Mesothelin-targeting high affinity nanobodies and related products and uses

Abstract

This invention discloses a high-affinity nanobody targeting mesothelin, related products, and applications, belonging to the field of biomedical technology. The nanobody comprises a heavy chain variable region, which includes CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID NO.1; the amino acid sequence of CDR2 is shown in SEQ ID NO.2; and the amino acid sequence of CDR3 is shown in SEQ ID NO.3. This nanobody can specifically bind to mesothelin antigen with high affinity. Using it as an antigen-binding domain to construct chimeric antigen receptors and MSLN-VHH-CAR-NK cells, it exhibits excellent targeting effects and killing activity against mesothelin-positive tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to a mesothelin-targeting high-affinity nanobody and related products and applications. Background Technology

[0002] Cancer remains a serious threat to human health, especially recurrent and metastatic cancer. Targeted therapy and immunotherapy have brought benefits to the clinical treatment of many types of cancer, but further improvements are still needed to enhance overall response rates and treatment outcomes.

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

[0004] Besides being expressed in normal mesothelial cells of the pleura and peritoneum, low-level MSLN expression can also be observed in the normal epithelial cells of the ovarian surface epithelium, vaginal adventitia, testes, fallopian tubes, and tonsils. However, MSLN expression levels are significantly elevated in various 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 squamous cell carcinomas of various origins. Therefore, using MSLN as a target to treat MSLN-high expression-related tumors with chimeric antigen receptor-modified T cells (CAR-T) or NK cells (CAR-NK) is theoretically reasonable and feasible.

[0005] Nanobodies (Nb), or variable domain of heavy chain antibody (VHH), possess biological and pharmacological properties such as small size, high solubility, high stability, and strong tissue penetration. VHHs are resistant to chemical and enzymatic modifications and readily fuse with other domains, making them an important alternative to traditional antibodies. CAR-NK (chimeric antigen receptor natural killer) therapy, as an emerging direction in tumor immunotherapy, shows broad application prospects due to its unique biological characteristics and clinical advantages in safety and accessibility, especially in the treatment of solid tumors where it is significantly superior to CAR-T. CAR-NK therapy is expected to develop rapidly in the next 5 years. However, existing MSLN-CAR-T therapies suffer from insufficient solid tumor infiltration and the risk of cytokine storms, and CD28, being a T cell co-stimulatory domain, is incompatible with the NK signaling pathway.

[0006] Therefore, providing anti-mesothelin nanobodies with high penetration ability, high specificity and high affinity is of great significance for targeted therapy of malignant tumors that highly express mesothelin.

[0007] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a mesothelin-targeting high-affinity nanobody, related products, and applications. The nanobody possesses high affinity and can serve as the antigen-binding domain of a chimeric antigen receptor molecule in the preparation of CAR-NK cells, showing promising application prospects in tumor therapy.

[0009] The first aspect of the present invention provides a mesothelin-targeting high-affinity nanobody comprising a heavy chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown in SEQ ID NO.1; the amino acid sequence of CDR2 is shown in SEQ ID NO.2; and the amino acid sequence of CDR3 is shown in SEQ ID NO.3.

[0010] In one embodiment of the present invention, the amino acid sequence of the nanobody is shown in SEQ ID NO.4.

[0011] A second aspect of the present invention provides a nucleic acid molecule that encodes the above-mentioned nanobody.

[0012] A third aspect of the present invention provides a chimeric antigen receptor, the chimeric antigen receptor comprising a signal peptide, an antigen-binding domain, a hinge region, a transmembrane region, and a signal transduction domain; the antigen-binding domain comprising the aforementioned nanobody.

[0013] In one embodiment of the present invention, the signal peptide includes a CD8α signal peptide; and / or, the hinge region includes a CD8α hinge region; and / or, the transmembrane region includes a CD8α transmembrane region; and / or, the signal transduction domain includes an immune receptor tyrosine activation motif.

[0014] In one embodiment of the present invention, the signal transduction domain further includes a co-stimulatory molecule, the co-stimulatory molecule comprising 4-1BB.

[0015] A fourth aspect of the present invention provides a recombinant expression vector comprising the encoding gene of the chimeric antigen receptor described above.

[0016] In one embodiment of the present invention, the recombinant expression vector is a lentiviral vector, a retroviral vector, or an adeno-associated virus vector.

[0017] In one embodiment of the present invention, the recombinant expression vector is a lentiviral vector.

[0018] A fifth aspect of the present invention provides a chimeric antigen receptor immune cell, wherein the chimeric antigen receptor immune cell expresses the chimeric antigen receptor described above.

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

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

[0021] A sixth aspect of the present invention provides the use of a reagent in the in vitro preparation of activated immune cells, the reagent containing the coding sequence of the chimeric antigen receptor or the recombinant expression vector described above.

[0022] A seventh aspect of the present invention provides a pharmaceutical composition comprising the above-described chimeric antigen receptor immune cells.

[0023] The eighth aspect of the present invention provides the use of the above-mentioned nanobody, the above-mentioned nucleic acid molecule, the above-mentioned chimeric antigen receptor, the above-mentioned expression vector, the above-mentioned chimeric antigen receptor immune cell, or the above-mentioned pharmaceutical composition in the preparation of a tumor therapeutic drug.

[0024] In one embodiment of the present invention, the tumor is a tumor that highly expresses mesothelin.

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

[0026] In one embodiment of the present invention, the reagent is used for ELISA detection, flow cytometry detection, or circulating tumor cell detection.

[0027] Compared with the prior art, the technical effects achieved by the present invention are as follows: This invention uses recombinant protein from the extracellular region of MSLN as an antigen to immunize alpacas in order to construct a VHH phage library. After bio-screening, anti-MSLN nanobodies were obtained. These nanobodies can specifically bind to mesothelin antigen with high affinity. Using them as antigen-binding domains, chimeric antigen receptors and MSLN-VHH-CAR-NK cells were constructed, which showed good targeting effect and killing activity against mesothelin-positive tumors. Attached Figure Description

[0028] Figure 1 This is an affinity curve of the anti-mesothelin nanobody detected using Biacore. Figure 2 This is a graph showing the effector-to-target ratio of M-1-3 CAR-NK-92 as detected by the lactate dehydrogenase release assay. Figure 3 This is a bar chart showing the secretion level of interleukin-10 (IL-10) in M-1-3 CAR-NK-92 cells; Figure 4 This is a bar chart showing the secretion level of M-1-3 CAR-NK-92 Granzyme B. Figure 5 This is a bar chart showing the secretion level of interferon-γ (IFN-γ) in M-1-3 CAR-NK-92 cells. Detailed Implementation

[0029] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0030] The technical solution of the present invention is illustrated below through specific embodiments. It should be understood that the one or more steps mentioned in the present invention do not preclude the existence of other methods and steps before or after the combined steps, or that other methods and steps may be inserted between these explicitly mentioned steps. It should also be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Unless otherwise stated, the numbering of each method step is only for the purpose of identifying each method step, and not for limiting the order of each method or limiting the scope of the present invention. Changes or adjustments to their relative relationships, without substantial changes to the technical content, can also be considered as within the scope of the present invention.

[0031] The raw materials and instruments used in the examples are not subject to any specific restrictions on their source; they can be purchased from the market or prepared according to conventional methods known to those skilled in the art.

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

[0033] The "heavy chain antibody" described in this article refers to antibodies derived from camelid or cartilaginous fish. Compared to the aforementioned four-chain antibodies, heavy chain antibodies lack the light chain and heavy chain constant region 1 (CH1), containing only two heavy chains composed of a variable region (VHH) and other constant regions. The variable region is connected to the constant region via a hinge-like structure. Each heavy chain of camelid heavy chain antibodies contains one variable region (VHH) and two constant regions (CH2 and CH3), while each heavy chain of cartilaginous fish heavy chain antibodies contains one variable region and five constant regions (CH1-CH5). The antigen-binding fragment of heavy chain antibodies includes VHH and single-chain heavy chain antibodies. By fusing with the constant region of human IgG Fc, heavy chain antibodies can possess the CH2 and CH3 regions of human IgG Fc.

[0034] A binding molecule containing two or more single-domain antibodies is a multivalent single-domain antibody; a binding molecule containing two or more single-domain antibodies with different specificities is a multispecific single-domain antibody. Multivalent or multispecific single-domain antibodies are linked together by a linker. The linker typically consists of 1-15 amino acids selected from G and S.

[0035] In this article, heavy chain antibodies and antibodies are used to distinguish different combinations of antibodies. Due to their structural similarities, the structural descriptions of antibodies below, except for those involving light chains, also apply to heavy chain antibodies.

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

[0037] The term "variable" refers to the wide variation in certain segments within a variable domain within the antibody sequence. Variable domains mediate antigen binding and define the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed across all amino acids spanned by the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) (present in both light and heavy chain variable domains): HCDR1, HCDR2, and HCDR3 in the heavy chain variable domain (simply referred to as CDR1, CDR2, and CDR3 in heavy chain antibodies) and LCDR1, LCDR2, and LCDR3 in the light chain variable domain. The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of both the natural heavy and light chains each contain four FR regions (FR1, FR2, FR3, and FR4), which mostly adopt a β-sheet conformation and are linked by three HVRs that form a ring connection and, in some cases, part of a β-sheet structure. The HVRs in each chain are held together very closely by the FR regions and, together with the HVRs of the other chain, contribute to the formation of the antibody's antigen-binding site. Typically, the structure of the variable region in the light chain is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, and the structure of the variable region in the heavy chain is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. The constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as antibody involvement in antibody-dependent cell-mediated cytotoxicity.

[0038] The “Fc region” (crystallizable fragment region), or “Fc domain”, or simply “Fc”, refers to the C-terminal region of an antibody heavy chain that mediates the binding of immunoglobulins to host tissues or factors, including binding to Fc receptors on various cells of the immune system (e.g., effector cells) or to the first component (C1q) of the classical complement system. In IgG, IgA, and IgD antibody isotypes, the Fc region consists of two identical protein fragments from the CH2 and CH3 domains of the two antibody heavy chains; the Fc regions of IgM and IgE contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as the sequence segment from the amino acid residue at position C226 or P230 of the heavy chain to the carboxyl terminus, where the numbering is based on the EU index, as in Kabat. Each heavy chain of a camelid heavy chain antibody contains one variable region (VHH) and two constant regions (CH2 and CH3). By fusing with the constant regions of human IgG Fc, the heavy chain antibody can possess the CH2 and CH3 regions of human IgG Fc.

[0039] An "antibody fragment" comprises a portion of a complete antibody, preferably the antigen-binding region and / or variable region of the complete antibody. Antibody fragments are preferably antigen-binding fragments of the antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules; scFv-Fc fragments; multispecific antibodies formed from antibody fragments; and any fragment whose half-life should be increased through chemical modification or incorporation into liposomes. Digestion of an antibody with papain produces two identical antigen-binding fragments called "Fab" fragments and a residual "Fc" fragment, the name reflecting its ease of crystallization. Fab fragments consist of a complete light chain and a variable domain (VH) of the heavy chain, and a first constant domain (CH1) of the heavy chain. Each Fab fragment is monovalent in terms of antigen binding, i.e., it has a single antigen-binding site. Treatment of an antibody with pepsin produces a larger F(ab')2 fragment, which roughly corresponds to two Fab fragments linked by disulfide bonds, possessing different antigen-binding activities and still capable of cross-linking antigens. The Fab' fragment differs from the Fab fragment due to the addition of several additional residues (including one or more cysteine ​​residues from the antibody hinge region) at the carboxyl terminus of the CH1 domain. The F(ab')2 antibody fragment was originally generated as a pair of Fab' fragments, with a hinge cysteine ​​residue between them. Other chemical conjugations of antibody fragments are also known. The Fc fragment contains the carboxyl-terminal portions of two heavy chains held together by disulfide bonds. The effector function of the antibody is determined by the sequence in the Fc region, which is also recognized by Fc receptors (FcRs) found on certain cell types. Antigen-binding fragments of heavy chain antibodies include VHH and single-chain heavy chain antibodies.

[0040] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a dimer of a tightly bound, non-covalently linked heavy chain variable domain and a light chain variable domain. Six hypervariable rings (three from the heavy chain and three from the light chain) protrude from the folds of these two domains, contributing the amino acid residues for antigen binding and conferring antigen-binding specificity to the antibody. However, even a single variable domain (or half an Fv containing only three antigen-specific HVRs) can recognize and bind antigens, although with lower affinity than a complete binding site. A "single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment containing antibody VH and VL domains linked together into a single polypeptide chain. Preferably, the sFv polypeptide also contains a polypeptide linker between the VH and VL domains, allowing the sFv to form the desired antigen-binding structure. The Fv of a heavy chain antibody is VHH.

[0041] Antibodies in this article also include “chimeric” antibodies, wherein a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided they exhibit the desired biological activity.

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

[0043] In this document, 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, microbodies, affinity molecules, target-binding regions of receptors, cell adhesion molecules, ligands, enzymes, cytokines, and chemokines. In this document, the term "antibody" includes monoclonal antibodies (including full-length antibodies having the immunoglobulin Fc region), antibody compositions with multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies), biantibodies and single-chain molecules, and antibody fragments, especially antigen-binding fragments, such as Fab, F(ab')2, and Fv. In this document, the terms "immunoglobulin" (Ig) and "antibody" are used interchangeably.

[0044] The “mesothelin-binding molecule” described herein comprises an anti-mesothelin single-domain antibody, wherein the complementarity-determining region (CDR) of the single-domain antibody comprises CDR1, CDR2 and CDR3, wherein CDR1 comprises the sequence shown in SEQ ID NO:1, CDR2 comprises the sequence shown in SEQ ID NO:2 and CDR3 comprises the sequence shown in SEQ ID NO:3.

[0045] The 4-1BB (also known as CD137 or TNFRSF9) described in this article is a glycosylated type I membrane protein, first discovered in 1989. It is a very important co-stimulatory receptor on T cells and other immune cells and belongs to the TNRSF protein family.

[0046] In this article, "Kd" stands for Koff, which represents the dissociation rate constant, indicating the speed of intermolecular dissociation, and is measured in seconds (s). -1 In Biacore's affinity assay, a larger Koff value indicates a slower rate of RU decrease and a flatter curve slope. Therefore, high affinity is characterized by rapid binding and slow separation.

[0047] In this article, "Ka" stands for Kon, which is the association rate constant, representing the speed at which molecules bind together, and its unit is M. -1 ∙S -1 In Biacore's affinity assay, a larger Kon value indicates a shorter time to reach maximum RU and a steeper curve slope.

[0048] The "KD" mentioned in this article represents the dissociation constant, a specific type of equilibrium constant used to measure the tendency of a larger substance to separate (dissociate) from another smaller component. It is the reciprocal of the association constant, and its unit is mol / L (M) or nmol / L (nM). The smaller the KD value, the stronger the binding affinity between the two substances. KD = Kd / Ka.

[0049] Example 1 This embodiment constructs a phage nanobody library and uses ELISA for preliminary screening. The specific steps are as follows: (1) Construction of phage nanobody library Alpaca were immunized five times with mature recombinant mesothelin protein (Human MSLN / Mesothelin Protein, Kaika Biotechnology). After serum titer was detected by ELISA, peripheral blood was collected to isolate lymphocytes, total RNA was extracted, and then reverse transcribed into cDNA. The VHH gene was then amplified using nested PCR. The nested PCR steps for obtaining the IgG2 and IgG3 heavy chain variable region sequences (heavy chain variable region VHH) are as follows: 1) A pair of specific nested outer primers were designed for the first round of PCR amplification using cDNA as a template. The amplified region was the alpaca heavy chain antibody gene, with product sizes of 750 bp and 900 bp, respectively. The 750 bp PCR product was recovered by gel electrophoresis. 2) Nested inner primers were designed for the second round of PCR amplification using the 750 bp first-round PCR product as a template. The amplified product was the heavy chain antibody variable region VHH fragment, with a product size of 500 bp. The second-round PCR product was purified and recovered using a PCR product purification kit. The target gene VHH and the vector pComb3xss were digested with SfiI enzyme. The digested VHH and pComb3xss were then ligated with T4 DNA ligase, and the VHH target gene was cloned into pHEN1 phage particles. The particles were then electroporated and transformed into TG1 competent cells to construct a VHH gene library.

[0050] (2) Screening of phage nanobody libraries Library screening was performed using phage display technology, and precise single-clone phage ELISA was used to ensure the specificity and binding strength of each clone. Phages that specifically bind to MSLN protein were obtained, and several mesothelin peptide nanobody sequences were obtained by sequencing. Finally, the amino acid sequence of the nanobody that binds to MSLN with high affinity was verified as shown in SEQ ID NO.4 (QLQLVESGGGLVQPGGSLRLSCAASGRIDSSYAMGWARQAPGNEREFVAGITWIGGTTYYTDSVKGRFTISKDNTKNMMYLQMDSLKPEDTAVYYCAACPRLSGPDYASAETYGCWGQGTQVTVSSAHHSEDPHGQAGQ), with screening number M-1-3. The nanobody contains a heavy chain variable region, which includes CDR1, CDR2 and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID NO.1 (SYAMG); the amino acid sequence of CDR2 is shown in SEQ ID NO.2 (GITWIGGTTYYTDSVKG); and the amino acid sequence of CDR3 is shown in SEQ ID NO.3 (CPRLSGPDYASAETYGC).

[0051] Example 2 This embodiment describes the prokaryotic expression and purification of the anti-mesothelin nanobody (M-1-3) screened in Example 1, followed by determination of antibody affinity. The specific steps are as follows: (1) Construct a prokaryotic expression vector with the M-1-3 VHH sequence carrying the His tag.

[0052] The M-1-3 protein-coding gene sequence with added Nde I and Xho I restriction sites (SEQ ID) NO.5: CATATGCAGCTGCAGCTGGTGGAAAGCGGTGGTGGCCTGGTTCAGCCGGGTGGTAGCCTGCGCCTGAGTTTGTGCAGCAAGCGGTCGTATTGATAGTAGCTATGCAATGGGTTGGGCACGCCAGGCCCCGGGTAATGAACGTGAATTTGTGGCCGGTATTACCTGGATTGGTGGTACAACCTATTATACCGATAGCGTTAAAGGCCGCTTTACCATTAGT AAAGATAATACCAAAAACATGATGTACCTGCGAATGGATAGCCTGAAACCGGAAGATACCGCAGTGTATTATTGTGCAGCATGCCCGCGTCTGAGCGGCCCGGATTATGCAAGCGCCGAAACCTATGGCTGCTGGGGCCAGGGTACACAGGTTACCGTGAGCAGCGCACATCATAGCGAAGATCCGCATGGTCAGGCAGGCCAGCTCGAG) is recombined into Nde through base synthesis I and Xho Transform DH5α competent cells into the pET-22b vector linearized with I endonuclease.

[0053] Single clones were picked from transformation plates and incubated overnight at 37°C with shaking. The PCR loading system is shown in Table 1. The PCR conditions were: 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; and storage at 4°C. Single clones whose PCR product band size met the expectations were sent to a sequencing company for sequencing identification, and the single clones with correct sequencing results were stored.

[0054] Table 1: PCR sample loading system

[0055] (2) M-1-3 VHH prokaryotic expression and purification Transformation: Extract the correctly sequenced plasmid, add it to E. coli competent cells BL21(DE3), incubate on ice for 30 min, heat shock at 42℃ for 60 s, continue incubation on ice for 3 min, add 500 μl LB medium and incubate at 37℃ for 30 min, spread on a plate containing 100 µg / ml ampicillin, and incubate at 37℃ overnight.

[0056] PCR identification: Single clones were picked from the transformation plate and cultured overnight at 37°C with shaking. The PCR loading system and reaction conditions were as described above. Single clones whose PCR product band size met the expectations were stored and detected by agarose gel electrophoresis.

[0057] Induction of expression: BL21(DE3) containing M-1-3 VHH plasmid was cultured at 37℃ with shaking. When the OD value reached 0.6, IPTG was added to a final concentration of 1 mM. The culture was continued at 37℃ with shaking overnight to induce the expression of the fusion protein. After the expression was completed, the cells were centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the cells were collected.

[0058] Protein purification: Cell lysis: The cells were dissolved in binding buffer (0.5 mol / L NaCl, 5 mmol / L imidazole, 20 mmol / L Tris-Cl, pH 8.0), sonicated, and centrifuged at 4℃ and 10000 rpm for 10 min to collect the supernatant crude protein. Equilibration: Pack the Ni-NTA packing material into the column and wash the column with binding buffer to equilibrate it; Combined: Allow crude protein to flow naturally through the equilibrated column packing and collect the effluent; Equilibration: Wash the Ni-NTA column with 10 column volumes of binding buffer; Washing: Wash the Ni-NTA column with 6 column volumes of washing buffer (0.5 mol / L NaCl, 60 mmol / L imidazole, 20 mmol / L Tris-Cl, pH 8.0) and collect the effluent; Elution: Elute with elution buffer (0.5 mol / L NaCl, 250 mmol / L imidazole, 20 mmol / L Tris-Cl, pH 8.0) and collect the eluent.

[0059] Polyacrylamide gel electrophoresis (PAGE) detection: Polyacrylamide gels were prepared, and crude protein and effluent components were processed separately for PAGE detection. The results showed that the fusion protein was purified; SDS-PAGE analysis revealed a clear band near the theoretical molecular weight, indicating that the fusion protein was successfully purified.

[0060] Dialysis: The purified fraction was dialyzed into protein preservation buffer (PBS, 300 mM NaCl, 10% Glycerol, 0.3% SKL, pH 7.4), concentrated, filtered and sterilized; protein concentration was quantified using a protein quantification kit, antibody purity was determined by SEC-HPLC, and stored at -80°C.

[0061] In addition, in this embodiment, the purified VHH antibody was also subjected to affinity determination using the Biacore T200. Biacore is a bioanalytical sensing technology based on surface plasmon resonance (SPR), which can detect and track the entire process of binding and dissociation between molecules in solution and molecules immobilized on the chip surface, record it in the form of a sensing map, and provide kinetic and affinity data.

[0062] During the assay, the M-1-3 VHH antibody was immobilized onto the chip surface. The mobile phase consisted of a solution containing different concentrations of mesothelin protein. The assay results are as follows: Figure 1 As shown, the antibody's affinity reaches the nanomolar level, with KD = 1.79E-10 M and Ka = 2.56E+05 M. -1 ∙S -1 Kd = 4.58E-04 S -1 .

[0063] Example 3 This embodiment prepares a lentiviral vector expressing a chimeric antigen receptor (M-1-3 CAR) targeting MSLN.

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

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

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

[0067] The amino acid sequences (SEQ ID NO.7) of the CD8α hinge region and transmembrane region are as follows: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC.

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

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

[0070] The specific preparation method is as follows:

[0071] Table 2: PCR sample loading system

[0072] Single colonies were picked from plates and incubated overnight at 37°C with shaking. The PCR loading system is shown in Table 3. The primers used were as follows: P1: ATGGCCTTACCAGTGACCGC; P2: TTACTGCAGAATTCACGCGT Table 3: PCR sample loading system

[0073] PCR conditions were as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 60 s, 35 cycles; 72℃ extension for 5 min, and storage at 4℃. Single clones whose PCR product band size met expectations, as determined by agarose gel electrophoresis, were sent to a sequencing company for sequencing identification. The plasmid pGV852-M-1-3-CAR, which was correctly sequenced, was extracted.

[0074] Example 4 In this embodiment, the lentiviral vector pGV852-M-1-3-CAR prepared in Example 3 was subjected to lentiviral packaging, concentration, and titer detection, including the following steps: (1) Lentiviral packaging 48 hours before transfection, HEK293T cells in logarithmic growth phase were digested with trypsin and adjusted to approximately 5 × 10⁶ cells / day with medium containing 10% serum. 6 Cells were reseeded in 10 cm cell culture dishes and cultured at 37°C in a 5% CO2 incubator. They were ready for transfection when the cell density reached 70%–80%. Replace the medium with 2% serum 1 hour before transfection; Add the prepared DNA solutions (20 μg pGV852-M-1-3-CAR plasmid, 15 μg pHelper 1.0 vector plasmid, and 10 μg pHelper 2.0 vector plasmid) to a sterile centrifuge tube, mix them thoroughly with the corresponding volumes of transfection reagent, adjust the total volume to 1 mL of transfection system, and incubate at room temperature for 15 min. The transfection system was slowly added dropwise to the culture medium of HEK293T cells, mixed well, and cultured in a cell culture incubator containing 5% CO2 at 37°C. After 6-8 hours, the culture medium containing the transfection system mixture was discarded, and the cells were washed once with 10 mL of PBS. Slowly add 12 mL of cell culture medium containing 2% serum, and incubate at 37°C in a 5% CO2 incubator for 48 h. Collect the viral supernatant.

[0075] (2) Lentiviral concentration Collect the supernatant of HEK293T cells 48 h after transfection, centrifuge at 4000 g for 10 min at 4℃ to remove cell debris and impurities; The supernatant was filtered through a 0.45 μm filter into a 40 mL ultracentrifuge tube and centrifuged at 25,000 rpm and 4 °C for 2 h. The supernatant was discarded, and the virus pellet was dissolved and resuspended in 1640 medium (containing 10% FBS). After being fully dissolved, centrifuge at 10,000 rpm for 5 minutes. Then, take the supernatant and divide it into 50 μL portions, store them in the finished product tubes, and store them at 80°C.

[0076] (3) Lentiviral titer detection HEK293T adherent cells were seeded in 24-well plates at a density of 1.5 × 10⁶ cells per well. 5 100 μL of cells; Prepare 4 sterile EP tubes and add 90 μL of serum-free culture medium to each tube; Add 11 μL of the virus stock solution to be tested to the first tube, mix well, then add 10 μL to the second tube, and continue the same operation until the last tube. Select the required cell wells, add all the diluted virus solution, and incubate at 37°C in an incubator containing 5% CO2. After 24 hours, aspirate the supernatant from the cell wells and add 2000 μL of complete culture medium. Fluorescence expression was observed 72 hours after infection.

[0077] The titer calculation formula is as follows: Titer (TU / mL) = Total number of cells × Proportion of fluorescent cells / Infection volume (mL).

[0078] Example 5 This embodiment uses the lentivirus-transduced NK cells prepared in Example 4, and includes the following steps: To enhance virus adsorption, culture plates were coated with retrotronectin (20 μg / mL), and NK-92 cells were prepared into 3-5 × 10⁶ cells using infection medium. 4 For each well of a 6-well plate, add 4 mL of the suspension per mL to achieve a plate coating of approximately 15-30%. Lentiviral virus was added at a multiplicity of infection (MOI) of 300, and polybrene was added to a final concentration of 5 μg / mL. After 16 hours, the cells from each well were collected into a clean 1.5 ml EP tube, centrifuged at 2000 rpm for 2 min, the supernatant was removed, and the medium was replaced with complete medium. After gently mixing, the cells were returned to the culture plate for further culture. After 72 hours, GFP-positive cells were sorted by flow cytometry and cultured in 1640 medium (containing 10% FBS) with 1 μg / mL puromycin. The expression rate of chimeric antigen receptor in NK-92 cells was detected by flow cytometry. The infection efficiency of NK-92 cells was 99.19%, indicating that the stable M-1-3 CAR-NK-92 cell line was successfully constructed and obtained through sorting.

[0079] Example 6 This embodiment performs an in vitro cytotoxicity experiment on the 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 As target cells, the target cell concentration was adjusted to 1×10⁻⁶. 5 / mL, take 100μL and inoculate it into a white 96-well plate, i.e., 1×10 4 Each cell.

[0080] (2) Effector cell inoculation M-1-3 CAR-NK-92 and control NK-92 cells were used as effector cells. Cell counts were calculated according to E:T ratios of 0.625:1, 1.25:1, 2.5:1, 5:1, and 10:1. The corresponding cells were collected (triple-well packs), centrifuged, and resuspended in 300 μL of 1640 complete medium. The cells were then added to the corresponding wells, gently shaken, and incubated for 24 h.

[0081] (3) Detection method 45 minutes before the experimental endpoint, add 20 μL of 10xlysis to the target cell maximum release group; 10 minutes before the experimental endpoint, place the 96-well plate on the plate shaking surface and shake for 5 minutes, then centrifuge at 1000g for 3 minutes. Transfer 50 μL of supernatant from each well to a new 96-well plate, add 50 μL of the mixed substrate (CytoTox 96® Non-Radioactive CytotoxicityAssay) to each well, incubate at room temperature in the dark for 30 minutes, add 50 μL of stop solution, and detect the absorbance at 490 nm.

[0082] (4) The formula for calculating CAR-NK kill efficiency is: Kill efficiency % = (Experimental group - Control group) / Maximum release group × 100% The results are as follows Figure 2 As shown, the M-1-3 CAR-NK-92 cells constructed in this invention exhibit cytotoxic activity against MKN-45 tumor cells expressing MSLN. The killing efficiency was approximately 20.38±1.17% at an effector-to-target ratio of 0.625:1, approximately 31.89±0.22% at an effector-to-target ratio of 1.25:1, approximately 49.10±3.49% at an effector-to-target ratio of 2.5:1, and approximately 76.82±1.85% at an effector-to-target ratio of 5:1. These efficiencys were significantly higher than those of the control group NC CAR-NK-92 (14.17±0.21%, 18.61±0.47%, 26.80±0.39%, and 45.78±0.64%), indicating that the M-1-3 CAR-NK-92 cells constructed in this invention possess specific tumor-killing effects.

[0083] Example 7 This embodiment examines the secretion of IL-10, Granzyme B, and IFN-γ from the M-1-3 CAR-NK-92 cytokines constructed in Example 5. (1) Co-culture experiment Collect MKN-45 MSLN Cells were washed once with dilution buffer, centrifuged at 1000 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in 1640 complete culture medium. Cell counts were performed, and the cells were finally diluted to 1×10⁶. 5 A concentration of 100 μL / mL was added to each well of a 96-well plate. Collect NC CAR-NK-92 and M-1-3 CAR-NK-92 cells (effective cells), wash the cells once with dilution buffer, centrifuge at 1000 rpm for 3 min, discard the supernatant, resuspend the cells in 1640 complete medium, count the cells, and finally dilute the cells to 5 × 10⁻⁶. 5 concentration per mL; Add 100 μL of effector cells to the corresponding 96-well plate and co-culture at 37°C and 5% CO2 for 24 h; Centrifuge at 1000 rpm for 5 min, collect the supernatant and detect cytokines.

[0084] (2) Cytokine detection The sample was serially diluted using standard diluent (e.g., 1:2). Take N 1.5mL EP tubes (N = number of samples + number of controls) Add 40 μL of mixed beads to each tube (sample, negative control); and add 50 μL of the corresponding test reagent (sample, control) and 50 μL of PE Detection Reagent to each tube. After shaking to mix thoroughly, incubate at room temperature in the dark for 3 hours.

[0085] Add 1 mL of wash buffer to each tube, gently blow it to mix, and centrifuge at 200 g for 5 minutes.

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

[0087] The samples were analyzed by flow cytometry.

[0088] (3) Results Analysis The secretion results of IL-10, Granzyme B, and IFN-γ factors are as follows: Figure 3 , Figure 4 and Figure 5 As shown, with MKN-45 MSLN After co-culturing the target cells, the IL-10 secreted by M-1-3 CAR-NK-92 cells was approximately 4677.3±101.6 pg / mL, GranzymeB was approximately 181.8±2.6 μg / mL, and IFN-γ was approximately 6189.7±293.2 pg / mL, all of which were significantly higher than those of the control group NC CAR-NK-92 cells (3414.2±40.5 pg / mL, 121.2±2.1 μg / mL, and 2729.1±25.0 pg / mL, respectively).

[0089] In summary, this invention screens and prepares high-affinity anti-mesothelin nanobodies that can efficiently and specifically bind to mesothelin. Using mesothelin as an antigen-binding domain, chimeric antigen receptors and CAR-NK cells are constructed. The resulting CAR-NK-92 cells exhibit significant killing activity and specificity against mesothelin-positive tumor cells and can secrete tumor-killing cytokines. This indicates that the nanobodies of this invention can be effectively applied to immunotherapy and are of great significance for the development of tumor therapeutic drugs.

[0090] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention 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, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3, characterized in that, The amino acid sequence of CDR1 is shown in SEQ ID NO.1; the amino acid sequence of CDR2 is shown in SEQ ID NO.2; and the amino acid sequence of CDR3 is shown in SEQ ID NO.

3.

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

4.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the nanobody as described in 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 according to claim 1 or 2.

5. The chimeric antigen receptor according to claim 4, characterized in that, The signal peptide includes a CD8α signal peptide; and / or, the hinge region includes a CD8α hinge region; and / or, the transmembrane region includes a CD8α transmembrane region; and / or, the signal transduction domain includes an immune receptor tyrosine activation motif.

6. The chimeric antigen receptor according to claim 5, characterized in that, The signal transduction domain further includes a co-stimulatory molecule, which includes 4-1BB.

7. A recombinant expression vector, characterized in that, The recombinant expression vector contains the encoding gene of the chimeric antigen receptor as described in any one of claims 4-6.

8. The recombinant expression vector according to claim 7, characterized in that, The recombinant expression vector is a lentiviral vector, a retroviral vector, or an adeno-associated virus vector.

9. The recombinant expression vector according to claim 7, characterized in that, The recombinant expression vector is a lentiviral vector.

10. A chimeric antigen receptor immune cell, characterized in that, The chimeric antigen receptor immune cell expresses the chimeric antigen receptor as described in any one of claims 4-6, wherein the chimeric antigen receptor immune cell is a T cell, NK cell, or macrophage.

11. The chimeric antigen receptor immune cell according to claim 10, characterized in that, The chimeric antigen receptor immune cells are NK cells.

12. The application of the reagent in the in vitro preparation of activated immune cells, characterized in that, The reagent contains the coding sequence of the chimeric antigen receptor as described in any one of claims 4-6 or the recombinant expression vector as described in any one of claims 7-9.

13. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the chimeric antigen receptor immune cells as described in claim 10 or 11.

14. The use of the nanobody of claim 1 or 2, the nucleic acid molecule of claim 3, the chimeric antigen receptor of any one of claims 4-6, the expression vector of any one of claims 7-9, the chimeric antigen receptor immune cell of claim 10 or 11, or the pharmaceutical composition of claim 13 in the preparation of a tumor therapeutic agent; wherein the tumor is a tumor that highly expresses mesothelin, and the tumor that highly expresses mesothelin is gastric cancer, pancreatic cancer, ovarian cancer, malignant pleural mesothelioma, lung adenocarcinoma, triple-negative breast cancer, or cholangiocarcinoma.

15. The use of the nanobody according to claim 1 or 2 in the preparation of mesothelin detection reagent.

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