CN116496400A - Anti-c-Met and anti-MSLN antibodies, their dual chimeric antigen receptors and applications thereof - Google Patents

Abstract

The invention relates to the technical field of biology and medicine, in particular to an anti-c-Met and anti-MSLN antibody, a double chimeric antigen receptor thereof and application thereof. The anti-MSLN antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequences set forth in SEQ ID NOs: 1. SEQ ID NO:2 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3, the light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 6. SEQ ID NO:7 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in figure 8. The anti-c-Met antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14. SEQ ID NO:15 and SEQ ID NO:16, HCDR1, HCDR2 and HCDR3, said light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 19. SEQ ID NO:20 and SEQ ID NO:21, LCDR1, LCDR2 and LCDR3. The invention also relates to the use of said antibodies and chimeric antigen receptors that specifically bind to c-Met and anti-MSLN for the treatment of cancer.

Description

Anti-c-Met and anti-MSLN antibodies, their dual chimeric antigen receptors and applications thereof

technical field

The invention relates to the technical fields of biology and medicine, in particular to anti-c-Met and anti-MSLN antibodies, their double chimeric antigen receptors and applications thereof.

Background technique

The use of autologous T cells expressing chimeric antigen receptor (CAR) for tumor cell immunotherapy is a promising strategy, as demonstrated by CAR-T cells targeting CD19 for B-cell malignancies. A CAR is a synthetic receptor consisting of an antigen-binding domain, usually a single-chain fragment variable (scFv) from a monoclonal antibody, connected by a hinge or spacer to a Transmembrane domain and intracellular signaling domain from the T cell receptor complex, which contains CD3 zeta (CD3ζ) and co-stimulatory signaling domains. CAR-T cells mediate the killing of tumor cells through the binding of CAR-T single-chain antibodies to specific target antigens on the surface of cancer cells without presenting antigens in the major histocompatibility complex (MHC). After directly binding to a specific target antigen, CAR-T cells are activated through the function of the intracellular signaling domain. As an antigen recognition domain, scFv initiates and determines the intensity of T cell activation, providing specificity in an MHC-independent manner. In general, 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.

c-Met, also known as hepatocyte growth factor (HGF) receptor, belongs to the family of receptor tyrosine kinases (RTKs). The combination of HGF and c-Met can regulate a variety of downstream signaling pathways to participate in a variety of physiological processes, including cell proliferation and survival, apoptosis and angiogenesis; when the HGF/c-Met signaling pathway is abnormal, it will lead to tumors happened. Therefore c-Met may be a potential target for tumor therapy.

Mesothelin (MSLN) is a glycoprotein found on the cell surface and in serum. Its gene encodes a 69kDa precursor protein, which is enzymatically hydrolyzed into a membrane-bound protein that retains about 40KD at the C-terminal during the maturation process. mature mesothelin. After hydrolysis, about 30KD of the N-terminus is a fragment of megakaryocyte-promoting factor (MPF), which is released from the cells 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, and it is basically not expressed in other tissues and cells. However, MSLN was found to be significantly overexpressed in a variety of tumor types, including 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 plays a certain role in promoting the proliferation, invasion, metastasis and other characteristics of tumor cells. The non-expression and low expression of MSLN in normal cells determines its potential advantage as a specific target for targeted therapy of tumors.

At present, the combined use of c-Met and MSLN as a target in the treatment of pancreatic cancer is still less researched and applied, especially the application of combining chimeric antigen receptors and whether and how it can be effectively applied to treat tumors more efficiently and cancer remain to be further developed.

Contents of the invention

In order to solve the above technical problems, the present invention provides an anti-c-Met and anti-MSLN antibody, a dual chimeric antigen receptor comprising a c-Met antigen-binding domain and an MSLN antigen-binding domain, and applications thereof.

In one aspect, the present invention provides an anti-MSLN antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises SEQ ID NO: 1, HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 2 and SEQ ID NO: 3, the light chain variable region comprises LCDR1 shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively , LCDR2 and LCDR3.

Further, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO:4 and a light chain variable region as shown in SEQ ID NO:9. Further, the antibody or its antigen-binding fragment is a monoclonal antibody, chimeric antibody, humanized antibody, fully human antibody, Fab, Fab', Fv fragment, F(ab') ₂ , scFv or di-scFv . Further, the antibody or antigen-binding fragment thereof is scFv. Further, the antibody or antigen-binding fragment thereof further comprises a linker connecting the heavy chain variable region and the light chain variable region. Further, the linker has an amino acid sequence as shown in SEQ ID NO:11. Further, the antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO:12.

In another aspect, the present invention provides an anti-c-Met antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises SEQ ID NO HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, said light chain variable region comprises respectively shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21 LCDR1, LCDR2, and LCDR3.

Further, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO:17 and a light chain variable region as shown in SEQ ID NO:22. Further, the antibody or its antigen-binding fragment is a monoclonal antibody, chimeric antibody, humanized antibody, fully human antibody, Fab, Fab', Fv fragment, F(ab') ₂ , scFv or di-scFv . Further, the antibody or antigen-binding fragment thereof is scFv. Further, the antibody or antigen-binding fragment thereof further comprises a linker connecting the heavy chain variable region and the light chain variable region. Further, the linker has the amino acid sequence shown in SEQ ID NO:24. Further, the antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO:25.

In another aspect, the present invention provides a chimeric antigen receptor comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, wherein the extracellular antigen binding domain is composed of MSLN antigen Combining domain composition, the MSLN antigen binding domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID HCDR1, HCDR2 and HCDR3 shown in NO: 3, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or wherein The extracellular antigen binding domain consists of a c-Met antigen binding domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises respectively HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, the light chain variable region comprises SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 20 and SEQ ID NO: 16 ID NO: LCDR1, LCDR2 and LCDR3 shown in 21.

Further, the MSLN antigen-binding domain comprises a heavy chain variable region as shown in SEQ ID NO:4 and a light chain variable region as shown in SEQ ID NO:9. Further, the MSLN antigen-binding domain further comprises a linker connecting the heavy chain variable region and the light chain variable region. Further, the linker has an amino acid sequence as shown in SEQ ID NO:11. Further, the MSLN antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO:12.

Further, the c-Met antigen-binding domain comprises a heavy chain variable region as shown in SEQ ID NO: 17 and a light chain variable region as shown in SEQ ID NO: 22. Further, the c-Met antigen-binding domain further comprises a linker connecting the heavy chain variable region and the light chain variable region. Further, the linker has an amino acid sequence as shown in SEQ ID NO:24. Further, the c-Met antigen-binding domain includes the amino acid sequence shown in SEQ ID NO:25.

Further, the transmembrane domain has an amino acid sequence as shown in SEQ ID NO:27. Further, the intracellular signaling domain includes 4-1BB co-stimulatory signal molecule and human CD3ζ signaling domain. Further, the 4-1BB co-stimulatory signal molecule has the amino acid sequence shown in SEQ ID NO:28. Further, the human CD3ζ signaling domain has an amino acid sequence as shown in SEQ ID NO:29. Further, the dual chimeric antigen receptor also includes a hinge region connecting the extracellular antigen binding domain and the transmembrane domain. Further, the hinge region has an amino acid sequence as shown in SEQ ID NO:30. Further, the dual chimeric antigen receptor also includes a signal peptide at its N-terminus. Further, the signal peptide has the amino acid sequence shown in SEQ ID NO:31. Further, the chimeric antigen receptor has an amino acid sequence as shown in SEQ ID NO:33 or SEQ ID NO:34.

In another aspect, the present invention provides a dual chimeric antigen receptor comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain.

Further, the extracellular antigen-binding domain consists of a c-Met antigen-binding domain and an MSLN antigen-binding domain.

Further, the MSLN antigen-binding domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in 3, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively. Further, the MSLN antigen-binding domain comprises a heavy chain variable region as shown in SEQ ID NO:4 and a light chain variable region as shown in SEQ ID NO:9. Further, the MSLN antigen-binding domain further comprises a linker connecting the heavy chain variable region and the light chain variable region. Further, the linker has an amino acid sequence as shown in SEQ ID NO:11. Further, the MSLN antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO:12.

Further, the c-Met antigen binding domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID HCDR1, HCDR2 and HCDR3 shown in NO: 16, the light chain variable region includes LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively. Further, the c-Met antigen-binding domain comprises a heavy chain variable region as shown in SEQ ID NO: 17 and a light chain variable region as shown in SEQ ID NO: 22. Further, the c-Met antigen-binding domain further comprises a linker connecting the heavy chain variable region and the light chain variable region. Further, the linker has the amino acid sequence shown in SEQ ID NO:24. Further, the c-Met antigen-binding domain includes the amino acid sequence shown in SEQ ID NO:25.

Further, the extracellular antigen-binding domain also includes a linker connecting the c-Met antigen-binding domain and the MSLN antigen-binding domain. Further, the linker connecting the c-Met antigen-binding domain and the MSLN antigen-binding domain has the amino acid sequence shown in SEQ ID NO:24. Further, the transmembrane domain has an amino acid sequence as shown in SEQ ID NO:27. Further, the intracellular signaling domain includes 4-1BB co-stimulatory signal molecule and human CD3ζ signaling domain. Further, the 4-1BB co-stimulatory signal molecule has the amino acid sequence shown in SEQ ID NO:28. Further, the human CD3ζ signaling domain has an amino acid sequence as shown in SEQ ID NO:29. Further, the dual chimeric antigen receptor also includes a hinge region connecting the extracellular antigen binding domain and the transmembrane domain. Further, the hinge region has an amino acid sequence as shown in SEQ ID NO:30. Further, the dual chimeric antigen receptor also includes a signal peptide at its N-terminus. Further, the signal peptide has the amino acid sequence shown in SEQ ID NO:31. Further, the dual chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO:35. Further, the dual chimeric antigen receptor also contains the amino acid sequence of hIL21 at its C-terminus. Further, the dual chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO:36.

In another aspect, the invention provides an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof or a dual chimeric antigen receptor as described herein.

In another aspect, the present invention provides an expression vector comprising a nucleic acid molecule as described herein.

In another aspect, the present invention provides a host cell comprising an expression vector as described herein. Further, the host cells include immune cells. Further, the immune cells include T cells, B cells, NK cells, monocytes, macrophages or dendritic cells or any combination thereof.

In another aspect, the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector or host cell as described herein, and optionally a pharmaceutically acceptable carrier and/or or excipients.

In another aspect, the invention provides a kit comprising an antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector or host cell as described herein.

In another aspect, the invention provides an anti-c-Met antibody or antigen-binding fragment thereof, a c-Met chimeric antigen receptor, a nucleic acid molecule encoding the same, an expression vector, a host cell, a pharmaceutical composition or Use of the kit in preparing medicines for preventing and/or treating c-MET overexpressed cancer. Further, the cancer includes pancreatic cancer.

In another aspect, the present invention provides anti-MSLN antibodies or antigen-binding fragments thereof, MSLN chimeric antigen receptors, nucleic acid molecules encoding them, expression vectors, host cells, pharmaceutical compositions or kits as described herein in the preparation Use in a medicament for preventing and/or treating cancers overexpressing MSLN. Further, the cancer includes pancreatic cancer.

In another aspect, the present invention provides anti-c-Met antibodies and anti-MSLN antibodies or antigen-binding fragments thereof, c-Met and MSLN dual chimeric antigen receptors, nucleic acid molecules encoding the same, expression vectors, Use of the host cell, pharmaceutical composition or kit in the preparation of medicines for preventing and/or treating cancers overexpressing c-MET and MSLN. Further, the cancer includes pancreatic cancer.

definition

As used herein, the term "antibody" refers to an immunoglobulin antibody capable of specifically binding to a target (such as carbohydrates, polynucleotides, lipids, polypeptides, etc.) through at least one antigen recognition site located in the variable region of an immunoglobulin molecule. Globulin molecule. As used herein, the term includes not only intact polyclonal or monoclonal antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, but also antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2 , Fv fragments, single chain (e.g. scFv, di-scFv, (scFv)2) and domain antibodies (including e.g. shark and camel antibodies), and fusion proteins comprising antibodies, and any other modification comprising an antigen recognition site Type of immunoglobulin molecules.The antibodies of the present invention are not limited by any particular method of producing antibodies.

As used herein, the term "antigen-binding fragment" includes antigenic compound-binding fragments of these antibodies, including Fab, F(ab') ₂ , Fd, Fv, scFv, antibody minimal recognition units, and combinations of these antibodies and fragments. Single-chain derivatives, such as scFv-Fc, etc., are preferably scFv.

The term "scFv" means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker, which retains the ability to bind antigen. Such scFv molecules may have the general structure: _NH2 -VL-linker-VH-COOH or _NH2 -VH-linker-VL-COOH. As used herein, the linker used in scFv is not limited, it can be any linker well known in the art for linking VH and VL in scFv. As an example, the transmembrane domain can have a sequence such as SEQ ID NO: 11 (eg, as used herein for linking the VH and VL of an anti-MSLN antibody) and SEQ ID NO: 24 (eg, as used herein for linking an anti-c-Met antibody). The amino acid sequences shown in VH and VL).

As used herein, the term "chimeric antigen receptor" or "CAR" generally refers to a group of polypeptides, usually two in the simplest embodiment, which, when in immune effector cells, provide the cell with the Specific for target cells (usually cancer cells) and generate intracellular signals. In some embodiments, the CAR comprises at least one extracellular antigen-binding domain (such as a VHH, scFv, or portion thereof), a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain"). ”) comprising a functional signaling domain derived from a stimulatory molecule and/or a co-stimulatory molecule.

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 chimeric antigen receptors. As an example, the transmembrane domain may have the amino acid sequence shown in SEQ ID NO:27. 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 chimeric antigen receptors. As an example, the intracellular signaling domain can comprise a 4-1BB co-stimulatory signaling molecule and a human CD3ζ signaling domain. Further, the 4-1BB co-stimulatory signal molecule may have an amino acid sequence as shown in SEQ ID NO:28. Further, the human CD3ζ signaling domain may have an amino acid sequence as shown in SEQ ID NO:29. Further, the chimeric antigen receptor may also include a hinge region connecting the extracellular antigen-binding domain and the transmembrane domain. As used herein, the hinge region used in a chimeric antigen receptor is not limited, and it may be a hinge region well known in the art for linking an extracellular antigen-binding domain and a transmembrane structure in a chimeric antigen receptor. any hinge region of the domain. As an example, the hinge region may have the amino acid sequence shown in SEQ ID NO:30. Further, the chimeric antigen receptor also includes a signal peptide at its N-terminal. As used herein, the signal peptide used in chimeric antigen receptors 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 the amino acid sequence shown in SEQ ID NO:31.

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, named CDR1, CDR2 and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, eg as defined in the Kabat numbering system, the Chothia numbering system or the IMGT numbering system. For a given antibody, those skilled in the art will readily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it is directed against. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (KD) for that interaction. In the present invention, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.

In some embodiments, the present invention also provides variants of an anti-c-Met (or anti-MSLN) antibody as described herein, which is identical to the amino acid sequence of an anti-c-Met (or anti-MSLN) antibody as described herein have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity and substantially retain the antibody from which they were derived The biological function (such as the biological activity of specifically binding to c-MET (or MSLN)).

More specifically, the variant differs from an anti-c-Met (or anti-MSLN) antibody as described herein by only one or more (e.g., at most 20, at most 15, at most 10, at most 5 Conservative substitution of one or at most one amino acid) Conservative substitution of amino acid residues.

As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by an adenine, or both a position in each of the polypeptides is occupied by lysine), then the molecules are identical at that position.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the expected properties of the protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions for amino acid residues with amino acid residues that have similar side chains, e.g., are physically or functionally similar (e.g., have similar size, shape, charge, chemical properties, including Substitution of residues with the ability to form covalent or hydrogen bonds, etc.).

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When the vector is capable of achieving expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phage such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, papillomaviruses, Polyoma vacuolar virus (eg 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 an origin of replication.

As used herein, the term "host cell" refers to cells that can be used to introduce vectors, 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. A host cell can include a single cell or a population of cells. Host cells can include immune cells. Immune cells include T cells, B cells, NK cells, monocytes, macrophages or dendritic cells or any combination thereof.

The introduction of vectors into host cells can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. Another method is to use _MgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient compatible with the subject and the active ingredient pharmacologically and/or physiologically, These are well known in the art and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives.

As used herein, the term "prevention" refers to methods performed to prevent or delay the occurrence of a disease or disorder or symptom (eg, a disease associated with high expression of c-Met and MSLN) in a subject. As used herein, the term "treatment" refers to a method performed to obtain a beneficial or desired clinical result. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction in extent of disease, stabilization (i.e., no longer worsening) of disease state, delay or slowing of disease progression, amelioration or palliation of disease status, and relief of symptoms (whether partial or total), whether detectable or not. In addition, "treating" can also refer to prolonging survival as compared to expected survival if not receiving treatment.

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, human) has a disease associated with high expression of c-Met and MSLN.

As used herein, the terms "cancer overexpressing c-MET", "cancer overexpressing MSLN", "cancer overexpressing c-MET and MSLN" and "diseases associated with high expression of c-Met and MSLN" mean It means that the expression level of c-MET and/or MSLN in diseased cells of a subject, such as cancer cells, is higher than the expression level of c-MET and/or MSLN in normal healthy cells of the same subject. In some instances, "cancers that overexpress c-MET and MSLN" and "diseases associated with high expression of c-Met and MSLN" are used interchangeably. In specific embodiments, for example, cancers that overexpress c-MET and/or MSLN can include pancreatic cancer.

Description of drawings

Figure 1 shows the results of high-throughput flow screening of c-MET and MSLN scFv antibody sequences.

Figure 2 shows that c-Met and MSLN scFv sequences screened out by flow cytometric analysis recognize recombinant proteins with different tags. Panel A is c-Met and panel B is MSLN. The screened scFv antibody sequence was transiently transfected into 293T cells, harvested after 24 hours, and incubated with 1ug c-Met-hFc and MSLN-hFc antigens respectively, and the secondary antibody was PE anti-human IgG.

Figure 3 shows the verification of the binding ability of c-MET and MSLN scFv antibody sequences to antigen (c-Met and MSLN). (Figure A) c-Met (scFv) antibody sequence negative control group, (Figure B) c-Met (scFv) antibody sequence experimental group; (Figure C) MSLN (scFv) antibody sequence negative control group, (Figure D) MSLN (scFv) antibody sequence experiment group. Panel A, c-Met (scFv) antibody sequence was incubated with antigen MSLN; panel B, c-Met (scFv) antibody sequence was incubated with antigen c-Met. Panel C, MSLN (scFv) antibody sequence was incubated with natural antigen c-Met; Panel D, MSLN (scFv) antibody sequence was incubated with antigen MSLN.

Figure 4 shows a schematic diagram of the four CAR structures targeting c-Met and MSLN.

Figure 5 shows the verification of the binding of the four CAR plasmids to the antigen. Panel A, Flow cytometry verification of c-Met chimeric antigen receptor (CAR) plasmid. The CAR plasmid was transiently transfected into 293T cells, and 1×10 ⁶ cells were collected 24 hours later, and detected by c-Met antigen. Panel B, Flow cytometry verification of the MSLN chimeric antigen receptor (CAR) plasmid. The CAR plasmid was transiently transfected into 293T cells, and 1×10 ⁶ cells were collected 24 hours later, and detected using MSLN antigen. Panel CD, flow cytometry verification of c-MET and MSLN common chimeric antigen receptor (CAR) plasmid. The CAR plasmid was transiently transfected into 293T cells, and 1×10 ⁶ cells were collected 24 hours later, and detected using c-Met antigen and MSLN antigen respectively. EF, Flow cytometry validation of a common chimeric antigen receptor (CAR) plasmid expressing human IL-21 targeting c-Met and MSLN. The CAR plasmid was transiently transfected into 293T cells, and 1×10 ⁶ cells were collected 24 hours later, and detected using c-Met antigen and MSLN antigen respectively.

Figure 6 shows the results of lactate dehydrogenase (LDH) release experiments. (Figure A) Normal T cells, c-MET-CAR-T cells, MSLN-CAR-T cells, Tandem-CAR-T cells and Tandem-CAR(IL-21)-T cells at different effect-target ratios (20 /1, 10/1, 5/1 and 1/1) in vitro killing effect on target cell Capan-2. (Panel B) In vitro killing effect of 5 effector T cells on target cell PANC-1 at different effector-target ratios. The line graph represents the mean ± standard deviation (the effect-to-target ratio is 20/1), n≥3, *p<0.05, ****p<0.0001. Statistical analysis was performed using Two-Way ANOVA with Tukey's method for multiple comparisons.

Figure 7 shows the secretion levels of cytokines IFN-γ, TNF-α, IL-6 and IL-21 after CAR-T cells were co-cultured with Capan-2. The effector T cells were co-cultured with Capan-2 for 18 hours according to the effector-target ratio of 10:1, the histogram represents the mean ± standard deviation, n≥3, *p<0.05, **p<0.01, ***p< 0.001, ****p<0.0001, Statistical analysis was performed using One-Way ANOVA, and Tukey's method was used for multiple comparisons.

Figure 8 shows the secretion levels of cytokines IFN-γ, TNF-α, IL-6 and IL-21 after CAR-T cells were co-cultured with pancreatic cancer cells PANC-1. The effector T cells were co-cultured with PANC-1 for 18 hours according to the ratio of effector-target ratio = 10:1, the histogram represents the mean ± standard deviation, n≥3, statistical analysis was performed using One-Way ANOVA, and Tukey's method was used for multiple Compare.

Fig. 9 shows the flow cytometry detection results of the expression of c-MET on the surface of the MET-OE PANC-1 cell line (A) and the expression of MSLN on the surface of the MSLN-OE PANC-1 cell line (B). Histograms represent mean ± standard deviation, n≥3, ****p<0.0001, statistical analysis was performed using paired-sample t-test.

Figure 10 shows the results of the LDH release assay of MET-OE PANC-1 cells and MSLN-OE PANC-1 cells. (Figure A) Normal T cells, c-MET-CAR-T cells, MSLN-CAR-T cells, Tandem-CAR-T cells and Tandem-CAR(IL-21)-T cells at different effect-target ratios (20 /1, 10/1, 5/1 and 1/1) in vitro killing effect on target cell MET-OE PANC-1; (Figure B) five effector T cells on target cell MSLN-OE In vitro killing effect of PANC-1. The line graph represents the mean ± standard deviation (the effect-to-target ratio is 20/1), n≥3, **p<0.01, ****p<0.0001. Statistical analysis was performed using Two-Way ANOVA, and Tukey's method was used for multiple comparisons.

Figure 11 shows the in vivo verification results of CAR-T cells on pancreatic cancer mouse xenografts. (Figure A) In vivo experiment flow chart; (Figure B) Tumor size display in the control and experimental groups, a: Normal T cell group, b: c-MET-CAR-T cell group, c: MSLN-CAR-T cell Group, d: Tandem-CAR-T cell group and e: Tandem-CAR(IL-21)-T cell group; (Panel C) mouse body weight change curve; (Panel D) tumor growth curve. The line graph represents the mean ± standard deviation, statistics on the tumor size on d28, n=4, *p<0.05, **p<0.01, ****p<0.0001

Figure 12 shows the HE staining results (100×) of hearts, lungs, livers, kidneys and spleens of mice in four CAR-T cell groups.

Detailed ways

The present invention will be further described below in conjunction with 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 technical field.

The primers used to construct the plasmids in the examples are shown in the table below:

The hIL21 sequence is shown in SEQ ID NO:32.

Example 1: Preparation of anti-c-Met antibody and anti-MSLN antibody

1. Preparation of c-Met and MSLN highly expressed stable transfected cell lines and recombinant proteins

First, human c-Met and human MSLN template plasmids (both purchased from Beijing Sino Biological Science and Technology Co., Ltd.; Cat. No.: Mesothelin cDNA ORF Clone in Cloning Vector, Human (HG13128-G), c-MET cDNA ORF Clone in Cloning Vector , Human (HG10692-M)) based on c-Met and MSLN gene extracellular segment sequence (hc-Met, hMSLN) was amplified, and the antigen expression vector (Huazhi Tiancheng; Cat: hfc, mfc) was digested . Then, the amplified hc-Met and hMSLN genes were connected to the hfc and mfc vectors respectively through the Gibson assembly system (Huazhi Tiancheng; Cat: HZGB15-1) to construct hc-met antigen expression plasmids (hc-Met-hfc, hc - Met-mfc, hMSLN-hfc, hMSLN-mfc). In 293T cells, use the hc-Met or hMSLN antigen expression plasmid to carry out lentivirus packaging through the three-plasmid lentivirus packaging system, collect and concentrate the virus after 48 hours; The medium was screened to obtain high-expression cell lines, which were fixed with 4% paraformaldehyde; the fixed high-expression cell lines were frozen at -80°C for mouse immunization. The antigen expression plasmid was constructed, and the expression plasmid was transiently transfected into 293F cells, and cultured in suspension for 7 days; the supernatant of the cells was collected by high-speed centrifugation, and the supernatant was purified by protein A/G column to obtain c-Met and MSLN natural conformation antigens respectively. Concentration, AKATA purification, BCA quantitative aliquots were stored at -80°C for screening antibody sequences.

2. Enrichment and isolation of positive B cells from immunized mice

After intraperitoneal injection of c-Met and MSLN high-expression cell lines, Balb/C mice (5-7 weeks old, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were immunized three times, and blood was taken from the tail vein, and ELISA was used to The antibody titer in mouse serum was detected by adsorption assay (Elisa), which showed that all immunized mice produced a relatively ideal immune response. After 30 days of immunization, the mouse spleen was taken, and B lymphocytes were enriched by magnetic bead sorting technology, and positive B lymphocytes expressing high-affinity antibodies were obtained based on microfluidic technology.

3. Screening of high-affinity and high-specificity c-Met and MSLN scFv

Based on the self-optimized single-cell reverse transcription technology, the mRNA of a single positive B cell is reverse-transcribed, and the antibody variable region sequence of the B cell is amplified by combining nested PCR, temperature gradient PCR and landing PCR technology, and the antibody sequence is cloned to the plasmid to complete the preparation of the scFv library. The above-mentioned c-Met and MSLN-scFv antibody plasmid libraries were packaged into 293T cells using a lentiviral packaging system to obtain c-Met and MSLN scFv single-chain antibody 293T cell display libraries. Based on the flow cytometric sorting technique, the 293T cells displaying high-affinity and high-specificity scFv sequences on the surface were sorted and enriched. The genome of the enriched positive 293T cells was extracted and the scFv sequence was amplified, re-cloned into the LentiCMV-DP3-HA-Puro vector (purchased from Huazhi Tiancheng) and single clones were picked. The monoclonal plasmid was transfected into 293T cells, and high-affinity and high-specificity antibody sequences were screened by flow cytometry. The results of flow cytometry analysis showed that 9 high-affinity and high-specificity c-Met antibody sequences were screened (Fig. 1A); 4 high-affinity and high-specificity MSLN antibody sequences were screened (Fig. 1B). The screened positive sequences were further transfected into 293T cells, and the cells were harvested and incubated with c-Met and MSLN recombinant proteins with different labels. The results of flow cytometry analysis showed that the screened sequences could recognize c-Met and MSLN proteins respectively (Fig. 2). Considering whether the obtained antibody sequence can specifically recognize the recombinant protein, we used flow cytometry to verify the binding ability of the screened scFv antibody sequence to different antigens (c-MET and MSLN). The experimental results showed that c-Met (scFv) could not bind to the antigen MSLN, but could well bind to the antigen c-Met (Figure 3A and 3B); similarly, MSLN (scFv) could not bind to the antigen c-Met, but could Binding well to the antigen MSLN (Fig. 3C and 3D).

Embodiment 2: Construction of the chimeric antigen receptor (CAR) plasmid targeting c-Met and MSLN

In this example, four kinds of CAR plasmids were constructed, including: c-Met-CAR, MSLN-CAR, c-Met-MSLN-CAR (i.e. tandem CAR) and c-Met-MSLN-CAR co-expressing IL-21 (i.e. tandem CAR). CAR(IL-21)) (FIG. 4).

The c-Met scFv sequence (clone number: 2-2e-5) and the MSLN scFv sequence (clone number: 2-3c-11) with high affinity and high specificity after screening and verification in Example 1 were used to construct The corresponding CAR plasmid. Chimeric antigen receptor (CAR) is composed of scFv antibody, transmembrane domain of human CD8, 4-1BB co-stimulatory signaling molecule and human CD3ζ signaling domain. The CAR basic plasmid HZ-WQ012: pCDH-EF1α-hCAR-T2A-GFP was successfully constructed on the expression vector constructed by Huazhi Tiancheng Biotechnology Co., Ltd. At the same time, pCDH-EF1α-hCAR-T2A-GFP was used as the backbone vector to construct the CAR plasmid pCDH-EF1α-hCAR-F2A-hIL21-T2A-GFP and c-Met(scFv)-hCAR- T2A-GFP, MSLN(scFv)-hCAR-T2A-GFP and c-Met-GS-MSLN(scFv)-hCAR-T2A-GFP. In addition, c-Met(scFv)-GS-MSLN(scFv)-hCAR-F2A-hIL21-T2A-GFP was constructed using pCDH-EF1α-hCAR-F2A-hIL21-T2A-GFP as the backbone vector.

Example 3: Verification of CAR plasmids targeting c-Met and MSLN

Design sequencing primers, send the successfully identified CAR plasmids to Beijing Qingke Bio-Sequencing Co., Ltd. for Sanger sequencing, compare the obtained sequencing sequences, and select the clone number with the correct sequence. In 293T cells, the sequenced correct c-Met and MSLN chimeric antigen receptor (CAR) plasmids were transiently transfected. After 24 hours, the cells were collected and incubated with the antigens c-Met and MSLN respectively for flow cytometric detection. The results showed that the four constructed CARs could specifically recognize and bind c-Met and MSLN antigens (Figure 5).

Example 4: In vitro killing experiment of CAR-T cells on pancreatic cancer cells

1. Results of lactate dehydrogenase (LDH) release experiment

The expressions of c-MET and MSLN proteins on the surface of pancreatic cancer cell lines and normal pancreatic cells were detected by flow cytometry, and the pancreatic cancer cell line with high expression (Capan-2) and pancreatic cancer cell with low expression were screened out strain (PANC-1). In this experiment, we selected these two cell lines as the experimental subjects to conduct in vitro killing experiments of CAR-T cells to evaluate the killing effect of CAR-T cells on these two types of cells. Effector T cells were co-cultured with Capan-2 cells and PANC-1 cells for 18 hours according to different effector-target ratios to obtain cell supernatants, and the amount of LDH in the supernatants was detected. The experimental results showed that compared with ordinary T cells, the cell supernatants of the four CAR-T cells we constructed co-cultured with Capan-2 (highly expressing c-MET and MSLN) for 18 hours contained more LDH (p <0.001); while the amount of LDH contained in the cell supernatant of the four CAR-T cells co-cultured with PANC-1 (basically not expressing c-MET and MSLN) for 18 hours was not significantly increased (compared with ordinary T cells There was no statistical difference) (Figure 6). This shows that compared with ordinary T cells, the four CAR-T cells we constructed have significant killing activity against the pancreatic cancer cell line Capan-2 that highly expresses c-MET and MSLN, but against the pancreatic cancer cell line Capan-2 that does not express c-MET and MSLN The pancreatic cancer cell line PANC-1 cells, whose killing activity is similar to that of ordinary T cells, did not produce additional or stronger killing effects. In addition, the tandem CAR(IL-21)-T cells produced the most LDH in the cell supernatant after co-cultured with Capan-2 among the four CAR-T cells, suggesting that this kind of CAR (IL-21)-T cells can recognize two tumor antigens. Tandem CAR has stronger killing activity on Capan-2 cells.

2. Cytokine secretion test results

When CAR-T cells recognize and bind to corresponding tumor antigens on the surface of tumor cells, CAR-T cells are activated and produce a variety of anti-tumor cytokines (such as IFN-γ and TNF-α) and inflammatory factors (such as IL- 6). In order to ensure that ordinary T cells and the four types of CAR-T cells are in a good proliferation and activity state, we choose to grow these effector T cells for about 10-12 days, and respectively combine these effector T cells with pancreatic cancer cells Capan-2 (highly expressed c- MET and MSLN) were co-cultured for 18 hours according to the effect-to-target ratio of 10:1, and the secretion of cytokines was detected in the cell supernatant. We found that compared with ordinary T cells, the secretion of IFN-γ and TNF-α in the four CAR-T cells we constructed was significantly increased (p<0.001) (Fig. 7A, B). And tandem CAR-T cells and tandem CAR(IL-21)-T cells with tandem structure secreted more IFN-γ and TNF-α (p<0.001). In addition, the tandem CAR(IL-21)-T cells had the most IFNγ and TNFα among the four CAR-T cells. These experimental results suggest that the CAR-T cells we constructed have a strong killing effect on Capan-2 cells, and the tandem CAR-T cells have stronger anti-tumor activity. In tandem CAR-T cells, tandem CAR-T cells have stronger killing activity than tandem CAR(IL-21)-T cells; the difference between the two is that the latter is activated It can additionally release the cytokine IL-21, which is also an anti-tumor cytokine, and the presence of this cytokine may further improve the anti-tumor ability of CAR-T cells. The experimental results suggest that among the four CAR-T cells, tandem CAR(IL-21)-T cells may have better anti-tumor activity as tandem CAR and fourth-generation CAR.

CAR-T cells will inevitably produce IL-6 during the anti-tumor process. Our experimental results showed that in the process of killing Capan-2 cells by effector T cells, compared with the normal T cell group, the IL-6 produced by the four CAR-T cell groups in the process of killing tumor cells were all increased (p< 0.001), which increased by about 2-3 times, but there was no statistical difference in the production of IL-6 among the four CAR-T cell groups (Fig. 7C). The results showed that when the four CAR-T cells killed tumor cells, there was no significant difference in the release of IL-6 from CAR-T cells with different structures.

IL-21 is a member of the IL-2 cytokine superfamily and is an anti-tumor cytokine. In the four kinds of CAR-T cells we constructed, only the intracellular domain of the tandem CAR (IL-21) was additionally added with the IL-21 receptor gene, theoretically the tandem CAR (IL-21)-T cells were activated Additional IL-21 can then be released. The experimental results showed that the secretion of IL-21 in the four CAR-T cell groups was higher than that in the control group; in addition, the secretion of IL-21 in the tandem CAR(IL-21)-T cell group was much higher than that in the other groups. (FIG. 7D). The experimental results also proved that after the tandem CAR (IL-21)-T cells were activated, the IL-21 receptor gene in the CAR structure could be normally expressed and additionally secrete more IL-21.

After about 10-12 days of growth, these effector T cells were co-cultured with pancreatic cancer cells PANC-1 (the cell surface basically does not express c-MET and MSLN) for 18 hours according to the effector-target ratio of 10:1. The secretion of cytokines in the cell supernatant. The results showed that there was no significant difference in the secretion of anti-tumor cytokines (IFN-γ, TNF-α and IL-21) and IL-6 among the five effector T cells (p>0.05) ( FIG. 8 ). This is mainly due to the fact that c-MET and MSLN proteins are not expressed on the surface of PANC-1 cells, and the four CAR-T cells are not fully activated when co-cultured with PANC-1 cells. Therefore, compared with ordinary T cells, these CAR-T cells - T cells did not produce more IFN-γ, TNF-α, IL-21 and IL-6.

4. Construction of MET-OE PANC-1 cells and MSLN-OE PANC-1 cells

In order to further prove that the CAR-T cells we constructed have the targeting ability when killing tumor cells, we need to knock down or overexpress the target genes (MET and MSLN) on the same pancreatic tumor cell line. The c-MET and MSLN proteins are basically not expressed on the surface of PANC-1 cells. Therefore, we constructed the corresponding MET-OE lentivirus and MSLN-OE lentivirus by overexpressing the target gene, and constructed them by lentiviral transfection MET-OE PANC-1 cell line and MSLN-OEPANC-1 cell line. In addition, since the antibiotic selection tags of the MET-OE plasmid and MSLN-OE plasmid stored in our laboratory are both puromycin, it is impossible to construct PANC-1 cells that overexpress c-MET and MSLN at the same time, and by sequentially transfecting MET -OE lentivirus and MSLN-OE lentivirus may not be able to obtain ideal overexpression cell lines. Therefore, in the end, we chose two lentiviruses to transfect PANC-1 cells separately to construct MET-OE PANC-1 cell line and MSLN-OE PANC-1 cell line. The c-MET and MSLN protein expression levels on the surface of these two constructed PANC-1 cells were detected by flow cytometry to prove whether we have successfully constructed MET-OE PANC-1 cell lines and MSLN-OE PANC-1 cells strain. The results of flow cytometry showed that the expression rate of c-MET on the surface of MET-OE PANC-1 cells was about 49.7%, and the expression rate on the surface of MSLN-OE PANC-1 cells was about 56.8% (Figure 9). Finally, we successfully constructed MET-OE PANC-1 cell line (high expression of c-MET protein on the surface) and MSLN-OE PANC-1 cell line (high expression of MSLN protein on the surface). These two lines of cells will be used in recovery experiments later.

5. Reply to the experimental results

The CAR-T cells were co-cultured with the constructed MET-OE PANC-1 cells and MSLN-OE PANC-1 cells for 18 hours, and the supernatant was collected for LDH release detection. We found that the original common T cells and the four CAR-T cells had similar and weak killing abilities against PANC-1 cells, and there was no statistical difference in the release level of LDH. However, in experiments using MET-OE PANC-1 cells (high expression of c-MET protein on the surface) or MSLN-OE PANC-1 cells (high expression of MSLN protein on the surface) as target cells, we found that compared with ordinary T cells, Three CAR-T cells in each group had significant killing activity against these cell lines (Figure 10). Figure 10A shows that c-MET-CAR-T cells, tandem CAR-T cells and tandem CAR(IL-21)-T cells all have a strong killing effect on MET-OE PANC-1 cells; however, MSLN-CAR - The killing effect of T cells on MET-OE PANC-1 cells is only similar to that of ordinary T cells. Figure 10B shows that MSLN-CAR-T cells, tandem CAR-T cells and tandem CAR(IL-21)-T cells all have a strong killing effect on MSLN-OE PANC-1 cells; however, c-MET-CAR - The killing effect of T cells on MSLN-OE PANC-1 cells is only similar to that of ordinary T cells. Reversion experiments once again proved that the four kinds of CAR-T cells we constructed had targeted killing of tumor cells, that is, compared with tumor cells with low expression of c-MET and/or MSLN on the cell surface, CAR-T cells were more effective in killing tumor cells. Tumor cells with high surface expression of c-MET and/or MSLN have stronger killing activity.

Example 5: In vivo verification of CAR-T cells on transplanted tumors in mice with pancreatic cancer

Experimental animals: 4-6 week old female NOD-SCID female mice were purchased from Jiangsu Jicui Biotechnology Co., Ltd. They were bred in the SPF animal experiment center of the GLP laboratory building in Lanzhou University Medical Campus. This animal experiment has been approved by the Experimental Animal Ethics Committee of the Second Hospital of Lanzhou University (02021-667), and the experimental design and experimental operation procedures are in compliance with animal welfare requirements.

Experimental cells: pancreatic cancer cell line Capan-2.

Establishment of mouse pancreatic cancer CDX model:

1) 4-6 week old female NOD-SCID female mice, select the left side of the back near the thigh, after povidone iodine disinfection, use a 1mL syringe to absorb 100uL of tumor cell suspension, insert the needle at an angle of 45 degrees, and turn 15 degrees after piercing the skin. Lift the skin slightly with the needle tip at an angle of 10 degrees. After confirming that it is subcutaneous, continue to insert the needle for about 1 cm, gently push in the tumor cell suspension to make the subcutaneous bulge appear, and withdraw the needle slowly;

2) Capan-2 cells grow relatively slowly and form tumors in about 2 weeks, which is counted as day d0. The body weight of the mice is weighed, and the length and width of the tumor are measured with a vernier caliper to calculate the tumor volume. Tumor volume (mm ³ )=length×width ² ×0.5.

3) The mice were randomly divided into 5 groups (common T group, c-MET-CAR-T group, MSLN-CAR-T group, tandem CAR-T group and tandem CAR(IL-21)-T group), each Group of 5. The corresponding effector T cells were injected into the tail vein on d0 and d7 respectively, with a volume of ^100uL of cell suspension.

Treatment of effector T cells: After taking out from the liquid nitrogen tank, resuscitate by conventional methods, add complete T cell medium, resuspend effector T cells, and control the growth density at about 1× ¹⁰⁶ cells/mL, and culture the cells at 37°C Incubate in the box for 12-24 hours. When injecting mice, effector T cells need to be washed twice with PBS and resuspended in PBS to ensure a cell density of 5×10 ⁶ cells/(100-200uL), placed in an ice box, and the tail vein injection of mice should be completed as soon as possible .

4) Tumor volumes were weighed and measured on d0 day, d7 day, d14 day, d21 day and d28 day respectively.

5) Draw a curve graph of mouse body weight change and a curve graph of tumor volume change.

1. CAR-T cells have a significant inhibitory effect on mouse models of pancreatic cancer

We have verified the killing effect of c-MET-CAR-T cells, MSLN-CAR-T cells, tandem CAR-T cells and tandem CAR(IL-21)-T cells on pancreatic cancer cell Capan-2 in vitro . In order to further verify its anti-tumor effect in vivo, we successfully established a mouse pancreatic cancer CDX model by subcutaneous tumorigenesis of Capan-2 cells. About 2 weeks after tumor formation, the mice were randomly divided into 5 groups, with 4 mice in each group, and 5 kinds of effector T cells were injected into the tail vein of the mice, and an additional one was added on the 7th day, during which the tumor size was measured every 7 days and mouse body weight (Fig. 11A). The experimental results showed that the c-MET-CAR-T cell, MSLN-CAR-T cell, tandem CAR-T cell and tandem CAR(IL-21)-T cell groups had smaller tumor volumes compared with the normal T group , and the tumor volume of mice in the tandem CAR(IL-21)-T group shrunk most significantly, followed by the tandem CAR-T group (Fig. 11B and 14D). In addition, although the tumor volume of the c-MET-CAR-T cell group and the MSLN-CAR-T cell group still increased by the end of the experiment, compared with the normal T group, no matter the tumor growth rate or the final tumor volume Both are significantly slower and smaller in size. In addition, the weight gain of the mice in the four experimental groups was significantly higher than that in the normal T group ( FIG. 11C ).

2. CAR-T cells have no obvious toxicity to important organs

Off-target effects in CAR-T cell therapy are common complications. Although CAR-T cells are targeted, these antigens may also be expressed in normal organs of the human body. Therefore, CAR-T cells may accidentally injure normal organs or tissues. In order to clarify whether CAR-T cells can damage the normal organs of mice, we took some important organs (heart, lung, liver, kidney and spleen) of the mice in the four experimental groups for HE staining. Inflammation and necrosis did not appear (Figure 12). The experimental results suggest that the 4 CAR-T cells constructed in this study have good safety.

In the description of the specification, descriptions with reference to the terms "one embodiment", "specific embodiment", "example" and the like mean that a specific feature, structure, material or characteristic described in connection with the embodiment or example is included in at least one embodiment of the present invention In an embodiment or example. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The above content is only an example and description of the present invention. Those skilled in the art will make various modifications or supplements to the described specific embodiments or replace them in similar ways, as long as they do not deviate from the invention or exceed this right. The scope defined in the claims should all belong to the protection scope of the present invention.

Claims (10)

1. An anti-MSLN antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequences set forth in SEQ ID NOs: 1. SEQ ID NO:2 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3, the light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 6. SEQ ID NO:7 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown in figure 8,

preferably, the antibody or antigen binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO:4 and a heavy chain variable region as set forth in SEQ ID NO:9 and a light chain variable region as shown in,

preferably, the antibody or antigen binding fragment thereof is a monoclonal antibody, chimeric antibody, humanized antibody, fully human antibody, fab ', fv fragment, F (ab') ₂ An scFv or di-scFv,

preferably, the antibody or antigen binding fragment thereof is an scFv,

preferably, the antibody or antigen binding fragment thereof further comprises a linker connecting the heavy chain variable region and the light chain variable region,

Preferably, the linker has the sequence as set forth in SEQ ID NO:11, and a sequence of the amino acids shown in the formula (I),

preferably, the antibody or antigen binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO:12, and a polypeptide having the amino acid sequence shown in FIG. 12.

2. An anti-c-Met antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14. SEQ ID NO:15 and SEQ ID NO:16, HCDR1, HCDR2 and HCDR3, said light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 19. SEQ ID NO:20 and SEQ ID NO:21 LCDR1, LCDR2 and LCDR3,

preferably, the antibody or antigen binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO:17 and a heavy chain variable region as set forth in SEQ ID NO:22, a light chain variable region as shown in figure 22,

preferably, the antibody or antigen binding fragment thereof is a monoclonal antibody, chimeric antibody, humanized antibody, fully human antibody, fab ', fv fragment, F (ab') ₂ An scFv or di-scFv,

preferably, the antibody or antigen binding fragment thereof is an scFv,

preferably, the antibody or antigen binding fragment thereof further comprises a linker connecting the heavy chain variable region and the light chain variable region,

preferably, the linker has the sequence as set forth in SEQ ID NO:24, and a sequence of the amino acids shown in the specification,

Preferably, the antibody or antigen binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO:25, and a polypeptide comprising the amino acid sequence shown in seq id no.

3. A chimeric antigen receptor comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain,

wherein the extracellular antigen-binding domain consists of a MSLN antigen-binding domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequences set forth in SEQ ID NOs: 1. SEQ ID NO:2 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3, the light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 6. SEQ ID NO:7 and SEQ ID NO:8 LCDR1, LCDR2 and LCDR3, or

Wherein the extracellular antigen-binding domain consists of a c-Met antigen-binding domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequences as set forth in SEQ ID NOs: 14. SEQ ID NO:15 and SEQ ID NO:16, HCDR1, HCDR2 and HCDR3, said light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 19. SEQ ID NO:20 and SEQ ID NO:21 LCDR1, LCDR2 and LCDR3,

preferably, the MSLN antigen binding domain comprises the amino acid sequence as set forth in SEQ ID NO:4 and a heavy chain variable region as set forth in SEQ ID NO:9 and a light chain variable region as shown in,

Preferably, the MSLN antigen binding domain further comprises a linker connecting the heavy chain variable region and the light chain variable region,

preferably, the linker has the sequence as set forth in SEQ ID NO:11, and a sequence of the amino acids shown in the formula (I),

preferably, the MSLN antigen binding domain comprises the amino acid sequence as set forth in SEQ ID NO:12, the amino acid sequence shown in the specification,

preferably, the c-Met antigen binding domain comprises the amino acid sequence as set forth in SEQ ID NO:17 and a heavy chain variable region as set forth in SEQ ID NO:22, a light chain variable region as shown in figure 22,

preferably, the c-Met antigen binding domain further comprises a linker connecting the heavy chain variable region and the light chain variable region,

preferably, the linker has the sequence as set forth in SEQ ID NO:24, and a sequence of the amino acids shown in the specification,

preferably, the c-Met antigen binding domain comprises the amino acid sequence as set forth in SEQ ID NO:25, and a sequence of the amino acids shown in the formula II,

preferably, the transmembrane domain has the amino acid sequence as set forth in SEQ ID NO:27, and a sequence of the amino acid sequence shown in seq id no,

preferably, the intracellular signaling domain comprises a 4-1BB costimulatory signaling molecule and a human CD3 zeta signaling domain,

preferably, the 4-1BB costimulatory signaling molecule has the sequence as set forth in SEQ ID NO:28, and a sequence of the amino acid sequence shown in the specification,

preferably, the human CD3 zeta signaling domain has the sequence as set forth in SEQ ID NO:29, and a nucleotide sequence shown in the formula (I),

Preferably, the dual chimeric antigen receptor further comprises a hinge region linking the extracellular antigen binding domain and the transmembrane domain,

preferably, the hinge region has the sequence set forth in SEQ ID NO:30, and a sequence of the amino acid shown in the specification,

preferably, the dual chimeric antigen receptor further comprises a signal peptide at its N-terminus,

preferably, the signal peptide has the sequence as set forth in SEQ ID NO:31, and a sequence of the amino acids shown in the formula,

preferably, the chimeric antigen receptor has the amino acid sequence as set forth in SEQ ID NO:33 or SEQ ID NO:34, and a nucleotide sequence shown in seq id no.

4. A dual chimeric antigen receptor comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain,

wherein the extracellular antigen-binding domain consists of a c-Met antigen-binding domain and a MSLN antigen-binding domain,

wherein the MSLN antigen binding domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequences set forth in SEQ ID NOs: 1. SEQ ID NO:2 and SEQ ID NO:3, HCDR1, HCDR2 and HCDR3, the light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 6. SEQ ID NO:7 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown in figure 8,

wherein the c-Met antigen binding domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14. SEQ ID NO:15 and SEQ ID NO:16, HCDR1, HCDR2 and HCDR3, said light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 19. SEQ ID NO:20 and SEQ ID NO:21 LCDR1, LCDR2 and LCDR3,

Preferably, the MSLN antigen binding domain comprises the amino acid sequence as set forth in SEQ ID NO:4 and a heavy chain variable region as set forth in SEQ ID NO:9 and a light chain variable region as shown in,

preferably, the MSLN antigen binding domain further comprises a linker connecting the heavy chain variable region and the light chain variable region,

preferably, the linker has the sequence as set forth in SEQ ID NO:11, and a sequence of the amino acids shown in the formula (I),

preferably, the MSLN antigen binding domain comprises the amino acid sequence as set forth in SEQ ID NO:12, the amino acid sequence shown in the specification,

preferably, the c-Met antigen binding domain comprises the amino acid sequence as set forth in SEQ ID NO:17 and a heavy chain variable region as set forth in SEQ ID NO:22, a light chain variable region as shown in figure 22,

preferably, the c-Met antigen binding domain further comprises a linker connecting the heavy chain variable region and the light chain variable region,

preferably, the linker has the sequence as set forth in SEQ ID NO:24, and a sequence of the amino acids shown in the specification,

preferably, the c-Met antigen binding domain comprises the amino acid sequence as set forth in SEQ ID NO:25, and a sequence of the amino acids shown in the formula II,

preferably, the extracellular antigen-binding domain further comprises a linker connecting the c-Met antigen-binding domain and the MSLN antigen-binding domain,

preferably, the linker has the sequence as set forth in SEQ ID NO:24, and a sequence of the amino acids shown in the specification,

Preferably, the transmembrane domain has the amino acid sequence as set forth in SEQ ID NO:27, and a sequence of the amino acid sequence shown in seq id no,

preferably, the intracellular signaling domain comprises a 4-1BB costimulatory signaling molecule and a human CD3 zeta signaling domain,

preferably, the 4-1BB costimulatory signaling molecule has the sequence as set forth in SEQ ID NO:28, and a sequence of the amino acid sequence shown in the specification,

preferably, the human CD3 zeta signaling domain has the sequence as set forth in SEQ ID NO:29, and a nucleotide sequence shown in the formula (I),

preferably, the dual chimeric antigen receptor further comprises a hinge region linking the extracellular antigen binding domain and the transmembrane domain,

preferably, the hinge region has the sequence set forth in SEQ ID NO:30, and a sequence of the amino acid shown in the specification,

preferably, the dual chimeric antigen receptor further comprises a signal peptide at its N-terminus,

preferably, the signal peptide has the sequence as set forth in SEQ ID NO:31, and a sequence of the amino acids shown in the formula,

preferably, the dual chimeric antigen receptor has the amino acid sequence as set forth in SEQ ID NO:35, and a sequence of the amino acid shown in the formula (I),

preferably, the dual chimeric antigen receptor further comprises the amino acid sequence of hIL21 at its C-terminus,

preferably, the dual chimeric antigen receptor has the amino acid sequence as set forth in SEQ ID NO:36, and a nucleotide sequence shown in seq id no.

5. An isolated nucleic acid molecule encoding the antibody or antigen binding fragment thereof of claim 1 or 2, the chimeric antigen receptor of claim 3, or the dual chimeric antigen receptor of claim 4.

6. An expression vector comprising the nucleic acid molecule of claim 5.

7. A host cell comprising the expression vector of claim 6,

preferably, the host cell comprises an immune cell,

preferably, the immune cells comprise T cells, B cells, NK cells, monocytes, macrophages or dendritic cells or any combination thereof.

8. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of claim 1 or 2, the nucleic acid molecule of claim 5, the expression vector of claim 6 or the host cell of claim 7, and optionally a pharmaceutically acceptable carrier and/or excipient.

9. A kit comprising the antibody or antigen-binding fragment thereof of claim 1 or 2, the nucleic acid molecule of claim 5, the expression vector of claim 6, or the host cell of claim 7.

10. Use of an antibody or antigen binding fragment thereof according to claim 1 or 2, a chimeric antigen receptor according to claim 3, a dual chimeric antigen receptor according to claim 4, a nucleic acid molecule according to claim 5, an expression vector according to claim 6, a host cell according to any one of claims 7, a pharmaceutical composition according to claim 8 or a kit according to claim 9 for the preparation of a medicament for the prevention and/or treatment of cancers overexpressing c-MET and MSLN,

Preferably, the cancer comprises pancreatic cancer.