Mesothelin-targeted high-affinity nano antibody as well as related products and application of mesothelin-targeted high-affinity nano antibody

Abstract

The invention discloses a mesothelin-targeted high-affinity nano antibody as well as a related product and application thereof, and belongs to the technical field of biological medicines. The nano antibody comprises a heavy chain variable region, the heavy chain variable region comprises CDR1, CDR2 and CDR3, and the amino acid sequence of the CDR1 is as shown in SEQ ID NO.1; the amino acid sequence of the CDR2 is as shown in SEQ ID NO. 2; and the amino acid sequence of the CDR3 is as shown in SEQ ID NO.3. The nano antibody can be specifically combined with mesothelin antigen and has high affinity; when being used as an antigen binding structural domain to construct a chimeric antigen receptor and an MSLN-VHH-CAR-NK cell, the MSLN-VHH-CAR-NK cell has a very good targeting effect and killing activity on mesothelin positive tumors.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedicine, in particular to a mesothelin-targeted high-affinity nanobody and related products and applications. BACKGROUND

[0002] Tumors still pose a serious threat to human health, especially recurrent and metastatic tumors. Targeted therapy and immunotherapy have brought clinical treatment to a variety of types of cancer, but further improvements are still needed to improve overall response rate and treatment effect.

[0003] Human mesothelin (MSLN) gene is located on chromosome 16 (16P13.3), contains 17 exons, cDNA is about 2138 bp long, contains an open reading frame of 1884 bp, encodes a precursor protein containing 628 amino acids, with a molecular weight of about 69 kDa. The precursor protein can be hydrolyzed by furin protease into two parts: a 40 kDa size, cell membrane-bound mature mesothelin and a 31 kDa size, soluble megakaryocyte colony-stimulating factor. Mature MSLN is a glycosylphosphatidylinositol-anchored cell surface glycoprotein, which can be divided into three consecutive regions: region I (Ser296-Leu390), region II (Glu391-Tyr486) and region III (Pro487-Ser598). Region I is the membrane distal region, which has protein binding sites such as MUC16, CA125, etc., and it is related to tumor proliferation and invasion, and is also the binding site of many immunotherapeutic drugs targeting MSLN-expressing tumors. Region III is the membrane proximal region, which can effectively prevent ineffective targeting binding caused by mesothelin shedding.

[0004] In addition to the expression of MSLN in normal mesothelial cells of the pleura and peritoneum, low levels of MSLN expression can also be observed in normal epithelial cells of the ovarian surface epithelium, vaginal adventitia, testis, and fallopian tube and tonsil. However, the expression level of MSLN is significantly increased in a variety of malignant tumors, including gynecological tumors such as ovarian cancer (44.4-97.3%), triple-negative breast cancer (67%), endometrial cancer (45.5-77%) and cervical cancer (42.4%); digestive system cancers such as pancreatic cancer (80-85%), gastric cancer (44-78%) and cholangiocarcinoma (22%); malignant pleural mesothelioma (45-100%), lung adenocarcinoma (39-69%) and some other squamous cell carcinomas of different origins. Therefore, it is theoretically reasonable and feasible to use MSLN as a target for the treatment of MSLN-expressing related tumors by chimeric antigen receptor engineered T cells (CAR-T) or NK cells (CAR-NK).

[0005] Nanobody (Nb), i.e. variable domain of heavy-chain antibody (VHH), has the biological and pharmacological characteristics of small volume, high solubility, high stability, strong tissue penetration ability, etc. VHHs are resistant to chemical and enzymatic modification and are easy to fuse with other domains, making them an important substitute for traditional antibodies. CAR-NK (chimeric antigen receptor natural killer cell) therapy, as an emerging direction of tumor immunotherapy, has shown broad application prospects due to its unique biological characteristics and clinical advantages in safety and accessibility, especially in the field of solid tumor treatment, which is significantly superior to CAR-T. It is expected that CAR-NK therapy will develop rapidly in the next five years. However, the existing MSLN-CAR-T therapy has the problems of insufficient solid tumor infiltration and cytokine storm risk, and CD28 is a T cell costimulatory domain, which is incompatible with the NK signaling pathway.

[0006] Therefore, it is of great significance to provide anti-mesothelin nanobodies with high penetration ability, high specificity and high affinity for targeted treatment of malignant tumors with high expression of mesothelin.

[0007] The information disclosed in this BACKGROUND section is only for the purpose of increasing the understanding of the general background of the application and should not be taken as an acknowledgment or any form of suggestion that this information forms prior art that is already known in this field. SUMMARY

[0008] The purpose of the present application is to overcome the shortcomings of the prior art and provide a mesothelin-targeted high-affinity nanobody and related products and applications. The nanobody has high affinity and can be used as an antigen-binding domain of a chimeric antigen receptor molecule to prepare CAR-NK cells, which has good application prospects in tumor treatment.

[0009] The first aspect of the present application provides a mesothelin-targeted high-affinity nanobody, which comprises a heavy chain variable region, the heavy chain variable region comprising CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 being shown as SEQ ID NO. 1; the amino acid sequence of CDR2 being shown as SEQ ID NO. 2; and the amino acid sequence of CDR3 being shown as SEQ ID NO. 3.

[0010] In an embodiment of the present application, the amino acid sequence of the nanobody is shown as SEQ ID NO. 4.

[0011] The second aspect of the present application provides a nucleic acid molecule, which encodes the above-mentioned nanobody.

[0012] The third aspect of the present application provides a chimeric antigen receptor, which comprises a signal peptide, an antigen binding domain, a hinge region, a transmembrane region and a signal transduction domain; the antigen binding domain comprises the nanobody.

[0013] In an embodiment of the present application, the signal peptide comprises a CD8a signal peptide; and / or, the hinge region comprises a CD8a hinge region; and / or, the transmembrane region comprises a CD8a transmembrane region; and / or, the signal transduction domain comprises an immunoreceptor tyrosine-based activation motif.

[0014] In an embodiment of the present application, the signal transduction domain further comprises a costimulatory molecule, which comprises 4-1BB.

[0015] The fourth aspect of the present application provides a recombinant expression vector, which comprises a coding gene of the chimeric antigen receptor.

[0016] In an embodiment of the present application, the recombinant expression vector is a lentivirus vector, a retrovirus vector or an adeno-associated virus vector.

[0017] In an embodiment of the present application, the recombinant expression vector is a lentivirus vector.

[0018] The fifth aspect of the present application provides a chimeric antigen receptor immune cell, which expresses the chimeric antigen receptor.

[0019] In an embodiment of the present application, the chimeric antigen receptor immune cell is a T cell, a B cell, an NK cell, a mast cell or a macrophage.

[0020] In an embodiment of the present application, the chimeric antigen receptor immune cell is an NK cell.

[0021] The sixth aspect of the present application provides a use of a reagent in preparing an activated immune cell in vitro, which comprises a coding sequence of the chimeric antigen receptor or the recombinant expression vector.

[0022] The seventh aspect of the present application provides a pharmaceutical composition, which comprises the chimeric antigen receptor immune cell.

[0023] The eighth aspect of the present application provides a use of the nanobody, the nucleic acid molecule, the chimeric antigen receptor, the expression vector, the chimeric antigen receptor immune cell or the pharmaceutical composition in preparing a tumor treatment drug.

[0024] In an embodiment of the present application, the tumor is a tumor with high expression of mesothelin.

[0025] The ninth aspect of the present application provides the use of the above-mentioned nanobody in the preparation of a mesothelin detection reagent.

[0026] In an embodiment of the present application, the reagent is used in ELISA detection, flow cytometry detection or circulating tumor cell detection.

[0027] Compared with the prior art, the present application achieves the following technical effects: The present application uses MSLN extracellular region recombinant protein as an antigen, immunizes a llama, constructs a VHH phage library, and obtains an anti-MSLN nanobody after biological panning, which can specifically bind to a mesothelin antigen and has high affinity; the nanobody is used as an antigen binding domain to construct a chimeric antigen receptor and MSLN-VHH-CAR-NK cell, and the MSLN-VHH-CAR-NK cell has good targeting effect and killing activity on mesothelin positive tumors. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is a Biacore detection affinity curve graph of an anti-mesothelin nanobody; Figure 2 is a M-1-3 CAR-NK-92 effector-target ratio graph detected by lactate dehydrogenase release experiment; Figure 3 is a column graph of M-1-3 CAR-NK-92 cell interleukin-10 (IL-10) secretion level; Figure 4 is a column graph of M-1-3 CAR-NK-92 cell granzyme B secretion level; Figure 5 is a column graph of M-1-3 CAR-NK-92 cell interferon-γ (IFN-γ) secretion level. DETAILED DESCRIPTION

[0029] Unless otherwise clearly indicated, throughout the specification and claims, the term "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element or group of elements but not the exclusion of any other element or group of elements.

[0030] The technical solutions of the present application are described below through specific examples. It should be understood that one or more steps mentioned in the present application do not exclude the existence of other methods and steps before or after the combination steps, or other methods and steps can be inserted between the explicitly mentioned steps. It should also be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. Unless otherwise specified, the numbering of each method step is only for the purpose of identifying each method step, and is not intended to limit the arrangement order of each method or to limit the scope of the implementation of the present application. Changes or adjustments of the relative relationship can also be considered as the scope of the implementation of the present application without substantial technical content changes.

[0031] The raw materials and instruments used in the examples are not specifically limited in source, and can be purchased in the market or prepared according to the conventional methods well known to those skilled in the art.

[0032] As used herein, the terms "single domain antibody", "heavy chain variable region domain of heavy chain antibody", "VHH", "nanobody", "single variable domain" are used interchangeably and all refer to a single domain polypeptide or protein that specifically recognizes and binds to an antigen. The single domain antibody is the variable region of a heavy chain antibody. Generally, a single domain antibody contains three CDRs and four FRs. The single domain antibody is the smallest functional antigen binding fragment. Generally, after obtaining an antibody naturally lacking a light chain and a heavy chain constant region 1 (CH1), the variable region of the heavy chain of the antibody is cloned to construct a single domain antibody consisting of only one heavy chain variable region.

[0033] The "heavy chain antibody" described herein is an antibody derived from a Camelidae organism or a Chondrichthyes organism. Compared with the above-mentioned 4-chain antibody, the heavy chain antibody lacks a light chain and a heavy chain constant region 1 (CH1) and only contains 2 heavy chains consisting of a variable region (VHH) and other constant regions, and the variable region is connected to the constant region through a hinge region-like structure. Each heavy chain of the Camelidae heavy chain antibody contains 1 variable region (VHH) and 2 constant regions (CH2 and CH3), and each heavy chain of the Chondrichthyes heavy chain antibody contains 1 variable region and 5 constant regions (CH1-CH5). The antigen binding fragment of the heavy chain antibody includes VHH and single chain heavy chain antibody. By fusion with the constant region of human IgG Fc, the heavy chain antibody can have CH2 and CH3 of human IgG Fc.

[0034] The binding molecule containing two or more single domain antibodies is a multivalent single domain antibody; the binding molecule containing two or more single domain antibodies with different specificities is a multispecific single domain antibody. The multivalent single domain antibody or the multispecific single domain antibody connects multiple single domain antibodies through a linker. The linker usually consists of 1-15 amino acids selected from G and S.

[0035] Herein, the heavy chain antibody and the antibody are intended to distinguish different combination modes of the antibody. Due to the similarity of the structures of the two, the following description of the structure of the antibody is applicable to the heavy chain antibody except for the light chain.

[0036] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domains of the heavy or light chain of an antibody. The variable domains of the heavy chain and light chain can be referred to as "VH" and "VL", respectively. These domains are generally the most variable parts of an antibody (relative to other antibodies of the same type) and contain the antigen binding sites.

[0037] The term "variable" refers to the broad variability of certain segments of the variable domains among antibodies of the same type. The variable domains mediate antigen binding and define specificity of a particular antibody for a particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. Instead, it is concentrated in three segments called hypervariable regions (HVRs) both in the light chain and the heavy chain variable domains, i.e., HCDR1, HCDR2, HCDR3 in the variable domain of the heavy chain (which can be referred to as CDR1, CDR2, CDR3 in heavy chain antibodies) and LCDR1, LCDR2, and LCDR3 in the variable domain of the light chain. The more highly conserved portions of the variable domains are referred to as framework regions (FR). The variable domains of the heavy and light chains each comprise four FR regions, FR1, FR2, FR3, and FR4, mostly from a beta-sheet format, connected by three HVRs that form loops connecting, and in some cases forming part of, the beta-sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen binding site of antibodies. In general, the structure of the light chain variable region is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, and the structure of the heavy chain variable region is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. The constant domains are not involved directly in binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0038] An "Fc region" (fragment, crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates the binding of the immunoglobulin to host tissues or factors including binding to Fc receptors (FcR) on various cells of the immune system (e.g., effector cells) or to the first component (Clq) of the classical complement system. In IgG, IgA and IgD antibody isotypes, the Fc region is composed of two identical protein fragments from the CH2 and CH3 domains of each of the two heavy chains; the Fc region of IgM and IgE comprises three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. While the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined as spanning from an amino acid residue at position C226 or P230 of the heavy chain to the carboxy-terminus, wherein the numbering is according to the EU index as in Kabat. Each heavy chain of a camelid heavy chain antibody comprises one variable region (VHH) and two constant regions (CH2 and CH3). By fusion with the constant region of human IgG Fc, the heavy chain antibody can have CH2 and CH3 of human IgG Fc.

[0039] An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen binding and / or variable region of the intact antibody. The antibody fragment is preferably an antigen binding fragment of an antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; scFv-Fc fragments; multi-specific antibodies formed from antibody fragments; and any fragments that are capable of increasing the half-life by chemical modification or by incorporation into liposomes. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, which contains the antibody's epitope binding sites. The Fab fragment consists of an entire light chain and the variable region of a heavy chain, and one heavy chain constant region domain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen combining site. Pepsin treatment yields a F(ab')2 fragment that roughly corresponds to two disulfide linked Fab fragments having different antigen binding activities and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having an additional few residues at the carboxy terminus of the CH1 domain, including the one or more cysteines from the antibody hinge region. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. The Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector function of antibodies is determined by sequences in the Fc region, which region is also the region recognized by Fc receptors (FcR) found on certain types of cells. The antigen binding fragments of heavy chain antibodies include VHH and single chain heavy chain antibodies.

[0040] "Fv" is the minimum antibody fragment that contains a complete antigen- recognition and binding site. This fragment consists of a dimer of one heavy- and one light-chain variable domain connected by a linker. From the folding of these two domains, six hypervariable loops (3 loops each from the H and L chain) are observed, which contribute most of the amino acid residues involved in antigen binding and confer antigen binding specificity to antibodies. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) is sufficient for antigen recognition, although at a lower affinity than the entire binding site. "Single-chain Fv" also abbreviated as "sFv" or "scFv" are antibody fragments that comprise the VHand VLdomains of antibody, connected by a linker peptide. Preferably, the sFv polypeptides further comprise a polypeptide linker between the VHand VLdomains, which enables the sFv to form the desired structure for antigen binding. Fv of heavy chain antibodies is VHH.

[0041] Antibodies herein also include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical with, or homologous to, corresponding sequences in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with, or homologous to, corresponding sequences in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

[0042] Herein, a chimeric antigen receptor (CAR) contains an optional signal peptide sequence, a mesothelin binding molecule comprising an anti-mesothelin single domain antibody sequence, a hinge region, a transmembrane region, an intracellular co-stimulatory domain, and an intracellular signaling domain.

[0043] Herein, an "antigen binding molecule" is a protein that specifically binds to an antigen, including but not limited to, antibodies, antigen-binding fragments of antibodies, heavy chain antibodies, nanobodies, minibodies, affibodies, target binding regions of receptors, cell adhesion molecules, ligands, enzymes, cytokines, and chemokines. Herein, the term "antibody" includes monoclonal antibodies (including full length antibodies having an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies), diabodies, and single chain molecules, as well as antibody fragments, particularly antigen-binding fragments, such as Fab, F(ab')2, and Fv. Herein, the terms "immunoglobulin" (Ig) and "antibody" are used interchangeably.

[0044] A "mesothelin binding molecule" described herein comprises an anti-mesothelin single domain antibody, the complementarity determining regions CDRs of which comprise CDR1, CDR2, and CDR3, wherein CDR1 comprises the sequence set forth in SEQ ID NO: 1, CDR2 comprises the sequence set forth in SEQ ID NO: 2, and CDR3 comprises the sequence set forth in SEQ ID NO: 3.

[0045] 4-1BB (also known as CD137 or TNFRSF9) described herein is a glycosylated type I membrane protein, first discovered in 1989, which is a very important costimulatory receptor on T cells and other immune cells, belonging to the TNRSF protein family.

[0046] The "Kd" described herein, i.e. Koff, represents the dissociation rate constant, which represents the speed of dissociation between molecules, and the unit is S -1 In the Biacore assay for determining affinity, the larger the Koff represents the slower the rate of RU decline, and the flatter the curve slope. Therefore, high affinity is manifested as fast association and slow dissociation.

[0047] The "Ka" described herein, i.e. Kon, is the association rate constant, which represents the speed of association between molecules, and the unit is M -1 ∙S -1 In the Biacore assay for determining affinity, the larger the Kon represents the shorter the time to reach the maximum RU, and the steeper the curve slope.

[0048] The "KD" described herein represents the dissociation constant (dissociation constant, KD), which is a specific type of equilibrium constant, used to measure the tendency of a larger object to separate (dissociate) from another smaller component, which is the inverse of the association constant, and the unit is mol / L (M) or nmol / L (nM). The smaller the KD value indicates the stronger the binding ability of the two substances. KD = Kd / Ka.

[0049] Example 1 This example constructs a phage nanobody library and uses ELISA for preliminary screening, and the specific steps are as follows: (1) Construction of phage nanobody library The mature mesothelin recombinant protein (Human MSLN / Mesothelin Protein, Kailv Biological Technology Co., Ltd.) was used to immunize the lama for 5 times, and the serum titer was detected by ELISA. Then, the peripheral blood was extracted to separate the lymphocytes, extract the total RNA, and then reverse transcribe the cDNA. Then, the VHH gene was amplified by nested PCR. The IgG2 and IgG3 heavy chain variable region sequences (heavy chain variable region VHH) were obtained by the following steps: 1) a pair of specific nested outer primers were designed, and the cDNA was used as the template for the first round of PCR amplification. The amplification region was the llama heavy chain antibody gene, and the product size was 750 bp and 900 bp, respectively; the 750 bp PCR product was recovered by gel electrophoresis; 2) the nested inner primers were designed, and the 750 bp first round PCR product was used as the template for the second round of PCR amplification. The amplification product was the heavy chain antibody variable region VHH fragment, and the product size was 500 bp; the second round of PCR product was purified and recovered using a PCR product purification kit. The VHH target gene and the vector pComb3xss were digested by SfiI enzyme. The digested VHH and pComb3xss were connected by T4 DNA ligase. The VHH target gene was cloned into the pHEN1 phagemid, and was electroporated into the TG1 competent cells to construct the VHH gene library.

[0050] (2) Screening of phage nanobody library The library was screened by phage display technology, and the specificity and binding strength of each clone were ensured by precise monoclonal phage ELISA. The phage capable of specifically binding to MSLN protein was obtained, and several mesothelin polypeptide nanobody sequences were obtained by sequencing. The amino acid sequence of the nanobody with high affinity to MSLN was finally verified as SEQ ID NO. 4 (QLQLVESGGGLVQPGGSLRLSCAASGRIDSSYAMGWARQAPGNEREFVAGITWIGGTTYYTDSVKGRFTISKDNTKNMMYLQMDSLKPEDTAVYYCAACPRLSGPDYASAETYGCWGQGTQVTVSSAHHSEDPHGQAGQ), and the screening number was M-1-3. The nanobody comprises a heavy chain variable region, the heavy chain variable region comprises CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is as shown in SEQ ID NO. 1 (SYAMG); the amino acid sequence of CDR2 is as shown in SEQ ID NO. 2 (GITWIGGTTYYTDSVKG); and the amino acid sequence of CDR3 is as shown in SEQ ID NO. 3 (CPRLSGPDYASAETYGC).

[0051] Example 2 The present embodiment carries out prokaryotic expression and purification of the anti-mesothelin nanobody (M-1-3) screened in Example 1, and carries out determination of antibody affinity. The specific steps are as follows: (1) Construction of M-1-3 VHH sequence prokaryotic expression vector with His tag.

[0052] The M-1-3 protein coding gene sequence (SEQ ID NO. 5: CATATGCAGCTGCAGCTGGTGGAAAGCGGTGGTGGCCTGGTTCAGCCGGGTGGTAGCCTGCGCCTGAGTTGTGCAGCAAGCGGTCGTATTGATAGTAGCTATGCAATGGGTTGGGCACGCCAGGCCCCGGGTAATGAACGTGAATTTGTGGCCGGTATTACCTGGATTGGTGGTACAACCTATTATACCGATAGCGTTAAAGGCCGCTTTACCATTAGTAAAGATAATACCAAAAACATGATGTACCTGCAGATGGATAGCCTGAAACCGGAAGATACCGCAGTGTATTATTGTGCAGCATGCCCGCGTCTGAGCGGCCCGGATTATGCAAGCGCCGAAACCTATGGCTGCTGGGGCCAGGGTACACAGGTTACCGTGAGCAGCGCACATCATAGCGAAGATCCGCATGGTCAGGCAGGCCAGCTCGAG) added with Nde I and Xho I restriction enzyme sites is synthesized by base synthesis, recombined into an Nde I and Xho I endonuclease linearized pET-22b vector, and transformed into DH5α competent cells.

[0053] A single colony is picked from the transformation plate, cultured at 37°C overnight, and the PCR sample addition system is shown in Table 1. The PCR conditions are: 95°C pre-denaturation for 3 min; 95°C denaturation for 30 s, 50°C annealing for 30 s, 72°C extension for 30 s, 35 cycles; 72°C extension for 5 min, 4°C storage. The PCR product band size is detected by agarose gel electrophoresis, and the single colony with the expected size is sent to a sequencing company for sequencing identification. The single colony with correct sequencing results is stored.

[0054] Table 1: PCR sample addition system

[0055] (2) M-1-3 VHH prokaryotic expression and purification Transformation: extract the sequencing correct plasmid, add to E. coli competent BL21 (DE3), ice bath 30 min, 42℃ heat shock 60 s, continue ice bath 3 min, add 500 μl LB medium 37℃ incubate 30 min, spread on plate containing 100 μg / ml ampicillin, 37℃ culture overnight.

[0056] PCR identification: pick single colony on the transformation plate, 37℃ shake culture overnight, PCR sample addition system and reaction conditions are as described above, save the single colony whose PCR product band size is consistent with the expected after agarose gel electrophoresis detection.

[0057] Induced expression: 37℃ shake culture BL21 (DE3) containing M-1-3 VHH plasmid, when OD value reaches 0.6, add IPTG to final concentration of 1 mM, continue to shake culture at 37℃ overnight, induce fusion protein expression, after expression, centrifuge at 4000 rpm for 10 min, discard supernatant, collect bacterial cells.

[0058] Protein purification: Bacterial lysis: bacterial cells are dissolved with binding buffer (0.5 mol / L NaCl, 5 mmol / L imidazole, 20 mmol / L Tris-Cl, pH8.0), ultrasonic broken, centrifuged at 10000 rpm for 10 min at 4℃ to collect supernatant crude protein; Equilibrium: take Ni-NTA filler to pack column, wash column with binding buffer to balance; Binding: let crude protein flow through the balanced column filler naturally, collect effluent; Equilibrium: wash Ni-NTA column with 10 times column volume of binding buffer; Washing: wash Ni-NTA column with 6 times column volume of washing buffer (0.5 mol / L NaCl, 60 mmol / L imidazole, 20 mmol / L Tris-Cl, pH8.0), collect effluent; Elution: elute with elution buffer (0.5 mol / L NaCl, 250 mmol / L imidazole, 20 mmol / L Tris-Cl, pH8.0), collect effluent.

[0059] Polyacrylamide gel electrophoresis detection: configure polyacrylamide gel, sample preparation for crude protein and effluent components respectively, polyacrylamide gel electrophoresis detection. The results show that the fusion protein is purified, SDS-PAGE electrophoresis analysis appears obvious band near the theoretical molecular weight, which can be initially judged that the fusion protein is successfully purified.

[0060] Dialysis: dialyze the purified components into protein storage buffer (PBS, 300 mM NaCl, 10% Glycerol, 0.3% SKL, pH 7.4), concentrate, filter sterilization; protein concentration is quantified by protein quantification kit, antibody purity is determined by SEC-HPLC, and is stored at -80°C.

[0061] In addition, the affinity of the purified VHH antibody in this embodiment is also determined by Biacore T200. Biacore is a biological analysis sensing technology developed based on surface plasmon resonance (SPR), which can detect and track the entire change process of the combination and dissociation of molecules in the solution with molecules fixed on the chip surface in the form of a sensing graph, and provide kinetic and affinity data.

[0062] In the determination process, the M-1-3 VHH antibody is solidified to the chip surface, and the mobile phase is a solution containing different concentrations of mesothelin protein. The determination result is shown in Figure 1 The affinity of the antibody reaches the nanomolar level, KD = 1.79E-10 M, Ka = 2.56E+05 M -1 ∙S -1 , Kd = 4.58E-04 S -1 .

[0063] Example 3 In this embodiment, a lentiviral vector expressing a chimeric antigen receptor targeting MSLN (M-1-3 CAR) is prepared.

[0064] First, a lentiviral vector pGV852 M-1-3 CAR carrying the M-1-3 CAR chimeric antigen receptor is constructed, including a CD8α signal peptide, a nanobody against mesothelin (M-1-3 VHH), a CD8α hinge region, a transmembrane region and an immunoreceptor tyrosine-based activation motif (CD3ζ).

[0065] The amino acid sequence (SEQ ID NO. 6) of the signal peptide is: MALPVTALLLPLALLLHAARP.

[0066] The amino acid sequence of M-1-3 VHH is shown in SEQ ID NO. 4.

[0067] The amino acid sequence (SEQ ID NO. 7) of the CD8α hinge region and the transmembrane region is: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC.

[0068] 4-1BB intracellular region amino acid sequence (SEQ ID NO. 8) is: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0069] CD3ζ amino acid sequence (SEQ ID NO. 9) is: RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0070] A specific preparation method is as follows:

[0071] Table 2: PCR loading system

[0072] The single clone was picked on the plate and cultured at 37℃ overnight. The PCR loading system is shown in Table 3. The primers used are as follows: P1: ATGGCCTTACCAGTGACCGC; P2: TTACTGCAGAATTCACGCGT Table 3: PCR loading system

[0073] The PCR conditions are as follows: pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 30 s, annealing at 50℃ for 30 s, extension at 72℃ for 60 s, 35 cycles; extension at 72℃ for 5 min, and preservation at 4℃. The single clone with the expected band size of the PCR product was sent to a sequencing company for sequencing identification. The single clone pGV852-M-1-3-CAR plasmid with correct sequencing results was extracted.

[0074] Example 4 In this example, the lentivirus vector pGV852-M-1-3-CAR prepared in Example 3 was subjected to lentivirus packaging, concentration and titer detection, including the following steps: (1) Lentivirus packaging Before transfection, the logarithmic growth phase HEK293T cells were trypsinized and adjusted to about 5×10 6 cells with culture medium containing 10% serum, and then inoculated in a 10 cm cell culture dish and cultured in a 37℃, 5% CO2 incubator. When the cell density reached 70%-80%, it could be used for transfection; The medium was replaced with a medium containing 2% serum 1 h before transfection; The prepared DNA solutions (pGV852-M-1-3-CAR plasmid 20 μg, pHelper 1.0 vector plasmid 15 μg, and pHelper 2.0 vector plasmid 10 μg) were added to a sterilized centrifuge tube, mixed uniformly with the corresponding volume of transfection reagent, and the total volume of the transfection system was adjusted to 1 mL. Incubation was performed at room temperature for 15 min; The transfection system was slowly added to the culture medium of the HEK293T cells, mixed uniformly, and cultured in a 37℃, 5% CO2 cell incubator. After 6-8 h, the culture medium containing the transfection system mixture was discarded, and 10 mL of PBS was added for washing once; Slowly add 12 mL of cell culture medium containing 2% serum, and culture in a 37℃, 5% CO2 incubator for 48 h. Collect the virus supernatant.

[0075] (2) Lentivirus concentration Collect the supernatant of HEK293T cells 48 h after transfection, centrifuge at 4000 g for 10 min at 4°C to remove cell debris and impurities; Filter the supernatant with a 0.45 μm filter into a 40 mL ultracentrifuge tube, centrifuge at 25000 rpm at 4°C for 2 h, discard the supernatant, and resuspend the virus precipitate with 1640 culture medium (containing 10% FBS); After complete dissolution, centrifuge at high speed for 5 min at 10000 rpm, and take the supernatant to be divided into 50 μL each in a finished product tube, and stored at 80°C.

[0076] (3) Lentivirus titer detection Sow HEK293T adherent cells in a 24-well plate, 1.5×10 5 cells per well, volume 100 μL; Prepare 4 sterile EP tubes, add 90 μL of serum-free medium to each tube; Take 11 μL of virus stock solution to be determined and add it to the first tube, mix well, take 10 μL and add it to the second tube, continue the same operation until the last tube; Select the required cell wells, add all the diluted virus solutions, and place them in a 37°C incubator with a 5% CO2 concentration; After 24 h, aspirate the supernatant in the cell wells, and add 2000 μL of complete culture medium; After 72 h of infection, observe the fluorescence expression.

[0077] The titer calculation formula is as follows: titer (TU / mL) = total number of cells x fluorescence cell ratio / infection volume (mL).

[0078] Example 5 This example uses lentivirus prepared in Example 4 to transduce NK cells, including the following steps: Coat the culture plate with Retronectin (20 μg / mL) to enhance virus adsorption, use the infection solution to prepare an NK-92 cell suspension of 3-5×10 4 cells / mL, add 4 mL to each well of a 6-well plate to achieve a plating amount of about 15-30%; Add lentivirus according to a multiplicity of infection (MOI) of 300, and add polybrene to a final concentration of 5 μg / mL; After 16h, the cells in each well were collected into a clean 1.5 ml EP tube, centrifuged at 2000 rpm for 2 min, the supernatant was removed, replaced with complete culture medium, mixed gently and then returned to the culture plate for continuous culture; After 72h, the GFP positive cells were sorted by flow cytometry and cultured in 1640 medium (containing 10% FBS) containing 1 μg / mL puromycin; The chimeric antigen receptor expression rate of NK-92 cells was detected by flow cytometry, and the infection efficiency of NK-92 cells was 99.19%, indicating that the M-1-3 CAR-NK-92 stable cell strain was successfully constructed and obtained by sorting.

[0079] Example 6 This example carries out cell in vitro toxicity experiment on M-1-3 CAR-NK-92 cells constructed in Example 5, including the following steps: (1) Target cell inoculation MKN-45 cells overexpressing MSLN (MKN-45 MSLN ) were used as target cells, and the target cell concentration was adjusted to 1×10 5 / mL, 100 μL of which was inoculated into a white 96-well plate, i.e. 1×10 4 cells.

[0080] (2) Effect cell inoculation M-1-3 CAR-NK-92 and control NK-92 cells were used as effector cells, and the cell amount was calculated according to E:T=0.625:1, 1.25:1, 2.5:1, 5:1, 10:1, respectively. The corresponding cells (three duplicate wells) were taken, centrifuged, and resuspended in 300 μL of 1640 complete culture medium, and then added to the corresponding wells, and the cells were gently shaken and cultured in an incubator for 24 h.

[0081] (3) Detection method At 45 min before the end of the experiment, 20 ul of 10xlysis was added to the maximum release group of target cells; 10 minutes before the end of the experiment, the 96-well plate was placed on the plate shock period for 5 minutes, and centrifuged at 1000g for 3 minutes. 50 μL of supernatant was taken from each well and transferred to a new 96-well plate, 50 uL of mixed substrate (CytoTox 96®Non-Radioactive Cytotoxicity Assay) was added to each well, and incubated at room temperature for 30 min in the dark. 50 uL of stop solution was added, and the optical absorption value was detected at 490 nm.

[0082] (4) The calculation formula of CAR-NK killing efficiency is: Killing efficiency % = (experimental group-blank group) / maximum release group x 100% Results as shown in Figure 2 Figure 6, the M-1-3 CAR-NK-92 cells constructed by the application have killing activity on MKN-45 tumor cells expressing MSLN, and the killing efficiency is about 20.38±1.17% when the effector-target ratio is 0.625:1, about 31.89±0.22% when the effector-target ratio is 1.25:1, about 49.10±3.49% when the effector-target ratio is 2.5:1, and about 76.82±1.85% when the effector-target ratio is 5:1, which is significantly higher than the 14.17±0.21%, 18.61±0.47%, 26.80±0.39% and 45.78±0.64% of the control group NC CAR-NK-92, indicating that the M-1-3 CAR-NK-92 cells constructed by the application have specific tumor killing effect.

[0083] Example 7 This example detects the secretion of cytokines IL-10, Granzyme B and IFN-γ of the M-1-3 CAR-NK-92 cells constructed in Example 5. (1) Co-culture experiment Collect MKN-45 MSLN cells, wash the cells once with dilution buffer, centrifuge at 1000 rpm for 3 min, discard the supernatant, resuspend the cells in 1640 complete culture medium, count, and finally dilute the cells to a concentration of 1×10 5 / mL, 100 μL per well into a 96-well plate; Collect NC CAR-NK-92, M-1-3 CAR-NK-92 cells (effector cells), wash the cells once with dilution buffer, centrifuge at 1000 rpm for 3 min, discard the supernatant, resuspend the cells in 1640 complete culture medium, count, and finally dilute the cells to a concentration of 5×10 5 / mL; Add 100 μL of effector cells to the corresponding 96-well plate, and co-culture at 37°C, 5% CO2 for 24 h; Centrifuge at 1000 rpm for 5 min, collect the supernatant and detect the cytokines.

[0084] (2) Cytokine detection Dilute the sample with the standard diluent (dilution ratio such as 1:2) Take N 1.5 mL EP tubes (N = sample number + control number) Add 40 μL of mixed beads to each tube (sample, negative control), and add 50 μL of the corresponding test agent (sample, control) and 50 μL of PE Detection Reagent to each tube, shake well to mix, and incubate at room temperature for 3 hours in the dark.

[0085] Add 1 mL wash buffer to each tube, gently blow evenly after 200g centrifugation for 5 minutes.

[0086] Carefully aspirate the supernatant, and add 200 μL wash buffer to each tube, and resuspend the precipitate.

[0087] The sample is subjected to flow cytometry detection.

[0088] (3) Result analysis IL-10, Granzyme B, IFN-γ factor secretion results are shown in Figure 3 , Figure 4 and Figure 5 After co-cultured with MKN-45 MSLN target cells, the IL-10 secreted by M-1-3 CAR-NK-92 cells was about 4677.3±101.6 pg / mL, Granzyme B was about 181.8±2.6 μg / mL, and IFN-γ was about 6189.7±293.2 pg / mL, which were significantly higher than the control group NC CAR-NK-92, 3414.2±40.5 pg / mL, 121.2±2.1 μg / mL and 2729.1±25.0 pg / mL.

[0089] In summary, the nanobodies with high affinity against mesothelin are screened and prepared, which can be efficiently and specifically combined with mesothelin, and are used as antigen binding domains to construct chimeric antigen receptors and CAR-NK cells. The obtained CAR-NK-92 cells have obvious killing activity and specificity to mesothelin-positive tumor cells, and can secrete tumor-killing cytokines, indicating that the nanobodies of the application can be effectively applied to immunotherapy, and have important significance for developing tumor treatment drugs.

[0090] The foregoing description of specific exemplary embodiments of the application is intended to be illustrative only and is not intended to limit the application to the precise forms described. Many modifications and variations are possible in light of the above teachings without departing from the spirit or essential characteristics of the application. The exemplary embodiments were chosen and described in order to explain the principles of the application and its practical application and to allow others skilled in the art to understand the application for various exemplary embodiments with various modifications being suited to the particular use contemplated. The scope of the application is intended to be defined by the claims and their equivalents.

Claims

1. A mesothelin-targeting high-affinity nanobody comprising a heavy chain variable region, the heavy chain variable region comprising a CDR1, a CDR2 and a CDR3, characterized in that, the amino acid sequence of the CDR1 is shown as SEQ ID NO. 1; the amino acid sequence of the CDR2 is shown as SEQ ID NO. 2; and the amino acid sequence of the CDR3 is shown as SEQ ID NO.

3.

2. The Nanobody according to claim 1, characterized in that, the amino acid sequence of the nanobody is shown as SEQ ID NO.

4.

3. A nucleic acid molecule, characterized in that, the nucleic acid molecule encodes the nanobody of claim 1 or 2.

4. A chimeric antigen receptor, characterized in that, the chimeric antigen receptor comprises a signal peptide, an antigen binding domain, a hinge region, a transmembrane region and a signal transduction domain; the antigen binding domain comprises the nanobody of claim 1 or 2; preferably, the signal peptide comprises a CD8α signal peptide; and / or, the hinge region comprises a CD8α hinge region; and / or, the transmembrane region comprises a CD8α transmembrane region; and / or, the signal transduction domain comprises an immunoreceptor tyrosine-based activation motif; more preferably, the signal transduction domain further comprises a costimulatory molecule, the costimulatory molecule comprising 4-1BB.

5. A recombinant expression vector, characterized in that, the recombinant expression vector comprises a gene encoding the chimeric antigen receptor of claim 4; preferably, the recombinant expression vector is a lentiviral vector, a retroviral vector or an adeno-associated viral vector; more preferably, the recombinant expression vector is a lentiviral vector.

6. A chimeric antigen receptor immune cell, comprising: the chimeric antigen receptor immune cell expresses the chimeric antigen receptor of claim 4, preferably, the chimeric antigen receptor immune cell is a T cell, a B cell, an NK cell, a mast cell or a macrophage; more preferably, the chimeric antigen receptor immune cell is an NK cell.

7. Use of an agent for preparing activated immune cells in vitro, characterized in that, the agent contains a sequence encoding the chimeric antigen receptor of claim 4 or the recombinant expression vector of claim 5.

8. A pharmaceutical composition, characterized by, the pharmaceutical composition comprises the chimeric antigen receptor immune cell of claim 6. 9.Use of the nanobody of claim 1 or 2, the nucleic acid molecule of claim 3, the chimeric antigen receptor of claim 4, the expression vector of claim 5, the chimeric antigen receptor immune cell of claim 6 or the pharmaceutical composition of claim 7 in the preparation of a tumor treatment drug. preferably, the tumor is a mesothelin-high tumor. 10.Use of the nanobody of claim 1 or 2 in the preparation of a mesothelin detection reagent.

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