CN118234755A - Chimeric antigen receptor specifically binding to MSLN and its application - Google Patents

Abstract

Bispecific antigen binding molecules that specifically bind to MSLN and NKG2D ligands (NKG2DL), and bispecific chimeric antigen receptors comprising the bispecific antigen binding molecules, modified immune cells expressing the bispecific chimeric antigen receptors, and methods for preparing the modified immune cells. Modified immune cells that co-express MSLN-specific CAR and other bioactive molecules (e.g., PD-1 antibodies and/or rIL-15, or NKG2D or active fragments), and methods for preparing the modified immune cells. These bispecific antigen binding molecules, CARs, and immune cells are used to prevent and/or treat diseases associated with the expression of mesothelin, such as malignant pleural mesothelioma, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, and the like.

Description

Chimeric antigen receptor specifically binding to MSLN and its application Technical Field

The present invention relates to the field of biomedicine, in particular, the present invention relates to bispecific antigen binding molecules that specifically bind to MSLN and NKG2D ligands (NKG2DL), and bispecific chimeric antigen receptors comprising the bispecific antigen binding molecules, modified immune cells expressing the bispecific chimeric antigen receptors, and methods for preparing the modified immune cells. The present invention also relates to modified immune cells that co-express MSLN-specific CAR and other bioactive molecules (e.g., PD-1 antibodies and/or rIL-15, or NKG2D or active fragments), and methods for preparing the modified immune cells. The present invention also relates to these bispecific antigen binding molecules, CARs and immune cells for preventing and/or treating diseases associated with the expression of mesothelin, such as malignant pleural mesothelioma, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, etc.

Background technique

Mesothelin (MSLN), the precursor protein of MSLN is hydrolyzed by protease into 31kDa megakaryocyte-potentiating factor (MPF) and 40kDa mesothelin. Previous studies have shown that CA125/MUC16 is a ligand of mesothelin, which binds to mesothelin through the repeated fragments of the extracellular functional region at the N-terminus and participates in cell adhesion. MSLN has a highly specific expression. It is lowly expressed in mesothelial cells in the peritoneal cavity, pleural cavity and pericardial cavity in normal tissues, and is highly expressed in solid tumors such as malignant pleural mesothelioma, pancreatic cancer, lung cancer, breast cancer, and ovarian cancer. It is especially highly expressed in malignant mesothelioma (85%-90%), pancreatic cancer (80%-85%), ovarian epithelial cancer (60%-65%) and lung cancer (60%-65%), and is related to cell proliferation, cell adhesion function and anti-apoptosis process. These biological characteristics suggest that MSLN could serve as an ideal tumor therapeutic target with multiple indications.

NKG2D is a member of the NKG2 family, which includes seven proteins (A, B, C, D, E, F, H) and is a type II C-lectin-like receptor. NKG2D is expressed on all NK cells, CD8+T cells, and the vast majority of NKT cells and γδT cells, and is an important activating receptor. NKG2D can directly recognize ligand molecules expressed on the surface of tumor cells without antigen presentation, and then activate or co-stimulate immune effector cells, thereby playing a killing role on tumor cells. One type of NKG2D ligand is MHC class I chain-related molecules A/B (MIC-A/B), and the other type of ligand is UL16-binding proteins (ULBPs). NKG2D ligands (NKG2DL) are almost not expressed on healthy tissue cells, and are not expressed or expressed very little in normal cells. However, when cells are infected or cancerous, the expression of these ligands will increase significantly. When the NKG2D receptor binds to its ligand, it will activate immune effector cells and cause killing effects. NKG2DL is mainly expressed on most epithelial tumor cells, such as ovarian cancer, colon cancer, etc., and is rarely expressed on normal cells.

As the incidence of cancer increases year by year, traditional treatments such as surgery, radiotherapy and chemotherapy are not effective in tumor treatment, and effective tumor treatment methods are urgently needed in clinic. Chimeric antigen receptor (CAR)-T cell therapy is considered to be one of the most promising cancer treatment methods and has become a new hope for human beings to fight cancer. It cultivates immune cells collected from the patient in vitro, transduces specific exogenous genes in vitro, and then amplifies them in vitro and infuses them back into the patient's body to achieve the purpose of treating tumors in a non-MHC restricted manner. CAR-T cell therapy has achieved significant therapeutic effects in the treatment of hematological malignancies, with a complete remission rate of over 90% for relapsed and refractory B-cell leukemia. Solid tumors account for about 90% of all malignant tumors, and there is a large demand for therapeutic drugs. However, the current therapeutic effect of CAR-T cell therapy in solid tumors is still insufficient. The complex tumor microenvironment of solid tumors greatly limits the therapeutic effect of CAR-T cell therapy in solid tumors. How to enhance the efficacy of CAR-T cell therapy in solid tumors is the focus of current research.

Summary of the invention

In this application, the inventors first developed a fully human antibody with low immunogenicity that can specifically recognize/bind to MSLN. On this basis, the present invention further designed and constructed an MSLN/NKG2DL bispecific CAR and immune cells that co-express MSLN-specific CAR and NKG2DL-specific binding molecules, and improved the killing of cells expressing tumor antigens and reduced their off-target toxicity to a certain extent by dual targeting MSLN and NKG2DL. The present invention also designed and constructed a CAR targeting MSLN that co-expresses PD-1 antibodies and/or rIL-15. By co-expressing PD-1 antibodies, the binding of PD-1 to PD-L1 is blocked, and T cells are reactivated, thereby enhancing the immune response; co-expressing rIL-15 can promote the proliferation and activation of T and NK cells, and enhance the tumor killing effect of CAR-T cells.

The CAR of the present invention can direct immune effector cell specificity and reactivity to cells expressing MSLN or simultaneously expressing MSLN and NKG2DL (e.g., malignant pleural mesothelioma, pancreatic cancer, lung cancer, breast cancer, ovarian cancer) in a non-MHC restricted manner so that they are eliminated. Therefore, the CAR targeting MSLN or simultaneously targeting MSLN and NKG2DL of the present invention has the potential for preventing and/or treating MSLN-positive tumors such as malignant pleural mesothelioma, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, and has great clinical value.

Bispecific Antigen Binding Molecules

The first aspect of the present invention provides a bispecific antigen-binding molecule, which comprises a first antigen-binding domain capable of specifically binding to MSLN and a second antigen-binding domain capable of specifically binding to NKG2D ligand (NKG2DL); wherein the first antigen-binding domain capable of specifically binding to MSLN comprises a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein,

(1) the VH comprises: the following three heavy chain CDRs defined according to the Kabat numbering system: CDR-H1 with a sequence of SEQ ID NO: 3 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 4 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 5 or a variant thereof; and/or the VL comprises: the following three light chain CDRs defined according to the Kabat numbering system: CDR-L1 with a sequence of SEQ ID NO: 6 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 7 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 8 or a variant thereof;

or

(2) the VH comprises: the following three heavy chain CDRs defined according to the IMGT numbering system: CDR-H1 with a sequence of SEQ ID NO: 9 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 10 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 11 or a variant thereof; and/or the VL comprises: the following three light chain CDRs defined according to the IMGT numbering system: CDR-L1 with a sequence of SEQ ID NO: 12 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 13 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 8 or a variant thereof;

or

(3) the VH comprises: the following three heavy chain CDRs defined according to the Chothia numbering system: CDR-H1 with a sequence of SEQ ID NO: 14 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 15 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 5 or a variant thereof; and/or the VL comprises: the following three light chain CDRs defined according to the Chothia numbering system: CDR-L1 with a sequence of SEQ ID NO: 6 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 7 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 8 or a variant thereof;

Wherein, the variant described in any one of (1), (2) and (3) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

In certain embodiments, the VH and/or VL further comprises framework regions (FRs) from human immunoglobulins.

In certain embodiments, the VH comprises a sequence as shown in SEQ ID NO: 1 or a variant thereof; wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

In certain embodiments, the VL comprises a sequence as shown in SEQ ID NO: 2 or a variant thereof; wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

In certain embodiments, the first antigen binding domain is selected from a full-length antibody, a Fab fragment, a Fab' fragment, a F(ab)' ₂ fragment, a F(ab)' ₃ fragment, a single-chain antibody (e.g., scFv, di-scFv or (scFv) ₂ ), a miniantibody, a disulfide-stabilized Fv protein (dsFv) and a single domain antibody (sdAb, nanobody).

In certain embodiments, the first antigen-binding domain is a single-chain antibody, which comprises, from its N-terminus to its C-terminus:

(1) VH-L1 comprising the sequence shown in SEQ ID NO: 1 or a variant thereof-VL comprising the sequence shown in SEQ ID NO: 2 or a variant thereof; or

(2) VL-L1 comprising the sequence shown in SEQ ID NO: 2 or a variant thereof-VH comprising the sequence shown in SEQ ID NO: 1 or a variant thereof;

Wherein, the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

In some embodiments, the L1 is a polypeptide. In some embodiments, the L1 comprises one or more (e.g., 1, 2, or 3) sequences as shown in (GmS)n, wherein m is selected from an integer of 1-6, and n is selected from an integer of 1-6. In some embodiments, m is 3, 4, or 5. In some embodiments, n is 1 or 2. In some embodiments, the L1 has a sequence of SEQ ID NO: 30.

In certain embodiments, the single-chain antibody comprises the sequence shown in SEQ ID NO: 18 or a variant thereof, which has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 18, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions.

In certain embodiments, the second antigen-binding domain capable of specifically binding to a NKG2D ligand (NKG2DL) comprises NKG2D or its ligand-binding domain.

In certain embodiments, the second antigen binding domain comprises the full-length sequence of NKG2D, for example, the sequence shown in SEQ ID NO:21 or a variant thereof, wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or several amino acid substitutions, deletions or additions (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

In certain embodiments, the second antigen binding domain comprises a ligand binding domain of NKG2D, such as an extracellular antigen binding domain. In certain embodiments, the second antigen binding domain comprises a sequence as shown in SEQ ID NO: 20 or a variant thereof, wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions.

In certain embodiments, the first antigen binding domain and the second antigen binding domain are connected by a linker L2.

In certain embodiments, the first antigen binding domain is a single chain antibody.

In certain embodiments, the L2 is a polypeptide. In certain embodiments, the L2 comprises one or more (e.g., 1, 2, or 3) sequences as shown in (GmS)n, wherein m is selected from an integer of 1-6, and n is selected from an integer of 1-6. In certain embodiments, m is 3, 4, or 5. In certain embodiments, n is 1 or 2. In certain embodiments, the L2 has a sequence of SEQ ID NO: 32.

In certain embodiments, the first antigen binding domain is linked to the N-terminus or C-terminus of the second antigen binding domain through the L2.

In certain embodiments, the bispecific antigen binding molecule comprises the sequence shown in SEQ ID NO:22 or a variant thereof, which has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:22, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions.

Preparation of bispecific antigen-binding molecules

The bispecific antigen binding molecules of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant technology. For example, a DNA molecule encoding the bispecific antigen binding molecule is obtained by chemical synthesis or PCR amplification, the resulting DNA molecule is inserted into an expression vector, and then transfected into a host cell. Then, the transfected host cell is cultured under specific conditions and the bispecific antigen binding molecules of the present invention are expressed.

Therefore, the second aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a bispecific antigen binding molecule of the present invention.

The third aspect of the present invention provides a vector (e.g., a cloning vector or an expression vector) comprising an isolated nucleic acid molecule as described above. In certain embodiments, the vector of the present invention is, for example, a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR/Cas9 vector or a viral vector; preferably, the vector is an expression vector; preferably, the vector is an episomal vector; preferably, the vector is a viral vector; more preferably, the viral vector is a lentiviral vector, an adenoviral vector or a retroviral vector.

A fourth aspect of the present invention provides a host cell comprising the isolated nucleic acid molecule or vector as described above. Such host cells include, but are not limited to, prokaryotic cells such as Escherichia coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).

In another aspect, the present invention also relates to a method for preparing the bispecific antigen binding molecule of the present invention, comprising culturing the host cell as described above under conditions allowing protein expression, and recovering the bispecific antigen binding molecule from the cultured host cell culture.

Bispecific chimeric antigen receptor

The present invention relates to CAR targeting MSLN and NKG2DL, which features include non-MHC restricted MSLN and NKG2DL recognition capabilities, which endow immune cells (e.g., T cells, NK cells, monocytes, macrophages or dendritic cells) expressing the CAR with the ability to recognize cells expressing MSLN and NKG2DL (e.g., tumor cells) independently of antigen processing and presentation.

Therefore, the fifth aspect of the present invention provides a bispecific chimeric antigen receptor, which comprises a bispecific antigen binding domain, a spacer domain, a transmembrane domain and an intracellular signaling domain.

I. Extracellular antigen binding domain

The antigen binding domain contained in the bispecific chimeric antigen receptor of the present invention confers the ability of the CAR to recognize MSLN and NKG2DL.

In certain embodiments, the bispecific antigen binding domain comprises the bispecific antigen binding molecule described in the first aspect.

In certain embodiments, the bispecific antigen binding domain comprises a first antigen binding domain capable of specifically binding to MSLN and a second antigen binding domain capable of specifically binding to NKG2D ligand (NKG2DL); wherein the first antigen binding domain and the second antigen binding domain are as defined in the first aspect.

In certain embodiments, the first antigen binding domain is a single chain antibody, and the first antigen binding domain is connected to the N-terminus or C-terminus of the second antigen binding domain via a linker L2.

In certain embodiments, the bispecific antigen-binding domain comprises the sequence shown in SEQ ID NO:22 or a variant thereof, which has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:22, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions.

II. Transmembrane domain

The transmembrane domain included in the bispecific chimeric antigen receptor of the present invention can be any protein structure known in the art, as long as it can be thermodynamically stable in a cell membrane (particularly a eukaryotic cell membrane). The transmembrane domain suitable for the bispecific CAR of the present invention can be derived from a natural source. In such embodiments, the transmembrane domain can be derived from any membrane-bound or transmembrane protein. Alternatively, the transmembrane domain can be a synthetic non-naturally occurring protein segment, such as a protein segment mainly comprising hydrophobic residues such as leucine and valine.

In certain embodiments, the transmembrane domain is a transmembrane region selected from the following proteins: α, β or ζ chain of T cell receptor, CD28, CD45, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD152, CD154 and PD-1 and any combination thereof. In certain preferred embodiments, the transmembrane domain is a transmembrane region selected from the following proteins: CD8α, CD28, CD4, PD-1, CD152 and CD154. In certain embodiments, the transmembrane domain comprises a CD8α transmembrane region as shown in SEQ ID NO:42 or a CD28 transmembrane region as shown in SEQ ID NO:44.

III. Spacer Domain

The bispecific chimeric antigen receptor of the present invention comprises a spacer domain located between the extracellular antigen binding domain and the transmembrane domain.

In certain embodiments, the spacer domain comprises the CH2 and CH3 regions of an immunoglobulin (e.g., IgG1 or IgG4). In such embodiments, without being bound by a particular theory, it is believed that CH2 and CH3 extend the antigen binding domain of the bispecific CAR from the cell membrane of the cell expressing the bispecific CAR and can more accurately mimic the size and domain structure of a natural TCR.

In certain embodiments, the spacer domain comprises a hinge domain. A hinge domain can be an amino acid segment usually found between two domains of a protein, which can allow the protein to be flexible and allow one or two domains to move relative to each other. Therefore, the hinge domain can be any amino acid sequence as long as it can provide this flexibility of the extracellular antigen binding domain and its mobility relative to the transmembrane domain.

In certain embodiments, the hinge domain is a hinge region or a portion thereof of a naturally occurring protein. In certain embodiments, the spacer domain is selected from a hinge domain and/or an immunoglobulin (e.g., IgG1 or IgG4) CH2 and CH3 region. In certain embodiments, the hinge domain comprises a hinge region of CD8α, IgG4, PD-1, CD152, or CD154. In certain embodiments, the hinge domain comprises a CD8α hinge region having a sequence as shown in SEQ ID NO:38 or an IgG4 hinge region having a sequence as shown in SEQ ID NO:40.

IV. Signal Peptide

In certain embodiments, the bispecific CAR of the present invention may further include a signal peptide SP1 at its N-terminus. Typically, a signal peptide is a polypeptide sequence that targets a sequence connected thereto to a desired site. In certain embodiments, the signal peptide can target the bispecific CAR connected thereto to the secretory pathway of the cell and allow the bispecific CAR to be further integrated and anchored into the lipid bilayer. Signal peptides that can be used for CAR are known to those skilled in the art. In certain embodiments, the signal peptide SP1 includes a heavy chain signal peptide (eg, a heavy chain signal peptide of IgG1), a granulocyte-macrophage colony stimulating factor receptor 2 (GM-CSFR2) signal peptide, an IL2 signal peptide, or a CD8α signal peptide. In certain preferred embodiments, the signal peptide SP1 is selected from a CD8α signal peptide. In certain exemplary embodiments, the signal peptide SP1 includes an amino acid sequence shown in SEQ ID NO:34.

In certain embodiments, the bispecific CAR of the present invention can also be co-expressed with another bioactive molecule (e.g., an antigen binding molecule targeting NKG2D ligand (NKG2DL), or a PD1/PD-L1 pathway inhibitor and an IL-15 agonist). The other bioactive molecule may have its own proprietary signal peptide, which is named signal peptide SP2 to distinguish it from the signal peptide in the previous paragraph. Signal peptide SP2 guides the transport of other bioactive molecules to a specific site in the cell or outside the cell membrane. The signal peptide SP2 may be the same or different from the signal peptide SP1 described in the previous paragraph. Preferably, the signal peptide SP2 may be different from the signal peptide SP1 described in the previous paragraph.

V. Intracellular signaling domain

The intracellular signaling domain contained in the bispecific CAR of the present invention is involved in transducing the signal generated by the binding of the bispecific CAR of the present invention to MSLN and/or NKG2DL into the interior of the immune effector cell, activating at least one normal effector function of the immune effector cell expressing the bispecific CAR, or enhancing the secretion of at least one cytokine (e.g., IL-2, IFN-γ) of the immune effector cell expressing the bispecific CAR.

In certain embodiments, the intracellular signaling domain comprises a primary signaling domain and/or a co-stimulatory signaling domain.

In certain embodiments, the primary signaling domain may be any intracellular signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM). In certain embodiments, the primary signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM). In certain embodiments, the primary signaling domain comprises an intracellular signaling domain of a protein selected from the group consisting of CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b, or CD66d. In certain embodiments, the primary signaling domain comprises an intracellular signaling domain of CD3ζ.

In certain embodiments, the costimulatory signaling domain may be an intracellular signaling domain from a costimulatory molecule. In certain embodiments, the costimulatory signaling domain comprises an intracellular signaling domain of a protein selected from the group consisting of CARD11, CD2, CD7, CD27, CD28, CD30, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD270 (HVEM), CD278 (ICOS) or DAP10.

In certain embodiments, the co-stimulatory signaling domain is selected from the intracellular signaling domain of CD28, or the intracellular signaling domain of CD137 (4-1BB), or a combination of fragments of both.

In certain embodiments, the intracellular signaling domain comprises a co-stimulatory signaling domain. In certain embodiments, the intracellular signaling domain comprises two or more co-stimulatory signaling domains. In such embodiments, the two or more co-stimulatory signaling domains may be the same or different.

In certain embodiments, the intracellular signaling domain comprises a primary signaling domain and at least one co-stimulatory signaling domain. The primary signaling domain and at least one co-stimulatory signaling domain can be connected in series to the carboxyl terminus of the transmembrane domain in any order.

In certain embodiments, the intracellular signaling domain may include the intracellular signaling domain of CD3 zeta and the intracellular signaling domain of CD137. In certain exemplary embodiments, the intracellular signaling domain of CD3 zeta includes the amino acid sequence shown in SEQ ID NO: 48. In certain exemplary embodiments, the intracellular signaling domain of CD137 includes the amino acid sequence shown in SEQ ID NO: 46.

In certain exemplary embodiments, the intracellular signaling domain of the chimeric antigen receptor has the sequence shown in SEQ ID NO:50.

VI. Full-length CAR

The present invention provides a chimeric antigen receptor capable of specifically binding to MSLN and NKG2DL, wherein the chimeric antigen receptor comprises a bispecific antigen binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain from its N-terminus to its C-terminus. In certain preferred embodiments, the intracellular signaling domain is a co-stimulatory signaling domain and a primary signaling domain from the N-terminus to the C-terminus.

In certain embodiments, preferably, the spacer domain comprises a hinge region of CD8 (eg, CD8α) or IgG4 (eg, a hinge region whose sequence is shown in SEQ ID NO: 38 or 40).

In certain embodiments, the transmembrane domain comprises the transmembrane region of CD8 (eg, CD8α) or CD28 (eg, the transmembrane region whose sequence is shown in SEQ ID NO: 42 or 44).

In certain embodiments, the intracellular signaling domain comprises a primary signaling domain and a co-stimulatory signaling domain, wherein the primary signaling domain comprises the intracellular signaling domain of CD3ζ (e.g., the sequence shown in SEQ ID NO:48), and the co-stimulatory signaling domain comprises the intracellular signaling domain of CD137 (4-1BB) (e.g., the sequence shown in SEQ ID NO:46); more preferably, the intracellular signaling domain of the chimeric antigen receptor has the sequence shown in SEQ ID NO:50.

In certain preferred embodiments, the chimeric antigen receptor comprises, from its N-terminus to its C-terminus, the signal peptide SP1, a bispecific antigen binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain (from the N-terminus to the C-terminus are a co-stimulatory signaling domain and a primary signaling domain).

In certain embodiments, the signal peptide SP1 comprises a heavy chain signal peptide of IgG1 or a CD8α signal peptide (e.g., a signal peptide having a sequence as shown in SEQ ID NO: 34). In certain exemplary embodiments, the bispecific CAR of the present invention comprises a sequence as shown in SEQ ID NO: 23 or a variant thereof, wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution.

VII. Co-expression of Bispecific CAR and Additional Biologically Active Molecules

In some cases, the bispecific CAR described in the fifth aspect of the present invention can also be co-expressed with another biologically active molecule. The self-cleaving peptide can prevent the amino acids from forming covalent bonds during the translation process and maintain the translation to continue, so that the translation product is "self-cleaved", thereby separating the bispecific chimeric antigen receptor of the present invention from another biologically active molecule. Therefore, when the bispecific CAR described in the fifth aspect of the present invention can also be co-expressed with another biologically active molecule, the chimeric antigen receptor that can specifically bind to MSLN and NKG2DL becomes an independent CAR having an extracellular antigen binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain, while the other biologically active molecules can be secreted outside the cell or expressed as a membrane-forming chimeric polypeptide or protein. As the immune cells expressing the bispecific CAR proliferate and enrich in the tumor microenvironment, the other biologically active molecules are enriched in the tumor microenvironment and synergize with the bispecific CAR to exert an anti-tumor effect.

In certain embodiments, the additional biologically active molecule comprises a PD1/PD-L1 pathway inhibitor and an IL-15 agonist.

In certain embodiments, the nucleic acid sequence encoding the bispecific CAR is connected to the nucleic acid sequence of another biologically active molecule through the nucleic acid sequence of the self-cleaving peptide. The bispecific CAR can be at the N-terminus or C-terminus of another biologically active molecule. In certain exemplary embodiments, the bispecific CAR is at the 5' end of another biologically active molecule. Any self-cleaving peptide that can cause the fusion protein to be cleaved into two independent proteins can be applied to the present invention. In certain exemplary embodiments, the self-cleaving peptide is P2A, preferably having the sequence shown in SEQ ID NO: 27, and its nucleotide sequence can be optimized according to the needs of genetic recombination. In such embodiments, the fusion protein comprising a bispecific CAR and another biologically active molecule has the following structure:

N'-signal peptide SP1--extracellular antigen binding domain that specifically binds to MSLN and NKG2DL--spacer domain--transmembrane domain--intracellular signal transduction domain--self-cleaving peptide--signal peptide SP2--another biologically active molecule-C', wherein the signal peptide SP2 is the same as or different from the signal peptide SP1.

In certain embodiments, the signal peptide SP2 at the N-terminus of the additional biologically active molecule is an IL2 signal peptide (eg, as shown in SEQ ID NO: 36).

In certain embodiments, the PD1/PD-L1 pathway inhibitor and the IL-15 agonist included in the additional biologically active molecule may be further linked via a self-cleaving peptide.

In certain embodiments, the PD1/PD-L1 pathway inhibitor is selected from an anti-PD-1 or PD-L1 antibody or an antigen-binding fragment thereof; preferably, the anti-PD-1 or PD-L1 antibody or an antigen-binding fragment thereof is a single-chain antibody (e.g., scFv); preferably, the anti-PD-1 single-chain antibody comprises the amino acid sequence shown in SEQ ID NO:52 or a variant thereof, wherein the variant has a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:52, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions.

In certain embodiments, the IL-15 agonist is selected from a fusion protein comprising IL-15 and an IL-15 receptor α (IL-15Rα) Sushi domain; preferably, the IL-15 agonist comprises the amino acid sequence shown in SEQ ID NO: 54 or a variant thereof, wherein the variant has a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 54, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions.

Co-expression of MSLN-specific chimeric antigen receptor and additional bioactive molecules

The sixth aspect of the present invention also relates to the co-expression of MSLN-specific chimeric antigen receptors with additional biologically active molecules (e.g., antigen binding molecules targeting NKG2D ligands, or PD1/PD-L1 pathway inhibitors and IL-15 agonists).

The MSLN-specific chimeric antigen receptor comprises an antigen binding domain targeting MSLN, a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein the antigen binding domain targeting MSLN comprises the first antigen binding domain defined in the first aspect, and the spacer domain, the transmembrane domain and the intracellular signaling domain are as defined in the fifth aspect.

Similar to the bispecific CAR described in the fifth aspect, the MSLN-specific chimeric antigen receptor can be co-expressed with another biologically active molecule. By using a self-cleaving peptide, the chimeric antigen receptor that specifically binds to MSLN becomes an independent CAR having an extracellular antigen binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain, while the other biologically active molecules can be secreted outside the cell or expressed as a membrane-chimeric polypeptide or protein.

The additional biologically active molecules include antigen binding molecules targeting NKG2D ligand (NKG2DL), or include PD1/PD-L1 pathway inhibitors and IL-15 agonists.

In certain embodiments, the nucleic acid sequence encoding the MSLN-specific CAR is connected to the nucleic acid sequence of another biologically active molecule through the nucleic acid sequence of the self-cleaving peptide. The MSLN-specific CAR can be at the N-terminus or C-terminus of another biologically active molecule. In certain exemplary embodiments, the MSLN-specific CAR is at the 5' end of another biologically active molecule. Any self-cleaving peptide that can cause the fusion protein to be cleaved into two independent proteins can be applied to the present invention. In certain exemplary embodiments, the self-cleaving peptide is P2A, preferably having the sequence shown in SEQ ID NO: 27, and its nucleotide sequence can be optimized according to the needs of genetic recombination. In such embodiments, the fusion protein comprising the MSLN-specific CAR and another biologically active molecule has the following structure:

N'-signal peptide SP1--extracellular antigen binding domain that specifically binds to MSLN--spacer domain-transmembrane domain-intracellular signal transduction domain-self-cleaving peptide-optional signal peptide SP2--other biologically active molecules-C'. Wherein, the signal peptide SP2 may be optional depending on the other biologically active molecules. When the signal peptide SP2 is contained, the signal peptide SP2 is the same as or different from the signal peptide SP1.

In certain embodiments, the signal peptide SP2 at the N-terminus of the additional biologically active molecule is an IL2 signal peptide (e.g., as shown in SEQ ID NO: 36).

In certain embodiments, when the additional biologically active molecule is an antigen binding molecule that targets NKG2D ligand (NKG2DL), the N-terminus of the additional biologically active molecule does not comprise a signal peptide.

In certain embodiments, when the additional biologically active molecule is a PD1/PD-L1 pathway inhibitor and an IL-15 agonist, the N-terminus of the additional biologically active molecule comprises a signal peptide SP2.

In certain embodiments, the antigen binding molecule targeting NKG2D ligand (NKG2DL) comprises NKG2D or its ligand binding domain. In certain embodiments, the antigen binding molecule targeting NKG2D ligand comprises a sequence shown in SEQ ID NO: 20 or 21 or a variant thereof, wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution.

In certain embodiments, the antigen binding molecule targeting NKG2D ligand (NKG2DL) is optionally connected to a 4-1BB intracellular signaling domain (e.g., a sequence as shown in SEQ ID NO: 46) at its N-terminus. In such embodiments, the additional biologically active molecule comprises a 4-1BB intracellular signaling domain and an antigen binding molecule targeting NKG2D ligand (NKG2DL) from N-terminus to C-terminus. In certain embodiments, the additional biologically active molecule comprises an amino acid sequence as shown in SEQ ID NO: 66.

In certain embodiments, the PD1/PD-L1 pathway inhibitor and the IL-15 agonist included in the additional biologically active molecule may be further linked via a self-cleaving peptide.

In certain embodiments, the PD1/PD-L1 pathway inhibitor is selected from an anti-PD-1 or PD-L1 antibody or an antigen-binding fragment thereof; preferably, the anti-PD-1 or PD-L1 antibody or an antigen-binding fragment thereof is a single-chain antibody (e.g., scFv); preferably, the anti-PD-1 single-chain antibody comprises the amino acid sequence shown in SEQ ID NO: 52 or a variant thereof, wherein the variant has a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 52, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions.

In certain embodiments, the IL-15 agonist is selected from a fusion protein comprising IL-15 and an IL-15 receptor α (IL-15Rα) Sushi domain; preferably, the IL-15 agonist comprises the amino acid sequence shown in SEQ ID NO: 54 or a variant thereof, wherein the variant has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 54, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions.

Preparation of chimeric antigen receptors

Methods for generating chimeric antigen receptors and immune effector cells (e.g., T cells) comprising the chimeric antigen receptor are known in the art, and may include transfecting cells with at least one polynucleotide encoding CAR, and expressing the polynucleotides in cells. For example, the nucleic acid molecules encoding the CAR of the present invention may be included in an expression vector (e.g., a lentiviral vector), which can be expressed in a host cell such as a T cell to manufacture the CAR.

Therefore, the seventh aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the bispecific chimeric antigen receptor described in the fifth aspect.

Those skilled in the art will appreciate that, due to the degeneracy of the genetic code, a nucleotide sequence encoding a chimeric antigen receptor of the present invention may have a variety of different sequences. Therefore, unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence.

In certain exemplary embodiments, the nucleotide sequence encoding the bispecific chimeric antigen receptor of the fifth aspect is selected from: (1) the sequence shown in SEQ ID NO: 24 or a degenerate variant thereof; (2) a sequence substantially identical to the sequence described in (1), for example, a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to (1), or a sequence having one or more nucleotide substitutions compared to the sequence described in (1); and the sequence substantially retains at least one biological activity of the nucleotide sequence from which it is derived (for example, being able to encode the ability to direct the specificity and reactivity of immune effector cells to cells expressing MSLN and/or NKG2DL in a non-MHC restricted manner).

As described in the fifth aspect above, the bispecific CAR of the present invention can also be co-expressed with other biologically active molecules to synergistically exert anti-tumor effects.

Therefore, the eighth aspect of the present invention also provides a nucleic acid construct, which comprises a first nucleic acid sequence encoding the bispecific chimeric antigen receptor described in the fifth aspect, and further comprises a second nucleic acid sequence encoding another biologically active molecule.

In certain embodiments, the additional biologically active molecule encoded by the second nucleic acid sequence has anti-tumor activity. In certain embodiments, the additional biologically active molecule comprises a PD1/PD-L1 pathway inhibitor and an IL-15 agonist.

In certain embodiments, the additional biologically active molecule encoded by the second nucleotide sequence further comprises a signal peptide SP2 at its N-terminus. In certain embodiments, the signal peptide SP2 is different from the signal peptide SP1 contained in the bispecific chimeric antigen receptor encoded by the first nucleic acid sequence. In certain embodiments, the signal peptide SP2 at the N-terminus of the additional biologically active molecule is an IL2 signal peptide, and the IL2 signal peptide refers to a signal peptide sequence contained in the IL2 natural gene sequence, preferably, the IL2 natural gene is a human IL2 natural gene, and the IL2 signal peptide is a human IL2 signal peptide. In certain exemplary embodiments, the IL2 signal peptide comprises a sequence as shown in SEQ ID NO: 36.

In certain embodiments, the first nucleotide sequence is located upstream of the second nucleotide sequence.

In certain embodiments, the first nucleic acid sequence and the second nucleic acid sequence are connected by a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A, E2A, F2A, T2A or any combination thereof). In certain embodiments, the self-cleaving peptide is P2A (e.g., as shown in SEQ ID NO: 27). In certain exemplary embodiments, the sequence encoding the self-cleaving peptide is connected to the 3' end of the first nucleotide sequence and to the 5' end of the second nucleotide sequence.

In certain embodiments, the PD1/PD-L1 pathway inhibitor is selected from an anti-PD-1 or PD-L1 single-chain antibody, for example comprising the amino acid sequence shown in SEQ ID NO:52.

In certain embodiments, the IL-15 agonist comprises the sequence shown in SEQ ID NO:54.

In certain embodiments, the nucleic acid sequence encoding the PD1/PD-L1 pathway inhibitor and the nucleic acid sequence encoding the IL-15 agonist are linked by a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A, E2A, F2A, T2A or any combination thereof). In certain embodiments, the self-cleaving peptide is P2A (e.g., as shown in SEQ ID NO: 27).

In certain exemplary embodiments, the nucleic acid construct described in the eighth aspect comprises, in order from its 5' end to the 3' end: a nucleotide sequence encoding the signal peptide SP1, a nucleotide sequence encoding the bispecific antigen binding domain, a nucleotide sequence encoding the spacer domain, a nucleotide sequence encoding the transmembrane domain, a nucleotide sequence encoding the intracellular signaling domain, a nucleotide sequence encoding the self-cleaving peptide sequence, a nucleotide sequence encoding the signal peptide SP2, a nucleotide sequence encoding the PD1/PD-L1 pathway inhibitor, a nucleotide sequence encoding the self-cleaving peptide sequence, and a nucleotide sequence encoding the IL-15 agonist.

As described above in the sixth aspect, the MSLN-specific chimeric antigen receptor can be co-expressed with an antigen binding molecule targeting NKG2D ligand (NKG2DL) to synergistically exert an anti-tumor effect.

Therefore, the ninth aspect of the present invention also provides a nucleic acid construct, which comprises:

(1) a first nucleic acid sequence encoding a chimeric antigen receptor specific for MSLN; and

(2) a second nucleic acid sequence encoding another biologically active molecule, wherein the other biologically active molecule comprises an antigen binding molecule targeting NKG2D ligand (NKG2DL); wherein,

The MSLN-specific chimeric antigen receptor comprises an MSLN-targeting antigen binding domain, a spacer domain, a transmembrane domain and an intracellular signaling domain, and the MSLN-targeting antigen binding domain comprises the first antigen binding domain defined in the first aspect.

In certain embodiments, the antigen binding molecule targeting NKG2D ligand (NKG2DL) comprises NKG2D or its ligand binding domain. In certain embodiments, the antigen binding molecule targeting NKG2D ligand comprises a sequence shown in SEQ ID NO: 20 or 21 or a variant thereof, wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution.

In certain embodiments, the antigen binding molecule targeting NKG2D ligand (NKG2DL) is optionally connected to a 4-1BB intracellular signaling domain (e.g., a sequence as shown in SEQ ID NO: 46) at its N-terminus. In such embodiments, the additional biologically active molecule comprises a 4-1BB intracellular signaling domain and an antigen binding molecule targeting NKG2D ligand (NKG2DL) from N-terminus to C-terminus. In certain embodiments, the additional biologically active molecule comprises an amino acid sequence as shown in SEQ ID NO: 66.

In certain embodiments, the first nucleic acid sequence and the second nucleic acid sequence are connected by a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A, E2A, F2A, T2A or any combination thereof). In certain embodiments, the self-cleaving peptide is P2A; for example, the nucleotide sequence encoding the self-cleaving peptide is shown in SEQ ID NO: 28 or 29.

In certain embodiments, the transmembrane domain, the spacer domain, and the intracellular signaling domain comprised by the MSLN-specific chimeric antigen receptor encoded by the first nucleic acid sequence are as defined in the fifth aspect.

In certain embodiments, the MSLN-specific chimeric antigen receptor encoded by the first nucleic acid sequence further comprises a signal peptide SP1 at its N-terminus, and the signal peptide SP1 is as defined in the fifth aspect.

In certain embodiments, the additional biologically active molecule encoded by the second nucleotide sequence does not contain a signal peptide at its N-terminus. In certain embodiments, the additional biologically active molecule encoded by the second nucleotide sequence further contains a signal peptide SP2 at its N-terminus; preferably, the signal peptide SP2 is different from the signal peptide SP1 contained in the MSLN-specific chimeric antigen receptor encoded by the first nucleic acid sequence; preferably, the signal peptide SP2 at the N-terminus of the additional biologically active molecule is an IL2 signal peptide (e.g., as shown in SEQ ID NO: 36).

In certain embodiments, the nucleic acid construct comprises, from its 5' end to its 3' end, in sequence: a nucleotide sequence encoding the signal peptide SP1, a nucleotide sequence encoding the antigen binding domain targeting MSLN, a nucleotide sequence encoding the spacer domain, a nucleotide sequence encoding the transmembrane domain, a nucleotide sequence encoding the intracellular signaling domain, a nucleotide sequence encoding the self-cleaving peptide sequence, and a nucleotide sequence encoding the antigen binding molecule targeting NKG2D ligand (NKG2DL).

In certain embodiments, the signal peptide SP1 comprises a heavy chain signal peptide of IgG1 or a CD8α signal peptide (eg, a signal peptide whose sequence is shown in SEQ ID NO: 34).

In certain embodiments, the antigen binding domain targeting MSLN is selected from the first antigen binding domain defined in the first aspect (eg, comprising the sequence shown in SEQ ID NO: 18).

In certain embodiments, the spacer domain comprises a hinge region of CD8 (eg, CD8α) or IgG4 (eg, a hinge region whose sequence is shown in SEQ ID NO: 38 or 40).

In certain embodiments, the transmembrane domain comprises the transmembrane region of CD8 (eg, CD8α) or CD28 (eg, the transmembrane region whose sequence is shown in SEQ ID NO: 42 or 44).

In certain embodiments, the intracellular signaling domain comprises a primary signaling domain and a co-stimulatory signaling domain, wherein the primary signaling domain comprises the intracellular signaling domain of CD3ζ (e.g., the sequence shown in SEQ ID NO:48), and the co-stimulatory signaling domain comprises the intracellular signaling domain of CD137 (4-1BB) (e.g., the sequence shown in SEQ ID NO:46); more preferably, the intracellular signaling domain of the chimeric antigen receptor has the sequence shown in SEQ ID NO:50.

In certain embodiments, the self-cleaving peptide sequence is P2A, for example having the sequence shown in SEQ ID NO:27.

In certain embodiments, the antigen binding molecule targeting NKG2D ligand comprises NKG2D or its ligand-binding domain, for example, comprises the sequence shown in SEQ ID NO: 20 or 21.

In certain embodiments, the nucleic acid construct further comprises a nucleotide sequence encoding a 4-1BB intracellular signaling domain between the nucleotide sequence encoding the self-cleaving peptide sequence and the nucleotide sequence encoding the antigen binding molecule targeting NKG2D ligand (NKG2DL). In certain embodiments, the 4-1BB intracellular signaling domain has a sequence shown in SEQ ID NO: 46.

In certain exemplary embodiments, the nucleic acid construct comprises a nucleotide sequence selected from the group consisting of: (1) a sequence shown in SEQ ID NO: 26 or a degenerate variant thereof; (2) a sequence substantially identical to the sequence shown in any one of (1) (e.g., a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence shown in any one of (1), or a sequence having one or more nucleotide substitutions compared to the sequence shown in any one of (1)).

As described in the sixth aspect above, the MSLN-specific chimeric antigen receptor can be co-expressed with a PD1/PD-L1 pathway inhibitor and an IL-15 agonist to synergistically exert an anti-tumor effect.

Therefore, the tenth aspect of the present invention also provides a nucleic acid construct comprising:

(1) a first nucleic acid sequence encoding a chimeric antigen receptor specific for MSLN; and

(2) a second nucleic acid sequence encoding another biologically active molecule, wherein the other biologically active molecule comprises a PD1/PD-L1 pathway inhibitor and an IL-15 agonist; wherein,

The MSLN-specific chimeric antigen receptor comprises an MSLN-targeting antigen binding domain, a spacer domain, a transmembrane domain and an intracellular signaling domain, and the MSLN-targeting antigen binding domain comprises the first antigen binding domain defined in the first aspect.

In certain embodiments, the first nucleic acid sequence and the second nucleic acid sequence are linked by a nucleotide sequence encoding a self-cleaving peptide (eg, P2A, E2A, F2A, T2A, or any combination thereof).

In certain embodiments, the nucleotide sequence encoding the PD1/PD-L1 pathway inhibitor and the nucleotide sequence encoding the IL-15 agonist are linked by a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A, E2A, F2A, T2A or any combination thereof).

In certain embodiments, the self-cleaving peptide is P2A; for example, the nucleotide sequence encoding the self-cleaving peptide is shown in SEQ ID NO: 28 or 29.

In certain embodiments, the transmembrane domain, the spacer domain, and the intracellular signaling domain comprised by the MSLN-specific chimeric antigen receptor encoded by the first nucleic acid sequence are as defined in the fifth aspect.

In certain embodiments, the MSLN-specific chimeric antigen receptor encoded by the first nucleic acid sequence further comprises a signal peptide SP1 at its N-terminus, and the signal peptide SP1 is as defined in the fifth aspect.

In certain embodiments, the additional biologically active molecule encoded by the second nucleotide sequence further comprises a signal peptide SP2 at its N-terminus; preferably, the signal peptide SP2 is different from the signal peptide SP1 contained in the MSLN-specific chimeric antigen receptor encoded by the first nucleic acid sequence; preferably, the signal peptide SP2 at the N-terminus of the additional biologically active molecule is an IL2 signal peptide (for example, as shown in SEQ ID NO: 36).

In certain embodiments, the PD1/PD-L1 pathway inhibitor is selected from an anti-PD-1 or PD-L1 antibody or an antigen-binding fragment thereof; preferably, the anti-PD-1 or PD-L1 antibody or an antigen-binding fragment thereof is a single-chain antibody (e.g., scFv); preferably, the anti-PD-1 single-chain antibody comprises the amino acid sequence shown in SEQ ID NO:52 or a variant thereof, wherein the variant has a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:52, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions.

In certain embodiments, the IL-15 agonist is selected from a fusion protein comprising IL-15 and an IL-15 receptor α (IL-15Rα) Sushi domain; preferably, the IL-15 agonist comprises the amino acid sequence shown in SEQ ID NO: 54 or a variant thereof, wherein the variant has a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 54, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions.

In certain embodiments, the nucleic acid construct comprises, from its 5' end to its 3' end, a nucleotide sequence encoding the signal peptide SP1, a nucleotide sequence encoding the antigen binding domain targeting MSLN, a nucleotide sequence encoding the spacer domain, a nucleotide sequence encoding the transmembrane domain, a nucleotide sequence encoding the intracellular signaling domain, a nucleotide sequence encoding the self-cleaving peptide sequence, a nucleotide sequence encoding the signal peptide SP2, a nucleotide sequence encoding the PD1/PD-L1 pathway inhibitor, a nucleotide sequence encoding the self-cleaving peptide sequence, and a nucleotide sequence encoding the IL-15 agonist.

In certain embodiments, the signal peptide SP1 comprises a heavy chain signal peptide of IgG1 or a CD8α signal peptide (eg, a signal peptide whose sequence is shown in SEQ ID NO: 34).

In certain embodiments, the antigen binding domain targeting MSLN comprises the first antigen binding domain defined in the first aspect (eg, comprising the sequence shown in SEQ ID NO: 18).

In certain embodiments, the spacer domain comprises a hinge region of CD8 (eg, CD8α) or IgG4 (eg, a hinge region whose sequence is shown in SEQ ID NO: 38 or 40).

In certain embodiments, the transmembrane domain comprises the transmembrane region of CD8 (eg, CD8α) or CD28 (eg, the transmembrane region whose sequence is shown in SEQ ID NO: 42 or 44).

In certain embodiments, the intracellular signaling domain comprises a primary signaling domain and a co-stimulatory signaling domain, wherein the primary signaling domain comprises the intracellular signaling domain of CD3ζ (e.g., the sequence shown in SEQ ID NO:48), and the co-stimulatory signaling domain comprises the intracellular signaling domain of CD137 (4-1BB) (e.g., the sequence shown in SEQ ID NO:46); more preferably, the intracellular signaling domain of the chimeric antigen receptor has the sequence shown in SEQ ID NO:50.

In certain embodiments, the self-cleaving peptide sequence is P2A, for example having the sequence shown in SEQ ID NO:27.

In certain embodiments, the signal peptide SP2 comprises an IL2 signal peptide (eg, as shown in SEQ ID NO: 36).

In certain embodiments, the PD1/PD-L1 pathway inhibitor is selected from an anti-PD-1 or PD-L1 single-chain antibody, for example comprising the amino acid sequence shown in SEQ ID NO:52.

In certain embodiments, the IL-15 agonist comprises the sequence shown in SEQ ID NO:54.

In certain exemplary embodiments, the nucleic acid construct comprises a nucleotide sequence selected from the group consisting of: (1) a sequence shown in SEQ ID NO:57 or a degenerate variant thereof; (2) a sequence substantially identical to the sequence shown in any one of (1) (e.g., a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence shown in any one of (1), or a sequence having one or more nucleotide substitutions compared to the sequence shown in any one of (1)).

The eleventh aspect of the present invention provides a vector comprising the isolated nucleic acid molecule described in the seventh aspect, or the nucleic acid construct described in the eighth aspect, the ninth aspect or the tenth aspect.

In certain embodiments, the vector is selected from a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR/Cas9 vector, and a viral vector.

In certain embodiments, the vector is an expression vector.

In certain embodiments, the vector is an episomal vector.

In certain embodiments, the vector is a viral vector.

In certain exemplary embodiments, the viral vector is a lentiviral vector, an adenoviral vector, or a retroviral vector.

In certain embodiments, the vector is an episomal or non-integrating viral vector, such as an integration-defective retrovirus or lentivirus.

Engineered immune cells

The twelfth aspect of the present invention further provides a modified immune cell, which comprises and expresses the isolated nucleic acid molecule described in the seventh aspect of the present invention. The modified immune cell expresses the bispecific chimeric antigen receptor described in the fifth aspect.

The thirteenth aspect of the present invention further provides a modified immune cell, which comprises and expresses the nucleic acid construct described in the eighth aspect of the present invention. The modified immune cell expresses the bispecific chimeric antigen receptor described in the fifth aspect and the additional biologically active molecule, such as a PD1/PD-L1 pathway inhibitor and an IL-15 agonist.

The fourteenth aspect of the present invention also provides a modified immune cell, which comprises a nucleic acid construct expressing the ninth aspect of the present invention. The modified immune cell expresses a MSLN-specific chimeric antigen receptor and an antigen binding molecule targeting an NKG2D ligand (NKG2DL). In certain embodiments, the antigen binding molecule targeting an NKG2D ligand (NKG2DL) is optionally connected to a 4-1BB intracellular signaling domain at its N-terminus.

The fifteenth aspect of the present invention further provides a modified immune cell, which comprises and expresses the nucleic acid construct described in the tenth aspect of the present invention. The modified immune cell expresses a MSLN-specific chimeric antigen receptor, a PD1/PD-L1 pathway inhibitor, and an IL-15 agonist.

In certain embodiments, the immune cells are derived from T lymphocytes, NK cells, monocytes, macrophages or dendritic cells and any combination thereof; preferably, the immune cells are obtained from patients; alternatively, the immune cells are obtained from healthy donors; preferably, the immune cells are derived from T lymphocytes or NK cells.

In certain embodiments, the transcription or expression of one or two target genes among the genes related to immune exclusion (e.g., TRAC, TRBC, B2M, HLA-A, HLA-B or HLA-C) and genes of immune co-inhibitory pathways or signaling molecules (e.g., PD-1, CTLA-4 or LAG-3) of the engineered immune cells is inhibited; preferably, the transcription or expression of the target genes is inhibited by a method selected from gene knockout (e.g., CRISPR, CRISPR/Cas9), homologous recombination, and interfering RNA.

The present invention also provides a method for preparing modified immune cells, which comprises: (1) providing immune cells from a patient or a healthy donor; (2) introducing the isolated nucleic acid molecule described in the seventh aspect, or the nucleic acid construct described in the eighth aspect, the ninth aspect or the tenth aspect, or a vector containing them into the immune cells described in step (1) to obtain immune cells capable of expressing and optionally other biologically active molecules.

In certain embodiments, in step (1), the immune cells are pretreated, and the pretreatment includes sorting, activation and/or proliferation of the immune cells; more preferably, the pretreatment includes contacting the immune cells with anti-CD3 antibodies and anti-CD28 antibodies, thereby stimulating the immune cells and inducing their proliferation, thereby generating pretreated immune cells.

In certain embodiments, in step (2), the nucleic acid molecule or vector is introduced into the immune cells by viral infection.

In certain embodiments, in step (2), the nucleic acid molecule or vector is introduced into the immune cell by non-viral vector transfection, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, transposon vector system, CRISPR/Cas9 vector, TALEN method, ZFN method or electroporation method.

In certain embodiments, the method further comprises the step of expanding the immune cells obtained in step (2) after step (2).

Immune cell composition

In the sixteenth aspect, the present invention also provides an immune cell composition, which includes the modified immune cells of any of the foregoing aspects, and optional unmodified and/or unsuccessfully modified immune cells, which do not express the target CAR. Limited by the current level of technology and some unknown reasons, not all immune cells can express the target CAR after modification. Moreover, immune cells that do not express CAR also have certain biological activities, so the immune cell composition can contain immune cells that express and do not express the target CAR, and the immune cell composition can still meet the needs of clinical applications.

In certain embodiments, the engineered immune cells expressing the target CAR account for approximately 10%-100%, preferably 40%-80% of the total cell number of the immune cell composition.

In certain embodiments, the immune cell composition is cultured into an immune cell line. Therefore, in another aspect, the present invention also provides an immune cell line containing the immune cell composition.

In another aspect, the present invention provides a kit for preparing the modified immune cells described in any of the above aspects. In certain embodiments, the kit includes the isolated nucleic acid molecule described in the seventh aspect, or the nucleic acid construct described in the eighth aspect, the ninth aspect, or the tenth aspect, or a vector comprising them, and necessary solvents, such as sterile water, physiological saline, or cell culture fluid, such as LB culture fluid, such as EliteCell primary T lymphocyte culture system (Product No.: PriMed-EliteCell-024), and optionally, instructions for use.

Pharmaceutical composition

In the seventeenth aspect, the present invention provides a pharmaceutical composition comprising the bispecific antigen binding molecule described in the first aspect of the present invention, the bispecific chimeric antigen receptor described in the fifth aspect (including a CAR construct co-expressing a bispecific chimeric antigen receptor and another biologically active molecule), the isolated nucleic acid molecule described in the second aspect or the seventh aspect, the nucleic acid construct described in the eighth aspect or the ninth aspect or the tenth aspect, the vector described in the third aspect or the eleventh aspect, the host cell described in the fourth aspect, the modified immune cell described in the twelfth aspect, the thirteenth aspect, the fourteenth aspect or the fifteenth aspect, or the immune cell composition described in the sixteenth aspect, and a pharmaceutically acceptable carrier and/or excipient.

In certain embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent, such as a drug with anti-tumor activity (e.g., anti-PD1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, pemetrexed, cisplatin, paclitaxel, gemcitabine, capecitabine or FOLFIRINOX).

In certain embodiments, the bispecific antigen binding molecule of the first aspect, the bispecific chimeric antigen receptor of the fifth aspect (including a CAR construct in which the bispecific chimeric antigen receptor is co-expressed with another biologically active molecule), the isolated nucleic acid molecule of the second aspect or the seventh aspect, the nucleic acid construct of the eighth aspect or the ninth aspect or the tenth aspect, the vector of the third aspect or the eleventh aspect, the host cell of the fourth aspect, the modified immune cell of the twelfth aspect, the thirteenth aspect, the fourteenth aspect or the fifteenth aspect, or the immune cell composition of the sixteenth aspect and the additional pharmaceutically active agent can be administered simultaneously, separately or sequentially.

In certain embodiments, the pharmaceutical composition of the present invention comprises: the bispecific antigen binding molecule described in the first aspect.

In certain embodiments, the pharmaceutical composition of the present invention comprises: the isolated nucleic acid molecule of the seventh aspect, the nucleic acid construct of the eighth aspect, the ninth aspect or the tenth aspect, or a vector comprising them.

In certain embodiments, the pharmaceutical composition of the present invention comprises: the modified immune cell described in the twelfth aspect, the thirteenth aspect, the fourteenth aspect or the fifteenth aspect, or the immune cell composition described in the sixteenth aspect.

The bispecific antigen binding molecules described in the first aspect of the present invention, the bispecific chimeric antigen receptor described in the fifth aspect (including a CAR construct co-expressed with a bispecific chimeric antigen receptor and another bioactive molecule), the isolated nucleic acid molecules described in the second aspect or the seventh aspect, the nucleic acid construct described in the eighth aspect or the ninth aspect or the tenth aspect, the vector described in the third aspect or the eleventh aspect, the host cell described in the fourth aspect, the modified immune cell described in the twelfth aspect or the thirteenth aspect or the fourteenth aspect or the fifteenth aspect, or the immune cell composition described in the sixteenth aspect can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical composition of the present invention should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such injections can be sterile injection solutions. In addition, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze drying) for storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

The bispecific antigen binding molecules described in the first aspect of the present invention, the bispecific chimeric antigen receptor described in the fifth aspect (including a CAR construct co-expressed by a bispecific chimeric antigen receptor and another bioactive molecule), the nucleic acid molecules described in the second aspect or the seventh aspect, the nucleic acid construct described in the eighth aspect or the ninth aspect or the tenth aspect, the carrier described in the third aspect or the eleventh aspect, the host cell described in the fourth aspect, the twelfth aspect or the thirteenth aspect or the fourteenth aspect or the fifteenth aspect The modified immune cell or the immune cell composition described in the sixteenth aspect can be administered by any suitable method known in the art, including but not limited to, oral, oral, sublingual, eyeball, local, parenteral, rectal, leaf sheath, endocytoplasmic reticulum groove, groin, bladder, local (such as, powder, ointment or drops), or nasal route. However, for many therapeutic uses, the preferred route of administration/mode is parenteral administration (e.g., intravenous injection or push injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The technician should understand that the route of administration and/or mode will change according to the intended purpose. In certain embodiments, the bispecific antigen binding molecule of the first aspect of the present invention, the bispecific chimeric antigen receptor of the fifth aspect (including a CAR construct in which the bispecific chimeric antigen receptor is co-expressed with another biologically active molecule), the isolated nucleic acid molecule of the second aspect or the seventh aspect, the nucleic acid construct of the eighth aspect or the ninth aspect or the tenth aspect, the vector of the third aspect or the eleventh aspect, the host cell of the fourth aspect, the modified immune cell of the twelfth aspect or the thirteenth aspect or the fourteenth aspect or the fifteenth aspect, or the immune cell composition of the sixteenth aspect is administered by intravenous injection or push injection.

The pharmaceutical composition of the present invention may include a "therapeutically effective amount" or a "preventive effective amount" of the bispecific antigen binding molecule described in the first aspect of the present invention, the bispecific chimeric antigen receptor described in the fifth aspect (including a CAR construct co-expressed with a bispecific chimeric antigen receptor and another biologically active molecule), the isolated nucleic acid molecule described in the second aspect or the seventh aspect, the nucleic acid construct described in the eighth aspect or the ninth aspect or the tenth aspect, the vector described in the third aspect or the eleventh aspect, the host cell described in the fourth aspect, the modified immune cell described in the twelfth aspect or the thirteenth aspect or the fourteenth aspect or the fifteenth aspect, or the immune cell composition described in the sixteenth aspect. "Preventive effective amount" refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease. "Therapeutically effective amount" refers to an amount sufficient to cure or at least partially prevent the disease and its complications in patients who already have the disease. The therapeutically effective amount of the bispecific antigen binding molecule described in the first aspect of the present invention, the bispecific chimeric antigen receptor described in the fifth aspect (including a CAR construct in which the bispecific chimeric antigen receptor is co-expressed with another biologically active molecule), the isolated nucleic acid molecule described in the second aspect or the seventh aspect, the nucleic acid construct described in the eighth aspect or the ninth aspect or the tenth aspect, the vector described in the third aspect or the eleventh aspect, the host cell described in the fourth aspect, the modified immune cell described in the twelfth aspect or the thirteenth aspect or the fourteenth aspect or the fifteenth aspect, or the immune cell composition described in the sixteenth aspect may vary according to the following factors: the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and gender, the method of administration of the drug, and other treatments administered simultaneously, etc.

Treatment methods and uses

On the other hand, the present invention provides a method for preventing and/or treating a disease associated with the expression of mesothelin in a subject (e.g., a human), the method comprising administering to a subject in need thereof an effective amount of the bispecific antigen binding molecule of the first aspect of the present invention, the bispecific chimeric antigen receptor of the fifth aspect (including a CAR construct in which the bispecific chimeric antigen receptor is co-expressed with another biologically active molecule), the isolated nucleic acid molecule of the second aspect or the seventh aspect, the nucleic acid construct of the eighth aspect or the ninth aspect or the tenth aspect, the vector of the third aspect or the eleventh aspect, the host cell of the fourth aspect, the modified immune cell of the twelfth aspect or the thirteenth aspect or the fourteenth aspect or the fifteenth aspect, the immune cell composition of the sixteenth aspect, or the pharmaceutical composition of the seventeenth aspect.

In certain embodiments, the disease associated with the expression of mesothelin is selected from a proliferative disease, such as a tumor. In certain embodiments, the disease associated with the expression of mesothelin is a non-tumor-related indication associated with the expression of mesothelin.

In certain embodiments, the tumor is a MSLN positive tumor. In certain embodiments, the tumor is a MSLN positive and NKG2DL positive tumor. In certain embodiments, the tumor is selected from solid tumors (e.g., malignant pleural mesothelioma, pancreatic cancer, lung cancer (e.g., squamous cell lung cancer), breast cancer, ovarian cancer (e.g., epithelial ovarian cancer)).

In certain embodiments, the method comprises administering to the subject an effective amount of the bispecific antigen binding molecule of the first aspect.

In certain embodiments, the method comprises administering to the subject an effective amount of the engineered immune cell of aspect 12, aspect 13, aspect 14, or aspect 15, or the immune cell composition of aspect 16.

In certain embodiments, the method comprises the following steps: (1) providing the subject with the immune cells required (e.g., T lymphocytes, NK cells, monocytes, macrophages, dendritic cells, or any combination of these cells); (2) introducing the isolated nucleic acid molecule described in the seventh aspect, or the nucleic acid construct described in the eighth aspect, the ninth aspect or the tenth aspect, or a vector containing them into the immune cells described in step (1); (3) administering the immune cells obtained in step (2) to the subject for treatment.

In certain embodiments, the method administers immune cells expressing a CAR of interest to the subject by dosing, e.g., administering a partial dose in divided doses, e.g., one, two, three or more times, e.g., administering a first percentage of the total dose on the first day of treatment, administering a second percentage of the total dose on a subsequent (e.g., second, third, fourth, fifth, sixth or seventh day or later) treatment day, e.g., administering a third percentage (e.g., the remaining percentage) of the total dose on a subsequent (e.g., third, fourth, fifth, sixth, seventh, eighth, ninth, tenth day or later) treatment day.

In certain embodiments, 10% of the total dose of cells is administered on the first day of treatment, 30% of the total dose of cells is administered on the second day, and the remaining 60% of the total dose of cells is administered on the third day.

In certain embodiments, 50% of the total dose of cells is administered on the first day of treatment, and 50% of the total dose of cells is administered on a subsequent (e.g., second, third, fourth, fifth, sixth or seventh or later) treatment day. In certain embodiments, 1/3 of the total dose of cells is administered on the first day of treatment, 1/3 of the total dose of cells is administered on a subsequent (e.g., second, third, fourth, fifth, sixth or seventh day or later) treatment day, and 1/3 of the total dose of cells is administered on a subsequent (e.g., third, fourth, fifth, sixth, seventh, eighth, ninth, tenth day or later) treatment day.

In certain embodiments, the total cell dose comprises 1×10 ⁷ to 10×10 ⁸ target CAR-positive immune cells, for example, comprises (1-5)×10 ⁷ to (5-10)×10 ⁸ target CAR-positive immune cells.

In certain embodiments, the physician may adjust the dosage or treatment regimen based on clinical circumstances such as the patient's condition, tumor size and stage, or combination therapy drugs.

In certain embodiments, the bispecific antigen binding molecule described in the first aspect of the present invention, the bispecific chimeric antigen receptor described in the fifth aspect (including a bispecific chimeric antigen receptor and a CAR construct co-expressed with another bioactive molecule), the separated nucleic acid molecule described in the second aspect or the seventh aspect, the nucleic acid construct described in the eighth aspect or the ninth aspect or the tenth aspect, the carrier described in the third aspect or the eleventh aspect, the host cell described in the fourth aspect, the transformed immune cell described in the twelfth aspect or the thirteenth aspect or the fourteenth aspect or the fifteenth aspect, the immune cell composition described in the sixteenth aspect, or the pharmaceutical composition described in the seventeenth aspect and other agents are administered in combination. In certain embodiments, the other agents include (i) an agent that increases the efficacy of cells containing CAR nucleic acids or CAR polypeptides (e.g., immune cells expressing CAR of the present invention, modified immune cells or immune cell compositions of the present invention); (ii) an agent that improves one or more side effects associated with the administration of cells containing CAR nucleic acids or CAR polypeptides (e.g., immune cells expressing CAR of the present invention, modified immune cells or immune cell compositions of the present invention); (iii) another pharmaceutically active agent with anti-tumor activity. These agents may be administered before, simultaneously with, or after administration of the bispecific antigen binding molecule of the first aspect of the invention, the bispecific chimeric antigen receptor of the fifth aspect (including a CAR construct in which a bispecific chimeric antigen receptor is co-expressed with another biologically active molecule), the isolated nucleic acid molecule of the second aspect or the seventh aspect, the nucleic acid construct of the eighth aspect or the ninth aspect or the tenth aspect, the vector of the third aspect or the eleventh aspect, the host cell of the fourth aspect, the modified immune cell of the twelfth aspect or the thirteenth aspect or the fourteenth aspect or the fifteenth aspect, the immune cell composition of the sixteenth aspect, or the pharmaceutical composition of the seventeenth aspect.

In certain embodiments, the above method further comprises administering to the subject a second therapy, which can be any therapy known for tumors, such as surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.

In certain embodiments, the second therapy can be applied separately or in combination with the above-described method; or, the second therapy can be applied simultaneously or sequentially with the above-described method.

In certain embodiments, the subject can be a mammal, such as a human.

In another aspect, the bispecific antigen binding molecule of the first aspect of the present invention, the bispecific chimeric antigen receptor of the fifth aspect (including the CAR construct co-expressed by the bispecific chimeric antigen receptor and another biologically active molecule), the isolated nucleic acid molecule of the second aspect or the seventh aspect, the nucleic acid construct of the eighth aspect or the ninth aspect or the tenth aspect, the vector of the third aspect or the eleventh aspect, the host cell of the fourth aspect, the modified immune cell of the twelfth aspect or the thirteenth aspect or the fourteenth aspect or the fifteenth aspect, the immune cell composition of the sixteenth aspect, or the pharmaceutical composition of the seventeenth aspect are provided for the preparation of a drug for the prevention and/or treatment of a disease related to the expression of mesothelin. The dosage, dosage form, route of administration, indication, combination therapy and other aspects of the aforementioned treatment methods can be applied to the use of the drug.

Abbreviations

CAR Chimeric Antigen Receptor

CDR Complementarity determining region in the variable region of an immunoglobulin

CDR-H1 Complementarity determining region 1 in the variable region of the immunoglobulin heavy chain

CDR-H2 Complementarity determining region 2 in the variable region of the immunoglobulin heavy chain

CDR-H3 Complementarity determining region 3 in the variable region of the immunoglobulin heavy chain

CDR-L1 Complementarity determining region 1 in the immunoglobulin light chain variable region

CDR-L2 Complementarity determining region 2 in the immunoglobulin light chain variable region

CDR-L3 Complementarity determining region 3 in the immunoglobulin light chain variable region

FR Antibody framework region: amino acid residues in the variable region of an antibody other than CDR residues

VH antibody heavy chain variable region

VL Antibody light chain variable region

Kabat The immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

IMGT is based on the international ImMunoGeneTics information system initiated by Lefranc et al. For the Immunoglobulin G (IMGT) numbering system, see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.

Chothia The immunoglobulin numbering system proposed by Chothia et al. is a classical rule for identifying CDR region boundaries based on the position of structural loop regions (see, eg, Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883).

IL-2 Interleukin 2

IFN Interferon

PCR polymerase chain reaction

FACS flow cytometry

K _D equilibrium dissociation constant

kon binding rate constant

kdis dissociation rate constant

Definition of Terms

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the molecular biology, microbiology, cell biology, biochemistry, immunology and other operating steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

As used herein, the term "antibody" refers to an immunoglobulin molecule that can specifically bind to a target (such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term includes not only complete polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chains (such as scFv, di-scFv, (scFv) ₂ ) and domain antibodies (including, for example, shark and camel antibodies), and fusion proteins including antibodies, and immunoglobulin molecules of any other modified configuration including antigen recognition sites. The antibodies of the present invention are not limited by any specific method for producing antibodies. Antibodies include antibodies of any type, such as IgG, IgA or IgM (or its subclass), and antibodies do not need to belong to any specific type. Depending on the amino acid sequence of the constant region of the heavy chain of the antibody, immunoglobulins can be assigned to different types. There are five major types of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to the different types of immunoglobulins are called α, δ, ε, γ, and μ, respectively. Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. The subunit structures and three-dimensional configurations of the different types of immunoglobulins are well known. The heavy chain constant region consists of four domains (CH1, hinge region, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domains are not directly involved in antibody binding to antigen, but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

The VH and VL regions of antibodies can also be subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each _VH and _VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain/light chain pair form an antigen binding site, respectively. The distribution of amino acids in each region or domain can follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883.

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 each of the variable regions of the heavy and light chains, 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 (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883) or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). 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 those skilled in the art (eg, see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

In the present invention, the CDRs contained in the antibodies or antigen-binding fragments thereof can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention are preferably determined by the Kabat, Chothia or IMGT numbering systems.

As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide of a fragment of an antibody, such as a polypeptide of a fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen bound by the full-length antibody and/or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include camelid Ig, Ig NAR, Fab fragment, Fab' fragment, F(ab)' ₂ fragment, F(ab)' ₃ fragment, Fd, Fv, scFv, di-scFv, (scFv) ₂ , minibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins ("dsFv") and single domain antibodies (sdAb, nanobodies) and polypeptides that contain at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

As used herein, the term "Fd" means an antibody fragment consisting of VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F(ab') ₂ fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab'fragment" means a fragment obtained after reducing the disulfide bonds linking two heavy chain fragments in the F(ab') ₂ fragment, consisting of a complete light chain and the Fd fragment (consisting of VH and CH1 domains) of the heavy chain.

As used herein, the term "Fv" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen binding site. It is generally believed that six CDRs confer antigen binding specificity to an antibody. However, even a variable region (e.g., a Fd fragment, which contains only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of a complete binding site.

As used herein, the term "Fc" means an antibody fragment formed by the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain of an antibody bound via a disulfide bond. The Fc fragment of an antibody has a variety of different functions but does not participate in antigen binding.

As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: _NH2 -VL-linker-VH-COOH or _NH2 -VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) ₄ can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also be present between the VH and VL of the scFv. In certain embodiments, the VH and VL domains may be positioned relative to each other in any suitable arrangement. For example, scFv comprising NH ₂ -VH-VH-COOH, NH _2- VL-VL-COOH. The scFv can form any possible engineering structure, single chain antibody (scFv), tandem antibody (tandem di-scFvs), bifunctional antibody, trifunctional antibody, tetrafunctional antibody, disulfide bond stabilized Fv protein, camel Ig, IgNAR, etc. In certain embodiments of the present invention, scFv can form di-scFv, which refers to two or more single scFvs connected in series to form an antibody. In certain embodiments of the present invention, scFv can form (scFv) ₂ , which refers to two or more single scFvs connected in parallel to form an antibody.

As used herein, the term "single-domain antibody (sdAb)" has the meaning generally understood by those skilled in the art, and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen as the full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single-domain antibodies are also called nanobodies.

Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody, and/or competes with the full-length antibody for specific binding to the antigen.

Antibody antigen-binding fragments (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antibody antigen-binding fragments can be screened for specificity in the same manner as for intact antibodies.

Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies but also antigen-binding fragments of antibodies.

As used herein, the expression "specific binding" or "specifically directed against" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. The strength or affinity of a specific binding interaction can be represented by the equilibrium dissociation constant ( _KD ) of the 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 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.

The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the speed of formation and dissociation of antigen binding sites/antigen complexes. Both "association rate constants" (ka or kon) and "dissociation rate constants" (kdis or koff) can be calculated by concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of kdis/kon is equal to the dissociation constant K _D (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473). K _D , kon and kdis values can be measured by any effective method. In certain embodiments, the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR). In addition, the dissociation constant can be measured using bioluminescence interferometry or Kinexa.

As used herein, the term "identity" is used to refer to the matching 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 (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions of the two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, the two sequences are compared when they are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change 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 of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

The compilation of the twenty conventional amino acids involved in this article follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S.Golub and D.R.Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. A vector may include a sequence that replicates directly and autonomously in a cell, or may include a sequence sufficient to allow integration into the host cell DNA. When a vector enables the expression of a protein encoded by an 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 viral vectors, etc. Non-limiting examples of viral vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector may contain a variety of elements for controlling expression, including, but not limited to, a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene. In addition, the vector may also contain a replication initiation site.

As used herein, the term "episomal vector" means that the vector is capable of replicating without being integrated into the host's chromosomal DNA and being gradually lost by dividing host cells, and also means that the vector replicates extrachromosomally or episomally.

As used herein, the term "viral vector" is broadly used to refer to a nucleic acid molecule (e.g., a transfer plasmid) that includes a virally derived nucleic acid element that typically facilitates transfer or integration of the nucleic acid molecule into the genome of a cell, or a viral particle that mediates nucleic acid transfer. In addition to the nucleic acid, the viral particle typically will include various viral components and sometimes host cell components.

The term "viral vector" can refer to a virus or viral particle capable of transferring nucleic acid into a cell, or to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and/or functional genetic elements primarily derived from viruses.

As used herein, the term "retroviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements primarily derived from retroviruses, or portions thereof.

As used herein, the term "lentiviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements derived primarily from a lentivirus or portions thereof (including LTR). In certain embodiments, the terms "lentiviral vector", "lentiviral expression vector" may be used to refer to a lentiviral transfer plasmid and/or an infectious lentiviral particle. When elements (e.g., cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc.) are referred to herein, it is understood that the sequences of these elements are present in the lentiviral particles of the present invention in the form of RNA and in the DNA plasmids of the present invention in the form of DNA.

As used herein, an "integration-defective" retrovirus or lentivirus refers to a retrovirus or lentivirus that has an integrase that cannot integrate the viral genome into the genome of a host cell. In certain embodiments, the integrase protein is mutated to specifically reduce its integrase activity. An integration-defective lentiviral vector can be obtained by modifying the pol gene encoding the integrase protein to produce a mutant pol gene encoding an integration-defective integrase. The integration-defective viral vector has been described in patent application WO 2006/010834, which is incorporated herein by reference in its entirety.

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 human cells, immune cells (such as T lymphocytes, NK cells, monocytes, macrophages or dendritic cells, etc.). Host cells can include single cells or cell colonies.

As used herein, the term "chimeric antigen receptor" or "CAR" refers to a recombinant polypeptide construct comprising at least one extracellular antigen binding domain, a spacer domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as "intracellular signaling domain"), which combines the antibody-based specificity for the target antigen (eg, MSLN) with the immune effector cell activation intracellular domain to exhibit specific immune activity for cells expressing the target antigen (eg, MSLN). In the present invention, the expression "immune effector cell expressing CAR" refers to an immune effector cell expressing CAR and having an antigen-specificity determined by the targeting domain of the CAR. Methods for making CARs (e.g., for cancer treatment) are known in the art, see, for example, Park et al., Trends Biotechnol., 29:550-557, 2011; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J. Hematol. Oncol., 6:47, 2013; PCT patent publications WO2012/079000, WO2013/059593; and U.S. Patent Publication 2012/0213783, all of which are incorporated herein by reference in their entirety.

As used herein, the term "extracellular antigen binding domain" refers to a polypeptide that is capable of specifically binding to an antigen or receptor of interest. The domain will be capable of interacting with cell surface molecules. For example, an extracellular antigen binding domain can be selected to recognize an antigen that is a target cell surface marker associated with a particular disease state.

As used herein, the term "intracellular signaling domain" refers to a protein portion that conducts effector signaling function signals and guides cells to perform specialized functions. Therefore, the intracellular signaling domain has the ability to activate at least one normal effector function of an immune effector cell expressing a CAR. For example, the effector function of a T cell can be a cytolytic activity or an auxiliary activity, including the secretion of cytokines.

As used herein, the term "primary signaling domain" refers to a portion of a protein that is capable of regulating the primary activation of a TCR complex in a stimulatory or inhibitory manner. Primary signaling domains that act in a stimulatory manner typically contain a signaling motif known as an immunoreceptor tyrosine-based activation motif (ITAM). Non-limiting examples of ITAMs containing primary signaling domains particularly useful in the present invention include those derived from TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD3 ζ, CD22, CD79a, CD79b, and CD66d.

As used herein, the term "costimulatory signaling domain" refers to the intracellular signaling domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules that provide the second signal required for the efficient activation and function of T lymphocytes after binding to an antigen, except for antigen receptors or Fc receptors. Non-limiting examples of the costimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD270 (HVEM), CD278 (ICOS), DAP10.

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 (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: sterile water, saline, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, preservatives. For example, pH regulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings generally understood by those skilled in the art, which can stabilize the desired activity of the active ingredient in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient includes a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or illness or symptom (e.g., a tumor) 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, compared to the expected survival period (if not receiving treatment), extending the survival period.

As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the term "subject" refers to a living organism including one in which an immune response can be elicited. In certain embodiments, the subject (e.g., a human) suffers from a tumor (e.g., a tumor associated with MSLN), or, is at risk of suffering from the above-mentioned disease.

As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, stop, or delay the occurrence of a disease (e.g., a tumor); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

As used herein, the term "immune cell" refers to a cell involved in an immune response, such as a cell involved in promoting immune effector functions. Examples of immune cells include T cells (e.g., α/β T cells and γ/δ T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived macrophages.

The immune cells of the present invention can be self/autologous ("self") or non-self ("non-self", e.g., allogeneic, isogenic, or allogeneic). As used herein, "self" refers to cells from the same subject; "allogeneic" refers to cells of the same species that are genetically different from the comparison cells; "isogenic" refers to cells from a different subject that are genetically identical to the comparison cells; and "allogeneic" refers to cells from a different species than the comparison cells. In a preferred embodiment, the cells of the present invention are allogeneic.

Exemplary immune cells that can be used for CAR described herein include T lymphocytes and/or NK cells. The term "T cell" or "T lymphocyte" is well known in the art and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes or activated T lymphocytes. T cells can be T helper (Th) cells, such as T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be helper T cells (HTL; CD4T cells) CD4T cells, cytotoxic T cells (CTL; CD8T cells), CD4CD8T cells, CD4CD8T cells or any other T cell subsets. In certain embodiments, T cells may include primary T cells and memory T cells.

It will be appreciated by those skilled in the art that other cells may also be used as immune cells with CAR as described herein. Specifically, immune cells also include NK cells, monocytes, macrophages or dendritic cells, NKT cells, neutrophils and macrophages. Immune cells also include progenitor cells of immune cells, wherein the progenitor cells can be induced in vivo or in vitro to differentiate into immune cells. Therefore, in certain embodiments, immune cells include progenitor cells of immune cells, such as hematopoietic stem cells (HSCs) contained in CD34+ cell populations derived from umbilical cord blood, bone marrow or flowing peripheral blood, which differentiate into mature immune cells after administration in a subject, or they can be induced in vitro to differentiate into mature immune cells.

As used herein, the term "modified immune cell" refers to an immune cell expressing any of the CARs described herein, or an immune cell into which any of the isolated nucleic acids or vectors described herein have been introduced. After a polynucleotide encoding a CAR polypeptide is introduced into a cell in a variety of ways, the CAR polypeptide can also be synthesized in situ in the cell. Alternatively, the CAR polypeptide can be produced extracellularly and then introduced into the cell. Methods for introducing polynucleotide constructs into cells are known in the art. In some embodiments, a stable transformation method can be used to integrate the polynucleotide construct into the genome of the cell. In other embodiments, a transient transformation method can be used to transiently express a polynucleotide construct, and the polynucleotide construct is not integrated into the genome of the cell. In other embodiments, a viral-mediated method can be used. Polynucleotides can be introduced into cells by any suitable method, such as recombinant viral vectors (e.g., retroviruses, adenoviruses), liposomes, and the like. Transient transformation methods include, for example, but not limited to, microinjection, electroporation, or microparticle bombardment. The polynucleotide may be included in a vector, such as a plasmid vector or a viral vector.

As used herein, the term "immune effector function" refers to the function or response of immune effector cells to enhance or promote immune attack on target cells (e.g., killing of target cells, or inhibiting their growth or proliferation). For example, the effector function of T cells can be cytolytic activity or auxiliary activity, including the secretion of cytokines.

Advantageous Effects of the Invention

The current therapeutic effect of CAR-T cell therapy in solid tumors is still insufficient, mainly because solid tumors have complex tumor microenvironments and high tumor heterogeneity. The present invention provides a bispecific CAR targeting MSLN and NKG2DL or an immune cell co-expressing MSLN-specific CAR and NKG2D or its active fragments, which improves the killing of cells expressing tumor antigens and reduces their off-target toxicity to a certain extent by dual targeting MSLN and NKG2DL. The present invention also provides an immune cell co-expressing MSLN-specific CAR and PD-1 antibody and rIL-15, which blocks the binding of PD-1 to PD-L1 by co-expressing PD-1 antibody, restores the activity of T cells, and thus enhances the immune response; at the same time, co-expressing rIL-15 can promote the proliferation and activation of T and NK cells, and enhance the tumor killing effect of CAR-T cells.

Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. According to the following detailed description of the accompanying drawings and preferred embodiments, various objects and advantages of the present invention will become practicable to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the structure of the chimeric antigen receptor constructed in Example 2.

Figure 2 shows the results of antigen expression detection of target cells in Experimental Example 5. Figure 2A: MSLN expression of NCI-H226 and SK-OV-3 cells; Figure 2B: MSLN expression of PANC-1 and A431; Figure 2C: NKG2DL expression of PANC-1 and SK-OV-3.

Figure 3 shows the results of the cytotoxic activity assay of CAR-T against target cells in Experimental Example 6. Figure 3A: The cytotoxicity of G16-PD1-rIL15 CAR and G16 CAR against NCI-H226-luc, SKOV-3-luc, and A431-luc; Figure 3B: The cytotoxicity of NK2-G-G16-10 CAR against PANC-1-luc and SKOV-3-luc; Figure 3C: The cytotoxicity of G16-P2A-NKG2D CAR against PANC-1-luc and SKOV-3-luc.

FIG. 4 shows the results of the cytokine release assay caused by CAR-T cells in Experimental Example 7.

FIG5 shows the killing ability of G16 CAR-T and G16-PD1-rIL15 CAR-T on SKOV-3 target cells in mice in Experimental Example 8.

FIG6 shows the killing ability of NK2-G-G16-10 CAR-T and G16-P2A-NKG2D CAR-T on PANC1 target cells in mice in Experimental Example 8.

Sequence information

The sequence information involved in the present invention is provided as follows:

Detailed ways

The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.

Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Compiled Molecular Biology Laboratory Manual, 3rd Edition, John Wiley & Sons, Inc., 1995. It is understood by those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the invention claimed.

Example 1: Preparation of specific single-chain antibody (scFv) binding to human MSLN

1) Phage library screening of MSLN scFv

Take 1.33mL of the fully human phage library and screen it with biotinylated MSLN and SV magnetic beads. The screening product is titrated by phage plating detection. Take 0.5mL of the first round of screening product and mix it with 0.5mL PBST, and perform the second and third rounds of screening according to the above steps.

2) ELISA detection of monoclonal phage

Single colonies were inoculated from phage panning plates into 96-well plates and monoclonal phages were detected by ELISA.

MSLN scFv sequencing: 158 monoclones were selected for sequencing based on the ELISA test results to obtain the scFv sequence. The forward and reverse primers used for sequencing were PKLT1F (SEQ ID NO: 61) and PKLT1R (SEQ ID NO: 62). The sequence results were analyzed using Sequcher software to obtain the scFv sequence G16, whose VH and VL sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and the CDR sequence information is shown in the following table.

Table 1: CDR sequences of scFv

3) Construction of scFv-Fc and analysis of antibody aggregates

The G16 scFv sequence was linked to the Fc (human IgG1) sequence and constructed in the TGEX-KAL vector, and then the expi293 cells were transfected for expression and purification of the scFv-Fc protein, and the Fc (human IgG1) sequence was shown in SEQ ID NO: 63. The results of the SEC analysis experiment showed that the area of the G16 antibody monomer peak (main peak) accounted for more than 85%.

Table 2: SEC data of scFv-Fc protein

4) MSLN cell binding assay

The binding ability of G16 scFv-Fc protein and positive control (i.e., HN1 scFv-Fc protein, HN1 scFv amino acid sequence and nucleotide sequence are shown in SEQ ID NO: 64 and 65, respectively) to three cell lines expressing MSLN was detected by flow cytometry. After gradient dilution of G16 scFv-Fc protein or HN1 scFv-Fc protein, three cell lines expressing MSLN were selected for staining and then detected by flow cytometry. The results are shown in the following table, and the affinity of G16 scFv to three MSLN-positive cells is significantly better than that of the positive control sequence HN1.

Table 3: Affinity determination results of candidate scFv-Fc for three types of MSLN positive cells

Example 2: Construction of lentiviral plasmid and viral packaging

2.1 Construction of lentiviral plasmid:

(1) The order of connection of the various parts of the chimeric antigen receptor

First, based on the scFv sequence in the above embodiment, CAR is further constructed. The structure of the CAR includes the following four types, and can be seen in Figure 1.

The first type: CAR targeting MSLN, named G16 CAR; the full-length amino acid sequence is SEQ ID NO: 58, the nucleotide sequence is SEQ ID NO:59;

Structure: N-signal peptide 1 (SEQ ID NO: 34)-G16 scFv (SEQ ID NO: 18)-spacer (CD8 Hinge) (SEQ ID NO: 38)-transmembrane domain (CD8TM) (SEQ ID NO: 42)-intracellular signaling domain (4-1BB-CD3ζ) (SEQ ID NO: 50).

The second type: a CAR targeting MSLN that co-expresses rIL-15 and PD1, named G16-PD1-rIL15 CAR; The full-length amino acid sequence is SEQ ID NO:56, and the nucleotide sequence is SEQ ID NO:57;

Structure: N-signal peptide 1 (SEQ ID NO:34)-G16 scFv (SEQ ID NO:18)-spacer (CD8Hinge) (SEQ ID NO:38)-transmembrane domain (CD8TM) (SEQ ID NO:42)-intracellular signaling domain (4-1BB-CD3ζ) (SEQ ID NO:50)-self-cleavage peptide P2A (SEQ ID NO:27)-signal peptide 2 (SEQ ID NO:36)-PD-1 scFv (SEQ ID NO:52)-self-cleavage peptide P2A (SEQ ID NO:27)-rIL15 (SEQ ID NO:54).

The third type: a CAR that dually targets NKG2DL and MSLN, named NK2-G-G16-10 CAR; full-length amino The amino acid sequence is SEQ ID NO: 23, and the nucleotide sequence is SEQ ID NO: 24;

Structure: N-signal peptide 1 (SEQ ID NO: 34) -NKG2D-2 extracellular antigen binding domain (SEQ ID NO: 20) -Linker2 (SEQ ID NO: 32) -G16 scFv (SEQ ID NO: 18) -spacer (IgG4Hinge) (SEQ ID NO: 40) -transmembrane domain (CD28TM) (SEQ ID NO: 44) -intracellular signaling domain (4-1BB-CD3ζ) (SEQ ID NO: 50).

Among them, the NKG2D-2 sequence is derived from SEQ ID No. 5 in patent CN109803983B.

The fourth type: CAR that dually targets NKG2DL and MSLN, named G16-P2A-NKG2D CAR; full-length The amino acid sequence is SEQ ID NO:25, and the nucleotide sequence is SEQ ID NO:26;

Structure: N-signal peptide 1 (SEQ ID NO: 34) -G16 scFv (SEQ ID NO: 18) -spacer (CD8Hinge) (SEQ ID NO: 38) -transmembrane domain (CD8TM) (SEQ ID NO: 42) -intracellular signaling domain (4-1BB-CD3ζ) (SEQ ID NO: 50) -self-cleaving peptide P2A (SEQ ID NO: 27) -4-1BB intracellular signaling domain (SEQ ID NO: 46) -NKG2D full length sequence (SEQ ID NO: 21).

Among them, the full-length sequence of NKG2D comes from NP_031386.2.

(2) After the codons of the scFv and rIL15 nucleotide sequences in the above structure were optimized, they were outsourced for synthesis and constructed into the Lenti-4-EF1a vector. Single clones were selected for culture and preservation, and finally the plasmids were extracted for sequencing. The bacterial solution with correct sequencing was used to prepare lentiviral plasmids.

2.2 Virus packaging:

Add the CAR lentiviral plasmid and transfection reagent mixture constructed above dropwise to 293T (ATCC) cells, gently shake the culture dish and mix thoroughly. Place the culture dish in a 37°C, 5% CO ₂ incubator; after 6 to 8 hours of culture, remove the culture medium containing the transfection reagent and replace it with fresh complete culture medium. After 48 hours of continuous culture, collect the supernatant of the culture medium containing the virus in the culture dish, filter it with a 0.45μm filter membrane, transfer it to a centrifuge tube, balance it, and centrifuge it at 20000×g at 4°C for 2 hours. After the centrifugation, carefully remove the liquid in the centrifuge tube in the biosafety cabinet, add 500μL PBS buffer to resuspend the precipitate, and store the virus at -80°C.

Experimental Example 3: CAR-T cell preparation

1) Isolation of primary T cells:

(1) Human PBMC cells were isolated using lymphocyte separation medium (GE), cultured in an incubator at 37°C and 5% _CO2 , 100 μL/mL of CD3 antibody and CD28 antibody were added, mixed thoroughly, and incubated at room temperature for 15 minutes.

(2) Remove the magnetic beads and mix thoroughly by pipetting up and down at least 5 times.

(3) Pipette 50 μL of magnetic beads/mL into the above sample, mix thoroughly, and incubate at room temperature for 10 minutes.

(4) Add complete medium to a total volume of 2.5 mL in the tube, insert the tube (with the lid open) into the magnet, and let it stand at room temperature for 5 minutes.

(5) After incubation, the tube remains in the magnet and is gently inverted to pour out the cells.

(6) Resuspend the cells in X-vivo 15 medium and add 10% FBS, 300 U/mL IL-2, 5 ng/mL IL-15 and 10 ng/mL IL-7.

2) T cell activation:

The cell density was adjusted to 1×10 ⁶ cells/mL, and cytokine and antibody complexes (configured at a final concentration of 300 U/mL of IL-2, 10 ng/mL of IL-7, 5 ng/mL of IL-15, 500 ng/mL of Anti-CD3 (OKT3), and 2 μg/mL of Anti-CD28) were added and cultured for 48 hours.

3) Viral infection:

(1) Calculate the required amount of virus according to MOI = 20. The calculation formula is as follows: Required amount of virus (mL) = (MOI * number of cells) / virus titer

(2) After taking out the virus from the -80°C refrigerator, quickly thaw it in a 37°C water bath. Add the amount of virus calculated above to a six-well plate, add DEAE with a final concentration of 5 μg/mL, mix thoroughly, seal the four sides of the six-well plate with sealing film, and centrifuge at 800×g for 1 hour.

(3) After centrifugation, remove the sealing film and place the six-well plate in a 37°C 5% CO ₂ incubator for 24 hours.

(4) Centrifuge at 250 × g for 10 min, remove the supernatant containing the virus, resuspend the cell pellet with fresh culture medium, transfer the cells to a new six-well plate, and continue culturing for 3-6 days before use.

Experimental Example 4: Detection of the positive rate of CAR-T cells

The nucleic acid sequence encoding CAR is expressed under the drive of the promoter, and the lentiviral transfected T cells are labeled with biotin-labeled MSLN antigen, then detected with fluorescently labeled streptavidin, and measured by flow cytometry to reflect the expression level of CAR on the surface of T cells. The CAR positivity of the CAR-T cells obtained in Example 3 was detected by the above method, and the FACS test results are shown in the following table. The results showed that the CAR positivity rate of all CAR-T cells was greater than 20%, indicating that CAR was successfully expressed after lentiviral transfection of effector cells, and T cells expressing MSLN-CAR chimeric antigen receptors were successfully constructed.

Table 4: CAR positive rate test results

Chimeric Antigen Receptor CAR positive rate (%) G16 67.32 G16-PD1-rIL15 29.85 NK2-G-G16-10 55.80 G16-P2A-NKG2D 39.04

Experimental Example 5: Detection of Antigen Expression in Target Cells

The antigen expression of target cells SK-OV-3, PANC-1, NCI-H226, and A431 was measured by flow cytometry using MSLN-biotin and PE-streptavidin antibodies. The results showed that NCI-H226 and SK-OV-3 cells highly expressed MSLN (Figure 2A), while PANC-1 and A431 cells did not express MSLN (Figure 2B); PANC-1 cells expressed NKG2DL at a low level, and SK-OV-3 cells basically did not express NKG2DL (Figure 2C).

Experimental Example 6: Evaluation of CAR-T's killing activity on target cells

The Luciferase gene was integrated into the genome of NCI-H226, SKOV-3, PANC-1 and A431 cells by lentiviral transduction, and NCI-H226, SKOV-3, PANC-1 and A431 cells that stably expressed Luciferase were obtained (named NCI-H226-luc, SKOV-3-luc, PANC-1-luc and A431-luc, respectively). NCI-H226-luc, SKOV-3-luc, PANC-1-luc and A431-luc cells were collected, centrifuged, resuspended, and the cell density was adjusted to 1×10 ⁵ cells/mL. The target cells were inoculated in a 96-well plate at a volume of 100 μL/well and allowed to stand in a 5% CO ₂ 37°C incubator for 30 min. CAR-T cells were collected by centrifugation and resuspended in 1640 medium with 10% FBS. G16-CAR, G16-PD1-rIL15 CAR, NK2-G-G16-10CAR, G16-P2A-NKG2D CAR and blank T cells without CAR transfection were used as effector cells and then added to 96-well plates containing NCI-H226-luc, SKOV-3-luc, PANC-1-luc and A431-luc at an E/T (effector cell/target cell) ratio of 0.5:1, 0.25:1 and 0.125:1, respectively (PANC-1 has a low expression of NKG2DL, so the E/T ratio was 1:1, 0.5:1, 0.25:1 and 0.125:1), 100 μL/well, and the final volume was filled to 200 μL/well, 5% CO ₂ Incubate in a 37°C incubator for 18 to 24 hours. After the incubation period, remove the plate from the incubator, add 20 μL of fluorescence detection reagent, and use an enzyme-labeled instrument to detect the fluorescence reading.

The results of CAR-T cell killing activity test are as follows: there is no difference between G16-PD1-rIL15 CAR and G16 CAR in killing NCI-H226-luc and SKOV-3-luc, and no killing of negative A431 cells (Figure 3A); when the effector cell/target cell ratio of NK2-G-G16-10 CAR is 1, the lysis rate of PANC-1-luc cells is as high as 98%, G16 CAR has no killing of PANC-1-luc cells, and NK2-G-G16-10 CAR has better killing of SKOV-3-luc cells than G16 CAR (Figure 3B); when the effector cell/target cell ratio of G16-P2A-NKG2D CAR is 1, the lysis rate of PANC-1-luc cells can reach 55%, G16 has no killing of PANC-1-luc cells, and G16-P2A-NKG2D There was no difference between CAR and G16 in killing SKOV-3-luc cells ( Figure 3C ).

Experimental Example 7: CAR-T cell cytokine release

Collect NCI-H226-luc, SKOV-3-luc, and A431-luc cells, adjust the cell density to 1×10 ⁵ /mL with culture medium, inoculate target cells in 96-well plates at 100 μL/well, and resuspend CAR-T cells with culture medium. G16-PD1-rIL15 CAR, G16-CAR, and blank T cells without CAR transfection are used as effector cells, and then added to the 96-well plate containing target cells at an E/T (effector cell/target cell) ratio of 1:1, 100 μL/well, and the final volume is supplemented to 200 μL/well, and cultured overnight in a 5% CO ₂ 37°C incubator. After the culture is completed, the well plate is removed from the incubator, centrifuged, and the supernatant is taken. The cytokine release is detected using an ELISA kit (IL2, IFN-γ). The test results are shown in Figure 4. For MSLN-positive NCI-H226-luc and SKOV-3-luc cells, the IL2 and IFN-γ secretions of G16-PD1-rIL15 CAR and G16-CAR were higher than those of the UTD group; for MSLN-negative A431-luc cells, no IL2 and IFN-γ were secreted by G16-PD1-rIL15 CAR and G16-CAR.

Experimental Example 8: In vivo model to evaluate the killing ability of CAR-T cells on target cells

To evaluate the anti-tumor ability of G16 CAR-T and G16-PD1-rIL15 CAR-T in vivo, we established a subcutaneous transplant tumor model of SKOV-3 ovarian cancer cells in B-NDG mice. When the transplant tumor volume reached about 100-150 mm ³ , 100 mg/kg of cyclophosphamide was intraperitoneally administered to eliminate residual immune cells. On D7, 5×10 ⁶ G16 CAR-T and G16-PD1-rIL15 CAR-T cells were injected through the tail vein, respectively, and virus-uninfected T cells (UTD) were used as negative controls. The growth of subcutaneous transplant tumors was observed and measured twice a week. The results showed that G16 CAR-T cells could significantly inhibit the growth of subcutaneous transplant tumors of tumor cells. G16-PD1-rIL15 CAR-T cells were able to completely eliminate tumor cells in mice, and no tumor recurrence was observed and there was no significant change in the weight of mice (Figure 5).

To evaluate the anti-tumor ability of NK2-G-G16-10 CAR-T and G16-P2A-NKG2D CAR-T in vivo, we established a B-NDG mouse subcutaneous transplant tumor model of PANC1-MSLN pancreatic cancer cells. When the transplant tumor volume reached about 100-150 mm ³ , 100 mg/kg of cyclophosphamide was intraperitoneally administered to eliminate residual immune cells. On D7, 5×10 ⁶ G16 CAR-T, NK2-G-G16-10 CAR-T cells, and G16-P2A-NKG2D CAR-T cells were injected through the tail vein, respectively, and virus-uninfected T cells (UTD) were used as negative controls. The growth of subcutaneous transplant tumors was observed and measured twice a week. The results showed that all CAR-T cells were able to significantly inhibit the growth of subcutaneous tumor cell transplants, while NK2-G-G16-10 CAR-T cells and G16-P2A-NKG2D CAR-T cells were able to inhibit the growth of subcutaneous tumor cell transplants more quickly and completely eliminate tumor cells in mice without significant changes in the weight of the mice (Figure 6).

Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims (44)

A bispecific antigen-binding molecule comprising a first antigen-binding domain capable of specifically binding to MSLN and a second antigen-binding domain capable of specifically binding to an NKG2D ligand (NKG2DL); wherein the first antigen-binding domain capable of specifically binding to MSLN comprises a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein, (1) the VH comprises: the following three heavy chain CDRs defined according to the Kabat numbering system: CDR-H1 with a sequence of SEQ ID NO: 3 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 4 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 5 or a variant thereof; and/or the VL comprises: the following three light chain CDRs defined according to the Kabat numbering system: CDR-L1 with a sequence of SEQ ID NO: 6 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 7 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 8 or a variant thereof; or (2) the VH comprises: the following three heavy chain CDRs defined according to the IMGT numbering system: CDR-H1 with a sequence of SEQ ID NO: 9 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 10 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 11 or a variant thereof; and/or the VL comprises: the following three light chain CDRs defined according to the IMGT numbering system: CDR-L1 with a sequence of SEQ ID NO: 12 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 13 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 8 or a variant thereof; or (3) the VH comprises: the following three heavy chain CDRs defined according to the Chothia numbering system: CDR-H1 with a sequence of SEQ ID NO: 14 or a variant thereof; CDR-H2 with a sequence of SEQ ID NO: 15 or a variant thereof; CDR-H3 with a sequence of SEQ ID NO: 5 or a variant thereof; and/or the VL comprises: the following three light chain CDRs defined according to the Chothia numbering system: CDR-L1 with a sequence of SEQ ID NO: 6 or a variant thereof; CDR-L2 with a sequence of SEQ ID NO: 7 or a variant thereof; CDR-L3 with a sequence of SEQ ID NO: 8 or a variant thereof; Among them, the variant described in any one of (1), (2) and (3) has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions. The bispecific antigen-binding molecule of claim 1, wherein the VH comprises the sequence as shown in SEQ ID NO: 1 or a variant thereof; the VL comprises the sequence as shown in SEQ ID NO: 2 or a variant thereof; wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions. The bispecific antigen-binding molecule of claim 1 or 2, wherein the first antigen-binding domain is selected from a full-length antibody, a Fab fragment, a Fab' fragment, a F(ab)' ₂ fragment, a F(ab)' ₃ fragment, a single-chain antibody (e.g., scFv, di-scFv or (scFv) ₂ ), a miniantibody, a disulfide-stabilized Fv protein (dsFv) and a single-domain antibody (sdAb, nanobody). The bispecific antigen-binding molecule of any one of claims 1 to 3, wherein the first antigen-binding domain is a single-chain antibody, and the single-chain antibody comprises, from its N-terminus to its C-terminus: (1) VH-L1 comprising the sequence shown in SEQ ID NO: 1 or a variant thereof-VL comprising the sequence shown in SEQ ID NO: 2 or a variant thereof; or (2) VL-L1 comprising the sequence shown in SEQ ID NO: 2 or a variant thereof-VH comprising the sequence shown in SEQ ID NO: 1 or a variant thereof; wherein the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions; Preferably, L1 is a polypeptide; preferably, L1 comprises one or several (e.g., 1, 2 or 3) sequences as shown in (GmS)n, wherein m is selected from an integer of 1-6, and n is selected from an integer of 1-6; preferably, m is 3, 4, or 5; preferably, n is 1 or 2; more preferably, L1 has the sequence of SEQ ID NO: 30; More preferably, the single-chain antibody comprises the sequence shown in SEQ ID NO: 18 or a variant thereof, which has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 18, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions. The bispecific antigen-binding molecule of any one of claims 1 to 4, wherein the second antigen-binding domain capable of specifically binding to an NKG2D ligand (NKG2DL) comprises NKG2D or its ligand-binding domain; Preferably, the second antigen-binding domain comprises the sequence shown in SEQ ID NO: 20 or 21 or a variant thereof, wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions. The bispecific antigen-binding molecule of any one of claims 1 to 5, wherein the first antigen-binding domain and the second antigen-binding domain are connected by a linker L2; Preferably, the first antigen binding domain is as defined in claim 4; Preferably, L2 is a polypeptide; preferably, L2 comprises one or several (e.g., 1, 2 or 3) sequences as shown in (GmS)n, wherein m is selected from an integer of 1-6, and n is selected from an integer of 1-6; preferably, m is 3, 4, or 5; preferably, n is 1 or 2; more preferably, L2 has the sequence of SEQ ID NO: 32; Preferably, the first antigen-binding domain is connected to the N-terminus or C-terminus of the second antigen-binding domain through the L2; Preferably, the bispecific antigen binding molecule comprises the sequence shown in SEQ ID NO:22 or a variant thereof, which has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:22, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the bispecific antigen binding molecule of any one of claims 1 to 6. A vector comprising the isolated nucleic acid molecule of claim 7; Preferably, the vector is selected from a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR/Cas9 vector or a viral vector; Preferably, the vector is an expression vector; Preferably, the vector is an episomal vector; Preferably, the vector is a viral vector; more preferably, the viral vector is a lentiviral vector, an adenoviral vector or a retroviral vector. A host cell comprising the isolated nucleic acid molecule of claim 7 or the vector of claim 8. A method for preparing the bispecific antigen-binding molecule of any one of claims 1 to 6, comprising culturing the host cell of claim 9 under conditions that allow protein expression, and recovering the bispecific antigen-binding molecule from the cultured host cell culture. A bispecific chimeric antigen receptor comprising a bispecific antigen binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain, wherein the bispecific antigen binding domain comprises the bispecific antigen binding molecule of any one of claims 1 to 6; Preferably, the bispecific antigen binding domain comprises a first antigen binding domain capable of specifically binding to MSLN and a second antigen binding domain capable of specifically binding to NKG2D ligand (NKG2DL); wherein the first antigen binding domain is as defined in any one of claims 1 to 4, and the second antigen binding domain is as defined in claim 5; Preferably, the first antigen-binding domain is a single-chain antibody, and the first antigen-binding domain is connected to the N-terminus or C-terminus of the second antigen-binding domain via a linker L2. The bispecific chimeric antigen receptor of claim 11, wherein the bispecific antigen binding domain comprises the sequence shown in SEQ ID NO: 22 or a variant thereof, wherein the variant has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 22, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions. The bispecific chimeric antigen receptor of claim 11 or 12, wherein the transmembrane domain is selected from the transmembrane region of the following proteins: α, β or ζ chain of T cell receptor, CD28, CD45, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD152, CD154 and PD-1; Preferably, the transmembrane domain is selected from the transmembrane regions of the following proteins: CD8α, CD28, CD4, PD-1, CD152 and CD154; Preferably, the transmembrane domain comprises a CD8α transmembrane region with a sequence as shown in SEQ ID NO:42 or a CD28 transmembrane region with a sequence as shown in SEQ ID NO:44. The bispecific chimeric antigen receptor of any one of claims 11 to 13, wherein the spacer domain is located between the antigen binding domain and the transmembrane domain, and the spacer domain is selected from the hinge domain and/or the CH2 and CH3 regions of an immunoglobulin (e.g., IgG1 or IgG4); Preferably, the hinge domain comprises the hinge region of CD8α, IgG4, PD-1, CD152 or CD154; more preferably, the hinge domain comprises the CD8α hinge region whose sequence is shown in SEQ ID NO:38 or the IgG4 hinge region whose sequence is shown in SEQ ID NO:40. The bispecific chimeric antigen receptor of any one of claims 11 to 14, wherein the intracellular signaling domain comprises a primary signaling domain and/or a co-stimulatory signaling domain; Preferably, the intracellular signaling domain comprises a co-stimulatory signaling domain and a primary signaling domain in sequence from the N-terminus to the C-terminus; Preferably, the intracellular signaling domain comprises a primary signaling domain and at least one co-stimulatory signaling domain; Preferably, the primary signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM); Preferably, the primary signaling domain comprises an intracellular signaling domain selected from the group consisting of CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b or CD66d; more preferably, the primary signaling domain comprises a CD3ζ intracellular signaling domain having a sequence as shown in SEQ ID NO: 48; Preferably, the co-stimulatory signaling domain comprises an intracellular signaling domain selected from the following proteins: CARD11, CD2, CD7, CD27, CD28, CD30, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD270 (HVEM), CD278 (ICOS) or DAP10; Preferably, the costimulatory signaling domain is selected from the intracellular signaling domain of CD28 or the intracellular signaling domain of CD137 (4-1BB) or a combination of fragments thereof; more preferably, the costimulatory signaling domain comprises the intracellular signaling domain of CD137 (4-1BB) whose sequence is shown in SEQ ID NO: 46; More preferably, the intracellular signaling domain sequence comprises the sequence shown in SEQ ID NO:50. The bispecific chimeric antigen receptor according to any one of claims 11 to 15, wherein the bispecific chimeric antigen receptor further comprises a signal peptide SP1 at its N-terminus; Preferably, the signal peptide SP1 comprises a heavy chain signal peptide (e.g., a heavy chain signal peptide of IgG1), a granulocyte-macrophage colony-stimulating factor receptor 2 (GM-CSFR2) signal peptide, an IL2 signal peptide, or a CD8α signal peptide; more preferably, the signal peptide SP1 comprises a sequence as shown in SEQ ID NO:34. The bispecific chimeric antigen receptor according to any one of claims 11 to 16, wherein the bispecific chimeric antigen receptor comprises, from its N-terminus to its C-terminus, a signal peptide SP1, a bispecific antigen binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain; Preferably, the signal peptide SP1 comprises a heavy chain signal peptide of IgG1 or a CD8α signal peptide (eg, a signal peptide having a sequence as shown in SEQ ID NO: 34); Preferably, the bispecific antigen-binding domain comprises the bispecific antigen-binding molecule of any one of claims 1 to 6 (eg, comprising the sequence shown in SEQ ID NO: 22); Preferably, the spacer domain comprises a hinge region of CD8 (eg, CD8α) or IgG4 (eg, a hinge region whose sequence is shown in SEQ ID NO: 38 or 40); Preferably, the transmembrane domain comprises the transmembrane region of CD8 (eg, CD8α) or CD28 (eg, the transmembrane region whose sequence is shown in SEQ ID NO: 42 or 44); Preferably, the intracellular signaling domain comprises a primary signaling domain and a costimulatory signaling domain, wherein the primary signaling domain comprises the intracellular signaling domain of CD3ζ (e.g., the sequence shown in SEQ ID NO: 48), and the costimulatory signaling domain comprises the intracellular signaling domain of CD137 (4-1BB) (e.g., the sequence shown in SEQ ID NO: 46); more preferably, the intracellular signaling domain of the chimeric antigen receptor has the sequence shown in SEQ ID NO: 50; Preferably, the bispecific chimeric antigen receptor comprises the sequence shown in SEQ ID NO: 23 or a variant thereof, wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the bispecific chimeric antigen receptor of any one of claims 11 to 17; Preferably, the isolated nucleic acid molecule comprises a nucleotide sequence selected from the following: (1) a sequence shown in SEQ ID NO:24 or a degenerate variant thereof; (2) a sequence substantially identical to the sequence shown in any one of (1) (e.g., a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence shown in any one of (1), or a sequence having one or more nucleotide substitutions compared to the sequence shown in any one of (1)). A nucleic acid construct comprising: (1) a first nucleic acid sequence encoding a chimeric antigen receptor specific for MSLN; and (2) a second nucleic acid sequence encoding another biologically active molecule, wherein the other biologically active molecule comprises an antigen binding molecule targeting NKG2D ligand (NKG2DL); wherein, The MSLN-specific chimeric antigen receptor comprises an MSLN-targeting antigen binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain, and the MSLN-targeting antigen binding domain comprises the first antigen binding domain defined in any one of claims 1-4. The nucleic acid construct of claim 19, wherein the antigen binding molecule targeting NKG2D ligand (NKG2DL) comprises NKG2D or its ligand-binding domain; Preferably, the antigen binding molecule targeting NKG2D ligand comprises the sequence shown in SEQ ID NO: 20 or 21 or a variant thereof, wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared with the sequence from which it is derived, or has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared with the sequence from which it is derived; preferably, the substitutions are conservative substitutions. The nucleic acid construct of claim 19 or 20, wherein the antigen binding molecule targeting NKG2D ligand (NKG2DL) is optionally connected to a 4-1BB intracellular signaling domain (e.g., a sequence as shown in SEQ ID NO: 46) at its N-terminus; Preferably, the additional biologically active molecule comprises the amino acid sequence shown in SEQ ID NO:66. The nucleic acid construct of any one of claims 19 to 21, wherein the first nucleic acid sequence and the second nucleic acid sequence are linked by a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A, E2A, F2A, T2A or any combination thereof); Preferably, the self-cleaving peptide is P2A; for example, the nucleotide sequence encoding the self-cleaving peptide is shown in SEQ ID NO: 28 or 29. The nucleic acid construct according to any one of claims 19 to 22, which has one or more of the following characteristics: (1) The transmembrane domain is as defined in claim 13; (2) The spacer domain is as defined in claim 14; (3) The intracellular signaling domain is as defined in claim 15; (4) the MSLN-specific chimeric antigen receptor encoded by the first nucleic acid sequence further comprises a signal peptide SP1 at its N-terminus, wherein the signal peptide SP1 is defined as in claim 16; (5) The additional biologically active molecule encoded by the second nucleotide sequence does not contain a signal peptide at its N-terminus or further contains a signal peptide SP2; preferably, the signal peptide SP2 is different from the signal peptide SP1 contained in the MSLN-specific chimeric antigen receptor encoded by the first nucleic acid sequence; preferably, the signal peptide SP2 at the N-terminus of the additional biologically active molecule is an IL2 signal peptide (for example, as shown in SEQ ID NO:36). The nucleic acid construct of any one of claims 19 to 23, wherein the nucleic acid construct comprises, from its 5' end to its 3' end, in order: a nucleotide sequence encoding the signal peptide SP1, a nucleotide sequence encoding the antigen binding domain targeting MSLN, a nucleotide sequence encoding the spacer domain, a nucleotide sequence encoding the transmembrane domain, a nucleotide sequence encoding the intracellular signaling domain, a nucleotide sequence encoding the self-cleaving peptide sequence, and a nucleotide sequence encoding the antigen binding molecule targeting NKG2D ligand (NKG2DL); Preferably, the signal peptide SP1 comprises a heavy chain signal peptide of IgG1 or a CD8α signal peptide (eg, a signal peptide having a sequence as shown in SEQ ID NO: 34); Preferably, the antigen binding domain targeting MSLN is selected from the first antigen binding domain defined in any one of claims 1 to 4 (eg, comprising the sequence shown in SEQ ID NO: 18); Preferably, the spacer domain comprises a hinge region of CD8 (eg, CD8α) or IgG4 (eg, a hinge region whose sequence is shown in SEQ ID NO: 38 or 40); Preferably, the transmembrane domain comprises the transmembrane region of CD8 (eg, CD8α) or CD28 (eg, the transmembrane region whose sequence is shown in SEQ ID NO: 42 or 44); Preferably, the intracellular signaling domain comprises a primary signaling domain and a costimulatory signaling domain, wherein the primary signaling domain comprises the intracellular signaling domain of CD3ζ (e.g., the sequence shown in SEQ ID NO: 48), and the costimulatory signaling domain comprises the intracellular signaling domain of CD137 (4-1BB) (e.g., the sequence shown in SEQ ID NO: 46); more preferably, the intracellular signaling domain of the chimeric antigen receptor has the sequence shown in SEQ ID NO: 50; Preferably, the self-cleaving peptide sequence is P2A, for example having the sequence shown in SEQ ID NO: 27; Preferably, the antigen binding molecule targeting NKG2D ligand comprises NKG2D or its ligand-binding domain, for example, comprises the sequence shown in SEQ ID NO: 20 or 21. The nucleic acid construct of claim 24, wherein the nucleic acid construct further comprises a nucleotide sequence encoding a 4-1BB intracellular signaling domain between the nucleotide sequence encoding the self-cleaving peptide sequence and the nucleotide sequence encoding the antigen-binding molecule targeting NKG2D ligand (NKG2DL); Preferably, the 4-1BB intracellular signaling domain has the sequence shown in SEQ ID NO: 46; Preferably, the nucleic acid construct comprises a nucleotide sequence selected from the following: (1) a sequence shown in SEQ ID NO: 26 or a degenerate variant thereof; (2) a sequence substantially identical to the sequence shown in any one of (1) (e.g., a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence shown in any one of (1), or a sequence having one or more nucleotide substitutions compared to the sequence shown in any one of (1)). A nucleic acid construct comprising: (1) a first nucleic acid sequence encoding a MSLN-specific chimeric antigen receptor or a bispecific chimeric antigen receptor according to any one of claims 11 to 17; and (2) a second nucleic acid sequence encoding another biologically active molecule, wherein the other biologically active molecule comprises a PD1/PD-L1 pathway inhibitor and an IL-15 agonist; wherein, The MSLN-specific chimeric antigen receptor comprises an MSLN-targeting antigen binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain, wherein the MSLN-targeting antigen binding domain comprises the first antigen binding domain defined in any one of claims 1-4. The nucleic acid construct of claim 26, wherein: (i) the first nucleic acid sequence and the second nucleic acid sequence are linked via a nucleotide sequence encoding a self-cleaving peptide (eg, P2A, E2A, F2A, T2A or any combination thereof); and / or (ii) the nucleotide sequence encoding the PD1/PD-L1 pathway inhibitor and the nucleotide sequence encoding the IL-15 agonist are connected via a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A, E2A, F2A, T2A or any combination thereof); Preferably, the self-cleaving peptide is P2A; for example, the nucleotide sequence encoding the self-cleaving peptide is shown in SEQ ID NO: 28 or 29. The nucleic acid construct of claim 26 or 27, which has one or more of the following characteristics: (1) The transmembrane domain is as defined in claim 13; (2) The spacer domain is as defined in claim 14; (3) The intracellular signaling domain is as defined in claim 15; (4) the MSLN-specific chimeric antigen receptor or bispecific chimeric antigen receptor encoded by the first nucleic acid sequence further comprises a signal peptide SP1 at its N-terminus, wherein the signal peptide SP1 is defined as in claim 16; (5) The additional biologically active molecule encoded by the second nucleotide sequence further comprises a signal peptide SP2 at its N-terminus; preferably, the signal peptide SP2 is different from the signal peptide SP1 contained in the MSLN-specific chimeric antigen receptor or bispecific chimeric antigen receptor encoded by the first nucleic acid sequence; preferably, the signal peptide SP2 at the N-terminus of the additional biologically active molecule is an IL2 signal peptide (for example, as shown in SEQ ID NO:36). The nucleic acid construct of any one of claims 26 to 28, wherein: (i) the PD1/PD-L1 pathway inhibitor is selected from an anti-PD-1 or PD-L1 antibody or an antigen-binding fragment thereof; preferably, the anti-PD-1 or PD-L1 antibody or an antigen-binding fragment thereof is a single-chain antibody (e.g., scFv); preferably, the anti-PD-1 single-chain antibody comprises the amino acid sequence shown in SEQ ID NO:52 or a variant thereof, wherein the variant has a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% with SEQ ID NO:52, or has a substitution, deletion or addition of one or more amino acids (e.g., substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids); preferably, the substitution is a conservative substitution; and / or (2) The IL-15 agonist is selected from a fusion protein comprising IL-15 and an IL-15 receptor α (IL-15Rα) Sushi domain; preferably, the IL-15 agonist comprises the amino acid sequence shown in SEQ ID NO:54 or a variant thereof, wherein the variant has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:54, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions. The nucleic acid construct of any one of claims 26 to 29, wherein the nucleic acid construct comprises, from its 5' end to its 3' end, a nucleotide sequence encoding the signal peptide SP1, a nucleotide sequence encoding the MSLN-targeting antigen binding domain or the bispecific antigen binding domain, a nucleotide sequence encoding the spacer domain, a nucleotide sequence encoding the transmembrane domain, a nucleotide sequence encoding the intracellular signaling domain, a nucleotide sequence encoding the self-cleaving peptide sequence, a nucleotide sequence encoding the signal peptide SP2, a nucleotide sequence encoding the PD1/PD-L1 pathway inhibitor, a nucleotide sequence encoding the self-cleaving peptide sequence, and a nucleotide sequence encoding the IL-15 agonist; Preferably, the signal peptide SP1 comprises a heavy chain signal peptide of IgG1 or a CD8α signal peptide (eg, a signal peptide having a sequence as shown in SEQ ID NO: 34); Preferably, the antigen binding domain targeting MSLN comprises the first antigen binding domain defined in any one of claims 1 to 4 (eg, comprising the sequence shown in SEQ ID NO: 18); Preferably, the bispecific antigen-binding domain comprises the bispecific antigen-binding molecule of any one of claims 1 to 6 (eg, comprising the sequence shown in SEQ ID NO: 22); Preferably, the spacer domain comprises a hinge region of CD8 (eg, CD8α) or IgG4 (eg, a hinge region whose sequence is shown in SEQ ID NO: 38 or 40); Preferably, the transmembrane domain comprises the transmembrane region of CD8 (eg, CD8α) or CD28 (eg, the transmembrane region whose sequence is shown in SEQ ID NO: 42 or 44); Preferably, the intracellular signaling domain comprises a primary signaling domain and a costimulatory signaling domain, wherein the primary signaling domain comprises the intracellular signaling domain of CD3ζ (e.g., the sequence shown in SEQ ID NO: 48), and the costimulatory signaling domain comprises the intracellular signaling domain of CD137 (4-1BB) (e.g., the sequence shown in SEQ ID NO: 46); more preferably, the intracellular signaling domain of the chimeric antigen receptor has the sequence shown in SEQ ID NO: 50; Preferably, the self-cleaving peptide sequence is P2A, for example having the sequence shown in SEQ ID NO: 27; Preferably, the signal peptide SP2 comprises an IL2 signal peptide (for example, as shown in SEQ ID NO: 36); Preferably, the PD1/PD-L1 pathway inhibitor is selected from an anti-PD-1 or PD-L1 single-chain antibody, for example comprising the amino acid sequence shown in SEQ ID NO: 52; Preferably, the IL-15 agonist comprises the sequence shown in SEQ ID NO:54. The nucleic acid construct of any one of claims 26 to 30, wherein the nucleic acid construct comprises a nucleotide sequence selected from the group consisting of: (1) a sequence as shown in SEQ ID NO: 57 or a degenerate variant thereof; (2) a sequence substantially identical to the sequence shown in any one of (1) (e.g., a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in any one of (1), or a sequence having one or more nucleotide substitutions compared to the sequence shown in any one of (1)). A vector comprising the isolated nucleic acid molecule of claim 18, or the nucleic acid construct of any one of claims 19 to 31; Preferably, the vector is selected from a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR/Cas9 vector, or a viral vector; Preferably, the vector is an expression vector; Preferably, the vector is an episomal vector; Preferably, the vector is a viral vector; more preferably, the viral vector is a lentiviral vector, an adenoviral vector or a retroviral vector. An engineered immune cell comprising the isolated nucleic acid molecule of claim 18; Preferably, the engineered immune cells express the bispecific chimeric antigen receptor according to any one of claims 11-17. A modified immune cell comprising the nucleic acid construct of any one of claims 19 to 25; Preferably, the modified immune cells express a chimeric antigen receptor encoded by the first nucleic acid sequence, and an antigen binding molecule targeting NKG2D ligand (NKG2DL) encoded by the second nucleic acid sequence; preferably, the antigen binding molecule targeting NKG2D ligand (NKG2DL) is optionally connected to a 4-1BB intracellular signaling domain at its N-terminus. A modified immune cell comprising the nucleic acid construct of any one of claims 26 to 31; Preferably, the modified immune cells express the chimeric antigen receptor encoded by the first nucleic acid sequence, and the PD1/PD-L1 pathway inhibitor and IL-15 agonist encoded by the second nucleic acid sequence. The modified immune cells according to any one of claims 33 to 35, wherein the immune cells are derived from T lymphocytes, NK cells, monocytes, macrophages or dendritic cells and any combination thereof; preferably, the immune cells are obtained from patients; alternatively, the immune cells are obtained from healthy donors; preferably, the immune cells are derived from T lymphocytes or NK cells. The modified immune cell according to any one of claims 33 to 36, wherein the transcription or expression of one or two target genes of the immune exclusion-related genes (e.g., TRAC, TRBC, B2M, HLA-A, HLA-B or HLA-C) and the genes of the immune co-inhibitory pathway or signaling molecules (e.g., PD-1, CTLA-4 or LAG-3) of the modified immune cell is inhibited; preferably, the transcription or expression of the target gene is inhibited by a method selected from gene knockout (e.g., CRISPR, CRISPR/Cas9), homologous recombination, and interfering RNA. A method for preparing modified immune cells, comprising: (1) providing immune cells from a patient or a healthy donor; (2) introducing the isolated nucleic acid molecule of claim 18 or a vector comprising the same into the immune cells of step (1) to obtain immune cells capable of expressing a bispecific chimeric antigen receptor; or, introducing the nucleic acid construct of any one of claims 19 to 31 or a vector comprising the same into the immune cells of step (1) to obtain immune cells capable of co-expressing a chimeric antigen receptor and another biologically active molecule; Preferably, in step (1), the immune cells are pretreated, and the pretreatment includes sorting, activation and/or proliferation of the immune cells; more preferably, the pretreatment includes contacting the immune cells with anti-CD3 antibodies and anti-CD28 antibodies, thereby stimulating the immune cells and inducing their proliferation, thereby generating pretreated immune cells; Preferably, in step (2), the nucleic acid molecule or vector is introduced into the immune cell by viral infection; Preferably, in step (2), the nucleic acid molecule or vector is introduced into the immune cell by non-viral vector transfection, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, transposon vector system, CRISPR/Cas9 vector, TALEN method, ZFN method or electroporation method; Preferably, after step (2), the method further includes a step of amplifying the immune cells obtained in step (2). An immune cell composition comprising the modified immune cells described in any one of claims 33-37; optionally, the composition also includes unmodified and/or unsuccessfully modified immune cells; preferably, the number of modified immune cells accounts for 10%-100%, more preferably 40%-80% of the total number of cells in the immune cell composition. A kit comprising the bispecific antigen binding molecule of any one of claims 1 to 6, or the isolated nucleic acid molecule of claim 7, or the vector of claim 8, or the host cell of claim 9, or the bispecific chimeric antigen receptor of any one of claims 11 to 17, or the isolated nucleic acid molecule of claim 18, or the nucleic acid construct of any one of claims 19 to 31, or the vector of claim 32; Preferably, the kit comprises the isolated nucleic acid molecule of claim 7 or a vector comprising the isolated nucleic acid molecule; the kit is used to prepare the bispecific antigen-binding molecule of any one of claims 1 to 6; Preferably, the kit comprises the isolated nucleic acid molecule of claim 18 or a vector comprising the isolated nucleic acid molecule; the kit is used to prepare the modified immune cell of claim 33 or an immune cell composition comprising the modified immune cell; Preferably, the kit comprises the nucleic acid construct of any one of claims 19-25 or a vector comprising the nucleic acid construct; the kit is used to prepare the modified immune cell of claim 34 or an immune cell composition comprising the modified immune cell. Preferably, the kit comprises the nucleic acid construct of any one of claims 26-31 or a vector comprising the nucleic acid construct; the kit is used to prepare the modified immune cell of claim 35 or an immune cell composition comprising the modified immune cell. A pharmaceutical composition comprising the bispecific antigen binding molecule of any one of claims 1 to 6, or the isolated nucleic acid molecule of claim 7, or the vector of claim 8, or the host cell of claim 9, or the bispecific chimeric antigen receptor of any one of claims 11 to 17, or the isolated nucleic acid molecule of claim 18, or the nucleic acid construct of any one of claims 19 to 31, or the vector of claim 32, or the modified immune cell of any one of claims 33 to 37, or the immune cell composition of claim 39, and a pharmaceutically acceptable carrier and/or excipient; Preferably, the pharmaceutical composition further comprises another pharmaceutically active agent, such as a drug having anti-tumor activity; preferably, the other pharmaceutically active agent comprises anti-PD1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, pemetrexed, cisplatin, paclitaxel, gemcitabine, capecitabine or FOLFIRINOX; Optionally, the bispecific antigen binding molecule, or isolated nucleic acid molecule, or vector, or host cell, or nucleic acid construct, or modified immune cell, or immune cell composition contained in the pharmaceutical composition can be administered simultaneously, separately or sequentially with the additional pharmaceutically active agent. Use of the bispecific antigen binding molecule of any one of claims 1 to 6, or the isolated nucleic acid molecule of claim 7, or the vector of claim 8, or the host cell of claim 9, or the bispecific chimeric antigen receptor of any one of claims 11 to 17, or the isolated nucleic acid molecule of claim 18, or the nucleic acid construct of any one of claims 19 to 31, or the vector of claim 32, or the modified immune cell of any one of claims 33 to 37, or the immune cell composition of claim 39, or the pharmaceutical composition of claim 41 in the preparation of a medicament for preventing and/or treating a disease associated with the expression of mesothelin; Preferably, the disease associated with the expression of mesothelin is selected from a proliferative disease, such as a tumor, or a non-tumor-related indication associated with the expression of mesothelin; Preferably, the tumor is an MSLN-positive tumor; Preferably, the tumor is a MSLN and NKG2DL positive tumor; Preferably, the tumor is selected from solid tumors; preferably, the solid tumor is selected from malignant pleural mesothelioma, pancreatic cancer, lung cancer (eg, lung squamous cell carcinoma), breast cancer, ovarian cancer (eg, ovarian epithelial carcinoma). A method for preventing and/or treating a disease associated with the expression of mesothelin in a subject (e.g., a human), the method comprising administering to a subject in need thereof an effective amount of the bispecific antigen binding molecule of any one of claims 1 to 6, or the isolated nucleic acid molecule of claim 7, or the vector of claim 8, or the host cell of claim 9, or the bispecific chimeric antigen receptor of any one of claims 11 to 17, or the isolated nucleic acid molecule of claim 18, or the nucleic acid construct of any one of claims 19 to 31, or the vector of claim 32, or the engineered immune cell of any one of claims 33 to 37, or the immune cell composition of claim 39, or the pharmaceutical composition of claim 41; Preferably, the disease associated with the expression of mesothelin is selected from a proliferative disease, such as a tumor, or a non-tumor-related indication associated with the expression of mesothelin; Preferably, the tumor is an MSLN-positive tumor; Preferably, the tumor is a MSLN and NKG2DL positive tumor; Preferably, the tumor is selected from solid tumors; preferably, the solid tumor is selected from malignant pleural mesothelioma, pancreatic cancer, lung cancer (such as lung squamous cell carcinoma), breast cancer, ovarian cancer (such as ovarian epithelial cancer); Preferably, the method further comprises administering to the subject a second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, virotherapy, adjuvant therapy, and any combination thereof. The method of claim 43, wherein the method comprises administering to the subject the engineered immune cell of any one of claims 33-37, or the immune cell composition of claim 39, or a pharmaceutical composition comprising the engineered immune cell or immune cell composition; Preferably, the method comprises the following steps: (1) providing the subject with the immune cells required; (2) introducing the isolated nucleic acid molecule of claim 18 or a vector comprising the same, or the nucleic acid construct of any one of claims 19 to 31 or a vector comprising the same into the immune cells of step (1); (3) administering the immune cells obtained in step (2) to the subject; Optionally, in step (3), the total dose of the immune cells contains 1×10 ⁷ to 10×10 ⁸ CAR-positive cells; Preferably, in step (3), the total dose of the immune cells is administered to the subject in divided doses.