Targeting recombinant vector and method for preparing CAR-M by using same

Abstract

The invention discloses a targeting recombinant vector and a method for preparing CAR-M. The targeting recombinant vector is used for preparing CAR-M. The amino acid sequence of the polypeptide motif is as shown in any one of SEQ ID NO: 11-21; the targeting recombinant vector comprises the polypeptide motif or the targeting polypeptide. The optimal design of adeno-associated virus AAV capsid protein peptide fragment modification realizes specific targeting of tumor-associated macrophages, so that the purpose of improving transfection efficiency is achieved; through comparison and selection of macrophage specific promoters in a gene sequence, in-vivo safety is synergistically realized, and toxic and side effects caused by off-target are reduced. After a non-viral gene delivery vector is modified by a polypeptide motif or a targeting polypeptide, specific and efficient recognition of macrophages is realized, and the macrophages are successfully transfected. By utilizing the TAM specific targeting polypeptide and the targeting recombinant vector disclosed by the invention, chimeric antigen receptor-tumor associated macrophages (CAR-TAM) can be generated in vivo and in vitro, an immune microenvironment of solid tumors can be remodeled, and malignant solid tumors can be treated.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a targeted recombinant vector and a method for preparing CAR-M using the same. It also relates to a polypeptide motif and a targeting polypeptide containing the same, a CAR-M prepared thereby, and the application of the CAR-M. Background Art

[0002] In recent years, the emergence of cell immunotherapy has brought new hope to cancer patients. Among them, the effectiveness of chimeric antigen receptor (CAR) T cells (CAR-T) has been supported by a large number of clinical trials, especially in hematological malignancies, showing very optimistic therapeutic effects, which has led to the approval of a number of CAR-T products targeting BCMA and CD19 by the FDA. However, (1) the complex and lengthy preparation process has increased the technical barriers of cell therapy, not only placing extremely high demands on the production process, but also increasing the product price, resulting in increased treatment costs for most patients. (2) In addition to the high cost involved in producing a customized T cell product for each patient, the complexity of such a customized product also makes it impossible for T cell products to be mass-produced in industry, limiting the wider use of this therapy in clinical practice. Compared with traditional first-line therapeutic drugs, such as small molecule drugs or monoclonal antibodies, this technology lacks competitiveness. The long production cycle also means that patients have to wait for a long treatment cycle, which complicates the development of the disease and increases the difficulty of treatment. Although the current general CAR-T technology has alleviated the above problems to a certain extent, industrial GMP mass production of CAR-T products has not yet been achieved. (3) Limited by the influence of the tumor microenvironment (TME), current cell therapy products have not shown ideal therapeutic effects on solid tumors. On the one hand, immune cells such as T cells find it difficult to infiltrate the TME, resulting in insufficient numbers of CAR-T cells in the tumor. On the other hand, the phenotypic diversity of tumors or the absence of specific antigens on the tumor surface (immune escape) or the interaction between immune tolerance and tumor-associated immunosuppressive cells (MDSC / TAM) increase the difficulty of developing CAR-T products and also inhibit the widespread application of products in various solid tumors. (4) Currently, a large amount of preclinical and clinical trial data show that there are significant toxic side effects in the body. In order to solve the above bottlenecks and expand the application of CAR technology in solid tumors, researchers have focused on other more practical immune cells such as NK and macrophages Mφ. Among them, tumor-associated macrophages (TAM), which are highly related to TME and treatment resistance, are particularly noteworthy.

[0003] Macrophages and other myeloid-derived suppressor cells (MDSCs) are considered to be important components of the tumor immunosuppressive microenvironment and important practitioners of immunosuppressive effects. Among them, TAMs, which are widely infiltrated in solid tumors, not only account for more than 50% of the total number of tumor interstitial immune cells, providing an ideal target for treatment, but also play an important role in the occurrence, development and maintenance of the TME immunosuppressive microenvironment of tumors. In addition, macrophages are the central effectors and regulators of the innate immune system. After activation, macrophages also have functions such as phagocytosis, cytotoxicity, secretion of proinflammatory factors, and presentation of antigens to T cells. They have great potential in activating anti-tumor innate immunity and adaptive immune responses. Therefore, applying CAR technology to TAMs and achieving in situ implementation of CAR-TAMs through in vivo implementation can, to a certain extent, solve the current clinical challenges faced by CAR-T: (1) complex in vitro process flow and high production process technology barriers, (2) poor versatility, high personalization requirements, and large in vivo toxic side effects, and (3) TME limitations. This is expected to provide new ideas for the clinical treatment of solid tumors. Currently, a small number of researchers have conducted research on CAR-Ms, but most are in the conceptual verification stage and have not completely solved the above challenges. The in vivo implementation of CAR-M technology is still challenging, and it is difficult for common gene delivery vectors to successfully infect TAMs. Morrissey et al. first verified the successful modification of CAR gene structure in macrophages and the effects of different intracellular signaling domains on CAR-M-specific phagocytosis (MA Morrissey, et al. Chimeric antigen receptors that triggerphagocytosis. eLife, 7 (2018), p.e36688, 10.7554 / eLife.36688). However, the main conclusions were verified by the interaction between antibody-coupled magnetic beads and CAR-M cells. There was insufficient data for mutual verification between CAR-M and target cells, and the technology was not implemented in vivo.Subsequently, Klichinsky et al. used the second-generation CAR structure to modify macrophages, and by reinfusing CAR-M cells, they confirmed for the first time that CAR-modified macrophages can infiltrate solid tumors and phagocytize cancer cells (M. Klichinsky, et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nat Biotechnol, 38(8)(2020), 947-953, 10.1038 / s41587-020-0462-y). The preliminary results of the clinical trial disclosed in March 2022 also demonstrated the safety of this therapy in the treatment of HER2-positive metastatic ovarian cancer patients (ClinicalTrials.gov Identifier: NCT04660929). This technology still uses the traditional in vitro CAR-T process flow, and has not solved the production process barriers and the specific and efficient implementation of CAR-M technology in vivo. Another clinical trial (NCT05007379), initiated by Centre Oscar Lambret in France, is evaluating the in vitro therapeutic efficacy and immune responses of CAR-Ms against HER2-positive tumors by constructing tumor organoids from patients with breast cancer at various stages of progression. While these studies have demonstrated some anti-tumor activity in solid breast cancer tumors, they are based on CAR-T technology for producing engineered immune cells in vitro, and the production of CAR-Ms still requires a lengthy and complex preparation process. Meanwhile, Kim's team in South Korea, through the commercialization of Polyplus, is developing a CAR-M-based system. -Macrophage kit, for the first time, achieved the purpose of in vivo editing of CAR-M in a tumor-bearing mouse model (Mikyung Kang.et al.Nanocomplex-Mediated In Vivo Programming toChimeric Antigen Receptor-M1 Macrophages for Cancer Therapy.Advanced Materials.33(43)(2021),e2103258,10.1002 / adma.202103258), further verifying the feasibility of in vivo implementation of CAR-M technology. However, this technology is based on a mouse tumor animal model and lacks the support of human-related tumor data. In addition, it is limited by the design of the gene delivery vector, and its in vivo infection effect is not ideal. It does not have a good inhibitory effect on mouse tumors, and its in vivo safety has not been well resolved. Therefore, it is still a huge challenge to solve the technical bottlenecks faced by clinical CAR-T technology, screen gene vectors that can efficiently target and infect M2 macrophages, and realize efficient CAR-M technology in vivo to achieve effective inhibition of solid tumors.

[0004] Prior art CN108025024A (modified monocytes / macrophages expressing chimeric antigen receptors and their uses, published on May 11, 2018, i.e., Nat Biotechnol, 38 (2020), 947–953, DOI: 10.1038 / s41587-020-0462-y patent family) expresses chimeric antigen receptors in monocytes, macrophages, or dendritic cells. The modified cells are recruited to the tumor microenvironment, where they act as effective immune effectors by infiltrating tumors and killing target cells. The invention includes modified cells and pharmaceutical compositions containing the modified cells for adoptive cell therapy and treatment of diseases or conditions associated with immunosuppression. It successfully used CAR technology to transform macrophages in vitro and infused them back into a mouse model of HER2-positive metastatic ovarian cancer to successfully shrink tumors in the animals and prolong their lifespan. However, this approach simply copies CAR-T technology and has the following disadvantages: ① The preparation process is cumbersome, resulting in a longer treatment cycle; ② The genetically modified cells are macrophage precursor cells (monocytes), and no research has been conducted on specific macrophages; ③ It is difficult to proliferate, expand, and transfect macrophages in vitro.

[0005] The technology involved in the prior art CN111925448A (preparation method for in vivo generation of CAR-macrophages and application in tumor immunotherapy, published on November 13, 2020) is similar to the technology in the literature Advanced Materials.33(43)(2021), DOI:10.1002 / adma.202103258 mentioned above, which uses non-viral nanocarriers to deliver chimeric antigen receptor plasmids to achieve CAR-M technology in vivo. The transgenic vector used in this scheme is a cationic polymer nanomaterial, which has low transfection efficiency for primary macrophages, lacks specific targeting to target cells, and has off-target and toxic side effects when used in vivo.

[0006] Mikyung Kang.et al. (Nanocomplex-Mediated In Vivo Programming to Chimeric Antigen Receptor-M1 Macrophages for Cancer Therapy. Advanced Materials. 33(43)(2021), DOI: 10.1002 / adma.202103258 https: / / onlinelibrary.wiley.com / doi / 10.1002 / adma.202103258) successfully transformed macrophages in vitro and verified the specific killing function of CAR-M cells by co-incubating them with tumor cells. In vivo experiments verified the safety of this technical solution in vivo. The transgenic vector used in this solution is a commercial cationic polymer nanomaterial (jetPEI), which has a low infection effect on M2 macrophages and does not achieve an ideal therapeutic effect on tumors. In addition, this implementation plan also studies M1 macrophages that are easily infected. Summary of the Invention

[0007] In order to solve the defects in the existing technology that transgenic vector tools cannot efficiently infect primary M2 macrophages, have low targeting, and chimeric antigen receptor-macrophages (CAR-M) are difficult to expand in vitro, the present invention provides a targeted recombinant vector and a method for preparing CAR-M using the same.

[0008] To solve the above technical problems, one of the technical solutions of the present invention is to provide a polypeptide motif, the amino acid sequence of which is shown in any one of SEQ ID NOs: 11 to 21.

[0009] In order to solve the above technical problems, the second technical solution of the present invention is to provide a targeting polypeptide, which includes the polypeptide motif as described in one of the technical solutions.

[0010] To solve the above technical problems, the third technical solution of the present invention is to provide a targeted recombinant vector comprising the polypeptide motif as described in one of the technical solutions, or the targeting polypeptide as described in the second technical solution.

[0011] In some preferred embodiments, the targeted recombinant vector is a viral vector or a non-viral vector.

[0012] Preferably, the non-viral vector includes a vector constructed of a cationic polymer, and the viral vector includes an adeno-associated virus.

[0013] In other preferred embodiments, the cationic polymer includes PBAE, PEI and liposome carriers; and / or the serotype of the adeno-associated virus is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11.

[0014] Preferably, the PBAE is HPBAE, and its molecular weight is, for example, 8,000-12,000; the PEI is -Macrophage; the liposome carrier is 2000; the adeno-associated virus is AAV2 or AAV9.

[0015] In other preferred embodiments, the nucleotide sequence encoding the targeting peptide is shown in SEQ ID NO: 22 to 32 and its corresponding RNA sequence, which is inserted into the genome of the adeno-associated virus to construct a recombinant adeno-associated virus.

[0016] Preferably, the nucleotide sequence encoding the targeting peptide is inserted into the gene sequence of the adeno-associated virus capsid protein.

[0017] More preferably, the genome of the recombinant adeno-associated virus further comprises: a nucleotide sequence encoding a CAR molecule; and / or a nucleotide sequence of a promoter specific to macrophages, brain glial cells, or monocytes.

[0018] In other preferred embodiments, the recombinant adeno-associated virus comprises one or more of the following conditions:

[0019] (1) The backbone virus serotype is AAV9;

[0020] (2) the capsid protein is VP1, VP2 and / or VP3 subunit;

[0021] (3) The promoter is a promoter specific to macrophages; preferably, it is a promoter as shown in the nucleotide sequence of SEQ ID NO: 1.

[0022] The fourth technical solution of the present invention is: to provide a method for preparing a chimeric antigen receptor-macrophage (CAR-M), which utilizes the targeted recombinant vector as described in the third technical solution of the present invention to introduce the nucleotide sequence encoding the CAR molecule into the macrophage, thereby preparing CAR-M.

[0023] Preferably, the CAR is a CAR molecule against hematological tumors or solid tumors, such as a CAR molecule against CD19, B-cell maturation antigen (BCMA) or CD22, or a CAR molecule against tight junction molecule Claudin18.2 (CLDN18.2), human epidermal growth factor receptor 2 (HER2), natural killer cell surface activation receptor D (natural killer group 2-member D, NKG2D), mesothelin or prostate membrane specific antigen (PMSA), or a CAR molecule against SIRPα.

[0024] In some preferred embodiments, the amino acid sequence of the anti-CD19 CAR molecule is shown in SEQ ID NO: 3 or 7, and the amino acid sequence of the anti-CLDN 18.2 CAR molecule is shown in SEQ ID NO: 5.

[0025] Preferably, the nucleotide sequence encoding the anti-CD19 CAR molecule is shown in SEQ ID NO: 2 or 6 or 9, the nucleotide sequence encoding the anti-CLDN 18.2 CAR molecule is shown in SEQ ID NO: 4 or 10, and the nucleotide sequence encoding the anti-SIRPα CAR molecule is shown in SEQ ID NO: 8.

[0026] In other preferred embodiments, the macrophages are tumor-associated macrophages; preferably, the tumor-associated macrophages are derived from bone marrow, or the tumor-associated macrophages are in situ tumor-associated macrophages in vivo; more preferably, they are M2 macrophages.

[0027] The fifth technical solution of the present invention is to provide a CAR-M prepared by the preparation method described in the fourth technical solution of the present invention.

[0028] The sixth technical solution of the present invention is to provide a use of the polypeptide motif described in one of the technical solutions of the present invention, the targeting polypeptide described in the second technical solution, or the targeted recombinant vector described in the third technical solution in the preparation of targeted CAR-M.

[0029] Preferably, the macrophages are tumor-associated macrophages; preferably, the tumor-associated macrophages are derived from bone marrow, or the tumor-associated macrophages are in situ tumor-associated macrophages in vivo; more preferably, they are M2 macrophages.

[0030] The seventh technical solution of the present invention is to provide the use of AAV9 in specifically targeting M2 macrophages or in preparing reagents that specifically target M2 macrophages.

[0031] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0032] The reagents and raw materials used in the present invention are commercially available.

[0033] The positive progress effect of the present invention is:

[0034] (1) The present invention provides a method for generating chimeric antigen receptor-tumor-associated macrophages (CAR-TAMs) in vivo. By injecting the above-screened AAV9 or its recombinant adeno-associated virus tTAM-rAAV into a patient or animal model, chimeric antigen receptor-tumor-associated macrophages (CAR-TAMs) can be generated in vitro, thereby reshaping the immune microenvironment of solid tumors and treating malignant solid tumors.

[0035] (2) The present invention provides a viral vector AAV9 and tTAM-rAAV with high targeting ability and high transfection efficiency for tumor-associated macrophages, which can be used to specifically edit tumor-associated macrophages or M2 macrophages in vivo and in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The morphological characteristics and flow cytometry identification of different types of macrophages. Figure 1 A shows the morphological characteristics of M1 and M2 macrophages. Figure 1 B is the flow cytometry identification results of different types of macrophages (DAPI-A:CD86, APC-A:CD163).

[0037] Figure 2 TAM was identified by flow cytometry (APC-A:CD163).

[0038] Figure 3 BMDM-M2 were transfected with different types of vectors.

[0039] Figure 4 The blank control was used to verify the effect of targeting BMDM-M2.

[0040] Figure 5This is to verify the effect of targeting peptide P1 (SEQ ID NO: 11) targeting BMDM-M2.

[0041] Figure 6 This is to verify the effect of targeting peptide P2 (SEQ ID NO: 12) targeting BMDM-M2.

[0042] Figure 7 This is to verify the effect of targeting peptide P3 (SEQ ID NO: 13) targeting BMDM-M2.

[0043] Figure 8 This is to verify the effect of targeting peptide P4 (SEQ ID NO: 14) targeting BMDM-M2.

[0044] Figure 9 This is to verify the effect of targeting peptide P5 (SEQ ID NO: 15) targeting BMDM-M2.

[0045] Figure 10 This is to verify the effect of targeting peptide P6 (SEQ ID NO: 16) targeting BMDM-M2.

[0046] Figure 11 This is to verify the effect of targeting peptide P7 (SEQ ID NO: 17) targeting BMDM-M2.

[0047] Figure 12 This is to verify the effect of targeting peptide P8 (SEQ ID NO: 18) targeting BMDM-M2.

[0048] Figure 13 This is to verify the effect of targeting peptide P9 (SEQ ID NO: 19) targeting BMDM-M2.

[0049] Figure 14 This is to verify the effect of targeting peptide P10 (SEQ ID NO: 20) targeting BMDM-M2.

[0050] Figure 15 This is to verify the effect of targeting peptide P11 (SEQ ID NO: 21) targeting BMDM-M2.

[0051] Figure 16 To screen the targeting peptides for semi-quantitative analysis of their targeting effect on BMDM-M2.

[0052] Figure 17 This figure shows the effect of AAV modified with targeting peptide and transfected with different vectors (lentivirus, jetPEI and PEI) into BMDM-M2.

[0053] Figure 18 To verify the specific phagocytosis of tumor cells by CAR-M2. Figure 18A is the fluorescence observation after CAR-M2 was co-incubated with K562-CD19-Luci-RFP cells and K562-RFP cells. Figure 18 B shows the phagocytic killing of K562-CD19-Luci-RFP cells and K562-RFP cells by CAR-M2 (GFP:CAR-M2, RFP:K562).

[0054] Figure 19 This image demonstrates AAV9's specific infection of M2 macrophages. Different macrophage cell types were used, including primary M1 / M2 cells and the RAW264.7 (murine) and THP-1 (human) cell lines. The results demonstrate that AAV9 specifically infects only M2 macrophages.

[0055] Figure 20 These are diagrams showing the therapeutic effects of various carriers. DETAILED DESCRIPTION

[0056] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0057] This invention combines the therapeutic potential of cell therapy with the convenience of traditional drug preparation and application to create a product that can be brought to a large number of patients. The present invention prepares a ready-to-use injectable reagent that prepares tumor-specific receptors for TAMs in situ on the tumor with minimal interference, restoring TAMs' ability to recognize, phagocytose, and present tumor cells, and achieving efficient inhibition of solid tumors. Compared to CAR-T therapy, it does not require patients to undergo chemotherapy pre-adaptation, nor does it require live leukocyte separation to obtain lymphocytes. This operation method solves some problems of existing CAR-T / CAR-M technology to a certain extent:

[0058] (1) Avoid the cumbersome in vitro operation process of existing CAR-T / CAR-M technology and shorten the treatment cycle;

[0059] (2) Reduce production costs and provide off-the-shelf CAR-M products for easy transportation and storage;

[0060] (3) Compared with the current CAR-M technology, it solves the problem of macrophages being difficult to expand in vitro;

[0061] (4) Solve the problem of specific targeting of TAM by current transgenic vector tools and solve the current deficiency of low transfection efficiency in primary macrophages;

[0062] (5) The in situ implementation of this technical solution can, to a certain extent, avoid the in vivo safety issues of existing technologies;

[0063] (6) A specific adeno-associated virus AAV9 and its recombinant adeno-associated virus tTAM-rAAV and a non-viral vector modified with a targeting peptide are used to transfect macrophages to achieve in situ CAR-M in tumors in vivo. This overcomes the difficulties of low transfection efficiency of primary macrophages by current gene delivery vectors and low safety in vivo application, and is expected to truly achieve a simple, safe and efficient solution to the problem of difficult-to-treat solid tumors.

[0064] The improvement of the present invention over the prior art is:

[0065] ① Implementing in situ chimeric antigen receptor-tumor-associated macrophage (CAR-TAM) therapy in vivo;

[0066] ② Screening AAV9 and selecting capsid protein-modified AAV9 to load the CAR gene sequence to achieve specific targeting of tumor-associated macrophages, thereby improving transfection efficiency;

[0067] ③ The optimized design of the modified peptide segment of the adeno-associated virus (AAV) capsid protein and the comparative selection of macrophage-specific promoters in the transgenic sequence work together to achieve the in vivo safety of the technology and reduce the toxic side effects caused by off-target effects.

[0068] The relationship between each invention point and each technical effect is:

[0069] ① In vivo tumor in situ chimeric antigen receptor-tumor-associated macrophage (CAR-TAM) therapy solves or avoids the problems of traditional CAR-M therapy with complicated procedures, high production process barriers, long treatment cycles, high costs, and the difficulty of expanding macrophages in vitro;

[0070] ② The selected AAV9 and its adeno-associated virus were used to load the CAR gene sequence, which greatly improved the transfection efficiency of tumor-associated macrophages and the safety of in vivo application;

[0071] ③Through the optimized design of the peptide modification of the adeno-associated virus (AAV) capsid protein, the comparative selection of macrophage-specific promoters in the transgenic sequence, and the coordinated combination of the two, a recombinant adeno-associated virus vector with excellent ability to target tumor-associated macrophages and perform efficient transfection was screened out.

[0072] ④The modification of non-viral vectors with TAM-specific targeting peptides improves the safety and infection efficiency issues currently faced by non-viral vectors and increases the therapeutic effect of CAR cells.

[0073] Preferred and more preferred scope of technical solutions

[0074] ① Selection of AAV serotype:

[0075] Widest range of applicability: Serotypes can be AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. Also included are recombinant serotypes such as Rec2 and Rec3 recently discovered from primate brains.

[0076] Preferably: AAV2 and AAV9.

[0077] More preferably: AAV9.

[0078] ②Types of AAV capsid proteins:

[0079] Widest Scope of Applicability: The capsid protein can be derived from any AAV serotype. The AAV capsid protein of the present invention can also be a chimeric capsid protein. For example, the AAV capsid protein of the present invention can include amino acid sequences from at least one, at least two, or at least three different AAV serotypes.

[0080] Preferably: capsid proteins of AAV2 and AAV9.

[0081] More preferably: the capsid protein of AAV9.

[0082] ③ Selection of TAM peptide amino acid sequence for AAV capsid protein modification:

[0083] Maximum scope of application: All peptides with an amino acid sequence of less than 15aa that have the ability to target macrophages, brain glial cells or monocytes.

[0084] Preferably: all peptides with an amino acid sequence of less than 15 aa that have the ability to target macrophages.

[0085] More preferably, any peptide with an amino acid sequence of less than 15 amino acids that has the ability to target tumor-associated macrophages or M2 macrophages, such as the peptides with amino acid sequences shown in SEQ ID NOs: 11 to 21. The nucleotide sequences encoding the peptides are shown in SEQ ID NOs: 22 to 32.

[0086] ④ Selection of the modification position and amount of the TAM peptide amino acid sequence on the surface of the adeno-associated virus capsid protein

[0087] Maximum scope of application: The TAM peptide sequence screened above can be modified in the amino acid sequence of any VP1, VP2 and / or VP3 subunit of the AAV viral capsid protein.

[0088] ⑤ A macrophage-specific promoter needs to be inserted into the AAV transgenic sequence. Selection of macrophage-specific promoter:

[0089] Widest scope of application: All promoters that are specific to macrophages, brain glial cells, or monocytes.

[0090] Preferably: all promoters that are specific to macrophages.

[0091] More preferably: any promoter specific to tumor-associated macrophages or M2 macrophages, preferably the promoter shown in the nucleotide sequence of SEQ ID NO: 1.

[0092] ⑥ A nucleic acid sequence for expressing chimeric antigen receptors needs to be inserted into the AAV transgenic sequence, including a gene encoding the extracellular domain of the chimeric antigen receptor (CAR), a gene encoding the transmembrane region of the chimeric antigen receptor, a gene encoding the intracellular signaling domain, and a fluorescent protein expression gene.

[0093] Regarding the sequence of this part, the present invention adopts the second-generation CAR-T sequence (anti-CD19, targeting malignant blood tumors) that has been clinically approved for use and the anti-hCLDN18.2 CAR-T sequence targeting solid tumors, or the anti-SIRPα CAR molecule that can target both solid tumors and blood tumors.

[0094] For example, the amino acid sequence of the anti-CD19 CAR molecule is shown in SEQ ID NO: 3 or 7, and the amino acid sequence of the anti-CLDN18.2 CAR molecule is shown in SEQ ID NO: 5.

[0095] The nucleotide sequence encoding the anti-CD19 CAR molecule is shown in SEQ ID NO: 2 or 6 or 9, the nucleotide sequence encoding the anti-CLDN 18.2 CAR molecule is shown in SEQ ID NO: 4 or 10, and the nucleotide sequence encoding the anti-SIRPα CAR molecule is shown in SEQ ID NO: 8.

[0096] ⑦Type of transfected cells

[0097] Maximum applicability: macrophages, monocytes, or dendritic cells.

[0098] Preferably: macrophages or monocytes.

[0099] More preferably: macrophages, particularly tumor-associated macrophages or M2 macrophages.

[0100] ⑧Route of administration

[0101] Widest application range: intratumoral (IT), intravenous (IV), intraperitoneal (IP), intramuscular (IM), subcutaneous (SC), epidural (E), intracerebral (IC), intracerebroventricular (ICV), intranasal (IN) and intradermal (ID), etc.

[0102] Preferably: intratumoral (IT), intravenous (IV), intraperitoneal (IP), subcutaneous (SC).

[0103] More preferably: intratumoral (IT), intravenous (IV), preferably intratumoral (IT) administration.

[0104] Example 1: Extraction and culture of bone marrow-derived macrophages

[0105] This example provides a method for extracting and culturing bone marrow-derived macrophages (BMDM), comprising the following steps:

[0106] ①Anesthetize and kill the mice, then soak them in 75% alcohol;

[0107] ②Dissect and remove the intact mouse hind leg bones, and wash them in 75% alcohol and PBS solution in sequence;

[0108] ③ Cut the two bones apart at the joint, then open both ends and rinse with PBS 2-3 times to flush out the cells;

[0109] ④ Centrifuge at 1400 rpm for 5 min and discard the supernatant;

[0110] ⑤ Then add 1 mL of red blood cell lysis buffer and lyse for 5 minutes, shaking from time to time to ensure sufficient lysis;

[0111] ⑥ Centrifuge at 1400 rpm for 5 min and discard the supernatant;

[0112] ⑦ Resuspend the 1640 culture medium and inoculate it into the culture dish;

[0113] ⑧After overnight incubation, stimulatory factors were added (BMDM-M1 cells: GM-CSF 40 ng / mL, BMDM-M2 cells: M-CSF 20 ng / mL), and the cells were incubated for 7 days. Then, induced for 24 / 48 hours (BMDM-M1 cells: GM-CSF 40 ng / mL + IFN-γ 50 ng / mL + LPS 100 ng / mL, BMDM-M2 cells: M-CSF 20 ng / mL + IL-4 20 ng / mL) to obtain BMDM-M1 cells and BMDM-M2 cells.

[0114] Figure 1The results show that bone marrow-derived monocytes can differentiate into two types of macrophages after induced differentiation culture under different conditions (M1 macrophages: spherical cells account for a larger proportion, other irregular-shaped cells are mostly short and thick with antennae, and highly express CD86; M2 macrophages: spherical cells account for a smaller proportion, other irregular-shaped cells are mostly slender, and highly express CD163).

[0115] Example 2: Isolation and culture of tumor-associated macrophages from tumor tissue

[0116] This embodiment provides a method for isolating and culturing tumor-associated macrophages in tumor tissue, comprising the following steps:

[0117] Primary tumors (6-8 mm in diameter) from MMTV-PyMT mice (C57BL / 6 background) were mechanically dissociated and digested with a commercial collagenase / hyaluronidase solution containing DNase I (4 U / ml) at 37°C for 45 minutes. After pulse centrifugation (450 × g), the supernatant (primarily immune cells) was collected. CD11b cells were enriched from the supernatant using CD11b microbeads, and tumor-associated macrophages (TAMs) were obtained by allowing CD11b cells to adhere to tissue culture dishes for 2 hours (non-adherent cells were removed by washing with PBS).

[0118] Figure 2 The results show that tumor-associated macrophages can be isolated by this method, and macrophages can be labeled and sorted using antibodies targeting macrophage-specific markers. The tumor-associated macrophages isolated from tumor tissue have the phenotype of M2 macrophages.

[0119] Example 3: Transfection of bone marrow-derived BMDM-M2 cells using different vectors

[0120] In order to verify whether transgenic technology can be used to obtain M2 macrophages or tumor-associated macrophages that can express chimeric antigen receptors, the present invention uses a lentiviral vector carrying a second-generation CAR gene sequence and an EGFP gene (constructed by Shandong Weizhen Biotechnology Co., Ltd.) to transfect bone marrow-derived BMDM-M2 cells, and uses flow cytometry to evaluate the efficiency of gene infection. In order to compare the differences in the transfection effects of different types of vectors on M2 cells, the present invention constructed liposomes containing the same CAR plasmid load. 2000 (Invitrogen, 11668027), cationic polymer PBAE (laboratory synthesis) and commercialization -Macrophage (Polyplus Transfection, #101000043) complex, and transfection experiments were performed on M2 macrophages under the same experimental conditions.

[0121] The construction method of the lentiviral vector carrying the second-generation CAR gene sequence and the EGFP gene is as follows:

[0122] CD19-CD3zeta CAR related information: Signal peptide: aa 1-21 CD8 (Uniprot Q96QR6 HUMAN) Extracellular antibody sequence: VL chain: aa 23-130 anti-CD19 CAR (Genbank AMZ04819) – GS linker: ggtggcggtggctcg ggcggtggtgggtcgggtggcggcggatct (SEQ ID NO: 33) – VH chain: aa 148-267 anti-CD19 CAR (Genbank AMZ04819) Stalk / Transmembrane: aa 138-206 CD8 (Uniprot Q96QR6 HUMAN) Cytosolic sequence: aa 52-164, Human TCR CD3 zeta chain (Uniprot P20963) Fluorophore: EGFP.

[0123] The construction method of the cationic polymer PBAE is as follows:

[0124] HPBAE polymers with varying Mw were synthesized via a controlled step-growth addition reaction. The molecular weight of HPBAE was modified by varying the stoichiometric ratio of the starting monomers. The specific process involved dissolving the monomers, such as triacrylates, diacrylates, and amines, in anhydrous DMSO (500 mg / mL). The reaction solution was heated to 90°C and allowed to react for varying times (3-48 hours). The molecular weight of the synthesized HPBAE polymer was monitored by high-temperature gel permeation chromatography (GPC). When the Mw reached 8,000-12,000, the reaction system was diluted to 100 mg / mL with DMSO to terminate the polymerization. Subsequently, different types of secondary amine end-capping agents were added according to specific molar ratios, and the reaction was stirred at room temperature for 48-72 hours to complete the end-capping reaction. The final polymer product was purified by three precipitations in diethyl ether, then dried under vacuum for 24-48 hours. The resulting product was stored at -20°C for subsequent studies.

[0125] Diacrylate monomers include: 1,4-butanediol diacrylate, 1,3-butanediol diacrylate, poly(ethylene glycol) diacrylate, bisphenol A ethoxylate diacrylate, and bisphenol A glycerol diacrylate. Triacrylate monomers include trimethylolpropane triacrylate. The molar ratio of triacrylate to diacrylate is 0.3:1-2:1. Secondary amine monomers primarily include: 3-morpholinopropylamine, 1,3-diaminopropane, 4-amino-1-butanol, and 5,4-amino-1-pentanol. The reaction molar amount of the secondary amine monomers is 1-2 mol.

[0126] Figure 3 The results show that bone marrow-derived BMDM-M2 cells can be successfully transfected using a variety of vectors. -Macrophage, 2000 and HPBAE have similar infection efficiencies, but 2000 and -Macrophage is more toxic than HPBAE, affecting cell proliferation and promoting cell death.

[0127] Example 4: Verification of Specific Phagocytosis of Tumor Cells by CAR-M2

[0128] In order to verify whether the CAR-M2 cells expressing chimeric antigen receptors (obtained from Example 3) have restored the phagocytic ability of tumor cells and whether the phagocytic killing process is specific, the present invention co-incubated CAR-M2 cells with K562-RFP cells (Shandong Weizhen Biotechnology Co., Ltd., CL852678) and photographed and observed using a Nikon fluorescence microscope and a Nano Live holographic label-free live cell 3D imaging system. The results showed that CAR-M2 cells expressing chimeric antigen receptors can phagocytose and kill tumor cells, and only K562-CD19-RFP cells expressing the CD19 target are effectively killed, proving that the phagocytic killing process is specific.

[0129] Figure 18 The results show that after co-incubation with K562-CD19-Luci-RFP cells expressing the CD19 target, the CAR-M2 cells can successfully and effectively phagocytose and kill the K562-CD19-Luci-RFP target cells. However, after co-incubation with K562-RFP cells that do not express the CD19 target, the CAR-M2 cells cannot effectively phagocytose and kill the K562-CD19-Luci-RFP target cells, and the target cells even proliferate. This example demonstrates the specificity of CAR-M2 in phagocytosis and killing of tumor cells.

[0130] Example 5: Screening of TAM peptides targeting tumor-associated macrophages

[0131] The present invention utilizes phage display technology to screen TAMpeptide sequences with excellent targeting to tumor-associated macrophages. Specifically,

[0132] ① Use Error-prone PCR (EP-PCR) technology to randomly generate a nucleic acid sequence library encoding TAM peptides or a PhD-7 heptapeptide random library, a PhD-10 decapeptide random library, or a PhD-12 dodecapeptide random library;

[0133] ②Then the above coding sequences were constructed on the M13 phage genome to construct the TAM peptide M13 phage library;

[0134] ③ Then, the M2 macrophages / tumor-associated macrophages obtained in Example 1 / 2 were mixed with 1*10 9 PFU TAMpeptide M13 phage were co-incubated for 2 h;

[0135] ④Wash away unbound phages using PBS and RPMI 1640 medium;

[0136] ⑤ Treat cells in the culture system with rabbit anti-M13 phage (Sigma) and goat anti-rabbit FITC (Sigma) antibodies;

[0137] ⑥ Use MACSQ flow cytometer to analyze the cells and screen out the peptide TAMpeptide-1 representing the group with the highest signal.

[0138] The amino acid sequences of the TAM peptides are shown in SEQ ID NOs: 11 to 21. These TAM peptides can give the recombinant vector of the present invention good targeting properties, allowing the vector to specifically recognize and target M2 macrophages and TAMs, thereby reducing the uptake of non-target cells to a certain extent, thereby improving the infection efficiency of target cells, reducing the dosage of the vector during treatment, and improving safety.

[0139] Example 6: Determination of affinity of TAM targeting peptides for tumor-associated macrophages or M2 macrophages

[0140] The present invention uses phage display technology to screen out TAM peptides that specifically target tumor-associated macrophages, and then modifies the N-terminus with the fluorescent molecule 5-FAM to obtain 11 peptides with fluorescent signals. The peptides are then added to tumor-associated macrophages for co-incubation. After washing the cells, the targeting of the peptides to the cells is observed using a fluorescence microscope, and the intensity of the fluorescent signal is used to semi-quantify the affinity of the peptides for tumor-associated macrophages. Specifically,

[0141] ① According to Example 1 and Example 2, primary M2 macrophages were obtained;

[0142] ② Plate M2 cells into 96-well plates, with 50,000 cells per well, and culture in a cell culture incubator at 37°C and 5% CO2 for 24 hours;

[0143] ③ Eleven targeting peptides were added to the cell culture medium at different concentrations (0.1 μM-5 μM) and incubated with the cells for 3-6 hours, with three replicates added for each concentration;

[0144] ④ The cells were then washed three times with PBS to remove free peptides that were not targeted to the cell surface;

[0145] ⑤ Use a fluorescence microscope to observe the fluorescence signal intensity of each well to determine the targeting effect of the targeting peptide on the cells. Take fluorescence photos of the cells at 100 times and 200 times magnification respectively, and compare them with PBS without peptide added as the control group. The results are as follows: Figure 4-Figure 15 As shown;

[0146] ⑥ Adjust the fluorescence exposure time and gain to fixed values, re-photograph the target peptides P1-P11, and use the fluorescence microscope's built-in software (Olympus image analysis software) and Image J software to analyze the signal value of the obtained fluorescence images to obtain semi-quantitative fluorescence signal intensity, such as Figure 16 .

[0147] according to Figure 4-Figure 15 , we can clearly see the targeting effect of the targeting peptide on M2 cells. The more cells are lit up, the stronger the fluorescence signal is, indicating that the targeting effect of the peptide on the cells is better and the affinity is higher. Figure 16 The semi-quantitative analysis of the fluorescence signal intensity shows that the targeting peptides P10, P5 and P4 show higher signal values, indicating that the three peptides have good affinity for M2 cells and can target M2 macrophages well, thereby providing feasibility for the next step to improve the infection efficiency of target cells, reduce the amount of vector used during treatment, and improve safety.

[0148] Example 7: Screening of AAV serotypes targeting tumor-associated macrophages or M2 macrophages

[0149] In order to screen out AAV serotypes with excellent targeting for tumor-associated macrophages or M2 macrophages, the present invention uses AAVs of different serotypes to infect M2 macrophages and tumor-associated macrophages TAMs isolated from tumor tissue, and screens out an AAV serotype - AAV9 - with excellent targeting for tumor-associated macrophages or M2 macrophages, and this serotype only specifically infects M2 macrophages.

[0150] Figure 19 The AAV9 specific infection of primary M2 macrophages verification diagram shows that AAV9 successfully infected primary M2 macrophages, while M1 and macrophage cell lines were not successfully infected.

[0151] Example 8: Selection of TAM peptide modification position and modification amount on the surface of adeno-associated virus capsid protein

[0152] In order to confirm and screen the optimal modification design of the adeno-associated virus capsid protein for targeting TAM peptide, in this example, the gene sequence encoding the TAM peptide was inserted into the coding genes of the AAV virus capsid protein VP1, VP2 and VP3 subunits, and the macrophage-specific promoter F4 / 80 promoter gene sequence (SEQ ID NO: 1), the second-generation anti-CD19 CAR-T gene sequence and the EGFP gene sequence were inserted into the transgenic sequence of the adeno-associated virus to construct three recombinant adeno-associated viruses with different capsid protein modifications, tTAM-rAAV-VP1, tTAM-rAAV-VP2, and tTAM-rAAV-VP3 (constructed by Wuhan Vinocell Biotechnology Co., Ltd.), and the three recombinant adeno-associated viruses were used to transfect the above-mentioned bone marrow-derived BMDM-M2 cells and tumor-associated macrophages, and analyzed by MACSQ flow cytometer.

[0153] In order to confirm the optimal modification design of TAM peptide in adeno-associated virus capsid protein, the present invention inserted the gene sequence encoding TAM peptide into different positions of the nine variable regions (VRs) of AAV virus capsid protein, constructed several different envelope plasmids, and used these envelope plasmids and AAV-EGFP plasmid to package # recombinant adeno-associated viruses tTAM-rAAV-ALSCATPLRNFW (SEQ ID NO: 20)-EGFP with different capsid protein modifications. These recombinant adeno-associated viruses were used to transfect the above-mentioned bone marrow-derived BMDM-M2 cells and tumor-associated macrophages, and analyzed using MACSQ flow cytometer.

[0154] Example 9: Comparative verification of the efficiency of tTAM-rAAV-ALSCATPLRNFW (SEQ ID NO: 20)-EGFP, lentiviral vector (positive control), HPBAE@pCAR, and jetPEI@pCAR in in vitro transfection of bone marrow-derived BMDM-M2 cells and tumor-associated macrophages ( Figure 17 ).

[0155] In order to compare the transfection efficiency of tTAM-rAAV-VP1 and currently available transgenic vectors in bone marrow-derived BMDM-M2 cells and tumor-associated macrophages in vitro, the present invention co-incubated these four transgenic vectors with bone marrow-derived BMDM-M2 cells and tumor-associated macrophages, and analyzed them using a MACSQ flow cytometer.

[0156] Example 10: Establishment and treatment of mouse tumor model

[0157] The mice used in this example were C57BL / 6J mice, and the cells of murine colorectal cancer MC38 expressing human CLDN18.2 were inoculated to establish a tumor-bearing mouse model.

[0158] C57BL / 6 mice were anesthetized by intraperitoneal injection of 0.3% sodium pentobarbital and the surgical site was disinfected with 70% ethanol. 2 × 10 6 MC38-hClaudin18.2 cells (produced by Shanghai Model Organisms Science Co., Ltd.) were inoculated subcutaneously in mice. 3Day 0 was designated as Day 0. The mice were randomly divided into 6 groups with 8 mice in each group, 3 of which were used for anti-tumor experimental studies and 5 for survival observation. The 7 groups were administered with (1) normal saline, (2) jetPEI@pCAR-hClaudin18.2, (3) HPBAE@pCAR-hClaudin18.2, (4) tTAM-rAAV-hClaudin18.2, (5) empty AAV9, (6) AAV9-CAR CLDN18.2 and (7) HPBAE, respectively. The second-generation anti-CD19 CAR sequence (nucleotide sequence SEQ ID NO: 2; amino acid sequence SEQ ID NO: 3) in the above-mentioned recombinant adeno-associated virus transgenic sequence was replaced with an anti-hClaudin18.2 CAR sequence (nucleotide sequence SEQ ID NO: 4) that targets Claudin18.2, which is highly expressed in various solid tumors such as primary gastric cancer, metastatic gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, and lung cancer (sequence source: Nanjing Kaidi Medical Technology Co., Ltd. patent CN113416260B, constructed by Shanghai Shenggong Bioengineering Technology Service Co., Ltd.). The administration time is Day 4, 8, 12, 16, 20, and 24. The dosage is pDNA 1.2 mg / kg, 1×10 11 GC / mouse. Mice were sacrificed on Day 28, and tumors were harvested for evaluation of antitumor efficacy. Survival was monitored up to 60 days.

[0159] Figure 20 The tumor-bearing mouse treatment diagram shows the therapeutic effects of various gene delivery vectors on MC38 tumor mice expressing Hcldn18.2. The results show that the screened AAV9 and the designed non-viral vectors exhibited significant tumor-suppressing effects.

[0160] Specific promoter F4 / 80 promoter nucleotide sequence:

[0161]

[0162] Anti-CD19 CAR Nucleotide sequence:

[0163]

[0164] Anti-CD19 CAR Amino acid sequence: 486 aa

[0165] MALPVTALLLPLALLLHAARPEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYSSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:3)

[0166] hClaudin18.2 CAR nucleotide sequence:

[0167] atggccctgcccgtcaccgctctgctgctgccccttgctctgcttcttcatgcagcaaggccggacgtggtcatgacgcagtcaccggattcactggcagtaagtcttggggagcgagcgacaatcaactgcaaatctagccagagtcttcttaactccggtaatcagaagaattacttgacttggtaccaacagaagccaggacagccgcctaagcttctgatctactgggcgtctacacgcgagtcaggtgtacctgatcgattctccgggagtggatccggcacggatttcacgctcacgataagtagcttgcaagctgaggatgttgctgtctactattgccagaatacatatagttttcc

[0168] actgacgttcggacaagggacgaagctggaaataaaagggggtggagggtccggcggcgggggatcaggcggcggtgggtccgat

[0169] gtgcaattggtagaatccggtggcggtctggtacaacctggcggatctttgaggctgtcctgcgccgcgagtggttttacattctcaagcttt

[0170] ggcatgcattgggttaggcaagcccctggtaagggtctggagtgggtcgccttcatttcttctggaagtcatacgatttattacgcggattct

[0171] gtcaaagggcgcttctctatctcaagggataacgcgaagaatacactttttctccaaatgaatagcttgagagcggaggacacagccgttt

[0172] attactgcgctcgcttccagtacggcaatagctttgactattgggggcaaggtactttggttactgtctcatctaccacgacgccagcgccg

[0173] cgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgca

[0174] gtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttat

[0175] caccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagat

[0176] ggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtac

[0177] cagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggac

[0178] cctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggccta

[0179] cagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc(SEQ ID NO:4)

[0180] Amino acid sequence of hClaudin18.2 CAR:

[0181] TLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAG<​TCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRV

[0183] KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY

[0184] NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:5)

[0185] Anti-CD19 CAR2 nucleotide sequence:

[0186] ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTCCT

[0187] GCTGATCCCCGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTG

[0188] GGCGACAGGGTGACCATCAGCTGCAGGGCCAGCCAGGACATCAGCAAGTACCTGA

[0189] ACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAG

[0190] CAGGCTGCACAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGAC

[0191] TACAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCC

[0192] AGCAGGGCAACACCCTGCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAC

[0193] CGGCAGCACCAGCGGCAGCGGCAAGCCCGGCAGCGGCGAGGGCAGCACCAAGGG

[0194] CGAGGTGAAGCTGCAGGAGAGCGGCCCCGGCCTGGTGGCCCCCAGCCAGAGCCTG

[0195] AGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGA

[0196] TCAGGCAGCCCCCCAGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGCGA

[0197] GACCACCTACTACAACAGCGCCCTGAAGAGCAGGCTGACCATCATCAAGGACAAC

[0198] AGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCA

[0199] TCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTACTGG

[0200] GGCCAGGGCACCAGCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGAGGTGCAG

[0201] CTGCAGCAGAGCGGCGCCGAGCTGGTGAAGCCCGGCGCCAGCGTGAAGATGAGCT

[0202] GCAAGGCCAGCGGCTACACCTTCACCAGCTACAACATGCACTGGGTGAAGCAGAC

[0203] CCCCGGCCAGGGCCTGGAGTGGATCGGCGCCATCTACCCCGGCAACGGCGACACC

[0204] AGCTACAACCAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGAGCAGC

[0205] AGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGC

[0206] GAGGACAGCGCCGACTACTACTGCGCCAGGAGCAACTACTACGGCAGCAGCTACT

[0207] GGTTCTTCGACGTGTGGGGCGCCGGCACCACCGTGACCGTGAGCAGCGGCAGCAC

[0208] CAGCGGCGGCGGCAGCGGCGGCGGCAGCGGCGGCGGCGGCAGCAGCGACATCGT

[0209] GCTGACCCAGAGCCCCGCCATCCTGAGCGCCAGCCCCGGCGAGAAGGTGACCATG

[0210] ACCTGCAGGGCCAGCAGCAGCGTGAACTACATGGACTGGTACCAGAAGAAGCCCG

[0211] GCAGCAGCCCCAAGCCCTGGATCTACGCCACCAGCAACCTGGCCAGCGGCGTGCC

[0212] CGCCAGGTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGG

[0213] GTGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCTTCAACCCCC

[0214] CCACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGAGAGCAAGTACGGCCCCCC

[0215] CTGCCCCCCCTGCCCCATGTTCTGGGTGCTGGTGGTGGTGGGCGGCGTGCTGGCCT

[0216] GCTACAGCCTGCTGGTGACCGTGGCCTTCATCATCTTCTGGGTGAAGAGGGGCAGG

[0217] AAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGGCCCGTGCAGACCACCC

[0218] AGGAGGAGGACGGCTGCAGCTGCAGGTTCCCCGAGGAGGAGGAGGGCGGCTGCG

[0219] AGCTGAGGGTGAAGTTCAGCAGGAGCGCCGACGCCCCCGCCTACCAGCAGGGCCA

[0220] GAACCAGCTGTACAACGAGCTGAACCTGGGCAGGAGGGAGGAGTACGACGTGCT

[0221] GGACAAGAGGAGGGCAGGGACCCCGAGATGGGCGGCAAGCCCAGGAGGAAGA

[0222] ACCCCCAGGAGGGCCTGTACAACGAGCTGCAGAAGGACAAGATGGCCGAGGCCT

[0223] ACAGCGAGATCGGCATGAAGGGCGAGAGGAGGGGCAAGGGCCACGACGGCC

[0224] TGTACCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCA

[0225] GGCCCTGCCCCCCAGG(SEQ ID NO:6)

[0226] Anti-CD19 CAR2:

[0227] MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSSASLGDRVTISCRASQDISKYLNWYQQ

[0228] KPDGTVKLLIYHTSRLHSGVPRSRFSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTF

[0229] GGGTCLOTHESGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDY

[0230] GVSWIRQPPRKGLEWLGVIWGSETTYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDT

[0231] AIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGGGGSEVQLQQSGAELVKPGASVKMS

[0232] CKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSST

[0233] AYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGSTSGGGSGGGSG

[0234] GGGSSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLA

[0235] SGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKESKYGPPC

[0236] PPCPMFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEED

[0237] GCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR

[0238] DPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:7)

[0239] CAR-SIRPα RNA sequence

[0240] AUGGAGCCCGCCGGCCCGGCCCCUGGCCGCCUAGGGCCGCUGCUGCUCUGCCUG

[0241] CUGCUCUCCGCGUCCUGUUUCUGUACAGGAGCCACGGGGAAGGAACUGAAGGUG

[0242] ACUCAGCCUGAGAAAUCAGUGUCUGUUGCUGCUGGGGAUUCGACCGUUCUGAA

[0243] CUGCACUUUGACCUCCUUGUUGCCGGUGGGACCCAUAUAGGUGGUACAGAGGAG

[0244] UAGGGCCAAGCCGCGCUGUUGAUCUACAGUUUCGCAGGAGAAUACGUUCCUCGA

[0245] AUUAGAAAUGUUUCAGAUACUACUAAGAGAAACAAUAUGGGACUUUCCAUCCG

[0246] UAUCAGUAAUGUCACCCCAGCAGCAGAUGCUGGCAUCUACUACUGUGUGAAGUUCCA

[0247] GAAAGGAUCAUCAGAGCCUGACACAGAAUACAAUCUGGAGGGGGAACAGAGGG

[0248] UCUAUGUACUCGCCAAACCUUCUCCACCGGAGGUAUCCGGCCCAGCAGACAGGG

[0249] GCAUACCUGACCAGAAAGUACUUCACCUGGCAAGUCUCAUGGCCUUCUCUCCCCC

[0250] GGAAUAUCACCCUGAAGUGGUUCAAAGAUGGGGCAAGAACUCCACCCUUGGAG

[0251] ACCACCGUGAACCCUAGUGGAAAGAAUGUCUCCUACAACACAUCUCCAGCACAGUC

[0252] AGGGUGGUACUAAACUCCAUGGAUGUAAUUCUAAGGUCAUCUGCGAGGUAGC

[0253] CCACAUCACCUUGGAUAGAAGCCCUCUUCGUGGGGAUGCUAACCUCUUACUU

[0254] CAUCCGAGUUUCACCACCACGUGAAGGUCACCCAACAGUCCCCGACGUCAAUGAA

[0255] CCAGGUGAACCUCACCUGCCGGGCUGAGAGGUUCUACCCCGAGGAUCUCCAGCU

[0256] GAUCUGGCUGGAGAAUGGAAACGUAUCACGGAAUGACACGCCCAAGAAUCUCA

[0257] CAAGAACACGGAGGGACCUAUAAUUACACAAGCUUGUUCCUGGUGAACUCA

[0258] UCUGCUCAUAGAGAGGACGUGGUGUUCACGUGCCAGGUGUAAGCACGACCAACA

[0259] GCCAGCGAUCACCCGAAACCAUACCGUGCUGGGAUUUGCCCACUCGAGUGAUCA

[0260] AGGGAGCAUGCAAACCUUCCCUGUGAGACCUACUACUACCAAGCCAGUGCUGCG

[0261] AACUCCCUCACCUGUGCACCCUACCGGGACAUCUCAGCCCCAGAGACCAGAAGA

[0262] UUGUCGGCCCCGUGGCUCAGUGAAGGGGACCGGAUUGGACUUCGCCUGUGAUA

[0263] UUUACAUCUGGGCACCCUUGGCCGGAAUCUGCGUGGCCCUUCUGCUUGA

[0264] UCAUCACUCUCAUCUGCUACCACACUUGUUCCUGGCGCUGACAUCGGCUUUGC

[0265] UGCUGGCCCUGAUCUUCAUUACUCUCCUGUUCUCUCAGCAGGAGUGCAGACAUG

[0266] CUGCCAACCUGCAGGACCCCAACCAGCUCUACAAUGAGCUCAAUCUAGGGCGAA

[0267] GAGAGGAAUAUGACGUCUUGGAGAAGAAGCGGGCUCGGGAUCCAGAGAUGGGA

[0268] GGCAAACAGCAGAGGAGGAGGAACCCCCAGGAAGGCGUAUACAAUGCACUGCA

[0269] GAAAGACAAGAUGGCAGAAGCCUACAGUGAGAUCGGCACAAAAGGCGAGAGGC

[0270] GGAGAGGCAAGGGGCACGAUGGCCUUUACCAGGGUCUCAGCACUGCCACCAAGG

[0271] ACACCUAUGAUGCCCUGCAUAUGCAGACCCUGGCCCCUCGC(SEQ ID NO:8)

[0272] CAR-hCD47 RNA sequence

[0273] AUGGCCCUGCCCGUCACCGCUCUGCUGCUGCCCCUUGCUCUGCUUCUUCAUGCA

[0274] GCAAGGCCGAUGGAGCCCGCCGGCCCGGCCCCCGGCCGCCUCGGGCCGCUGCUC

[0275] UGCCUGCUGCUCGCCGCGUCCUGCGCCUGGUCAGGAGUGGCGGGUGAGGAGGAG

[0276] CUGCAGGUGAUUCAGCCUGACAAGUCCGUGUUGGUUGCAGCUGGAGAGACAGC

[0277] CACUCUGCGCUGCACUGCGACCUCUCUGAUCCCUGUGGGGCCCAUCCAGUGGUU

[0278] CAGAGGAGCUGGACCAGGCCGGGAAUUAAUCUACAAUCAAAAAGAAGGCCACU

[0279] UCCCCCGGGUAACAACUGUUUCAGACCUCACAAAGAGAAACAACAUGGACUUUU

[0280] CCAUCCGCAUCGGUAACAUCACCCCAGCAGAUGCCGGCACCUACUACUGUGUGA

[0281] AGUUCCGGAAAGGGAGCCCCGAUGACGUGGAGUUUAAGUCUGGAGCAGGCACU

[0282] GAGCUGUCUGUGCGCGCCAAACCCUCUGCCCCCGUGGUAUCGGGCCCUGCGGCG

[0283] AGGGCCACACCUCAGCACACAGUGAGCUUCACCUGCGAGUCCCACGGCUUCUCA

[0284] CCCAGAGACAUCACCCUGAAAUGGUUCAAAAAUGGGAAUGAGCUCUCAGACUUC

[0285] CAGACCAACGUGGACCCCGUAGGAGAGAGCGUGUCCUACAGCAUCCACAGCACA

[0286] GCCAAGGUGGUGCUGACCCGCGAGGACGUUCACUCUCAAGUCAUCUGCGAGGUG

[0287] GCCCACGUCACCUUGCAGGGGGACCCUCUUCGUGGGACUGCCAACUUGUCUGAG

[0288] ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCAUCGCGUCGCAGC

[0289] CCCUGUCCCUGCGCCCAGAGGCGUGCCGGCCAGCGGCGGGGGGCGCAGUGCACA

[0290] CGAGGGGGCUGGACUUCGCCUGUGAUAUCUACAUCUGGGCGCCCUUGGCCGGGA

[0291] CUUGUGGGGUCCUUCUCCUGUCACUGGUUAUCACCCUUUACUGCAAACGGGGCA

[0292] GAAAGAAAACUCCUGUAUAUAUUCAAACAACCAUUUAUGAGACCAGUACAAACU

[0293] ACUCAAGAGGAAGAUGGCUGUAGCUGCCGAUUUCCAGAAGAAGAAGAAGGAGGG

[0294] AUGUGAACUGAGAGUGAAGUUCAGCAGGAGCGCAGACGCCCCCGCGUACCAGCA

[0295] GGGCCAGAACCAGCUCUAUAACGAGCUCAAUCUAGGACGAAGAGAGGAGUACG

[0296] AUGUUUUGGACAAGAGACGUGGCCGGGACCCUGAGAUGGGGGGAAAGCCGAGA

[0297] AGGAAGAACCCUCAGGAAGGCCUGUACAAUGAACUGCAGAAAGAUAAGAUGGC

[0298] GGAGGCCUACAGUGAGAUUGGGAUGAAAGGCGAGCGCCGGGAGGGGCAAGGGGC

[0299] ACGAUGGCCUUUACCAGGGUCUCAGUACAGCCACCAAGGACACCUACGACGCCC

[0300] UUCACAUGCAGGCCCUGCCCCCUCGC(SEQ ID NO:9)

[0301] CAR-hClaudin18.2 RNA sequence

[0302] AUGGCCCUGCCCGUCACCGCUCUGCUGCUGCCUGCCCCUUGCUCUUCUUCAUGCA

[0303] GCAAGGCCGGACGUGGUCAUGACGCAGUCACCGGAUUCACUGGCAGUAAGUCUU

[0304] GGGGAGCGAGCGACAAUCAACUGCAAAUCUAGCCAGAGUCUCUUAACUCCGGU

[0305] AAUCAGAAGAAUUACUUGACUUGGUACCAACAGAAGCCAGGACAGCCGCCUAA

[0306] GCUUCUGAUCUACUGGGCGUCUACACGCGAGUCAGGUGUACCUGAUCGAUUCUC

[0307] CGGGAGUGGAUCCGGCACGGAUUUCACGCUCACGAUAAGUAGCUUGCAAGCUG

[0308] AGGAGUUGCUGUCUAUCAUUGCCAGAUACAUAUAUAUGUUUCCACUGACGUUC

[0309] GGACAAGGGACGAAGCUGGAAAUAAAAGGGGGUGGAGGGUCCGGCGGCGGGGGG

[0310] AUCAGGCGGCGGUGGUCCGAUUGUGCAAUUGGUAGAUCCGGGUGGCGGUCUGG

[0311] UACAACCUGGCGGAUCUUUGAGGCUGUCCUGCGCGCGAGUGGUUUUACAUUCU

[0312] CAAGCUUUGGCAUGCAUUGGGGUUAGGCAAGCCCCUGGUAAGGGUCUGGAGUGG

[0313] GUGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG

[0314] AGGGCGCUUCUCUAUCUCAAGGGAUAACGCGAAGAAAUCACUUUUUCUCCCAAA

[0315] UGAAUAGCUUGAGAGCGGAGGACACAGCCGUUUAUAUACUGCGCUCGCUUCCAG

[0316] UACGGCAUAGCUUUGACUAUUGGGGGCAAGGUACUUUGGUUACUCUCAUC

[0317] UACCACGACGCCAGCGCGCGACCACCAACACCGGCGCCCACCAUCGCGUCGCAG

[0318] CCCCUGUCCCUGCGCCCAGAGGCGUGCCGGCCAGCGGCGGGGGGCGCAGUGCAC

[0319] ACGAGGGGGCUGGACUUCGCCUGUGUAUAUCUACAUCUGGGGCGCCCUUGGCCGGG

[0320] ACUUGUGGGGUCCUUCCUCUGUCACUGGGUUAUCACCCUUUACUGCAAACGGGGC

[0321] AGAAAGAAACUCCUGUAUAUAUUCAAACAACCAUUUAUGAGACCAGUACAAAC

[0322] UACUCAAGAGGAAGAUGGCGUGAGCUGCCGAUUUCCAGAGAAGAAGAAGGAG

[0323] GAUGUGAACUGAGAGAGAGAUUCAGCAGGAGCGCAGACGCCCCCGCGUACCAGC

[0324] AGGGCCAGAACCAGCUCUAUAACGAGCUCAAUCUAGGACGAAGAGAGGAGUAC

[0325] GAUGUUUUGGACAAGAGACGUGGCCCGGGACCCUGAGAUGGGGGGAAAGCCGAG

[0326] AAGGAAGAACCCUCAGGAAGGCCUUGUACAAUGACUGCAGAAAGAUAAGAUGG

[0327] CGGAGGCCUACAGUGAGAUGGGAUGAAAGGCGAGCGCGGAGGGGGCAAGGGG

[0328] CACGAUGGCCUUUACCAGGGUCUCACAGACACCAAGGACACCUACGACGCCCUUCACAUGCAGGCCCUGCCCCCUCGC(SEQ ID NO:10)

[0329]

[0330]

[0331] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A polypeptide motif, characterized in that The amino acid sequence of the polypeptide motif is shown in any one of SEQ ID NOs: 11 to 21.

2. A targeting polypeptide, characterized in that: It comprises the polypeptide motif according to claim 1.

3. A targeted recombinant vector, characterized in that: The targeted recombinant vector comprises the polypeptide motif according to claim 1 or the targeting polypeptide according to claim 2; Preferably, the targeted recombinant vector is a viral vector or a non-viral vector; More preferably, the non-viral vector includes a vector constructed of a cationic polymer, and the viral vector includes adeno-associated virus.

4. The targeted recombinant vector according to claim 3, wherein The cationic polymer includes PBAE, PEI and liposome carriers; and / or the adeno-associated virus serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11; Preferably, the PBAE is HPBAE, and its molecular weight is, for example, 8,000-12,000; the PEI is -Macrophage; the liposome carrier is 2000; the adeno-associated virus is AAV2 or AAV9.

5. The targeted recombinant vector according to claim 3 or 4, characterized in that The nucleotide sequence encoding the targeting peptide is shown in SEQ ID NOs: 22 to 32 and the corresponding RNA sequence, which is inserted into the genome of the adeno-associated virus to construct a recombinant adeno-associated virus; Preferably, the nucleotide sequence encoding the targeting peptide is inserted into the gene sequence of the capsid protein of the adeno-associated virus; More preferably, the genome of the recombinant adeno-associated virus further comprises: a nucleotide sequence encoding a CAR molecule; and / or a nucleotide sequence of a promoter specific to macrophages, brain glial cells, or monocytes.

6. The targeted recombinant vector according to claim 5, wherein The recombinant adeno-associated virus comprises one or more of the following conditions: (1) The backbone virus serotype is AAV9; (2) the capsid protein is VP1, VP2 and / or VP3 subunit; (3) The promoter is a promoter specific to macrophages; preferably, it is a promoter as shown in the nucleotide sequence of SEQ ID NO:

1.

7. A method for preparing a targeted chimeric antigen receptor-macrophage CAR-M, characterized in that: Using the targeted recombinant vector according to any one of claims 3 to 6, the nucleotide sequence encoding the CAR molecule is introduced into macrophages, thereby producing CAR-M; Preferably, the CAR is a CAR molecule against hematological tumors or solid tumors, such as a CAR molecule against CD19, BCMA or CD22, or a CAR molecule against CLDN 18.2, HER2, NKG2D, Mesothelin or PMSA, or a CAR molecule against SIRPα; More preferably, the amino acid sequence of the anti-CD19 CAR molecule is shown in SEQ ID NO: 3 or 7, and the amino acid sequence of the anti-CLDN18.2 CAR molecule is shown in SEQ ID NO: 5; preferably, the nucleotide sequence encoding the anti-CD19 CAR molecule is shown in SEQ ID NO: 2 or 6 or 9, the nucleotide sequence encoding the anti-CLDN 18.2 CAR molecule is shown in SEQ ID NO: 4 or 10, and the nucleotide sequence encoding the anti-SIRPα CAR molecule is shown in SEQ ID NO:

8.

8. The preparation method according to claim 7, wherein The macrophages are tumor-associated macrophages; Preferably, the tumor-associated macrophages are derived from bone marrow, or the tumor-associated macrophages are in situ tumor-associated macrophages in vivo; More preferably, they are M2 macrophages.

9. A targeted CAR-M prepared by the preparation method according to claim 7 or 8.

10. Use of the polypeptide motif according to claim 1, the targeting polypeptide according to claim 2, or the targeted recombinant vector according to any one of claims 3 to 6 in the preparation of a targeted CAR-M; Preferably, the macrophages are tumor-associated macrophages; preferably, the tumor-associated macrophages are derived from bone marrow, or the tumor-associated macrophages are in situ tumor-associated macrophages in vivo; more preferably, they are M2 macrophages.

11. Use of AAV9 in specifically targeting M2 macrophages or in preparing reagents that specifically target M2 macrophages.

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