A nanobody targeting mesothelin and related products and uses

Abstract

The application discloses a nanobody targeting mesothelin and related products and application, and belongs to the technical field of biological medicine. The nanobody comprises a heavy chain variable region, the heavy chain variable region comprises CDR1, CDR2 and CDR3, 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. The nanobody can be specifically combined with the mesothelin antigen, has high affinity, is used as an antigen combining domain to construct a chimeric antigen receptor and MSLN-VHH-CAR-NK cell, and has 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 nanobody targeting mesothelin 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 improvement is still needed to improve the 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 targeted 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 alternative to 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 risk of cytokine storm, 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 acknowledgement 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 deficiencies of the prior art and provide a nanobody targeting mesothelin 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 nanobody targeting mesothelin, 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 encoding 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 gene encoding the chimeric antigen receptor.

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

[0017] In an embodiment of the present application, the recombinant expression vector is a lentiviral 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, the reagent comprising 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:

[0028] 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. The nanobody 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

[0029] Figure 1 is a Biacore detection affinity curve graph of the anti-mesothelin nanobody;

[0030] Figure 2 is a MSLN CAR-NK-92 effector-target ratio graph detected by lactate dehydrogenase release experiment;

[0031] Figure 3 is a column graph of the interleukin-10 (IL-10) secretion level of CAR-NK-92 cells;

[0032] Figure 4 is a column graph of the interferon-γ (IFN-γ) secretion level of CAR-NK-92 cells. DETAILED DESCRIPTION

[0033] Unless otherwise defined, the terms "comprises", "comprising", "includes", "including", "contains", "containing" or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises, includes or contains a list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process or method.

[0034] The technical solutions of the present application are described below through specific examples. It should be understood that the one or more steps mentioned in the present application do not exclude other methods and steps before and after the combination steps, or other methods and steps can be inserted between these explicitly mentioned steps. It should also be understood that these examples are only used to illustrate the present application and do not limit the scope of the present application. Unless otherwise stated, 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, without substantial technical content changes, can also be considered as the implementation scope of the present application.

[0035] The starting materials and instruments employed in the examples are not particularly limited in their source, and can be purchased on the market or prepared according to conventional methods well known to those skilled in the art.

[0036] As used herein, the terms "single domain antibody", "heavy chain variable region domain of a 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. 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. A single domain antibody is constructed by cloning the variable region of the heavy chain of an antibody after obtaining the antibody naturally lacking the light chain and the heavy chain constant region 1 (CH1) so that the single domain antibody consists of only one heavy chain variable region.

[0037] A "heavy chain antibody" as described herein is an antibody derived from a Camelid or a Chondrichthyan. In contrast to the above-mentioned 4-chain antibodies, a heavy chain antibody lacks a light chain and a heavy chain constant region 1 (CH1) and comprises only two heavy chains consisting of a variable region (VHH) and other constant regions, the variable region being connected to the constant region by a hinge-like structure. Each heavy chain of a Camelid heavy chain antibody comprises one variable region (VHH) and two constant regions (CH2 and CH3), each heavy chain of a Chondrichthyan heavy chain antibody contains one variable region and five constant regions (CH1-CH5). Antigen binding fragments of a heavy chain antibody include VHH and single chain heavy chain antibodies. By fusion with the constant region of a human IgG Fc, a heavy chain antibody can have CH2 and CH3 of a human IgG Fc.

[0038] Binding molecules comprising two or more single domain antibodies are multivalent single domain antibodies; binding molecules comprising two or more single domain antibodies of different specificity are multispecific single domain antibodies. Multivalent single domain antibodies or multispecific single domain antibodies comprise multiple single domain antibodies connected by linkers. The linkers typically consist of 1-15 amino acids selected from G and S.

[0039] In the present context, heavy chain antibodies and antibodies are intended to distinguish between different combinations of antibodies. Due to the similarity in structure, the following description of the structure of an antibody applies to heavy chain antibodies as well, except where indicated otherwise.

[0040] 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.

[0041] The term "variable" refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies, and are used to confer antigen binding specificity and particular antigenic specificity of a particular antibody. However, the variability is not evenly distributed throughout the variable domains of antibodies; 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 region 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 region of the light chain. The more highly conserved portions of variable domains are called the framework regions (FRs). The variable domains of the naturally occurring heavy and light chains each comprise four FR regions, FR1, FR2, FR3, and FR4, mostly from beta-sheet formation, connected by three HVRs. 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. Generally, the structure of a light chain variable region is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, and the structure of a heavy chain variable region is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. The constant domains do not participate 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.

[0042] The "Fc region" (fragment, crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of the heavy chain of an antibody that mediates the binding of the immunoglobulin to host tissues or factors including binding to Fc receptors located 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 to stretch from an amino-terminus at heavy chain position C226 or P230 to the carboxy-terminus of the heavy chain, with numbering 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.

[0043] "Antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding and / or variable region of the intact antibody. Antibody fragments preferably are antigen binding fragments of antibodies. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; scFv-Fc fragments; multispecific antibodies formed from antibody fragments; and any fragments capable of increasing the half-life of the antibody by chemical modification or by incorporation into a liposome. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, which contains the antibody's epitope recognition site for its Fc receptors. Fab fragments consist 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 that 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 functions of antibodies are determined in the Fc region, which is also the region recognized by Fc receptors (FcR) found on certain types of cells. Antigen binding fragments of heavy chain antibodies include VHH and single chain heavy chain antibodies.

[0044] "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 in tight, non-covalent association. From the folding of these two domains emanate the three pairs of HVRs (H1, H2, and H3 on the heavy chain, and L1, L2, and L3 on the light chain) that contribute the amino acid residues for the antigen binding site. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. "Single-chain Fv" also is abbreviated "sFv" or "scFv" and is a antibody fragment comprising the VHand VLdomains of antibody, linked by a polypeptide linker as a single polypeptide chain. Preferably, the sFv polypeptide further comprises 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.

[0045] Antibodies herein also include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical with or homologous to a corresponding sequence in a 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 a corresponding sequence 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.

[0046] Herein, a chimeric antigen receptor (CAR) contains an optional signal peptide sequence, a mesothelin binding molecule containing a mesothelin binding single domain antibody sequence, a hinge region, a transmembrane region, an intracellular co-stimulatory domain, and an intracellular signaling domain.

[0047] Herein, an "binding molecule" of an antigen 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.

[0048] 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.

[0049] 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 co-stimulatory receptor on T cells and other immune cells, belonging to the TNRSF protein family.

[0050] "Kd" described herein, i.e., Koff, represents the dissociation rate constant, representing the speed of dissociation between molecules, with the unit of S -1 In Biacore assay affinity experiments, the larger the Koff represents the slower the rate of RU drop, and the flatter the curve slope. Therefore, high affinity is represented by fast association and slow dissociation.

[0051] "Ka" or Kon, as used herein, is the association rate constant, which represents the speed of intermolecular association, and the unit is M -1 ·S -1 In the Biacore assay, the greater the Kon, the shorter the time to reach the maximum RU, and the steeper the curve slope.

[0052] "KD" as used herein represents the dissociation constant, which is a specific type of equilibrium constant that measures the tendency of a larger object to separate from another smaller component, and is the reciprocal of the association constant, with the unit of mol / L (M) or nmol / L (nM). The smaller the KD value, the stronger the binding ability of the two substances. KD = Kd / Ka.

[0053] Example 1

[0054] This example constructs a phage nanobody library and uses ELISA for preliminary screening, and the specific steps are as follows:

[0055] (1) Construction of phage nanobody library

[0056] After immunizing the alpaca 5 times with the mature mesothelin recombinant protein (Human MSLN / Mesothelin Protein, Kaikang Biological), and detecting the serum titer by ELISA, peripheral blood is extracted to separate lymphocytes, total RNA is extracted, and then cDNA is reverse transcribed, and then the VHH gene is amplified by nested PCR. The steps of obtaining IgG2 and IgG3 heavy chain variable region sequences (heavy chain variable region VHH) by nested PCR are as follows: 1) design a pair of specific nested outer primers, and use cDNA as a template for first-round PCR amplification, and the amplification region is the alpaca heavy chain antibody gene, and the product size is 750 bp and 900 bp, respectively; through DNA gel electrophoresis, the 750 bp PCR product is recovered by cutting the gel; 2) design a nested inner primer, and use the 750 bp first-round PCR product as a template for second-round PCR amplification, and the amplification product is the heavy chain antibody variable region VHH fragment, and the product size is 500 bp; the second-round PCR product is purified and recovered using a PCR product purification kit. The VHH target gene and the vector pComb3xss are digested with SfiI enzyme, and the digested VHH and pComb3xss are ligated with T4 DNA ligase, and then the VHH target gene is cloned into the pHEN1 phagemid, and then it is electroporated into TG1 competent cells to construct a VHH gene library.

[0057] (2) Screening of phage nanobody library

[0058] The library screening was performed by phage display technology, and the specificity and binding strength of each clone were ensured by precise monoclonal phage ELISA, to obtain phages capable of specifically binding to MSLN protein. Sequencing was performed to obtain several sequences of mesothelin polypeptide nanobodies, and finally the amino acid sequence of the nanobody with high affinity to MSLN was verified, such as SEQ ID NO. 4 (QVQLVESGGGMVQPGGSLRLSCAASGFNLDYYGVGWFRQAPGKEREGISCIRGSGEITDYLDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATRQSLGYCSDYVNWYKYWGQGTQVTVSSAHHSEDPHGQAGQ), and the screening number was M-1-40. 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 shown as SEQ ID NO. 1 (YYGVG); the amino acid sequence of CDR2 is shown as SEQ ID NO. 2 (CIRGSGEITDYLDSVKG); and the amino acid sequence of CDR3 is shown as SEQ ID NO. 3 (RQSLGYCSDYVNWYKY).

[0059] Example 2

[0060] In this embodiment, the anti-mesothelin nanobody (M-1-40) screened in Example 1 was subjected to prokaryotic expression and purification, and the antibody affinity was determined. The specific steps are as follows:

[0061] (1) Construct a prokaryotic expression vector of M-1-40 VHH sequence with His tag.

[0062] M-1-40 protein coding gene sequence (SEQ ID NO. 5: CATATGCAGGTTCAGCTGGTTGAAAGCGGTGGTGGCATGGTGCAGCCGGGTGGTAGCCTGCGCCTGAGCTGTGCAGCAAGCGGCTTTAATCTGGATTATTATGGTGTGGGCTGGTTTCGCCAGGCCCCGGGTAAAGAACGTGAAGGCATTAGTTGCATTCGTGGCAGTGGCGAAATTACCGATTATCTGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGTGATAATGCCAAAAATACCGTTTATCTGCAGATGAATAGCCTGAAACCGGAAGATACCGCCGTGTATTATTGCGCCACCCGCCAGAGTCTGGGCTATTGCAGCGATTATGTGAATTGGTATAAATATTGGGGTCAGGGTACACAGGTGACCGTGAGTAGTGCACATCATAGCGAAGATCCGCATGGTCAGGCCGGCCAG CTCGAG ) by base synthesis, recombined into Nde I and Xho I endonuclease linearized pET-22b vector, and transformed into DH5a competent cells.

[0063] Single colonies were picked from the transformation plate and cultured overnight at 37°C with shaking. The PCR sample loading system is shown in Table 1, and the PCR conditions were as follows: 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 4°C storage. The PCR product band size was detected by agarose gel electrophoresis, and the single colony with the expected band size was sent to a sequencing company for sequencing identification. The single colony with correct sequencing results was preserved.

[0064] Table 1: PCR sample loading system

[0065]

[0066]

[0067] (2) M-1-40 VHH prokaryotic expression and purification

[0068] Transformation: The sequencing correct plasmid was extracted and added to E. coli competent BL21(DE3), ice bath for 30 min, 42℃ heat shock for 60 s, continue ice bath for 3 min, add 500 μl LB medium 37℃ incubate for 30 min, spread on the plate containing 100 μg / ml ampicillin, 37℃ culture overnight.

[0069] PCR identification: Single colony was picked from the transformation plate, 37℃ shaking culture overnight, PCR sample addition system and reaction conditions were as previously described, single colony which PCR product band size was consistent with the expected was saved after agarose gel electrophoresis detection.

[0070] Induced expression: BL21(DE3) containing M-1-40VHH plasmid was cultured at 37℃, when OD value reached 0.6, IPTG was added to a final concentration of 1 mM, 37℃ continue shaking culture overnight, induced fusion protein expression, after expression, 4000 rpm centrifugal 10 min, discard supernatant, collect bacterial cells.

[0071] Protein purification:

[0072] Bacterial lysis: bacterial cells were dissolved with binding buffer(0.5 mol / L NaCl, 5 mmol / L imidazole, 20 mmol / L Tris-Cl, pH 8.0), ultrasonic broken, 4℃, 10000 rpm centrifugal 10 min to collect supernatant crude protein;

[0073] Equilibrium: take Ni-NTA filler to fill column, wash column with binding buffer;

[0074] Binding: crude protein was naturally flowed through the column filler after equilibrium, collect effluent;

[0075] Equilibrium: Ni-NTA column was washed with 10 times column volume of binding buffer;

[0076] Washing: Ni-NTA column was washed with 6 times column volume of washing buffer(0.5 mol / L NaCl, 60 mmol / L imidazole, 20 mmol / L Tris-Cl, pH 8.0), collect effluent;

[0077] Elution: eluted with elution buffer(0.5 mol / L NaCl, 250 mmol / L imidazole, 20 mmol / L Tris-Cl, pH 8.0), collect effluent.

[0078] Polyacrylamide gel electrophoresis detection: polyacrylamide gel was configured, sample was prepared for crude protein and effluent components respectively, and polyacrylamide gel electrophoresis detection was performed. The results showed that the fusion protein was purified, SDS-PAGE electrophoresis analysis showed obvious bands near the theoretical molecular weight, which could be preliminarily judged that the fusion protein was successfully purified.

[0079] Dialysis: The purified components were dialyzed into protein storage buffer (PBS, 300 mM NaCl, 10% Glycerol, 0.3% SKL, pH 7.4), concentrated, filtered to remove bacteria, and the protein concentration was quantified using a protein quantification kit, the antibody purity was determined by SEC-HPLC, and the protein was stored at -80°C.

[0080] In addition, the affinity of the purified VHH antibody was determined by Biacore T200 in this example. Biacore is a biological analysis sensing technology developed based on surface plasmon resonance (SPR), which can detect and track the entire process of the binding and dissociation of molecules in the solution with molecules fixed on the chip surface, record it in the form of a sensing graph, and provide kinetic and affinity data.

[0081] During the determination process, the M-1-40 VHH antibody was immobilized on the chip surface, and the mobile phase was a solution containing different concentrations of mesothelin protein. The determination results are shown in Figure 1 The affinity of the antibody reached the sub-nanomolar level, with KD = 4.84E-10 M, Ka = 2.28E+05 M -1 ·S -1 , Kd = 1.10E-04 S -1 .

[0082] Example 3

[0083] In this example, a lentiviral vector expressing a chimeric antigen receptor targeting MSLN (MSLN CAR) was prepared.

[0084] First, a lentiviral vector pGV852 MSLN CAR carrying the MSLN CAR chimeric antigen receptor was constructed, including a CD8α signal peptide, an anti-MSLN VHH, a CD8α hinge region, a transmembrane region, and an immunoreceptor tyrosine activation motif (CD3ζ).

[0085] The amino acid sequence of the signal peptide (SEQ ID NO. 6) is: MALPVTALLLPLALLLHAARP.

[0086] The amino acid sequence of the anti-MSLN VHH is shown in SEQ ID NO. 4.

[0087] The amino acid sequence of the CD8α hinge region and the transmembrane region (SEQ ID NO. 7) is:

[0088] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC.

[0089] 4-1BB intracellular region amino acid sequence (SEQ ID NO. 8) is:

[0090] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0091] CD3ζ amino acid sequence (SEQ ID NO. 9) is:

[0092] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0093] The specific preparation method is as follows:

[0094]

[0095] Table 2: PCR loading system

[0096]

[0097]

[0098] The single clone was picked on the plate and cultured at 37℃ overnight. The PCR loading system is shown in Table 3, and the primers used are as follows: P1: ATGGCCTTACCAGTGACCGC; P2: TTACTGCAGAATTCACGCGT

[0099] Table 3: PCR loading system

[0100]

[0101] 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 PCR product band size of the single clone meeting the expectation is detected by agarose gel electrophoresis, and the single clone pGV852 MSLN-CAR plasmid with correct sequencing result is extracted.

[0102] Example 4

[0103] In this example, the lentivirus packaging, concentration and titer detection of the lentivirus vector pGV852 MSLN-CAR prepared in Example 3 are carried out, including the following steps:

[0104] (1) Lentivirus packaging

[0105] Forty-eight hours before transfection, the logarithmically growing 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%, the cells were used for transfection;

[0106] One hour before transfection, the medium was replaced with a medium containing 2% serum;

[0107] The prepared DNA solutions (pGV852 MSLN-CAR plasmid 20 μg, pHelper1.0 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, and then incubated at room temperature for 15 min;

[0108] The transfection system was slowly added into the culture solution of HEK293T cells, mixed, and cultured at 37°C in a cell incubator with 5% CO2. After 6-8 hours, the culture medium containing the transfection system mixture was discarded, and 10 mL of PBS solution was added for cleaning once.

[0109] Slowly add 12 mL of cell culture medium containing 2% serum, and culture at 37°C in a 5% CO2 incubator for 48 hours. Collect the virus supernatant.

[0110] (2) Lentivirus concentration

[0111] Collect the supernatant of HEK293T cells after transfection for 48 hours, centrifuge at 4°C and 4000g for 10 minutes, and remove the cell debris and impurities.

[0112] Filter the supernatant with a 0.45 μm filter into a 40 mL ultracentrifuge tube, centrifuge at 25000 rpm and 4°C for 2 hours, discard the supernatant, and resuspend the virus precipitate with 1640 culture medium (containing 10% FBS).

[0113] After complete dissolution, centrifuge at high speed for 10000 rpm for 5 minutes, take the supernatant and divide it into 50 μL per part, and store it in the product tube at 80°C.

[0114] (3) Lentivirus titer detection

[0115] Spread HEK293T adherent cells in a 24-well plate, 1.5 x 10 5 cells per well, and the volume is 100 μL;

[0116] Prepare 4 sterile EP tubes, and add 90 μL of serum-free culture medium to each tube;

[0117] Take 11 μL of the virus stock solution to be tested and add it to the first tube, mix well, take 10 μL and add it to the second tube, and continue the same operation until the last tube;

[0118] Select the required cell wells, add all the diluted virus solutions, and place them in a 37°C incubator with a 5% CO2 concentration;

[0119] After 24 hours, aspirate the supernatant in the cell wells, and add 2000 μL of complete culture medium;

[0120] After 72 hours of infection, observe the fluorescence expression.

[0121] The titer calculation formula is as follows: titer (TU / mL) = total number of cells x fluorescence cell proportion / infection volume (mL).

[0122] Example 5

[0123] This embodiment uses lentivirus prepared in Example 4 to transduce NK cells, including the following steps:

[0124] Coat the culture plate with Retronectin (20 μg / mL) to enhance virus adsorption, use the infection solution to prepare a suspension of NK-92 cells at 3-5 x 104 / mL, add 4 mL per well of a 6-well plate to achieve a plating amount of about 15-30%;

[0125] Add lentivirus at a multiplicity of infection (MOI) of 300, and add polybrene to a final concentration of 5 μg / mL;

[0126] After 16 h, collect the cells in each well into a clean 1.5 ml EP tube, centrifuge at 2000 rpm for 2 min, remove the supernatant, replace it with complete culture medium, mix gently and then return it to the culture plate for continued culture;

[0127] After 72 h, use flow cytometry to detect the chimeric antigen receptor expression rate of NK-92 cells, and the infection efficiency of NK-92 cells is 48.1%, indicating that the MSLN CAR NK-92 cells are successfully constructed.

[0128] Example 6

[0129] This embodiment performs an in vitro cytotoxicity experiment on the MSLN CAR-NK-92 cells constructed in Example 5, including the following steps:

[0130] (1) Target cell inoculation

[0131] MKN-45 cells were used as target cells, and the target cell concentration was adjusted to 1 x 10 5 / mL, and 100 μL was inoculated into a white 96-well plate, i.e., 1 x 10 4 cells.

[0132] (2) Effect cell inoculation

[0133] MSLN CAR-NK-92 and control NK 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, and 10:1, respectively. The corresponding cells (three replicate 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.

[0134] (3) Detection method

[0135] 45 minutes before the experimental endpoint, add 20 μL of 10xlysis to the target cell maximum release group; 10 minutes before the endpoint, place the 96-well plate on a 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, and add 50 μL of the mixed substrate (CytoTox) to each well. Non-Radioactive Cytotoxicity Assay: Incubate at room temperature in the dark for 30 min, add 50 μL of stop solution, and detect the light absorbance at 490 nm.

[0136] (4) The formula for calculating CAR-NK kill efficiency is:

[0137] Kill efficiency % = (Experimental group - Control group) / Maximum release group × 100%

[0138] The results are as follows Figure 2 As shown, the M-1-40CAR-NK-92 cells constructed in this invention exhibit cytotoxic activity against MKN-45 tumor cells expressing MSLN. The killing efficiency was approximately 10.06±0.13% at an effector-to-target ratio of 0.625:1, approximately 17.16±0.60% at an effector-to-target ratio of 1.25:1, approximately 31.56±1.42% at an effector-to-target ratio of 2.5:1, and approximately 67.56±2.38% at an effector-to-target ratio of 5:1. These efficiencys were significantly higher than those of the control group NC CAR-NK-92 (4.55±1.75%, 10.45±0.96%, 22.04±1.06%, and 46.85±1.43%, respectively), indicating that the MSLN CAR-NK-92 cells constructed in this invention possess specific tumor-killing effects.

[0139] Example 7

[0140] This embodiment examines the secretion of cytokines IL-10, TNF-α, and IFN-γ from the M-1-40CAR-NK-92 cytokine constructed in Example 5.

[0141] (1) Co-culture experiment

[0142] Collect MKN-45 cells, wash them once with dilution buffer, centrifuge at 1000 rpm for 3 min, discard the supernatant, resuspend the cells in 1640 complete culture medium, count the cells, and finally dilute the cells to 1×10⁻⁶. 5 A concentration of 100 μL / mL was added to each well of a 96-well plate.

[0143] Collect NC CAR-NK-92, SCFV(X)CAR-NK-92, SCFV(Y)CAR-NK-92, and M-1-40CAR-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 them, and finally dilute the cells to 5 × 10⁻⁶. 5 concentration per mL;

[0144] Add 100 μL of effector cells to the corresponding 96-well plate and co-culture at 37°C and 5% CO2 for 24 h;

[0145] Centrifuge at 1000 rpm for 5 min, collect the supernatant and detect cytokines.

[0146] (2) Cytokine detection

[0147] The sample was serially diluted using standard diluent (e.g., 1:2).

[0148] Take N 1.5mL EP tubes (N = number of samples + number of controls)

[0149] 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.

[0150] Add 1 mL of wash buffer to each tube, gently mix, and centrifuge at 200 g for 5 minutes.

[0151] Carefully aspirate the supernatant and add 200 μL of wash buffer to each tube, then resuspend the precipitate.

[0152] The samples were analyzed by flow cytometry.

[0153] (3) Results Analysis

[0154] The secretion results of IL-10 and IFN-γ factors are as follows: Figure 3 , Figure 4 As shown, after co-culturing with MKN-45 target cells expressing MSLN, the IL-10 secreted by M-1-40CAR-NK-92 cells was approximately 2951.3±35.0 pg / mL, and the IFN-γ was approximately 2694.3±97.6 pg / mL, both significantly higher than those of the control group NC CAR-NK-92 (2554.5±10.1 pg / mL and 2304.2±42.8 pg / mL, respectively).

[0155] In summary, the nanobodies with high affinity to mesothelin are screened and prepared, can be efficiently and specifically combined with mesothelin, 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 tumor cells positive to mesothelin, and can secrete cytokines for killing tumor, indicating that the nanobodies can be effectively applied to immunotherapy, and have important significance for developing tumor treatment drugs.

[0156] 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 are chosen and described in order to explain the principles of the application and its practical application to thereby enable others skilled in the art to best utilize the application and various embodiments with various modifications as are suited to the particular use contemplated. It is intended that the scope of the application be defined by the claims and their equivalents.

Claims

1. A nanobody targeting mesothelin, comprising a heavy chain variable region, said heavy chain variable region comprising 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 is a 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 cells express the chimeric antigen receptor as described in any one of claims 4-6.

11. The chimeric antigen receptor immune cell according to claim 10, characterized in that, The chimeric antigen receptor immune cells are T cells, B cells, NK cells, mast cells, or macrophages.

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

13. 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.

14. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises chimeric antigen receptor immune cells as described in any one of claims 10-12.

15. 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 any one of claims 10-12, or the pharmaceutical composition of claim 14 in the preparation of a tumor therapeutic drug, wherein the tumor is a tumor that highly expresses mesothelin, and the tumor that highly expresses mesothelin is gastric cancer, pancreatic cancer, ovarian cancer, epithelioid mesothelioma, lung adenocarcinoma, triple-negative breast cancer, cholangiocarcinoma, endometrioid adenocarcinoma, or acute myeloid leukemia.

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

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