6, or the amino acid sequence of the GAL4-VP64 is as shown in SEQ ID NO: 6.

[0033] SEQ ID NO: 6:

[0034] MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVSAAAGGSGGSGGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDFDFDLDMLGS

[0035] In a second aspect, the present invention provides an isolated nucleic acid molecule encoding the synNotch receptor according to the present invention.

[0036] According to some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule encoding the extracellular domain is shown in SEQ ID NO: 7.

[0037] SEQ ID NO: 7:

[0038]

[0039] According to some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule encoding the transmembrane domain is shown in SEQ ID NO: 8.

[0040] SEQ ID NO: 8:

[0041]

[0042] According to some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule encoding the intracellular domain is shown in SEQ ID NO: 9.

[0043] SEQ ID NO: 9:

[0044] atgaagctgctgagcagcatcgagcaggcctgtgacatctgccggctgaagaaactgaagtgcagcaaagaaaagcccaagtgcgccaagtgcctgaagaacaactgggagtgccggtacagccccaagaccaagagaagccccctgaccagagccca cctgaccgaggtggaaagccggctggaaagactggaacagctgtttctgctgatcttcccacgcgaggacctggacatgatcctgaagatggacagcctgcaggacatcaaggccctgctgaccggcctgttcgtgcaggacaacgtgaacaaggacg ccgtgaccgacagactggccagcgtggaaaccgacatgcccctgaccctgcggcagcacagaatcagcgccaccagcagcagcgaggaaagcagcaacaagggccagcggcagctgacagtgtctgctgctgcaggcggaagcggaggctctggcgga tctgatgccctggacgacttcgacctggatatgctgggcagcgacgccctggatgattttgatctggacatgctgggatctgacgctctggacgatttcgatctcgacatgttgggatcagatgcactggatgactttgacctggacatgctcggatca

[0045] According to some preferred embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule encoding the synNotch receptor according to the present invention is shown as SEQ ID NO: 10.

[0046] SEQ ID NO: 10:

[0047]

[0048] In a third aspect, the present invention provides a recombinant vector comprising the nucleic acid molecule according to the present invention.

[0049] The recombinant vector of the present invention can be obtained by carrying the nucleic acid molecule according to the present invention using expression vectors known in the art, such as eukaryotic expression vectors, prokaryotic expression vectors, viruses or bacteriophages, etc., preferably plasmid expression vectors.

[0050] In a fourth aspect, the present invention provides an engineered macrophage comprising a first nucleic acid molecule or a first recombinant vector, wherein the first nucleic acid molecule is the nucleic acid molecule encoding the synNotch receptor according to the present invention, and the first recombinant vector is a recombinant vector comprising the nucleic acid molecule encoding the synNotch receptor according to the present invention.

[0051] According to some embodiments of the present invention, the engineered macrophages are derived from RAW264.7 mouse mononuclear macrophages, THP-1 cells, J774A.1 cells, mouse bone marrow-derived macrophages or human peripheral blood mononuclear cells. Preferably, the engineered macrophages are derived from RAW264.7 mouse mononuclear macrophages.

[0052] According to some embodiments of the present invention, the engineered macrophages further comprise a second nucleic acid molecule or a second recombinant vector, wherein the second nucleic acid molecule comprises a nucleic acid molecule encoding a fusion protein of a cytokine and an anti-PD-L1 nanobody; and the second recombinant vector comprises the second nucleic acid molecule.

[0053] According to some embodiments of the invention, the cytokine is selected from one or more of IL-12, IL-2, TNF-α and INF-γ.

[0054] Preferably, the cytokine is IL-12, that is, the second nucleic acid molecule comprises a nucleic acid molecule encoding a fusion protein of IL-12 (interleukin-12) and an anti-PD-L1 nanobody (IL-12-PD-L1 Nb fusion protein).

[0055] More preferably, the IL-12 comprises IL-12p40 and IL-12p35 and a first connecting peptide connecting the two.

[0056] Further preferably, the amino acid sequence of the IL-12p40 is as shown in SEQ ID NO: 11, and the nucleotide sequence of the nucleic acid molecule encoding the IL-12p40 is as shown in SEQ ID NO: 12.

[0057] SEQ ID NO: 11:

[0058] MCPQKLTISWFAIVLLVSPLMAMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRRYYNSSCSKWACVPCRVRS

[0059] SEQ ID NO: 12:

[0060]

[0061] Preferably, the amino acid sequence of the IL-12p35 is as shown in SEQ ID NO: 13, and the nucleotide sequence of the nucleic acid molecule encoding the IL-12p35 is as shown in SEQ ID NO: 14.

[0062] SEQ ID NO: 13:

[0063] MVSVPTASPSASSSSSQCRSSMCQSRYLLFLATLALLNHLSLARVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDDHEDITRDQTSTLKTCLPLELHKNESCLATRE TSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA

[0064] SEQ ID NO: 14:

[0065] atggtcagcgttccaacagcctcaccctcggcatccagcagctcctctcagtgccggtccagcatgtgtcaatcacgctacctcctctttttggccacccttgccctcctaaaccacctcagtttggccagggtcattccagtctctggacctgccaggtgtcttagccagtcccga aacctgctgaagaccacagatgacatggtgaagacggccagagaaaaactgaaacattattcctgcactgctgaagacatcgatcatgaagacatcacacgggaccaaaccagcacattgaagacctgtttaccactggaactacacaagaacgagagttgcctggctactagagag acttcttccacaacaagagggagctgcctgcccccacagaagacgtctttgatgatgaccctgtgccttggtagcatctatgaggacttgaagatgtaccagacagagttccaggccatcaacgcagcacttcagaatcacaaccatcagcagatcattctagacaagggcatgctg gtggccatcgatgagctgatgcagtctctgaatcataatggcgagactctgcgccagaaacctcctgtgggagaagcagacccttacagagtgaaaatgaagctctgcatcctgcttcacgccttcagcacccgcgtcgtgaccatcaacagggtgatgggctatctgagctccgcc

[0066] In the present invention, the first connecting peptide can adopt a flexible linker commonly used in the art. Preferably, the amino acid sequence of the first connecting peptide is as shown in SEQ ID NO: 15, and the nucleotide sequence of the nucleic acid molecule encoding the first connecting peptide is as shown in SEQ ID NO: 16.

[0067] SEQ ID NO: 15:GGGGSGGGGSGGGLASGGS

[0068] SEQ ID NO: 16:

[0069] ggcggcggcgggagtggcggcgggggttctggcggaggcctcgctagcggtggctcc

[0070] According to some preferred embodiments of the present invention, the amino acid sequence of the IL-12 is shown as SEQ ID NO: 17, and the nucleotide sequence of the nucleic acid molecule encoding the IL-12 is shown as SEQ ID NO: 18.

[0071] SEQ ID NO: 17:

[0072] MCPQKLTISWFAIVLLVSPLMAMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNM DLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFL VEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGGGGSGGGGSGGGLASGGSMVSVPTASPSASSSSSQCRSSMCQSRYLLFLATLALLNHLSLARVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDH EDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA

[0073] SEQ ID NO: 18:

[0074]

[0075] According to some embodiments of the present invention, the amino acid sequence of the anti-PD-L1 nanobody (PD-L1 Nb) comprises the amino acid sequence as shown in SEQ ID NO: 19, or the amino acid sequence of the anti-PD-L1 nanobody is as shown in SEQ ID NO: 19. The nucleotide sequence of the nucleic acid molecule encoding the anti-PD-L1 nanobody comprises the nucleotide sequence as shown in SEQ ID NO: 20, or the nucleotide sequence of the nucleic acid molecule encoding the anti-PD-L1 nanobody is as shown in SEQ ID NO: 20.

[0076] SEQ ID NO: 19:

[0077] MAQVQLVETGGGLVQPGGSLRLSCTASGFTFSMHAMTWYRQAPGKQRELVAVITSHGDRANYTDSVRGRFTISRDNTKNMVYLQMNSLKPEDTAVYYCNVPRYDSWGQGTQVTVSSGGLPETGGHHHHHH

[0078] SEQ ID NO: 20:

[0079] atggcccaggtgcagctggtggagaccggcggaggcctggtgcagccagaggaagcctgaggctgagctgcacagcctccggcttcaccttcagcatgcacgccatgacctggtacagacaggcccccggcaagcagagggagctggtggctgtgatcacctcccacggcgacagagccaactacaccgatagc gtgagaggcaggttcaccatctccagggacaacacaaagaacatggtgtacctgcagatgaactccctgaagcccgaggataccgccgtgtactactgcaacgtgcccaggtacgacagctggggccagggcacccaggtgacagtgtcctccggcggcctgcccgagacaggaggacaccaccaccaccatcac

[0080] According to some embodiments of the present invention, the anti-PD-L1 nanobody is connected to the IL-12p35 domain of the IL-12 via a second connecting peptide.

[0081] In the present invention, the second connecting peptide can adopt a flexible linker commonly used in the art. Preferably, the amino acid sequence of the second connecting peptide consists of n amino acid sequences (GGGGS) as shown in SEQ ID NO: 21, wherein n is an integer from 1 to 5. More preferably, the amino acid sequence of the second connecting peptide is as shown in SEQ ID NO: 22, and the nucleotide sequence of the nucleic acid molecule encoding the second connecting peptide is as shown in SEQ ID NO: 23.

[0082] SEQ ID NO: 22: GGGGSGGGGSGGGGS

[0083] SEQ ID NO: 23: ggaggaggaggctccggaggaggcggatctggcggaggaggctct

[0084] According to some preferred embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule encoding the IL-12-PD-L1 Nb fusion protein is shown in SEQ ID NO: 24.

[0085] SEQ ID NO: 24:

[0086]

[0087] According to some embodiments of the present invention, the second nucleic acid molecule further comprises an upstream activation sequence (UAS) and a promoter at the 5' end of the nucleic acid molecule encoding the IL-12-PD-L1 Nb fusion protein. The second nucleic acid molecule further comprises a polyA sequence at the 3' end of the nucleic acid molecule encoding the IL-12-PD-L1 Nb fusion protein.

[0088] Preferably, the nucleotide sequence of the upstream activation sequence comprises the nucleotide sequence shown in SEQ ID NO: 25, or the nucleotide sequence of the upstream activation sequence is as shown in SEQ ID NO: 25.

[0089] SEQ ID NO: 25:

[0090] ggagcactgtcctccgaacgtcggagcactgtcctccgaacgtcggagcactgtcctccgaacgtcggagcactgtcctccgaacg

[0091] Preferably, the promoter is a minimal CMV promoter (miniCMV). More preferably, the nucleotide sequence of the minimal CMV promoter is shown in SEQ ID NO: 26.

[0092] SEQ ID NO: 26:

[0093] taggcgtgtacggtgggaggcctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagc

[0094] According to some preferred embodiments of the present invention, the second nucleic acid molecule comprises, from the 5' end to the 3' end, an upstream activation sequence (UAS), a minimal CMV promoter (miniCMV), a nucleic acid molecule encoding an IL-12-PD-L1 Nb fusion protein, and a polyA sequence.

[0095] According to a most preferred embodiment of the present invention, the nucleotide sequence of the second nucleic acid molecule is as shown in SEQ ID NO: 27.

[0096] SEQ ID NO: 27:

[0097]

[0098] In a fifth aspect, the present invention provides a pharmaceutical composition for preventing and / or treating cancer, comprising the engineered macrophages according to the present invention and a pharmaceutically acceptable excipient.

[0099] According to some embodiments of the present invention, the cancer is selected from one or more of melanoma, liver cancer, lung cancer, pancreatic cancer, brain cancer, breast cancer and ovarian cancer. Preferably, the cancer is melanoma.

[0100] In a sixth aspect, the present invention provides use of the engineered macrophages according to the present invention in the preparation of a medicament for preventing and / or treating cancer.

[0101] According to some embodiments of the present invention, the cancer is selected from one or more of melanoma, liver cancer, lung cancer, pancreatic cancer, brain cancer, breast cancer and ovarian cancer. Preferably, the cancer is melanoma.

[0102] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0103] The engineered macrophages provided by the present invention can specifically recognize TRP1-positive cells through the synNotch receptor while retaining the ability of macrophages to target tumors. In addition, after the extracellular domain of the synNotch receptor recognizes the corresponding antigen (TRP1), the transmembrane domain of the synNotch receptor is cleaved, and the intracellular domain of the synNotch receptor falls off and binds to the upstream activation sequence of the second nucleic acid molecule, thereby initiating the expression of the IL-12-PD-L1 Nb fusion protein, thereby specifically secreting the IL-12-PD-L1 Nb fusion protein into the tumor. In addition, PD-L1 Nb not only acts as an immune checkpoint blocker, but can also bind to PD-L1-positive cells in the tumor, thereby preventing the IL-12-PD-L1 Nb fusion protein from spreading out of the tumor site.

[0104] Experimental verification shows that the homing ability of the engineered macrophages provided by the present invention is not affected, and they have good specific targeting effects on both subcutaneous tumors and metastatic tumors. In addition, the engineered macrophages provided by the present invention can effectively inhibit the growth of subcutaneous tumors and metastatic tumors without producing obvious side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0106] Figure 1 The structure of the synNotch receptor of the present invention and the mechanism of action of the synNotch receptor controlling the expression of the mCherry recombinant vector are shown.

[0107] Figure 2 The figure shows the transfection results of RAW264.7 cells obtained by flow cytometry in the present invention.

[0108] Figure 3 Mac showing the invention synNotch Mechanism of action for recognition of TRP1 on B16F10 cells and expression of mCherry in response.

[0109] Figure 4 Mac showing the invention synNotch Results of specific response to B16F10 cells. A is Mac synNotch Schematic diagram of specific response to TRP1 positive cells; B is Mac synNotch Fluorescence imaging after co-incubation with different cells; C is Mac synNotch Flow cytometric quantification of mCherry positivity after co-incubation with different cells.

[0110] Figure 5 Mac showing the invention synNotch A is a fluorescent image of the main organs and tumors of mice after injection of different drugs; B is a quantitative image of the distribution of Mac in the main organs and tumors of mice; C is a quantitative image of Mac synNotch Quantitative diagram of the main organs and tumor distribution in mice.

[0111] Figure 6 Mac showing the invention synNotch A is a fluorescence imaging diagram of the main organs of mice after injection of different drugs; B is a quantitative diagram of the distribution ratio of Mac in the main organs of mice without metastasis; C is a quantitative diagram of the distribution ratio of Mac in the main organs of mice with metastasis; D is a quantitative diagram of Mac synNotch Quantitative plot of the distribution percentage of major organs in mice with metastatic tumors.

[0112] Figure 7 The EGFP-positive and mCherry-positive Mac synNotch The accumulation of EGFP-positive Mac synNotch Accumulation in mice; B shows the mCherry-positive Mac synNotch Accumulation in mice.

[0113] Figure 8The construction and immunoblotting results of the IL-12-PD-L1 Nb fusion protein of the present invention are shown. A shows the structure of the IL-12-PD-L1 Nb fusion protein of the present invention; B is an immunoblot of the IL-12-PD-L1 Nb fusion protein of the present invention.

[0114] Fig. 9 The ELISA test results of the IL-12-PD-L1 Nb fusion protein of the present invention stimulating spleen cells to secrete IFN-γ are shown.

[0115] Fig.10 The results of co-incubation of IL-12p40 or IL-12-PD-L1 Nb fusion protein with B16F10 cells are shown. A is the result of co-incubation of IL-12p40 with B16F10 cells; B is the result of co-incubation of IL-12-PD-L1 Nb fusion protein with B16F10 cells.

[0116] Fig.11 Mac showing the invention TA99+IL-12-Nb mechanism of action.

[0117] Fig.12 Mac showing the invention TA99+IL-12-Nb IL-12-PD-L1 Nb secretion after response to B16F10 cells.

[0118] Fig.13 Mac showing the invention TA99+IL-12-Nb The secreted IL-12-PD-L1 Nb can activate splenocytes to secrete IFN-γ.

[0119] Fig.14 The survival conditions of mice in different experimental groups in Example 5 of the present invention are shown. A shows the changes in tumor volume of mice in different experimental groups; B shows the survival rates of mice in different experimental groups; and C shows the changes in body weight of mice in different experimental groups.

[0120] Fig.15 Figure 5 shows the changes in the tumor microenvironment of mice in different experimental groups in Example 5 of the present invention. A shows the IFN-γ level in the tumors of mice in different experimental groups; B shows the CD8 + The number of T cells; C shows the number of NKT cells in the tumors of mice in different experimental groups; D shows the number of NK cells in the tumors of mice in different experimental groups.

[0121] Fig.16 The survival conditions of mice in different experimental groups in Example 6 of the present invention are shown. A shows the radiation rate of mice in different experimental groups; B shows the survival rate of mice in different experimental groups; and C shows the weight changes of mice in different experimental groups.

[0122] Fig.17 The survival conditions of mice in different experimental groups in Example 7 of the present invention are shown. A shows the content of IL-12 in the serum of mice in different experimental groups; B shows the content of IFN-γ in the serum of mice in different experimental groups; C shows the spleen weight of mice in different experimental groups; D shows the liver water content of mice in different experimental groups; and E shows the body weight of mice in different experimental groups. DETAILED DESCRIPTION

[0123] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting the scope of the present invention.

[0124] The sequence of the various elements in the following embodiments is as follows:

[0125] TA99 scFv: amino acid sequence as shown in SEQ ID NO: 1;

[0126] CD8α signal peptide: amino acid sequence as shown in SEQ ID NO: 2;

[0127] MYC-tag: amino acid sequence as shown in SEQ ID NO: 3;

[0128] Extracellular domain: amino acid sequence as shown in SEQ ID NO: 4, encoding nucleic acid as shown in SEQ ID NO: 7;

[0129] Notch core: amino acid sequence as shown in SEQ ID NO: 5, encoding nucleic acid as shown in SEQ ID NO: 8;

[0130] GAL4-VP64: the amino acid sequence is shown in SEQ ID NO: 6, and the encoding nucleic acid is shown in SEQ ID NO: 9;

[0131] synNotch receptor: the encoding nucleic acid is shown in SEQ ID NO: 10;

[0132] IL-12p40: the amino acid sequence is shown in SEQ ID NO: 11, and the encoding nucleic acid is shown in SEQ ID NO: 12;

[0133] IL-12p35: the amino acid sequence is shown in SEQ ID NO: 13, and the encoding nucleic acid is shown in SEQ ID NO: 14;

[0134] First connecting peptide: the amino acid sequence is shown in SEQ ID NO: 15, and the encoding nucleic acid is shown in SEQ ID NO: 16;

[0135] IL-12: the amino acid sequence is shown in SEQ ID NO: 17, and the encoding nucleic acid is shown in SEQ ID NO: 18;

[0136] PD-L1 Nb: the amino acid sequence is shown in SEQ ID NO: 19, and the encoding nucleic acid is shown in SEQ ID NO: 20;

[0137] Second connecting peptide: the amino acid sequence is shown in SEQ ID NO: 22, and the encoding nucleic acid is shown in SEQ ID NO: 23;

[0138] IL-12-PD-L1 Nb fusion protein: the encoding nucleic acid is shown in SEQ ID NO: 24;

[0139] Upstream activating sequence (UAS): the encoding nucleic acid is shown in SEQ ID NO: 25;

[0140] Minimal CMV promoter (miniCMV): the encoding nucleic acid is shown in SEQ ID NO: 26;

[0141] IL-12-PD-L1 Nb fusion protein whose expression is controlled by synNotch receptor: the encoding nucleic acid is shown in SEQ ID NO: 27.

[0142] The construction of each recombinant vector in the following examples was commissioned to General Biotech (Anhui) Co., Ltd. The cells used were purchased from Xiangya Hospital.

[0143] Example 1: SynNotch-engineered macrophages specifically respond to TRP1 + B16F10 cells

[0144] In order to explore whether the synNotch receptor can specifically respond to TRP1-positive cells, the vector pCMV-HA was double-digested with 5'BbSI and 3'NotI, and then SEQ ID NO: 10 was inserted into the restriction site to construct a recombinant vector containing the synNotch receptor nucleic acid. The specific construction process was entrusted to General Bio (Anhui) Co., Ltd. In addition, a recombinant vector for the expression of mCherry controlled by the synNotch receptor was constructed. In order to mark the recombinant vector for the expression of mCherry controlled by the synNotch receptor, the recombinant vector also contained a nucleic acid for stably expressing EGFP. Specifically, the vector pcDNA3.1-EGFP was double-digested with 5'DraIII and 3'BstBI, and then SEQ ID NO: 26 was inserted into the restriction site to construct the recombinant vector CMV-EGFP-5×UAS-miniCMV-mCherry for the expression of mCherry controlled by the synNotch receptor. The specific construction process was entrusted to General Bio (Anhui) Co., Ltd.

[0145] SEQ ID NO: 28:

[0146] ggagcactgtcctccgaacgtcggagcactgtcctccgaacgtcggagcactgtcctccgaacgtcggagcactgtcctccgaacggagcatgtcctccgaacgtcggagcactgtcctccgaacgactagttaggcgtgtacggtgggaggcctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcctctcgacattcgttggatcgccaccatggtgagcaagggcgaggaggataacatggccatcatcaaggagttcatgcgcttcaaggtgcacatggagggctccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctcagttcatgtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtggtgaccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggtgaagctgcgcggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggcctcctccgagcggatgtaccccgaggacggcgccctgaagggcgagatcaagcagaggctgaagctgaaggacggcggccactacgacgctgaggtcaagaccacctacaaggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttggacatcacctcccacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggcatggacgagctgtacaagtag

[0147] Figure 1 The structure of the synNotch receptor and a schematic diagram of the recombinant vector that controls the expression of mCherry are shown. When the synNotch receptor recognizes the corresponding antigen, its intracellular domain GAL4-VP64 will bind to the UAS sequence and initiate the expression of downstream mCherry.

[0148] To construct synNotch engineered macrophages (Mac synNotch ), the two recombinant vectors were transfected into RAW264.7 mouse mononuclear macrophages (obtained from Xiangya Hospital) using jetOPTIMUs (Polyplus) at a ratio of 1:1. 24 hours after transfection, the cells were blocked with PBS solution containing 2% BSA for 1 hour, and then incubated with CoraLite® Plus 647-coupled anti-MYC Tag antibody (Proteintech) at room temperature for 1 hour. After washing with PBS, the transfection status of RAW264.7 cells was analyzed by flow cytometry. Figure 2 As shown in Figure 2, more than 20% of the cells simultaneously expressed the MYC-tag, also known as the synNotch receptor, and EGFP. This indicates that RAW264.7 cells have been successfully transformed into Mac synNotch .

[0149] Mac synNotch Mechanisms for recognizing specific cells Figure 3 Mac synNotch The extracellular part of the synNotch receptor (i.e. Figure 3 After TA99 in the synNotch receptor recognizes TRP1, the Notch core of the synNotch receptor is cleaved by γ-secretase, and then the intracellular part of the synNotch receptor GAL4-VP64 (i.e. Figure 3 TF in the cell will fall off and enter the nucleus to bind to the UAS sequence and activate the downstream mCherry (i.e. Figure 3 The expression of RFP in

[0150] To verify the Mac synNotch Specificity for TRP1-positive cells, Mac synNotch The cells were co-incubated with TRP1-positive B16F10 cells or TRP1-negative 4T1, 3T3, LLC, or CT26 cells for 48 h, and then Mac was analyzed by flow cytometry and confocal imaging. synNotch Expression of mCherry. Figure 4 As shown, when co-incubated with B16F10 cells, almost all Mac synNotchWhen co-cultured with 4T1, CT26, 3T3 or LLC cells, almost all Mac synNotch Neither of them express mCherry. This indicates that Mac synNotch Only TRP1-positive cells will be recognized.

[0151] Example 2: Mac synNotch Able to home to and specifically respond to B16F10 tumors

[0152] To explore Mac synNotch To test the ability of homing to subcutaneous tumors, 1×10 6 B16F10 cells. When the tumor grows to 200 mm 3 After that, 5×10 6 Mac labeled with near-infrared fluorescent probe VT680 synNotch Or untreated macrophages (Mac) were injected into mice via tail vein injection. The PBS group was injected with an equal volume of PBS solution. synNotch The method of Mac was as follows: 1 g of VivoTrack 680 (VT680, PerkinElmer) was dissolved in 1.2 mL of PBS solution, and then 2.5 × 10 8 Mac synNotch After incubation at room temperature in the dark for 15 minutes, add 10-15 mL of PBS solution and wash the cells three times to obtain VT680-labeled Mac. synNotch or Mac.

[0153] 72 hours after macrophage injection, the main organs and tumors of the mice were obtained, and the fluorescence of VT680 in each organ and tumor was analyzed by a small animal imaging system (PerkinElmer, model: IVIS SPECTRUM CT). Figure 5 As shown, Mac and Mac synNotch In mice, the biodistribution is similar, with the largest amount accumulated in the liver, followed by the tumor, and less accumulation in other organs. This indicates that synNotch engineering does not affect the ability of macrophages to home to tumors.

[0154] To explore Mac synNotch To evaluate the ability of homing metastases, 1×10 6 7 days after tumor cell injection, 5×10 6 VT680-branded Mac synNotchOr Mac was injected into mice via tail vein injection. Mice of the same age that were not injected with B16F10 tumor cells were also injected with 5×10 6 A VT680-labeled Mac was used to observe the accumulation of macrophages in normal lungs. 72 hours after the injection of macrophages, the main organs and tumors of the mice were obtained, and then the fluorescence of VT680 in each organ and tumor was analyzed by a small animal imaging system (PerkinElmer, model: IVIS SPECTRUM CT). Figure 6 As shown, Mac and Mac synNotch In the B16F10 metastatic tumor model mice, the biodistribution was similar, with the largest amount accumulated in the liver, followed by the lungs, and less accumulation in other organs. synNotch There is a higher accumulation in the lungs of B16F10 metastatic tumor model mice, which indicates that macrophages also have a good targeting effect on metastatic tumors.

[0155] To explore Mac synNotch In vivo activation in mice, 6-week-old C57BL / 6 mice were injected with 5×10 5 7 days after tumor cell injection, 5×10 6 Mac synNotch The macrophages were injected into the mice through the tail vein. 72 hours after the macrophage injection, the main organs and tumors of the mice were obtained and prepared into single cell suspensions, and then the Mac in each organ and tumor was analyzed by flow cytometry. synNotch Response situation. Figure 7 As shown, EGFP-positive Mac synNotch Mainly distributed in the liver and tumors, while other organs accumulate Mac synNotch Less. And only Mac accumulated in the tumor synNotch Almost 100% of them expressed mCherry, while synNotch engineered macrophages distributed in other organs did not express mCherry. This indicates that synNotch engineered macrophages only activate synNotch receptors in B16F10 tumors, but not in normal tissues.

[0156] Example 3: Construction and functional verification of IL-12-PD-L1 Nb fusion protein

[0157] To verify the successful construction of IL-12-PD-L1 Nb fusion protein, a His tag was added to the C-terminus of PD-L1 Nb of IL-12-PD-L1 Nb fusion protein. Figure 8A shows the schematic diagram of the structure of IL-12-PD-L1 Nb fusion protein. IL-12 is divided into two parts, IL-12p40 and IL-12p35, which are connected by the first connecting peptide, and PD-L1 Nb is connected to IL-12p35 by the second connecting peptide. According to the standard transfection method of Lipo3000 provided by the manufacturer, the recombinant vector CMV-EGFP-5×UAS-miniCMV-IL12-Nb containing the nucleic acid encoding the IL-12-PD-L1 Nb fusion protein or the recombinant vector CMV-EGFP-5×UAS-miniCMV-IL12 containing the nucleic acid encoding IL-12 was transfected into HEK293T cells by Lipo3000 (Thermo Fisher). The control group represents HEK293T cells that were not transfected with the recombinant vector. Among them, the recombinant vector CMV-EGFP-5×UAS-miniCMV-IL12-Nb is obtained by replacing the mCherry sequence in the recombinant vector CMV-EGFP-5×UAS-miniCMV-mCherry in Example 1 with the encoding nucleic acid of IL-12-PD-L1 Nb (SEQ ID NO: 24), and the recombinant vector CMV-EGFP-5×UAS-miniCMV-IL12 is obtained by replacing the mCherry sequence in the recombinant vector CMV-EGFP-5×UAS-miniCMV-mCherry in Example 1 with the encoding nucleic acid of IL-12 (SEQ ID NO: 18). The specific construction process was entrusted to General Biotechnology (Anhui) Co., Ltd.

[0158] 24 hours after transfection, the cells were lysed and the proteins were collected, and then the expression of IL-12-PD-L1 Nb fusion protein in the cells was detected by immunoblotting. The specific method is as follows:

[0159] After washing with PBS, the cells were resuspended in RIPA lysis buffer containing 1 mM phenylmethylsulfonyl fluoride protease inhibitor (PSMF). The protein amount was determined using the BCA protein assay kit. Equal amounts of protein (20 μg) were separated on SDS-PAGE gels and then electroblotted onto PVDF membranes and blocked for 1 hour with Tris-buffered saline (TBS) containing 0.05% Tween-20 and 5% nonfat dry milk. Next, the membranes were incubated with primary antibodies against IL-12 (1: 1000 dilution), His-tag (1: 1000) or GAPDH (1: 1000 dilution) overnight, followed by incubation with horseradish peroxidase (HRP)-labeled secondary antibodies (1: 5000 dilution) for 1 hour. Protein signals were imaged using enhanced chemiluminescence (ECL) reagents on a chemiluminescence imaging system (ChemiDoc XRS+).

[0160] like Figure 8 As shown in Figure B, the molecular weight of the IL-12-PD-L1 Nb fusion protein increased by about 15 KDa compared to IL-12, and only the IL-12-PD-L1 Nb fusion protein contained a His tag, which indicated the successful expression of the IL-12-PD-L1 Nb fusion protein.

[0161] In order to explore whether fusion PD-L1 Nb would affect the function of IL-12 itself, the recombinant vector containing IL-12-PD-L1 Nb fusion protein or IL-12 encoding nucleic acid was transfected into HEK293T cells by Lipo3000 (Thermo Fisher Scientific), and then the cell culture medium within 24-48 hours of transfection was collected. At this time, the cell culture medium contained IL-12-PD-L1 Nb fusion protein or IL-12 secreted by HEK293T cells. The two cell culture media were mixed with fresh culture medium in different proportions. Specifically, the total amount of culture medium was fixed to 1 mL, and the volume of culture medium containing IL-12-PD-L1 Nb fusion protein or IL-12 was as follows: Fig. 9 As indicated, the remaining volume of the medium was supplemented with fresh DMEM complete medium. Then, the medium mixture was incubated with splenocytes extracted from the spleen of C57BL / 6 mice at 37°C for 2-12 hours to activate the splenocytes. After 48 hours of stimulation, the secretion of IFN-γ was tested by ELISA. Fig. 9 As shown in the figure, both IL-12 and IL-12-PD-L1 Nb fusion proteins can effectively stimulate splenocytes to secrete IFN-γ, which indicates that IL-12 fusion with PD-L1 Nb does not affect its own function.

[0162] In order to explore whether PD-L1 Nb can bind the fusion protein to PD-L1-positive B16F10 cells, the p35 part on the IL-12-PD-L1 Nb fusion protein was replaced with EGFP, and then the recombinant vector containing the nucleic acid encoding this fusion protein was transfected into HEK293T cells using Lipo3000 (Thermo Fisher Scientific). The cell culture medium within 24-48 hours of transfection was collected. At this time, the cell culture medium contained the p40-EGFP-PD-L1 Nb fusion protein secreted by HEK293T cells. This cell culture medium was incubated with B16F10 cells at 37°C for 2-12 hours, and then the fluorescence changes on the B16F10 cells were observed by confocal imaging. Fig.10As shown in B, green fluorescence appears on the B16F10 cell membrane after 2 hours of co-incubation, indicating that the p40-EGFP-PD-L1 Nb fusion protein binds to the B16F10 cells, and as time goes by, more p40-EGFP-PD-L1 Nb fusion proteins are attached to the B16F10 cells. However, when PD-L1 Nb is not present on the fusion protein, the fusion protein will not bind to the B16F10 cells even after 12 hours of co-incubation ( Fig.10 A). This indicates that PD-L1 Nb is able to anchor the fusion protein to PD-L1-positive B16F10 cells.

[0163] Example 4: SynNotch engineered macrophages secrete IL-12-PD-L1 Nb fusion protein after recognizing B16F10 cells

[0164] In order to prepare synNotch engineered macrophages (Mac TA99+IL-12-Nb ), the mCherry encoding nucleic acid on the recombinant vector expressing mCherry controlled by the synNotch receptor was replaced with the IL-12-PD-L1 Nb fusion protein encoding nucleic acid (SEQ ID NO: 24). The two recombinant vectors were transfected into RAW264.7 mouse mononuclear macrophages using jetOPTIMUs (Polyplus) at a ratio of 1:1, thereby preparing synNotch engineered macrophages capable of secreting IL-12-PD-L1 Nb fusion protein in response to B16F10 cells (the specific method is the same as in Example 1). Fig.11 Shows Mac TA99+IL-12-Nb Mechanism of action, Mac TA99+IL-12-Nb After recognizing TRP1 on B16F10 cells, the IL-12-PD-L1 Nb fusion protein is activated. PD-L1 Nb can bind to PD-L1 on B16F10 cells, blocking the PD-L1 / PD-1 signaling pathway while anchoring the IL-12-PD-L1 Nb fusion protein on B16F10 cells. IL-12 then activates T cells to secrete IFN-γ.

[0165] To verify the Mac TA99+IL-12-Nb Can Mac TA99+IL-12-Nb The cells were co-incubated with TRP1-positive B16F10 cells or TRP1-negative 4T1, LLC, 3T3 or CT26 cells for 48 h, and the secretion of IL-12 was analyzed by ELISA. TA99+IL-12-Nb Not co-incubated with cancer cells. Fig.12 As shown, Mac TA99+IL-12-Nb IL-12-PD-L1 Nb fusion protein was only secreted in large quantities when co-incubated with B16F10 cells, while the secretion of IL-12 was only slightly increased when co-incubated with TRP1-negative cells.

[0166] Verification of Mac by transwell experiment TA99+IL12-Nb Can the secreted IL-12-PD-L1 Nb fusion protein activate splenocytes? First, plate Mac TA99+IL-12-Nb cells and 4T1, LLC, 3T3, CT26 or B16F10 cells, and spleen cells were plated in the upper chamber 48 hours later and cultured for another 48 hours. TA99+IL-12-Nb The supernatant was collected and tested for IFN-γ by ELISA. Fig.13 As shown, only when co-incubated with B16F10 cells will the spleen cells in the upper chamber be activated to secrete IFN-γ. Fig.13 Mac IL-12-Nb Mac TA99+IL12-Nb .

[0167] Example 5: Mac TA99+IL-12-Nb Therapy can inhibit the growth of B16F10 subcutaneous tumors

[0168] To investigate the anti-tumor ability of synNotch-engineered macrophages, 1×10 6 Seven days after tumor cell implantation, the mice were divided into six groups: the first group of mice were injected with PBS solution through the tail vein (PBS group); the second group of mice were injected with 5×10 6 untreated RAW264.7 cells (Mac group); mice in the third group were injected with 5×10 6 RAW264.7 cells (Mac TA99 group); mice in group 4 were injected with 5×10 6 RAW264.7 cells (Mac IL-12-Nb The fifth group of mice was injected with 5×10 6 RAW264.7 cells (Mac TA99+IL-12 group); mice in group 6 were injected with 5×10 6RAW264.7 cells (Mac TA99+IL-12-Nb Each group of mice was injected with drugs once on the 7th, 12th and 17th days, and the tumor size, body weight change and survival rate of the mice were recorded every day. Fig.14 As shown, Mac TA99+IL-12-Nb The tumor growth of mice in the Mac group was the slowest, their body weight remained stable, and their survival rate was the highest. TA99+IL-12-Nb The therapy can effectively hinder the growth of B16F10 tumors and improve the survival rate of mice.

[0169] To explore Mac TA99+IL-12-Nb To investigate the effect of the therapy on the tumor microenvironment, the tumors of mice in each group were obtained 20 days after tumor implantation, and the content of cytokines in the tumors was analyzed by ELISA, or single cell suspensions were prepared and the number of immune cells in the tumors was analyzed by flow cytometry. Fig.15 As shown, compared with the other groups, Mac TA99+IL-12-Nb The level of IFN-γ in the tumor of the group was the highest ( Fig.15 A), and CD8 + The number of T cells, NK cells, and NKT cells increased the most ( Fig.15 BD). This shows that Mac TA99+IL-12-Nb Therapy can effectively reshape the immunosuppressive tumor microenvironment into a pro-inflammatory tumor microenvironment.

[0170] Example 6: Mac TA99+IL-12-Nb Therapy can inhibit the growth of B16F10 metastases

[0171] To explore Mac TA99+IL-12-Nb To treat metastatic tumors, 6-week-old C57BL / 6 mice were injected with 5×10 5 luciferase-expressing B16F10 (B16F10-luc) cells. Seven days after tumor cell implantation, mice were divided into six groups: the first group of mice were injected with 100 μL PBS solution through the tail vein (PBS group); the second group of mice were injected with 5×10 6 untreated RAW264.7 cells (Mac group); mice in the third group were injected with 5×10 6 RAW264.7 cells (Mac TA99 group); mice in group 4 were injected with 5×10 6RAW264.7 cells (Mac IL-12-Nb group); the fifth group of mice were injected with 5×10 6 RAW264.7 cells (Mac TA99+IL-12 group); mice in group 6 were injected with 5×10 6 RAW264.7 cells (Mac TA99+IL-12-Nb The mice in each group were injected with drugs once on the 7th, 12th and 17th days, and the weight changes and survival rates of the mice were recorded every day. The extent of metastasis was observed by chemiluminescence imaging on the 5th, 10th, 15th, 25th and 30th days. The specific method was as follows: each mouse was intraperitoneally injected with 100 μL of 30 mg / mL D-luciferin sodium salt solution, and 10 minutes later, the chemiluminescence in the mice was detected by the small animal imaging system (PerkinElmer, model: IVISSPECTRUM CT). Fig.16 As shown, Mac TA99+IL-12-Nb The progression of metastatic tumors in the mice in the Mac group was significantly restricted, the survival rate of the mice was significantly increased, and the weight of the mice remained basically unchanged. TA99+IL-12-Nb The therapy was also effective in controlling the progression of metastases.

[0172] Example 7: Mac TA99+IL-12-Nb The treatment has no significant side effects

[0173] To explore Mac TA99+IL-12-Nb To determine whether the therapy would cause side effects, 1×10 6 B16F10 cells. Seven days after tumor cell implantation, the mice were divided into five groups: the first group of mice were injected with 100 μL PBS solution by tail vein (PBS group); the second group of mice were injected with 5 μg IL-12 recombinant protein by tail vein (IL-12 group); the third group of mice were injected with 5×10 6 RAW264.7 cells (Mac CMV-IL-12 group); mice in group 4 were injected with 5×10 6RAW264.7 cells (Mac TA99+IL-12 group); the fifth group of mice were injected with 5×10 6 RAW264.7 cells (Mac TA99+IL-12-Nb The blood and organs of mice in each group were collected and analyzed 10 days after the cancer cells were injected. Fig.17 As shown, compared with the PBS group, the IL-12 group and Mac CMV-IL-12 The levels of IL-12 and IFN-γ in the serum of mice in the group were significantly increased ( Fig.17 AB), accompanied by a significant decrease in body weight ( Fig.17 E), Increased spleen weight ( Fig.17 C), and increased water content in the liver ( Fig.17 D). These changes indicate that both groups of mice had significant side effects. TA99+IL-12 Groups and Macs TA99+IL-12-Nb The indicators of mice in the group were closer to those of PBS, especially Mac TA99+IL-12-Nb The indicators of mice in the Mac TA99+IL-12-Nb The therapy can effectively reduce the side effects caused by IL-12 therapy.

[0174] The above are only several exemplary embodiments of the present invention, and are not intended to limit the present invention in any form. Although the present invention is disclosed as above with preferred embodiments, they are not intended to limit the present invention. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, can make some changes or modifications to obtain equivalent or equivalent embodiments using the above disclosed technical content, which fall within the scope of the present invention.

Claims

1. A synNotch receptor comprising an extracellular domain, a transmembrane domain and an intracellular domain, wherein: The extracellular domain comprises a single-chain variable fragment that targets a tumor-specific antigen; Wherein, the tumor-specific antigen is selected from one or more of TRP1, CD19, CD20, CD133, HER2, EGFR, mesothelin, IL13RA2, c-MET and EphA2; preferably, the tumor-specific antigen is TRP1.

2. The synNotch receptor according to claim 1, wherein The single-chain variable fragment is a TA99 single-chain antibody; preferably, the amino acid sequence of the TA99 single-chain antibody comprises the amino acid sequence shown in SEQ ID NO: 1, or the amino acid sequence of the TA99 single-chain antibody is shown in SEQ ID NO: 1; Preferably, the extracellular domain further comprises a membrane targeting signal peptide and a peptide tag; More preferably, the membrane targeting signal peptide is selected from one or more of a CD8α signal peptide, an immunoglobulin heavy chain signal peptide, an immunoglobulin light chain signal peptide and a GM-CSF signal peptide; further preferably, the membrane targeting signal peptide is a CD8α signal peptide; further preferably, the amino acid sequence of the CD8α signal peptide is as shown in SEQ ID NO: 2; More preferably, the peptide tag is selected from one or more of a MYC-tag, a 6×His-tag and a Flag-tag; further preferably, the peptide tag is a MYC-tag; further preferably, the amino acid sequence of the MYC-tag is as shown in SEQ ID NO: 3; Particularly preferably, the extracellular domain comprises a CD8α signal peptide, a MYC-Tag and a TA99scFv which are operably linked in sequence; most preferably, the amino acid sequence of the extracellular domain is as shown in SEQ ID NO:

4.

3. The synNotch receptor according to claim 1 or 2, wherein The transmembrane domain comprises a Notch core; preferably, the amino acid sequence of the Notch core comprises the amino acid sequence shown in SEQ ID NO: 5, or the amino acid sequence of the Notch core is as shown in SEQ ID NO: 5; Optionally, the intracellular domain comprises a transcription factor; preferably, the transcription factor is selected from one or more of GAL4-VP64, GAL4-p65-GR, GAL4-VPR and GAL4-MiniVPR; more preferably, the transcription factor is GAL4-VP64; further preferably, the amino acid sequence of GAL4-VP64 comprises the amino acid sequence shown in SEQ ID NO: 6, or the amino acid sequence of GAL4-VP64 is as shown in SEQ ID NO:

6.

4. An isolated nucleic acid molecule encoding the synNotch receptor according to any one of claims 1 to 3; preferably, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO:

10. A recombinant vector comprising the nucleic acid molecule according to claim 4 .

6. An engineered macrophage comprising a first nucleic acid molecule or a first recombinant vector, wherein: The first nucleic acid molecule is the nucleic acid molecule according to claim 4, and the first recombinant vector is the recombinant vector according to claim 5; Preferably, the engineered macrophages are derived from RAW264.7 mouse mononuclear macrophages, THP-1 cells, J774A.1 cells, mouse bone marrow-derived macrophages or human peripheral blood mononuclear cells; Preferably, the engineered macrophages are derived from RAW264.7 mouse mononuclear macrophages; Optionally, the engineered macrophage further comprises a second nucleic acid molecule or a second recombinant vector, wherein the second nucleic acid molecule comprises a nucleic acid molecule encoding a fusion protein of a cytokine and an anti-PD-L1 nanobody; the second recombinant vector comprises the second nucleic acid molecule; Preferably, the cytokine is selected from one or more of IL-12, IL-2, TNF-α and INF-γ; more preferably, the cytokine is IL-12.

7. The engineered macrophage according to claim 6, wherein The IL-12 comprises IL-12p40 and IL-12p35 and a first connecting peptide connecting the two; Preferably, the amino acid sequence of the IL-12p40 is as shown in SEQ ID NO: 11, and the nucleotide sequence of the nucleic acid molecule encoding the IL-12p40 is as shown in SEQ ID NO: 12; Preferably, the amino acid sequence of the IL-12p35 is as shown in SEQ ID NO: 13, and the nucleotide sequence of the nucleic acid molecule encoding the IL-12p35 is as shown in SEQ ID NO: 14; Preferably, the amino acid sequence of the first connecting peptide is as shown in SEQ ID NO: 15, and the nucleotide sequence of the nucleic acid molecule encoding the first connecting peptide is as shown in SEQ ID NO: 16; Preferably, the amino acid sequence of the anti-PD-L1 nanobody comprises the amino acid sequence as shown in SEQ ID NO: 17, or the amino acid sequence of the anti-PD-L1 nanobody is as shown in SEQ ID NO: 17; the nucleotide sequence of the nucleic acid molecule encoding the anti-PD-L1 nanobody comprises the nucleotide sequence as shown in SEQ ID NO: 18, or the nucleotide sequence of the nucleic acid molecule encoding the anti-PD-L1 nanobody is as shown in SEQ ID NO: 18; Optionally, the anti-PD-L1 nanobody is connected to the IL-12p35 of the IL-12 via a second connecting peptide; preferably, the amino acid sequence of the second connecting peptide consists of n amino acid sequences (GGGGS) as shown in SEQ ID NO: 19, wherein n is an integer from 1 to 5; more preferably, the amino acid sequence of the second connecting peptide is as shown in SEQ ID NO: 20, and the nucleotide sequence of the nucleic acid molecule encoding the second connecting peptide is as shown in SEQ ID NO: 21; Particularly preferably, the nucleotide sequence of the nucleic acid molecule encoding the fusion protein of IL-12 and anti-PD-L1 nanobody is as shown in SEQ ID NO:

22.

8. The engineered macrophage according to claim 6 or 7, wherein: The second nucleic acid molecule further comprises an upstream activation sequence and a promoter at the 5' end of the nucleic acid molecule encoding the fusion protein of IL-12 and anti-PD-L1 nanobody; the second nucleic acid molecule further comprises a polyA sequence at the 3' end of the nucleic acid molecule encoding the fusion protein of IL-12 and anti-PD-L1 nanobody; Preferably, the nucleotide sequence of the upstream activation sequence comprises the nucleotide sequence as shown in SEQ ID NO: 23, or the nucleotide sequence of the upstream activation sequence is as shown in SEQ ID NO: 23; Preferably, the promoter is a minimal CMV promoter; more preferably, the nucleotide sequence of the minimal CMV promoter is shown in SEQ ID NO: 24; Further preferably, the second nucleic acid molecule comprises, from the 5' end to the 3' end, an upstream activation sequence, a minimal CMV promoter, a nucleic acid molecule encoding an IL-12-PD-L1 Nb fusion protein, and a polyA sequence; most preferably, the nucleotide sequence of the second nucleic acid molecule is as shown in SEQ ID NO:

25.

9. A pharmaceutical composition for preventing and / or treating cancer, comprising the engineered macrophage according to any one of claims 6 to 8 and a pharmaceutically acceptable excipient; Preferably, the cancer is selected from one or more of melanoma, liver cancer, lung cancer, pancreatic cancer, brain cancer, breast cancer and ovarian cancer; more preferably, the cancer is melanoma.

10. Use of the engineered macrophage according to any one of claims 6 to 8 in the preparation of a medicament for preventing and / or treating cancer; Preferably, the cancer is selected from one or more of melanoma, liver cancer, lung cancer, pancreatic cancer, brain cancer, breast cancer and ovarian cancer; more preferably, the cancer is melanoma.

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