Synnotch-based engineered macrophages and uses thereof

Abstract

The application 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. The application also provides an engineered macrophage based on the synNotch receptor. The synNotch receptor of the application can specifically recognize a TRP1 antigen on the surface of a tumor cell. The engineered macrophage of the application has good specific targeting effect on both subcutaneous tumors and metastatic tumors, can effectively inhibit tumor growth and has no side effects.

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 leukocyte distributed throughout the body and participate in many of the body's life processes. 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, macrophages can account for up to 70% of the mass. Most opinions believe that most macrophages in tumor sites originate from bone marrow and peripheral blood mononuclear cells. Under the action of chemokines and cytokines secreted by tumor-associated stromal cells and tumor cells, they are recruited to the tumor site and transformed into tumor-associated macrophages. In addition to the primary tumor, macrophages are also one of the first immune cells recruited to the metastatic niche, providing favorable conditions for the metastasis and colonization of tumor cells.

[0003] Synthetic Notch (synNotch) receptors are engineered from the natural Notch protein. Researchers have not only modified the extracellular portion of the Notch protein to enable it to recognize specific antigens, but also the intracellular portion of the Notch protein, allowing for the manipulation of downstream responses following antigen recognition. SynNotch receptors typically consist of three components: an extracellular antigen recognition domain, responsible for antigen recognition; a transmembrane Notch core, which is cleaved by γ-secretase on the cell membrane upon antigen recognition, releasing the intracellular portion of synNotch into the cytoplasm; and an intracellular artificial transcription factor, which, after cleavage of the Notch core, enters the cell nucleus and triggers the expression of specific genes. Therefore, cells engineered with synNotch can not only recognize specific tumor cells but also tailor their responses to these cells. However, synNotch receptors are currently primarily used in cells that are less susceptible to tumor infiltration, such as T cells, limiting their potential application 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, due to the lack of pre-existing anti-tumor immunity, immune checkpoint blockade is less effective in immunologically "cold" tumors. 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 been suboptimal in clinical trials due to limited intratumoral accumulation and stimulation of lymphocytes in healthy tissues, resulting in adverse reactions.

[0005] Clinically used immune checkpoint blockers primarily take the form of antibodies. However, due to their large molecular weight and complex post-translational modifications, antibodies are difficult to express using recombinant vectors or to form fusion proteins with other proteins. Nanobodies, on the other hand, are typically only 15 kDa in size and are easily expressed using recombinant vectors and can be easily fused with other proteins. Summary of the Invention

[0006] To address the aforementioned challenges in the existing technology, the inventors, after extensive technical and practical exploration, have developed a therapy based on synNotch-engineered macrophages. These synNotch-engineered macrophages are capable of recognizing specific tumor antigens and secreting a fusion protein of IL-12 and an anti-PD-L1 nanobody (PD-L1 Nb). This therapy is suitable not only for primary tumors but also for metastatic tumors, eliciting a strong anti-tumor immune response without significant side effects.

[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 CD8α signal peptide, immunoglobulin heavy chain signal peptide, immunoglobulin light chain signal peptide and GM-CSF signal peptide. More preferably, the membrane targeting signal peptide is 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 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 that are operably linked in sequence.

[0023] More preferably, the amino acid sequence of the extracellular domain is 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. Expression vectors known in the art include eukaryotic expression vectors, prokaryotic expression vectors, viruses or bacteriophages, and 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 shown as SEQ ID NO: 11, and the nucleotide sequence of the nucleic acid molecule encoding the IL-12p40 is shown as 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 shown as SEQ ID NO: 13, and the nucleotide sequence of the nucleic acid molecule encoding the IL-12p35 is shown as 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 be 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 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 IL-12 via a second connecting peptide.

[0081] In the present invention, the second connecting peptide can be 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, where n is an integer from 1 to 5. More preferably, the amino acid sequence of the second connecting peptide is shown in SEQ ID NO: 22, and the nucleotide sequence of the nucleic acid molecule encoding the second connecting peptide is shown in SEQ ID NO: 23.

[0082] SEQ ID NO: 22:GGGGGSGGGGSGGGGS

[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 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 shown as 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 retain their tumor-targeting ability while also being able to specifically recognize TRP1-positive cells through the synNotch receptor. Furthermore, 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 is shed and binds to the upstream activation sequence of a second nucleic acid molecule, thereby initiating the expression of the IL-12-PD-L1 Nb fusion protein. This fusion protein is then specifically secreted into the tumor. Furthermore, PD-L1 Nb not only acts as an immune checkpoint blocker but also binds to PD-L1-positive cells within the tumor, thereby preventing the IL-12-PD-L1 Nb fusion protein from spreading beyond the tumor site.

[0104] Experimental verification demonstrates that the engineered macrophages provided by the present invention maintain their homing ability and exhibit excellent specific targeting effects on both subcutaneous tumors and metastatic tumors. Furthermore, the engineered macrophages provided by the present invention can effectively inhibit the growth of subcutaneous tumors and metastatic tumors without producing significant side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[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 analysis according to the present invention.

[0108] Figure 3 Mac showing the present invention synNotch Mechanism of action by which TRP1 is recognized on B16F10 cells and responds to mCherry expression.

[0109] Figure 4 Mac showing the present invention synNotch Results of specific response to B16F10 cells. 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 positive rate after co-incubation with different cells.

[0110] Figure 5 Mac showing the present invention synNotch Able to effectively home to subcutaneous tumors. Among them, A is the fluorescence imaging of the main organs and tumors of mice after injection of different drugs; B is the quantitative diagram of the distribution of Mac in the main organs and tumors of mice; C is the Mac synNotch Quantitative diagram of the distribution ratio of major organs and tumors in mice.

[0111] Figure 6 Mac showing the present invention synNotch Able to effectively home to metastatic tumors. Among them, A is the fluorescence imaging of the main organs of mice after injection of different drugs; B is the quantitative diagram of the distribution ratio of Mac in the main organs of mice without metastatic tumors; C is the quantitative diagram of the distribution ratio of Mac in the main organs of mice with metastatic tumors; D is the quantitative diagram of Mac synNotch Quantitative graph of the distribution ratio 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. Figure A shows the structure of the IL-12-PD-L1 Nb fusion protein of the present invention; Figure B is an immunoblot image of the IL-12-PD-L1 Nb fusion protein of the present invention.

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

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

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

[0117] Figure 12 Mac showing the present invention TA99+IL-12-Nb IL-12-PD-L1 Nb is secreted in response to B16F10 cells.

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

[0119] Figure 14 The figures show the survival of mice in different experimental groups in Example 5 of the present invention. A shows the changes in tumor volume of mice in different experimental groups; B shows the survival rate of mice in different experimental groups; and C shows the changes in body weight of mice in different experimental groups.

[0120] Figure 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 tumor 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] Figure 16 The figure shows the survival status of mice in different experimental groups in Example 6 of the present invention. 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 change of mice in different experimental groups.

[0122] Figure 17 The figure shows the survival status of mice in different experimental groups in Example 7 of the present invention. A shows the IL-12 content in the serum of mice in different experimental groups; B shows the IFN-γ content 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 will be further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, 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 shown in SEQ ID NO: 1;

[0126] CD8α signal peptide: amino acid sequence is 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: amino acid sequence as shown in SEQ ID NO: 6, encoding nucleic acid as shown in SEQ ID NO: 9;

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

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

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

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

[0135] IL-12: amino acid sequence as shown in SEQ ID NO: 17, encoding nucleic acid as 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: amino acid sequence as shown in SEQ ID NO: 22, encoding nucleic acid as 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 Bio (Anhui) Co., Ltd. The cells used were purchased from Xiangya Hospital.

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

[0144] To investigate whether the synNotch receptor specifically responds to TRP1-positive cells, the pCMV-HA vector was double-digested with 5' BbSI and 3' NotI, and SEQ ID NO: 10 was inserted into the restriction sites to construct a recombinant vector containing the synNotch receptor nucleic acid. This construction was commissioned by General Biotech (Anhui) Co., Ltd. Furthermore, a recombinant vector expressing mCherry under the control of the synNotch receptor was constructed. To tag this recombinant vector, the recombinant vector also contained nucleic acid stably expressing EGFP. Specifically, the pcDNA3.1-EGFP vector was double-digested with 5' DraIII and 3' BstBI, and SEQ ID NO: 26 was inserted into the restriction sites to construct the recombinant vector CMV-EGFP-5×UAS-miniCMV-mCherry expressing mCherry under the control of the synNotch receptor. This construction was commissioned by General Biotech (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 for synNotch receptor-controlled mCherry expression are shown. When the synNotch receptor recognizes the corresponding antigen, its intracellular domain GAL4-VP64 binds to the UAS sequence and initiates downstream mCherry expression.

[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-conjugated 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, more than 20% of the cells simultaneously expressed MYC-tag (synNotch receptor) and EGFP. This indicates that RAW264.7 cells were successfully transformed into Mac synNotch .

[0149] Mac synNotch Mechanisms for recognizing specific cells Figure 3 Mac synNotch on the extracellular portion of the synNotch receptor (i.e. Figure 3 After TA99 in the synNotch receptor recognizes TRP1, the Notch core of the synNotch receptor will be cut 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 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 hours, and then Mac was analyzed by flow cytometry and confocal imaging. synNotch mCherry expression. 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 determine the ability of homing to subcutaneous tumors, 1×10 6 B16F10 cells. When the tumor grows to 200mm 3 After that, 5×10 6 Mac synNotch The VT680-labeled macrophages (MACs) were injected into the mice via tail vein. The PBS group was injected with an equal volume of PBS solution. synNotch The Mac method 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 harvested, and the fluorescence of VT680 in each organ and tumor was analyzed using 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 accumulating in the liver, followed by tumors, and less accumulation in other organs. This suggests that synNotch engineering does not affect the ability of macrophages to home to tumors.

[0154] To explore Mac synNotch To investigate the ability of homing metastatic tumors, 1×10 6 7 days after the injection of tumor cells, 5×10 6 A VT680-branded Mac synNotchor Mac were injected into mice via tail vein. Mice of the same age without B16F10 tumor cells were also injected with 5×10 6 VT680-labeled Macs were used to observe the accumulation of macrophages in normal lungs. 72 hours after macrophage injection, the main organs and tumors of the mice were harvested, and the fluorescence of VT680 in each organ and tumor was analyzed using a small animal imaging system (PerkinElmer, model: IVIS SPECTRUM CT). Figure 6 As shown, Mac and Mac synNotch In B16F10 metastatic tumor model mice, the biodistribution is similar, with the largest amount accumulated in the liver, followed by the lungs, while the amount accumulated in other organs is relatively small. 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 the injection of tumor cells, 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. The Mac synNotch Response situation. Figure 7 As shown, EGFP-positive Mac synNotch Mac is 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 the synNotch-engineered macrophages 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 the IL-12-PD-L1 Nb fusion protein. Figure 8A shows a schematic diagram of the IL-12-PD-L1 Nb fusion protein structure. IL-12 consists of two components, IL-12p40 and IL-12p35, connected by a first linker peptide. PD-L1 Nb is linked to IL-12p35 via a second linker peptide. HEK293T cells were transfected with either 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 using Lipo3000 (Thermo Fisher Scientific) according to the manufacturer's standard Lipo3000 transfection protocol. A 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 Bio (Anhui) Co., Ltd.

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

[0159] After washing with PBS, cells were resuspended in RIPA lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (PSMF) protease inhibitor. Protein quantity was determined using a BCA protein assay kit. Equal amounts of protein (20 μg) were separated on SDS-PAGE gels and electrophoretically transferred to PVDF membranes. The membranes were blocked for 1 hour with Tris-buffered saline (TBS) containing 0.05% Tween-20 and 5% nonfat dry milk. Next, the membranes were incubated overnight with primary antibodies against IL-12 (1:1000 dilution), His-tag (1:1000 dilution), or GAPDH (1:1000 dilution), followed by incubation with horseradish peroxidase (HRP)-conjugated 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 is approximately 15 kDa larger than that of IL-12, and only the IL-12-PD-L1 Nb fusion protein contains a His tag, indicating the successful expression of the IL-12-PD-L1 Nb fusion protein.

[0161] To investigate whether fusion of PD-L1 Nb affects the function of IL-12 itself, recombinant vectors containing IL-12-PD-L1 Nb fusion protein or IL-12 encoding nucleic acid were transfected into HEK293T cells using Lipo3000 (Thermo Fisher Scientific), and then the cell culture medium was collected within 24-48 hours of transfection. 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 at 1 mL, and the volume of culture medium containing IL-12-PD-L1 Nb fusion protein or IL-12 was as follows: Figure 9 The remaining volume of the culture medium was replenished with fresh DMEM complete medium. The culture medium mixture was then incubated with splenocytes extracted from the spleen of C57BL / 6 mice at 37°C for 2-12 hours to activate the splenocytes. IFN-γ secretion was measured by ELISA 48 hours after stimulation. Figure 9 As shown in the results, both IL-12 and IL-12-PD-L1 Nb fusion proteins can effectively stimulate splenocytes to secrete IFN-γ, which indicates that the fusion of IL-12 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 of 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 was collected within 24-48 hours of transfection. 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. Figure 10As shown in Figure 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 has bound to the B16F10 cells. As time goes by, more p40-EGFP-PD-L1 Nb fusion protein is 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 ( Figure 10 A). This demonstrates that PD-L1 Nb is able to anchor the fusion protein to PD-L1-positive B16F10 cells.

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

[0164] In order to prepare synNotch engineered macrophages (Macrophages) that can secrete IL-12-PD-L1 Nb fusion protein in response to B16F10 cells, TA99+IL-12-Nb ), the nucleic acid encoding mCherry on the recombinant vector expressing mCherry under the control of the synNotch receptor was replaced with the nucleic acid encoding the IL-12-PD-L1 Nb fusion protein (SEQ ID NO: 24). The two recombinant vectors were transfected into RAW264.7 mouse mononuclear macrophages using jetOPTIMUs (Polyplus) at a 1:1 ratio, thereby generating synNotch-engineered macrophages capable of secreting the IL-12-PD-L1 Nb fusion protein in response to B16F10 cells (the specific method was the same as in Example 1). Figure 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 binds to PD-L1 on B16F10 cells, blocking the PD-L1 / PD-1 signaling pathway and anchoring the IL-12-PD-L1 Nb fusion protein to B16F10 cells. IL-12 then activates T cells to secrete IFN-γ.

[0165] To verify the Mac TA99+IL-12-Nb Can B16F10 cells secrete IL-12-PD-L1 Nb fusion protein and convert 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 hours, and the secretion of IL-12 was analyzed by ELISA. TA99+IL-12-Nb Not co-incubated with cancer cells. Figure 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] Verify Mac by transwell experiment TA99+IL12-Nb Can the secreted IL-12-PD-L1 Nb fusion protein activate splenocytes? First, plate Mac in the lower chamber TA99+IL-12-Nb cells and 4T1, LLC, 3T3, CT26 or B16F10 cells, and 48 hours later, spleen cells were plated in the upper chamber and cultured for another 48 hours. TA99+IL-12-Nb The supernatant was collected and the IFN-γ content was tested by ELISA. Figure 13 As shown, only when the splenocytes in the upper chamber are co-incubated with B16F10 cells will they be activated to secrete IFN-γ. Figure 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 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 6RAW264.7 cells (Mac) were transfected with a recombinant vector containing the synNotch receptor and a recombinant vector expressing the IL-12-PD-L1 Nb fusion protein controlled by the synNotch receptor. TA99+IL-12-Nb Each group of mice was injected with drugs once on the 7th, 12th and 17th day, and the tumor size, body weight change and survival rate of the mice were recorded every day. Figure 14 As shown, Mac TA99+IL-12-Nb The tumor growth of mice in the Mac group was the slowest, their weight remained stable, and their survival rate was the highest. TA99+IL-12-Nb The therapy can effectively inhibit 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, tumors of mice in each group were harvested 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. Figure 15 As shown, compared with the other groups, Mac TA99+IL-12-Nb The level of IFN-γ in the tumor of group 1 was the highest ( Figure 15 A), and CD8 + The numbers of T cells, NK cells, and NKT cells increased the most ( Figure 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 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) were transfected with a recombinant vector containing the synNotch receptor and a recombinant vector expressing the IL-12-PD-L1 Nb fusion protein controlled by the synNotch receptor. 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 of the mice was detected by a small animal imaging system (PerkinElmer, model: IVISSPECTRUM CT). Figure 16 As shown, Mac TA99+IL-12-Nb The progression of metastatic tumors in the mice of 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 can also effectively control the progression of metastatic tumors.

[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 produce side effects, 1×10 6 Seven days after tumor cell implantation, the mice were divided into five groups: the first group received a tail vein injection of 100 μL PBS solution (PBS group); the second group received a tail vein injection of 5 μg IL-12 recombinant protein (IL-12 group); the third group received a tail vein injection of 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) were transfected with a recombinant vector containing the synNotch receptor and a recombinant vector expressing the IL-12-PD-L1 Nb fusion protein controlled by the synNotch receptor. 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. Figure 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 ( Figure 17 AB), accompanied by a significant loss of weight ( Figure 17 E), increased spleen weight ( Figure 17 C), and increased liver water content ( Figure 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 group A were closer to those in group B, especially the Mac TA99+IL-12-Nb The indicators of mice in the Mac group were the most normal. TA99+IL-12-Nb The therapy can effectively reduce the side effects of IL-12 therapy.

[0174] The above descriptions are merely exemplary embodiments of the present invention and are not intended to limit the present invention in any way. Although the present invention is disclosed above with reference to preferred embodiments, they are not intended to limit the present invention. Any person skilled in the art who, without departing from the scope of the present invention, may make slight changes or modifications to the above-disclosed technical contents to obtain equivalent or equivalent embodiments falls within the scope of the present invention.

Claims

1. A synNotch receptor, which consists of an extracellular domain, a transmembrane domain and an intracellular domain, wherein: The amino acid sequence of the extracellular domain is shown in SEQ ID NO: 4; The transmembrane domain comprises a Notch core, the N-terminus of the Notch core is connected to the C-terminus of the extracellular domain, the C-terminus of the Notch core is connected to the N-terminus of the intracellular domain, and the amino acid sequence of the Notch core is shown in SEQ ID NO: 5; The intracellular domain is the transcription factor GAL4-VP64, and the amino acid sequence of GAL4-VP64 is shown in SEQ ID NO:

6.

2. An isolated nucleic acid molecule encoding the synNotch receptor according to claim 1.

3. The nucleic acid molecule according to claim 2, wherein The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

10. A recombinant vector comprising the nucleic acid molecule according to claim 2 or 3.

5. 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 2 or 3, and the first recombinant vector is the recombinant vector according to claim 4.

6. The engineered macrophage according to claim 5, wherein 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.

7. The engineered macrophage according to claim 5, wherein 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; and the second recombinant vector comprises the second nucleic acid molecule.

8. The engineered macrophage according to claim 7, wherein The cytokine is selected from one or more of IL-12, IL-2, TNF-α and INF-γ.

9. The engineered macrophage according to claim 8, wherein The IL-12 comprises IL-12p40, IL-12p35 and a first connecting peptide connecting the two.

10. The engineered macrophage according to claim 9, wherein The amino acid sequence of the IL-12p40 is shown in SEQ ID NO: 11, and the nucleotide sequence of the nucleic acid molecule encoding the IL-12p40 is shown in SEQ ID NO:

12.

11. The engineered macrophage according to claim 9, wherein The amino acid sequence of the IL-12p35 is shown in SEQ ID NO: 13, and the nucleotide sequence of the nucleic acid molecule encoding the IL-12p35 is shown in SEQ ID NO:

14.

12. The engineered macrophage according to claim 9, wherein The amino acid sequence of the first connecting peptide is shown in SEQ ID NO: 15, and the nucleotide sequence of the nucleic acid molecule encoding the first connecting peptide is shown in SEQ ID NO:

16.

13. The engineered macrophage according to claim 9, wherein 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.

14. The engineered macrophage according to claim 9, wherein The anti-PD-L1 nanobody is connected to the IL-12p35 of the IL-12 via a second connecting peptide.

15. The engineered macrophage according to claim 14, wherein 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.

16. The engineered macrophage according to claim 15, wherein The amino acid sequence of the second connecting peptide is shown in SEQ ID NO: 20, and the nucleotide sequence of the nucleic acid molecule encoding the second connecting peptide is shown in SEQ ID NO:

21.

17. The engineered macrophage according to claim 16, wherein The nucleotide sequence of the nucleic acid molecule encoding the fusion protein of IL-12 and anti-PD-L1 nanobody is shown in SEQ ID NO:

22.

18. The engineered macrophage according to claim 17, 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.

19. The engineered macrophage according to claim 18, wherein The nucleotide sequence of the upstream activation sequence comprises the nucleotide sequence shown in SEQ ID NO: 23, or the nucleotide sequence of the upstream activation sequence is shown in SEQ ID NO:

23.

20. The engineered macrophage according to claim 18, wherein The promoter is the minimal CMV promoter.

21. The engineered macrophage according to claim 20, wherein The nucleotide sequence of the minimal CMV promoter is shown in SEQ ID NO:

24.

22. The engineered macrophage according to any one of claims 18 to 21, wherein 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.

23. The engineered macrophage of claim 22, wherein The nucleotide sequence of the second nucleic acid molecule is shown in SEQ ID NO:

25.

24. A pharmaceutical composition for preventing and / or treating cancer, comprising the engineered macrophage according to any one of claims 5 to 23 and a pharmaceutically acceptable excipient.

25. The pharmaceutical composition according to claim 24, wherein The cancer is melanoma.

26. Use of the engineered macrophage according to any one of claims 5 to 23 in the preparation of a medicament for preventing and / or treating melanoma.

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