WO2023131063A1

WO2023131063A1 - Chimeric antigen receptors specifically binding to msln and use thereof

Info

Publication number: WO2023131063A1
Application number: PCT/CN2022/143718
Authority: WO
Inventors: 朱雁林; 常建辉; 吴子明; 蔡珍珍; 王江漫; 闫忠辉; 欧阳学农; 葛均友
Original assignee: 成都科伦精准生物科技有限公司
Priority date: 2022-01-10
Filing date: 2022-12-30
Publication date: 2023-07-13

Abstract

A bispecific antigen-binding molecule specifically binding to MSLN and NKG2D ligand (NKG2DL), a bispecific chimeric antigen receptor containing the bispecific antigen-binding molecule, a modified immune cell expressing the bispecific chimeric antigen receptor, and a method for preparing the modified immune cell. A modified immune cell that co-express an MSLN-specific CAR and an additional bioactive molecule, such as a PD-1 antibody and/or RIL-15, or NKG2D or an active fragment, and a method for preparing the modified immune cell. The bispecific antigen-binding molecules, CAR and immune cells are used for preventing and/or treating diseases related to the expression of mesothelin, such as malignant pleural mesothelioma, pancreatic cancer, lung cancer, breast cancer, ovarian cancer and other cancers.

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 a bispecific antigen-binding molecule specifically binding to MSLN and NKG2D ligand (NKG2DL), and a bispecific chimeric antigen comprising the bispecific antigen-binding molecule Receptors, engineered immune cells expressing said bispecific chimeric antigen receptors, and methods for preparing said engineered immune cells. The present invention also relates to engineered immune cells that co-express MSLN-specific CARs and additional bioactive molecules (such as PD-1 antibodies and/or rIL-15, or NKG2D or active fragments), and the preparation of said engineered immune cells cell method. The present invention also relates to the use of these bispecific antigen binding molecules, CARs and immune cells for the prevention and/or treatment of 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 repeat fragment of the N-terminal extracellular domain and participates in cell adhesion. MSLN has highly specific expression, low expression in mesothelial cells of peritoneal cavity, pleural cavity and pericardial cavity in normal tissues, high expression in malignant pleural mesothelioma, pancreatic cancer, lung cancer, breast cancer, ovarian cancer and other solid tumors Expression, especially in malignant mesothelioma (85%-90%), pancreatic cancer (80%-85%), ovarian epithelial cancer (60%-65%) and lung cancer (60%-65%) , related to cell proliferation, cell adhesion function and anti-apoptotic process. These biological characteristics suggest that MSLN can serve as an ideal target for tumor therapy 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 most 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 exerting a killing effect on tumor cells. One type of ligand of NKG2D is MHC class I chain-related molecules A/B (MHC class I chain-related molecules A/B, MIC-A/B), and the other type of ligand is UL16-binding proteins (UL16-binding proteins ,ULBPs). NKG2D ligands (NKG2DL) are hardly 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 NKG2D Binding of a receptor to its ligand activates immune effector cells, causing killing. NKG2DL is mainly expressed on most epithelial tumor cells, such as ovarian cancer and colon cancer, and rarely expressed on normal cells.

As the incidence of cancer increases year by year, traditional methods of surgery, radiotherapy, and chemotherapy are ineffective in tumor treatment, and there is an urgent need for clinically effective tumor treatments. 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 patients in vitro, transduces specific exogenous genes in vitro, amplifies them in vitro and infuses them back into patients, so as to achieve the purpose of treating tumors in a non-MHC-restricted manner. CAR-T cell therapy has achieved remarkable efficacy in the treatment of hematologic malignancies, and the complete remission rate for relapsed and refractory B-cell leukemia exceeds 90%. Solid tumors account for about 90% of all malignant tumors, and their therapeutic drugs are in great demand. 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.

Contents 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 designs and constructs MSLN/NKG2DL bispecific CAR and immune cells that co-express MSLN-specific CAR and NKG2DL-specific binding molecules, and improves the killing of tumor antigen-expressing cells by dual targeting MSLN and NKG2DL And reduce its off-target toxicity to a certain extent. The present invention also designs and constructs a MSLN-targeted CAR that co-expresses PD-1 antibody and/or rIL-15, and blocks the combination of PD-1 and PD-L1 by co-expressing PD-1 antibody to restore the activity of T cells , so as to enhance the immune response; co-expression of 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 is capable of directing immune effector cell specificity and reactivity to cells expressing MSLN or both MSLN and NKG2DL in a non-MHC-restricted manner (e.g. malignant pleural mesothelioma, pancreatic cancer, lung cancer, breast cancer, ovarian cancer) thereby causing it to be cleared. Therefore, the CAR targeting MSLN or simultaneously targeting MSLN and NKG2DL of the present invention has the potential to be used for the prevention and/or treatment of MSLN-positive tumors such as malignant pleural mesothelioma, pancreatic cancer, lung cancer, breast cancer, and ovarian cancer, and has great potential. 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 includes: the following three heavy chain CDRs defined according to the Kabat numbering system: the sequence is CDR-H1 of SEQ ID NO: 3 or a variant thereof; the sequence is SEQ ID NO: 4 or a variant thereof CDR-H2; the sequence is CDR-H3 of SEQ ID NO: 5 or a variant thereof; and/or, the VL includes: the following three light chain CDRs defined according to the Kabat numbering system: the sequence is SEQ ID NO: 6 CDR-L1 of a variant thereof; CDR-L2 of SEQ ID NO: 7 or a variant thereof; CDR-L3 of SEQ ID NO: 8 or a variant thereof;

or

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

or

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

Wherein, the variant described in any one of (1), (2), (3) has one or several amino acid substitutions, deletions or additions (such as 1, 2 or substitution, deletion or addition of 3 amino acids); preferably, the substitution is a conservative substitution.

In certain embodiments, the VH and/or VL further comprise 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 , or have one or several amino acid substitutions, deletions or additions (eg, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, Said 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 , or have one or several amino acid substitutions, deletions or additions (eg, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, Said 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 ), minibodies, disulfide-stabilized Fv proteins (dsFv) and single domain antibodies (sdAb, nanobodies).

In certain embodiments, the first antigen-binding domain is a single-chain antibody, and the single-chain antibody includes in order from its N-terminus to its C-terminus:

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

wherein said 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, or have one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); preferably, said substitutions are conservative substitutions.

In certain embodiments, said L1 is a polypeptide. In some embodiments, the L1 comprises one or several (eg, 1, 2 or 3) sequences shown as (GmS)n, wherein m is selected from an integer of 1-6, and n is selected from 1 An integer of -6. In certain embodiments, m is 3, 4, or 5. In certain embodiments, n is 1 or 2. In certain embodiments, said L1 has the 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 having at least 70%, at least 75%, at least 80%, compared to SEQ ID NO: 18 %, 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 sequence, or a substitution, deletion, or addition of one or several amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids Substitution, deletion or addition); Preferably, the substitution is a conservative substitution.

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

In certain embodiments, the second antigen-binding domain comprises the full-length sequence of NKG2D, for example comprising the sequence shown in SEQ ID NO: 21 or a variant thereof which has an 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, or have one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions) compared to the sequence from which it is derived , deletion or addition); preferably, the substitution is a conservative substitution.

In certain embodiments, the second antigen-binding domain comprises a ligand-binding domain of NKG2D, eg, an extracellular antigen-binding domain. In certain embodiments, the second antigen binding domain comprises the sequence set forth in SEQ ID NO: 20 or a variant thereof having 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% of the sequence identity, or have 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 Preferably, the substitutions are conservative substitutions.

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

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

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

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

In certain embodiments, the bispecific antigen binding molecule comprises the sequence set forth in SEQ ID NO: 22 or a variant thereof having 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 sequences, or substitutions, deletions or additions of one or several amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or Substitution, deletion or addition of 10 amino acids); preferably, the substitution is a conservative substitution.

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, for example, by genetic engineering and recombination techniques. For example, a DNA molecule encoding the bispecific antigen-binding molecule is obtained by chemical synthesis or PCR amplification, inserted into an expression vector, and then transfected into a host cell. Then, the transfected host cells are cultured under specific conditions, and express the bispecific antigen-binding molecule of the present invention.

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

A third aspect of the present invention provides a vector (such as a cloning vector or an expression vector) comprising the 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 It 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 E. 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, which comprises, under conditions that allow protein expression, culturing the host cell as described above, and extracting from the cultured host cell culture The bispecific antigen binding molecule is recovered.

bispecific chimeric antigen receptor

The present invention relates to CARs targeting MSLN and NKG2DL, characterized by non-MHC-restricted recognition of MSLN and NKG2DL, which confers on immune cells (e.g., T cells, NK cells, monocytes, macrophages, or tree cells) expressing the CAR. dendritic cells) have the ability to recognize MSLN- and NKG2DL-expressing cells (eg, tumor cells) independently of antigen processing and presentation.

Therefore, the fifth aspect of the present invention provides a bispecific chimeric antigen receptor comprising 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 endows the CAR with the ability to recognize MSLN and NKG2DL.

In certain embodiments, the bispecific antigen binding domain comprises the bispecific antigen binding molecule of 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 an 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-terminal or C-terminal of the second antigen-binding domain through a linker L2.

In certain embodiments, the bispecific antigen binding domain comprises the sequence set forth in SEQ ID NO: 22 or a variant thereof having 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 a sequence with 100% identity, or a substitution, deletion or addition of one or several amino acids (for example, 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 contained 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 cell membranes (especially eukaryotic cell membranes). The transmembrane domains of bispecific CARs suitable for use in the invention may be derived from natural sources. In such embodiments, the transmembrane domain may be derived from any membrane-bound or transmembrane protein. Alternatively, the transmembrane domain may be a synthetic non-naturally occurring protein segment, eg, a protein segment comprising predominantly hydrophobic residues such as leucine and valine.

In certain embodiments, the transmembrane domain is a transmembrane region of a protein selected from the group consisting of alpha, beta, or zeta chains of T cell receptors, 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 of a protein selected from the group consisting of 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 spacer domain contained in the bispecific chimeric antigen receptor of the present invention is 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 (eg, IgGl 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 out of the cell membrane of the cell expressing the bispecific CAR and may more accurately mimic the native Size and domain structure of TCRs.

In certain embodiments, the spacer domain comprises a hinge domain. A hinge domain may be a stretch of amino acids typically found between two domains of a protein that may allow the protein to be flexible and allow movement of one or both domains relative to each other. Thus, the hinge domain may be any amino acid sequence that provides such flexibility of the extracellular antigen binding domain and such mobility relative to the transmembrane domain.

In certain embodiments, the hinge domain is the hinge region or portion thereof of a naturally occurring protein. In certain embodiments, the spacer domain is selected from a hinge domain and/or the CH2 and CH3 regions of an immunoglobulin (eg, IgGl or IgG4). In certain embodiments, the hinge domain comprises the 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 comprise a signal peptide SP1 at its N-terminus. Typically, a signal peptide is a polypeptide sequence that targets the sequence to which it is linked to a desired site. In certain embodiments, the signal peptide can target the bispecific CAR linked thereto to the secretory pathway of the cell and allow further integration and anchoring of the bispecific CAR into the lipid bilayer. Signal peptides that can be used in CAR are known to those skilled in the art. In certain embodiments, the signal peptide SP1 comprises a heavy chain signal peptide (such as an IgG1 heavy chain signal peptide), a granulocyte-macrophage colony-stimulating factor receptor 2 (GM-CSFR2) signal peptide, an IL2 signal peptide , or CD8α signal peptide. In certain preferred embodiments, the signal peptide SP1 is selected from CD8α signal peptides. In certain exemplary embodiments, the signal peptide SP1 comprises the amino acid sequence shown in SEQ ID NO:34.

In certain embodiments, the bispecific CAR of the present invention can also be combined with other biologically active molecules (such as antigen-binding molecules targeting NKG2D ligand (NKG2DL), or PD1/PD-L1 pathway inhibitors and IL-15 agonist) co-expression. The other bioactive molecule may have its own signal peptide, which is named signal peptide SP2 to distinguish it from the signal peptide in the previous paragraph. The signal peptide SP2 directs the transport of additional bioactive molecules to specific sites within the cell or outside the cell membrane. The signal peptide SP2 may be the same as 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 domains

The intracellular signaling domain contained in the bispecific CAR of the present invention is involved in transducing the signal generated by the combination of the bispecific CAR of the present invention with MSLN and/or NKG2DL into the immune effector cells, activating the expression of bispecific At least one normal effector function of the immune effector cells of the CAR, or enhanced secretion of at least one cytokine (eg, IL-2, IFN-γ) of the immune effector cells 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 activation motif (ITAM). In certain embodiments, the primary signaling domain comprises an immunoreceptor tyrosine 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 the intracellular signaling domain of CD3ζ.

In certain embodiments, the co-stimulatory signaling domain may be an intracellular signaling domain from a co-stimulatory molecule. In certain embodiments, the co-stimulatory 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 thereof.

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 may be tandem to the carboxy-terminus of the transmembrane domain in any order.

In certain embodiments, the intracellular signaling domain may comprise an intracellular signaling domain of CD3ζ and an intracellular signaling domain of CD137. In certain exemplary embodiments, the intracellular signaling domain of the CD3ζ comprises the amino acid sequence shown in SEQ ID NO:48. In certain exemplary embodiments, the intracellular signaling domain of the CD137 comprises 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. The chimeric antigen receptor comprises a bispecific antigen binding domain, a spacer domain, and a transmembrane structure from its N-terminus to its C-terminus. Domain, intracellular signaling domain. In certain preferred embodiments, wherein 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 having a sequence as shown in SEQ ID NO: 38 or 40).

In certain embodiments, the transmembrane domain comprises a transmembrane region of CD8 (e.g., CD8α) or CD28 (e.g., a transmembrane region having a sequence as set forth 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 an intracellular signaling domain of CD3ζ (e.g., as sequence shown in SEQ ID NO:48), the co-stimulatory signaling domain comprises the intracellular signaling domain of CD137 (4-1BB) (for example, as the sequence shown in SEQ ID NO:46); more preferably, The intracellular signaling domain of the chimeric antigen receptor has a sequence shown in SEQ ID NO:50.

In some preferred embodiments, the chimeric antigen receptor comprises the signal peptide SP1, the bispecific antigen binding domain, the spacer domain, the transmembrane domain, the intracellular Signal transduction domains (from N-terminal to C-terminal are co-stimulatory signaling domains and primary signaling domains).

In certain embodiments, the signal peptide SP1 comprises an IgG1 heavy chain signal peptide or a CD8α signal peptide (for example, a signal peptide whose sequence is shown in SEQ ID NO: 34). In certain exemplary embodiments, the bispecific CAR of the invention comprises the sequence shown in SEQ ID NO: 23 or a variant thereof having 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, or a substitution, deletion or addition of one or several amino acids (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. Coexpressed Bispecific CARs and Additional Bioactive 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 amino acids from forming covalent bonds during translation and maintain translation to continue, so that the translation product is "self-cleaved", thereby enabling the bispecific chimeric antigen receptor of the present invention and additional biological activity molecular separation. 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 capable of specifically binding to MSLN and NKG2DL becomes an independent extracellular antigen-binding structure Domain, spacer domain, transmembrane domain, and intracellular signaling domain CAR, while other biologically active molecules can be secreted outside the cell or express membrane-forming chimeric polypeptides or proteins. With the expansion and enrichment of immune cells expressing bispecific CARs in the tumor microenvironment, additional bioactive molecules are enriched in the tumor microenvironment, and synergistically exert anti-tumor effects with bispecific CARs.

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 linked to the nucleic acid sequence of another biologically active molecule via the nucleic acid sequence of the self-cleaving peptide. The bispecific CAR can be at the N- or C-terminus of another biologically active molecule. In certain exemplary embodiments, the bispecific CAR is 5' to the additional biologically active molecule. Any self-cleaving peptide capable of causing the fusion protein to be cleaved into two separate proteins can be used in the present invention. In some 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 gene recombination. In such embodiments, the fusion protein comprising the bispecific CAR and the additional biologically active molecule has the following structure:

N'-signal peptide SP1--extracellular antigen-binding domain that specifically binds MSLN and NKG2DL--spacer domain-transmembrane domain-intracellular signaling domain-self-cleavage peptide-signal peptide SP2--other Bioactive Molecules - C'. Wherein the signal peptide SP2 is the same 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, the PD1/PD-L1 pathway inhibitor and IL-15 agonist included in the additional bioactive 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 antigen-binding fragment thereof; preferably, the anti-PD-1 or PD-L1 antibody or its The antigen-binding fragment is a single-chain antibody (such as scFv); preferably, the anti-PD-1 single-chain antibody comprises the amino acid sequence shown in SEQ ID NO:52 or a variant thereof, and the variant is identical to SEQ ID NO:52 Compared to having 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% , sequences of at least 98%, at least 99%, or 100% identity, or substitutions, deletions, or additions of one or several amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions); preferably, said substitutions are conservative substitutions.

In certain embodiments, the IL-15 agonist is selected from a fusion protein comprising IL-15 and IL-15 receptor alpha (IL-15Rα) Sushi domain; preferably, the IL-15 agonist comprises The amino acid sequence shown in SEQ ID NO:54 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91% compared to SEQ ID NO:54 %, 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, or have one or several amino acids Substitutions, deletions or additions (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions); preferably, all The substitutions described are conservative substitutions.

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

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

The MSLN-specific chimeric antigen receptor comprises an MSLN-targeted antigen-binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain, and the MSLN-targeted antigen-binding domain comprises the first aspect The first antigen-binding domain defined in, the spacer domain, transmembrane domain and 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 with an extracellular antigen-binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain, while another Biologically active molecules can be secreted outside the cell or express membrane-forming chimeric polypeptides or proteins.

Said additional bioactive molecules comprise antigen binding molecules targeting NKG2D ligand (NKG2DL), or comprise PD1/PD-L1 pathway inhibitors and IL-15 agonists.

In certain embodiments, the nucleic acid sequence encoding the MSLN-specific CAR is linked to the nucleic acid sequence of another biologically active molecule via the nucleic acid sequence of the self-cleaving peptide. The MSLN-specific CAR can be at the N- or C-terminus of another biologically active molecule. In certain exemplary embodiments, the MSLN-specific CAR is 5' to the additional biologically active molecule. Any self-cleaving peptide capable of causing the fusion protein to be cleaved into two separate proteins can be used in the present invention. In some 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 gene recombination. In such embodiments, the fusion protein comprising the MSLN-specific CAR and the additional biologically active molecule has the following structure:

N'-signal peptide SP1--extracellular antigen-binding domain specifically binding to MSLN--spacer domain-transmembrane domain-intracellular signaling domain-self-cleavage peptide-optional signal peptide SP2--in addition Bioactive Molecule-C'. Wherein, depending on other biologically active molecules, the signal peptide SP2 may be optional. 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, when the additional biologically active molecule is an antigen binding molecule targeting 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 NKG2D ligand (NKG2DL)-targeted antigen binding molecule comprises NKG2D or a ligand-binding domain thereof. In certain embodiments, the antigen-binding molecule targeting NKG2D ligand comprises the sequence shown in SEQ ID NO: 20 or 21, or a variant thereof having 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, or have one or several amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions) compared to the sequence from which it is derived or addition); Preferably, the substitution is a conservative substitution.

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

Preparation of Chimeric Antigen Receptors

Methods for generating chimeric antigen receptors and immune effector cells (e.g., T cells) comprising the chimeric antigen receptors are known in the art and may include transfecting cells with at least one polynucleotide encoding a CAR, and The polynucleotide is expressed in the cell. For example, a nucleic acid molecule encoding a CAR of the present invention can be included in an expression vector (eg, a lentiviral vector) capable of being expressed in a host cell, such as a T cell, to produce 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 of the fifth aspect.

Those skilled in the art understand that due to the degeneracy of the genetic code, the nucleotide sequence encoding a chimeric antigen receptor of the present invention may have many different sequences. Thus, unless otherwise stated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.

In certain exemplary embodiments, the nucleotide sequence encoding the bispecific chimeric antigen receptor described in the fifth aspect is selected from: (1) the sequence shown in SEQ ID NO: 24 or its degeneracy variant; (2) a sequence that is substantially identical to the sequence described in (1), e.g., has 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% , a sequence of at least 99%, or 100% sequence identity, or a sequence having one or more nucleotide substitutions compared to the sequence described in (1); and said sequence substantially retains the sequence from which it was derived At least one biological activity of the nucleotide sequence (eg, capable of encoding 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 mentioned 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 the first nucleic acid sequence encoding the bispecific chimeric antigen receptor described in the fifth aspect, and further comprises the first nucleic acid sequence encoding another biologically active molecule. Two nucleic acid sequences.

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-terminal of the additional bioactive molecule is an IL2 signal peptide, and the IL2 signal peptide refers to the signal peptide sequence contained in the IL2 natural gene sequence. Preferably, the IL2 natural gene is Human IL2 natural gene, the IL2 signal peptide is a human IL2 signal peptide. In certain exemplary embodiments, the IL2 signal peptide comprises the sequence shown in SEQ ID NO:36.

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

In some 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 self-cleaving peptide is P2A (e.g., as set forth in SEQ ID NO: 27). In certain exemplary embodiments, the sequence encoding the self-cleaving peptide is linked to the 3' end of the first nucleotide sequence and is linked to the 5' end of the second nucleotide sequence.

In some embodiments, the PD1/PD-L1 pathway inhibitor is selected from 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 encoded by a self-cleaving peptide (such as P2A, E2A, F2A, T2A or any of them) Combination) nucleotide sequence connection. In certain embodiments, the self-cleaving peptide is P2A (e.g., as set forth in SEQ ID NO: 27).

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

As described in the sixth aspect above, 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 comprising:

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

(2) a second nucleic acid sequence encoding an additional biologically active molecule comprising an antigen-binding molecule targeting NKG2D ligand (NKG2DL); wherein,

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

In some 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 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, spacer domain, and intracellular signaling domain contained in 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 said second nucleotide sequence does not comprise a signal peptide at its N-terminus. 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 that described in the first nucleic acid sequence. The signal peptide SP1 contained in the encoded MSLN-specific chimeric antigen receptor; 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 nucleic acid construct sequentially comprises from its 5' end to its 3' end: a nucleotide sequence encoding the signal peptide SP1, a nucleoside encoding the MSLN-targeted antigen-binding domain Acid sequence, nucleotide sequence encoding the spacer domain, nucleotide sequence encoding the transmembrane domain, nucleotide sequence encoding the intracellular signaling domain, encoding the self-cleavage peptide sequence The nucleotide sequence of the nucleotide sequence encoding the antigen-binding molecule targeting NKG2D ligand (NKG2DL).

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

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

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

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 a ligand of NKG2D comprises NKG2D or a ligand-binding domain thereof, for example comprising the sequence shown in SEQ ID NO: 20 or 21.

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

In some exemplary embodiments, the nucleic acid construct comprises a nucleotide sequence selected from the group consisting of: (1) the sequence shown in SEQ ID NO: 26 or a degenerate variant thereof; (2) and (1) The sequence shown in any of (1) has 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%, A sequence having at least 99%, or 100% sequence identity, 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, MSLN-specific chimeric antigen receptors can be co-expressed with PD1/PD-L1 pathway inhibitors and IL-15 agonists to synergistically exert anti-tumor effects.

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

(2) A second nucleic acid sequence encoding an additional bioactive molecule comprising a PD1/PD-L1 pathway inhibitor and an IL-15 agonist; wherein,

In some 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 encoded by a self-cleaving peptide (such as P2A, E2A, F2A, T2A or any combination thereof) nucleotide sequence linkage.

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 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 that described in the first nucleic acid sequence. The signal peptide SP1 contained in the encoded MSLN-specific chimeric antigen receptor; 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 nucleic acid construct sequentially comprises the nucleotide sequence encoding the signal peptide SP1, the nucleotide sequence encoding the MSLN-targeted antigen binding domain from its 5' end to the 3' end sequence, the nucleotide sequence encoding the spacer domain, the nucleotide sequence encoding the transmembrane domain, the nucleotide sequence encoding the intracellular signaling domain, the self-cleaving peptide sequence encoding Nucleotide sequence, nucleotide sequence encoding the signal peptide SP2, nucleotide sequence encoding the PD1/PD-L1 pathway inhibitor, nucleotide sequence encoding the self-cleavage peptide sequence, encoding the Nucleotide sequences of IL-15 agonists.

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

In certain embodiments, the signal peptide SP2 comprises an IL2 signal peptide (e.g., as set forth in SEQ ID NO: 36).

In some exemplary embodiments, the nucleic acid construct comprises a nucleotide sequence selected from the group consisting of: (1) the sequence shown in SEQ ID NO: 57 or a degenerate variant thereof; (2) and (1) The sequence shown in any of (1) has 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%, A sequence having at least 99%, or 100% sequence identity, 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 of the seventh aspect, or the nucleic acid construct of the eighth, ninth or tenth aspect.

In some embodiments, the vector is selected from DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR/Cas9 vectors, and viral vectors.

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 also provides a modified immune cell that expresses the isolated nucleic acid molecule described in the seventh aspect of the present invention. The engineered immune cell expresses the bispecific chimeric antigen receptor described in the fifth aspect.

The thirteenth aspect of the present invention also provides a modified immune cell that expresses the nucleic acid construct described in the eighth aspect of the present invention. The engineered immune cells express the bispecific chimeric antigen receptor of the fifth aspect and the additional bioactive molecules, such as PD1/PD-L1 pathway inhibitors and IL-15 agonists.

The fourteenth aspect of the present invention also provides a modified immune cell that expresses the nucleic acid construct described in the ninth aspect of the present invention. The engineered immune cells express MSLN-specific chimeric antigen receptors and antigen-binding molecules targeting NKG2D ligand (NKG2DL). In certain embodiments, the NKG2D ligand (NKG2DL)-targeted antigen binding molecule optionally has a 4-1BB intracellular signaling domain linked to its N-terminus.

The fifteenth aspect of the present invention also provides a modified immune cell that expresses the nucleic acid construct described in the tenth aspect of the present invention. The engineered immune cells express MSLN-specific chimeric antigen receptors as well as PD1/PD-L1 pathway inhibitors and IL-15 agonists.

In some 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; optionally Preferably, the immune cells are obtained from healthy donors; preferably, the immune cells are derived from T lymphocytes or NK cells.

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

The present invention also provides a method for preparing modified immune cells, which includes: (1) providing immune cells from patients or healthy donors; (2) using the isolated nucleic acid molecules described in the seventh aspect, or the eighth aspect , the nucleic acid constructs described in the ninth aspect or the tenth aspect, or vectors containing them are introduced into the immune cells described in step (1), so as to obtain immune cells capable of expressing and optionally additional bioactive molecules.

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

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

In some embodiments, in step (2), the nucleic acid molecule or vector is introduced into the immune cells through non-viral vector transfection, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, Microinjection, transposon vector systems, CRISPR/Cas9 vectors, TALEN approach, ZFN approach or electroporation approach.

In certain embodiments, after step (2), a step of expanding the immune cells obtained in step (2) is further included.

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 preceding aspects, and optionally unmodified and/or unsuccessfully modified immune cells, These unengineered and/or unsuccessfully engineered immune cells do not express the CAR of interest. Limited by the current level of technology and some unknown reasons, not all immune cells can be engineered to express the target CAR. 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 CAR of interest account for about 10%-100% of the total number of cells in the immune cell composition, preferably 40%-80%.

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 comprising the immune cell composition.

In another aspect, the present invention provides a kit for preparing the engineered immune cells described in any of the above aspects. In certain embodiments, the kit includes the isolated nucleic acid molecule of the seventh aspect, or the nucleic acid construct of the eighth, ninth, or tenth aspect, or a vector comprising them, and the necessary Solvents, such as sterile water, normal saline, or cell culture fluid, such as LB culture fluid, such as EliteCell primary T lymphocyte culture system (product number: PriMed-EliteCell-024), and optionally, instructions for use .

pharmaceutical composition

In the seventeenth aspect, the present invention provides a pharmaceutical composition, which contains the bispecific antigen-binding molecule described in the first aspect of the present invention, the bispecific chimeric antigen receptor (including bispecific antigen receptor) described in the fifth aspect CAR constructs co-expressed with specific chimeric antigen receptors and other biologically active molecules), the isolated nucleic acid molecules described in the second aspect or the seventh aspect, the eighth aspect or the ninth aspect or the tenth aspect The nucleic acid construct, the vector described in the third aspect or the eleventh aspect, the host cell described in the fourth aspect, the modified DNA described in the twelfth aspect, the thirteenth aspect, the fourteenth aspect or the fifteenth aspect The immune cells of the invention, or the immune cell composition described in the sixteenth aspect, and pharmaceutically acceptable carriers and/or excipients.

In certain embodiments, the pharmaceutical composition further comprises additional pharmaceutically active agents, such as drugs with anti-tumor activity (such as anti-PD1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, Pemet Trecet, 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 (comprising bispecific chimeric antigen receptor and another biologically active molecule co-expressed CAR construct), 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 third aspect or the eleventh aspect The vector described in the fourth 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 described in the sixteenth aspect The composition and the additional pharmaceutically active agent may be administered simultaneously, separately or sequentially.

In certain embodiments, the pharmaceutical composition of the present invention comprises: the bispecific antigen-binding molecule of 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 or the ninth aspect or the tenth aspect, or a vector comprising them.

In some 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 sixteenth aspect immune cell composition.

The bispecific antigen-binding molecules described in the first aspect of the present invention, the bispecific chimeric antigen receptors described in the fifth aspect (including the construction of CARs co-expressed with bispecific chimeric antigen receptors and other biologically active molecules) body), 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 second 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 powder for injection and concentrated solution for injection), inhalation, spray, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. Pharmaceutical compositions of the invention should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is injection. Such injections can be sterile injectable solutions. In addition, sterile injectable solutions can be prepared as sterile lyophilized powder (eg, by vacuum drying or freeze-drying) for ease of storage and use. Such sterile lyophilized powders can be dispersed in suitable carriers before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (such as 0.9% (w/v) NaCl), Dextrose solution (eg 5% glucose), surfactant containing solution (eg 0.01% polysorbate 20), pH buffer solution (eg 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 receptors described in the fifth aspect (including the construction of CARs co-expressed with bispecific chimeric antigen receptors and other biologically active molecules) body), 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 second 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 obtained by Administration by any suitable method known in the art, including but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical ( eg, powder, ointment, or drops), or nasal route. However, for many therapeutic uses, the preferred route/mode of administration is parenteral (eg, intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will understand that the route and/or manner of administration will vary depending on 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 bispecific chimeric antigen receptor and another biological active molecule co-expressed CAR construct), 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 third aspect or the eleventh aspect The vector according to the aspect, the host cell according to the fourth aspect, the modified immune cell according to the twelfth aspect or the thirteenth aspect or the fourteenth aspect or the fifteenth aspect, or the sixteenth aspect The immune cell composition is given by intravenous injection or bolus injection.

The pharmaceutical composition of the present invention may include "therapeutically effective amount" or "preventively 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 ( Comprising 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 eighth aspect or the ninth aspect or the tenth aspect The nucleic acid construct described in the third aspect or the carrier described in 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 described A modified immune cell, or the immune cell composition described in the sixteenth aspect. "Prophylactically effective amount" refers to an amount sufficient to prevent, arrest, or delay the onset of a disease. A "therapeutically effective amount" refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. The bispecific antigen-binding molecules described in the first aspect of the present invention, the bispecific chimeric antigen receptors described in the fifth aspect (including the construction of CARs co-expressed with bispecific chimeric antigen receptors and other biologically active molecules) body), 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 second aspect The treatment of 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 The effective amount may vary depending on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered at the same time, etc. .

Treatment and Use

In another aspect, the present invention provides a method for preventing and/or treating diseases associated with the expression of mesothelin in a subject (such as a human), the method comprising submitting to a subject in need thereof or administering an 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 (comprising bispecific chimeric antigen receptor and another biologically active molecule co-expressed CAR construct), 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 third aspect or the eleventh aspect The vector described in the fourth 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, the immune cell described in the sixteenth aspect composition, or the pharmaceutical composition described in 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 expression of mesothelin is a non-tumor-related indication associated with 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 (eg, malignant pleural mesothelioma, pancreatic cancer, lung cancer (eg, lung squamous cell carcinoma), breast cancer, ovarian cancer (eg, 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 the twelfth aspect, the thirteenth aspect, the fourteenth aspect, or the fifteenth aspect, or The immune cell composition of the sixteenth aspect.

In some embodiments, the method includes the following steps: (1) providing the immune cells (such as 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 comprising them into the step ( 1) the immune cells; (3) administering the immune cells obtained in step (2) to the subject for treatment.

In certain embodiments, the method administers the immune cells expressing the CAR of interest to the subject by dose-fractionation, e.g., one, two, three or more divided doses, e.g., on the first day of treatment. A first percentage of the total dose administered on one day and a second percentage of the total dose administered on a subsequent (e.g., second, third, fourth, fifth, sixth, or seventh day or later) treatment day , such as on subsequent (e.g. third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or later) treatment days a third percentage of the total dose (e.g., remaining percentage).

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

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

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

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 (comprising bispecific chimeric antigen receptor and another A CAR construct co-expressed with biologically active molecules), 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 third aspect or the tenth aspect The vector of one 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 sixteenth aspect The immune cell composition of the invention, or the pharmaceutical composition of the seventeenth aspect is administered in combination with another agent. In certain embodiments, the additional reagents include (i) increasing cells comprising a CAR nucleic acid or CAR polypeptide (e.g., immune cells expressing a CAR of the invention, engineered immune cells or immune cell compositions of the invention) (ii) improving one or more of the effects associated with administration of cells comprising CAR nucleic acid or CAR polypeptide (such as immune cells expressing CAR of the present invention, engineered immune cells or immune cell compositions of the present invention) Agents for various side effects; (iii) Additional pharmaceutically active agents with antitumor activity. These agents can be administered after the administration of the bispecific antigen-binding molecules described in the first aspect of the present invention, the bispecific chimeric antigen receptors described in the fifth aspect (including bispecific chimeric antigen receptors and other biologically active molecules) co-expressed CAR construct), 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 third aspect or the eleventh aspect The vector described in the fourth 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, the immune cell described in the sixteenth aspect composition, or the pharmaceutical composition of the seventeenth aspect before, simultaneously or after administration.

In certain embodiments, the methods described above further comprise 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, virotherapy, adjuvant therapy and any combination thereof.

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

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

In another aspect, there are provided the bispecific antigen binding molecules described in the first aspect of the present invention, the bispecific chimeric antigen receptors described in the fifth aspect (comprising bispecific chimeric antigen receptors and other biological active molecule co-expressed CAR construct), 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 third aspect or the eleventh aspect The vector described in the 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, the sixteenth aspect Use of the immune cell composition, or the pharmaceutical composition described in the seventeenth aspect, in the preparation of a medicament for preventing and/or treating diseases related to the expression of mesothelin. The dosage, dosage form, route of administration, indications, combination therapy and other aspects of the aforementioned treatment methods can be applied to the use of the drug.

Acronym

CAR chimeric antigen receptor

CDR Complementarity Determining Regions in Immunoglobulin Variable Regions

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

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

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

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

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

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

FR Antibody framework region: the amino acid residues in the antibody variable region except CDR residues

VH Antibody heavy chain variable region

VL antibody light chain variable region

Kabat 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.

(IMGT)), see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.

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

IL-2 Interleukin 2

IFN Interferon

PCR polymerase chain reaction

FACS Flow Cytometry Fluorescence Sorting

_KD equilibrium dissociation constant

kon association 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. Moreover, the operational steps of molecular biology, microbiology, cell biology, biochemistry, immunology, etc. used herein are all routine steps widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

As used herein, the term "antibody" refers to an immunoglobulin antibody capable of specifically binding to a target (such as carbohydrates, polynucleotides, lipids, polypeptides, etc.) through at least one antigen recognition site located in the variable region of an immunoglobulin molecule. Globulin molecule. As used herein, the term includes not only intact polyclonal or monoclonal antibodies, but also fragments thereof (e.g. Fab, Fab', F(ab')2, Fv), single chains (e.g. scFv, di-scFv, (scFv ) ₂ ) and domain antibodies (including eg shark and camel antibodies), as well as fusion proteins comprising antibodies, and immunoglobulin molecules comprising any other modified configuration of an antigen recognition site. The antibodies of the present invention are not limited by any particular method of producing antibodies. Antibodies include antibodies of any class, such as IgG, IgA, or IgM (or subclasses thereof), and antibodies need not be of any particular class. Depending on the amino acid sequence of the constant region of the antibody heavy chains, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. The heavy chain constant region consists of 4 domains (CH1, hinge region, CH2 and CH3). Each light chain is composed 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-antigen binding, but exhibit a variety of effector functions, such as mediating immunoglobulin interactions with host tissues or factors, including various cells of the immune system (e.g., effector cells) and classical complement Binding of the first component (C1q) of the 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 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from amino-terminus to carboxy-terminus. The variable regions (VH and VL) of each heavy chain/light chain pair form the antigen binding site, respectively. The allocation of amino acids in each region or domain can follow Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987and 1991)), or Chothia & Lesk (1987) J.Mol.Biol.196:901- 917; Definition by 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. The variable regions of the heavy and light chains each contain three CDRs, named 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, those skilled in the art will readily identify the CDRs defined by each numbering system. Also, the correspondence between different numbering systems is well known to those skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

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

As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region 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 the same antigen to which the full-length antibody binds, and/or Or compete 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. Can be obtained by recombinant DNA techniques. or by enzymatic or chemical cleavage of intact antibodies to produce antigen-binding fragments of antibodies. Non-limiting examples of antigen-binding fragments include camelid Ig, Ig NAR, Fab fragments, Fab' fragments, F(ab)' ₂ fragments, F(ab )' ₃ fragments, Fd, Fv, scFv, di-scFv, (scFv) ₂ , minibodies, diabodies, tribodies, tetrabodies, disulfide bond stabilized Fv proteins ("dsFv") and single constructs Domain antibodies (sdAbs, Nanobodies) and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding capacity on the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136 middle.

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 a fragment comprising two An antibody fragment of a Fab fragment; the term "Fab'fragment" means the fragment obtained after reduction of the disulfide bond linking the two heavy chain fragments in the F(ab') ₂ fragment, consisting of an intact light chain and the Fd of the heavy chain Fragment (consisting of VH and CH1 domains).

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 capable of forming a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a variable region (such as the Fd fragment, which contains only three CDRs specific for an antigen) is capable of recognizing and binding antigen, although perhaps with a lower affinity than the full binding site.

As used herein, the term "Fc" means that the second and third constant regions of the first heavy chain of an antibody are combined with the second and third constant regions of the second heavy chain via disulfide bonds. Antibody fragments. The Fc fragment of an antibody has a variety of different functions, but is not involved in antigen binding.

As used herein, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are linked by a linker (see, e.g., Bird et al., Science 242:423 -426 (1988); Huston et al, Proc. 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 the repeated GGGGS amino acid sequence 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, described by Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001) Cancer Immunol. In some cases, there may also be a disulfide bond 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 _NH2 -VH-VH-COOH, _NH2- VL-VL-COOH. The scFv can form any engineering possible 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 scFv connected in series to form an antibody. In certain embodiments of the present invention, scFv can form (scFv) ₂ , which refers to the parallel connection of two or more single scFv to form an antibody.

As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art, which refers to a single monomer variable antibody domain (such as a single heavy chain variable antibody domain) region) that retains the ability to specifically bind to the same antigen to which the full-length antibody binds (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 maintains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen.

Antigen-binding fragments of antibodies (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 techniques or enzymatic or chemical cleavage methods) ), and antigen-binding fragments of antibodies are screened for specificity in the same manner as for whole antibodies.

Herein, unless the context clearly dictates otherwise, when the term "antibody" is referred to, it includes not only whole antibodies but also antigen-binding fragments of antibodies.

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

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

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

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the expected properties of the protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions for amino acid residues with amino acid residues that have similar side chains, e.g., are physically or functionally similar (e.g., have similar size, shape, charge, chemical properties, including Ability to form covalent or hydrogen bonds, etc.) Families of amino acid residues having similar side chains have been defined in the art. These families include those 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), non-polar side chains (such as alanine, valine, leucine, isoleucine amino acid, proline, phenylalanine, methionine), beta branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine) amino acids. Therefore, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying amino acid conservative 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 writing of the twenty conventional amino acids referred to herein follows conventional usage. See, e.g., 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 sequences that replicate autonomously directly in a cell, or may include sequences sufficient to permit integration into the host cell DNA. When the vector is capable of achieving expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phage such as lambda phage or M13 phage and viral vectors. Non-limiting examples of viral vectors include, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, Vascular viruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication.

As used herein, episomal in the term "episomal vector" means that the vector is capable of replication without being integrated into the chromosomal DNA of the host and is not gradually lost by dividing host cells, and also means that the vector is extrachromosomal or episomal copy.

As used herein, the term "viral vector" is used broadly to refer to a nucleic acid molecule (such as a transfer plasmid) that includes virus-derived nucleic acid elements that typically facilitate the transfer or integration of the nucleic acid molecule into the genome of a cell, or mediate the transfer of the nucleic acid of virus particles. Virions will typically include, in addition to nucleic acid, various viral components and sometimes host cell components.

The term "viral vector" may 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 derived primarily from viruses.

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

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

As used herein, an "integration-deficient" retrovirus or lentivirus refers to a retrovirus or lentivirus that has an integrase that is unable to integrate the viral genome into the genome of a host cell. In certain embodiments, an integrase protein is mutated to specifically reduce its integrase activity. An integration-deficient lentiviral vector can be obtained by modifying the pol gene encoding an integrase protein to generate a mutant pol gene encoding an integration-deficient integrase. Such integration-defective viral vectors have been described in patent application WO 2006/010834, which is hereby incorporated 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) cells, NK cells, monocytes, macrophages or dendritic cells, etc.). A host cell can include a single cell or a population of cells.

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

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

As used herein, the term "intracellular signaling domain" refers to the portion of a protein that transduces effector function signals and directs the cell to perform a specialized function. Thus, the intracellular signaling domain has the ability to activate at least one normal effector function of a CAR-expressing immune effector cell. For example, the effector function of a T cell can be cytolytic activity or helper activity, including secretion of cytokines.

As used herein, the term "primary signaling domain" refers to the portion of a protein that is capable of modulating the primary activation of the TCR complex in a stimulatory or inhibitory manner. Primary signaling domains that act in a stimulatory manner generally contain signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs). Non-limiting examples of ITAMs containing primary signaling domains particularly useful in the present invention include those derived from TCRzeta, FcRgamma, FcRbeta, CD3gamma, CD3delta, CD3epsilon, CD3zeta, 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 other than antigen receptors or Fc receptors that provide secondary signals required for efficient activation and function of T lymphocytes after binding to antigens. Non-limiting examples of such 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 compatible with the subject and the active ingredient pharmacologically and/or physiologically, These are well known in the art (see e.g. Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: sterile water, physiological saline, pH adjusters, surfactants , adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, preservatives. For example, pH adjusting agents 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 to maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (eg, buffered saline), alcohols and polyols (eg, glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizer has the meaning generally understood by those skilled in the art, and it can stabilize the desired activity of the active ingredient in the medicine, 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 dry whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (eg, aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (eg 0.9% (w/v) NaCl), dextrose solutions (eg, 5% dextrose), solutions containing surfactants (eg, 0.01% polysorbate 20), pH buffered solutions (eg, phosphate buffered saline), Ringer's solutions, and any combination thereof.

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

As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the term "subject" is meant to include living organisms in which an immune response can be elicited. In certain embodiments, the subject (eg, a human) has, or is at risk of having, a tumor (eg, a tumor associated with MSLN).

As used herein, the term "effective amount" refers to an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing a disease (for example, a tumor) refers to an amount sufficient to prevent, arrest, or delay the occurrence of a disease (for example, a tumor); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent an existing disease The patient's disease and the amount of its complications. Determining such an effective amount is well within the capability of those skilled in the art. For example, amounts effective for therapeutic use will depend on the severity of the disease being treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered concomitantly etc.

As used herein, the term "immune cell" refers to a cell involved in an immune response, eg, involved in promoting immune effector functions. Examples of immune cells include T cells (such as 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 invention may be self/autologous ("self") or non-self ("non-self", eg, allogeneic, syngeneic 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 comparative cell; The same cell from a different subject; "allogeneic" refers to a cell from a different species than the compared cell. In preferred embodiments, the cells of the invention are allogeneic.

Exemplary immune cells that can be used in the CARs described herein include T lymphocytes and/or NK cells. The terms "T cells" or "T lymphocytes" are art recognized and are intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes or activated T lymphocytes. T cells may be T helper (Th) cells, such as T helper 1 (Th1) or T helper 2 (Th2) cells. The T cells may be helper T cells (HTL; CD4 T cells) CD4 T cells, cytotoxic T cells (CTL; CD8 T cells), CD4CD8 T cells, CD4CD8 T cells or any other subset of T cells. In certain embodiments, T cells can include naive T cells and memory T cells.

Those skilled in the art will appreciate that other cells can also be used as immune cells with a 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 to differentiate into immune cells in vivo or in vitro. Accordingly, in certain embodiments, the immune cells include progenitor cells of immune cells, such as hematopoietic stem cells (HSCs) contained within a population of CD34+ cells derived from cord blood, bone marrow, or flowing peripheral blood, which are administered to a subject differentiated into mature immune cells, or it can be induced to differentiate into mature immune cells in vitro.

As used herein, the term "modified immune cell" refers to an immune cell expressing any one of the CARs described herein, or introduced with any one of the isolated nucleic acids or vectors described herein. After the polynucleotide encoding the CAR polypeptide can be introduced into the cell by various methods, 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, transient transformation methods can be used to transiently express polynucleotide constructs and the polynucleotide constructs are not integrated into the genome of the cell. In other embodiments, virus-mediated methods can be used. Polynucleotides can be introduced into cells by any suitable means, such as recombinant viral vectors (eg, retroviruses, adenoviruses), liposomes, and the like. Transient transformation methods include, for example but are not limited to, microinjection, electroporation, or particle bombardment. A 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 an immune effector cell that enhances or promotes an immune attack on a target cell (eg, killing of a target cell, or inhibiting its growth or proliferation). For example, the effector function of a T cell can be cytolytic activity or helper activity, including secretion of cytokines.

Beneficial Effects of the Invention

At present, the therapeutic effect of CAR-T cell therapy in solid tumors is still insufficient, mainly because solid tumors have complex tumor microenvironment 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 an active fragment thereof, which improves the killing of cells expressing tumor antigens by dual targeting MSLN and NKG2DL and in a certain reduce its off-target toxicity. The present invention also provides immune cells that co-express MSLN-specific CAR and PD-1 antibody and rIL-15, by co-expressing PD-1 antibody, blocking the combination of PD-1 and PD-L1, and restoring the activity of T cells, Thereby enhancing the immune response; at the same time, the co-expression of 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 with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention, rather than limiting the scope of the present invention. Various objects and advantages of this invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of preferred embodiments.

Description of drawings

Figure 1 shows the structure of the chimeric antigen receptor constructed in Example 2.

FIG. 2 shows the detection results of antigen expression 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 assaying the killing activity of CAR-T on target cells in Experimental Example 6. Figure 3A: The killing results of G16-PD1-rIL15 CAR and G16 CAR on NCI-H226-luc, SKOV-3-luc, A431-luc; Figure 3B: NK2-G-G16-10 CAR on PANC-1-luc, Killing results of SKOV-3-luc; Figure 3C: Killing results of G16-P2A-NKG2D CAR on PANC-1-luc and SKOV-3-luc.

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

Figure 5 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.

Figure 6 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, but not to limit it.

Unless otherwise specified, the molecular biology experiment methods and immunoassay methods used in the present invention are basically with reference to J.Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M.Ausubel et al., Refinement Molecular Biology Laboratory Manual, 3rd Edition, John Wiley & Sons, Inc., 1995 described in the method. Those skilled in the art understand that the examples describe the present invention by way of example and are not intended to limit the scope of the claimed invention.

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

1) Phage library screening for MSLN scfv

Take 1.33mL of fully human phage library and screen it with biotinylated MSLN and SV magnetic beads, and the screened products are tested for phage titration by plating. Take 0.5 mL of the first-round panning product and mix it with 0.5 mL of PBST, and carry out the second and third rounds of screening according to the above steps.

2) ELISA detection of monoclonal phage

Inoculate a single colony from the phage panning product titer plate into a 96-deep well plate, and detect monoclonal phage by ELISA.

MSLN scFv sequencing: According to the ELISA detection results, 158 single clones were selected for sequencing to obtain the scfv sequence. The forward and reverse primers used for sequencing were: PKLT1F (SEQ ID NO: 61), PKLT1R (SEQ ID NO: 62) . Sequcher software was used to analyze the sequence results to obtain the scfv sequence G16, its 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 table below.

Table 1: CDR sequences of scFv

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

The G16 scfv sequence was connected with the Fc (human IgG1) sequence and constructed in the TGEX-KAL vector, and then transfected into expi293 cells to express and purify the scFv-Fc protein. The Fc (human IgG1) sequence is shown in SEQ ID NO:63. The experimental results of SEC analysis showed that the G16 antibody monomer peak (main peak) area accounted for more than 85%.

Table 2: SEC data of scFv-Fc proteins

4) MSLN cell binding assay

Detect G16 scFv-Fc protein and positive control (that is, HN1 scFv-Fc protein, HN1 scFv amino acid sequence and nucleotide sequence respectively as shown in SEQ ID NO:64 and 65) and 3 kinds of expression MSLN by flow cytometry Binding capacity of the cell line. After gradient dilution of G16 scFv-Fc protein or HN1 scFv-Fc protein, three kinds of cell lines expressing MSLN were selected for staining, and then detected by flow cytometry. The results are shown in the table below, the affinity of G16 scFv to the three MSLN positive cells was significantly better than that of the positive control sequence HN1.

Table 3: Affinity determination results of candidate scFv-Fc to 3 kinds of MSLN positive cells

Example 2: Construction of lentiviral plasmid and viral packaging

2.1 Construction of lentiviral plasmid:

(1) Connection sequence of each part of chimeric antigen receptor

First, based on the scFv sequences in the above examples, CAR was further constructed. The structure of the CAR includes the following four types, which can be seen in FIG. 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 (CD8Hinge) (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 co-expressing 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-cleaving peptide P2A (SEQ ID NO:27)-rIL15 (SEQ ID NO:54).

The third type: a CAR dual targeting NKG2DL and MSLN, named NK2-G-G16-10 CAR; full-length ammonia 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) - Hinge (IgG4 Hinge) (SEQ ID NO:40) -Transmembrane Domain (CD28™) (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 the patent CN109803983B.

The fourth type: dual-targeting NKG2DL and MSLN CAR, 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-cleavage 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 is derived from NP_031386.2.

(2) The scFv and rIL15 nucleotide sequence codons in the above structure are codon-optimized and outsourced to synthesize and build into the Lenti-4-EF1a vector, pick a single clone for culture and preservation, and finally extract the plasmid for sequencing, and sequence the correct Bacterial broth was used to prepare lentiviral plasmids.

2.2 Virus packaging:

Add the above-constructed CAR lentiviral plasmid and transfection reagent mixture dropwise to 293T (ATCC) cells, shake the culture dish gently, and mix well. Place the culture dish in a 37°C, 5% CO ₂ incubator; after culturing for 6-8 hours, remove the medium containing the transfection reagent and replace it with fresh complete medium. After continuous cultivation for 48 hours, the medium supernatant containing the virus in the culture dish was collected, filtered with a 0.45 μm filter membrane, transferred to a centrifuge tube, and after balancing, centrifuged at 20,000 × g for 2 hours at 4°C. After centrifugation, in a biological safety cabinet, carefully suck out the liquid in the centrifuge tube, add 500 μL of PBS buffer to resuspend the pellet, and store the virus at -80°C.

Experimental Example 3: Preparation of CAR-T cells

1) Isolation of primary T cells:

(1) Human PBMC cells were isolated by lymphocyte separation medium (GE), cultured in an incubator at 37°C and 5% CO ₂ , added 100 μL/mL of CD3 antibody and CD28 antibody, mixed well, and placed at room temperature Incubate for 15 minutes.

(2) Take out the magnetic beads, pipette up and down at least 5 times with a pipette gun, and mix well.

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

(4) Add complete culture medium to a total volume of 2.5mL in the tube, insert the tube (capped) into the magnetic pole, and let stand at room temperature for 5 minutes.

(5) After the incubation, the tube remains in the magnetic pole, gently inverted, and the cells in the tube are poured out.

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

2) Activation of T cells:

Adjust the cell density to 1×10 ⁶ cells/mL, add cytokines and antibody complexes (according to the final concentration of 300U/mL IL-2, 10ng/mL IL-7, 5ng/mL IL-15, 500ng/mL Anti - CD3 (OKT3), 2 μg/mL Anti-CD28 configuration), continuous culture for 48 hours.

3) Virus infection:

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

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

(3) After the centrifugation, the sealing film was torn off, and the six-well plate was placed in an incubator at 37° C. with 5% CO ₂ , and cultured for 24 hours.

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

Experimental Example 4: Positive rate detection of CAR-T cells

The nucleic acid sequence encoding CAR was expressed under the drive of the promoter, and the lentiviral transfected T cells were labeled with biotin-labeled MSLN antigen, then detected with fluorescently-labeled streptavidin, and determined by flow cytometry, Reflect the expression level of CAR on the surface of T cells. The CAR-positive rate of the CAR-T cells obtained in Example 3 was detected by the above method, and the FACS detection results are shown in the table below. The results showed that the CAR-positive rate of all CAR-T cells was greater than 20%, indicating that the effector cells were transfected with lentivirus, CAR was successfully expressed, and T cells expressing MSLN-CAR chimeric antigen receptor were successfully constructed.

Table 4: The positive rate detection results of CAR

嵌合抗原受体chimeric antigen receptor	CAR的阳性率(％)Positive rate of CAR (%)
G16G16	67.3267.32
G16-PD1-rIL15G16-PD1-rIL15	29.8529.85
NK2-G-G16-10NK2-G-G16-10	55.8055.80
G16-P2A-NKG2DG16-P2A-NKG2D	39.0439.04

Experimental example 5: Antigen expression detection of target cells

Antigen expression of target cells SK-OV-3, PANC-1, NCI-H226, A431 was determined by flow cytometry using MSLN-biotin and PE-streptavidin antibodies. The results showed that both NCI-H226 and SK-OV-3 cells highly expressed MSLN (Fig. 2A), while PANC-1 and A431 cells did not express MSLN (Fig. 2B); PANC-1 cells expressed NKG2DL lower, SK-OV- 3 cells essentially did not express NKG2DL (Fig. 2C).

Experimental example 6: Evaluation of the killing activity of CAR-T on target cells

The Luciferase gene was integrated into the genomes of NCI-H226, SKOV-3, PANC-1 and A431 cells by lentiviral transduction to obtain NCI-H226, SKOV-3, PANC-1 and A431 cells that can stably express Luciferase (named NCI-H226-luc, SKOV-3-luc, PANC-1-luc and A431-luc, respectively). Collect NCI-H226-luc, SKOV-3-luc, PANC-1-luc and A431-luc cells, centrifuge, resuspend the cells, adjust the cell density to 1×10 ⁵ cells/mL, and inoculate at 100 μL/well The target cells were placed in a 96-well plate in a 5% CO ₂ incubator at 37° C. for 30 minutes. Collect CAR-T, collect by centrifugation and resuspend CAR-T cells in 1640 medium with 10% FBS, G16-CAR, G16-PD1-rIL15 CAR, NK2-G-G16-10CAR, G16-P2A-NKG2D CAR and untransformed Blank T cells transfected with CAR were used as effector cells, and then added to cells containing NCI-H226-luc and SKOV-3-luc according to the ratio of E/T (effector cells/target cells) of 0.5:1, 0.25:1, and 0.125:1, respectively. , PANC-1-luc, A431-luc in 96-well plates (the expression of NKG2DL in PANC-1 is low, so the E/T ratio is 1:1, 0.5:1, 0.25:1, 0.125:1), 100 μL /well, the final volume was made up to 200 μL/well, and cultured in 5% CO ₂ 37°C incubator for 18-24h. After the incubation, take the well plate out of the incubator, add 20 μL of fluorescence detection reagent, and use a microplate reader to detect the fluorescence reading.

The results of the CAR-T killing activity test are as follows, G16-PD1-rIL15 CAR and G16 CAR have no difference in killing NCI-H226-luc and SKOV-3-luc, and have no killing effect on negative A431 cells (Figure 3A); NK2-G -G16-10 CAR has a lysis rate of 98% on PANC-1-luc cells when the effector cell/target cell ratio is 1, G16 CAR has no killing effect on PANC-1-luc cells, NK2-G-G16-10 CAR The killing effect on SKOV-3-luc cells is better than that of G16 CAR (Figure 3B); G16-P2A-NKG2D CAR has a lysis rate of 55% on PANC-1-luc cells when the effector cell/target cell ratio is 1, G16 did not kill PANC-1-luc cells, and G16-P2A-NKG2D CAR did not kill SKOV-3-luc cells differently from G16 (Fig. 3C).

Experimental Example 7: Release of cytokines from CAR-T cells

Collect NCI-H226-luc, SKOV-3-luc, and A431-luc cells, use culture medium to adjust the cell density to 1×10 ⁵ cells/mL, inoculate target cells in a 96-well plate at 100 μL/well, and culture CAR-T cells were resuspended in the base, G16-PD1-rIL15 CAR, G16-CAR, and blank T cells not transfected with CAR were used as effector cells, and then added to the In a 96-well plate containing target cells, 100 μL/well, the final volume was made up to 200 μL/well, and cultured overnight in a 5% CO ₂ 37° C. incubator. After the culture, the well plate was taken out from the incubator, centrifuged, and the supernatant was taken, and the release of cytokines was detected using ELISA kits (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 UTD group; for MSLN In negative A431-luc cells, G16-PD1-rIL15 CAR and G16-CAR did not secrete IL2 and IFN-γ.

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

In order to evaluate the anti-tumor ability of G16 CAR-T and G16-PD1-rIL15 CAR-T in vivo, we established a B-NDG mouse subcutaneous xenograft model of SKOV-3 ovarian cancer cells. When the volume of the transplanted tumor reached about 100-150 mm ³ , 100 mg/kg of cyclophosphamide was administered intraperitoneally to remove 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 xenografts was observed and measured twice a week. The results showed that G16 CAR-T cells could significantly inhibit the growth of subcutaneously transplanted tumor cells. G16-PD1-rIL15 CAR-T cells were able to completely eliminate tumor cells in mice, and no tumor recurrence was seen and the body weight of mice did not change significantly (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 xenograft model of PANC1-MSLN pancreatic cancer cells. When the volume of the transplanted tumor reached about 100-150 mm ³ , 100 mg/kg of cyclophosphamide was administered intraperitoneally to remove residual immune cells. On D7, 5×10 ⁶ G16 CAR-T cells, 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 control. The growth of subcutaneous xenografts was observed and measured twice a week. The results showed that all CAR-T cells can significantly inhibit the growth of subcutaneously transplanted tumor cells, while NK2-G-G16-10 CAR-T cells and G16-P2A-NKG2D CAR-T cells can inhibit tumor cells more rapidly The growth of subcutaneously transplanted tumors, and the tumor cells in the mice can be completely eliminated, and the body weight of the mice has no significant change ( FIG. 6 ).

Although the specific implementation of the present invention has been described in detail, those skilled in the art will understand that: according to all the teachings that have been published, various modifications and changes can be made to the details, and these changes are all within the protection scope of the present invention . The full scope of the invention is given by the claims appended hereto and any equivalents thereof.

Claims

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 antigen-binding domain capable of specifically binding to MSLN The sex-binding first antigen binding domain comprises a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein,

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

or

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

or

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

Wherein, the variant described in any one of (1), (2), (3) has one or several amino acid substitutions, deletions or additions (such as 1, 2 or substitution, deletion or addition of 3 amino acids); preferably, the substitution is a conservative substitution.
The bispecific antigen-binding molecule of claim 1, wherein the VH comprises a sequence as shown in SEQ ID NO: 1 or a variant thereof; the VL comprises a sequence as shown in SEQ ID NO: 2 or a variant thereof variant; wherein said 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, or a substitution, deletion, or addition of one or several amino acids compared to the sequence from which it was derived ( For example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); 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 the group consisting of full-length antibodies, Fab fragments, Fab' fragments, F(ab)' 2 fragments, F(ab) ' 3 fragments, single chain antibodies (eg scFv, di-scFv or (scFv) 2 ), minibodies, disulfide stabilized Fv proteins (dsFv) and single domain antibodies (sdAb, nanobodies).
The bispecific antigen-binding molecule according to any one of claims 1-3, wherein the first antigen-binding domain is a single-chain antibody, and the single-chain antibody comprises in sequence from its N-terminus to its C-terminus:

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

wherein said 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, or have one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions;

Preferably, said L1 is a polypeptide; preferably, said L1 comprises one or several (eg 1, 2 or 3) sequences shown as (GmS)n, wherein m is selected from an integer of 1-6 , n is an integer selected from 1-6; preferably, m is 3, 4, or 5; preferably, n is 1 or 2; more preferably, the L1 has the sequence of SEQ ID NO: 30;

More preferably, said single chain antibody comprises the sequence shown in SEQ ID NO: 18 or a variant thereof, said variant having at least 70%, at least 75%, at least 80%, at least Sequences that are 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 , or have a substitution, deletion or addition of one or several amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletion or addition); preferably, the substitution is a conservative substitution.
The bispecific antigen-binding molecule according to any one of claims 1-4, wherein the second antigen-binding domain capable of specifically binding to an NKG2D ligand (NKG2DL) comprises NKG2D or its ligand-binding domain ( ligand-binding domain);

Preferably, the second antigen binding domain comprises the sequence shown in SEQ ID NO: 20 or 21 or a variant thereof having 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 , or have one or several amino acid substitutions, deletions or additions (eg, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, Said substitutions are conservative substitutions.
The bispecific antigen-binding molecule of any one of claims 1-5, wherein the first antigen-binding domain and the second antigen-binding domain are linked by a linker L2;

Preferably, said first antigen binding domain is as defined in claim 4;

Preferably, said L2 is a polypeptide; preferably, said L2 comprises one or several (eg 1, 2 or 3) sequences shown as (GmS)n, wherein m is selected from an integer of 1-6 , n is an integer selected from 1-6; preferably, m is 3, 4, or 5; preferably, n is 1 or 2; more preferably, the L2 has the sequence of SEQ ID NO: 32;

Preferably, the first antigen-binding domain is connected to the N-terminal or C-terminal 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 having 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 sequence, or has one or several amino acid substitutions, deletions or additions (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitution, deletion or addition); preferably, said substitution is a conservative substitution.
An isolated nucleic acid molecule comprising a nucleotide sequence encoding the bispecific antigen binding molecule of any one of claims 1-6.
A carrier comprising the isolated nucleic acid molecule of claim 7;

Preferably, the vector is selected from DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR/Cas9 vectors or viral vectors;

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.
The method for preparing the bispecific antigen-binding molecule of any one of claims 1-6, comprising culturing the host cell of claim 9 under conditions that allow protein expression, and culturing the host cell from the cultured host cell recovering the bispecific antigen binding molecule.
A bispecific chimeric antigen receptor comprising a bispecific antigen binding domain, a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein said bispecific antigen binding domain comprises claim 1- 6. The bispecific antigen-binding molecule according to any one of the claims;

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 The first antigen-binding domain is as defined in any one of claims 1-4, 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-terminal or C-terminal of the second antigen-binding domain through 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, and the variant is the same as SEQ ID NO: 22 compared 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% identical sequences, or have one or several 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 according to claim 11 or 12, wherein the transmembrane domain is selected from the transmembrane regions of the following proteins: α, β or ζ chains of T cell receptors, 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 as shown in SEQ ID NO:42 or a CD28 transmembrane region as shown in SEQ ID NO:44.
The bispecific chimeric antigen receptor according to any one of claims 11-13, 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 (eg, IgG1 or IgG4);

Preferably, the hinge domain comprises a hinge region of CD8α, IgG4, PD-1, CD152 or CD154; more preferably, the hinge domain comprises a CD8α hinge region as shown in SEQ ID NO:38 or a sequence such as IgG4 hinge region shown in SEQ ID NO:40.
The bispecific chimeric antigen receptor according to any one of claims 11-14, 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 sequentially from the N-terminal to the C-terminal;

Preferably, said intracellular signaling domain comprises a primary signaling domain and at least one co-stimulatory signaling domain;

Preferably, said primary signaling domain comprises an immunoreceptor tyrosine activation motif (ITAM);

Preferably, the primary signaling domain comprises an intracellular signaling domain selected from the following proteins: CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b or CD66d; more preferably, the The primary signal transduction domain comprises a CD3ζ intracellular signal transduction domain 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 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 thereof; more preferably, the co-stimulatory The signaling domain comprises a CD137 (4-1BB) intracellular signaling domain 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 11-15, wherein the bispecific chimeric antigen receptor further comprises a signal peptide SP1 at its N-terminal;

Preferably, the signal peptide SP1 comprises a heavy chain signal peptide (eg heavy chain signal peptide of IgG1), granulocyte-macrophage colony-stimulating factor receptor 2 (GM-CSFR2) signal peptide, IL2 signal peptide, or CD8α signal peptide Peptide; more preferably, the signal peptide SP1 comprises the sequence shown in SEQ ID NO:34.
The bispecific chimeric antigen receptor according to any one of claims 11-16, wherein the bispecific chimeric antigen receptor comprises signal peptide SP1, bispecific antigen binding domain, spacer domain, transmembrane domain, intracellular signaling domain;

Preferably, the signal peptide SP1 comprises an IgG1 heavy chain signal peptide or a CD8α signal peptide (for example, a signal peptide whose sequence is shown in SEQ ID NO: 34);

Preferably, the bispecific antigen binding domain comprises the bispecific antigen binding molecule of any one of claims 1-6 (for example, comprising the sequence shown in SEQ ID NO: 22);

Preferably, the spacer domain comprises a hinge region of CD8 (such as CD8α) or IgG4 (for example, a hinge region whose sequence is shown in SEQ ID NO: 38 or 40);

Preferably, the transmembrane domain comprises a transmembrane region of CD8 (such as CD8α) or CD28 (for example, a sequence such as the transmembrane region shown in SEQ ID NO: 42 or 44);

Preferably, the intracellular signaling domain comprises a primary signaling domain and a co-stimulatory signaling domain, wherein the primary signaling domain comprises an intracellular signaling domain of CD3ζ (e.g., as SEQ ID NO: 48), the co-stimulatory signaling domain comprises the intracellular signaling domain of CD137 (4-1BB) (for example, as the sequence shown in SEQ ID NO: 46); more preferably, the chimeric The intracellular signaling domain of the 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 having 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 , or have one or several amino acid substitutions, deletions or additions (eg, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; preferably, Said substitutions are conservative substitutions.
An isolated nucleic acid molecule comprising a nucleotide sequence encoding the bispecific chimeric antigen receptor of any one of claims 11-17;

Preferably, the isolated nucleic acid molecule comprises a nucleotide sequence selected from the following: (1) the sequence shown in SEQ ID NO: 24 or a degenerate variant thereof; (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% compared to the sequence shown in any one of (1)) , 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 A sequence with 100% sequence identity, 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 an MSLN-specific chimeric antigen receptor; and

(2) a second nucleic acid sequence encoding an additional biologically active molecule comprising an antigen-binding molecule targeting NKG2D ligand (NKG2DL); wherein,

The MSLN-specific chimeric antigen receptor comprises an MSLN-targeted antigen-binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain, and the MSLN-targeted antigen-binding domain comprises claim 1 - the first antigen binding domain as defined in any one of 4.
The nucleic acid construct of claim 19, wherein the antigen-binding molecule targeting NKG2D ligand (NKG2DL) comprises NKG2D or its ligand-binding domain (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 having 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, or a substitution, deletion or addition of one or several amino acids (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.
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 at its N-terminus (for example, as sequence shown in SEQ ID NO:46);

Preferably, said additional biologically active molecule comprises the amino acid sequence shown in SEQ ID NO:66.
The nucleic acid construct according to any one of claims 19-21, wherein the first nucleic acid sequence and the second nucleic acid sequence are encoded by nucleosides encoding self-cleavage peptides (such as P2A, E2A, F2A, T2A or any combination thereof) Acid sequence linkage;

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 19-22, which has one or more of the following features:

(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, and the signal peptide SP1 is as defined in claim 16;

(5) The other 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 first nucleic acid sequence The signal peptide SP1 contained in the encoded MSLN-specific chimeric antigen receptor; 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 according to any one of claims 19-23, wherein the nucleic acid construct comprises sequentially from its 5' end to its 3' end: a nucleotide sequence encoding the signal peptide SP1, encoding the target The nucleotide sequence of the antigen binding domain to MSLN, the nucleotide sequence encoding the spacer domain, the nucleotide sequence encoding the transmembrane domain, the nucleotide sequence encoding the intracellular signaling domain acid sequence, the nucleotide sequence encoding the self-cleaving peptide sequence, the nucleotide sequence encoding the antigen-binding molecule targeting NKG2D ligand (NKG2DL);

Preferably, the signal peptide SP1 comprises an IgG1 heavy chain signal peptide or a CD8α signal peptide (for example, a signal peptide whose sequence is 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 1-4 (for example, comprising the sequence shown in SEQ ID NO: 18);

Preferably, the spacer domain comprises a hinge region of CD8 (such as CD8α) or IgG4 (for example, a hinge region whose sequence is shown in SEQ ID NO: 38 or 40);

Preferably, the transmembrane domain comprises a transmembrane region of CD8 (such as CD8α) or CD28 (for example, a sequence such as the transmembrane region shown in SEQ ID NO: 42 or 44);

Preferably, the intracellular signaling domain comprises a primary signaling domain and a co-stimulatory signaling domain, wherein the primary signaling domain comprises an intracellular signaling domain of CD3ζ (e.g., as SEQ ID NO: 48), the co-stimulatory signaling domain comprises the intracellular signaling domain of CD137 (4-1BB) (for example, as the sequence shown in SEQ ID NO: 46); more preferably, the chimeric The intracellular signaling domain of the 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 (ligand-binding domain), for example comprising the sequence shown in SEQ ID NO: 20 or 21.
The nucleic acid construct according to claim 24, wherein the nucleic acid construct has a nucleotide sequence between the nucleotide sequence encoding the self-cleaving peptide sequence and the antigen-binding molecule encoding the targeting NKG2D ligand (NKG2DL) A nucleotide sequence encoding a 4-1BB intracellular signaling domain is further included between the sequences;

Preferably, the 4-1BB intracellular signaling domain has a 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) shown in any one of (1) The sequence is substantially identical compared to a sequence (e.g., 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, 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) the first nucleic acid sequence encoding the MSLN-specific chimeric antigen receptor or encoding the bispecific chimeric antigen receptor described in any one of claims 11-17; and

(2) A second nucleic acid sequence encoding an additional bioactive molecule comprising a PD1/PD-L1 pathway inhibitor and an IL-15 agonist; wherein,

The MSLN-specific chimeric antigen receptor comprises an MSLN-targeted antigen-binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain, and the MSLN-targeted antigen-binding domain comprises claim 1 - the first antigen binding domain as defined in any one of 4.
The nucleic acid construct of claim 26, wherein:

(i) the first nucleic acid sequence and the second nucleic acid sequence are connected by a nucleotide sequence encoding a self-cleaving peptide (such as 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 encoded by a self-cleaving peptide (such as P2A, E2A, F2A, T2A or any of them) combination) of nucleotide sequences connected;

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 features:

(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-terminal, and the signal peptide SP1 is as defined in claim 16 ;

(5) The other 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 MSLN specificity encoded by the first nucleic acid sequence. The signal peptide SP1 contained in the sexual chimeric antigen receptor or the bispecific chimeric antigen receptor; preferably, the signal peptide SP2 at the N-terminus of the additional biologically active molecule is an IL2 signal peptide (for example, as SEQ ID NO:36 shown).
The nucleic acid construct of any one of claims 26-28, 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 Single-chain antibody (such as scFv); Preferably, the anti-PD-1 single-chain antibody comprises the amino acid sequence shown in SEQ ID NO:52 or a variant thereof, and the variant has 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% , sequences with at least 99%, or 100% identity, or substitutions, deletions, or additions of one or several amino acids (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;

and / or

(2) the IL-15 agonist is selected from a fusion protein comprising IL-15 and IL-15 receptor α (IL-15Rα) Sushi domain; preferably, the IL-15 agonist comprises SEQ ID NO: The amino acid sequence shown in 54 or its variant, described variant has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% compared with SEQ ID NO:54 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical, or have one or several amino acid substitutions, deletions, or Addition (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions); preferably, the substitution is Conservative substitutions.
The nucleic acid construct according to any one of claims 26-29, wherein, from its 5' end to its 3' end, the nucleic acid construct comprises a nucleotide sequence encoding the signal peptide SP1, encoding the targeting The antigen binding domain of MSLN or the nucleotide sequence of the bispecific antigen binding domain, the nucleotide sequence encoding the spacer domain, the nucleotide sequence encoding the transmembrane domain, the encoding the The nucleotide sequence of the intracellular signaling domain, the nucleotide sequence encoding the self-cleaving peptide sequence, the nucleotide sequence encoding the signal peptide SP2, and the core encoding the PD1/PD-L1 pathway inhibitor A nucleotide sequence, a nucleotide sequence encoding the self-cleaving peptide sequence, a nucleotide sequence encoding the IL-15 agonist;

Preferably, the signal peptide SP1 comprises an IgG1 heavy chain signal peptide or a CD8α signal peptide (for example, a signal peptide whose sequence is 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 1-4 (for example, 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 1-6 (for example, comprising the sequence shown in SEQ ID NO: 22);

Preferably, the spacer domain comprises a hinge region of CD8 (such as CD8α) or IgG4 (for example, a hinge region whose sequence is shown in SEQ ID NO: 38 or 40);

Preferably, the transmembrane domain comprises a transmembrane region of CD8 (such as CD8α) or CD28 (for example, a sequence such as the transmembrane region shown in SEQ ID NO: 42 or 44);

Preferably, the intracellular signaling domain comprises a primary signaling domain and a co-stimulatory signaling domain, wherein the primary signaling domain comprises an intracellular signaling domain of CD3ζ (e.g., as SEQ ID NO: 48), the co-stimulatory signaling domain comprises the intracellular signaling domain of CD137 (4-1BB) (for example, as the sequence shown in SEQ ID NO: 46); more preferably, the chimeric The intracellular signaling domain of the 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 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 according to any one of claims 26-30, wherein the nucleic acid construct comprises a nucleotide sequence selected from the group consisting of: (1) the sequence shown in SEQ ID NO:57 or its degenerate variants (2) a sequence that is substantially identical to the sequence shown in any one of (1) (for example, has at least 50%, at least 55%, at least 60% compared with the sequence shown in any one of (1) %, 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%, A sequence with at least 97%, at least 98%, at least 99%, or 100% sequence identity, or a sequence with one or more nucleotide substitutions compared to the sequence shown in any of (1)) .
A carrier comprising the isolated nucleic acid molecule of claim 18, or the nucleic acid construct of any one of claims 19-31;

Preferably, the vector is selected from DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR/Cas9 vectors, or viral vectors;

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 cell expresses 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 19-25;

Preferably, the engineered immune cell expresses the chimeric antigen receptor encoded by the first nucleic acid sequence, and the antigen-binding molecule targeting NKG2D ligand (NKG2DL) encoded by the second nucleic acid sequence; preferably, The antigen-binding molecule targeting NKG2D ligand (NKG2DL) optionally has a 4-1BB intracellular signaling domain linked to its N-terminus.
A modified immune cell comprising the nucleic acid construct of any one of claims 26-31;

Preferably, the engineered 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 cell according to any one of claims 33-35, wherein the immune cell is derived from T lymphocytes, NK cells, monocytes, macrophages or dendritic cells and any combination thereof; preferably Preferably, the immune cells are obtained from a patient; alternatively, the immune cells are obtained from a healthy donor; preferably, the immune cells are derived from T lymphocytes or NK cells.
The modified immune cell according to any one of claims 33-36, wherein the immune exclusion-related genes (for example, TRAC, TRBC, B2M, HLA-A, HLA-B or HLA - C) transcription or expression of one or both target genes of genes of immune co-inhibitory pathways or signaling molecules (for example, PD-1, CTLA-4 or LAG-3) is inhibited; preferably, said target The transcription or expression of the gene is inhibited by a method selected from gene knockout (for example, CRISPR, CRISPR/Cas9), homologous recombination, 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 step (1) described immune cells to obtain immune cells capable of expressing bispecific chimeric antigen receptors; or, introducing the nucleic acid construct according to any one of claims 19-31 or a vector comprising it into the step (1) said immune cells to obtain immune cells capable of co-expressing chimeric antigen receptors and additional biologically active molecules;

Preferably, in step (1), the immune cells are pretreated, and the pretreatment includes sorting, activation and/or proliferation of immune cells; more preferably, the pretreatment includes combining immune cells with anti-CD3 contacting the antibody with an anti-CD28 antibody, thereby stimulating said immune cells and inducing their proliferation, thereby generating preconditioned immune cells;

Preferably, in step (2), the nucleic acid molecule or vector is introduced into immune cells by virus infection;

Preferably, in step (2), the nucleic acid molecule or vector is introduced into the immune cells through 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), a step of expanding the immune cells obtained in step (2) is also included.
An immune cell composition, comprising the modified immune cell according to any one of claims 33-37; optionally, the composition further comprises unmodified and/or unsuccessfully modified immune cells; preferably, the The modified immune cells account for 10%-100% of the total number of cells in the immune cell composition, more preferably 40%-80%.
A kit comprising the bispecific antigen-binding molecule of any one of claims 1-6, or the isolated nucleic acid molecule of claim 7, or the carrier of claim 8, or the vector of claim 8 The host cell described in 9, or the bispecific chimeric antigen receptor described in any one of claims 11-17, or the isolated nucleic acid molecule described in claim 18, or the described nucleic acid molecule described in any one of claims 19-31 described nucleic acid construct, or the carrier described in 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 nucleic acid molecule of any one of claims 1-6. antigen binding molecules;

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 engineered immune cell of claim 33 or comprises the An immune cell composition of the engineered immune cell;

Preferably, the kit comprises the nucleic acid construct according to any one of claims 19-25 or a vector comprising the nucleic acid construct; the kit is used to prepare the modified immune cell according to claim 34 Or an immune cell composition comprising said engineered immune cell.

Preferably, the kit comprises the nucleic acid construct according to any one of claims 26-31 or a vector comprising the nucleic acid construct; the kit is used to prepare the modified immune cell according to claim 35 Or an immune cell composition comprising said engineered immune cell.
A pharmaceutical composition comprising the bispecific antigen-binding molecule of any one of claims 1-6, or the isolated nucleic acid molecule of claim 7, or the carrier of claim 8, or the carrier of claim 9 said host cell, or the bispecific chimeric antigen receptor described in any one of claims 11-17, or the isolated nucleic acid molecule described in claim 18, or the described bispecific chimeric antigen receptor described in any one of claims 19-31 Nucleic acid construct, or the carrier according to claim 32, or the modified immune cell according to any one of claims 33-37, or the immune cell composition according to claim 39, and a pharmaceutically acceptable carrier and/or excipients;

Preferably, the pharmaceutical composition further comprises another pharmaceutically active agent, such as a drug with anti-tumor activity; preferably, the other pharmaceutically active agent includes 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 engineered immune cell, or immune cell composition contained in the pharmaceutical composition may be Simultaneously, separately or sequentially with said additional pharmaceutically active agent.
The bispecific antigen binding molecule of any one of claims 1-6, 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 11-17, or the isolated nucleic acid molecule of claim 18, or the nucleic acid construct of any one of claims 19-31, or the nucleic acid molecule of any one of claims 19-31, or The carrier according to claim 32, or the transformed immune cell according to any one of claims 33-37, or the immune cell composition according to claim 39, or the pharmaceutical composition according to claim 41, during preparation Use in a medicament for the prevention and/or treatment of diseases associated with the expression of mesothelin;

Preferably, the disease associated with the expression of mesothelin is selected from proliferative diseases, such as tumors, or non-tumor-related indications associated with the expression of mesothelin;

Preferably, said tumor is a MSLN positive tumor;

Preferably, said 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 diseases associated with the expression of mesothelin in a subject (such as a human), said method comprising administering an effective amount of any of claims 1-6 to a subject in need thereof. One of the bispecific antigen binding molecules, or the isolated nucleic acid molecule of claim 7, or the vector of claim 8, or the host cell of claim 9, or any of claims 11-17 One of the bispecific chimeric antigen receptors, or the isolated nucleic acid molecule of claim 18, or the nucleic acid construct of any one of claims 19-31, or the carrier of claim 32 , or the modified immune cell according to any one of claims 33-37, or the immune cell composition according to claim 39, or the pharmaceutical composition according to claim 41;

Preferably, the disease associated with the expression of mesothelin is selected from proliferative diseases, such as tumors, or non-tumor-related indications associated with the expression of mesothelin;

Preferably, said tumor is a MSLN positive tumor;

Preferably, said 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 the group consisting of surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, Complementary therapies 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 said engineered immune cell or immune cell composition;

Preferably, the method comprises the following steps: (1) providing immune cells required by the subject; (2) applying the isolated nucleic acid molecule of claim 18 or a vector comprising it, or any of claims 19-31 One of the nucleic acid constructs or a vector comprising the same is introduced into the immune cells described in step (1); (3) administering the immune cells obtained in step (2) to the subject;

Optionally, in step (3), the total dose of immune cells comprises 1×10 7 to 10×10 8 CAR-positive cells;

Preferably, in step (3), the total dose of immune cells is administered to the subject in divided doses.