CN116063530B

CN116063530B - Antibody for resisting mesothelin and application thereof

Info

Publication number: CN116063530B
Application number: CN202211209311.XA
Authority: CN
Inventors: 狄升蒙; 石磊; 陈鑫; 冯冬歌; 余学军
Original assignee: Huadao Shanghai Biopharma Co ltd
Current assignee: Huadao Shanghai Biopharma Co ltd
Priority date: 2021-12-29
Filing date: 2021-12-29
Publication date: 2023-09-29
Anticipated expiration: 2041-12-29
Also published as: CN114276454A; CN114276454B; CN116063531B; CN116063530A; CN116063532B; CN116063531A; CN116063532A; CN116063529B; CN116063529A

Abstract

The application provides an antibody for resisting mesothelin and application thereof. The amino acid sequence of CDR3 of said antibody comprises the sequence shown in SEQ ID NO. 10. The anti-mesothelin antibody can specifically bind to mesothelin antigen, has better affinity, is used as an antigen binding domain to construct chimeric antigen receptor and CAR-T cells, and has obvious killing activity on mesothelin positive tumor cells.

Description

Antibody for resisting mesothelin and application thereof

The application relates to a divisional application of a patent application with the application number of 202111643701.3 (the application date of the original application is 2021, 12 and 29 days), and the application name is an anti-mesothelin nano antibody and application thereof.

Technical Field

The application belongs to the technical field of biological medicines, and particularly relates to a high-affinity antibody for anti-mesothelin and application thereof.

Background

Mesothelin (MSLN) is a cell surface glycoprotein, the cDNA of MSLN contains an open reading frame of 1884bp, and has 17 exons in total, and encodes a precursor protein consisting of 628 amino acids and having a relative molecular mass of about 69kDa, which is glycosylated and cleaved by furin to yield 2 proteins having a relative molecular mass of about 40kDa and 31kDa, respectively, the hydrophobic sequence at the carboxy-terminus being substituted by glycosyl phosphatidylinositol and anchored to the cell membrane, and having a molecular weight of about 40kDa, called mesothelin; the amino-terminal portion is released into the blood or further degraded and has a molecular weight of about 31kDa, known as megakaryocyte-stimulating factor (MPF) (see: chang, K. & Pantan, I.molecular cloning of mesothelin, a differentiation antigen present on mesothelium, mesothesium, and ovarian cancer.proceedings of the National Academy of Sciences of the United States of America 93,136-140, doi:10.1073/pnas.93.1.136 (1996)). Current studies indicate that MSLN is involved in regulating cell survival, proliferation and migration and is able to combat killing of some cytotoxic drugs by cells (Tang, z., qian, M. & Ho, m.the role of mesothelin in tumor progression and targeted therapy.anti-cancer agents in medicinal chemistry 13,276-280, doi:10.2174/1871520611313020014 (2013).).

MSLN is a differentiation antigen that is highly expressed in tumor tissues such as pancreatic cancer, malignant mesothelioma, ovarian cancer, and non-small cell lung cancer (see: baldo, P. & Cecco, S.Amatuximab and novel agents targeting mesothelin for solid turs.Oncotargetsand therapy 10,5337-5353, doi:10.2147/ott.s145105 (2017)). Immunohistochemical staining indicated: MSLN expression was positive in most pancreatic cancer tissues (75% -100%), whereas MSLN expression was negative in most chronic pancreatitis tissues (80% -100%) and almost all normal pancreatic tissues (see: baruch, A.C.et al. Immunochemistry study of the expression of mesothelin in fine-needle aspirationbiopsy specimens of pancreatic adenochemistry, diagnostic cytochemistry 35,143-147, doi:10.1002/dc.20594 (2007); ibrahim, D.A..Abouhashem, N.S. diagnostic value of IMP3 and mesothelin in differentiating pancreatic ductal adenocarcinoma from chronic pancreitis. Pathology, research and practice 212,288-293, doi:10.1016/j.prp.2016.01.007 (2016)). Epithelial malignant pleural mesothelioma (Epithelial malignant intercostal mesothelioma, MPM) ubiquitously expresses mesothelin, whereas sarcomatoid pleural mesothelioma does not express mesothelin, most serous epithelial ovarian cancers and related primary peritoneal cancers express mesothelin.

MSLN also exists in part of normal tissues, using mouse anti-human mesothelin antibody K1 developed by the Pastan group, strong K1 reactivity has been demonstrated in mesothelial cells of the peritoneum, pleura and pericardial cavity, but at lower levels than that achieved in malignant tissues (see: chang K, pai L H, bata J K, et al. Classification of the anti (CAK 1) recognized by monoclonal antibody K1 present on ovarian cancers and normal mesothelium, [ J ]. Cancer Research,1992,52 (1): 181-186.); weak K1 responses were detected in oviduct epithelium, tracheal basal epithelium, and tonsil epithelium; mesothelin can be found in various layers of the cornea, however, K1 response was not detected in most normal tissues such as liver, kidney, spleen, bone marrow, lymph node, thymus, myocardium, tongue, skeletal muscle, skin, cerebral cortex, cerebellum, spinal cord, peripheral nerve, pituitary gland, adrenal gland, salivary gland, breast, thyroid gland, parathyroid gland, testis, prostate, epididymis, cervical epithelium, lung parenchyma, esophagus, small intestinal epithelium, colon epithelium, bladder epithelium, gall bladder epithelium, etc. (see: chang K, pai L H, batra J K, et al, characterization of the anti (CAK 1) recognized by monoclonal antibody K1 present on ovarian cancers and normal mesothelium..J ]. Cancer Research,1992,52 (1): 181-186.).

Current studies indicate that MSLN can be targeted for treatment (see: argani, p., et al, mesothelin is overexpressed in the vast majority of ductal adenocarcinomas of the pancreas: identification of a new Pancreatic Cancer marker by Serial Analysis of Gene Expression (SAGE). Clin Cancer Res,2001.7 (12): 3862-8), pastan and colleagues use a soluble antibody fragment of an anti-mesothelin antibody conjugated to an immunotoxin to treat patients with mesothelin positive tumors, demonstrating that this approach has sufficient safety and certain clinical activity in Pancreatic Cancer treatment (see: hassan R, et al, preclinical evaluation of MORAb-009,a chimeric antibody targeting tumor-associated mesothelin. Cancer immune. 2007;7:20-29;Hassan R,et al.Phase I Study of SS1P,a Recombinant Anti-Mesothelin Immunotoxin Given as a Bolus i.infusions to Patients with Mesothelin-Expressing Mesothelioma, ovarian, and pancric Cancer Res.2007;13 (17): 5144-5149.) in Ovarian Cancer, this treatment strategy produces a slight response according to RECIST criteria and completely alleviates the condition in another patient.

Stroms et al constructed TCR-T cells expressing recognition MSLN, which induced massive death of pancreatic cancer cells in vitro experiments, and significantly prolonged survival in vivo experiments in tumor-bearing mice (see: stroms, I.M. et al T Cells Engineered against a Native Antigen Can Surmount Immunologic and Physical Barriers to Treat Pancreatic Ductal Adenocarpioma. Cancer cell 28,638-652, doi:10.1016/j. Ccell.2015.09.022 (2015)). However, this study was performed only in vivo in mice and no clinical study was performed. In 2015, researchers at the university of pennsylvania have completed an example of a clinical trial (NCT 01897415) of MSLN-targeted CAR-T cells targeted to pancreatic ductal adenocarcinoma patients that controlled tumor growth in the patients, but reduced the therapeutic effect of CAR-T due to graft versus host reactions, resulting in very brief time to control tumor growth (see: deSelm, c.j., tano, z.e., varghese, A.M.) and Adeumbili, p.s.car T-cell therapy for pancreatic caner. Journal of surgical oncology 116,63-74, doi:10.1002/jso.24627 (2017). A clinical trial report was also published by the research team at the university of pennsylvania in 2018, and 6 refractory pancreatic ductal adenocarcinoma metastasis and recurrence patients were evaluated using the meso-CAR-T targeted therapy, and the results showed that 2 patients had stable disease and 4 patients had developed disease progression, and that none of the 6 patients had adverse effects of cytokine release syndrome or neurological symptoms (see: beatty, g.l.et al activity of Mesothelin-Specific Chimeric Antigen Receptor T Cells Against PancreaticCarcinoma Metastases in a Phase 1Trial.Gastroenterology 155,29-32, doi:10.1053/j.gastro.2018.03.029 (2018)).

In conclusion, the mesothelin can be used as a target point of tumor treatment, and the anti-mesothelin antibody with high specificity and affinity is provided, so that the method has important significance in the field of tumor treatment.

Disclosure of Invention

In order to overcome the defects and actual needs of the prior art, the application provides a high-affinity antibody for anti-mesothelin and application thereof. The antibody has high affinity, can be used as an antigen binding domain of a chimeric antigen receptor molecule to prepare the CAR-T cell, and the obtained CAR-T cell has good application prospect in the aspect of tumor treatment.

In order to achieve the above purpose, the application adopts the following technical scheme:

in a first aspect, the present application provides an anti-mesothelin nanobody, the amino acid sequence of CDR1 of the nanobody comprises the sequence shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 or SEQ ID No.4, the amino acid sequence of CDR2 of the nanobody comprises the sequence shown in SEQ ID No.5, SEQ ID No.6 or SEQ ID No.7, and the amino acid sequence of CDR3 of the nanobody comprises the sequence shown in SEQ ID No.8, SEQ ID No.9 or SEQ ID No. 10.

According to the application, an unimmunized alpaca is immunized by using a mesothelin recombinant protein, a phage display nano antibody library is constructed, and the mesothelin antibody is screened according to the phage display nano antibody library, so that a monoclonal antibody capable of specifically binding to the mesothelin antigen is obtained.

SEQ ID NO.1：GFTFRNYG。

SEQ ID NO.2：GFTFSRYG。

SEQ ID NO.3：GRTFSTYS。

SEQ ID NO.4：GFTFRRYG。

SEQ ID NO.5：IFSDGST。

SEQ ID NO.6：ISWIGGST。

SEQ ID NO.7：IYSDGST。

SEQ ID NO.8：ATDPGSGTTTLHRPFGS。

SEQ ID NO.9：AATKSEWRDPDY。

SEQ ID NO.10：ATDPGSGTTTLHRSFGS。

Preferably, the amino acid sequence of CDR1 of the nanobody comprises the sequence shown in SEQ ID NO.1, the amino acid sequence of CDR2 comprises the sequence shown in SEQ ID NO.3, and the amino acid sequence of CDR3 comprises the sequence shown in SEQ ID NO. 8.

Preferably, the amino acid sequence of CDR1 of the nanobody comprises the sequence shown in SEQ ID NO.2, the amino acid sequence of CDR2 comprises the sequence shown in SEQ ID NO.3, and the amino acid sequence of CDR3 comprises the sequence shown in SEQ ID NO. 8.

Preferably, the amino acid sequence of CDR1 of the nanobody comprises the sequence shown in SEQ ID NO.3, the amino acid sequence of CDR2 comprises the sequence shown in SEQ ID NO.6, and the amino acid sequence of CDR3 comprises the sequence shown in SEQ ID NO. 9.

Preferably, the amino acid sequence of CDR1 of the nanobody comprises the sequence shown in SEQ ID NO.2, the amino acid sequence of CDR2 comprises the sequence shown in SEQ ID NO.7, and the amino acid sequence of CDR3 comprises the sequence shown in SEQ ID NO. 10.

Preferably, the amino acid sequence of CDR1 of the nanobody comprises the sequence shown in SEQ ID NO.4, the amino acid sequence of CDR2 comprises the sequence shown in SEQ ID NO.7, and the amino acid sequence of CDR3 comprises the sequence shown in SEQ ID NO. 8.

Preferably, the heavy chain variable region of the nanobody further comprises: the framework region 1 (FR 1) shown in SEQ ID No.11 or SEQ ID No.12, SEQ ID No.13, SEQ ID No.14, SEQ ID No.15, SEQ ID No.16 or SEQ ID No.17, the framework region 2 (FR 2) shown in SEQ ID No.18, SEQ ID No.19, SEQ ID No.20 or SEQ ID No.21, the framework region 3 (FR 3) shown in SEQ ID No.22 and the framework region 4 (FR 4) shown in SEQ ID No. 22.

SEQ ID NO.11：QVQLVESGGGLVQPGGSLRLSCAAS。

SEQ ID NO.12：QVQLVESGGGLVPAGGSLRLSCAAS。

SEQ ID NO.13：MGWARQVPGQGLEWVSG。

SEQ ID NO.14：MGWARQVPGKELEWVSG。

SEQ ID NO.15：MGWFRQAPGKEREPVAV。

SEQ ID NO.16：MGWARQVPGKGLEWVSG。

SEQ ID NO.17：MGWARQVSGKGLEWVSG。

SEQ ID NO.18：

YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC。

SEQ ID NO.19：

YYADSVKGRFTISRDNAQNTVYLQMNSLKPEDTAVYYC。

SEQ ID NO.20：

YYADSVKGRFTISRDYAKNTLYLQMNSLKPADTAVYYC。

SEQ ID NO.21：

YYADSVKGRFTISRDNAKNTVYLQMNTLKPEDTAVYYC。

SEQ ID NO.22：WGQGTQVTVSS。

Preferably, the heavy chain variable region of the nanobody comprises the amino acid sequence shown as SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.26 or SEQ ID NO. 27.

SEQ ID NO.23：

QVQLVESGGGLVQPGGSLRLSCAASGFTFRNYGMGWARQVPGQGLEWVSGIFSDGSTYYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDPGSGTTTLHRPFGSWGQGTQVTVSS。

SEQ ID NO.24：

QVQLVESGGGLVQPGGSLRLSCAASGFTFSRYGMGWARQVPGKELEWVSGIFSDGSTYYADSVKGR FTISRDNAQNTVYLQMNSLKPEDTAVYYCATDPGSGTTTLHRPFGSWGQGTQVTVSS。

SEQ ID NO.25：

QVQLVESGGGLVPAGGSLRLSCAASGRTFSTYSMGWFRQAPGKEREPVAVISWIGGSTYYADSVKGR FTISRDYAKNTLYLQMNSLKPADTAVYYCAATKSEWRDPDYWGQGTQVTVSS。

SEQ ID NO.26：

QVQLVESGGGLVQPGGSLRLSCAASGFTFSRYGMGWARQVPGKGLEWVSGIYSDGSTYYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDPGSGTTTLHRSFGSWGQGTQVTVSS。

SEQ ID NO.27：

QVQLVESGGGLVQPGGSLRLSCAASGFTFRRYGMGWARQVSGKGLEWVSGIYSDGSTYYADSVKG RFTISRDNAKNTVYLQMNTLKPEDTAVYYCATDPGSGTTTLHRPFGSWGQGTQVTVSS。

According to the application, a phage display technology is utilized to screen a mesothelin immune camel VHH library, and the obtained nano antibody has high affinity and important application prospect in the aspect of constructing a chimeric antigen receptor of targeting mesothelin.

In a second aspect, the application provides a nucleic acid molecule comprising a gene encoding the anti-mesothelin nanobody of the first aspect.

In a third aspect, the application provides a chimeric antigen receptor comprising a signal peptide, an antigen binding domain comprising a nanobody of anti-mesothelin of the first aspect, a hinge region, a transmembrane region and a signal transduction domain.

Preferably, the signal peptide comprises a CD8 a signal peptide.

Preferably, the hinge region comprises a CD8 a hinge region.

Preferably, the transmembrane region comprises any one or a combination of at least two of a CD8 a transmembrane region, a CD28 transmembrane region or a DAP10 transmembrane region.

Preferably, the signal transduction domain comprises an immunoreceptor tyrosine activation motif (cd3ζ).

Preferably, the signal transduction domain further comprises a co-stimulatory molecule comprising any one or a combination of at least two of 4-1BB, the intracellular domain of CD28, OX40, ICOS or the intracellular domain of DAP 10.

Preferably, the chimeric antigen receptor comprises a CD8 a signal peptide, a nanobody against mesothelin according to the first aspect, a CD8 a hinge region, a CD8 a transmembrane region and an immunoreceptor tyrosine activation motif.

In a fourth aspect, the present application provides an expression vector comprising a gene encoding the chimeric antigen receptor of the third aspect.

Preferably, the expression vector is any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector comprising a gene encoding the chimeric antigen receptor according to the third aspect, preferably a lentiviral vector.

In a fifth aspect, the present application provides a recombinant lentivirus comprising the expression vector of the fourth aspect.

Preferably, the recombinant lentivirus is prepared from mammalian cells transfected with the expression vector and helper plasmid of the fourth aspect.

In a sixth aspect, the present application provides a chimeric antigen receptor immune cell expressing the chimeric antigen receptor of the third aspect.

Preferably, the chimeric antigen receptor immune cell comprises the recombinant lentivirus of the expression vector and/or the substrate aspect of the fourth aspect.

Preferably, the chimeric antigen receptor immune cells comprise any one or a combination of at least two of T cells, B cells, NK cells, mast cells or macrophages.

In a seventh aspect, the application provides a pharmaceutical composition comprising the chimeric antigen receptor immune cell of the sixth aspect.

Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

Preferably, the auxiliary materials comprise any one or a combination of at least two of carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, bacteriostats or buffers.

In an eighth aspect, the application provides the use of the anti-mesothelin nanobody of the first aspect, the nucleic acid molecule of the second aspect, the chimeric antigen receptor of the third aspect, the expression vector of the fourth aspect, the recombinant lentivirus of the fifth aspect, the chimeric antigen receptor immune cell of the sixth aspect or the pharmaceutical composition of the seventh aspect in the preparation of a medicament for treating a tumor.

Preferably, the tumor comprises a mesothelin-expressing tumor.

Compared with the prior art, the application has at least the following beneficial effects:

(1) The application uses the recombinant protein of mesothelin to immunize the non-immunized alpaca to construct phage display nano antibody library, and screens the anti-mesothelin antibody according to the phage display nano antibody library, the obtained nano antibody can specifically bind to the mesothelin antigen and has better affinity, and the KD (M) is respectively 2.83 multiplied by 10 as known by the determination of the antibody affinity ^-8 、1.01×10 ^-8 、1.41×10 ^-8 、6.34×10 ^-8 And 5.82×10 ^-8 ；

(2) The anti-mesothelin nano antibody provided by the application has better affinity, is used as an antigen binding domain to construct a chimeric antigen receptor, and is used for preparing T cells, wherein the CAR-T cells have killing activity on mesothelin positive tumor cells, and can efficiently secrete cytokines IFN-gamma and TNF-alpha after being co-cultured with the mesothelin positive cells.

Drawings

FIG. 1A is a graph showing the affinity of Biacore for detection of anti-mesothelin nanobody VHH-1 in example 2;

FIG. 1B is a graph showing the affinity of Biacore for detection of anti-mesothelin nanobody VHH-3 in example 2;

FIG. 1C is a graph showing the affinity of Biacore for detection of anti-mesothelin nanobody VHH-9 in example 2;

FIG. 1D is a graph showing the affinity of Biacore for detection of anti-mesothelin nanobody VHH-28 in example 2;

FIG. 1E is a graph showing the affinity of Biacore for detection of anti-mesothelin nanobody VHH-29 in example 2;

FIG. 2 is a graph showing the results of FACS detection of the anti-mesothelin nanobody recognizing mesothelin antigen in example 3;

FIG. 3 is a map of a chimeric antigen receptor lentiviral vector targeting mesothelin of example 4;

FIG. 4 is a schematic diagram of the structure of a chimeric antigen receptor expressing mesothelin according to example 4;

FIG. 5 is a graph showing the results of flow assay of the expression rate of chimeric antigen receptor of T lymphocytes in example 6;

FIG. 6A is a FACS detection of T cells and CAR-T cell phenotype (CD 3) in example 6 ⁺ CD4 ⁺ ) The obtained result graph;

FIG. 6B is a FACS detection of T cells and CAR-T cell phenotype (CD 3) in example 6 ⁺ CD8 ⁺ ) The obtained result graph;

FIG. 7 is a graph of the killing effect of CAR-T cells on 293T cells as described in example 7;

FIG. 8 is a graph showing the killing effect of CAR-T cells on pancreatic cancer cells AsPC1-Mesothelin in example 7;

FIG. 9 is a graph showing the killing effect of CAR-T cells on pancreatic cancer cells PANC1-Mesothelin in example 7;

FIG. 10 is a bar graph of TNF alpha secretion by CAR-T cells in example 8;

FIG. 11 is a bar graph of IFNγ secretion by CAR-T cells in example 8.

Detailed Description

The following embodiments are further described with reference to the accompanying drawings, but the following examples are merely simple examples of the present application and do not represent or limit the scope of the application, which is defined by the claims.

The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.

Example 1

In this example, phage nanobody libraries were constructed and panning were performed and primary screening was performed using ELISA, as follows:

(1) Construction of phage nanobody library

Immunizing alpaca with Mesothelin (MSLN) extracellular recombinant protein, ELISA detecting serum titer, and extracting peripheral blood; separating lymphocytes, extracting total RNA, performing reverse transcription to obtain cDNA, and amplifying VHH genes by nested PCR; insertion of VHH Gene into pShort phagemid, electric transformation of SU320+Separating and purifying the phage by PEG8000/NaCI precipitation method after the competent cells are amplified to obtain an antibody library; adjusting concentration, packaging, and freezing in refrigerator at-80deg.C;

(2) Screening of phage nanobody libraries

Firstly, taking 293T cells and an antibody library for co-incubation to carry out negative screening, and then taking supernatant to respectively incubate with 293T-MSLN cells (mesothelin positive) and 293T cells; washing for 4 times by adopting a pre-cooled PT buffer solution at 4 ℃; infecting NEB alpha 5F' cells, adding helper phage, and culturing overnight; coating a plate by a Drop method, and counting the enrichment degree the next day; separating and purifying the phage by PEG8000/NaCI precipitation method, and then carrying out the next round of screening; after enrichment, the VHH region was amplified using the obtained phage as a template, and subjected to second generation sequencing to obtain 5 anti-mesothelin nanobodies, the amino acid sequences of which were respectively designated VHH-1, VHH-3, VHH-9, VHH-28 and VHH-29 as shown in SE1 ID No.22, SE1 ID No.23, SE1 ID No.24, SE1 ID No.25 and SE1 ID No. 26.

Example 2

This example expresses and purifies the anti-mesothelin nanobody (VHH-mIgG 2a Fc nanobody) selected in example 1 and determines the affinity of the antibody. In order to further identify the antibodies obtained by screening, it was necessary to express the antibodies by mammalian cells, and therefore, a plasmid vector carrying a mouse Fc tag-expressing VHH, designated C-4pCP. Stuffer-mCg a-FC, was constructed first, comprising the following steps:

(1) The VHH fragment was amplified using PCR, the reaction system of which is shown in Table 1, and the amplification procedure is shown in Table 2;

TABLE 1

TABLE 2

(2) The enzyme digestion system is shown in Table 3, the enzyme digestion temperature is 37 ℃ and the time is 6 hours, and the carrier after enzyme digestion is usedPurifying by using a PCR purification kit, dissolving the recovered DNA in 45 mu L of water, and detecting the concentration of the DNA;

TABLE 3 Table 3

Reagent(s)	Usage amount
		C-4 pCP.Stuffer-mCg2a-FC	5μg
10 Xenzyme cutting Buffer (10 Xreaction Buffer)	5μL
		FspA I	2μL
PfI 23II	2μL
		ddH ₂ O	Make up to 50 mu L

(3) Connecting the PCR amplification product into an enzyme-digested linearization vector by adopting a homologous recombination mode, wherein the system is shown in a table 4, and the condition is that the water bath is carried out for 30min at 37 ℃;

TABLE 4 Table 4

Reagent(s)	Volume (mu L)
		Exnase II	1
2×Exnase II buffer	2
		Linearization carrier (linearized vector)	4
Insert (Insert fragment)	3

(4) Adding all homologous recombination reaction systems into DH5 alpha competent cells, and transforming DH5 alpha competent cells under the transformation conditions shown in Table 5;

TABLE 5

Procedure	Temperature (temperature)	Time
			Ice bath	0℃	5min
Heat shock	42℃	1min
			Ice bath	0℃	3min
Adding 500 μl of LB medium, and shaking culturing at 220rpm	37℃	1.5h
			mu.L was pipetted and spread on LB/Amp plates	37℃	Overnight

(5) The transformation plate is selected to be pre-identified by monoclonal PCR, and the conditions of a PCR identification system are shown in Table 6; the conditions are that the pre-denaturation is carried out for 3min at 95 ℃; denaturation at 95℃for 30s, annealing at 55℃for 30s, elongation at 72℃for 30s,35 cycles; extending at 72 ℃ for 5min, preserving at 4 ℃, and sending to a sequencing company for sequencing identification, wherein the sequencing result meets the expectations, and successfully constructing a plasmid vector with the mouse Fc tag for expressing VHH in the examples of the specification.

TABLE 6

293E cells were passaged approximately 24h prior to plasmid transfection to a cell density of approximately 0.6X10 ⁶ cells/mL; when the cell density is 1.2X10 ⁶ cell/mL, activity>At 95%, 293E cells were transfected with PEI at a ratio of 150. Mu.g DNA/100mL 293E, wherein the ratio of plasmid DNA to PEI was 1:2;

37℃、130rpm、8％ CO ₂ shaking culture for 6 days, collecting cell culture supernatant at 3000rpm for 30min, filtering the collected supernatant containing target antibody with 0.45 μm filter, and adding sample to purification column MabSelect ^TM SuRe ^TM The column was washed with 5 times PBS, the protein eluted with 0.1M Gly-HCl (pH 3.0) and neutralized with 1/10 volume of Tris-HCl at pH 8.5, followed by dialysis overnight at 4℃and then determination of A with Nanodrop 2000 ₂₈₀ The antibody purity was determined by SEC-HPLC.

In addition, in this example, the affinity of the purified VHH antibody was also determined by Biacore.

Biacore is a bioanalytical sensing technology developed based on surface plasmon resonance (surface Plasmon resonance, SPR) and can detect and track the whole change process of binding and dissociating molecules in a solution and molecules fixed on the surface of a chip, record the whole change process in the form of a sensor map and provide dynamics and affinity data.

In the measurement, the antibodies were immobilized on the chip surface, the mobile phase was a solution containing antigen (mesothelin), and the measurement results are shown in Table 7 and FIGS. 1A to 1E, wherein the affinities of the 5 antibodies reach sub-nanomolar levels of 2.83E-8, 1.01E-8, 1.41E-8, 6.34E-8 and 5.82E-8, respectively.

TABLE 7

Nanobody	Ka(1/Ms)	Kd(1/s)	KD(M)
				VHH-1(SEQ ID NO.23)	5.53E+04	1.56E-03	2.83E-8
VHH-3(SEQ ID NO.24)	6.54E+04	6.6E-04	1.01E-8
				VHH-9(SEQ ID NO.25)	4.95E+04	6.98E-03	1.41E-8
VHH-28(SEQ ID NO.26)	6.83E+04	4.33E-03	6.34E-8
				VHH-29(SEQ ID NO.27)	6.25E+04	3.64E-03	5.82E-8

Example 3

This example describes a flow assay for the anti-mesothelin nanobody of the example.

293T (Mesothelin-), 293T-Mesothelin cells were mixed with purified anti-Mesothelin nanobodies, incubated for 30min in an ice bath, then incubated with APC-labeled goat anti-mouse IgG antibodies for 30min, and examined using a flow cytometer, as shown in FIG. 2, indicating that the anti-Mesothelin nanobodies of the present application recognize Mesothelin antigens on the cell surface.

Example 4

This example prepares lentiviral vectors expressing a chimeric antigen receptor targeting MSLN (MSLN CAR).

First, a lentiviral vector pSIN03 MSLN CAR carrying a chimeric antigen receptor of MSLN CAR was constructed, the vector profile of which is shown in FIG. 3, and the schematic of the chimeric antigen receptor of which is shown in FIG. 4, comprising a CD8 alpha signal peptide, an anti-mesothelin nanobody (anti-MSLN VHH), a CD8 alpha hinge region, a transmembrane region and an immunoreceptor tyrosine activation motif (CD 3 zeta).

Wherein the amino acid sequence of the signal peptide (SEQ ID NO. 28) is:

MALPVTALLLPLALLLHAARP。

the amino acid sequence of the anti-MSLN VHH is shown as SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26 or SEQ ID NO. 27.

The amino acid sequences of the CD 8. Alpha. Hinge region and the transmembrane region (SEQ ID NO. 29) are:

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC。

the amino acid sequence of the intracellular region of 4-1BB (SEQ ID NO. 30) is:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL。

the CD3 zeta amino acid sequence (SEQ ID NO. 31) is:

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

the preparation method comprises the following steps:

(1) A PCR reaction system was prepared according to table 8 (reagents in table were derived from TOYOBO inc.), nanobody fragments of each anti-mesothelin were amplified, and the reaction was performed according to the PCR procedure shown in table 9, with primer sequences:

MS1-F(SEQ ID NO.32)：

tgccgctggccttgctgctccacgccgccaggccgcaggtgcagctggtggag；

MS1-R(SEQ ID NO.33)：cgctggcgtcgtggtgctagacactgtcacctg；

MS9-R(SEQ ID NO.34)：cgctggcgtcgtggtagagctcactgtcacctg。

TABLE 8

Reagent(s)	Volume (mu L)
		10×buffer	5
2mM dNTP	5
		25mM MgSO ₄	3
10μM MF1-F	1
		10 mu M MS1-R or MS9-R	1
Template DNA (cDNA clone)	1
		Sterile deionized water (PCR grade water)	33
KOD-Plus-Neo high-fidelity PCR enzyme	1

TABLE 9

(2) A PCR reaction system was prepared according to Table 10, and the resulting amplified product was preceded by a CD 8. Alpha. Signal peptide, and reacted according to the PCR procedure shown in Table 9, using the primers:

BamH-CD8αsig-F(SEQ ID NO.35)：

GCTGCAGGTCGACTCTAGAGGATCCCGCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGC；

table 10

After the reaction is finished, the PCR product is subjected to 1% agarose gel electrophoresis, the fragment of about 500bp is recovered, and the quantity is fixed by an ultraviolet absorption method;

(3) A PCR reaction system was prepared according to Table 11, and after completion of the preparation, a PCR reaction was performed according to the PCR procedure shown in Table 9 to amplify the CD 8. Alpha. Range-TM-41 BB-CD3Z fragment, using the primers as follows:

CD8αH-F(SEQ ID NO.36)：ACCACGACGCCAGCGCCGCGAC；

Vector-R(SEQ ID NO.37)：TCGATAAGCTTGATATCG；

TABLE 11

Reagent(s)	Volume (mu L)
		10×buffer	5
2mM dNTP	5
		25mM MgSO ₄	3
10 mu M upstream primer CD8 alpha H-F	1
		10 mu M downstream primer Vector-R	1
Template DNA (HD CD19 CAR)	1
		Sterile deionized water (PCR grade water)	33
KOD-Plus-Neo high-fidelity PCR enzyme	1

After the PCR is finished, carrying out 1% agarose gel electrophoresis, recovering fragments about 780bp, and quantifying by an ultraviolet absorption method;

(4) Carrying out BamHI and EcoRI double digestion on 5 μg of HD SIN03 CD19CAR plasmid constructed in a laboratory, and recovering the vector after water bath reaction for 2h at 37 ℃;

the 3 fragments recovered above and the vector backbone were linked by recombinase, the recombination reaction system is shown in Table 12, and after completion of the preparation, the mixture was subjected to a water bath reaction at 37℃for 0.5h, and transformed into E.coli stbl3 competent cells by a conventional method.

Table 12

Reagent(s)	Usage amount
		HD CD19 CAR	184.54ng
CD8αsingal MSLN VHH	31.32ng
		CD8αhinge-TM-41BB-CD3Z	29.72ng
5×CE buffer	2μL
		Exnase ^TM II	1μL
Sterile deionized water (PCR grade water)	Make up to 10 mu L

And selecting a monoclonal from the solid culture medium, culturing overnight, carrying out PCR identification, preparing a PCR reaction system as shown in table 13, carrying out a PCR procedure as shown in table 14, selecting a positive clone after the PCR is finished, and further carrying out sequencing identification, wherein the sequencing result meets the expectations.

TABLE 13

Reagent(s)	Volume (mu L)
		Taq PCR Master Mix	10
10μM F Seq-trEF1a-F	1
		10μM R Vector-R	1
Template DNA bacterial liquid	1
		Sterile deionized water (PCR grade water)	7

TABLE 14

Example 5

In this example, lentiviral packaging, concentration and titer detection were performed on the lentiviral vector HD SIN03-MSLN CAR prepared in example 4, comprising the steps of:

(1) Lentivirus package

At 1.6X10 ⁷ Cell count 293T cells were seeded in 15cm dishes at 37℃with 5% CO ₂ The virus is prepared for packaging after overnight culture, and the culture medium is DMEM containing 10% of fetal calf serum; the lentiviral vector 14.5. Mu.g HD SIN03-MSLN CAR, 16.7. Mu.g helper plasmid pMDLg-RRE, 16.7. Mu.g helper plasmid pRSV-REV and 6.5. Mu.g envelope plasmid VSVg were dissolved in 2mL serum-free DMEM medium and mixed well;

163.2. Mu.g PEI (1. Mu.g/. Mu.L) was dissolved in 2mL serum-free DMEM medium, vortexed at 1000rpm for 5 seconds and incubated at 25℃for 5min; adding PEI mixed solution into the DNA mixed solution, immediately vortex mixing or gently mixing after adding, and incubating for 20min at 25 ℃ to form a transfection complex; then, 4mL of the transfection complex is added into 25mL of DMEM medium containing 293T cells in a dropwise manner, and after 4 hours, the fresh medium is replaced; after 48 hours, collecting the virus liquid supernatant;

(2) Lentivirus concentration

Filtering the virus supernatant with a 0.45 μm filter membrane, collecting the filtered virus supernatant into a 50mL centrifuge tube, adding 1/4 PEG-NaCl virus concentrate, mixing the mixture upside down, and standing the mixture at 4 ℃ overnight; centrifuging at 4 ℃ at 3500rpm for 30min; removing supernatant, adding appropriate amount of RPMI 1640 medium (containing 10% FBS), and dissolving the resuspended virus precipitate; split charging 50 μl of the concentrated lentiviral suspension into each portion, storing in a finished tube, and storing at-80deg.C;

(3) Lentivirus titer detection

500 mu L K562 cells (1X 10) ⁵ Individual cells) were inoculated into 24-well plates, and the concentrated lentiviruses were added to the cell suspension in volumes of 1. Mu.L, 0.2. Mu.L and 0.04. Mu.L, respectively, and polybrene was added to a final concentration of 5. Mu.g/mL, 37℃at 5% CO ₂ After overnight incubation, fresh medium was changed;

after 72h infection, 400 Xg was centrifuged for 5min, the supernatant was discarded to collect cells, 100. Mu.L PBS+2% FBS was added to resuspend the cells, 0.5. Mu.g MSLN-his protein was added, and the cells were incubated on ice for 30min; flow ofAfter washing 1 time with buffer (PBS containing 2% FBS), 100. Mu.L buffer is added to resuspend cells, APC anti-His antibody is added, and incubation is performed on ice for 30min; after 2 times of washing with the flow buffer, 300 mu L of the flow buffer is added to resuspend cells, and a flow cytometer is adopted to detect the infection efficiency; the titer was calculated as follows: titer (TU/mL) =cell number (10 ⁵ ) X positive rate/viral volume (mL).

Example 6

This example uses lentivirus transduced T lymphocytes prepared in example 5, comprising the steps of:

(1) Diluting the anti-human CD3 antibody and the anti-human CD28 antibody with PBS (phosphate buffered saline) to obtain final concentrations of 1 mug/mL and 0.5 mug/mL respectively, coating an orifice plate, and standing at 4 ℃ in a refrigerator overnight; discarding the antibody coating liquid in the pore plate, and washing the plate with 1mL PBS;

(2) Human PBMC were conditioned to a density of 1X 10 with T cell culture medium (X-VIVO+10% FBS+300U/mL IL-2) ⁶ Per mL, inoculated into CD3 and CD28 antibody coated well plates for 24h activation; the activated T cells were collected and the cell density was adjusted to 1X 10 ⁶ Lentivirus was added per mL at a multiplicity of infection (multiplicity of infection, MOI) of 10, polybrene was added to a final concentration of 5 μg/mL; at 37℃with 5% CO ₂ Replacing fresh culture medium after overnight culture in the environment, and carrying out passage every 3 days;

(3) 5 days after T cell infection, 3X 10 cells were taken ⁵ Centrifuging at 4 ℃ for 5min at 400 Xg, discarding the supernatant, and washing once with streaming buffer; adding 100 mu L of buffer solution to resuspend the cells, adding 0.5 mu g of MSLN-his protein, and incubating on ice for 30min; adding buffer solution for cleaning for 1 time, adding 100 mu L of buffer solution for resuspension of cells, adding APC anti-His antibody, and incubating on ice for 30min; after 2 washes with buffer, 300 μl buffer was added to resuspend cells;

the expression rate of chimeric antigen receptor of T lymphocytes was measured by flow cytometry, and as a result, as shown in fig. 5, the infection efficiency of each group of CAR-T cells was: 41.1%, 41.8%, 39.2%, 47.1% and 36.4%, indicating successful construction of CAR-T cells.

In addition, the flow cytometer is used to detect lymphocyte phenotypes in this example, including the steps of:

(1) 5 days after T cell infection, 3X 10 cells were taken ⁵ Centrifuging at 4deg.C and 400×g for 5min, discarding supernatant, and washing with PBS (containing 2% FBS by mass) buffer solution once;

(2) Cells were resuspended in 50. Mu.L of buffer, 1. Mu.L of FITC-labeled Anti-CD3 Ab, percp-Cy5.5 labeled Anti-CD4Ab and PE-Cy7 labeled Anti-CD8 Ab were added and incubated on ice for 30min; after washing twice with buffer, cells were resuspended in 300. Mu.L of buffer and examined for cell phenotype using flow cytometry, as shown in FIGS. 6A and 6B, CD3 ⁺ CD4 ⁺ Cell population was 40% or so, CD3 ⁺ CD8 ⁺ The cell population was about 60%.

Example 7

In this example, an in vitro toxicity test of CAR-T cells was performed, comprising the steps of:

(1) Target cell inoculation

293-GPF-luci (MSLN negative, MSLN) ^- ) AsPC1-MSLN-luci, PANC1-MSLN-luci as target cells, and the concentration of the target cells was adjusted to 2X 10 ⁵ 50. Mu.L of the solution was inoculated into a white 96-well plate;

(2) Effector cell seeding

MSLN CAR-T and control T cells are effector cells, and CAR-T cells and control T cells are added into a 96-well plate according to the effective target ratio of 0.3:1 and 1:1;

(3) Each group was set with 3 duplicate wells and the average of 3 duplicate wells was taken, wherein each experimental group and each control group were as follows:

experimental group: each target cell + CAR-T;

control group: inoculating only target cells;

(4) The detection method comprises the following steps:

after 18h of co-culture of effector cells with target cells, 100. Mu.L of Steady-Reagents (Promega, cat#E2520), after 5min of reaction, the bioluminescence signal was detected using a multifunctional microplate reader;

(5) The CAR-T killing efficiency calculation formula is as follows:

killing efficiency% = (control-experimental)/control x 100%

The results are shown in figures 7-9, and the CAR-T cells constructed by the application have no killing effect on MSLN negative 293T cells and have killing activity on MSLN positive tumor cells, so that the CAR-T cells constructed by the application have high-efficiency tumor killing activity and high specificity.

Example 8

In this example, the secretion of the CAR-T cytokines TNF- α and IFN- γ was detected using the kits Human TNF- α ELISAKIT (co-products, cat# EK 182-96) and Human IFN- γ ELISA Kit (co-products, cat# EK 180-96), respectively.

1. Cell culture supernatant

Cell cultures with an effective target ratio of 1:1 were centrifuged at 400 Xg for 10min to remove sediment, and the supernatant was stored at-80℃for examination.

2. Reagent preparation

All reagents, samples were returned to 25℃before testing, and if crystallization occurred in the concentrated reagents, the incubation was performed at 37℃until the crystals were completely dissolved, and 1 Xof wash solution and 1 Xof assay buffer were prepared according to the instructions of use.

3. Standard substance and sample preparation

Standard substance: standard stock was 2-fold diluted with 5%1640 medium for a total of 8 dilution gradients, including zero concentration.

Sample: samples were diluted in ratio using 5%1640 medium.

4. Detection step

(1) Soaking the ELISA plate: adding 300 mu L of 1 Xwashing liquid, standing and soaking for 30s, discarding the washing liquid, and beating the micro-pore plate on water-absorbing paper;

(2) Adding a standard substance: standard wells were filled with 100 μl of 2-fold diluted standard and blank wells were filled with 100 μl of standard diluent (serum/plasma samples);

(3) Adding a sample: sample wells were added with 100 μl of cell culture supernatant;

(4) Adding a detection antibody: 50. Mu.L of diluted detection antibody (1:100 dilution) was added to each well;

(5) Incubation: sealing plates by using sealing plates, vibrating at 300rpm, and incubating at 25 ℃ for 2 hours;

(6) Washing: liquid was discarded, and 300. Mu.L of wash solution was added to wash the plate 6 times per well;

(7) And (3) enzyme adding and incubation: mu.L of diluted horseradish peroxidase-labeled streptavidin (1:100 dilution) was added to each well;

(8) Incubation: using a new sealing plate membrane sealing plate, oscillating at 300rpm, incubating at 25 ℃ for 45min, and washing;

(9) And (3) color development of the substrate: 100 mu L of chromogenic substrate TMB is added into each hole, and incubated for 20min at 25 ℃ in the dark;

(10) Adding a stop solution: 100 mu L of stop solution is added to each well;

(11) Detecting and reading: within 30min, performing dual-wavelength detection by using an enzyme-labeled instrument, and measuring the OD value at the maximum absorption wavelength of 450nm and the reference wavelength; the OD value after calibration was 450nm minus the measurement at the reference wavelength.

TNF- α, IFN- γ factor secretion results are shown in FIGS. 10 and 11, respectively, wherein spontaneous is a single CAR-T cell group, no cytokines were detected; no cytokines were detected in the CAR-T cell co-cultured group with 293T cells; after co-culture with target cells that overexpress MSLN, the secreted TNF- α by the CAR-T cells exceeds 500pg/mL and IFN- γ exceeds 3000pg/mL. The CAR-T cell constructed by the application releases cytokines to MSLN positive tumor cells, and has no obvious cytokine secretion to MSLN negative cells.

In conclusion, the nano antibody with high affinity for resisting mesothelin can be screened and prepared, can be combined with mesothelin in a high-efficiency manner, is used as an antigen binding domain to construct a chimeric antigen receptor and a CAR-T cell, and the obtained CAR-T cell has obvious killing activity and specificity for mesothelin positive tumor cells and can secrete cytokines for killing tumors, so that the nano antibody can be effectively applied to immunotherapy, and has important significance for developing tumor therapeutic drugs.

The applicant declares that the above is only a specific embodiment of the present application, but the scope of the present application is not limited thereto, and it should be apparent to those skilled in the art that any changes or substitutions that are easily conceivable within the technical scope of the present application disclosed by the present application fall within the scope of the present application and the disclosure.

Claims

1. A nano antibody aiming at anti-mesothelin, which is characterized in that the amino acid sequence of CDR1 of the antibody is shown as SEQ ID NO. 2;

the amino acid sequence of CDR2 of the antibody is shown as SEQ ID NO. 7;

the amino acid sequence of CDR3 of the antibody is shown as SEQ ID NO. 10.

2. A nucleic acid molecule encoding the anti-mesothelin nanobody of claim 1.

3. A chimeric antigen receptor, wherein the chimeric antigen receptor comprises a signal peptide, an antigen binding domain, a hinge region, a transmembrane region, and a signal transduction domain;

the antigen binding domain comprises the nanobody of claim 1 directed against mesothelin.

4. The chimeric antigen receptor according to claim 3, wherein the signal peptide comprises a CD8 a signal peptide.

5. The chimeric antigen receptor according to claim 3, wherein the hinge region comprises a CD8 a hinge region.

6. The chimeric antigen receptor according to claim 3, wherein the transmembrane region comprises any one or a combination of at least two of a CD8 a transmembrane region, a CD28 transmembrane region, or a DAP10 transmembrane region.

7. The chimeric antigen receptor according to claim 3, wherein the signaling domain comprises an immunoreceptor tyrosine activation motif.

8. The chimeric antigen receptor according to claim 3, wherein the signaling domain further comprises a co-stimulatory molecule comprising any one or a combination of at least two of 4-1BB, CD28 intracellular region, OX40, ICOS or DAP10 intracellular region.

9. The chimeric antigen receptor according to claim 3, wherein the chimeric antigen receptor comprises a CD8 a signal peptide, the anti-mesothelin nanobody of claim 1, a CD8 a hinge region, a CD8 a transmembrane region, and an immunoreceptor tyrosine activation motif.

10. An expression vector comprising a gene encoding the chimeric antigen receptor of any one of claims 3-9.

11. The expression vector according to claim 10, wherein the expression vector is any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector comprising a gene encoding the chimeric antigen receptor according to any one of claims 3 to 9.

12. The expression vector of claim 11, wherein the expression vector is a lentiviral vector comprising a gene encoding the chimeric antigen receptor of any one of claims 3-9.

13. A recombinant lentivirus comprising the expression vector of any one of claims 10-12.

14. A chimeric antigen receptor immune cell, characterized in that it expresses the chimeric antigen receptor of any one of claims 3 to 9.

15. The chimeric antigen receptor immune cell according to claim 14, comprising the expression vector of any one of claims 10-12 and/or the recombinant lentivirus of claim 13.

16. The chimeric antigen receptor-immune cell according to claim 14, wherein the chimeric antigen receptor-immune cell comprises any one or a combination of at least two of T cells, B cells, NK cells, mast cells or macrophages.

17. A pharmaceutical composition comprising the chimeric antigen receptor immune cell of any one of claims 14-16.

18. The pharmaceutical composition of claim 17, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant.

19. The pharmaceutical composition of claim 18, wherein the adjuvant comprises any one or a combination of at least two of a carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, co-solvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH regulator, antioxidant, bacteriostat, or buffer.

20. Use of the nanobody against mesothelin according to claim 1, the nucleic acid molecule according to claim 2, the chimeric antigen receptor according to any one of claims 3-9, the expression vector according to any one of claims 10-12, the recombinant lentivirus according to claim 13, the chimeric antigen receptor immune cell according to any one of claims 14-16 or the pharmaceutical composition according to any one of claims 17-19 for the preparation of a medicament for the treatment of a tumor;

the tumor is mesothelin-expressing tumor.