Antibodies targeting mesothelin, chimeric antigen receptors, and uses thereof

Abstract

Disclosed herein are anti-mesothelin antibodies and antigen binding fragments, chimeric antigen receptors ("CARs") having the anti-mesothelin antibodies and antigen binding fragments ("mesothelin CARs"), and genetically modified immune effector cells having such mesothelin CARs. Also provided herein are polynucleotides encoding the anti-mesothelin antibodies and antigen binding fragments and the mesothelin CARs. Also provided herein are compositions comprising the anti-mesothelin antibodies and antigen binding fragments and the mesothelin CARs. The disclosure also relates to the use of the anti-mesothelin antibodies and antigen binding fragments and the genetically modified immune effector cells having such mesothelin CARs in the treatment of cancer.

Description

Technical Field

[0001] This invention relates to molecular biology, cell biology, and immuno-oncology. Specifically, this invention provides anti-mesothelin antibodies, chimeric antigen receptors (CARs) comprising such anti-mesothelin antibodies (“mesothelin CARs”), genetically engineered immune effector cells expressing such mesothelin CARs, and their use in the treatment of tumors or cancers. Background Technology

[0002] Cell-based immunotherapy is a potentially curative therapy in cancer treatment. T cells and other immune cells can be modified to target tumor antigens by introducing genetic material encoding artificial or synthetic receptors targeting antigens (e.g., chimeric antigen receptors (CARs) specific to selected antigens). CAR-T therapy has recently achieved clinical success in treating certain hematologic malignancies. However, applying CAR-expressing T-cell therapies to solid tumors faces several obstacles that must be overcome to achieve clinical benefit. Therefore, there is a need for novel therapeutic strategies for designing CARs to treat cancer, particularly solid tumors, that can induce potent tumor clearance with minimal toxicity and immunogenicity. Mesothelin is expressed in various human cancers, such as mesothelioma, pancreatic cancer, and ovarian cancer. However, current therapies targeting mesothelin have only achieved limited success. Therefore, there remains an unmet need for other mesothelin-targeting therapeutic options. The compositions and methods presented in this article address these needs and offer other related advantages. Summary of the Invention

[0003] This disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to mesothelin (e.g., human dermothelin), comprising: (a) a light chain variable region (VL) comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) having amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively; or variants thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the LCDR; and / or (b) a heavy chain variable region (VH) comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3) having amino acid sequences of SEQ ID NOs: 4, 5, and 6; or variants thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the HCDR.

[0004] In some embodiments, the antibodies and antigen-binding fragments provided herein comprise LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3, wherein: (a) LCDR1, HCDR2, and HCDR3 have amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively; and (b) HCDR1, HCDR2, and HCDR3 have amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0005] In some embodiments, the antibody or antigen-binding fragment thereof provided herein comprises: (a) VL, having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 7; and / or (b) VH, having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 8.

[0006] In some embodiments, the antibodies and antigen-binding fragments provided herein comprise VL and VH, wherein VL and VH have the amino acid sequences of SEQ ID NO: 7 and 8, respectively.

[0007] In some implementations, the antibodies and antigen-binding fragments provided herein are monoclonal antibodies or antigen-binding fragments.

[0008] In some implementations, the antibodies and antigen-binding fragments provided herein are monoclonal antibodies or antigen-binding fragments.

[0009] In some implementations, the antibodies and their antigen-binding fragments provided herein are selected from the group consisting of IgG1 antibodies, IgG2 antibodies, IgG3 antibodies and IgG4 antibodies.

[0010] In some implementations, the antibodies and their antigen-binding fragments provided herein are selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, single-domain antibodies (sdAb), and heavy-chain antibodies (HCAb).

[0011] In some embodiments, the antibody and its antigen-binding fragment provided herein are scFv having the amino acid sequence of SEQ ID NO: 9.

[0012] This article also provides a chimeric antigen receptor (CAR) that specifically binds to mesothelin, comprising from the N-terminus to the C-terminus: (a) a mesothelin-binding domain containing the antibody or its antigen-binding fragment provided herein; (b) a transmembrane domain; and (c) a cytoplasmic domain.

[0013] In some implementations of the CAR provided herein, the transmembrane domain is derived from CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, TCR α chain, TCR β chain or TCR ζ chain, CD3ε, CD45, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 or CD154.

[0014] In some implementations of the CAR provided herein, the transmembrane domain includes the CD8 transmembrane region.

[0015] In some embodiments of the CAR provided herein, the transmembrane domain is a CD8 transmembrane region having the amino acid sequence of SEQ ID NO: 15 and the nucleotide sequence of SEQ ID NO: 16.

[0016] In some implementations of the CAR provided herein, the cytoplasmic domain includes signal transduction domains derived from CD3ζ, FcRγ, FcγRIIa, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, DAP10, DAP12, or any combination thereof.

[0017] In some implementations of the CAR provided herein, the cytoplasmic domain also includes a co-stimulatory domain derived from CD28, 4-1BB (CD137), OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD2, CD7, LIGHT, GITR, TLR, DR3, CD43, or any combination thereof.

[0018] In some embodiments of the CAR provided herein, the cytoplasmic domain comprises a CD3ζ signaling domain and a 4-1BB co-stimulatory domain. In some embodiments, the cytoplasmic domain is a CD3ζ signaling domain having the amino acid sequence of SEQ ID NO: 19 and the nucleotide sequence of SEQ ID NO: 20. In some embodiments, the co-stimulatory domain is a 4-1BB having the amino acid sequence of SEQ ID NO: 17 and the nucleotide sequence of SEQ ID NO: 18.

[0019] In some embodiments, the CAR provided herein further comprises a CD8 hinge domain located between the mesothelin-binding domain and the transmembrane domain. In some embodiments, the hinge domain is a CD8 hinge domain having the amino acid sequence of SEQ ID NO: 13 and the nucleotide sequence of SEQ ID NO: 14.

[0020] In some embodiments, the CAR provided herein also includes a signal peptide domain derived from CD8 at its N-terminus. In some embodiments, the signal peptide domain is CD8 having the amino acid sequence of SEQ ID NO: 11 and the nucleotide sequence of SEQ ID NO: 12.

[0021] In some implementations, the CAR provided herein contains the amino acid sequence of SEQ ID NO: 10.

[0022] This document also provides polynucleotides encoding antibodies or antigen-binding fragments thereof provided herein, as well as CARs. In some embodiments, the polynucleotides are mRNA.

[0023] This article also provides vectors containing the polynucleotides provided herein.

[0024] This article also provides cells containing the CAR provided herein or polynucleotides encoding the CAR provided herein.

[0025] In some implementations, the cells provided herein are immune effector cells.

[0026] In some implementations, the cells provided herein are derived from cells isolated from peripheral blood or bone marrow.

[0027] In some implementations, the cells provided herein are derived from cells differentiated in vitro from stem cells or progenitor cells, wherein the stem cells or progenitor cells are selected from the group consisting of T cell progenitor cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotent progenitor cells, embryonic stem cells, and induced pluripotent cells.

[0028] In some implementations, the cells provided herein are T cells or NK cells.

[0029] In some implementations, the cells provided herein are cytotoxic T cells, helper T cells, γδ T cells, CD4+ / CD8+ double-positive T cells, CD4+ T cells, CD8+ T cells, CD4- / CD8- double-negative T cells, CD3+ T cells, naive T cells, effector T cells, helper T cells, memory T cells, regulatory T cells, Th0 cells, Th1 cells, Th2 cells, Th3 (Treg) cells, Th9 cells, Th17 cells, Thαβ helper cells, Tfh cells, stem cell-like memory TSCM cells, central memory TCM cells, effector memory TEM cells, or effector memory TEMRA cells.

[0030] In some embodiments, the recombinant cell expression fusion protein provided herein comprises a first domain for activating antigen-presenting cells (APCs) and a second domain for activating immune effector cells, wherein: (i) the first domain comprises (a) a ligand or receptor-binding fragment thereof of an activating receptor for APCs, or (b) an antibody or antigen-binding fragment thereof of an activating receptor for APCs; and (ii) the second domain comprises (a) a co-stimulatory receptor or a functional fragment thereof of an immune effector cell, (b) a co-stimulatory ligand or receptor-binding fragment thereof of an immune effector cell, or (c) an antibody or antigen-binding fragment thereof of a co-stimulatory receptor for an immune effector cell.

[0031] In some embodiments, the first domain comprises an anti-CD40 antibody or an antigen-binding fragment thereof. In some embodiments, the first domain comprises an anti-CD40 scFv. In some embodiments, the first domain comprises an anti-CD40 scFv having the amino acid sequence SEQ ID NOs: 38-48. In some embodiments, the second domain is an antibody binding to CD28 or an antigen-binding fragment thereof. In some embodiments, the second domain comprises a CD28 cytoplasmic domain. In some embodiments, the second domain comprises a CD28 cytoplasmic domain having the amino acid sequence SEQ ID NO: 36. In some embodiments, the second domain comprises a CD28 scFv. In some embodiments, the second domain comprises a CD28 scFv having the amino acid sequence SEQ ID NO: 62.

[0032] In some embodiments, the N-terminus of the first domain of the fusion protein is linked to the C-terminus of the second domain of the fusion protein. In some embodiments, the N-terminus of the second domain of the fusion protein is linked to the C-terminus of the first domain of the fusion protein. In some embodiments, the first and second domains of the fusion protein are linked via a linker. In some embodiments, the linker is a hinge derived from CD8, CD28, (GGGGS)n (n=1, 2, 3, 4, 5…) or IgG4m. In some embodiments, the linker is a CD28 hinge having the amino acid sequence of SEQ ID NO: 25.

[0033] In some embodiments, the fusion protein has at least 85%, 90%, 95%, 98%, 99%, or 100% identity with the amino acid sequence selected from the group consisting of SEQ ID NOs: 49-59 and 63-73. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 68.

[0034] This article also provides pharmaceutical compositions comprising the antibody or antigen-binding fragment provided herein, the CAR provided herein, the polynucleotide provided herein, the vector provided herein, and / or the cell provided herein, and pharmaceutically acceptable excipients.

[0035] This article also provides the use of the antibodies or antigen-binding fragments, CARs, polynucleotides, vectors and / or cells provided herein in the preparation of medicaments for the treatment of cancer.

[0036] This article also provides information on the use of antibodies or antigen-binding fragments, CARs, polynucleotides, vectors, and / or cells provided herein in cancer therapy.

[0037] This article also provides methods for treating cancer in subjects in need, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment provided herein, a CAR provided herein, a polynucleotide provided herein, a vector provided herein, and / or a cell provided herein.

[0038] In some embodiments of the methods provided herein, the cells or cell populations are autologous relative to the subject.

[0039] In some implementations, the methods provided herein also include obtaining cells from a subject.

[0040] In some implementations, the methods provided herein also include administering additional therapy to the subject.

[0041] In some implementations of the methods provided herein, the subjects are human.

[0042] In some implementations of the methods provided herein, the cancer is a cancer expressing mesothelin.

[0043] In some implementations of the methods provided herein, the cancer is a solid tumor or a blood cancer.

[0044] In some implementations of the methods provided herein, the cancer is kidney cancer, lung cancer, urothelial carcinoma, breast cancer, brain tumor, thyroid cancer, leukemia, lymphoma, and / or multiple myeloma.

[0045] In some implementations of the methods provided herein, the cancers include renal cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, glioblastoma, undifferentiated thyroid carcinoma, B-cell non-Hodgkin lymphoma, and / or chronic lymphocytic leukemia.

[0046] This article also provides methods for preparing the cells provided herein, which include transferring polynucleotides encoding antibody or antigen-binding fragments provided herein, polynucleotides encoding CARs provided herein, polynucleotides provided herein, and / or vectors provided herein into the cells.

[0047] In some embodiments, the method is used to prepare cells capable of expressing CARs that specifically bind to mesothelin, and the method includes transferring the polynucleotides provided herein into the cells.

[0048] In some embodiments, the method is used to prepare cells capable of expressing a mesothelin-specific CAR and a fusion protein comprising a first domain for activating antigen-presenting cells (APCs) and a second domain for activating immune effector cells; and the method includes transferring the polynucleotide of claim 18 or 19 and the polynucleotide encoding the fusion protein into the cells, wherein:

[0049] (i) The first domain of the fusion protein comprises (a) a ligand or receptor-binding fragment of an APC-activating receptor, or (b) an antibody or antigen-binding fragment of an APC-activating receptor; and

[0050] (ii) The second domain of the fusion protein contains (a) a co-stimulatory receptor of an immune effector cell or a functional fragment thereof, (b) a co-stimulatory ligand of an immune effector cell or a receptor-binding fragment thereof, or (c) an antibody that binds to the co-stimulatory receptor of an immune effector cell or an antigen-binding fragment thereof.

[0051] In some implementations, the polynucleotide is transferred via electroporation.

[0052] In some embodiments, the polynucleotide is transferred via viral transduction. In some embodiments, the method includes viral transduction using lentivirus, retrovirus, adenovirus, or adeno-associated virus.

[0053] In some embodiments, a transposon system is used to transfer the polynucleotide. In some embodiments, the transposon system is Sleeping Beauty or PiggyBac.

[0054] In some embodiments, gene editing is used to transfer the polynucleotide. In some embodiments, a CRISPR-Cas system, a ZFN system, or a TALEN system is used to transfer the polynucleotide.

[0055] In some embodiments, the cells are selected from the group consisting of T cells, NK cells, NKT cells, macrophages, neutrophils, and granulocytes.

[0056] Other aspects and advantages of this disclosure will become apparent to those skilled in the art from the following detailed description, in which only exemplary embodiments of the disclosure are shown and described. It should be understood that this disclosure is capable of having other different embodiments, and that certain details thereof can be modified in various obvious respects without departing from this disclosure. Therefore, the drawings and descriptions should be considered illustrative in nature and not restrictive. 4. Brief description of the attached diagram

[0058] Figure 1 Flow cytometry results of MOLM14 stably transfected cell lines overexpressing human MSLN (huMSLN) were detected using an anti-MSLN antibody.

[0059] Figure 2 ELISA screening results. The values ​​are the difference between the MOLM14-huMSLN reading and the background cell MOLM14-muMSLN reading. Clones marked with a gray background (clone numbers: 100-186) were selected as candidate clones for subsequent PCR amplification of scFv sequences and sequencing.

[0060] Figure 3A Schematic diagram of the lentiviral vector structure of pUTCK-anti-MSLN scFv.BBZ. Figure 3B Schematic diagram of membrane-type LACOSTIM (mLACO) and soluble LACOSTIM (sLACO).

[0061] Figure 4 A549 cells, A549 cells electroporated with 1 μg MSLN mRNA, and A549 cells electroporated with 10 μg MSLN mRNA were used. MSLN expression levels were stained and detected by flow cytometry after 24 hours.

[0062] Figure 5 Flow cytometry results of staining anti-MSLN CAR-T cells and anti-MSLN / LACOSTIM CAR-T cells using MSLN-Fc antigen and anti-human IgG-Fc antibody. The staining results indicate that CAR molecules are effectively expressed on both anti-MSLN CAR-T cells and anti-MSLN / LACOSTIM CAR-T cells.

[0063] Figure 6Flow cytometry results of staining anti-MSLN CAR-T cells and anti-MSLN / LACOSTIM CAR-T cells using CD40-Fc antigen and anti-human IgG-Fc antibody. Staining results showed that LACOSTIM expression levels were very high in the A4025-MH150 CAR-T cells expressing LACOSTIM on the cell membrane. No LACOSTIM molecules were detected on the cell membrane of the MH150-25-9.3 CAR-T cells expressing secreted LACOSTIM, indicating that secreted LACOSTIM molecules may exist in a soluble form extracellularly.

[0064] Figure 7 The killing effect of control UTD cells and three types of anti-MSLN CAR-T cells co-incubated with A549 cells and A549 target cells electroporated with 1 μg and 10 μg MSLN mRNA under the condition of E:T=3:1 was measured.

[0065] Figure 8 The killing effect of control UTD cells and three types of anti-MSLN CAR-T cells co-incubated with A549 cells and A549 target cells electroporated with 1 μg and 10 μg MSLN mRNA under the condition of E:T=1:1 was measured.

[0066] Figure 9 The killing effect of control UTD cells and three types of anti-MSLN CAR-T cells co-incubated with A549 cells and A549 target cells electroporated with 1 μg and 10 μg MSLN mRNA under the condition of E:T=0.3:1 was investigated.

[0067] Figure 10 The killing effect of ASPC1 target cells co-incubated with control UTD cells and three types of anti-MSLN CAR-T cells under three conditions: E:T=3:1, E:T=1:1 and E:T=0.3:1.

[0068] Figure 11 The IL2 release levels of UTD cells and three types of anti-MSLN CAR-T cells were detected by ELISA after co-incubation with A549 cells, A549 target cells electroporated with 1 μg and 10 μg MSLN mRNA, and ASPC1 target cells.

[0069] Figure 12The IFN-γ release levels of UTD cells and three types of anti-MSLN CAR-T cells were detected by ELISA after co-incubation with A549 cells, A549 target cells electroporated with 1 μg and 10 μg MSLN mRNA, and ASPC1 target cells.

[0070] Figures 13-22 Under the condition of E:T=3:1, control UTD cells and three types of anti-MSLN CAR-T cells were compared with HCC70 ( Figure 13 ), SH-SY5Y ( Figure 14 ), Huh-7 ( Figure 15 ), U87 ( Figure 16 ), U251 ( Figure 17 A375 Figure 18 ), 293T ( Figure 19 ), PC3 ( Figure 20 Supt1 () Figure 21 ) and Molm14 ( Figure 22 IncuCyte analysis results of the killing effect of co-incubation with target cells.

[0071] Figure 23 Results of experiments evaluating the tumor-bearing mouse model of CAR-T cell tumor-killing efficacy.

[0072] Figure 24 Dose-dependent expansion of CAR-T cells in the peripheral blood of tumor-bearing mice after drug administration.

[0073] Figure 25 The level of sLACO secretion in the peripheral blood of tumor-bearing mice after administration of CAR-T cells.

[0074] Figure 26 Results of experiments evaluating the tumor-bearing mouse model of CAR-T cell tumor-killing efficacy.

[0075] Figure 27 The proliferation of CAR-T cells in mice was detected by flow cytometry.

[0076] Figure 28 Results of sLACO secretion levels in mouse peripheral blood detected by ELISA. Detailed Implementation

[0077] Although various embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many modifications, alterations, and substitutions can be made by those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0078] This disclosure provides novel antibodies comprising antigen-binding fragments that specifically bind mesothelin (e.g., human dermochrome). Furthermore, this disclosure provides chimeric antigen receptors (CARs) comprising such antibodies or antigen-binding fragments that specifically bind mesothelin (e.g., human dermochrome); and engineered immune effector cells (e.g., T cells) and cell populations (e.g., CAR-T) that recombinantly express CARs that specifically bind mesothelin (e.g., human dermochrome). Pharmaceutical compositions comprising therapeutically effective amounts of such antibodies or antigen-binding fragments are also disclosed herein, as are pharmaceutical compositions comprising therapeutically effective amounts of cells or cell populations. The use of such pharmaceutical compositions in the treatment of diseases and conditions associated with mesothelin expression (e.g., cancers expressing mesothelin) and related treatment methods are also disclosed herein.

[0079] 5.1 Definition

[0080] Unless otherwise defined herein, scientific and technical terms used in this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms. Generally, the nomenclature and techniques described herein relating to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization are well-known and commonly used nomenclature and techniques in the art.

[0081] This disclosure provides novel antibodies comprising antigen-binding fragments that specifically bind to mesothelin (e.g., human mesothelin). The term "mesothelin" includes any variant or isotype of mesothelin naturally expressed by cells. Therefore, the antibodies described herein can cross-react with mesothelin from species other than humans (e.g., cynomolgus monkey mesothelin). Alternatively, the antibodies may be specific to human mesothelin and not exhibit any cross-reactivity with other species. Mesothelin or any variants and isotypes thereof can be isolated from cells or tissues that naturally express them, or recombinantly generated using techniques well known in the art and / or those described herein.

[0082] As used herein, the term "antibody" and its grammatical equivalents refer to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or any combination thereof, through at least one antigen-binding site, wherein the antigen-binding site is typically located within a variable region of the immunoglobulin molecule. The term as used herein encompasses intact polyclonal antibodies, intact monoclonal antibodies, single-domain antibodies (sdAbs; e.g., camelid antibodies, alpaca antibodies), single-chain Fv (scFv) antibodies, heavy-chain antibodies (HCAbs), light-chain antibodies (LCAbs), multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, and any other modified immunoglobulin molecule containing an antigen-binding site (e.g., bivariate domain immunoglobulin molecules), provided that the antibody exhibits the desired biological activity. Antibodies also include, but are not limited to, mouse antibodies, camel antibodies, chimeric antibodies, humanized antibodies, and human antibodies. Antibodies can be any of the five major immunoglobulin classes—IgA, IgD, IgE, IgG, and IgM—or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), depending on the type of heavy chain constant domains they possess, which are respectively designated α, δ, ε, γ, and μ. Unless otherwise explicitly stated, the term "antibody" as used herein includes the "antigen-binding fragment" of a complete antibody. The term "antigen-binding fragment" as used herein refers to a portion or segment of a complete antibody, which is the antigen-determining variable region of the complete antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, linear antibodies, single-chain antibody molecules (e.g., scFv), heavy chain antibodies (HCAb), light chain antibodies (LCAb), disulfide-linked scFv (dsscFv), biantibodies, triantibodies, tetraantibodies, small antibodies, bivariate domain antibodies (DVD), single variable domain antibodies (sdAb; e.g., camelid antibodies, alpaca antibodies), single variable domain (VHH) of heavy chain antibodies, and bispecific or multispecific antibodies formed from antibody fragments.

[0083] As used herein, the term "humanized antibody" refers to a form of non-human (e.g., mouse) antibody containing a minimum non-human sequence, such as a specific immunoglobulin chain, a chimeric immunoglobulin, or a fragment thereof. Typically, humanized antibodies are human immunoglobulins. In some cases, residues in the Fv framework region of a human immunoglobulin are replaced by corresponding residues from a non-human species antibody. In some cases, residues in the CDR are replaced by CDR residues from a non-human species (e.g., mouse, rat, hamster, camel) having the desired specificity, affinity, and / or binding capacity. Humanized antibodies can be further modified by further substituting additional residues within the Fv framework region and / or the replaced non-human residues to improve and optimize the antibody's specificity, affinity, and / or binding capacity. As used herein, the term "human antibody" refers to an antibody produced by a human, or an antibody prepared using any technique known in the art that has an amino acid sequence corresponding to that of a human-produced antibody.

[0084] When referring to antibodies, the term "heavy chain" refers to a polypeptide chain of approximately 50-70 kDa, where the amino-terminal portion contains a variable region of approximately 120 to 130 or more amino acids, and the carboxyl-terminal portion contains a constant region. Based on the amino acid sequence of the heavy chain's constant region, it can be one of five different types, called α, δ, ε, γ, and μ. The different heavy chains vary in size: α, δ, and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with light chains, these different types of heavy chains produce the five well-known antibody classes: IgA, IgD, IgE, IgG, and IgM, including four subclasses of IgG: IgG1, IgG2, IgG3, and IgG4. The heavy chain can be the human heavy chain.

[0085] When referring to antibodies, the term "light chain" refers to a polypeptide chain of approximately 25 kDa, in which the amino-terminal portion contains a variable region of approximately 100 to approximately 110 or more amino acids, and the carboxyl-terminal portion contains a constant region. The length of a light chain is approximately 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, light chains are classified into two distinct types, called κ and λ. Light chain amino acid sequences are well-known in the art. Light chains can be human light chains.

[0086] The term "variable domain" or "variable region" refers to a portion of the antibody's light or heavy chain, typically located at the amino terminus of the light or heavy chain. The heavy chain is approximately 120 to 130 amino acids long, while the light chain is approximately 100 to 110 amino acids long. These domains are involved in the binding of each specific antibody to its specific antigen and determine its specificity. Variable domains exhibit extensive sequence differences between different antibodies. Sequence variations are concentrated in the CDR, while smaller variations within the variable domain are called frame regions (FRs). The CDRs of both the light and heavy chains are primarily responsible for the antibody-antigen interaction. The amino acid position numbers used in this article follow the EU numbers in Kabat et al. (1991), *Sequences of Immunologically Related Proteins* (US Department of Health and Human Services, Washington, D.C.), 5th edition. Variable regions can be human variable regions.

[0087] CDR refers to one of the three hypervariable regions (H1, H2, or H3) within the non-framework region of the VH β-sheet framework of an immunoglobulin (Ig or antibody), or one of the three hypervariable regions (L1, L2, or L3) within the non-framework region of the VL β-sheet framework of an antibody. Therefore, CDR is a variable region sequence scattered within the framework region sequence. CDR regions are well known to those skilled in the art and have been defined using various methods / systems. These systems and / or definitions have been developed and refined over many years, including Kabat, Chothia, IMGT, AbM, and Contact. For example, Kabat defined the region with the highest hypervariability within the antibody variable (V) domain (Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat, Adv. Prot. Chem. 32:1-75 (1978)). Chothia's definition, based on the location of the structural loop region, defines the CDR region sequence as residues that do not belong to the conserved β-sheet framework and are therefore capable of adapting to different conformations (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). Both terminologies are widely accepted in the art. Furthermore, the IMGT system is based on sequence variability and location within the variable region structure. The AbM definition is a compromise between Kabat and Chothia. The Contact definition is based on the analysis of existing antibody crystal structures. Software programs (e.g., abYsis) for analyzing antibody sequences and determining CDRs are available and well-known to those skilled in the art. The location of CDRs within typical antibody variable domains has been determined through comparison of numerous structures (Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); Morea et al., Methods 20:267-279 (2000)). Because different antibodies have different numbers of residues in their hypervariable regions, in typical variable domain numbering schemes, additional residues relative to typical positions are usually numbered by adding a, b, c, etc. next to the residue number (Al-Lazikani et al., ibid. (1997)). This nomenclature is also well known to those skilled in the art.

[0088] For example, CDRs defined according to Kabat (high-level) or Chothia (structure) naming are listed in the table below.

[0089]

[0090] The residue numbering follows the nomenclature of Kabat et al. mentioned above.

[0091] The residue numbering follows the nomenclature of Chothia et al. mentioned above.

[0092] One or more CDRs can be covalently or non-covalently incorporated into a molecule to create immunoadhesins. Immunoadhesins can incorporate CDRs as part of a larger polypeptide chain, covalently link CDRs to another polypeptide chain, or non-covalently incorporate CDRs. CDRs enable immunoadhesins to bind to specific target antigens. For example, CDR regions can be analyzed using the abYsis website.

[0093] Therefore, unless otherwise stated, a CDR or individually designated CDR (e.g., LCDR1, LCDR2, LCDR3) of a given antibody or its region (e.g., its variable region) should be understood to cover the complementarity-determining region (or the specific complementarity-determining region) defined according to any of the foregoing schemes or other known schemes. For example, when a specific CDR (e.g., HCDR3) is stated to contain the amino acid sequence of the corresponding CDR in a given VH or VL region amino acid sequence, it should be understood that such CDR has the sequence of the corresponding CDR (e.g., HCDR3) within the variable region defined according to any of the foregoing schemes or other known schemes. In some embodiments, a specific CDR sequence is specified, but it should be understood that the antibody provided may include CDRs as described in any of the other foregoing numbering schemes or other numbering schemes known to those skilled in the art. Similarly, unless otherwise stated, a FR or individually designated FR (e.g., VH FR1, VH FR2, VH FR3, VH FR4) of a given antibody or its region (e.g., its variable region) should be understood to cover the frame region (or the specific frame region) defined according to any known scheme.

[0094] The terms “epitope” and “antigenic determinant” are used interchangeably herein to refer to a site on the surface of a target molecule where an antibody or antigen-binding fragment binds, such as a localized region on the surface of an antigen. Target molecules may include proteins, peptides, nucleic acids, carbohydrates, or lipids. An epitope with immunogenic activity is a portion of the target molecule that elicits an immune response in an animal. An antigenically active epitope in a target molecule is a portion of the target molecule where an antibody binds, which can be determined by any method well known in the art, including, for example, immunoassay. An antigenic epitope is not necessarily immunogenic. Epitopes typically consist of chemically active surface groups (e.g., amino acids or sugar side chains) in a molecule and have specific three-dimensional structural features and specific charge characteristics. The term “epitope” includes linear epitopes and conformational epitopes. The region in a target molecule (e.g., a polypeptide) that contributes to the formation of an epitope can be a continuous amino acid in the polypeptide, or the epitope can be formed by two or more discontinuous regions of the target molecule. An epitope may or may not be a three-dimensional surface feature of the target molecule. Epitopes formed by consecutive amino acids (also known as linear epitopes) are usually retained after protein denaturation, while epitopes formed by ternary folding (also known as conformational epitopes) are usually lost after protein denaturation. Epitopes typically contain at least 3, more often at least 5, 6, 7, or 8-10 amino acids with a unique spatial conformation.

[0095] As used herein, the term "specific binding" refers to a polypeptide or molecule interacting with an epitope, protein, or target molecule more frequently, more rapidly, for a longer duration, with a higher affinity, or a combination of these characteristics compared to alternative substances (including related and unrelated proteins). The binding moiety (e.g., antibody) that specifically binds to a target molecule (e.g., an antigen) can be identified, for example, by immunoassay, ELISA, SPR (e.g., Biacore), or other techniques known to those skilled in the art. Typically, a specific response will be at least twice the background signal or noise, and can exceed 10 times the background. For a discussion of antibody specificity, see, for example, Paul (ed.), *Basic Immunology*, 2nd ed., Raven Press, New York, pp. 332-336. The binding affinity of the binding moiety of a specific target molecule to the target molecule can be higher than its affinity to different molecules. In some embodiments, the binding affinity of the binding moiety specifically binding to the target molecule may be at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 times higher than its affinity for different molecules. In some embodiments, the binding moiety specifically binding to a particular target molecule binds to different molecules with extremely low affinity, such that the binding is undetectable using assays described herein or known in the art. In some embodiments, "specific binding" refers to, for example, the binding moiety binding to a molecular target with a KD of about 0.1 mM or less. In some embodiments, "specific binding" refers to a peptide or molecule binding to a target with a KD of about 10 µM or less, or about 1 µM or less. In some embodiments, "specific binding" refers to a peptide or molecule binding to a target with a KD of about 0.1 µM or less, about 0.01 µM or less, or about 1 nM or less. Because of sequence identity among homologous proteins in different species, specific binding can include peptides or molecules that recognize proteins or targets in more than one species. Similarly, because of homology in certain regions of the polypeptide sequences of different proteins, specific binding can include peptides or molecules that recognize more than one protein or target. It should be understood that in some embodiments, the binding portion (e.g., an antibody) that specifically binds to a first target may or may not specifically bind to a second target. Therefore, “specific binding” does not necessarily require (although it may include) exclusive binding, i.e., binding to only a single target. Thus, in some embodiments, the binding portion (e.g., an antibody) may specifically bind to more than one target. For example, in some cases, an antibody may contain two identical antigen-binding sites, each of which specifically binds to the same epitopes on two or more proteins. In some alternative embodiments, the antibody may be bispecific and contain at least two antigen-binding sites with different specificities.

[0096] As used herein, the term “binding affinity” generally refers to the total strength of all non-covalent interactions between the binding moiety and the target molecule (e.g., an antigen). Binding to the target molecule is a reversible process, and binding affinity is typically expressed as the equilibrium dissociation constant (KD). KD is the ratio of the dissociation rate (koff or kd) to the association rate (kon or ka). The lower the KD of the binding pair, the higher the affinity. Various methods for measuring binding affinity are known in the art, any of which may be used for the purposes of this disclosure. Specific illustrative embodiments include the following. In some embodiments, “KD” or “KD value” can be measured by assays known in the art (e.g., binding assays). KD can be measured by radiolabeled antigen binding assays (RIA) (Chen et al., (1999) J. Mol. Biol. 293:865-881). KD or KD values ​​can also be measured using Biacore via surface plasmon resonance determination, for example using BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ); or via biolayer interferometry, for example using the Octet QK384 system (ForteBio, Menlo Park, CA).

[0097] In this document, the term "variant" as used in connection with a protein or polypeptide having a specific sequence characteristic ("reference protein" or "reference polypeptide") refers to a different protein or polypeptide that, compared to the reference protein or polypeptide, has one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid substitutions, deletions, and / or additions. Changes in the amino acid sequence can be amino acid substitutions. Changes in the amino acid sequence can be conserved amino acid substitutions. Functional fragments or functional variants of the protein or polypeptide retain the basic structural and functional properties of the reference protein or polypeptide.

[0098] The terms “polypeptide,” “peptide,” and “protein,” used interchangeably herein, and their grammatical equivalents, refer to amino acid polymers of any length, which may be linear or branched. They may include non-natural or modified amino acids, or be interrupted by non-amino acid breaks. Polypeptides, peptides, or proteins may also undergo, for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation or modification.

[0099] The terms “polynucleotide”, “nucleic acid”, and their grammatical equivalents, used interchangeably herein, refer to nucleotide polymers of any length and include DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogues, or any substrate that can be incorporated into the polymer by DNA or RNA polymerases.

[0100] The terms “identical,” “percentage of identity,” and their grammatical equivalents, used herein in the context of two or more polynucleotides or peptides, refer to two or more sequences or subsequences being identical, or having a specified percentage of identical nucleotide or amino acid residues, when compared and aligned for maximum correspondence (with gaps introduced where necessary). No conserved amino acid substitutions are considered part of the sequence identity. The percentage of identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software known in the art for obtaining amino acid or nucleotide sequence alignments are available, including but not limited to BLAST, ALIGN, Megalign, BestFit, GCG WisconsinPackage, and variants thereof. In some embodiments, the two polynucleotides or peptides provided herein are substantially identical, meaning that when compared and aligned for maximum correspondence, they are measured to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleotide or amino acid residue identity, using sequence comparison algorithms or by visual inspection. In some embodiments, identity exists within an amino acid sequence region of at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues, or any integer value therebetween. In some embodiments, identity exists within a region longer than 60-80 residues, for example, at least about 80-100 residues; and in some embodiments, the sequences are substantially identical in length across the entire length of the compared sequences, such as the coding region of a target protein or antibody. In some embodiments, identity exists within a nucleotide sequence region of at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases, or any integer value therebetween. In some embodiments, identity exists within a region longer than 60-80 bases, for example, at least about 80-1000 or more bases; and in some embodiments, the sequences are substantially identical in length across the entire length of the compared sequences, such as the nucleotide sequence encoding a target protein.

[0101] As used herein, the term "vector" and its grammatical equivalents refer to a carrier for carrying genetic material (e.g., a polynucleotide sequence) that can be introduced into a host cell and replicated and / or expressed therein. Vectors that can be used include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include selectable sequences or markers capable of stable integration into the host cell chromosome. Furthermore, a vector may include one or more selectable marker genes and appropriate expression control sequences. For example, the selectable marker genes may provide resistance to antibiotics or toxins, compensate for nutritional deficiencies, or supply critical nutrients absent in the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, etc., well-known in the art. When two or more polynucleotides are to be co-expressed, both polynucleotides may be inserted into a single expression vector or into separate expression vectors, for example. For single-vector expression, the encoding polynucleotide may be operatively ligated to a common expression control sequence or to different expression control sequences, such as an inducible promoter and a constitutive promoter. The introduction of the polynucleotide into the host cell can be confirmed using methods well-known in the art. Those skilled in the art will understand that polynucleotides are expressed in an amount sufficient to produce the desired product (such as the anti-mesothelin antibody or antigen-binding fragment described herein), and will also understand that expression levels can be optimized using methods well known in the art to obtain adequate expression.

[0102] When referring to cells, the term "genetic engineering" or its grammatical equivalents means altering the genetic material of a cell in a way that is not typically present in naturally occurring cells. Genetic alterations include, for example, the introduction of expressible polynucleotides, other additions, mutations / alterations, deletions, and / or other disruptions to the function of cellular genes. Such modifications can, for example, be made in the coding regions of genes and their functional segments. Additional modifications can, for example, be made in non-coding regulatory regions, where these modifications alter gene expression.

[0103] As used herein, the terms “transfer,” “transduction,” “transfection,” and their grammatical equivalents refer to the process of introducing exogenous polynucleotides into host cells. “Transferred,” “transfected,” or “transduced” cells are cells that have been transferred, transduced, or transfected with exogenous polynucleotides. These cells include primary test cells and their progeny. Polynucleotides can be “transferred” into host cells using any type of method known in the art, including, for example, chemical, physical, or biological methods. Polynucleotides are typically “transduced” into host cells using viruses. In contrast, polynucleotides are typically “transfected” into host cells using non-viral methods. These terms are sometimes used interchangeably, and their meaning in different contexts will be readily understood by those skilled in the art.

[0104] As used herein, the term "encoding" and its syntactic equivalents refer to the inherent property of a specific nucleotide sequence in a polynucleotide or nucleic acid (e.g., gene, cDNA, or mRNA) to act as a template in biological processes to synthesize other polymers and macromolecules having defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences, and the resulting biological properties. Thus, if the transcription and translation of mRNA corresponding to a gene produces a protein, then that gene encodes that protein. Unless otherwise stated, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may include introns.

[0105] "Isolated" polypeptides, peptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions are polypeptides, peptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions in a form not found in nature. Isolated polypeptides, peptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions include those substances that have been purified to the point that they are no longer in their naturally occurring form. In some embodiments, the isolated polypeptides, peptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions are substantially pure.

[0106] As used herein and as understood in the art, the term "immune effector cell" and its grammatical equivalents refer to cells derived from the hematopoietic system that play a direct role in the immune response against a target, such as a pathogen, cancer cell, or foreign substance. Immune effector cells include T cells, B cells, natural killer (NK) cells, NKT cells, macrophages, granulocytes, neutrophils, eosinophils, mast cells, and basophils.

[0107] The term "stimulation" of immune effector cells refers to a primary response induced by the binding of a stimulating molecule to its homologous ligand, thereby mediating signal transduction events in immune effector cells that can alter the expression of certain genes and / or reorganize the cytoskeleton. The term "stimulating molecule" refers to a molecule on immune effector cells that, upon binding to a homologous ligand normally present on APCs, mediates signal transduction to promote the maturation, differentiation, proliferation, and / or activation of immune effector cells. For example, the TCR / CD3 complex, as a T cell stimulating molecule, triggers T cell activation. The ligand of the stimulating molecule, or "stimulating ligand," refers to a ligand normally present on APCs that can bind to a stimulating molecule on immune effector cells to mediate a primary response of the immune effector cells, including but not limited to maturation, differentiation, activation, initiation of an immune response, and proliferation. Stimulating ligands are known in the art and include, for example, MHC class I molecules loaded with peptides, anti-CD3 antibodies, hyperactive anti-CD28 antibodies, and hyperactive anti-CD2 antibodies.

[0108] As used herein and as understood in the art, a “co-stimulatory signal” refers to a signal from a co-stimulatory receptor (e.g., CD28 or 4-1BB) that, in combination with a primary signal (e.g., TCR / CD3), promotes optimal clonal expansion, differentiation, and effector function of immune effector cells (e.g., T cells). As used herein and as understood in the art, a “co-stimulatory receptor” for immune effector cells refers to a molecule on immune effector cells that specifically binds to a “co-stimulatory ligand” to mediate a co-stimulatory response of the immune effector cells, such as enhancing the activation or proliferation of immune effector cells. Co-stimulatory receptors for immune effector cells include, but are not limited to, CD28, 4-1BB, ICOS, CD27, OX40, DAP10, CD30, 2B4, CD2, LIGHT, GITR, TLR, DR3, and CD43. A “functional fragment” of a co-stimulatory receptor refers to a fragment of the co-stimulatory receptor that retains the function of mediating co-stimulatory signals and stimulating immune effector cells. In some embodiments, the functional fragment of the co-stimulatory receptor retains the co-stimulatory domain of the co-stimulatory receptor. In some embodiments, the co-stimulatory domain is a cytoplasmic domain of the co-stimulatory receptor. In some embodiments, signaling from co-stimulatory receptors of immune effector cells (e.g., T cells) lowers the activation threshold of immune effector cells. In some embodiments, signaling from T cell co-stimulatory receptors enhances efficient cytokine production (by enhancing transcriptional activity and messenger RNA stability), cell cycle progression, survival, metabolic regulation, and TCR signaling events required for T cell responses.

[0109] As used herein and as understood in the art, a “co-stimulatory ligand” refers to a molecule that specifically binds to a homologous co-stimulatory receptor on immune effector cells, thereby providing a signal in addition to the primary signal provided by the stimulatory molecule, mediating responses in immune effector cells, including but not limited to proliferation, activation, and differentiation. Co-stimulatory ligands can be present on APCs (e.g., dendritic cells). Co-stimulatory ligands include, but are not limited to, CD58, mesothelin, CD83, CD80, CD86, CD137L (4-1BBL), CD252 (OX40L), CD275 (ICOS-L), CD54 (ICAM-1), CD49a, CD112 (PVRL2), CD150 (SLAM), CD155 (PVR), CD265 (RANK), CD270 (HVEM), TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153 (CD30L), CD48, CD160, CD200R (OX2R), and CD44. The "receptor-binding fragment" of a co-stimulatory ligand refers to the ligand fragment that retains the ability to bind to its receptor.

[0110] The term “treatment” and its grammatical equivalents used in this article in connection with a disease or condition or a subject suffering from a disease or condition refer to the action of inhibiting, eliminating, reducing, and / or improving symptoms, symptom severity, and / or symptom frequency associated with the disease or condition being treated. For example, when referring to cancer or tumor, the term “treatment” and its grammatical equivalents refer to the action of reducing the severity of cancer or tumor, or delaying or slowing the progression of cancer or tumor, including: (a) inhibiting the growth of cancer or tumor, or preventing its development; (b) causing the regression of cancer or tumor; or (c) delaying, improving, or minimizing one or more symptoms associated with the presence of cancer or tumor.

[0111] As used herein, the term "administration" and its grammatical equivalents refer to the act of delivering or having a therapeutic agent or pharmaceutical composition delivered to a subject by methods described herein or known in the art. Therapeutic agents can be compounds, peptides, antibodies, cells, or cell populations. Administering a therapeutic agent or pharmaceutical composition includes prescribing for delivery of the therapeutic agent or pharmaceutical composition to a subject. Exemplary forms of administration include oral dosage forms, such as tablets, capsules, syrups, and suspensions; injectable dosage forms, such as intravenous (IV), intramuscular (IM), or intraperitoneal (IP); percutaneous dosage forms, including creams, gels, powders, or patches; oral dosage forms; inhaled powders, sprays, suspensions, and rectal suppositories.

[0112] As used herein, the terms "effective amount," "therapeutic effective amount," and their grammatical equivalents refer to an amount of medicine administered to a subject, either alone or as part of a pharmaceutical composition, in a single dose or as part of a series of doses, that would produce any detectable positive effect on any symptom, aspect, or characteristic of a disease, symptom, or condition when administered to the subject. Therapeutic effective amounts can be determined by measuring the relevant physiological effects. The exact amount required varies from subject to subject, depending on the subject's age, weight and general condition, the severity of the condition being treated, the clinician's judgment, etc. In any individual case, a person skilled in the art can determine the appropriate "effective amount" using routine laboratory methods.

[0113] The terms “pharmaceuticalally acceptable carrier” or “pharmaceuticalally acceptable excipient” refer to materials suitable for administration to an individual with an active agent without causing undesirable biological effects or interacting with any other component of the pharmaceutical composition in a harmful manner.

[0114] As used herein, the term "subject" refers to any animal (e.g., a mammal) that will receive a specific treatment, including but not limited to humans, non-human primates, canines, felines, rodents, etc. Subjects may be human. Subjects may suffer from a specific disease or condition.

[0115] The term “self” as used in this article refers to any material derived from the same individual that will subsequently be reintroduced into that individual.

[0116] The term “allogeneic” as used in this article refers to grafts derived from different animals of the same species.

[0117] Scope: Throughout this disclosure, various aspects of the invention are described in terms of scope. It should be understood that the use of scope is for convenience and brevity only and should not be construed as a rigid limitation on the scope of the invention. Therefore, a description of a scope should be considered as specifically disclosing all possible sub-scopes within that scope and their respective values. For example, a description of a scope such as 1 to 6 should be considered as specifically disclosing sub-scopes such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and their respective values, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the scope.

[0118] This document describes exemplary genes and peptides with reference to GenBank numbers, GI numbers, and / or SEQ ID NO. It should be understood that those skilled in the art can readily identify homologous sequences by referring to sequence sources, including but not limited to GenBank (ncbi.nlm.nih.gov / genbank / ) and EMBL (embl.org / ).

[0119] 5.2 Anti-mesothelin antibody and antigen-binding fragment

[0120] This document provides antibodies or antigen-binding fragments thereof that specifically bind to mesothelin (e.g., human mesothelin). In some embodiments, this document provides anti-mesothelin antibodies. In some embodiments, the antibody is an IgA, IgD, IgE, IgG, or IgM antibody. In some embodiments, the antibody is an IgA antibody. In some embodiments, the antibody is an IgD antibody. In some embodiments, the antibody is an IgE antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgM antibody. In some embodiments, the antibody provided herein may be an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG4 antibody.

[0121] In some embodiments, this document provides an antigen-binding fragment for an anti-mesothelin antibody. In some embodiments, the antigen-binding fragment provided herein may be a single-domain antibody (sdAb), a heavy-chain antibody (HCAb), Fab, Fab', F(ab')2, Fv, a single-chain variable fragment (scFv), or (scFv)2. In some embodiments, the antigen-binding fragment for an anti-mesothelin antibody is a single-domain antibody (sdAb). In some embodiments, the antigen-binding fragment for an anti-mesothelin antibody is a heavy-chain antibody (HCAb). In some embodiments, the antigen-binding fragment for an anti-mesothelin antibody is Fab. In some embodiments, the antigen-binding fragment for an anti-mesothelin antibody is Fab'. In some embodiments, the antigen-binding fragment for an anti-mesothelin antibody is F(ab')2. In some embodiments, the antigen-binding fragment for an anti-mesothelin antibody is Fv. In some embodiments, the antigen-binding fragment for an anti-mesothelin antibody is scFv. In some embodiments, the antigen-binding fragment for an anti-mesothelin antibody is a disulfide-linked scFv [(scFv)2]. In some implementations, the antigen-binding fragment of the anti-mesothelin antibody is a biantibody (dAb).

[0122] In some embodiments, the anti-mesothelin antibodies or antigen-binding fragments provided herein include recombinant antibodies or antigen-binding fragments. In some embodiments, the anti-mesothelin antibodies or antigen-binding fragments provided herein include monoclonal antibodies or antigen-binding fragments. In some embodiments, the anti-mesothelin antibodies or antigen-binding fragments provided herein include polyclonal antibodies or antigen-binding fragments. In some embodiments, the anti-mesothelin antibodies or antigen-binding fragments provided herein include camelid antibodies or antigen-binding fragments (e.g., camel, dromedary camel, and llama). In some embodiments, the anti-mesothelin antibodies or antigen-binding fragments provided herein include chimeric antibodies or antigen-binding fragments. In some embodiments, the anti-mesothelin antibodies or antigen-binding fragments provided herein include humanized antibodies or antigen-binding fragments. In some embodiments, the anti-mesothelin antibodies or antigen-binding fragments provided herein include human antibodies or antigen-binding fragments. In some embodiments, the present invention provides anti-mesothelin human scFv.

[0123] In some embodiments, the anti-mesothelin antibody or antigen-binding fragment provided herein is isolated. In some embodiments, the anti-mesothelin antibody or antigen-binding fragment provided herein is substantially pure.

[0124] In some embodiments, the anti-mesothelin antibody or antigen-binding fragment provided herein comprises a multispecific antibody or antigen-binding fragment. In some embodiments, the anti-mesothelin antibody or antigen-binding fragment provided herein comprises a bispecific antibody or antigen-binding fragment. In some embodiments, a bispecific T-cell adaptor (BiTE) is provided herein. A BiTE is a bispecific antibody that binds to a T-cell antigen (e.g., CD3) and a tumor antigen. BiTEs have been shown to induce targeted lysis of target tumor cells, thus offering significant therapeutic potential for cancer and other conditions. In some embodiments, a BiTE specifically binds to CD3 and mesothelin is provided herein. In some embodiments, a BiTE comprises the anti-mesothelin antibody or antigen-binding fragment provided herein. In some embodiments, a BiTE comprises the anti-mesothelin scFv provided herein.

[0125] In some embodiments, the anti-mesothelin antibody or antigen-binding fragment provided herein includes a monovalent antigen-binding site. In some embodiments, the anti-mesothelin antibody or antigen-binding fragment includes a monospecific binding site. In some embodiments, the anti-mesothelin antibody or antigen-binding fragment includes a bivalent binding site.

[0126] In some embodiments, the anti-mesothelin antibody or antigen-binding fragment is a monoclonal antibody or antigen-binding fragment. Monoclonal antibodies can be prepared by any method known to those skilled in the art. An exemplary method is to screen protein expression libraries, such as phage display libraries or ribosome display libraries. Phage display is described, for example, in Ladner et al., U.S. Patent No. 5,223,409; Smith (1985) Science 228:1315-1317; and WO 92 / 18619. In some embodiments, recombinant monoclonal antibodies are isolated from a phage display library expressing the variable region or CDR of the desired species. Phage library screening can be performed using various techniques known in the art.

[0127] In some embodiments, monoclonal antibodies are prepared using hybridoma methods known to those skilled in the art. For example, mice, rats, rabbits, hamsters, or other suitable host animals are immunized using hybridoma methods as described above. In some embodiments, immunization is performed on lymphocytes in vitro. In some embodiments, the immunoantigen is a human protein or a fragment thereof.

[0128] Following immunization, lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol. Hybridoma cells are selected using specialized culture media known in the art; unfused lymphocytes and myeloma cells cannot survive the selection process. Hybridomas producing monoclonal antibodies against selected antigens can be identified by a variety of methods, including but not limited to immunoprecipitation, Western blotting, and in vitro binding assays (e.g., flow cytometry, FACS, ELISA, SPR (e.g., Biacore), and radioimmunoassay). Once hybridoma cells producing antibodies with the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution or other techniques. Hybridomas can be propagated in vitro using standard methods or in vivo as ascites tumors. Monoclonal antibodies can be purified from culture media or ascites fluid according to standard methods in the art, including but not limited to affinity chromatography, ion exchange chromatography, gel electrophoresis, and dialysis.

[0129] In some embodiments, monoclonal antibodies are prepared using recombinant DNA techniques known to those skilled in the art. For example, polynucleotides encoding the antibody are isolated from mature B cells or hybridoma cells, for instance, by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the antibody heavy and light chains, and their sequences are determined using standard techniques. Subsequently, the isolated polynucleotides encoding the heavy and light chains are cloned into a suitable expression vector; when said expression vector is transfected into host cells that do not normally produce immunoglobulins (e.g., *E. coli*, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells), monoclonal antibodies are produced.

[0130] In some embodiments, recombinant DNA technology is used to modify monoclonal antibodies to generate alternative antibodies. In some embodiments, the light and heavy chain constant domains of mouse monoclonal antibodies are replaced with constant regions of human antibodies to generate chimeric antibodies. In some embodiments, constant regions are truncated or removed to generate desired antibody fragments of monoclonal antibodies. In some embodiments, site-directed or high-density mutagenesis of variable regions is used to optimize the specificity and / or affinity of monoclonal antibodies.

[0131] In some embodiments, the anti-mesothelin antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment. Various methods for generating humanized antibodies are known in the art. Methods for achieving high-affinity binding of humanized antibodies are also known in the art. Non-limiting examples of such methods include high-frequency mutation of the variable region and selection of cells expressing such high-affinity antibodies (affinity maturation). In addition to using a display library, non-human animals, such as rodents, can be immunized with a designated antigen (e.g., recombinant mesothelin or its epitopes). In some embodiments, rodent antigen-binding fragments (e.g., mouse antigen-binding fragments) can be generated and isolated using methods known in the art and / or disclosed herein. In some embodiments, mice can be immunized with an antigen (e.g., recombinant mesothelin or its epitopes).

[0132] In some embodiments, anti-mesothelin antibodies or antigen-binding fragments are human antibodies or antigen-binding fragments. Human antibodies can be prepared using various techniques known in the art. In some embodiments, human antibodies are generated from immortalized human B lymphocytes immunized in vitro. In some embodiments, human antibodies are generated from lymphocytes isolated from an immunized individual. In any case, cells that produce antibodies against the target antigen can be generated and isolated. In some embodiments, human antibodies are selected from a phage library expressing human antibodies. Alternatively, phage display technology can be used to generate human antibodies and antibody fragments in vitro from an immunoglobulin variable region gene library of an unimmunized donor. Techniques for generating and using antibody phage libraries are well known in the art. Once antibodies are identified, affinity maturation strategies known in the art, including but not limited to chain shuffling and site-directed mutagenesis, can be employed to generate human antibodies with higher affinity. In some embodiments, human antibodies are generated in transgenic mice containing human immunoglobulin loci. After immunization, these mice are able to generate a complete human antibody library in the absence of endogenous immunoglobulin production.

[0133] The specific CDR sequences defined herein are generally based on combinations defined by Kabat. However, it should be understood that references to heavy chain CDRs or multiple CDRs and / or light chain CDRs or multiple CDRs for a particular antibody encompass all CDR definitions known to those skilled in the art.

[0134] The anti-mesothelin antibody or antigen-binding fragments provided in this article include the MH150 clone, whose sequence characteristics are described below.

[0135] In some embodiments, the anti-mesothelin antibody or antigen-binding fragments (e.g., for human mesothelin) provided herein comprise one, two, three, four, five, and / or six CDRs of any of the antibodies described herein. In some embodiments, the anti-mesothelin antibody or antigen-binding fragments provided herein comprise a VL, which comprises one, two, and / or three LCDRs from Table 1. In some embodiments, the anti-mesothelin antibody or antigen-binding fragments provided herein comprise a VH, which comprises one, two, and / or three HCDRs from Table 2. In some embodiments, the anti-mesothelin antibody or antigen-binding fragments provided herein comprise one, two, and / or three LCDRs from Table 1 and one, two, and / or three HCDRs from Table 2.

[0136] Table 1. Amino acid sequence of the CDR (LCDR) of the light chain of anti-mesothelin antibody.

[0137]

[0138] Table 2. Amino acid sequence of the heavy chain variable region CDR (HCDR) of anti-mesothelin antibody

[0139]

[0140] In some embodiments, the anti-mesothelin antibody or its antigen-binding fragment comprises a humanized antibody or its antigen-binding fragment. In some embodiments, the anti-mesothelin antibody or its antigen-binding fragment comprises LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and / or HCDR3 from the antibodies or antigen-binding fragments described herein. In some embodiments, the anti-mesothelin antibody or its antigen-binding fragment comprises a variant of the anti-mesothelin antibody or its antigen-binding fragment described herein. In some embodiments, the variant of the anti-mesothelin antibody or its antigen-binding fragment includes 1 to 30 amino acid substitutions, additions, and / or deletions in the anti-mesothelin antibody or its antigen-binding fragment. In some embodiments, the variant of the anti-mesothelin antibody or its antigen-binding fragment includes 1 to 25 amino acid substitutions, additions, and / or deletions in the anti-mesothelin antibody or its antigen-binding fragment. In some embodiments, the variant of the anti-mesothelin antibody or its antigen-binding fragment includes 1 to 20 substitutions, additions, and / or deletions in the anti-mesothelin antibody or its antigen-binding fragment. In some embodiments, variants of the anti-mesothelin antibody or its antigen-binding fragment contain 1 to 15 substitutions, additions, and / or deletions. In some embodiments, variants of the anti-mesothelin antibody or its antigen-binding fragment contain 1 to 10 substitutions, additions, and / or deletions. In some embodiments, variants of the anti-mesothelin antibody or its antigen-binding fragment contain 1 to 5 conserved amino acid substitutions, additions, and / or deletions. In some embodiments, variants of the anti-mesothelin antibody or its antigen-binding fragment contain 1 to 3 amino acid substitutions, additions, and / or deletions. In some embodiments, the amino acid substitutions, additions, and / or deletions are conserved amino acid substitutions. In some embodiments, the one or more conserved amino acid substitutions are located in the CDR of the antibody or its antigen-binding fragment. In some embodiments, the one or more conserved amino acid substitutions are not located in the CDR of the antibody or its antigen-binding fragment. In some embodiments, the one or more conserved amino acid substitutions are located in the frame region of the antibody or its antigen-binding fragment.

[0141] In some embodiments, this document provides antibodies or antigen-binding fragments thereof that specifically bind to mesothelin (e.g., human dermothelin) and comprise a light chain variable region (VL), said VL comprising: (1) a light chain CDR1 (LCDR1) having the amino acid sequence of SEQ ID NO: 1; (2) a light chain CDR2 (LCDR2) having the amino acid sequence of SEQ ID NO: 2; or (3) a light chain CDR3 (LCDR3) having the amino acid sequence of SEQ ID NO: 3; or variants having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the LCDR. In some embodiments, said variants have about 5 amino acid substitutions, additions, and / or deletions in the LCDR.

[0142] In some embodiments, this document provides antibodies or antigen-binding fragments thereof that specifically bind to mesothelin (e.g., human dermothelin) and include a heavy chain variable region (VH) comprising: (1) a heavy chain CDR1 (HCDR1) having the amino acid sequence of SEQ ID NO: 4; (2) a heavy chain CDR2 (HCDR2) having the amino acid sequence of SEQ ID NO: 5; or (3) a heavy chain CDR3 (HCDR3) having the amino acid sequence of SEQ ID NO: 6; or variants having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the HCDR. In some embodiments, the variants have up to about 5 amino acid substitutions, additions, and / or deletions in the HCDR.

[0143] In some embodiments, this document provides antibodies or antigen-binding fragments thereof that specifically bind mesothelin (e.g., human dermochrome) and have VL and VH. In some embodiments, VL and VH are linked by a linker. The linker may be a flexible linker or a rigid linker. In some embodiments, the linker has an amino acid sequence of (GGGGS)n, where n = 1, 2, 3, 4, or 5 (SEQ ID NO: 21). For example, the linker may have the amino acid sequence GGGGS. In some embodiments, the linker has an amino acid sequence of (EAAAK)n, where n = 1, 2, 3, 4, or 5. For example, the linker may have the amino acid sequence EAAAK (SEQ ID NO: 22). In some embodiments, the linker has an amino acid sequence of (PA)nP, where n = 1, 2, 3, 4, or 5 (SEQ ID NO: 23). In some embodiments, the linker has the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 24).

[0144] In some embodiments, this document provides antibodies or antigen-binding fragments thereof that specifically bind mesothelin (e.g., human cortisol) having VL and VH, wherein VL comprises LCDR1, LCDR2, and LCDR3, VH comprises HCDR1, HCDR2, and HCDR3, and wherein LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 have amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively; or variants thereof having up to about 5 amino acid substitutions, additions, and / or deletions in CDR.

[0145] In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to mesothelin (e.g., human dermochrome) having a VL, said VL comprising: (1) LCDR1 having the amino acid sequence of SEQ ID NO: 1; (2) LCDR2 having the amino acid sequence of SEQ ID NO: 2; or (3) LCDR3 having the amino acid sequence of SEQ ID NO: 3. The VL may comprise LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively. In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to mesothelin having a VH, said VH comprising: (1) HCDR1 having the amino acid sequence of SEQ ID NO: 4; (2) HCDR2 having the amino acid sequence of SEQ ID NO: 5; or (3) HCDR3 having the amino acid sequence of SEQ ID NO: 6. The VH may comprise HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively. In some embodiments, this document provides antibodies or antigen-binding fragments thereof that specifically bind mesothelin (e.g., human dermothelin), comprising: (a) a VL comprising the amino acid sequences having SEQ ID NOs: 1, 2 and 3, respectively; and (b) a VH comprising the amino acid sequences having SEQ ID NOs: 4, 5 and 6, respectively.

[0146] In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to mesothelin (e.g., human dermochrome), comprising a VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 with the amino acid sequence of SEQ ID NO: 7. In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to mesothelin (e.g., human dermochrome), comprising a VH having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 with the amino acid sequence of SEQ ID NO: 8.

[0147] Table 3. Amino acid sequences of the light chain variable region (VL) and heavy chain variable region (VH) of anti-mesothelin antibodies.

[0148]

[0149] In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to mesothelin (e.g., human dermothelin), comprising: (a) a VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 with the amino acid sequence of SEQ ID NO: 7; and (b) a VH having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 with the amino acid sequence of SEQ ID NO: 8.

[0150] In some embodiments, this document provides antibodies or antigen-binding fragments thereof that specifically bind mesothelin (e.g., human cortisol) having VL and VH, wherein VL and VH have the amino acid sequences of SEQ ID NO: 7 and 8, respectively.

[0151] In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to mesothelin (e.g., human dermothelin), comprising: (a) a VL comprising an LCDR of a VL having the amino acid sequence of SEQ ID NO: 7; and / or (b) a VH comprising an HCDR of a VH having the amino acid sequence of SEQ ID NO: 8.

[0152] In some embodiments, this document provides antibodies or antigen-binding fragments thereof that specifically bind mesothelin (e.g., human cortisol) having VL and VH, wherein VL comprises LCDR1, LCDR2, and LCDR3 from VL having the amino acid sequence of SEQ ID NO: 7, and VH comprises HCDR1, HCDR2, and HCDR3 from VH having the amino acid sequence of SEQ ID NO: 8.

[0153] In some embodiments, the anti-mesothelin antibody or its antigen-binding fragment provided herein is an scFv named MH150 (SEQ ID NO: 9). In some embodiments, the anti-mesothelin antibody or its antigen-binding fragment provided herein has at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of the same amino acid sequence as SEQ ID NO: 9. In some embodiments, the anti-mesothelin antibody or its antigen-binding fragment provided herein has a VL from MH150 (SEQ ID NO: 7). In some embodiments, it has a VH from MH150 (SEQ ID NO: 8). It may have both a VL and a VH from MH150. In some embodiments, it has a VL containing an LCDR from MH150 VL (SEQ ID NO: 7). In some embodiments, it has a VH containing an HCDR from MH150 VH (SEQ ID NO: 8). It may have VL and VH comprising VL and VH from MH150, respectively, an LCDR and an HCDR. In some embodiments, it is a variant of MH150. The MH150 variant may have a VL as an MH150 VL variant, which has up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO: 7. The MH150 variant may have a VH as an MH150 VH variant, which has up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO: 8. The substitutions, additions and / or deletions may be located in the HCDR or LCDR. In some embodiments, they are not located in the CDR. In some embodiments, the MH150 variant has up to about 5 conserved amino acid substitutions. In some embodiments, the MH150 variant has up to 3 conserved amino acid substitutions.

[0154] In some embodiments, this document also provides antibodies or antigen-binding fragments that compete with the aforementioned antibody or antigen-binding fragments for binding to mesothelin (e.g., human dermochrome). An antibody that “competes with another antibody for binding to a target” refers to an antibody that inhibits (partially or completely inhibits) the binding of another antibody to a target. Whether two antibodies compete for binding to a target, i.e., whether and to what extent one antibody inhibits the binding of the other antibody to a target, can be determined using known competition assays, such as BIACORE surface plasmon resonance (SPR) analysis. In some embodiments, an anti-mesothelin antibody or antigen-binding fragment competes with another antibody or antigen-binding fragment and inhibits its binding to mesothelin by at least 50%, 60%, 70%, 80%, 90%, or 100%. Competition assays can be performed, for example, as described in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi: 10.H01 / pdb.prot4277, or in Chapter 11 of Ed Harlow and David Lane, Using Antibodies (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1999).

[0155] In some implementations, this document provides an antibody or antigen-binding fragment that competes with MH150 for binding to mesothelin.

[0156] This disclosure also envisions other variants and equivalents substantially homologous to the recombinant, monoclonal, chimeric, humanized, and human antibodies or antibody fragments thereof described herein. In some embodiments, it is desirable to enhance the binding affinity of the antibody. In some embodiments, it is desirable to modulate the biological properties of the antibody, including but not limited to specificity, thermostability, expression level, effector function, glycosylation, immunogenicity, and / or solubility. Those skilled in the art will understand that amino acid alterations can modify the post-translational processes of an antibody, for example, by changing the number or location of glycosylation sites or altering membrane anchoring characteristics.

[0157] A mutation can be a substitution, deletion, or insertion of one or more nucleotides encoding an antibody or polypeptide, resulting in an alteration of the amino acid sequence relative to the native antibody or polypeptide sequence. In some embodiments, an amino acid substitution is the result of replacing one amino acid with another amino acid having similar structure and / or chemical properties, such as replacing leucine with serine, i.e., a conserved amino acid substitution. Insertions or deletions can range from about 1 to 5 amino acids. In some embodiments, the substitution, deletion, or insertion includes fewer than 25, 20, 15, 10, 5, 4, 3, or 2 amino acid substitutions relative to the parent molecule. In some embodiments, biologically useful and / or relevant amino acid sequence variations can be determined by systematically making insertions, deletions, or substitutions in the sequence and testing the activity of the resulting variant protein relative to the parent protein.

[0158] It is known in the art that constant regions of antibodies mediate a variety of effector functions, and these effector functions may vary depending on the antibody isotype. For example, the binding of the complement C1 component to the Fc region of an IgG or IgM antibody bound to an antigen activates the complement system. Activation of complement is crucial for opsonization and lysis of cellular pathogens. Activation of complement also stimulates inflammatory responses and may be involved in autoimmune hypersensitivity reactions. Furthermore, the Fc region of an antibody can bind to cells expressing Fc receptors (FcRs). Several Fc receptors exist that are specific to different classes of antibodies, including IgG receptors (γ receptors), IgE receptors (ε receptors), IgA receptors (α receptors), and IgM receptors (μ receptors). Binding of antibodies to cell surface Fc receptors triggers a variety of important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (a process known as antibody-dependent cell-mediated cytotoxicity or ADCC), release of inflammatory mediators, placental transport, and control of immunoglobulin production. In some embodiments, the anti-mesothelin antibody or its antigen-binding fragment described herein comprises at least one constant region of a human IgA antibody. In some embodiments, the anti-mesothelin antibody or its antigen-binding fragment described herein comprises at least one constant region of a human IgD antibody. In some embodiments, the anti-mesothelin antibody or its antigen-binding fragment described herein comprises at least one constant region of a human IgE antibody. In some embodiments, the anti-mesothelin antibody or its antigen-binding fragment described herein comprises at least one constant region of a human IgG antibody. In some embodiments, the anti-mesothelin antibody or its antigen-binding fragment described herein comprises at least one constant region of a human IgM antibody. In some embodiments, the anti-mesothelin antibody or its antigen-binding fragment described herein comprises at least one constant region of a human IgG1 antibody. In some embodiments, the anti-mesothelin antibody or its antigen-binding fragment described herein comprises at least one constant region of a human IgG2 antibody. In some embodiments, the anti-mesothelin antibody or its antigen-binding fragment described herein comprises at least one constant region of a human IgG3 antibody. In some embodiments, the anti-mesothelin antibody or its antigen-binding fragment described herein comprises at least one constant region of a human IgG4 antibody.

[0159] In some embodiments, at least one or more constant regions in the anti-mesothelin antibody or antigen-binding fragment described herein have been modified or deleted. In some embodiments, the antibody comprises modifications to one or more of the three heavy chain constant regions (CH1, CH2, or CH3) and / or light chain constant regions (CL). In some embodiments, the heavy chain constant region of the modified antibody comprises at least one human constant region. In some embodiments, it comprises more than one human constant region. In some embodiments, the modification of the constant region comprises the addition, deletion, or substitution of one or more amino acids in one or more regions. In some embodiments, one or more regions are partially or completely deleted from the constant region of the modified antibody. In some embodiments, the entire CH2 domain (ΔCH2 construct) is removed from the antibody. In some embodiments, the deleted constant region is replaced with a short amino acid spacer region that provides partial molecular flexibility typically conferred by the deleted constant region. In some embodiments, the modified antibody comprises a CH3 domain directly fused to the hinge region of the antibody. In some embodiments, the modified antibody comprises a peptide spacer region inserted between the hinge region and the modified CH2 and / or CH3 domains.

[0160] In some embodiments, the anti-mesothelin antibody or its antigen-binding fragment comprises an Fc region. In some embodiments, the Fc region is fused via a hinge. The hinge may be an IgG1 hinge, an IgG2 hinge, or an IgG3 hinge. The amino acid sequences of the Fc regions of human IgG1, IgG2, IgG3, and IgG4 are known to those skilled in the art. In some cases, Fc regions with amino acid variations have been identified in natural antibodies. In some embodiments, the modified antibody (e.g., the modified Fc region) provides altered effector functions, which in turn affect the biological characteristics of the antibody. For example, in some embodiments, the deletion or inactivation of the constant region (through point mutation or other means) reduces the Fc receptor binding of the modified antibody during circulation. In some embodiments, constant region modification reduces the immunogenicity of the antibody. In some embodiments, constant region modification increases the serum half-life of the antibody. In some embodiments, constant region modification reduces the serum half-life of the antibody. In some embodiments, constant region modification reduces or eliminates ADCC and / or complement-dependent cytotoxicity (CDC) of the antibody. In some embodiments, replacing specific amino acids in the human IgG1 Fc region with corresponding IgG2 or IgG4 residues reduces the effector function (e.g., ADCC and CDC) of the modified antibody. In some embodiments, the antibody does not have one or more effector functions (e.g., an "effect-free" antibody). In some embodiments, the antibody does not have ADCC activity and / or does not have CDC activity. In some embodiments, the antibody does not bind to Fc receptors and / or complement factors. In some embodiments, the antibody does not have effector function. In some embodiments, constant region modification increases or enhances the ADCC and / or CDC of the antibody. In some embodiments, the constant region is modified to eliminate disulfide bonds or oligosaccharide moieties. In some embodiments, the constant region is modified to add / replace one or more amino acids, thereby providing one or more cytotoxic, oligosaccharide, or carbohydrate linkage sites. In some embodiments, the anti-mesothelin antibody or its antigen-binding fragment comprises a variant Fc region that, compared to the native Fc region, is engineered to have substitutions at specific amino acid positions. In some embodiments, the anti-mesothelin antibody or its antigen-binding fragment described herein comprises a constant region of the IgG1 heavy chain, which, according to EU designations, comprises one or more amino acid substitutions selected from the group consisting of K214R, L234A, L235E, G237A, D356E, and L358M. In some embodiments, according to EU designations, the constant region of the IgG1 heavy chain comprises one or more amino acid substitutions selected from the group consisting of K214R, L234A, L235E, G237A, A330S, P331S, D356E, and L358M.In some embodiments, according to EU designations, the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, C226S, C229S, and P238S. In some embodiments, according to EU designations, the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, D356E, and L358M. In some embodiments, according to EU designations, the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of S131C, K133R, G137E, G138S, Q196K, I199T, N203D, K214R, C226S, C229S, and P238S.

[0161] In some embodiments, the variant may include the addition of amino acid residues to the amino and / or carboxyl termini of the antibody or peptide. The length of the added amino acid residues may range from one residue to one hundred or more residues. In some embodiments, the variant contains an N-terminal methionine residue. In some embodiments, the variant contains an additional peptide / protein (e.g., an Fc region) to form a fusion protein. In some embodiments, the variant is engineered to be detectable and may contain a detectable marker and / or protein (e.g., a fluorescent tag or enzyme).

[0162] The variant antibodies or antigen-binding fragments described herein can be generated using methods known in the art, including but not limited to site-directed mutagenesis, alanine scanning mutagenesis, and PCR mutagenesis.

[0163] In some embodiments, variants of the anti-mesothelin antibody or antigen-binding fragment disclosed herein may retain the ability to bind mesothelin to a degree similar to, the same as, or greater than that of the parent antibody or antigen-binding fragment. In some embodiments, the amino acid sequence of the variant and the parent antibody or antigen-binding fragment may have at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater identity. In some embodiments, variants of the anti-mesothelin antibody or antigen-binding fragment comprise the amino acid sequence of the parent anti-mesothelin antibody or antigen-binding fragment having one or more conserved amino acid substitutions. Conserved amino acid substitutions are known in the art, which include replacing an amino acid having certain physical and / or chemical properties with another amino acid having the same or similar chemical or physical properties.

[0164] In some embodiments, variants of the anti-mesothelin antibody or antigen-binding fragment comprise the amino acid sequence of the parent antibody or antigen-binding fragment having one or more non-conservative amino acid substitutions. In some embodiments, the one or more non-conservative amino acid substitutions do not interfere with or inhibit one or more biological activities (e.g., mesothelin binding) of the variant. In some embodiments, the one or more conserved amino acid substitutions and / or non-conservative amino acid substitutions can enhance the biological activity of the variant, such that the biological activity of the functional variant is increased relative to the parent binding moiety.

[0165] In some implementations, the variants may have 1, 2, 3, 4 or 5 amino acid substitutions in the CDRs of the binding site (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3).

[0166] In some embodiments, the anti-mesothelin antibody or antigen-binding fragment described herein is naturally or through intervention and is chemically modified. In some embodiments, the anti-mesothelin antibody or antigen-binding fragment is chemically modified by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization of known protecting / blocking groups, proteolytic cleavage, and / or linkage to cellular ligands or other proteins. Any of a variety of chemical modifications can be implemented using known techniques. The anti-mesothelin antibody or antigen-binding fragment may comprise one or more amino acid analogs (including, for example, non-natural amino acids) and other modifications known in the art.

[0167] In some embodiments, an anti-mesothelin antibody or its antigen-binding fragment (e.g., an antibody) binds to mesothelin (e.g., human dermothelin) with a dissociation constant (KD) of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, about 0.1 nM or less, 50 pM or less, 10 pM or less, or 1 pM or less. In some embodiments, an anti-mesothelin antibody or its antigen-binding fragment binds to mesothelin (e.g., human dermothelin) with a KD of about 20 nM or less. In some embodiments, an anti-mesothelin antibody or its antigen-binding fragment binds to mesothelin (e.g., human dermothelin) with a KD of about 10 nM or less. In some embodiments, an anti-mesothelin antibody or its antigen-binding fragment binds to mesothelin (e.g., human dermothelin) with a KD of about 1 nM or less. In some embodiments, the anti-mesothelin antibody or its antigen-binding fragment binds to mesothelin (e.g., human dermothelin) at a KD of about 0.5 nM or less. In some embodiments, the anti-mesothelin antibody or its antigen-binding fragment binds to mesothelin (e.g., human dermothelin) at a KD of about 0.1 nM or less. In some embodiments, the anti-mesothelin antibody or its antigen-binding fragment binds to mesothelin (e.g., human dermothelin) at a KD of about 50 pM or less. In some embodiments, the anti-mesothelin antibody or its antigen-binding fragment binds to mesothelin (e.g., human dermothelin) at a KD of about 25 pM or less. In some embodiments, the anti-mesothelin antibody or its antigen-binding fragment binds to mesothelin (e.g., human dermothelin) at a KD of about 10 pM or less. In some embodiments, the anti-mesothelin antibody or its antigen-binding fragment binds to mesothelin (e.g., human dermothelin) at a KD of about 1 pM or less. In some embodiments, the dissociation constant of the binder (e.g., antibody) to mesothelin is determined by immobilizing the mesothelin protein on a Biacore chip and allowing the binder to flow through the chip. In some embodiments, the dissociation constant of the binder (e.g., antibody) to mesothelin is determined by capturing the binder with an anti-human IgG antibody on a Biacore chip and allowing soluble mesothelin to flow through the chip.

[0168] The physical, chemical, and / or biological properties of the anti-mesothelin antibody or antigen-binding fragment disclosed herein can be analyzed using various methods known in the art. In some embodiments, the ability of the anti-mesothelin antibody to bind to mesothelin (e.g., human dermothelin) is detected. Binding assays include, but are not limited to, SPR (e.g., Biacore), ELISA, and FACS. Furthermore, the antibody's solubility, stability, thermal stability, viscosity, expression level, expression quality, and / or purification efficiency can be evaluated.

[0169] Epitope mapping is a method for identifying binding sites, regions, or epitopes on target proteins by which antibodies bind. Various methods for mapping epitopes on target proteins are known in the art. These methods include mutagenesis (including but not limited to shotgun mutagenesis, site-directed mutagenesis, and alanine scanning), domain or fragment scanning, peptide scanning (e.g., Pepscan technology), display methods (e.g., phage display, microbial display, and ribosome / mRNA display), methods involving proteolysis and mass spectrometry, and structural determinations (e.g., X-ray crystallography and NMR). In some embodiments, the anti-mesothelin antibody or antigen-binding fragment described herein is characterized by assays including but not limited to N-terminal sequencing, amino acid analysis, HPLC, mass spectrometry, ion-exchange chromatography, and papain digestion.

[0170] In some embodiments, an anti-mesothelin antibody or antigen-binding fragment is conjugated to or partially conjugated to a cytotoxic agent. In some embodiments, it is conjugated to a cytotoxic agent to form an ADC (antibody-drug conjugate). In some embodiments, the cytotoxic portion is a chemotherapeutic agent, including but not limited to methotrexate, doxorubicin / doxorubicin, melphalan, mitomycin C, chlorambucil, docalimcin, daunorubicin, pyrrolobenzodiazepine (PBD), or other intercalating agents. In some embodiments, the cytotoxic portion is a microtubule inhibitor, including but not limited to aurestatin, maytansine compounds (e.g., DM1 and DM4), and tubulolysin. In some embodiments, the cytotoxic portion is an enzymatically active toxin or fragment thereof derived from bacteria, fungi, plants, or animals, including but not limited to diphtheria toxin A chain, non-bound active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, α-arbusculin, tung oil protein, caryophyllin protein, pokeweed protein (PAPI, PAPII, and PAP-S), bitter melon inhibitor, lactudin, croton toxin, soapwort inhibitor, white tree toxin protein, mitogellin, restrictocin, phenomycin, enomycin, and trichothecenes. In some embodiments, the antibody is conjugated to one or more small molecule toxins, such as kazimidoxam, maytansine compounds, trichothecenes, and CC1065.

[0171] In some embodiments, the anti-mesothelin antibody or antigen-binding fragment described herein is conjugated to a detectable substance or molecule that makes the formulation available for diagnosis and / or detection. Detectable substances may include, but are not limited to: enzymes, such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; prosthetic groups, such as biotin and flavin; fluorescent materials, such as umbelliferone, luciferin, fluorescein isothiocyanate (FITC), rhodamine, tetramethylrhodamine isothiocyanate (TRITC), dichlorotriazine aminofluorescein, dansyl chloride, anthocyanin (Cy3), and phycoerythrin; bioluminescent materials, such as luciferase; and radioactive materials, such as 212Bi, 14C, 57Co, 51Cr, 67Cu, 18F, 68Ga, 67Ga, 153Gd, 159Gd, 68 Ge, 3H, 166Ho, 131I, 125I, 123I, 121I, 115In, 113In, 112In, 111In, 140La, 177Lu, 54Mn, 99Mo, 32P, 103Pd, 149Pm, 142Pr, 186Re, 188Re, 105Rh, 97Ru, 35S, 47Sc, 75Se, 153Sm, 113Sn, 117Sn, 85Sr, 99mTc, 201Ti, 133Xe, 90Y, 69Yb, 175Yb and 65Zn; positron-emitting metals; and magnetic metal ions.

[0172] The anti-mesothelin antibody or antigen-binding fragment described herein may be attached to a solid support. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. In some embodiments, the immobilized anti-mesothelin antibody or antigen-binding fragment is used for immunoassay. In some embodiments, it is used to purify target antigens (e.g., human dermochrome).

[0173] 5.3 CAR, TCR and genetically engineered immune effector cells

[0174] The anti-mesothelin antibodies or antigen-binding fragments described herein can serve as part of a chimeric antigen receptor (CAR) or T-cell receptor (TCR), which can be expressed in immune effector cells for cancer therapy. Therefore, this article also provides CARs and TCRs that specifically bind mesothelin, immune effector cells expressing such CARs or TCRs, and the uses of such cells.

[0175] 5.3.1 TCR

[0176] This article presents a mesothelin-specific T-cell receptor (TCR) (“mesothelin TCR”). A TCR is an antigen-specific molecule responsible for recognizing antigenic peptides presented by APCs or MHC products on the surface of any nucleated cell. This system endows T cells with the potential to recognize all intracellular antigens expressed by the cell, including viral proteins; these antigens are processed into short peptides, bind to intracellular MHC molecules, and are delivered to the cell surface as peptide-MHC complexes. This system enables exogenous proteins (e.g., mutated cancer antigens or viral proteins) or aberrantly expressed proteins to serve as targets for T cells (e.g., Davis and Bjorkman (1988) Nature 334:395-402; Davis et al. (1998) Annu. Rev. Immunol. 16:523-544).

[0177] Therefore, in some embodiments, this document provides a TCR comprising an anti-mesothelin antibody or antigen-binding fragment as described herein. The anti-mesothelin antibody or antigen-binding fragment can be any anti-mesothelin antibody or antigen-binding fragment described herein. For illustrative purposes, in some embodiments, the TCR provided herein may comprise an anti-mesothelin antibody or antigen-binding fragment having VL and VH, wherein VL comprises LCDR1, LCDR2, and LCDR3, VH comprises HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 have the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively.

[0178] In some implementations, the TCR provided herein may contain an anti-mesothelin antibody or antigen-binding fragment as an scFv named MH150.

[0179] 5.3.2 CAR

[0180] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid protein or polypeptide containing a binding moiety (e.g., an antibody) that links to signaling or activation domains of immune cells (e.g., T cells). In some implementations, CARs are synthetic receptors that retarget T cells to tumor surface antigens (Sadelain et al., Nat. Rev. Cancer 3(1):35-45 (2003); Sadelain et al., Cancer Discovery 3(4):388-398 (2013)). CARs can confer both antigen binding and immune cell activation functions to immune cells (e.g., T cells). Utilizing the antigen-binding properties of monoclonal antibodies, CARs can redirect T cell specificity and reactivity to selected targets in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition allows CAR-expressing T cells to recognize antigens independently of antigen processing, thereby bypassing a tumor escape mechanism.

[0181] A typical CAR molecule structure includes an extracellular antigen-binding domain (e.g., scFv), a transmembrane domain (TM), and an intracellular signal transduction domain. The extracellular antigen-binding domain of a CAR is usually derived from a monoclonal antibody (mAb) or from a receptor or its ligand. CAR binding to an antigen triggers phosphorylation of the immune receptor tyrosine activation motif (ITAM) in the intracellular domain, thereby initiating the signaling cascade required to induce cell lysis, cytokine secretion, and proliferation. Based on the presence or absence of intracellular co-stimulatory signals, CAR-expressing T cells (“CAR-T”) can be classified into three generations.

[0182] In some implementations, this document provides a CAR that specifically binds to mesothelin (“mesothelin CAR”). In some implementations, the CAR may be a “first-generation”, “second-generation”, or “third-generation” CAR (see, for example, Sadelain et al., Cancer Discov. 3(4):388-398 (2013); Jensen et al., Immunol. Rev. 257:127-133 (2014); Sharpe et al., Dis. Model Mech. 8(4):337-350 (2015); June et al. (2018), Science 359(6382):1361-1365).

[0183] First-generation CARs typically consist of an extracellular antigen-binding domain (e.g., a single-chain variable fragment (scFv)) fused to a transmembrane domain, which is fused to a cytoplasmic / intracellular domain of the T-cell receptor chain. First-generation CARs also typically have an intracellular domain derived from the CD3ζ chain, the primary signaling transducer from the endogenous T-cell receptor (TCR). First-generation CARs can provide de novo antigen recognition and induce activation of CD4+ and CD8+ T cells within a single fusion molecule via their CD3ζ chain signaling domain, independent of HLA-mediated antigen presentation. Second-generation CARs contain a cancer antigen-binding domain fused to an intracellular signaling domain capable of activating immune effector cells such as T cells, and a co-stimulatory domain designed to enhance the potency and persistence of immune effector cells such as T cells (Sadelain et al., Cancer Discov. 3:388-398 (2013)). Therefore, CAR design can combine antigen recognition with signal transduction, two functions physiologically performed by two separate complexes: the TCR heterodimer and the CD3 complex. "Second-generation" CARs include intracellular domains from various co-stimulatory receptors (e.g., CD28, 4-1BB, ICOS, OX40, etc.) in their cytoplasmic tails to provide additional signals to the cell. "Second-generation" CARs provide both co-stimulation (e.g., through the CD28 or 4-1BB domains) and activation (e.g., through the CD3ζ signaling domain). Studies have shown that "second-generation" CARs can improve the anti-tumor activity of T cells. In 2017, the FDA approved two anti-CD19 CAR-T cell products for the treatment of relapsed B-cell precursor acute lymphoblastic leukemia (B-ALL) and B-cell non-Hodgkin lymphoma. "Third-generation" CARs provide multiple co-stimuli (e.g., by simultaneously containing CD28 and 4-1BB domains) and activation (e.g., by containing the CD3ζ activation domain).

[0184] Therefore, this document provides a CAR that specifically binds to mesothelin, comprising, from the N-terminus to the C-terminus: (a) a mesothelin-binding domain comprising an anti-mesothelin antibody or its antigen-binding fragment provided herein; (b) a transmembrane domain; and (c) a cytoplasmic domain. The anti-mesothelin antibody or its antigen-binding fragment may be any anti-mesothelin antibody or its antigen-binding fragment described herein. For illustrative purposes, in some embodiments, the CAR provided herein may comprise an anti-mesothelin antibody or its antigen-binding fragment having VL and VH, wherein VL comprises LCDR1, CDR2, and CDR3, and VH comprises HCDR1, CDR2, and CDR3, and wherein LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 have the amino acid sequences of SEQ ID NO: 1, 2, 3, 4, 5, and 6, respectively.

[0185] In some embodiments, the CAR provided herein may comprise an anti-mesothelin antibody or an antigen-binding fragment thereof, wherein the anti-mesothelin antibody or the antigen-binding fragment thereof is an scFv named MH150.

[0186] In some embodiments, the transmembrane domain of the CAR provided herein comprises a hydrophobic α-helix spanning at least a portion of the membrane. Different transmembrane domains result in different receptor stabilizations. Upon antigen recognition, the receptor aggregates and the signal is transduced into the cell. In some embodiments, the transmembrane domain of the CAR provided herein may be derived from a protein or peptide naturally expressed in immune effector cells. A transmembrane domain derived from a protein or peptide means that the transmembrane domain comprises the entire transmembrane region of the protein or peptide or a fragment thereof. In some implementations, the CAR provided herein may have a transmembrane domain derived from CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, T cell receptor (TCR) α chain, TCR β chain or TCR ζ chain, CD3ε, CD45, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154 or other polypeptides expressed in immune effector cells. In some embodiments, the transmembrane domain of the CAR provided herein comprises transmembrane regions of CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, T cell receptor (TCR) α chain, TCR β chain or TCR ζ chain, CD3ε, CD45, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154 or other polypeptides expressed in immune effector cells.

[0187] In some embodiments, the transmembrane domain of the CAR provided herein is derived from CD8. In some embodiments, the transmembrane domain comprises a transmembrane region of CD8. In some embodiments, the transmembrane domain is derived from CD28. In some embodiments, the transmembrane domain comprises a transmembrane region of CD28. In some embodiments, the transmembrane domain is derived from CD3ζ. In some embodiments, the transmembrane domain comprises a transmembrane region of CD3ζ.

[0188] In some embodiments, the transmembrane domain may be synthetic, in which case it primarily comprises hydrophobic residues, such as leucine and valine. Optionally, the transmembrane domain may be derived from a polypeptide not naturally expressed in immune effector cells, provided that the transmembrane domain is capable of transducing signals generated by antigens bound to the CAR to intracellular signal transduction domains and / or co-stimulatory domains. In some embodiments, the ends of the transmembrane domain may comprise a phenylalanine-tryptophan-valine triplet. Optionally, a short oligopeptide or polypeptide linker, preferably 2 to 10 amino acids in length, may form the link between the CAR transmembrane domain and the cytoplasmic signal transduction domain. A glycine-serine duplex may provide a particularly suitable linker.

[0189] The cytoplasmic domains of the CARs described herein can be included within signal transduction domains that function in immune effector cells expressing CARs. These signal transduction domains can be derived, for example, from CD3ζ, Fc receptor γ, FcγRIIa, FcRβ (FcεR1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, or DAP12, or combinations thereof. Signal transduction domains derived from proteins or peptides refer to domains within those proteins or peptides responsible for activating immune effector cells (e.g., T cells), or fragments that retain their activating function. Generally, signal transduction domains induce persistence, migration, and / or effector functions in transduced immune effector cells (e.g., T cells) (Sharpe et al., Dis. Model Mech. 8:337-350 (2015); Finney et al., J. Immunol. 161:2791-2797 (1998); Krause et al., J. Exp. Med. 188:619-626 (1998)). The signal transduction domain of a protein or peptide can be its intracellular domain. In some embodiments, the signal transduction domain comprises intracellular domains of CD3ζ, FcRγ, FcγRIIa, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, DAP10, DAP12, or any combination thereof.

[0190] In some embodiments, the cytoplasmic domain of the CAR provided herein includes a signal transduction domain derived from CD3ζ. In some embodiments, the signal transduction domain includes an intracellular domain of CD3ζ.

[0191] In some embodiments, the cytoplasmic domain of the CAR provided herein further includes a co-stimulatory domain. In some embodiments, it further includes two co-stimulatory domains. Such co-stimulatory domains can enhance the activation of immune effector cells (e.g., T cells). The co-stimulatory signaling domain may be derived, for example, from CD28, 4-1BB (CD137), OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD2, CD7, LIGHT, TIGIT, GITR, TLR, DR3, or CD43. A co-stimulatory domain derived from a protein or peptide refers to a domain of that protein or peptide responsible for enhancing the activation of immune effector cells (e.g., T cells), or a fragment that retains its activation function. In some embodiments, the co-stimulatory domain of the CAR provided herein includes an intracellular domain of any of the above-mentioned molecules.

[0192] In some embodiments, the cytoplasmic domain of the CAR provided herein includes a co-stimulatory domain derived from CD28. In some embodiments, the co-stimulatory domain includes an intracellular domain of CD28. In some embodiments, the cytoplasmic domain includes a co-stimulatory domain derived from 4-1BB. In some embodiments, the co-stimulatory domain includes an intracellular domain of 4-1BB.

[0193] CARs comprising intracellular domains having costimulatory domains derived from 4-1BB, ICOS, or DAP-10 have been previously described (see U.S. Patent No. 7,446,190, which is incorporated herein by reference and also describes representative sequences of 4-1BB, ICOS, and DAP-10). In some embodiments, the cytoplasmic domain of the CAR may comprise two costimulatory domains derived from two costimulatory receptors, such as CD28 and 4-1BB (see Sadelain et al., Cancer Discov. 3(4):388-398 (2013)), CD28 and OX40, or other costimulatory ligand combinations disclosed herein.

[0194] The extracellular domain of a CAR can be fused with a leader peptide or signal peptide, which directs the nascent protein to the endoplasmic reticulum and subsequently translocates to the cell surface. It should be understood that once a polypeptide containing a signal peptide is expressed on the cell surface, the signal peptide is usually removed by proteolysis during the polypeptide's processing in the endoplasmic reticulum and translocation to the cell surface. Therefore, polypeptides such as CARs are typically expressed on the cell surface as mature proteins lacking a signal peptide, while the precursor form of the polypeptide includes the signal peptide. If the CAR requires glycosylation and / or anchoring to the cell membrane, the signal peptide or leader peptide may be necessary. The signal sequence or leader peptide is a peptide sequence typically present at the N-terminus of a newly synthesized protein and guides it into the secretory pathway. The signal peptide is covalently linked as a fusion protein to the N-terminus of the extracellular antigen-binding domain of the CAR. Any suitable signal peptide known in the art can be applied to CARs to enable their expression on the surface of immune cells (see Gierasch, Biochem. 28:923-930 (1989); von Heijne, J. Mol. Biol. 184(1):99-105 (1985)). Particularly useful signal peptides can be derived from cell surface proteins naturally expressed in immune cells as described herein, including any signal peptide of the polypeptides disclosed herein. Therefore, any suitable signal peptide can be used to direct CARs to expression on the surface of immune effector cells as described herein.

[0195] In some embodiments, the CAR may also include spacer regions or sequences that connect the domains of the CAR to each other. For example, spacer regions may be included between the signal peptide and the antigen-binding domain, between the antigen-binding domain and the transmembrane domain, between the transmembrane domain and the intracellular domain, and / or between domains within the intracellular domain (e.g., between stimulatory and costimulatory domains). The spacer regions may be flexible enough to allow the domains to interact with other peptides, for example, to make the antigen-binding domain oriented flexibly, thereby facilitating antigen recognition. The spacer regions may be, for example, the hinge region of IgG, a portion of the CH2CH3 (constant) region of an immunoglobulin and / or CD3, or other sequences suitable as spacer regions. In some embodiments, the CAR disclosed herein includes a hinge domain connecting the mesothelin-binding domain and the transmembrane domain. In some embodiments, the hinge domain includes a human CD8 hinge domain. In some embodiments, it includes a human CD28 hinge domain.

[0196] The following provides some exemplary molecules that can serve as the source of the CAR domains presented in this article.

[0197] CD3ζ contains three immune receptor tyrosine-based activation motifs (ITAMs) that transmit activation signals to cells (e.g., lymphoid lineage cells such as T cells) upon antigen binding. The CD3ζ polypeptide may have an amino acid sequence corresponding to the sequence of GenBank number NP_932170 (NP_932170.1, GI:37595565; see below), SEQ ID NO: 28, or a fragment thereof. In some embodiments, the CD3ζ signaling domain has the amino acid sequence of amino acids 52 to 164 of the CD3ζ polypeptide sequence provided below, or a fragment thereof sufficient to achieve signal transduction activity. For the domains within CD3ζ, see GenBank NP_932170, for example: signal peptide, amino acids 1 to 21; extracellular domain, amino acids 22 to 30; transmembrane region, amino acids 31 to 51; intracellular domain, amino acids 52 to 164. In some embodiments, the CAR may have a transmembrane domain derived from CD3ζ. The transmembrane domain may comprise a transmembrane region of CD3ζ (e.g., amino acids 31 to 51 of the sequence below) or a fragment thereof. In some embodiments, the cytoplasmic domain of the CAR may comprise a signal transduction domain derived from CD3ζ. In some embodiments, the signal transduction domain of CD3ζ may comprise an intracellular domain of CD3ζ (e.g., amino acids 52 to 164 of the sequence below) or a fragment thereof. It should be understood that, if desired, a shorter or longer CD3ζ sequence than a specifically defined domain may be included in the CAR.

[0198] 4-1BB. 4-1BB, also known as member 9 of the tumor necrosis factor receptor superfamily, acts as a ligand for tumor necrosis factor (TNF) and exhibits stimulatory activity. The 4-1BB polypeptide may have an amino acid sequence corresponding to the sequence in GenBank accession numbers P41273 (P41273.1, GI:728739) or NP_001552 (NP_001552.2, GI:5730095). In some embodiments, the CAR may comprise a transmembrane domain derived from 4-1BB.

[0199] CD8. Differentiation cluster 8 (CD8) is a transmembrane glycoprotein that acts as a co-receptor for the T-cell receptor (TCR). CD8 binds to the major histocompatibility complex (MHC) molecule and is specific for class I MHC proteins. In some embodiments, the CAR may comprise a transmembrane domain derived from CD8. The CD8 polypeptide may have an amino acid sequence corresponding to the sequence or a fragment thereof in GenBank accession number NP_001139345.1 (GI:225007536). For information on the domains within CD8, see GenBank accession NP_001139345.1, for example: signal peptide, amino acids 1 to 21; extracellular domain, amino acids 22 to 182; transmembrane domain, amino acids 183 to 203; intracellular domain, amino acids 204 to 235. In some embodiments, the CAR may comprise a hinge domain derived from CD8.

[0200] Therefore, for illustrative purposes, the CAR disclosed herein may include, from the N-terminus to the C-terminus, an anti-mesothelin antibody or antigen-binding fragment (e.g., the scFv disclosed herein), a hinge (e.g., the CD8 hinge), a transmembrane region (e.g., the CD8 transmembrane region), a co-stimulatory domain (e.g., the intracellular domain of 4-1BB), and a signal transduction domain (e.g., the T cell signal transduction domain of CD3ζ).

[0201] 5.4 LACOSTIM fusion protein

[0202] In some embodiments, the cells provided herein recombinantly express the fusion protein. In some embodiments, the cells are genetically modified to express the fusion protein. In some embodiments, the cells provided herein are genetically modified to express the fusion protein and the CAR provided herein.

[0203] In some embodiments, the fusion protein is a membrane protein. In some embodiments, the fusion protein is a soluble protein. In some embodiments, the fusion protein is a bispecific antibody.

[0204] In some embodiments, the fusion protein comprises a first domain for activating antigen-presenting cells (APCs) and a second domain for activating immune effector cells, wherein: (i) the first domain comprises (a) a ligand or receptor-binding fragment thereof of an activating receptor for APCs, or (b) an antibody or antigen-binding fragment thereof of an activating receptor for APCs; and (ii) the second domain comprises (a) a co-stimulatory receptor or a functional fragment thereof of an immune effector cell, (b) a co-stimulatory ligand or receptor-binding fragment thereof of an immune effector cell, or (c) an antibody or antigen-binding fragment thereof of a co-stimulatory receptor for an immune effector cell.

[0205] In some embodiments, the APC is selected from the group consisting of dendritic cells, macrophages, myeloid-derived suppressor cells, monocytes, B cells, T cells, and Langerhans cells. In some embodiments, the APC activation receptor is selected from the group consisting of CD40, CD80, CD86, CD91, DEC-205, and DC-SIGN. In some embodiments, the first domain comprises a ligand that binds to CD40, CD80, CD86, CD91, DEC-205, or DC-SIGN. In some embodiments, the first domain comprises an extracellular domain of the CD40 ligand (CD40L ECD). In some embodiments, the first domain comprises an antibody or an antigen-binding fragment thereof that binds to the APC activation receptor. In some embodiments, the first domain is an anti-CD40 antibody or an antigen-binding fragment thereof. In some embodiments, the first domain is a monoclonal antibody. In some embodiments, the first domain is a chimeric antibody, a humanized antibody, or a human antibody. In some embodiments, the first domain is Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, a single-chain antibody, a dual-variable-region antibody, a biantibody, a nanobody, or a single-variable-region antibody. In some embodiments, the anti-CD40 antibody or its antigen-binding fragment comprises a heavy-chain variable domain and / or a light-chain variable domain. In some embodiments, it is scFv.

[0206] In some embodiments, the second domain of the fusion protein provided herein is a co-stimulatory receptor of an immune effector cell or a functional fragment thereof. In some embodiments, the second domain of the fusion protein provided herein includes a cytoplasmic domain of the co-stimulatory receptor. In some embodiments, the co-stimulatory receptor is selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43. In some embodiments, the co-stimulatory receptor is CD28 or 4-1BB. In some embodiments, the co-stimulatory receptor is CD28. In some embodiments, the co-stimulatory receptor is 4-1BB. In some embodiments, the second domain further includes a transmembrane domain of the co-stimulatory receptor. In some embodiments, the second domain of the fusion protein provided herein includes a functional fragment of CD28, the functional fragment comprising a portion of an intracellular / cytoplasmic domain of CD28 capable of functioning as a co-stimulatory signal transduction domain. In some embodiments, the second domain includes a functional fragment of CD28. In some implementations, the second domain includes a CD28 extracellular domain, a CD28 transmembrane domain, and a CD28 intracellular domain.

[0207] In some embodiments, the second domain is a co-stimulatory ligand of an immune effector cell or a receptor-binding fragment thereof. In some embodiments, the co-stimulatory ligand is selected from the group consisting of CD58, mesothelin, CD83, CD80, CD86, CD137L, CD252, CD275, CD54, CD49a, CD112, CD150, CD155, CD265, CD270, TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153, CD48, CD160, CD200R, and CD44. In some embodiments, the second domain is an antibody that binds to a co-stimulatory receptor or an antigen-binding fragment thereof. In some embodiments, the co-stimulatory receptor is selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43. In some embodiments, the second domain is a monoclonal antibody. In some implementations, the second domain is a chimeric antibody, a humanized antibody, or a human antibody.

[0208] In some embodiments, the N-terminus of the first domain of the fusion protein is connected to the C-terminus of the second domain of the fusion protein. In some embodiments, the N-terminus of the second domain of the fusion protein is connected to the C-terminus of the first domain of the fusion protein. In some embodiments, the first and second domains of the fusion protein are connected via a linker. The linker can be a flexible linker or a rigid linker. In some embodiments, the linker has an amino acid sequence of (GGGGS)n, where n = 1, 2, 3, 4, or 5 (SEQ ID NO: 21). In some embodiments, the linker has an amino acid sequence of (EAAAK)n, where n = 1, 2, 3, 4, or 5 (SEQ ID NO: 22). In some embodiments, the linker has an amino acid sequence of (PA)nP, where n = 1, 2, 3, 4, or 5 (SEQ ID NO: 23). In some embodiments, the linker has an amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 24). In some embodiments, the linker is a CD8 hinge (SEQ ID NO: 13). In some embodiments, the connector is a CD28 hinge (SEQ ID NO: 25). In some embodiments, the connector is an IgG1 hinge (SEQ ID NO: 26). In some embodiments, the connector may be a trimerizing motif selected from the group consisting of T4 fibritin trimerizing motif (SEQ ID NO: 27), isoleucine zipper (SEQ ID NO: 28 or 29), GCN4II motif (SEQ ID NO: 30 or 31), Matrilin-1 motif (SEQ ID NO: 32 or 33), and XV type collagen trimerizing motif (SEQ ID NO: 34).

[0209] In some embodiments, the first domain of the fusion protein comprises CD40L ECD, and the second domain comprises a CD28 cytoplasmic domain. In some embodiments, the first domain comprises CD40L. In some embodiments, the first domain comprises CD40L ECD, and the second domain comprises an anti-CD28 antibody or an antigen-binding fragment thereof. In some embodiments, the first domain comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and the second domain comprises an anti-CD28 antibody or an antigen-binding fragment thereof.

[0210] In some embodiments, the fusion protein further includes a signal peptide domain. In some embodiments, the C-terminus of the signal peptide is linked to the N-terminus of the first domain. In some embodiments, the signal peptide comprises a signal peptide derived from CD8. In some embodiments, the signal peptide comprises a signal peptide derived from CD40L.

[0211] In some embodiments, the cellular recombinant expression provided herein comprises a fusion protein containing a first domain for activating antigen-presenting cells (APCs) and a second domain for activating immune effector cells, wherein: (i) the first domain comprises an anti-CD40 antibody or an antigen-binding fragment thereof; and (ii) the second domain comprises a CD28 transmembrane domain and a CD28 cytoplasmic domain. In some embodiments, the cells provided herein express the CAR provided herein and express the fusion protein. Expression of the fusion protein disclosed herein can not only promote the proliferation and activation of immune effector cells (e.g., T cells), but also stimulate the maturation and epitope expansion activity of antigen-presenting cells. In some embodiments, expression of the fusion protein in the genetically engineered T cells disclosed herein helps them overcome immunosuppression in the tumor microenvironment mediated by, for example, PD1 / PD-L1 signaling, regulatory T cells (Tregs), and TGF-β signaling, and enhances their antitumor activity. The fusion protein disclosed herein is also referred to as a lymphocyte-antigen-presenting cell costimulator (“LACOSTIM” or “LACO”). For example, the fusion proteins described herein include the lymphocyte-antigen-presenting cell costimulator described in U.S. Patent No. 11,667,723, the disclosure of which is incorporated herein by reference.

[0212] CD40: CD40 is a 48 kD transmembrane glycoprotein surface receptor and a member of the tumor necrosis factor receptor superfamily (TNFRSF). An exemplary amino acid sequence of human CD40 has been described (see, for example, accession number ALQ33424.1, GenBank NP_001241.1, GI:957949089), SEQ ID NO: 35 being the amino acid sequence of human CD40. CD40 was initially characterized as a co-stimulatory receptor expressed on APCs, playing a central role in B cell and T cell activation. The ligand of CD40, CD154 (also known as TRAP, T-BAM, CD40 ligand, or CD40L), is a type II integrated membrane protein. For information on the domains within CD40, see GenBank NP_001241.1, for example: signal peptide, amino acids 1 to 20; extracellular domain, positions 21 to 193; transmembrane domain, positions 194 to 215; intracellular domain, positions 216 to 277.

[0213] CD28: Differentiation cluster 28 (CD28) is a protein expressed on T cells and provides a co-stimulatory signal for T cell activation and survival. CD28 is a receptor for the CD80 (B7.1) and CD86 (B7.2) proteins. The CD28 polypeptide may have an amino acid sequence corresponding to the sequence of GenBank number P10747 (P10747.1, GI:115973) or NP_006130 (NP_006130.1, GI:5453611), the amino acid sequence of SEQ ID NO: 37, or a fragment thereof. For information on the domains within CD28, see GenBank NP_006130, e.g.: signal peptide, amino acids 1-18; extracellular domain, positions 19-152; transmembrane domain, positions 153-179; intracellular domain, positions 180-220.

[0214] In some embodiments, the first domain comprises an anti-CD40 antibody or an antigen-binding fragment thereof. In some embodiments, the first domain comprises an anti-CD40 scFv. In some embodiments, the first domain comprises an anti-CD40 scFv having the amino acid sequence of SEQ ID NOs: 38-48 listed in Table 4.

[0215] Table 4: CD40 scFv

[0216]

[0217] In some embodiments, the second domain is an antibody or antigen-binding fragment that binds to CD28. In some embodiments, the second domain comprises a CD28 cytoplasmic domain. In some embodiments, the second domain comprises a CD28 cytoplasmic domain having the amino acid sequence of SEQ ID NO: 36. In some embodiments, the second domain comprises a CD28 scFv. In some embodiments, the second domain comprises a CD28 scFv having VH (SEQ ID NO: 60) and / or VL (SEQ ID NO: 61). In some embodiments, the second domain comprises a CD28 scFv having the amino acid sequence of SEQ ID NO: 62.

[0218] Table 5: CD28 scFv

[0219]

[0220] In some embodiments, the fusion protein has at least 85%, 90%, 95%, 98%, 99%, or 100% identity with the amino acid sequence selected from the group consisting of SEQ ID NOs: 49-59 and 63-73. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 68.

[0221] 5.5 Polynucleotides and Carriers

[0222] This document also provides polynucleotides encoding the polypeptides described herein (e.g., anti-mesothelin antibodies or antigen-binding fragments, CARs that specifically bind mesothelin, or fusion proteins). The term "polynucleotide encoding a polypeptide" encompasses polynucleotides that include only the coding sequence of the polypeptide, as well as polynucleotides that include additional coding and / or non-coding sequences. The polynucleotides disclosed herein may be in RNA or DNA form. The DNA may be cDNA, genomic DNA, or synthetic DNA, and may be double-stranded or single-stranded. Single-stranded DNA may be a coding strand or a non-coding (antisense) strand. The polynucleotides disclosed herein may be mRNA.

[0223] This document also provides vectors and cells containing the polynucleotides described herein. In some embodiments, vectors containing the polynucleotides described herein are provided. The vector may be an expression vector. In some embodiments, the vectors provided herein contain a polynucleotide encoding the anti-mesothelin antibody described herein or an antigen-binding fragment thereof. In some embodiments, the vectors provided herein contain a polynucleotide encoding a polypeptide as part of the anti-mesothelin antibody described herein or an antigen-binding fragment thereof. In some embodiments, the vectors provided herein contain a polynucleotide encoding the CAR or TCR described herein. In some embodiments, the vectors provided herein contain a polynucleotide encoding a polypeptide as part of the CAR or TCR described herein. Various expression host / vector combinations can be employed. Available expression vectors for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papillomavirus, adenovirus, lentivirus, and cytomegalovirus. Available expression vectors for bacterial hosts include known bacterial plasmids, such as pCR1, pBR322, pMB9, and their derivatives from *Escherichia coli*, as well as plasmids with a wider host range, such as M13 and other filamentous single-stranded DNA bacteriophages.

[0224] This disclosure also provides host cells comprising the polypeptides described herein, polynucleotides encoding the polypeptides described herein, or vectors comprising such polynucleotides. In some embodiments, host cells comprising vectors containing the polynucleotides disclosed herein are provided. In some embodiments, the host cells provided herein comprise vectors containing polynucleotides encoding the anti-mesothelin antibody described herein or an antigen-binding fragment thereof. In some embodiments, the host cells provided herein comprise vectors containing polynucleotides encoding a polypeptide as part of the anti-mesothelin antibody described herein or an antigen-binding fragment thereof. In some embodiments, the host cells provided herein comprise polynucleotides encoding the anti-mesothelin antibody described herein or an antigen-binding fragment thereof. In some embodiments, the cells produce the anti-mesothelin antibody described herein or an antigen-binding fragment thereof. In some embodiments, the host cells provided herein comprise vectors containing polynucleotides encoding the CAR or TCR described herein. In some embodiments, the host cells provided herein comprise vectors containing polynucleotide molecules encoding a polypeptide as part of the CAR or TCR described herein. In some embodiments, the host cells provided herein comprise polynucleotides encoding the CAR or TCR described herein. In some embodiments, the host cells produce the mesothelin CAR or TCR described herein.

[0225] 5.6 cells

[0226] This document provides cells comprising the polynucleotides disclosed herein. In some embodiments, this document provides cells comprising polynucleotides encoding polypeptides disclosed herein. In some embodiments, this document provides cells comprising a vector having the polynucleotides disclosed herein. In some embodiments, this document provides cells recombinantly expressing the polypeptides disclosed herein. The polypeptide may be an anti-mesothelin antibody or an antigen-binding fragment, may be a mesothelin CAR, or may be a mesothelin TCR. In some embodiments, the cells provided herein may also express the fusion protein disclosed herein or comprise polynucleotides encoding the fusion protein disclosed herein. The fusion protein may be any LACOSTIM molecule disclosed herein.

[0227] In some embodiments, the cells provided herein express the CAR and the fusion protein disclosed herein. In some embodiments, the cells comprise a first polynucleotide encoding the fusion protein provided herein and a second polynucleotide encoding the CAR. In some embodiments, the cells express the TCR and the fusion protein disclosed herein. In some embodiments, the cells comprise a first polynucleotide encoding the fusion protein provided herein and a second polynucleotide encoding the TCR.

[0228] In some embodiments, the genetically engineered immune effector cells provided herein comprise a polynucleotide comprising a first fragment encoding a CAR and a second fragment encoding a fusion protein. In some embodiments, the first fragment encodes a TCR, and the second fragment encodes a fusion protein. In some embodiments, the polynucleotide has a first fragment and a second fragment from its N-terminus to its C-terminus. In some embodiments, it has a second fragment and a first fragment from its N-terminus to its C-terminus. In some embodiments, the cells provided herein comprise a polynucleotide having a first fragment encoding a CAR or TCR provided herein and a second fragment encoding a fusion protein disclosed herein. The first and second fragments can be linked by a nucleotide sequence encoding a linker. The linker can be a self-cleaving linker. In some embodiments, the first and second fragments are linked by a nucleotide sequence encoding a 2A peptide. In some embodiments, the 2A linker is a T2A peptide (e.g., SEQ ID NO: 99). In some embodiments, the linker is an F2A peptide (e.g., SEQ ID NO: 98). In some embodiments, the first fragment (encoding a CAR / TCR) is located at the 5' end of the second fragment (encoding a fusion protein). In some embodiments, the first fragment is located at the 3' end of the second fragment.

[0229] In some embodiments, cells co-expressing the first and second fragments have the amino acid or nucleoside sequences of SEQ ID NOs: 74-97. In some embodiments, the first and second fragments have the amino acid sequences listed in Table 6.

[0230] Table 6: CAR and LACOSTIM sequences in CAR-T cells co-expressing LACOSTIM

[0231]

[0232] In some embodiments, the cells provided herein are immune effector cells. In some embodiments, the immune effector cells are selected from the group consisting of T cells, B cells, natural killer (NK) cells, NKT cells, macrophages, granulocytes, neutrophils, eosinophils, mast cells, and basophils. In some embodiments, the immune effector cells provided herein are selected from the group consisting of T cells, NK cells, NKT cells, macrophages, neutrophils, and granulocytes. In some embodiments, the immune effector cells provided herein are T cells. In some embodiments, the immune effector cells provided herein are NK cells. In some embodiments, the immune effector cells provided herein are NKT cells. In some embodiments, the immune effector cells provided herein are macrophages. In some embodiments, the immune effector cells provided herein are neutrophils. In some embodiments, the immune effector cells provided herein are granulocytes.

[0233] In some embodiments, the immune effector cells provided herein may be genetically engineered. In some embodiments, the genetically engineered immune effector cells provided herein are isolated. In some embodiments, the genetically engineered immune effector cells provided herein are substantially pure.

[0234] Therefore, in some embodiments, this document provides immune effector cells recombinantly expressing the disclosed polypeptides (e.g., antibodies or CARs). This document also provides immune effector cells (e.g., T cells) comprising a polynucleotide encoding a disclosed polypeptide (e.g., antibody or CAR) or a vector having the disclosed polynucleotide. In some embodiments, this document provides immune effector cells (e.g., T cells) comprising a polynucleotide encoding an anti-mesothelin antibody or an antigen-binding fragment thereof disclosed herein. In some embodiments, this document provides immune effector cells (e.g., T cells) recombinantly expressing an anti-mesothelin antibody or an antigen-binding fragment thereof disclosed herein. In some embodiments, this document provides immune effector cells comprising a polynucleotide encoding a mesothelin CAR disclosed herein. In some embodiments, this document provides immune effector cells (e.g., T cells) recombinantly expressing a mesothelin CAR disclosed herein (e.g., mesothelin CAR-T cells). In some embodiments, this document provides immune effector cells comprising a polynucleotide encoding a mesothelin TCR disclosed herein. In some embodiments, this document provides immune effector cells (e.g., T cells) recombinantly expressing a mesothelin TCR disclosed herein (e.g., mesothelin TCR-T cells).

[0235] In some embodiments, the immune effector cells provided herein are T cells. The T cells may be cytotoxic T cells, helper T cells, γδ T cells, CD4+ / CD8+ double-positive T cells, CD4+ T cells, CD8+ T cells, CD4 / CD8 double-negative T cells, CD3+ T cells, naive T cells, effector T cells, memory T cells, regulatory T cells, Th0 cells, Th1 cells, Th2 cells, Th3 (Treg) cells, Th9 cells, Th17 cells, Thαβ helper cells, Tfh cells, stem cell-like memory TSCM cells, central memory TCM cells, effector memory TEM cells, effector memory TEMRA cells, or γδ T cells. In some embodiments, the T cells are cytotoxic T cells. In some embodiments, the T cells are genetically engineered. In some embodiments, the T cells provided herein are isolated. In some embodiments, the T cells provided herein are substantially pure.

[0236] In some implementations, the genetically engineered cells described herein are derived from cells isolated from a subject. As used herein, "derived from" genetically engineered cells refer to genetically engineered cells obtained by acquiring source cells and genetically manipulating them. Source cells can be from natural sources. For example, source cells can be primary cells isolated from a subject. The subject can be an animal or a human. Source cells can also be cells that have undergone in vitro passage or genetic manipulation.

[0237] In some embodiments, the genetically engineered cells provided herein are derived from cells isolated from humans. Immune effector cells (e.g., T cells) can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, T cell lines available in the art may be used. In some embodiments, the genetically engineered cells provided herein are derived from cells isolated from peripheral blood. In some embodiments, the genetically engineered cells provided herein are derived from cells isolated from bone marrow. In some embodiments, the genetically engineered cells provided herein are derived from cells isolated from peripheral blood mononuclear cells (PBMCs).

[0238] In some embodiments, the genetically engineered cells provided herein are derived from cells differentiated in vitro from stem cells or progenitor cells. In some embodiments, the stem cells or progenitor cells are selected from the group consisting of T cell progenitor cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotent progenitor cells, embryonic stem cells, and induced pluripotent cells. In some embodiments, the genetically engineered cells provided herein are derived from cells differentiated in vitro from T cell progenitor cells. In some embodiments, the genetically engineered cells provided herein are derived from cells differentiated in vitro from hematopoietic stem cells and progenitor cells. In some embodiments, the genetically engineered cells provided herein are derived from cells differentiated in vitro from hematopoietic pluripotent progenitor cells. In some embodiments, the genetically engineered cells provided herein are derived from cells differentiated in vitro from embryonic stem cells. In some embodiments, the genetically engineered cells provided herein are derived from cells differentiated in vitro from induced pluripotent cells.

[0239] In some embodiments, this document provides cell populations comprising the cells disclosed herein. The cells disclosed herein may comprise polynucleotides encoding the polypeptides disclosed herein, or recombinantly express the polypeptides disclosed herein. The polypeptides may be anti-mesothelin antibodies or antigen-binding fragments thereof, mesothelin CARs, or mesothelin TCRs. In some embodiments, the cells disclosed herein may also express the fusion proteins disclosed herein, or comprise polynucleotides encoding the fusion proteins disclosed herein. The fusion proteins may be any fusion proteins disclosed herein. In some embodiments, the cell populations provided herein express the CARs disclosed herein and the fusion proteins disclosed herein. In some embodiments, the cell populations provided herein comprise a first polynucleotide encoding the fusion protein disclosed herein and a second polynucleotide encoding the CAR. In some embodiments, the genetically engineered immune effector cells provided herein express the TCRs disclosed herein and the fusion proteins disclosed herein. In some embodiments, the cell populations provided herein comprise a first polynucleotide encoding the fusion protein disclosed herein and a second polynucleotide encoding the TCR. In some embodiments, the cell populations provided herein comprise a polynucleotide that comprises a first fragment encoding a CAR and a second fragment encoding a fusion protein. In some embodiments, the cell populations provided herein comprise a polynucleotide comprising a first segment encoding a TCR and a second segment encoding a fusion protein. In some embodiments, the polynucleotide has a first segment and a second segment from the N-terminus to the C-terminus. In some embodiments, the polynucleotide has a second segment and a first segment from the N-terminus to the C-terminus.

[0240] The cell population can be a homogeneous or heterogeneous population. In some embodiments, the cell population can be a heterogeneous population comprising any combination of the cells disclosed herein. In some embodiments, the cell population is derived from peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating lymphocytes (TILs), cytokine-induced killer cells (CIKs), lymphokine-activated killer cells (LAKs), or bone marrow-infiltrating lymphocytes (MILs). In some embodiments, the cell population provided herein is derived from PBMCs. In some embodiments, the cell population provided herein is derived from PBLs. In some embodiments, the cell population provided herein is derived from TILs. In some embodiments, the cell population provided herein is derived from CIKs. In some embodiments, the cell population provided herein is derived from LAKs. In some embodiments, the cell population provided herein is derived from MILs. The cell population can be genetically engineered to recombinantly express the polypeptides disclosed herein (e.g., antibodies or CARs). In some embodiments, the cell population provided herein contains a polynucleotide encoding a polypeptide disclosed herein (e.g., an antibody or CAR), or a vector containing the polynucleotides disclosed herein. In some embodiments, the cell populations provided herein contain polynucleotides encoding the disclosed anti-mesothelin antibody or antigen-binding fragment. In some embodiments, the cell populations provided herein recombinantly express the disclosed anti-mesothelin antibody or antigen-binding fragment. In some embodiments, the cell populations provided herein contain polynucleotides encoding the disclosed mesothelin CAR. In some embodiments, the cell populations provided herein recombinantly express the disclosed mesothelin CAR (e.g., mesothelin CAR-T cells). In some embodiments, the cell populations provided herein contain polynucleotides encoding the disclosed mesothelin TCR. In some embodiments, the cell populations provided herein recombinantly express the disclosed mesothelin TCR (e.g., mesothelin TCR-T cells).

[0241] 5.7 Pharmaceutical Compositions

[0242] This document also provides pharmaceutical compositions comprising the anti-mesothelin antibody or antigen-binding fragment disclosed herein and / or cells (e.g., CAR-T cells). In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the disclosed anti-mesothelin antibody or antigen-binding fragment and a pharmaceutically acceptable carrier. The antibody or fragment may be present at various concentrations. In some embodiments, the pharmaceutical compositions provided herein comprise a soluble anti-mesothelin antibody or antigen-binding fragment at a concentration of 1-1000 mg / ml. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the disclosed cells and a pharmaceutically acceptable carrier. Pharmaceutical compositions comprising the disclosed cells (e.g., CAR-T cells) may comprise a purified cell population. Those skilled in the art can readily determine the percentage of cells in the cell population using the various well-known methods described herein. The purity of a cell population comprising the genetically engineered cells provided herein can range from about 20% to about 100%.

[0243] In some embodiments, the pharmaceutical composition can be used in immunotherapy. In some embodiments, it can be used in immuno-oncology. In some embodiments, it can be used to inhibit tumor growth in a subject (e.g., a human patient). In some embodiments, it can be used to treat cancer in a subject (e.g., a human patient).

[0244] This document also provides kits for preparing pharmaceutical compositions containing the disclosed anti-mesothelin antibody or antigen-binding fragment. In some embodiments, the kit contains the disclosed anti-mesothelin antibody or antigen-binding fragment and a pharmaceutically acceptable carrier in one or more containers. In another embodiment, the kit may contain the disclosed anti-mesothelin antibody or antigen-binding fragment for administration to a subject. In a specific embodiment, the kit includes instructions for the preparation and / or administration of the antibody or antigen-binding fragment.

[0245] This document also provides kits for preparing the cells disclosed herein (e.g., T cells). In some embodiments, the kit comprises one or more vectors for generating genetically engineered cells (e.g., T cells) expressing the disclosed anti-mesothelin antibody or antigen-binding fragment and / or anti-mesothelin CAR. The kit can be used to generate genetically engineered immune effector cells (e.g., T cells) intended for administration to a compatible subject from autologous or non-autologous cells. In another embodiment, the kit may comprise the disclosed immune effector cells for administration to a subject. In a specific embodiment, the kit contains the immune effector cells in one or more containers and includes instructions for their preparation and / or administration.

[0246] In some embodiments, this document provides pharmaceutical compositions comprising the anti-mesothelin antibody, antigen-binding fragment, or cells provided herein, wherein said compositions are suitable for local application. In some embodiments, local application includes intratumoral injection, peritumoral injection, tumor-adjacent injection, lesion-specific injection, and / or injection into tumor-draining lymph nodes, or substantially any tumor-targeting injection, wherein the antitumor agent is expected to leak into primary lymph nodes near the targeted solid tumor.

[0247] Pharmaceutically acceptable carriers that can be used in the compositions provided herein include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and delayed absorption agents. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, intraspinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient (i.e., the anti-mesothelin antibody or antigen-binding fragment or immune effector cell provided herein) may be coated with a material to protect the active ingredient from acids and other natural conditions that could inactivate it.

[0248] This document also provides pharmaceutical compositions or formulations that improve the stability of anti-mesothelin antibodies or antigen-binding fragments to allow for long-term storage. In some embodiments, the pharmaceutical composition or formulation is stable for at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 5 years, or longer. In some embodiments, the pharmaceutical composition or formulation is stable when stored at 4°C, 25°C, or 40°C.

[0249] The pharmaceutical compositions disclosed herein may also include one or more of the following: buffer systems, preservatives, tension modifiers, chelating agents, stabilizers, antioxidants, wetting agents, emulsifiers, dispersants, and / or surfactants, as well as various combinations thereof. Those skilled in the art are well aware of the use of preservatives, isotonic agents, chelating agents, stabilizers, and surfactants in pharmaceutical compositions. See Remington, Pharmaceutical Science and Practice, 19th edition, 1995.

[0250] In some embodiments, the pharmaceutical composition is an aqueous formulation. Such formulations are typically solutions or suspensions, but may also include colloids, dispersions, emulsions, and multiphase materials. In some embodiments, the pharmaceutical compositions disclosed herein are lyophilized, and a solvent and / or diluent is added thereto prior to use by a physician or patient.

[0251] In the pharmaceutical compositions or formulations disclosed herein, the amount of active ingredient that can be combined with a carrier material can vary. In some embodiments, the amount of active ingredient that can be combined with a carrier material is the amount that produces a therapeutic effect. Generally, in percentage terms, this amount will be about 0.01% to about 99%, about 0.1% to about 70%, or about 1% to about 30% of the active ingredient combined with a pharmaceutically acceptable carrier.

[0252] The pharmaceutical compositions disclosed herein can be prepared using carriers that protect the active ingredient from rapid release, such as controlled-release formulations, including implants, transdermal patches, and microencapsulated delivery systems.

[0253] In some embodiments, the anti-mesothelin antibodies or antigen-binding fragments or immune effector cells (e.g., T cells) described herein can be formulated to ensure their proper distribution in vivo. For example, the blood-brain barrier (BBB) ​​repels many highly hydrophilic compounds. To ensure that the active ingredients described herein cross the BBB (if needed, e.g., for brain cancer), they can be formulated, for example, in liposomes.

[0254] 5.8 Methods and Applications

[0255] This disclosure also provides methods of using the disclosed anti-mesothelin antibodies or antigen-binding fragments, mesothelin CARs, mesothelin TCRs, polynucleotides encoding such anti-mesothelin antibodies or antigen-binding fragments and mesothelin CAR / TCRs, vectors containing such polynucleotides, cells expressing mesothelin CAR / TCRs, or pharmaceutical compositions containing such cells in the treatment of cancer. Without being bound by theory, the disclosed anti-mesothelin antibodies or antigen-binding fragments and cells expressing mesothelin CAR / TCRs can specifically target mesothelin-expressing cancer cells in vivo, thereby exerting a therapeutic effect of eliminating, lysing, and / or killing cancer cells. In some embodiments, the method includes administering a therapeutically effective amount of the disclosed anti-mesothelin antibody or antigen-binding fragment to a subject in need. In some embodiments, the method includes administering a therapeutically effective amount of the disclosed mesothelin CAR-T cells to a subject in need. In one embodiment, the method may include administering a therapeutically effective amount of the disclosed mesothelin CAR-T cells to a subject in need.

[0256] In some embodiments, this document provides a method of treating a tumor or cancer in a subject in need, comprising administering to the subject a therapeutically effective amount of the disclosed anti-mesothelin antibody or antigen-binding fragment. In some embodiments, this document provides the use of the said anti-mesothelin antibody or antigen-binding fragment in the treatment of tumors or cancer. In some embodiments, this document provides the use of the said anti-mesothelin antibody or antigen-binding fragment in the preparation of a medicament for the treatment of tumors or cancer.

[0257] In some embodiments, this document provides a method of treating a tumor or cancer in a subject in need, comprising administering to the subject a therapeutically effective amount of the disclosed immune effector cells (e.g., mesothelin CAR-T). In some embodiments, this document provides the use of said immune effector cells (e.g., mesothelin CAR-T) in the treatment of tumors or cancer. In some embodiments, this document provides the use of said immune effector cells in the preparation of medicaments for the treatment of tumors or cancer. In some embodiments, a cell population comprising the disclosed immune effector cells is used in the treatment. The cell population may be homogeneous or heterogeneous.

[0258] In some embodiments, this document provides a method of treating a tumor or cancer in a subject in need, comprising administering to the subject a therapeutically effective amount of the disclosed pharmaceutical composition. In some embodiments, this document provides the use of the pharmaceutical composition in treating a tumor or cancer. In some embodiments, this document provides the use of the pharmaceutical composition in the preparation of a medicament for treating a tumor or cancer.

[0259] The actual dose level of the active ingredient (i.e., the anti-mesothelin antibody, antigen-binding fragment, or immune effector cells provided herein) in the pharmaceutical compositions described herein can be altered to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response for a specific patient, composition, and route of administration, but is non-toxic to the patient. The selected dose level will depend on a variety of pharmacokinetic factors, including the activity of the specific composition described herein, route of administration, time of administration, excretion rate, duration of treatment, other drugs, compounds, and / or materials used in combination with the specific composition used, the age, sex, weight, condition, general health status, and medical history of the patient being treated, as well as similar factors known in the medical field.

[0260] Anti-mesothelin antibodies or antigen-binding fragments can be administered in sustained-release formulations, in which case a lower frequency of administration is required. Dosage and frequency vary depending on the half-life of the antibody or fragment in the patient. In therapeutic applications, sometimes relatively high doses are required at relatively short intervals until disease progression slows or ceases, and the patient's symptoms are partially or completely improved.

[0261] In some embodiments, the immune effector cells provided herein that recombinantly express the mesothelin CAR or TCR disclosed herein can be used in the treatment methods disclosed herein. When cell therapy is used, the cells provided herein may be administered at a rate of cells per kilogram of the subject's body weight (cells / kg). Depending on the route and site of administration, the cell dose may range from about 10^4 to about 10^10 cells / kg body weight, for example, about 10^5 to about 10^9, about 10^5 to about 10^8, about 10^5 to about 10^7, or about 10^5 to 10^6 cells / kg body weight. Generally, systemic administration uses a higher dose than regional administration, in which immune effector cells are administered to the tumor area. The precise determination of the effective dose may be based on individual factors of each subject, including, as described above, the specific subject's body type, age, sex, weight, and condition. Those skilled in the art can readily determine the dose based on the disclosure herein and their knowledge in the art.

[0262] The anti-mesothelin antibody or its antigen-binding fragment, immune effector cells, and pharmaceutical compositions provided herein may be administered to subjects by any method known in the art, including but not limited to pleural administration, intravenous administration, subcutaneous administration, intralymphatic administration, intratumoral administration, intramuscular administration, intradermal administration, intrathecal administration, intrapleural administration, intraperitoneal administration, intracranial administration, intraspinal administration, or other parenteral administration routes, such as by injection or infusion, or direct administration to the thymus. The phrase "parenteral administration" as used herein refers to administration methods other than enteral and local administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intracapsular, intrasacral, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. In some embodiments, subcutaneous administration is used. In some embodiments, intravenous administration is used. In some embodiments, oral administration is used. In one embodiment, the cells provided herein can be regionally delivered to a tumor using known methods, including but not limited to via hepatic or aortic pumps; limb, lung, or liver perfusion; portal vein; venous shunt; intracavitary or peritumoral veins, etc. In another embodiment, the cells provided herein can be administered systemically. In a preferred embodiment, the cells are administered regionally at the tumor site. The cells can also be administered intratumorally, for example by direct injection of the cells at the tumor site and / or tumor vascular system. For example, for malignant pleural diseases, mesothelioma, or lung cancer, intrapleural administration is preferred (see Adusumilli et al., Science Translational Medicine 6(261):261ra151 (2014)). Those skilled in the art can select an appropriate administration method based on the type of cancer to be treated and / or the location of the tumor. The cells can be introduced by injection or catheter. In one embodiment, the cells are administered to the pleura of a subject in need, for example using an intrapleural catheter. Optionally, an amplifying agent and / or a differentiating agent can be administered to the subject before, during, or after cell administration to increase the in vivo production of the cells provided herein.

[0263] The cells described in this article are typically proliferated in vitro prior to administration to subjects and may be expected to proliferate in vivo after administration to subjects (see Kaiser et al., Cancer Gene Therapy 22:72-78 (2015)). Cell proliferation should be accompanied by cell survival to allow for cell proliferation and persistence, such as T cells.

[0264] Diseases that can be treated with the anti-mesothelin antibodies or antigen-binding fragments, immune effector cells, or pharmaceutical compositions provided herein include any disease or condition related to mesothelin, as well as any disease or condition that specifically expresses mesothelin and / or has mesothelin as a therapeutic target (collectively, "mesothelin-related diseases or conditions"). For example, mesothelioma is a mesothelin-related disease.

[0265] Malignant mesothelioma is a type of cancer that occurs in the thin layer of cells (called mesothelium) that line the internal organs of the human body. There are three recognized types of mesothelioma. Pleural mesothelioma (e.g., malignant pleural mesothelioma or MPM) is the most common form of the disease, accounting for approximately 70% of cases, and occurs in the lining of the lungs, called the pleura. Peritoneal mesothelioma occurs in the lining of the abdominal cavity, called the peritoneum. Pericardial mesothelioma originates from the pericardium that covers the heart. In some embodiments, the anti-mesothelin antibodies or antigen-binding fragments thereof, cellular or pharmaceutical compositions disclosed herein can be used to treat mesothelioma. In some embodiments, the mesothelioma is pleural mesothelioma. In some embodiments, the mesothelioma is peritoneal mesothelioma. In some embodiments, the mesothelioma is pericardial mesothelioma.

[0266] Symptoms of pleural mesothelioma include, for example, lower back or lateral chest pain and shortness of breath. Other symptoms include difficulty swallowing, persistent cough, fever, weight loss, or fatigue. Some patients may also experience muscle weakness, loss of sensation, hemoptysis, swelling of the face and arms, and hoarseness. In the early stages of the disease (e.g., stage I mesothelioma), symptoms may be milder. Patients typically report persistent pain in a specific area of ​​the chest, weight loss, and fever.

[0267] Peritoneal mesothelioma originates in the abdomen, so symptoms typically include abdominal pain, weight loss, nausea, and vomiting. The cancer can also cause fluid accumulation in the abdominal cavity. Peritoneal mesothelioma originates in the abdomen and often spreads to other organs in that area, including the liver, spleen, or intestines. Severe abdominal pain is the most common initial complaint. Fluid accumulation in the abdominal cavity can also cause discomfort. Other symptoms of peritoneal mesothelioma may include difficulty defecating, nausea and vomiting, fever, and swelling of the feet.

[0268] Pericardial mesothelioma is the least common form of mesothelioma. It affects the heart. This rare mesothelioma cancer invades the pericardium. As the cancer progresses, the heart is unable to pump oxygen to the body as efficiently, causing the condition to deteriorate at an increasingly rapid rate. The most common symptoms associated with pericardial mesothelioma resemble those of a heart attack: nausea, chest pain, and shortness of breath.

[0269] In some embodiments, the methods provided herein can treat subjects with mesothelioma. In some embodiments, the methods can treat subjects at risk of developing mesothelioma. Subjects may be at risk of developing mesothelioma if they have been exposed to asbestos. Exposure to asbestos and inhalation of asbestos particles can cause mesothelioma. In most cases, subjects exposed to asbestos do not develop symptoms of mesothelioma until many years after exposure. In some embodiments, the methods provided herein can treat subjects suspected of having mesothelioma, for example, based on the presence of one or more of the symptoms described herein and / or a history of asbestos exposure. In some embodiments, subjects treatable with the methods disclosed herein may have precancerous conditions such as pleural plaques, benign mesothelioma, or mesothelial hyperplasia.

[0270] Another example of a disease or condition associated with mesothelin is pancreatic cancer. In some embodiments, the anti-mesothelin antibodies or antigen-binding fragments, cell or pharmaceutical compositions provided herein can be used to treat pancreatic cancer. Pancreatic cancers treatable by the methods described herein include, but are not limited to, pancreatic exocrine carcinomas and pancreatic endocrine carcinomas. Pancreatic exocrine carcinomas include, but are not limited to, adenocarcinoma, acinar cell carcinoma, adenosquamous carcinoma, colloid carcinoma, undifferentiated carcinoma with osteoclast-like giant cells, hepatoid carcinoma, intraductal papillary mucinous tumor, mucinous cystic tumor, pancreatoblastoma, serous cystadenoma, signet ring cell carcinoma, solid pseudopapillary tumor, pancreatic ductal carcinoma, and undifferentiated carcinoma. In some embodiments, pancreatic exocrine carcinoma is pancreatic ductal carcinoma. Pancreatic endocrine carcinomas include, but are not limited to, insulinomas and glucagonomas.

[0271] In some implementations, the pancreatic cancer is any one of early-stage pancreatic cancer, non-metastatic pancreatic cancer, primary pancreatic cancer, resected pancreatic cancer, advanced pancreatic cancer, locally advanced pancreatic cancer, metastatic pancreatic cancer, unresectable pancreatic cancer, pancreatic cancer in remission, recurrent pancreatic cancer, pancreatic cancer on adjuvant therapy, or pancreatic cancer on neoadjuvant therapy. In some implementations, the pancreatic cancer is locally advanced pancreatic cancer, unresectable pancreatic cancer, or metastatic pancreatic ductal carcinoma. In some implementations, the pancreatic cancer is resistant to gemcitabine-based therapy. In some implementations, the pancreatic cancer is unresponsive to gemcitabine-based therapy.

[0272] Ovarian cancer may also be associated with mesothelin expression. In some implementations, the anti-mesothelin antibodies or antigen-binding fragments, cellular or pharmaceutical compositions provided herein can be used to treat ovarian cancer. Ovarian cancer is classified according to tumor histology. Surface epithelial-stromal tumors (also known as ovarian epithelial carcinoma) are the most common type of ovarian cancer, including serous tumors (including serous papillary cystadenocarcinoma), endometrioid tumors, and mucinous cystadenocarcinoma.

[0273] The methods described in this article can be used to treat ovarian cancer at all stages, such as stage I, II, III, or IV. Staging can be performed, for example, during ovarian cancer resection. Ovarian cancer staging is as follows: Stage I cancer is confined to one or both ovaries. Stage II is if one or both ovaries are involved and the cancer has spread to the uterus and / or fallopian tubes or other parts of the pelvis. Stage III is if one or both ovaries are involved and the cancer has spread to lymph nodes or other parts of the pelvis but remains within the abdominal cavity (e.g., the intestines or the surface of the liver). Stage IV is if one or both ovaries are involved and the cancer has spread outside the abdominal cavity or into the liver.

[0274] In some embodiments, ovarian cancer is resistant to one or more chemotherapy agents. In some embodiments, ovarian cancer does not respond to said one or more chemotherapy agents.

[0275] Other cancers that can be treated with the anti-mesothelin antibodies or antigen-binding fragments, cells, or pharmaceutical compositions provided herein include, for example, brain cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, liver cancer, kidney cancer, lymphoma, leukemia, lung cancer (e.g., lung adenocarcinoma), melanoma, metastatic melanoma, mesothelioma, neuroblastoma, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, skin cancer, thymoma, sarcoma, non-Hodgkin lymphoma, Hodgkin lymphoma, uterine cancer, and any combination thereof.

[0276] In certain diseases and conditions, mesothelin is expressed in malignant cells and cancers. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematopoietic system cancer. In some embodiments, the cancer is renal cell carcinoma, lung cancer, urothelial carcinoma, breast cancer, brain tumor, thyroid cancer, leukemia, lymphoma, and / or multiple myeloma. In some embodiments, the cancer is renal cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, glioblastoma, undifferentiated thyroid carcinoma, B-cell non-Hodgkin lymphoma, and / or chronic lymphocytic leukemia.

[0277] In some implementations, the method includes identifying subjects who have, are suspected of having, or are at risk of developing mesothelin-related diseases or conditions. Therefore, this document provides a method for identifying subjects with diseases or conditions associated with elevated mesothelin expression and selecting them to receive treatment with the anti-mesothelin antibody or antigen-binding fragment, immune effector cells, or pharmaceutical compositions provided herein.

[0278] In some embodiments, subjects may be screened for diseases or conditions associated with elevated mesothelin expression, such as cancers that express mesothelin. In some embodiments, the method includes screening or detecting the presence of mesothelin-related diseases, such as tumors. Therefore, in some embodiments, samples may be obtained from patients suspected of having diseases or conditions associated with elevated mesothelin expression, and the level of mesothelin expression may be measured. In some embodiments, subjects who test positive for mesothelin-related diseases or conditions may be selected for treatment by the method of the present invention and may be administered a therapeutically effective amount of the anti-mesothelin antibody or antigen-binding fragment, immune effector cells, or pharmaceutical composition provided herein.

[0279] In cancer treatment, cancer cells or tumor cells in a subject may be eliminated, but any clinical improvement constitutes a benefit. Antitumor effects may manifest as a reduction in tumor volume, a decrease in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with cancer. Antitumor effects may also manifest as the ability of the cellular or pharmaceutical compositions provided herein to prevent the initial occurrence of tumors. In some embodiments, "antitumor effect" may manifest as a reduction in cancer-induced immunosuppression. Clinical improvement includes a reduction in the risk or rate of progression, or a reduction in the pathological consequences of cancer or tumors. It should also be understood that methods of treating cancer may include any effect that improves any signs or symptoms associated with cancer. Such signs or symptoms include, but are not limited to, reducing tumor burden, including inhibiting tumor growth, slowing the rate of tumor growth, shrinking tumors, reducing the number of tumors, and eliminating tumors, all of which can be measured using conventional tumor imaging techniques known in the art. Other signs or symptoms associated with cancer include, but are not limited to, fatigue, pain, weight loss, and other signs or symptoms associated with various cancers.

[0280] In some embodiments, the methods or uses provided herein can reduce tumor burden. Therefore, administration of the anti-mesothelin antibodies or antigen-binding fragments, cells, or pharmaceutical compositions disclosed herein can reduce the number of tumor cells in a subject, shrink the tumor, and / or eradicate the tumor. Methods for monitoring a patient's response to administration of the pharmaceutical compositions disclosed herein are known in the art and can be employed according to the methods disclosed herein.

[0281] In the methods disclosed herein, a therapeutically effective amount of the disclosed anti-mesothelin antibody or antigen-binding fragment, cell, or pharmaceutical composition is administered to a subject requiring cancer treatment. The subject may be a mammal. In some embodiments, the subject is a human. In some embodiments, these individuals do not have clinically measurable tumors. However, there is suspicion that they have disease near the original tumor site or at risk of progression via metastasis. This group can be further subdivided into high-risk and low-risk individuals. Subdivision is based on characteristics observed before or after initial treatment. These characteristics are clinically known in the art and are appropriately defined for different types of cancer. A typical characteristic of the high-risk subgroup is that the tumor has invaded adjacent tissues or shows lymph node involvement.

[0282] In some embodiments, the subject has persistent or recurrent disease, for example, after treatment with another mesothelin-specific antibody and / or mesothelin CAR-T and / or other therapies (including chemotherapy, radiotherapy, and / or hematopoietic stem cell transplantation (HSCT), such as allogeneic HSCT or autologous HSCT). In some embodiments, the administration is still effective in treating the subject even though the subject has developed resistance to another mesothelin-targeted therapy. In some embodiments, the subject has not yet relapsed but is identified as having a risk of relapse, such as a high risk of relapse, and therefore the compound or composition is administered prophylactically, for example, to reduce the likelihood of relapse or to prevent relapse.

[0283] In some implementations, the subject is eligible for transplantation, such as for hematopoietic stem cell transplantation (HSCT), including allogeneic or autologous HSCT. In some implementations, although the subject is eligible for transplantation, they have not received a transplant prior to the administration of mesothelin-binding molecules, including anti-mesothelin antibodies or antigen-binding fragments provided herein, immune effector cells, or pharmaceutical compositions. In some implementations, the subject is unsuitable for transplantation, such as for hematopoietic stem cell transplantation (HSCT), including allogeneic or autologous HSCT.

[0284] In some embodiments, the methods provided herein include adoptive cell therapy, wherein genetically engineered immune effector cells expressing a recombinant receptor containing a mesothelin-binding molecule (e.g., the mesothelin CAR provided herein) are administered to a subject. Such administration can promote the activation of said cells in a mesothelin-targeting manner (e.g., T-cell activation), thereby targeting and destroying cells of a disease or condition. Therefore, the methods and uses provided herein include methods and uses for adoptive cell therapy. In some embodiments, the method includes administering said cells or a composition containing said cells to a subject, for example, who has, is at risk of, or is suspected of having a disease, condition, or symptom. In some embodiments, said cells, cell populations, and compositions are administered to a subject with a specific disease or condition to be treated, for example, via adoptive cell therapy (e.g., adoptive T-cell therapy). In some embodiments, said cells or compositions are administered to a subject (e.g., a subject who has or is at risk of developing the disease or condition). In some aspects, said methods thereby treat, for example, by improving one or more symptoms of said disease or condition, such as by reducing tumor burden in cancers expressing mesothelin.

[0285] Methods of cell administration for adoptive cell therapy are known in the art and can be used in conjunction with the methods and compositions provided herein. For example, adoptive T-cell therapy is described in U.S. Patent Application Publication No. 2003 / 0170238 by Gruenberg et al.; U.S. Patent No. 4,690,915 by Rosenberg; Rosenberg (2011) Nat. Rev. Clin. Oncol. 8(10):577-85. See also Themeli et al. (2013) Nat. Biotechnol. 31(10):928-933; Tsukahara et al. (2013) Biochem. Biophys. Res. Commun. 438(1):84-9; Davila et al. (2013) PLoS ONE 8(4):e61338.

[0286] In some embodiments, cell therapy (e.g., adoptive cell therapy, adoptive T-cell therapy) is performed via autologous transfer, wherein cells are isolated and / or otherwise prepared from a subject who will receive the cell therapy or from a sample derived from that subject. Thus, in some embodiments, cells are derived from a subject requiring treatment (e.g., a patient) and administered to the same subject after isolation and processing.

[0287] In some embodiments, cell therapy (e.g., adoptive cell therapy, such as adoptive T-cell therapy) is performed via allogeneic transfer, wherein cells are isolated and / or otherwise prepared from a subject who is not intended to receive or ultimately receive the cell therapy (e.g., a first subject). In such embodiments, the cells are subsequently administered to a different subject of the same species (e.g., a second subject). In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0288] The anti-mesothelin antibodies or antigen-binding fragments, cells, or pharmaceutical compositions provided herein can be administered using medical devices known in the art. For example, in some embodiments, needle-free subcutaneous injection devices, such as those disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556, can be used. Examples of known implants and modules used for the purposes described herein include: an implantable microinfusion pump for dispensing drugs at a controlled rate, disclosed in U.S. Patent No. 4,487,603; a therapeutic device for transdermal drug administration, disclosed in U.S. Patent No. 4,486,194; a drug infusion pump for delivering drugs at a precise infusion rate, disclosed in U.S. Patent No. 4,447,233; a variable flow implantable infusion device for continuous drug delivery, disclosed in U.S. Patent No. 4,447,224; a permeable drug delivery system with a multi-compartmental structure, disclosed in U.S. Patent No. 4,439,196; and a permeable drug delivery system, disclosed in U.S. Patent No. 4,475,196. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are also known to those skilled in the art.

[0289] Combination therapy using agents with different mechanisms of action can produce additive or synergistic effects. Combination therapy allows the use of agents at lower doses than those used in monotherapy, thereby reducing toxic side effects and / or increasing the therapeutic index of the agents disclosed herein. Combination therapy can reduce the likelihood of the development of drug-resistant cancer cells. In some embodiments, the adjunctive therapy increases the therapeutic index of the cell or pharmaceutical composition described herein. In some embodiments, the adjunctive therapy reduces the toxicity and / or side effects of the cell or pharmaceutical composition described herein. In some embodiments, the anti-mesothelin antibody or antigen-binding fragment, cell, or pharmaceutical composition described herein may be administered in combination with the adjunctive therapy. In some embodiments, the adjunctive therapy may be surgical resection, radiotherapy, or chemotherapy.

[0290] Add-on therapy may be administered before, simultaneously with, or after administration of the anti-mesothelin antibody or antigen-binding fragment, cells, or pharmaceutical composition described herein. Combination administration may include administration in a single pharmaceutical formulation or using individual formulations together, or in either order consecutively, but typically within a timeframe that allows all active agents to exert their biological activity simultaneously. Those skilled in the art can readily determine appropriate regimens for combining the pharmaceutical compositions described herein with add-on therapy, including the timing and dosage of the add-on agents used in the combination therapy, based on the needs of the treated subject.

[0291] 5.9 Preparation method

[0292] For cells that generate recombinant expression of the antibodies or antigen-binding fragments disclosed herein, the chimeric antigen receptors disclosed herein, and / or the fusion proteins disclosed herein, one or more polynucleotides are introduced into target cells using a suitable expression vector. One or more polynucleotides encoding the fusion protein or CAR / TCR / BiTE and the fusion protein are transferred into target immune effector cells (e.g., T cells). The polynucleotides encoding the CAR / TCR / BiTE and the fusion protein may be located on different vectors or the same vector as needed. For example, the polynucleotides encoding the CAR or fusion protein disclosed herein may be cloned into a suitable vector (e.g., a viral vector) and introduced into target cells using known molecular biology techniques (see Ausubel et al., *Molecular Biology: A Laboratory Manual*, John Wiley and Sons, Baltimore, MD (1999)). Any vector suitable for expression in cells, particularly human cells, may be used. The vector contains appropriate expression elements, such as promoters that enable the encoded nucleic acid to be expressed in target cells. For retroviral vectors, cell activation can be optionally performed to improve transduction efficiency (see Parente-Pereira et al., J. Biol. Methods 1(2):e7 (doi:10.14440 / jbm.2014.30) (2014); Movassagh et al., Hum. Gene Ther. 11:1189-1200 (2000); Rettig et al., Mol. Ther. 8:29-41 (2003); Agarwal et al., J. Virol. 72:3720-3728 (1998); Pollok et al., Hum. Gene Ther. 10:2221-2236 (1998); Quinn et al., Hum. Gene Ther. 9:1457-1467 (1998); also see commercially available methods, such as Dynabeads). TM Human T-cell activation product, Thermo Fisher Scientific, Waltham, MA.

[0293] In one embodiment, the vector is a retroviral vector, such as a γ-retroviral vector or lentiviral vector for introducing the fusion protein and / or CAR, TCR, or BiTE into target cells. Retroviral vectors can be used for transduction to genetically modify cells to express the fusion protein and / or CAR, TCR, or BiTE. However, it should be understood that any suitable viral vector or non-viral delivery system can be used. Combinations of retroviral vectors with appropriate packaging cell lines are also suitable, wherein the capsid protein can be used to infect human cells. Various amphiphilic virus production cell lines are known in the art, including but not limited to PA12 (Miller et al., Mol. Cell. Biol. 5:431-437 (1985)), PA317 (Miller et al., Mol. Cell. Biol. 6:2895-2902 (1986)), and CRIP (Danos et al., Proc. Natl. Acad. Sci. USA 85:6460-6464 (1988)). Non-amphophilic particles are also suitable, such as particles pseudomorphized with VSVG, RD114, or GALV envelopes, as well as any other particles known in the art (Relander et al., Mol. Therap. 11:452-459 (2005)). Possible transduction methods also include direct co-culture of cells with production cells, or co-culturing cells directly with production cells (e.g., Bregni et al., Blood 80:1418-1422 (1992)), or culturing using only viral supernatant or concentrated vector stock, with or without appropriate growth factors and polycations (e.g., Xu et al., Exp. Hemat. 22:223-230 (1994); Hughes et al., J. Clin. Invest. 89:1817-1824 (1992)).

[0294] Other usable viral vectors include, for example, adenovirus vectors, lentiviral vectors and adeno-associated virus vectors, vaccinia virus, bovine papillomavirus-derived vectors or herpesviruses (e.g. EB virus) (see, for example, Miller, Hum. Gene Ther. 1(1):5-14 (1990); Friedman, Science 244:1275-1281 (1989); Eglitis et al., BioTechniques 6:608-614 (1988); Tolstoshev et al., Current Opin. Biotechnol. 1:55-61 (1990); Sharp, Lancet 337:1277-1278 (1991); Cornetta et al., Prog. Nucleic Acid Res. Mol. Biol. 36:311-322 (1989); Anderson, Science 226:401-409 (1984); Moen, Blood Cells 17:407-416 (1991); Miller et al., Biotechnology 7:980-990 (1989); Le Gal La Salle et al., Science 259:988-990 (1993); Johnson, Chest 107:77S-83S (1995)). Retroviral vectors are particularly well-established and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med. 323:370 (1990); Anderson et al., US Patent No. 5,399,346). Typically, the selected vectors exhibit high infection efficiency as well as stable integration and expression (see, for example, Cayouette et al., Human Gene Therapy 8:423-430 (1997); Kido et al., Current Eye Research 15:833-844 (1996); Bloomer et al., J. Virol. 71:6641-6649 (1997); Naldini et al., Science 272:263-267 (1996); Miyoshi et al., Proc. Natl. Acad. Sci. USA 94:10319-10323 (1997)).

[0295] Particularly useful vectors for expressing the fusion proteins disclosed herein and / or CAR / TCR / BiTE include those already used in human gene therapy. In one non-limiting embodiment, the vector is a retroviral vector. Expression in T cells or other immune effector cells, including engineered T cells, using retroviral vectors has been described (see Scholler et al., Sci. Transl. Med. 4:132-153 (2012); Parente-Pereira et al., J. Biol. Methods 1(2):e7(1-9) (2014); Lamers et al., Blood 117(1):72-82 (2011); Reviere et al., Proc. Natl. Acad. Sci. USA 92:6733-6737 (1995)). In one embodiment, the vector is an SGF retroviral vector, such as an SGF γ retroviral vector based on Moloney mouse leukemia virus. SGF vectors have been described previously (see, for example, Wang et al., Gene Therapy 15:1454-1459 (2008)).

[0296] The vectors used in this article employ promoters suitable for expression in specific host cells. These promoters can be inducible or constitutive promoters. In some embodiments, the promoter of the expression vector enables expression in stem cells (e.g., hematopoietic stem cells). In some embodiments, the promoter enables expression in immune effector cells (e.g., T cells). Non-viral vectors can also be used, provided the vector contains expression elements suitable for expression in target cells. Some vectors (e.g., retroviral vectors) can integrate into the host genome.

[0297] In some embodiments, this document provides methods for genetically engineering immune effector cells by transferring the polynucleotides described herein into them using non-viral delivery systems. For example, physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, liposome transfection, particle bombardment, microinjection, electroporation, etc. In some embodiments, RNA electroporation may be used (Van Driessche et al., Folia Histochemica et Cytobiologica 43:4:213-216 (2005)). In some embodiments, DNA transfection and transposons may be used. In some embodiments, the Sleeping Beauty system or the PiggyBac system may be used (e.g., Ivics et al., Cell 91(4):501-510 (1997); Cadiñanos et al., Nucleic Acids Research 35(12):e87 (2007)). Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, microbeads, and lipid systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as an in vitro and in vivo delivery carrier is a liposome (e.g., an artificial membrane vesicle).

[0298] In some implementations, this paper provides a method for genetically engineering immune effector cells by transferring the polynucleotides described herein into them using gene editing. If desired, targeted integration can be achieved using techniques such as nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), clustered regularly spaced short palindromic repeats (CRISPR), homologous recombination, non-homologous end joining, microhomologous end joining, and homologous end joining (Gersbach et al., Nucl. Acids Res. 39:7868-7878 (2011); Vasileva et al., Cell Death Dis. 6:e1831 (July 23, 2015); Sontheimer, Hum. Gene Ther. 26(7):413-424 (2015); Yao et al., Cell Research 27:801-814 (2017)).

[0299] The immune effector cells described herein can be obtained from the subject. Their sources include, but are not limited to, peripheral blood, cord blood, bone marrow, or other hematopoietic cell sources. Immune effector cells (e.g., T cells) can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, cell lines available in the art may be used. The immune effector cells described herein can be isolated using methods known in the art, including commercially available isolation methods (see, for example, Rowland-Jones et al., *LYMPHOCYTES: A PRACTICAL APPROACH*, Oxford University Press, New York (1999)). Various methods for isolating immune effector cells have been previously described and can be used, including but not limited to the use of donor peripheral lymphocytes (Sadelain et al., Nat. Rev. Cancer 3:35-45 (2003); Morgan et al., Science 314:126-129 (2006)), and the selective expansion of antigen-specific peripheral blood leukocytes in vitro using artificial antigen-presenting cells (AAPC) or dendritic cells (Dupont et al., Cancer Res. 65:5417-5427 (2005); Papanicolaou et al., Blood 102:2498-2505 (2003)).

[0300] In the treatment methods disclosed herein, the immune effector cells can be autologous or non-autologous relative to the subject receiving them. Autologous cells are isolated from the subject intended to receive the engineered cells. Optionally, cells can be obtained via apheresis, in which leukocytes are selectively removed from the drawn blood, recombined, and subsequently re-infused to a donor. Alternatively, allogeneic cells from a non-autologous donor who is not the subject can be used. For non-autologous donors, the cells are human leukocyte antigen (HLA) typing and matching according to methods known in the art to determine an appropriate level of compatibility. Cells can optionally be cryopreserved after isolation and / or genetic engineering, and after expansion of the genetically engineered cells (see Kaiser et al., ibid., 2015). Cell cryopreservation methods are well known in the art (see, for example, Freshney, CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUES, 4th ed., Wiley-Liss, New York (2000); Harrison and Rae, GENERAL TECHNIQUES OF CELL CULTURE, Cambridge University Press (1997)).

[0301] In some embodiments, isolated immune effector cells are genetically engineered in vitro to recombinantly express the fusion protein. In some embodiments, isolated immune effector cells are genetically engineered in vitro to recombinantly express the fusion protein and CAR / TCR / BiTE. In some embodiments, the immune effector cells provided herein are obtained by in vitro sensitization, wherein sensitization may occur before or after the immune effector cells are genetically engineered to recombinantly express the fusion protein disclosed herein. In embodiments where sensitized immune effector cells (e.g., T cells) are isolated from an in vivo source, it is evident that the genetic engineering is performed on already sensitized immune effector cells.

[0302] 5.10 Example

[0303] The following examples are provided to fully disclose and describe how to prepare and use the invention to those skilled in the art, and are not intended to limit the scope of what the inventors consider their invention, nor are they intended to represent all or only the experiments that have been performed. Efforts have been made to ensure the accuracy of the numerical values ​​used (e.g., amounts, temperatures, etc.), but certain experimental errors and deviations should be taken into account. Unless otherwise stated, parts are by weight, molecular weights are weight-average molecular weights, temperatures are expressed in degrees Celsius, and pressures are at or near atmospheric pressure. Standard abbreviations may be used, such as bp, base pair; kb, kilobase; pl, picoliter; s or sec, second; min, minute; h or hr, hour; aa, amino acid; nt, nucleotide; im, intramuscular; ip, intraperitoneal; sc, subcutaneous; etc.

[0304] Materials and methods

[0305] Primary human lymphocytes. Primary human CD4+ T cells and CD8+ T cells were isolated from healthy volunteer donors. T cells were stimulated with anti-CD3 / CD28 Dynabeads (Life Technologies, Grand Island, NY) and cultured in R10 medium supplemented with 100 IU / mL IL-2 [RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% HEPES, 1% GlutaMAX, 1% penicillin and streptomycin, 1% MEM NEAA and 1% sodium pyruvate].

[0306] Cell lines. The following cell lines were cultured in R10 medium and used in related experiments: A549 (human lung cancer cells), ASPC1 (human pancreatic tumor cells), H226 (human lung cancer cells), OVCAR3 (human adenocarcinoma cells), U87 (human malignant glioma cells), U251 (human malignant glioblastoma cells), Caski (human cervical cancer cells), SH-SY5Y (human neuroblastoma cells), Siha (human squamous cell carcinoma cells), HepG2 (human hepatocellular carcinoma cells), and PC3 (human prostate cancer cells). HCC70 (human breast cancer cells), THP1 (human leukemia cells), Jeko1 (human lymphoma cells), Nalm6 (human lymphoblastic leukemia cells), Molm14 (human myeloid leukemia cells), Raji (human lymphoma cells), RPMI8226 (human myeloma cells), A498 (human cancer cells), Huh-7 (hepatocellular carcinoma cells), A375 (malignant melanoma cells), 293T (human embryonic kidney (HEK) cells), and Supt1 (lymphoblastic lymphoma cells).

[0307] Lentiviral production and transduction. Lentiviral particles were produced from HEK293T cells transfected with transfer plasmids and packaging plasmids pRSV.REV, pMD2.G, and pMDLg.pRRE. Lentiviral particles were harvested after 24 and 48 hours and concentrated by ultracentrifugation.

[0308] T cells were stimulated with CD3 / CD28 Dynabeads on day 0. Lentiviral virus was added to the culture medium on day 1. Cells were replenished daily or every two days with R10 medium containing 100 IU / ml IL-2.

[0309] Production of anti-mesothelin CAR mRNA. In vitro transcription of mRNA was performed using the Ambion mMessage mMACHINE T7 Ultra kit (Life Technologies, Carlsbad, CA).

[0310] Electroporation of anti-mesothelin CAR mRNA. The mRNA was added to 0.1 ml of T cells (0.5 × 10^8 cells / ml) that had been washed twice and resuspended in OPTI-MEM. Electroporation was performed using a BTX830 (Harvard Apparatus BTX) in a 0.2 cm electroporation cuvette at 500 V for 0.7 ms.

[0311] Tumor killing assays were performed using the IncuCyte real-time cell analyzer. Tumor cells and T cells were washed twice with R10 medium and then resuspended in R10 medium. Tumor cells and CAR+ T cells were seeded at 4,000 cells / well in 96-well plates. Total integral intensity was recorded every 8 hours.

[0312] ELISA. 100,000 CAR-T cells and 100,000 tumor cells were co-cultured at 37°C for 24 hours. The supernatant was then collected and ELISA was performed according to the ELISA kit (R&D Systems) instructions to measure IL-2 and IFNγ secretion levels.

[0313] 5.10.1 Example 1: Construction of a stable MOLM14-huMSLN cell line

[0314] The full-length huMSLN gene sequence was cloned into the lentiviral vector pUTCK, packaged into lentivirus, and used to infect MOLM14 cells. On day 3 post-infection, cells were stained with an anti-huMSLN flow cytometry antibody. huMSLN antibody-positive MOLM14 cells were sorted by flow cytometry and further expanded in culture. Figure 1 As shown, the sorted MOLM14-huMSLN cell line expresses huMSLN protein at a high level.

[0315] 5.10.2 Example 2: Screening of anti-MSLN antibody phage display library

[0316] Based on an initial OD value of approximately 0.1, an appropriate amount of the original phage library was inoculated into a 500 ml Erlenmeyer flask containing 2×YT medium, and 100 μg / mL ampicillin and 2% glucose were added. The bacteria were cultured with shaking at 250 rpm and 37°C for approximately 3 hours, until the OD600 value reached approximately 0.5. Approximately 3×10^12 M13KO7 helper phages were added to the bacterial culture, and the culture was incubated at 37°C for 30 minutes. After 30 minutes, the infected bacterial culture was transferred to a 50 ml centrifuge tube and centrifuged at 2400g, 12°C for 30 minutes. The supernatant was discarded to eliminate the inhibitory effect of glucose on antibody expression.

[0317] Resuspend the precipitate in 600 ml of 2×YT solution and add ampicillin (100 μg / mL), kanamycin (60 μg / mL), and IPTG (100 μM) to a final concentration. Incubate overnight at 30°C with shaking at 200 rpm. The next day, collect the bacterial culture and centrifuge at 2400 g, 12°C for 30 minutes. After centrifugation, transfer 36 ml of the supernatant to a new 50 ml centrifuge tube, add 9 ml of PEG / NaCl, mix thoroughly, and place on ice for 1–2 hours. After 1–2 hours, centrifuge the mixture at 4000 rpm, 4°C for 30 minutes.

[0318] After centrifugation, the supernatant was gently discarded, and the phage pellet was resuspended in 3 ml of 1×PBS / 5% FBS solution and stored at 4°C for later use. Subsequently, the purified phage library was thoroughly mixed with MOLM14-huMSLN (resuspended in 5% FBS / PBS) and rotated at 10 rpm on a mixer in a 4°C refrigerator. After 1.5 hours, the mixture was washed with 5% FBS / PBS to remove as much supernatant as possible. The phage was then eluted with 500 μl of 0.1 M glycine at pH 2.2 and incubated at room temperature for 10 minutes. After centrifugation, the supernatant was transferred to a new tube, and 50 μl of Tris-HCl at pH 9.5 was added to neutralize the pH.

[0319] Add the eluted phages to 1×10^7 cells / 2.5 ml MOLM14 cells (resuspended in 5% FBS / PBS), mix thoroughly, and rotate at 10 rpm for approximately 0.5 hours to bind. Centrifuge, and repeat the above steps twice more.

[0320] After the final centrifugation, the supernatant was transferred to a new 15 ml tube and used to infect TG1. The panning process was repeated twice more, and the resulting clones were detected by whole-cell phage ELISA (results are shown in [link to results]). Figure 2 ). The scFv sequence was obtained by PCR amplification of positive clones. Figure 2 The gray background indicates clones identified by PCR. Finally, the scFv sequence identified by sequencing was cloned into the pUTCK-MSLN CAR vector ( Figure 3A ).

[0321] 5.10.3 Example 3: Tumor Cell Culture

[0322] Tumor cell lines were cultured in RPMI-1640 or DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics, and passaged every 2-3 days.

[0323] 5.10.4 Example 4: Electroporation of A549 tumor cell mRNA

[0324] 1. Collect A549 tumor cells and wash them three times with Opti-MEM medium.

[0325] 2. Resuspend the cells in Opti-MEM medium and adjust the cell concentration to 5×10e7 / ml.

[0326] 3. Gently mix the required volume of mRNA with 100 μl of tumor cells and immediately perform electroporation.

[0327] 4. Set the parameters on the BTX ECM 830 instrument: voltage 360 ​​V, pulse duration 1 ms.

[0328] 5. Add the mRNA / cell mixture to the BTX electroporation cup and tap gently to avoid creating air bubbles.

[0329] 6. Perform electroporation, then transfer the electroporated cells to 1 ml of preheated culture medium, mix well, and then incubate at 37°C.

[0330] 7. Protein expression levels were measured the day after electroporation. Results are shown below. Figure 4 A549 cells electroporated with 1 μg and 10 μg MSLN mRNA both showed different levels of expression.

[0331] 5.10.5 Example 5: Preparation of Lentiviral CAR-T Cells

[0332] Two candidate MSLN CAR sequences and three MSLN CAR / LACOSTIM sequences were cloned into the pUTCK vector (e.g., Figure 3A (As shown). Subsequently, plasmid extraction, lentivirus packaging, ultracentrifugation, and titer determination were performed. Finally, the cells were frozen at -80°C for later use. CAR-T cell preparation: On day 1, T cells were activated with anti-CD3 / CD28 Dynabeads, and the cell density was adjusted to 1×10^6 cells / ml. On day 2, lentivirus was added to the T cells at an MOI of 3, and the cells were cultured for another two days. On day 5, anti-CD3 / CD28 Dynabeads were removed, and cell counting, passage, and resuscitation were performed. On day 9, 3×10^5 T cells were taken from each group and stained with MSLN-Fc / anti-human IgG Fc and CD40-Fc / anti-human IgG Fc antibodies, respectively. The CAR positivity rate was detected by flow cytometry (e.g., CAR-T cell preparation). Figure 5 As shown), and evaluate the expression of LACOSTIM (e.g. Figure 6 (As shown).

[0333] Continue culturing CAR-T cells until about day 13. After harvesting, use them directly for functional experiments or freeze them for later use.

[0334] 5.10.6 Example 6: In vitro cytotoxicity detection of CAR-T cell preparations

[0335] 1. Twelve hours before co-culturing with CAR-T cells for cytotoxicity experiments, tumor cells were seeded into 96-well flat plates at a density of 3,000-10,000 cells / 100 μl per well.

[0336] 2. After 8-12 hours, allow the cells to fully adhere to the well surface. Dilute various anti-MSLN CAR-T cells to appropriate cell densities and co-incubate them with tumor cells at different effector-target ratios (e.g., E:T = 3:1, 1:1, and 0.3:1).

[0337] 3. Place the 96-well plate into the IncuCyte S3 instrument and set the scanning parameters.

[0338] 4. Three days after scanning, the total cumulative intensity of green fluorescence (GCU × μm² / well) was analyzed to calculate the tumor cell killing efficiency. Results are shown below. Figure 7-10 Compared to UTD cells, MH150 CAR-T cells and two types of MSLN CAR / LACOSTIM CAR-T cells, namely A4025-MH150 and MH150-25-9.3 CAR-T cells, showed little killing effect on A549 cells with low MSLN expression. However, they exhibited higher killing efficiency against A549 cells and ASPC1 cells with high MSLN expression. ELISA analysis of the supernatant after 24 hours of co-culturing tumor cells and CAR-T cells also confirmed these experimental results (see...). Figure 11 and Figure 12 ).

[0339] Furthermore, in subsequent studies, various tumor cells that do not express MSLN were screened as target cells, such as HCC70, SH-SY5Y, Huh-7, U87, U251, A375, and 293T (see [link to study]). Figure 13-22 Various candidate sequences exhibited good specificity.

[0340] 5.10.7 Example 7: In vivo tumor suppression detection in tumor-bearing animal models

[0341] Animal model experiments were conducted using MSLN CAR / LACOSTIM CAR-T cells (e.g., MH150-25-9.3 CAR-T cells). OVCAR3 cells in the expansion phase were subcutaneously injected into immunodeficient M-NSG mice at a dose of 10 × 10^6 cells per mouse to establish a subcutaneous OVCAR3 tumor-bearing model. When the tumor volume reached 70-100 mm³, the tumor-bearing mice were divided into three groups according to Table 7, and CAR-T cells were administered at doses of 1 × 10^6, 2 × 10^6, and 3 × 10^6 CAR-positive cells, respectively. Grouping and administration regimens are detailed in Table 7.

[0342] Table 7 Grouping scheme for animal model experiments

[0343]

[0344] Mice were fed under standardized conditions, and tumor volume, body weight, and survival status were recorded throughout the study. Results are shown in [link to study]. Figure 23 Mouse model experimental data showed that, after a single injection of CAR-T cells, both the medium-dose and high-dose groups exhibited significant tumor-killing efficacy compared to the low-dose group. In the high-dose group, complete tumor regression was observed in some mice on day 21 post-injection, and all mice achieved complete tumor regression by day 31. In contrast, tumor growth was significantly inhibited in a few mice in the low-dose group, while tumors continued to progress in other mice in the same group.

[0345] Detection of CAR-T cell changes in tumor-bearing animal models. After drug administration, the proportion of CAR+ T cells in the peripheral blood of tumor-bearing mice was detected by flow cytometry, and the expansion of CAR-T cells in mice was analyzed. Results are shown below. Figure 24 The results showed that after a single injection of CAR-T cells, the proportion of CAR-T cells in the peripheral blood of each dose group gradually increased, reaching a peak between days 21 and 28. This indicates that CAR-T cells undergo significant expansion during tumor killing.

[0346] Detection of sLACO secretion levels in tumor-bearing animal models. After drug administration, the sLACO secretion levels in the peripheral blood of tumor-bearing mice were detected using enzyme-linked immunosorbent assay (ELISA). Results are shown below. Figure 25 The results showed that after a single injection of CAR-T cells, the level of sLACO secretion in peripheral blood of each dose group gradually increased with the expansion of CAR-T cells, and the secretion level was roughly consistent with the expansion of CAR-T cells.

[0347] 5.10.8 Example 8: Comparison of in vivo experimental results of cells prepared using different production processes in tumor-bearing animal models

[0348] Animal model experiments were conducted using MSLN CAR / LACOSTIM CAR-T cells (e.g., MH150-25-9.3 CAR-T cells). Simultaneously, T cells (NTD) from the same donor were used as control T cells. OVCAR3 cells in the expansion phase were subcutaneously injected into immunodeficient M-NSG mice at a dose of 10 × 10^6 cells per mouse to establish an OVCAR3 subcutaneous tumor model. When the tumor size reached 70-100 mm³, the tumor-bearing mice were divided into three groups according to Table 8, and injected with NTD cells, CAR-T cell 1 (2 × 10^6 CAR-positive cells), and CAR-T cell 2 (2 × 10^6 CAR-positive cells), respectively. CAR-T cell 1 and CAR-T cell 2 were CAR-T cells prepared using the same donor and two different manufacturing processes. Grouping and administration regimens are shown in Table 8.

[0349] Table 8 Grouping scheme for animal model experiments

[0350]

[0351] *Process 1: Use human AB serum as a supplement to the amplification culture medium (e.g., Gemini, 100-912).

[0352] *Process 2: Use human platelet lysate as a supplement to the amplification medium (e.g., Compass Biomedical, PLSA, 081023).

[0353] Mice were continuously monitored, and tumor volume, body weight, and survival status were recorded. Results are shown below. Figure 26 Mouse model experiments showed that after a single injection of CAR-T cells, both CAR-T cells 1 and CAR-T cells 2 exhibited significant tumor-killing efficacy, and complete tumor regression was observed in some mice starting from day 21 post-injection. In contrast, tumors continued to grow in mice injected with control T cells.

[0354] Detection of CAR-T cell changes in tumor-bearing animal models. After drug administration, the number of CAR+ T cells in the peripheral blood of tumor-bearing mice in this embodiment was detected by flow cytometry, and the expansion of CAR-T cells in mice was analyzed. Results are shown below. Figure 27 The results showed that after a single injection of CAR-T cells, the number of CAR-T cells in the peripheral blood of different process groups gradually increased, reaching a peak on days 22-28. No expansion was observed in the T cell control group. This indicates that CAR-T cells undergo significant expansion during tumor killing.

[0355] Detection of sLACO secretion levels in tumor-bearing animal models. After drug administration, the sLACO secretion levels in the peripheral blood of tumor-bearing mice in this example were detected using enzyme-linked immunosorbent assay (ELISA). Results are shown below. Figure 28 The results showed that after a single injection of CAR-T cells, the level of sLACO secretion in peripheral blood gradually increased in different process groups, reaching a peak on days 14-22. The peak secretion occurred approximately 7 days earlier than the peak expansion of CAR-T cells.

[0356] While preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The invention is not intended to be limited to the specific embodiments provided in the specification. Although the invention has been described with reference to the foregoing specification, the description and illustration of embodiments herein are not intended to be construed in a limiting sense. Various modifications, alterations, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it should be understood that all aspects of the invention are not limited to the specific descriptions, configurations, or relative proportions listed herein, which depend on various conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in carrying out the invention. Therefore, the invention is contemplated to also cover any such alternatives, modifications, variations, or equivalents. The following claims are intended to define the scope of the invention and thereby cover the methods and structures within the scope of these claims and their equivalents.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to mesothelin, comprising: (a) A light chain variable region (VL) comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) having amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively; or having variants in the LCDR having up to about 5 amino acid substitutions, additions, and / or deletions; and / or (b) Heavy chain variable region (VH), which comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3) having amino acid sequences of SEQ ID NOs: 4, 5 and 6 respectively; or variants having up to about 5 amino acid substitutions, additions and / or deletions in HCDR.

2. The antibody or its antigen-binding fragment according to claim 1, comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3, wherein: (a) LCDR1, CDR2, and CDR3 have the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively; and (b) HCDR1, CDR2 and CDR3 have amino acid sequences of SEQ ID NOs: 4, 5 and 6, respectively.

3. The antibody or antigen-binding fragment thereof according to any one of claims 1-2, comprising: (a) A VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 7; and / or (b) A VH having at least 85%, at least 90%, at least 95%, at least 98% or 100% sequence identity with the amino acid sequence of SEQ ID NO:

8.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, comprising VL and VH, wherein VL and VH have the amino acid sequences of SEQ ID NOs: 7 and 8, respectively.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein the antibody or antigen-binding fragment is a monoclonal antibody or antigen-binding fragment.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the antibody or antigen-binding fragment is selected from the group consisting of IgG1 antibody, IgG2 antibody, IgG3 antibody and IgG4 antibody.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1-6, wherein the antibody is selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, single-domain antibody (sdAb) and heavy chain antibody (HCAb).

8. The antibody or antigen-binding fragment thereof according to any one of claims 1-7, wherein it is an scFv having the amino acid sequence of SEQ ID NO:

9.

9. A chimeric antigen receptor (CAR) that specifically binds to mesothelin, comprising, from the N-terminus to the C-terminus: (a) A mesothelin-binding domain comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-8; (b) Transmembrane domains; and (c) Cytoplasmic domain.

10. The CAR of claim 9, wherein the transmembrane domain is derived from CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, TCR α chain, TCR β chain or TCR ζ chain, CD3ε, CD45, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 or CD154.

11. The CAR according to any one of claims 9-10, wherein the transmembrane domain comprises a CD8 transmembrane region.

12. The CAR according to any one of claims 9-11, wherein the cytoplasmic domain comprises a signal transduction domain derived from CD3ζ, FcRγ, FcγRIIa, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, DAP10, DAP12, or any combination thereof.

13. The CAR according to any one of claims 9-12, wherein the cytoplasmic domain further comprises a co-stimulatory domain derived from CD28, 4-1BB (CD137), OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD2, CD7, LIGHT, GITR, TLR, DR3, CD43, or any combination thereof.

14. The CAR according to any one of claims 9-13, wherein the cytoplasmic domain comprises a CD3ζ signaling domain and a 4-1BB co-stimulatory domain.

15. The CAR according to any one of claims 9-14, further comprising a CD8 hinge domain located between the mesothelin-binding domain and the transmembrane domain.

16. The CAR according to any one of claims 9-15, further comprising a signal peptide domain derived from CD8 at its N-terminus.

17. The CAR according to any one of claims 9-16, comprising the amino acid sequence of SEQ ID NO:

10.

18. A polynucleotide encoding an antibody or antigen-binding fragment of any one of claims 1-8, or a CAR of any one of claims 9-17.

19. The polynucleotide of claim 18, wherein the polynucleotide is DNA or mRNA.

20. A carrier comprising the polynucleotide of claim 19.

21. A cell comprising an antibody or antigen-binding fragment according to any one of claims 1-8, a CAR according to any one of claims 9-17, a polynucleotide encoding a CAR according to any one of claims 18-19, or a vector according to claim 20.

22. The cell of claim 21, wherein the cell is an immune effector cell.

23. The cell according to any one of claims 21-22, wherein the cell is derived from cells isolated from peripheral blood or bone marrow.

24. The cell according to any one of claims 21-23, wherein the cell is derived from a cell differentiated in vitro from a stem cell or progenitor cell, wherein the stem cell or progenitor cell is selected from the group consisting of T cell progenitor cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotent progenitor cells, embryonic stem cells and induced pluripotent cells.

25. The cell according to any one of claims 21-24, wherein the cell is a T cell or an NK cell.

26. The cell according to any one of claims 21-25, wherein the cell is a cytotoxic T cell, helper T cell, γδ T cell, CD4+ / CD8+ double-positive T cell, CD4+ T cell, CD8+ T cell, CD4- / CD8- double-negative T cell, CD3+ T cell, naive T cell, effector T cell, helper T cell, memory T cell, regulatory T cell, Th0 cell, Th1 cell, Th2 cell, Th3 (Treg) cell, Th9 cell, Th17 cell, Thαβ helper cell, Tfh cell, stem cell-like memory TSCM cell, central memory TCM cell, effector memory TEM cell, or effector memory TEMRA cell.

27. The cell according to any one of claims 21-26, wherein the cell recombinantly expresses a fusion protein comprising a first domain for activating antigen-presenting cells (APCs) and a second domain for activating immune effector cells, wherein: (i) The first domain comprises (a) a ligand or receptor-binding fragment thereof that binds to the activating receptor of APC, or (b) an antibody or antigen-binding fragment thereof that binds to the activating receptor of APC; and (ii) The second domain comprises (a) a co-stimulatory receptor of an immune effector cell or a functional fragment thereof, (b) a co-stimulatory ligand of an immune effector cell or a receptor-binding fragment thereof, or (c) an antibody that binds to a co-stimulatory receptor of an immune effector cell or an antigen-binding fragment thereof.

28. The cell of claim 27, wherein the N-terminus of the first domain is connected to the C-terminus of the second domain.

29. The cell of claim 27, wherein the N-terminus of the second domain is connected to the C-terminus of the first domain.

30. The cell according to any one of claims 27-29, wherein the first and second domains are connected via a connector.

31. The cell according to any one of claims 27-30, wherein the APC is selected from the group consisting of dendritic cells, macrophages, myeloid-derived suppressor cells, monocytes, B cells, T cells and Langerhans cells.

32. The cell according to any one of claims 27-31, wherein the activation receptor of APC is selected from the group consisting of CD40, CD80, CD86, CD91, DEC-205 and DC-SIGN.

33. The cell of claim 32, wherein the first domain comprises a ligand or receptor-binding fragment of CD40, CD80, CD86, CD91, DEC-205 or DC-SIGN.

34. The cell of claim 33, wherein the first domain comprises a receptor-binding fragment of a CD40 ligand (CD40L).

35. The cell according to any one of claims 27-31, wherein the first domain comprises an antibody or an antigen-binding fragment thereof that binds to the APC activation receptor.

36. The cell of claim 35, wherein the first domain is an anti-CD40 antibody or an antigen-binding fragment thereof.

37. The cell according to any one of claims 27-36, wherein the immune effector cells are selected from the group consisting of T cells, NK cells, NKT cells, macrophages, neutrophils and granulocytes.

38. The cell according to any one of claims 27-37, wherein the second domain comprises the cytoplasmic domain of a co-stimulatory receptor.

39. The cell of claim 38, wherein the co-stimulatory receptor is selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3, and CD43.

40. The cell of claim 38, wherein the co-stimulatory receptor is CD28.

41. The cell according to any one of claims 27-37, wherein the second domain is a co-stimulatory ligand of an immune effector cell or a receptor-binding fragment thereof.

42. The cell of claim 41, wherein the co-stimulatory ligand is selected from the group consisting of CD58, CD70, CD83, CD80, CD86, CD137L, CD252, CD275, CD54, CD49a, CD112, CD150, CD155, CD265, CD270, TL1A, CD127, IL-4R, GITR-L, TIM-4, CD153, CD48, CD160, CD200R, and CD44.

43. The cell according to any one of claims 27-37, wherein the second domain is an antibody or an antigen-binding fragment thereof that binds to a co-stimulatory receptor.

44. The cell of claim 43, wherein the second domain is scFv.

45. The cell according to claim 43 or 44, wherein the co-stimulatory receptor is selected from the group consisting of CD28, 4-1BB, ICOS, CD27, OX40, DAP10, 2B4, CD30, CD2, LIGHT, GITR, TLR, DR3 and CD43.

46. ​​The cell according to claim 43 or 44, wherein the co-stimulatory receptor is CD28.

47. The cell of claim 27, wherein the first domain comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and the second domain comprises an anti-CD28 antibody or an antigen-binding fragment thereof.

48. The cell of claim 27, wherein the first domain comprises an anti-CD40 antibody or an antigen-binding fragment thereof, and the second domain comprises a CD28 cytoplasmic domain.

49. The cell according to any one of claims 47-48, wherein the fusion protein has at least 85%, 90%, 95%, 98% or 99% identity with the amino acid sequence selected from the group consisting of SEQ ID NOs:49-59 and 63-73.

50. The cell according to any one of claims 27-49, wherein it is an immune effector cell.

51. The cell of claim 50, wherein the cell is derived from cells isolated from peripheral blood or bone marrow.

52. The cell according to claim 50, wherein the cell is derived from a stem cell or progenitor cell differentiated in vitro, said stem cell or progenitor cell being selected from the group consisting of T cell progenitor cells, hematopoietic stem cells and progenitor cells, hematopoietic pluripotent progenitor cells, embryonic stem cells and induced pluripotent cells.

53. The cell according to any one of claims 27-52, wherein it is a T cell or an NK cell.

54. The cell according to claim 53, wherein it is a cytotoxic T cell, helper T cell, γδ T cell, CD4+ / CD8+ double-positive T cell, CD4+ T cell, CD8+ T cell, CD4 / CD8 double-negative T cell, CD3+ T cell, naive T cell, effector T cell, helper T cell, memory T cell, regulatory T cell, Th0 cell, Th1 cell, Th2 cell, Th3 (Treg) cell, Th9 cell, Th17 cell, Thαβ helper cell, Tfh cell, stem cell-like memory TSCM cell, central memory TCM cell, effector memory TEM cell, or effector memory TEMRA cell.

55. The cell according to claim 53, wherein it is a cytotoxic T cell.

56. A cell population comprising the cells of any one of claims 27-49, wherein the cell population is derived from peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor-infiltrating lymphocytes (TILs), cytokine-induced killer cells (CIKs), lymphokine-activated killer cells (LAKs), or bone marrow-infiltrating lymphocytes (MILs).

57. A pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment of any one of claims 1-8, a CAR of any one of claims 9-17, a polynucleotide of any one of claims 18-19, a carrier of claim 20, and / or a cell or cell population of any one of claims 21-56, and a pharmaceutically acceptable carrier or excipient.

58. Use of the antibody or antigen-binding fragment of any one of claims 1-8, the CAR of any one of claims 9-17, the polynucleotide of any one of claims 18-19, the vector of claim 20, the cell or cell population of any one of claims 21-56, and / or the pharmaceutical composition of claim 57 in the preparation of a medicament for treating cancer.

59. Use of the antibody or antigen-binding fragment of any one of claims 1-8, the CAR of any one of claims 9-17, the polynucleotide of any one of claims 18-19, the vector of claim 20, the cell or cell population of any one of claims 21-56, and / or the pharmaceutical composition of claim 57 in cancer treatment.

60. The use according to claim 58 or 59, wherein the cells, cell populations or pharmaceutical composition are used in combination with an adjunct therapy.

61. A method of treating cancer in a subject in need, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment of any one of claims 1-8, a CAR of any one of claims 9-17, a polynucleotide of any one of claims 18-19, a vector of claim 20, a cell or cell population of any one of claims 21-56, and / or a pharmaceutical composition of claim 57.

62. The method of claim 61, wherein the cell or cell group is autologous relative to the subject.

63. The method of claim 61, further comprising obtaining cells from the subject.

64. The method according to any one of claims 61-63, further comprising administering additional therapy to the subject.

65. The method according to any one of claims 61-64, wherein the subject is a human being.

66. The use according to any one of claims 58-60 or the method according to any one of claims 61-65, wherein the cancer is a cancer expressing mesothelin.

67. The use according to any one of claims 58-60 or the method according to any one of claims 61-65, wherein the cancer is a solid tumor or a hematologic cancer.

68. The use according to any one of claims 58-60 or the method according to any one of claims 61-65, wherein the cancer is renal cancer, lung cancer, urothelial carcinoma, breast cancer, brain tumor, thyroid cancer, leukemia, lymphoma and / or multiple myeloma.

69. The use according to any one of claims 58-60 or the method according to any one of claims 61-65, wherein the cancer includes renal cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, glioblastoma, undifferentiated thyroid carcinoma, B-cell non-Hodgkin lymphoma and / or chronic lymphocytic leukemia.

70. The use according to any one of claims 58-60 or the method according to any one of claims 61-65, wherein the cancer is mesothelioma.

71. The use according to any one of claims 58-60 or the method according to any one of claims 61-65, wherein the cancer is pleural mesothelioma, peritoneal mesothelioma or pericardial mesothelioma.

72. The use according to any one of claims 58-60 or the method according to any one of claims 61-65, wherein the cancer is pancreatic cancer.

73. The use according to any one of claims 58-60 or the method according to any one of claims 61-65, wherein the cancer is pancreatic ductal carcinoma.

74. The use according to any one of claims 58-60 or the method according to any one of claims 61-65, wherein the cancer is ovarian cancer.

75. The use according to any one of claims 58-60 or the method according to any one of claims 61-65, wherein the cancer is ovarian epithelial carcinoma.

76. A method for preparing cells according to any one of claims 21-55, comprising transferring a polynucleotide encoding an antibody or antigen-binding fragment according to any one of claims 1-8, a polynucleotide encoding a CAR according to any one of claims 9-17, a polynucleotide according to any one of claims 18-19, and / or a vector according to claim 20 into the cells.

77. The method of claim 76, wherein the method is used to prepare cells capable of expressing a CAR that specifically binds to mesothelin, and the method comprises transferring the polynucleotide of claim 18 or 19 into the cells.

78. The method according to any one of claims 76-77, wherein the method is used to prepare cells that express a mesothelin-specific CAR and a fusion protein comprising a first domain for activating antigen-presenting cells (APCs) and a second domain for activating immune effector cells; and the method comprises transferring the polynucleotide of claim 18 or 19 and the polynucleotide encoding the fusion protein into the cells, wherein: (i) The first domain of the fusion protein comprises (a) a ligand or receptor-binding fragment of an APC-activating receptor, or (b) an antibody or antigen-binding fragment of an APC-activating receptor; and (ii) The second domain of the fusion protein contains (a) a co-stimulatory receptor of an immune effector cell or a functional fragment thereof, (b) a co-stimulatory ligand of an immune effector cell or a receptor-binding fragment thereof, or (c) an antibody that binds to the co-stimulatory receptor of an immune effector cell or an antigen-binding fragment thereof.

79. The method according to any one of claims 76-78, wherein the polynucleotide is transferred by electroporation.

80. The method according to any one of claims 76-78, wherein the polynucleotide is transferred via viral transduction.

81. The method of claim 80, further comprising using lentivirus, retrovirus, adenovirus, or adeno-associated virus for viral transduction.

82. The method according to any one of claims 76-78, wherein the polynucleotide is transferred using a transposon system.

83. The method of claim 82, wherein the transposable subsystem is Sleeping Beauty or PiggyBac.

84. The method according to any one of claims 76-78, wherein gene editing is used to transfer the polynucleotide.

85. The method of claim 84, wherein the polynucleotide is transferred using a CRISPR-Cas system, a ZFN system, or a TALEN system.

86. The method according to any one of claims 76-85, wherein the cells are selected from the group consisting of T cells, NK cells, NKT cells, macrophages, neutrophils and granulocytes.

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