MSLN specific chimeric antigen receptor, CAR-T cell and application of MSLN specific chimeric antigen receptor

Abstract

The invention relates to the technical field of biology and medicine, in particular to an MSLN specific chimeric antigen receptor, a CAR-T cell and application of the MSLN specific chimeric antigen receptor. The MSLN specific chimeric antigen receptor comprises an MSLN antigen binding structural domain, a transmembrane structural domain and an intracellular signal transduction structural domain, wherein the MSLN antigen binding structural domain comprises an amino acid sequence as shown in SEQ ID NO: 1. After lymphocytes are modified by the MSLN specific chimeric antigen receptor disclosed by the invention, the killing ability of the lymphocytes on tumor cells is remarkably enhanced, and particularly, the MSLN specific chimeric antigen receptor has a remarkable directional killing effect on the tumor cells with high expression of MSLN.

Description

Technical Field

[0001] The present invention relates to the field of biology and medical technology, and in particular to an MSLN-specific chimeric antigen receptor, a CAR-T cell and applications thereof. Background Art

[0002] Tumor cell immunotherapy using autologous T cells expressing chimeric antigen receptors (CARs) is a promising strategy, as demonstrated by the use of CAR-T cells targeting CD19 to treat B-cell malignancies. CARs are synthetic receptors consisting of an antigen-binding domain, which is usually a single-chain fragment variable (scFv) from a monoclonal antibody, connected by a hinge or spacer to a transmembrane domain and an intracellular signaling domain from the T-cell receptor complex, which contains CD3 zeta (CD3ζ) and a co-stimulatory signaling domain. CAR-T cells mediate tumor cell killing by binding of the single-chain antibody of CAR-T to specific target antigens on the surface of cancer cells without the need for antigen presentation in the major histocompatibility complex (MHC). After direct binding to specific target antigens, CAR-T cells are activated through the function of the intracellular signaling domain. ScFv, as the antigen recognition domain, initiates and determines the strength of T-cell activation, providing specificity in an MHC-independent manner. Generally speaking, CAR-T cells with high-affinity scFv have stronger anti-tumor activity. Therefore, the preparation of high-affinity scFv is the basis for the construction of CAR-T.

[0003] Mesothelin (MSLN) is a glycoprotein found on the cell surface and in serum. Its gene encodes a 69kDa precursor protein, which is enzymatically hydrolyzed during maturation to retain a membrane-bound protein of about 40kDa at the C-terminus, which is mature mesothelin. After hydrolysis, the N-terminal fragment of about 30kDa, which is megakaryocyte promoting factor (MPF), falls off and is released outside the cell and exists in blood and urine. Under normal circumstances, the expression of MSLN is limited to mesothelial cells (peritoneum, pericardium and pleural cavity) and the expression level is low. It is basically not expressed in other tissues and cells. However, MSLN has been found to be significantly highly expressed in many tumor types, including gastric cancer, mesothelioma, pancreatic cancer, non-small cell lung cancer, lung adenocarcinoma, fallopian tube cancer, head and neck cancer, cervical cancer and ovarian cancer. Studies have shown that the abnormal expression of MSLN has a certain role in promoting the proliferation, invasion, metastasis and other characteristics of tumor cells. The fact that MSLN is not expressed and low in normal cells determines its potential advantage as a specific target for targeted therapy of tumors.

[0004] At present, the research and application of MSLN as a target in cancer treatment is still relatively rare, especially the application in combination with chimeric antigen receptors and whether and how it can be effectively used to more efficiently treat tumors and cancers still needs further development. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides an MSLN-specific chimeric antigen receptor, which comprises an MSLN antigen binding domain, a transmembrane domain and an intracellular signal transduction domain.

[0006] Furthermore, the MSLN antigen binding domain comprises the amino acid sequence shown in SEQ ID NO:1.

[0007] Furthermore, the transmembrane domain has an amino acid sequence as shown in SEQ ID NO:3.

[0008] Furthermore, the intracellular signaling domain comprises a 4-1BB co-stimulatory signal molecule and a human CD3ζ signaling domain, the 4-1BB co-stimulatory signal molecule having an amino acid sequence as shown in SEQ ID NO: 4, and the human CD3ζ signaling domain having an amino acid sequence as shown in SEQ ID NO: 5.

[0009] Furthermore, the MSLN-specific chimeric antigen receptor further comprises a hinge region connecting the MSLN antigen binding domain and the transmembrane domain, and the hinge region has an amino acid sequence as shown in SEQ ID NO:6.

[0010] Furthermore, the MSLN-specific chimeric antigen receptor further comprises a signal peptide at its N-terminus, and the signal peptide has an amino acid sequence as shown in SEQ ID NO:7.

[0011] Furthermore, the MSLN-specific chimeric antigen receptor has an amino acid sequence as shown in SEQ ID NO:8.

[0012] In another aspect, the present invention provides an isolated nucleic acid molecule encoding a MSLN-specific chimeric antigen receptor as described herein.

[0013] In another aspect, the present invention provides an expression vector comprising the isolated nucleic acid molecule as described herein.

[0014] In another aspect, the present invention provides a host cell comprising an expression vector as described herein. Further, the host cell comprises an immune cell, and further, the immune cell comprises a T cell, a B cell, a NK cell, a monocyte, a macrophage or a dendritic cell or any combination thereof.

[0015] In another aspect, the present invention provides the use of the MSLN-specific chimeric antigen receptor, isolated nucleic acid molecule, expression vector, host cell, pharmaceutical composition or kit as described herein in the preparation of a medicament for preventing and / or treating cancers that overexpress MSLN. Further, the cancers that overexpress MSLN include gastric cancer.

[0016] definition

[0017] As used herein, the term "chimeric antigen receptor" or "CAR" generally refers to a group of polypeptides, and in the simplest embodiment, there are usually two kinds, which, when in immune effector cells, provide cells for target cells (usually cancer cells) specificity, and produce intracellular signals. In some embodiments, CAR includes at least one extracellular antigen binding domain (such as VHH, scFv or part thereof), a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as "intracellular signaling domain"), which includes a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule.

[0018] As used herein, the transmembrane domain used in the chimeric antigen receptor is not limited, and it can be any transmembrane domain well known in the art for constructing a chimeric antigen receptor. As an example, the transmembrane domain can have an amino acid sequence as shown in SEQ ID NO: 3. As used herein, the intracellular signaling domain used in the chimeric antigen receptor is not limited, and it can be any intracellular signaling domain well known in the art for constructing a chimeric antigen receptor. As an example, the intracellular signaling domain can include a 4-1BB co-stimulatory signal molecule and a human CD3ζ signaling domain. Further, the 4-1BB co-stimulatory signal molecule can have an amino acid sequence as shown in SEQ ID NO: 4. Further, the human CD3ζ signaling domain can have an amino acid sequence as shown in SEQ ID NO: 5. Further, the chimeric antigen receptor can also include a hinge region connecting the extracellular antigen binding domain and the transmembrane domain. As used herein, the hinge region used in the chimeric antigen receptor is not limited, and it can be any hinge region well known in the art for connecting the extracellular antigen binding domain and the transmembrane domain in the chimeric antigen receptor. As an example, the hinge region may have an amino acid sequence as shown in SEQ ID NO: 6. Further, the chimeric antigen receptor further comprises a signal peptide at its N-terminus. As used herein, the signal peptide used in the chimeric antigen receptor is not limited, and it may be any signal peptide well known in the art for chimeric antigen receptors. As an example, the signal peptide may have an amino acid sequence as shown in SEQ ID NO: 7.

[0019] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. There are three CDRs in an antibody, designated as CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system. For a given antibody, a person skilled in the art will easily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to a person skilled in the art.

[0020] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. The strength or affinity of a specific binding interaction can be measured by the equilibrium dissociation constant (K) of the interaction. D ) indicates. In the present invention, the term "K D ” refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0021] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0022] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or other human cells. The host cell may include a single cell or a cell colony. The host cell may include an immune cell. The immune cell includes a T cell, a B cell, a NK cell, a monocyte, a macrophage or a dendritic cell or any combination thereof.

[0023] The vector can be introduced into the host cell using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0024] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with a subject and an active ingredient, which is well known in the art and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, preservatives.

[0025] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or condition or symptom (e.g., a disease associated with high expression of MSLN) in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical result. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or completely), whether detectable or undetectable. In addition, "treatment" can also refer to extending survival compared to the expected survival (if not receiving treatment).

[0026] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (eg, a human) suffers from a disease associated with high expression of MSLN.

[0027] As used herein, the terms "cancer overexpressing MSLN" and "disease associated with high expression of MSLN" mean that the expression of MSLN in the diseased cells of the subject, such as cancer cells, is higher than the expression of MSLN in the normal healthy cells of the same subject. In some cases, "cancer overexpressing MSLN" and "disease associated with high expression of MSLN" can be used interchangeably. In specific embodiments, for example, cancer overexpressing MSLN can include gastric cancer, ovarian cancer, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the structure of the chimeric antigen receptor (CAR) plasmid targeting MSLN is shown.

[0029] Figure 2 The expression efficiency detection of CAR targeting MSLN on the T cell surface in Example 3 of the present invention is shown.

[0030] Figure 3 The results of the CAR-T cell killing test on cancer cells are shown.

[0031] Figure 4 The anti-tumorigenic activity of MSLN-CAR-T in an ovarian cancer mouse model was demonstrated. DETAILED DESCRIPTION

[0032] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0033] Example 1: Preparation of antibodies that specifically bind to MSLN

[0034] 1.1 Vector construction

[0035] Using the plasmid containing the full-length gene of MSLN as a template, primers were designed to amplify the extracellular domain (ECD) gene of MSLN, and it was connected to the pcDNA3.1-His vector after double digestion with restriction endonucleases HindⅢ and KpnⅠ by homologous recombination. It was transformed into DH5α competent medium, coated with ampicillin-resistant plates, and cultured in a 37℃ incubator overnight. Single clones were picked for sequencing and identification, and the successfully constructed clones were expanded for culture and plasmid extraction.

[0036] 1.2 Expression and purification of MSLN recombinant protein

[0037] The successfully constructed recombinant plasmid pcDNA3.1-MSLN-His containing the MSLN extracellular domain gene was transfected into HEK293T cells. After transient transfection for 8 hours, the medium was replaced with 293freestyle medium and cultured for 5 days. The cell culture supernatant was collected and purified by affinity chromatography using NTA-Ni column to obtain high-purity recombinant protein MSLN-His.

[0038] 1.3 Animal immunization

[0039] 1 mg of purified MSLN-His recombinant protein was emulsified with an equal volume of Freund's complete adjuvant and the Alxa Bactrian camel was immunized subcutaneously in the neck for the first time. Then, 1 mg of the protein was emulsified with incomplete Freund's adjuvant and immunized three times in succession every 2 weeks. Peripheral anticoagulant and coagulated blood were collected on the 7th day after the shock immunization, and the MSLN-His recombinant protein was coated on a 96-well ELISA plate. The antibody titer was detected by indirect ELISA. The antibody titer against MSLN-His recombinant protein in the peripheral blood of the camel was 1:512000, indicating that the immunization effect was good and the subsequent antibody library construction could be carried out.

[0040] 1.4 Construction and panning of VHH phage antibody library

[0041] 1.4.1 Isolation of peripheral blood lymphocytes

[0042] 200 mL of peripheral anticoagulated blood was collected aseptically from the jugular vein of camels, diluted with an equal volume of sterile PBS, and then separated by centrifugation using Ficoll-Paque Plus lymphocyte separation medium (catalog 17144002, Cytiva) and lymphocyte separation tubes to obtain 8×10 8 The total RNA of the lymphocytes can be directly extracted or frozen at -80℃ for later use.

[0043] 1.4.2 VHH gene amplification

[0044] First, total RNA of lymphocytes was extracted according to the instructions (catalog 74134, RNeasy Plus Mini Kit, QIAGEN), and then reverse transcription was performed using a reverse transcription kit (catalog 18080051, SuperScript III First-Strand Synthesis System, Invitrogen) with RNA as a template to obtain cDNA. Then, nested PCR was used to amplify the VHH gene using cDNA as a template.

[0045] 1.4.3 Construction of VHH phage display vector

[0046] The product amplified in 1.4.2 and the phage display vector pMECS were double-digested with Pst I and Not I and recovered, and then ligated with T4 DNA ligase.

[0047] 1.4.4 Electroporation of ligation products into TG1 competent cells and harvesting of phage antibody library

[0048] The ligation product in 1.4.3 was added to the competent E. coli TG1 cells and electroporated into TG1. After electroporation, SOC medium was added immediately and cultured at 37°C, 200 rpm for 1 hour. Then, the culture was spread on LB / AMP-GLU plates. After culture at 37°C for 6 to 8 hours, the bacterial lawn was collected and 1 / 3 volume of 50% glycerol was added to obtain the prepared phage library.

[0049] 1.4.5 Determination of phage library diversity and capacity

[0050] The electroporation product was diluted 10-fold and then spread on LB / Amp-Glu plates. After culturing at 37°C for 12 h, the number of transformants was counted and the library capacity was 3.92×10 9 of phage libraries.

[0051] 1.4.6 Screening of specific nanoantibodies against MSLN protein

[0052] The prepared phage library was used as the antibody source and screened by phage display technology for three rounds. First, the purified MSLN-His recombinant protein (2 μg / mL) was coated on a 96-well ELISA plate, and the plate was blocked with 3% skim milk powder at 37°C for 1 h the next day. 1×10 10 Recombinant phage containing nano-antibodies were incubated at 37℃ for 1h and then washed 5 times with PBST. The phages bound to MSLN-His were eluted with 0.1M glycine (pH 1.5) and neutralized with 1M Tris-HCl (pH 8.0). The eluate was used to infect the host bacteria TG1 again and expanded for 3 rounds of screening. 96 clones were randomly selected from the plates of the third round of screening for expansion. The nano-antibodies that can specifically bind to the MSLN protein were identified by monoclonal ELISA. The results showed that 85 of the 96 clones were positive clones (P / N>3.0, P represents the OD450 value of the MSLN well, and N represents the OD450 value of the control well); the sequencing results of the positive clones were compared and analyzed, and a specific nano-antibody against MSLN was obtained, the gene sequence of which is shown in SEQ ID NO: 2, and the amino acid sequence is shown in SEQ ID NO: 1.

[0053] Example 2: Construction of CAR plasmid targeting MSLN

[0054] The plasmid containing the nanobody gene in 1.4.6 of Example 1 was used as a template to amplify the nanobody gene targeting MSLN and cloned into the lentiviral vector pSLCAR-BBz by homologous recombination to construct a second-generation CAR, which mainly comprises the following elements: CD8α signal peptide, MSLN antigen binding domain, CD8α hinge region, transmembrane domain of human CD8, 4-1BB costimulatory signal molecule and human CD3ζ signaling domain ( Figure 1 ).

[0055] Example 3: Preparation of CAR-T cells targeting MSLN

[0056] Peripheral blood of healthy donors was collected and lymphocytes were separated. CD3 / CD28 magnetic beads were used for stimulation for 48 hours to activate and amplify high-purity T cells. The CAR lentiviral plasmid constructed in the present invention was co-transfected with packaging plasmids psPAX2 and pMD2.G into HEK293T cells for 48 hours, and the culture supernatant was collected and concentrated by ultracentrifugation after filtration with a 0.45 μm filter to obtain the CAR lentivirus. The obtained CAR lentivirus was then used to infect the activated T cells to prepare CAR-T cells.

[0057] The expression efficiency of CAR targeting MSLN on the surface of T cells was detected by flow cytometry. Figure 2 As shown, it showed that the expression efficiency of CAR was 79.67%.

[0058] Example 4: CAR-T cell killing test on gastric cancer cells

[0059] In order to detect the MSLN targeting of MSLN-CAR-T in killing gastric cancer cells, the HGC-27 cell line was infected with MSLN overexpression lentivirus and screened multiple times with puromycin. The screened MSLN overexpression HGC-27 cells were subjected to MSLN flow cytometry detection, and the results showed that the extracellular MSLN expression of HGC-27-OE was significantly increased compared with that of HGC-27 cells (1.59% VS 84.8%). The results showed that the constructed MSLN overexpression HGC-27 cells can be used to verify the tumor killing targeting of the above-mentioned CAR-T cells.

[0060] The anti-tumor effect of CAR-T cells was detected by in vitro co-culture assay. MSLN-overexpressing HGC-27 cells in logarithmic growth phase and SKOV3 tumor cells naturally overexpressing MSLN (2×10 4 / well) were inoculated in a 96-well cell culture plate, and the prepared CAR-T cells were added according to the effector-target ratio (10:1). After co-culture for 24 hours, 3 μL of fluorescein potassium salt (15 mg / mL) was added to each well, mixed and incubated for 5 minutes, and then placed in a multifunctional microplate reader to detect the absorbance at 562 nm. The killing efficiency of CAR-T cells was calculated according to the absorbance values ​​of different treatment groups. The killing efficiency calculation formula was: killing efficiency % = (1-fluorescence value of experimental group / fluorescence value of negative control group) × 100%, where the negative control group was normally grown MSLN-overexpressing HGC-27 cells without the addition of CAR-T cells.

[0061] The results are as follows Figure 3 As shown, the killing efficiencies of CAR-MSLN targeting MSLN and irrelevant control CAR-CD19 targeting CD19 on MSLN-overexpressing HGC-27 cells and SKOV3 tumor cells were 74.20% and 5.06% and 82.34% and 6.21%, respectively, indicating that the CAR-T cells targeting MSLN prepared by the present invention have good specific killing activity against MSLN-positive tumor cells (P<0.0001).

[0062] Example 5: In vivo tumor killing activity of MSLN-CAR-T

[0063] Experimental methods

[0064] Male C57BL6 mice aged 6-8 weeks (purchased from Nanjing Junke Biotechnology Co., Ltd.) were raised in an animal room (room temperature 23±2°C, humidity 50%±10%), and mice were modeled using SKOV3 tumor cells using conventional methods. The appearance of hard nodules the size of rice grains in the armpits was used as the standard for successful modeling.

[0065] C57BL6 ovarian cancer model mice, tumor tissue block 95mm 3 At about 24 hours, the patients were randomly divided into 2 groups (conventional-T and MSLN-CAR-T). 5×10 6 The tumor volumes of tumor-bearing mice were measured on d0, d7, d14, d21, d28, and d35. Figure 4 As shown, it showed that the tumor growth of the MSLN-CAR-T treatment group was significantly inhibited.

[0066] The above contents are merely examples and explanations of the present invention. Those skilled in the art may make various modifications or additions to the described specific implementation schemes or replace them in a similar manner. As long as they do not deviate from the invention or exceed the scope defined by the claims, they shall all fall within the protection scope of the present invention.

Claims

1. A chimeric antigen receptor specific for MSLN, characterized in that: It contains MSLN antigen binding domain, transmembrane domain and intracellular signal transduction domain. Wherein, the MSLN antigen binding domain comprises the amino acid sequence shown in SEQ ID NO:

1.

2. The MSLN-specific chimeric antigen receptor according to claim 1, characterized in that The transmembrane domain has an amino acid sequence as shown in SEQ ID NO:

3.

3. The MSLN-specific chimeric antigen receptor according to claim 1, characterized in that The intracellular signaling domain comprises a 4-1BB co-stimulatory signal molecule and a human CD3ζ signaling domain, wherein the 4-1BB co-stimulatory signal molecule has an amino acid sequence as shown in SEQ ID NO: 4, and the human CD3ζ signaling domain has an amino acid sequence as shown in SEQ ID NO:

5.

4. The MSLN-specific chimeric antigen receptor according to claim 1, characterized in that The chimeric antigen receptor further comprises a hinge region connecting the MSLN antigen binding domain and the transmembrane domain, wherein the hinge region has an amino acid sequence as shown in SEQ ID NO: 6, The chimeric antigen receptor further comprises a signal peptide at its N-terminus, and the signal peptide has an amino acid sequence as shown in SEQ ID NO:

7.

5. The MSLN-specific chimeric antigen receptor according to claim 1, characterized in that The chimeric antigen receptor has an amino acid sequence as shown in SEQ ID NO:

8.

6. An isolated nucleic acid molecule, characterized in that Encoding the MSLN-specific chimeric antigen receptor according to any one of claims 1-5.

7. An expression vector, characterized in that: Comprising the nucleic acid molecule according to claim 6.

8. A host cell, characterized in that Comprising the expression vector according to claim 7.

9. The host cell according to claim 8, characterized in that The host cells include immune cells, The immune cells include one or more of T cells, B cells, NK cells, monocytes, macrophages and dendritic cells.

10. Use of the MSLN-specific chimeric antigen receptor according to any one of claims 1 to 5, the isolated nucleic acid molecule according to claim 6, the expression vector according to claim 7, or the host cell according to claim 8 or 9 in the preparation of a medicament for preventing and / or treating cancers that overexpress MSLN.

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