WO2023217062A1

WO2023217062A1 - Chimeric antigen receptor and use thereof

Info

Publication number: WO2023217062A1
Application number: PCT/CN2023/092659
Authority: WO
Inventors: 秦玉廓; 陆金华; 潘杰; 田龙果; 张�杰
Original assignee: 星尘生物科技(上海)有限公司
Priority date: 2022-05-10
Filing date: 2023-05-08
Publication date: 2023-11-16
Also published as: CN118510814A

Abstract

The present application relates to a chimeric antigen receptor, which comprises an extracellular antigen binding domain, wherein the extracellular antigen binding domain comprises a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain specifically binds to integrin. The present application also provides genetically engineered cells expressing the CAR and use of the cells in, for example, adoptive cell therapy.

Description

A kind of chimeric antigen receptor and its application

Technical field

This application relates to the technical field of tumor immunotherapy, specifically to a chimeric antigen receptor and its construction method and application.

Background technique

CAR-T, the full name is Chimeric Antigen Receptor T-Cell Immunotherapy, is a newly developed adoptive anti-tumor therapy. Genetically modified T cells express chimeric antigen receptors, usually consisting of a signaling domain, a transmembrane domain, and an extracellular antigen-binding domain, usually a single-chain variable fragment (scFv) from a monoclonal antibody, which can provide receptors to tumors. Body specific - Relevant antigen on target malignant cells. After binding to tumor-associated antigens through chimeric antigen receptors, CAR-T cells generate an immune response that is cytotoxic to malignant cells. Theoretically, CAR-T cells can specifically locate and eliminate tumor cells by interacting with tumor-associated antigens (TAA) expressed on the surface of tumor cells.

Human epidermal growth factor receptor 2 (also known as Her2/neu or ErbB-2) is a 185 kDa cytoplasmic transmembrane tyrosine kinase receptor. It is encoded by the c-erbB-2 gene and is a member of the HER family (Ross et al., 2003). The HER family generally regulates cell growth and survival, as well as adhesion, migration, differentiation and other cellular responses. Overexpression and/or amplification of Her2/neu has been observed during the development of various solid cancers including breast, gastric, gastric, colorectal, ovarian, pancreatic, endometrial, and non-small cell lung cancers. .

Integrins are a group of heterodimeric bidirectional transmembrane cell surface receptors (Hynes, Cell, 2002;110:673-87). Although most integrins are required for proper biological function, some of them are highly localized and significantly upregulated only in diseased tissues. Among them, integrin αvβ6, α(v)β(6) has been considered an important marker during tumorigenesis (Bandyopadhyay et al., Current drug targets, 2009;10:645-52), and has even been identified for challenge Prognostic indicators related to disease severity in malignant tumors, such as lung cancer (Elayadi et al., Cancer Res., 2007;67:5889-95), colorectal cancer (Bates, Cell Cycle, 2005;4:1350-2), Cervical cancer (Hazelbag et al., J Pathol., 2007;212:316-24) and gastric cancer (Zhang et al., Clin Oncol-UK, 2008;20:61-6).

Currently, the use of CAR-T or similar cellular immunotherapy to treat tumors still faces many challenges, such as how to improve the safety of products targeting certain targets, prevent tumor escape, and activate the synergy of multiple anti-cancer mechanisms.

Contents of the invention

The present application provides a CAR molecule targeting tumor antigens (such as HER2) and integrins (integrin αvβ6). The tumor antigen-targeting portion comes from anti-tumor antigen antibodies, while the integrin-targeting portion comes from integrin ligands. integrin table It reaches a variety of solid tumors and targets two targets at the same time, helping to prevent tumor escape; CAR targeting integrins helps inhibit the production of TGF-β, thereby reducing the immunity of the tumor microenvironment (TME). Inhibition; the sequence combination of anti-tumor antigens and integrins can reduce the affinity of the antibody and help reduce the recognition of normal cells expressing low levels of tumor antigens, thus improving the safety of products targeting tumor antigen targets.

In one aspect, the present application provides a chimeric antigen receptor comprising an extracellular antigen-binding domain, wherein the extracellular antigen-binding domain comprises a first antigen-binding domain and a second antigen-binding domain, wherein the third antigen-binding domain An antigen-binding domain specifically binds the integrin.

In certain embodiments, the antigen-binding domain includes a ligand or functional fragment thereof, or an antibody or antigen-binding fragment thereof.

In certain embodiments, wherein the first antigen binding domain includes an integrin ligand or functional fragment thereof, or an anti-integrin antibody or antigen binding fragment thereof.

In certain embodiments, wherein the antibody or antigen-binding fragment thereof includes a full-length antibody, Fab, single chain variable fragment (scFv), di-scFv, or single domain antibody (VHH).

In certain embodiments, wherein the first antigen binding domain is located upstream, downstream and/or intermediate of the second antigen binding domain.

In certain embodiments, the first antigen binding domain is directly or indirectly linked to the second antigen binding domain.

In some embodiments, the indirect connection includes connection through a linker.

In certain embodiments, the linker includes a peptide linker.

In certain embodiments, the N-terminus of the first antigen-binding domain is directly or indirectly connected to the C-terminus of the second antigen-binding domain.

In certain embodiments, the C-terminus of the first antigen-binding domain is directly or indirectly connected to the N-terminus of the second antigen-binding domain.

In certain embodiments, when the second antigen-binding domain is scFv, the first antigen-binding domain is located between VH and VL of the second antigen-binding domain, and the first antigen-binding domain is connected to the second antigen-binding domain respectively. VH and VL of the antigen-binding domain are directly or indirectly linked.

In certain embodiments, when the first antigen-binding domain is scFv, the second antigen-binding domain is located between VH and VL of the first antigen-binding domain, and the second antigen-binding domain is respectively connected to the first antigen-binding domain. VH and VL of the antigen-binding domain are directly or indirectly linked.

In certain embodiments, the extracellular antigen binding domain comprises an integrin ligand and an anti-tumor antigen scFv antibody.

In certain embodiments, the N-terminus of the integrin ligand is directly or indirectly linked to the C-terminus of the anti-tumor antigen scFv antibody.

In certain embodiments, the C-terminus of the integrin ligand is directly or indirectly linked to the N-terminus of the anti-tumor antigen scFv antibody.

In certain embodiments, the integrin ligand is located between the VH and VL of the anti-tumor antigen scFv antibody, and the integrin ligand is directly or indirectly connected to the VH and VL of the anti-tumor antigen scFv antibody, respectively.

In certain embodiments, the integrin ligand and the anti-tumor antigen scFv antibody are linked through a linker.

In certain embodiments, the N-terminus of the integrin ligand is connected to the C-terminus of the anti-tumor antigen scFv antibody through a linker.

In certain embodiments, the C-terminus of the integrin ligand is connected to the N-terminus of the anti-tumor antigen scFv antibody through a linker.

In certain embodiments, the two ends of the integrin are respectively connected to the VH and VL of the anti-tumor antigen scFv antibody through linkers.

In certain embodiments, the integrins include integrins aberrantly expressed on the surface of tumor cells.

In certain embodiments, the integrin is selected from one or more of αIIβ3, α8β1, α5β1, αvβ1, αvβ3, αvβ5, αvβ6 and αvβ8.

In certain embodiments, wherein the integrin is αvβ6.

In certain embodiments, wherein the first antigen-binding domain includes an αvβ6 ligand, or a functional fragment thereof, or an anti-αvβ6 antibody, or an antigen-binding fragment thereof.

In certain embodiments, the αvβ6 ligand or functional fragment thereof comprises the amino acid sequence shown in any one of SEQ ID NO: 4-5.

In certain embodiments, wherein the second antigen binding domain specifically binds a tumor antigen.

In certain embodiments, wherein the tumor antigen is selected from: HER2, MUCl, ROR1, AFP, FAP, MAGA, MUC16, EphA2, ErbB, PSCA, IL13Rα2, EPCAM, EGFR, EGFRVIII, PSMA, GPC3, CEA, GD2 , one or more of Mesothelin, PD-L1, CD133, AXL, DLL3, LMP1, MG7, PMEL, ROR2, VEGFR2, CD171, CLD18, FRα and cMet.

In certain embodiments, wherein the tumor antigen is HER2.

In certain embodiments, wherein the second antigen-binding domain includes an anti-HER2 antibody or antigen-binding fragment thereof.

In certain embodiments, wherein the anti-HER2 antibody or antigen-binding fragment thereof comprises an antibody heavy chain variable region (VH), wherein the VH further comprises the amino acid sequence set forth in SEQ ID NO:2.

In certain embodiments, wherein the anti-HER2 antibody or antigen-binding fragment thereof comprises an antibody heavy chain variable region (VL), wherein the VL further comprises the amino acid sequence set forth in SEQ ID NO: 1.

In certain embodiments, wherein the second antigen binding domain includes an anti-HER2 scFv antibody.

In certain embodiments, wherein the anti-HER2 scFv antibody comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 2 , the VL includes the amino acid sequence shown in SEQ ID NO:1.

In certain embodiments, wherein said VH and VL are connected by a linker.

In certain embodiments, the anti-HER2 scFv antibody comprises the amino acid sequence set forth in SEQ ID NO:3.

In certain embodiments, the N-terminus of the αvβ6 ligand is directly or indirectly linked to the C-terminus of an anti-HER2 scFv antibody.

In certain embodiments, from N-terminus to C-terminus, wherein the extracellular antigen binding domain sequentially comprises anti-HER2 VH-anti-HER2 VL-αvβ6 ligand, or anti-HER2 VL-anti-HER2 VH-αvβ6 ligand.

In certain embodiments, the C-terminus of the αvβ6 ligand is directly or indirectly connected to the N-terminus of the anti-HER2 scFv antibody.

In certain embodiments, from the N-terminus to the C-terminus, the extracellular antigen-binding domain sequentially comprises αvβ6 ligand-anti-HER2 VH-anti-HER2 VL, or αvβ6 ligand-anti-HER2 VL-anti-HER2 VH.

In certain embodiments, the αvβ6 ligand is located between the VH and VL of an anti-HER2 scFv antibody, and the second antigen binding domain is directly or indirectly connected to the VH and VL of the first antigen binding domain, respectively.

In certain embodiments, from N terminus to C terminus, wherein the extracellular antigen binding domain sequentially comprises anti-HER2 VH-αvβ6 ligand-anti-HER2 VL, or anti-HER2 VL-αvβ6 ligand-anti-HER2 VH.

In certain embodiments, the αvβ6 ligand is connected to the VH or VL of an anti-HER2 scFv antibody through a linker.

In certain embodiments, the linker comprises the amino acid sequence shown in SEQ ID NO: 14.

In certain embodiments, the extracellular antigen binding domain comprises the amino acid sequence shown in any one of SEQ ID NOs: 15-20.

In certain embodiments, the chimeric antigen receptor further comprises a transmembrane domain comprising a transmembrane domain derived from one or more proteins selected from the group consisting of: CD8, CD28, CD3ε (CD3e), 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L (CD154), TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, SLAM and their variants.

In certain embodiments, wherein the transmembrane domain comprises a transmembrane domain derived from CD28 or a variant thereof.

In certain embodiments, the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 10.

In certain embodiments, the chimeric antigen receptor further comprises an intracellular signaling domain comprising a cellular protein derived from one or more proteins selected from the group consisting of: Internal signaling domain: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FceRIγ, FceRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, DAP10, DAP-12 and a domain containing at least one ITAM.

In certain embodiments, wherein the intracellular signaling domain comprises a signaling domain derived from CD3ζ.

In certain embodiments, the intracellular signal transduction domain comprises the amino acid sequence shown in SEQ ID NO: 13.

In certain embodiments, wherein the chimeric antigen receptor comprises an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain.

In certain embodiments, the chimeric antigen receptor further comprises an intracellular costimulatory signaling domain comprising one or more proteins derived from the group consisting of: Intracellular costimulatory signaling domains: CD28, 4-1BB (CD137), CD27, CD2, CD7, CD8A, CD8B, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3 , 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40, MyD88 and their variants.

In certain embodiments, wherein the intracellular costimulatory signaling domain is derived from a costimulatory signaling domain of CD28 or a variant thereof.

In certain embodiments, wherein the intracellular costimulatory signaling domain comprises the amino acid sequence shown in SEQ ID NO: 11-12.

In certain embodiments, the chimeric antigen receptor further comprises a hinge region located between the transmembrane domain and the extracellular antigen binding domain, the hinge region comprising: Hinge region of one or more proteins from the following group: CD28, CD8, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8A, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT and their variants.

In certain embodiments, the hinge region comprises an IgG1 Fc domain or a variant thereof.

In certain embodiments, the hinge region comprises the amino acid sequence shown in any one of SEQ ID NOs: 8-9.

In certain embodiments, from the N-terminus to the C-terminus, the non-targeting fragments of the chimeric antigen receptor comprise extracellular antibodies. Protobinding domain, hinge region, transmembrane domain, intracellular costimulatory signaling domain and intracellular signaling domain.

In certain embodiments, from N-terminus to C-terminus, the hinge region, transmembrane domain, intracellular costimulatory signaling domain and intracellular signaling domain of the chimeric antigen receptor comprise SEQ ID NO: 21 The amino acid sequence shown.

In certain embodiments, the chimeric antigen receptor further comprises a signal peptide fragment, the C-terminus of the signal peptide fragment being connected to the N-terminus of the extracellular antigen-binding domain.

In certain embodiments, the signal peptide fragment includes a CD8α signal peptide fragment.

In certain embodiments, the signal peptide fragment comprises the amino acid sequence shown in SEQ ID NO:7.

In certain embodiments, the chimeric antigen receptor comprises the amino acid sequence set forth in any one of SEQ ID NOs: 22-27.

On the other hand, the present application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric antigen receptor described in the present application.

On the other hand, the present application also provides a construct comprising the nucleic acid molecule described in the present application.

In another aspect, the application also provides isolated one or more constructs comprising a nucleic acid molecule encoding a first chimeric antigen receptor and a nucleic acid molecule encoding a second chimeric antigen receptor, wherein the first Chimeric antigen receptors contain extracellular antigen-binding domains that specifically bind integrins.

In certain embodiments, the extracellular antigen-binding domain that specifically binds integrin includes an integrin ligand or functional fragment thereof, or an anti-integrin antibody or antigen-binding fragment thereof.

In certain embodiments, the extracellular antigen-binding domain that specifically binds integrin includes an integrin ligand or a functional fragment thereof, an anti-integrin scFv antibody or an antigen-binding fragment thereof, an anti-integrin VHH antibody or a functional fragment thereof. Antigen-binding fragments.

In certain embodiments, wherein the integrin is αVβ6.

In certain embodiments, the extracellular antigen-binding domain that specifically binds integrins includes an αVβ6 ligand or a functional fragment thereof, or an anti-αVβ6 antibody or an antigen-binding fragment thereof.

In certain embodiments, wherein the second chimeric antigen receptor comprises an extracellular antigen binding domain that specifically binds a tumor antigen.

In certain embodiments, the extracellular antigen-binding domain that specifically binds a tumor antigen includes a tumor antigen antibody or antigen-binding fragment thereof.

In certain embodiments, wherein the tumor antigen is HER2.

In certain embodiments, the extracellular antigen-binding domain that specifically binds a tumor antigen includes an anti-HER2 antibody or antigen-binding fragment thereof.

In certain embodiments, wherein the anti-HER2 antibody or antigen-binding fragment thereof comprises an antibody heavy chain variable region (VH), wherein the VH further comprises the amino acid sequence set forth in SEQ ID NO: 2.

In certain embodiments, wherein the extracellular antigen binding domain that specifically binds a tumor antigen includes an anti-HER2 scFv antibody.

In some embodiments, the VH and the VL are connected through a linker.

In certain embodiments, the first chimeric antigen receptor and the second chimeric antigen receptor each further comprise a transmembrane domain comprising one or more species selected from the group consisting of: Transmembrane domain of protein: CD8, CD28, CD3ε (CD3e), 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D , 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L (CD154), TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, SLAM and their variants.

In certain embodiments, the first chimeric antigen receptor and the second chimeric antigen receptor each further comprise an intracellular signaling domain, the intracellular signaling domain comprising: Intracellular signaling domain of one or more proteins in the group: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FceRIγ, FceRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, Simian immunodeficiency virus PBj14Nef, DAP10, DAP-12 and domains containing at least one ITAM.

In certain embodiments, the first chimeric antigen receptor and the second chimeric antigen receptor each comprise an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain.

In certain embodiments, the first chimeric antigen receptor and the second chimeric antigen receptor each further comprise an intracellular costimulatory signaling domain, the intracellular costimulatory signaling domain comprising: Intracellular costimulatory signaling domain of one or more proteins from the following group: CD28, 4-1BB (CD137), CD27, CD2, CD7, CD8A, CD8B, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40, MyD88 and their variants.

In certain embodiments, the first chimeric antigen receptor and the second chimeric antigen receptor each further comprise a hinge region located between the transmembrane domain and the extracellular antigen binding domain. Between, the hinge region comprises a hinge region derived from one or more proteins selected from the group consisting of: CD28, CD8, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8A, PD-1 , ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT and their variants.

In certain embodiments, from N-terminus to C-terminus, each of the first chimeric antigen receptor and the second chimeric antigen receptor Contains extracellular antigen-binding domain, hinge region, transmembrane domain, intracellular costimulatory signaling domain and intracellular signal transduction domain.

In certain embodiments, from N-terminus to C-terminus, the hinge region, transmembrane domain, intracellular costimulatory signaling domain and intracellular signaling domain comprise the amino acid sequence shown in SEQ ID NO: 21.

In some embodiments, from the N-terminus to the C-terminus, the first chimeric antigen receptor includes an extracellular antigen-binding domain that specifically binds to integrins, a hinge region, a transmembrane domain, and an intracellular costimulatory signal. transduction domain and intracellular signaling domain.

In some embodiments, from the N-terminus to the C-terminus, the second chimeric antigen receptor includes an extracellular antigen-binding domain that specifically binds a tumor antigen, a hinge region, a transmembrane domain, and an intracellular costimulatory signal. transduction domain and intracellular signaling domain.

In certain embodiments, the first chimeric antigen receptor and the second chimeric antigen receptor further comprise a signal peptide fragment, the C-terminus of the signal peptide fragment is connected to the N-terminus of the extracellular antigen binding domain. connect.

In certain embodiments, the first chimeric antigen receptor comprises the amino acid sequence set forth in any one of SEQ ID NOs: 29-30.

In certain embodiments, the second chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO: 28.

In certain embodiments, the nucleic acid molecule encoding a first chimeric antigen receptor and the nucleic acid molecule encoding a second chimeric antigen receptor are in the same construct.

In certain embodiments, the nucleic acid molecule encoding a first chimeric antigen receptor and the nucleic acid molecule encoding a second chimeric antigen receptor are in separate constructs.

In certain embodiments, the construct includes an expression vector.

In certain embodiments, the constructs include DNA vectors, RNA vectors, plasmids, and/or viral vectors.

In certain embodiments, the viral vectors include lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, or retroviral vectors.

On the other hand, the present application provides a cell comprising the nucleic acid molecule described in the present application or the construct described in the present application, and/or expressing the chimeric antigen receptor described in the present application.

In certain embodiments, the cells include immune effector cells.

In certain embodiments, the immune effector cells include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and/or peripheral Blood mononuclear cells.

In certain embodiments, the cells include mammalian cells.

In certain embodiments, the cells include human cells.

In certain embodiments, the cells include autologous or non-autologous immune effector cells.

In certain embodiments, the immune cells include modified immune effector cells.

In certain embodiments, the modified immune cells include cells that reduce immune rejection resulting from allogeneic cell therapy.

In certain embodiments, the cells include CAR-T cells or CAR-NK cells.

On the other hand, the present application provides a pharmaceutical composition, which contains the chimeric antigen receptor described in the present application, the nucleic acid molecule described in the present application, the construct described in the present application or the cell described in the present application, and optionally a pharmaceutically acceptable carrier or excipient.

On the other hand, the present application provides the chimeric antigen receptor described in the present application, the nucleic acid molecule described in the present application, the construct described in the present application, the cell described in the present application, or the pharmaceutical combination described in the present application. The use of the substance in the preparation of medicaments for the prevention and/or treatment of tumors or autoimmune diseases.

In certain embodiments, the tumor includes a solid tumor and/or a hematological tumor.

In certain embodiments, the tumors include breast cancer, gastric cancer, ovarian cancer, cervical cancer, urothelial cancer, esophageal cancer, bladder cancer, colorectal cancer, endometrial cancer, renal cancer, lung cancer, pancreatic cancer , head and neck cancer, sarcoma, glioblastoma, prostate cancer, and/or thyroid cancer.

In certain embodiments, the tumor comprises a HER2-positive tumor.

On the other hand, the present application provides the chimeric antigen receptor described in the present application, the nucleic acid molecule described in the present application, the construct described in the present application, the cell described in the present application, or the pharmaceutical combination described in the present application. The use of the substance in the preparation of a medicament for preventing and/or treating diseases or conditions in which HER2 is abnormally expressed.

In certain embodiments, the disease or condition in which HER2 expression is abnormal includes a disease or condition in which HER2 expression is upregulated.

In certain embodiments, the disease or disorder wherein the expression of HER2 is abnormal includes a tumor.

In certain embodiments, the tumor comprises a HER2-positive tumor.

On the other hand, the present application provides a method for preventing and/or treating tumors, which includes administering to a subject in need an effective amount of the chimeric antigen receptor described in the present application, the nucleic acid molecule described in the present application , the construct described in this application, the cell described in this application, and/or the pharmaceutical composition described in this application.

On the other hand, the present application provides a method for preventing and/or treating autoimmune diseases, which includes providing A subject is administered an effective amount of the chimeric antigen receptor described herein, the nucleic acid molecule described herein, the construct described herein, the cell described herein, and/or the nucleic acid molecule described herein Pharmaceutical compositions.

On the other hand, the present application provides a method for preventing and/or treating diseases or conditions with abnormal expression of HER2, which includes administering an effective amount of the chimeric antigen receptor described in the present application to a subject in need thereof. The present application The nucleic acid molecule described in this application, the construct described in this application, the cell described in this application, and/or the pharmaceutical composition described in this application.

The skilled person can readily discern other aspects and advantages of the present application from the detailed description below. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will realize, the contents of this application enable those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention covered by this application. Accordingly, the drawings and descriptions of the present application are illustrative only and not restrictive.

Description of the drawings

The specific features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates can be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

Figure 1 shows a schematic structural diagram of the CAR that binds the Her2 antigen sequence and recognizes the αvβ6 sequence described in this application.

Figure 2 shows the results of the CAR described in this application recognizing single or dual targets.

Figure 3 shows the expression of the CAR described in this application on the surface of T cells.

Figure 4 shows the killing results of the CAR-T cells described in this application on the tumor cell MDA-MB-468 (HER2 ^- αvβ6 ⁺ ) when the effect-to-target ratio is 1:1.

Figure 5 shows the level of cytokine release after co-culture of CAR-T cells and MDA-MB-468 cell line described in this application at an effect-to-target ratio of 0.1:1.

Figure 6 shows the killing results of the CAR-T cells described in this application on tumor cells NCI-H358 (HER2 ⁺ αvβ6 ⁺ ) when the effect-to-target ratio is 1:1.

Figure 7 shows the level of cytokine release after co-culture of CAR-T cells and NCI-H358 cell line described in this application at an effect-to-target ratio of 1:1.

Figure 8 shows a schematic diagram of the B-NDG mouse in vivo test described in this application.

Figure 9A shows the inhibition of tumor growth in B-NDG mice by the CAR-T cells described in this application.

Figure 9B shows the effect of CAR-T cells described in this application on the body weight of B-NDG mice.

Figure 10A shows the killing results of the CAR-T cells described in this application on tumor cells NCI-H358 (HER2 ⁺ αvβ6 ⁺ ) when the effect-to-target ratio is 3:1.

Figure 10B shows the killing results of the CAR-T cells described in this application on the tumor cell MDA-MB-468 (HER2 ^- αvβ6 ⁺ ) when the effect-to-target ratio is 3:1.

Figure 11 shows the results of the affinity detection of the CAR-T cells described in this application to the target.

Figure 12 shows the killing of SKMEL28 cells (which only weakly express HER2) by the CAR-T cells described in this application.

Figure 13 shows a schematic structural diagram of the CAR targeting both CEA and αvβ6 dual targets described in this application.

Figure 14 shows a schematic structural diagram of the CAR targeting both HER2 and αvβ6 dual targets described in this application.

Figure 15 shows the results that Y002 CAR and Y007 CAR can recognize single or dual targets as expected after expression.

Figure 16 shows the results that Y021 CAR and Y022 CAR can recognize single or dual targets as expected after expression.

Figure 17 shows the results of flow cytometric detection of CEA expression on the cell membrane of tumor cell lines SKOV3 and BxPC-3.

Figure 18 shows the killing results of Y002 CAR-T cells and Y007 CAR-T cells against the tumor cell line SKOV3 (CEA-αvβ6+) when the effect-to-target ratio is 1:1.

Figure 19 shows the cytokine release levels after Y002 CAR-T cells and Y007 CAR-T cells were co-cultured with the tumor cell line SKOV3 (CEA-αvβ6+) at an effect-to-target ratio of 1:1.

Figure 20 shows the killing results of Y002 CAR-T cells and Y007 CAR-T cells against the tumor cell line BxPC-3 (CEA+αvβ6+) when the effect-to-target ratio is 1:1.

Figure 21 shows the cytokine release levels after Y002 CAR-T cells and Y007 CAR-T cells were co-cultured with the tumor cell line BxPC-3 (CEA+αvβ6+) at an effect-to-target ratio of 1:1.

Figure 22 shows the killing results of Y021 CAR-T cells and Y022 CAR-T cells against the tumor cell line MDA-MB-468 (HER2-αvβ6+) when the effect-to-target ratio is 1:1.

Figure 23 shows the cytokine release levels after Y021 CAR-T cells and Y022 CAR-T cells were co-cultured with the tumor cell line MDA-MB-468 (HER2-αvβ6+) at an effect-to-target ratio of 1:1.

Figure 24 shows the killing results of Y021 CAR-T cells and Y022 CAR-T cells against the tumor cell line BxPC-3 (HER2+αvβ6+) when the effect-to-target ratio is 1:1.

Figure 25 shows the cytokine release levels after Y021 CAR-T cells and Y022 CAR-T cells were co-cultured with the tumor cell line BxPC-3 (HER2+αvβ6+) at an effect-to-target ratio of 1:1.

Detailed ways

The implementation of the invention of the present application will be described below with specific examples. Those familiar with this technology can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

Definition of Terms

In this application, the term "chimeric antigen receptor" or "CAR" generally refers to a group of polypeptides, usually two in the simplest embodiment, that when in immune effector cells, provide cellular protection against target cells. (usually cancer cells) and generate intracellular signals. In some embodiments, the CAR comprises at least one extracellular antigen-binding domain (e.g., VHH, scFv, or portion thereof), a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain"). ”), which comprise a functional signaling domain derived from a stimulatory molecule and/or a costimulatory molecule as defined below. In some embodiments, the set of polypeptides are in the same polypeptide chain (eg, comprise a chimeric fusion protein). In some embodiments, the set of polypeptides are discontinuous with each other, such as in different polypeptide chains. In some aspects, the set of polypeptides includes a dimerization switch that can couple the polypeptides to each other in the presence of a dimerizing molecule, for example, can couple an antigen-binding domain to an intracellular signaling domain. On the one hand, the stimulatory molecule of CAR is the ζ chain associated with the T cell receptor complex. In one aspect, the cytoplasmic signaling domain includes a primary signaling domain (eg, the primary signaling domain of CD3-ζ). In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In one aspect, the costimulatory molecule can be selected from 4-1BB (i.e. CD137), CD27, ICOS and/or CD28. In one aspect, a CAR comprises a chimeric fusion protein, which may comprise an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one aspect, a CAR includes a chimeric fusion protein, which can include an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain that includes functional signaling structures derived from costimulatory molecules. domains and functional signaling domains derived from stimulatory molecules. In one aspect, the CAR includes a chimeric fusion protein, which can include an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain that includes functionality derived from one or more costimulatory molecules. Sexual signaling domains and functional signaling domains derived from stimulatory molecules. In one aspect, the CAR includes a chimeric fusion protein, which may comprise an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain comprising at least two co-stimulatory domains derived from one or more The functional signaling domain of the molecule and the functional signaling domain derived from the stimulatory molecule. In one aspect, the CAR includes an optional leader sequence at the amino terminus (N-ter) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally excised from the antigen recognition domain (e.g., VHH) during cellular processing and localizes the CAR to the cell membrane.

In this application, the term "antibody" is generally used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific Antibodies), and antibody fragments, as long as they display the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854-4861). Antibodies can be murine, human, humanized, chimeric, or derived from other species.

A full-length antibody typically refers to an antibody composed of two "full-length antibody heavy chains" and two "full-length antibody light chains." A "full-length antibody heavy chain" is generally a polypeptide consisting, in the N-terminal to C-terminal direction, of an antibody heavy chain variable domain (VH), an antibody constant heavy chain domain 1 (CH1), and an antibody hinge region (HR). , consisting of antibody heavy chain constant domain 2 (CH2), and antibody heavy chain constant domain 3 (CH3), abbreviated as VH-CH1-HR-CH2-CH3; and in the case of antibodies of the IgE subclass, optionally Also included is the antibody heavy chain constant domain 4 (CH4). In some embodiments, a "full-length antibody heavy chain" is a polypeptide consisting of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction. "Full-length antibody light chain" is usually a polypeptide composed of an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL) in the N-terminal to C-terminal direction, abbreviated as VL-CL. The antibody light chain constant domain (CL) may be kappa or lambda. The two full-length antibody chains are linked together by an inter-polypeptide disulfide bond between the CL domain and the CH1 domain and between the hinge region of the full-length antibody heavy chain. Examples of typical full-length antibodies are natural antibodies such as IgG (eg, IgG1 and IgG2), IgM, IgA, IgD, and IgE).

In this application, the term "antigen-binding fragment" (also referred to herein as "targeting portion" or "antigen-binding portion") generally refers to the portion of an antibody molecule that contains the protein responsible for the specificity between the antibody and the antigen. Combined amino acids. The portion of an antigen that is specifically recognized and bound by an antibody is called an "epitope" as described above. An antigen-binding domain may typically comprise an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH); however, it does not necessarily need to comprise both. Fd fragments, for example, have two VH regions and typically retain some of the antigen-binding functionality of the intact antigen-binding domain. Examples of antigen-binding fragments of antibodies include (1) Fab fragments, which are monovalent fragments with VL, VH, constant light chain (CL), and CH1 domains; (2) F(ab') ₂ fragments, which have two fragments consisting of a hinge region. Bivalent fragment of two Fab fragments connected by a sulfur bridge; (3) Fd fragment with two VH and CH1 domains; (4) Fv fragment with VL and VH domains of one arm of the antibody, (5) dAb fragment (Ward et al., "Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli," Nature 341:544-546 (1989), which is incorporated by reference in its entirety), which has a VH domain; (6) Isolated complementarity determining regions (CDRs); (7) Single chain Fv (scFv), for example derived from scFV-library. Although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods through synthetic linkers that allow them to be prepared as a single protein chain in which the VL and VH regions pair to form a monovalent molecule ( Referred to as single-chain Fv (scFv)) (see, e.g., Huston et al., "Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli," Proc. Natl. Acad . Sci. USA 85:5879-5883 (1988)); and (8) VHH, "VHH" relates to the variable antigen binding domain of a heavy chain antibody from the family Camelidae (camel, dromedary, llama, alpaca, etc.) (See Nguyen VK et al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001, J Biotechnol., 74, 277-302 and review Vanlandschoot P. et al., 2011, Antiviral Research 92, 389- 407). VHH can also be called Nanobody (Nb) and/or single domain antibody body. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the function of the fragments is assessed in the same manner as intact antibodies.

In this application, the term "single domain antibody" or "VHH" generally refers to a class of antibodies that lacks the light chain of the antibody and only has the variable region of the heavy chain. In some cases, the single domain antibody can be from Bactrian camels, dromedary camels, alpacas, llamas, nurse sharks, giant star sharks or rays (for example, see Kang Xiaozhen et al., Chinese Journal of Bioengineering, 2018, 34( 12):1974-1984). For example, a single domain antibody can be derived from alpaca. Single domain antibodies can be composed of a heavy chain variable region (VH). The term "heavy chain variable region" generally refers to the amino-terminal domain of the heavy chain of the antigen-binding fragment. The heavy chain variable region can be further distinguished into hypervariable regions called complementarity determining regions (CDRs), which are interspersed with more conserved regions known as framework regions (FRs). Each heavy chain variable region can be composed of three CDRs and four FR regions, which can be arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The heavy chain variable region contains the binding domain that interacts with the antigen.

In this application, the term "single chain variable fragment" or "scFv" has its ordinary and conventional meaning and may include, but is not limited to, for example, the heavy chain (VH) variable region and light chain (VL) containing immunoglobulins. ) fusion proteins of variable regions, which are connected to each other with a short linker peptide. Without limitation, the linker may contain glycine (for flexibility) and hydrophilic amino acids such as serine or threonine (for solubility). The connector may connect the N-terminal of VH to the C-terminal of VL, or may connect the C-terminal of VH to the N-terminal of VL. In some alternatives, the ligand binding domain present on the CAR is a single chain variable fragment (scFv). The choice of linker can affect the solubility, expression and correct folding of the scFv. Peptide linkers can vary in length from 10 to 25 amino acids and typically include hydrophilic amino acids such as glycine (G) and serine (S). Hydrophilic sequences prevent the insertion of peptides within or between variable domains throughout the protein folding process. The most commonly used linker is the (Gly4Ser)n motif, where n is any integer from 1 to 5, because of its flexibility, neutral charge, and solubility. It is known that scFv can become dimers, trimers or tetramers depending on linker length, antibody sequence and other factors (Le Gall F. et al., 1999). Such a format is advantageous and has many possible clinical applications. In scFv-based bsAb formats there are tandem scFvs, which consist of two scFvs connected by a flexible peptide linker, such as a glycine-serine repeat motif in a tandem orientation. The well-known bispecific T cell engager (BiTE) technology is based on this format (Chames P. et al., 2009).

The CAR of the present application can be constructed into a VH-VL or VL-VH configuration with variations in the linker, hinge, transmembrane domain, costimulatory domain, and/or conduction domain, and the CAR still maintains its efficacy. In some embodiments, the scFv domain present on the CAR is specific for HER2 and/or αvβ6 present on the tumor cell.

The CAR of the present application may include linker residues added between the various domains for proper spacing and conformation of the molecule, such as a linker including an amino acid sequence that connects the VH domain and the VL domain and provides interaction with the two sub-binding domains. The functionally compatible spacer functions such that the resulting polypeptide retains specific binding affinity for the same target molecule as an antibody containing the same light and heavy chain variable regions. The CAR of the present application may contain one, two, three, four or five or more linkers. in special In other embodiments, the linker is about 1 to about 25 amino acids in length, about 5 to about 20 amino acids in length, or about 10 to about 20 amino acids in length, or any intervening length of amino acids. Illustrative examples of linkers include glycine polymers; glycine-serine polymers; glycine-alanine polymers; alanine-serine polymers; other flexible linkers known in the art, such as Whitlow linkers. Glycine and glycine-serine polymers are relatively unstructured and thus can serve as neutral tethers between domains of a fusion protein (eg, the CAR of the present application).

In this application, the term "complementarity determining region" (CDR) generally refers to the complementarity determining region within the variable region of an antigen-binding fragment. In this application, there are three CDRs in the heavy chain variable region, and the CDRs are named HCDR1, HCDR2 and HCDR3 for each variable region. The exact boundaries of these CDRs have been defined differently depending on the system. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991))) not only provides that it can be applied to any variable region of an antigen-binding fragment. A clear residue numbering system also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and co-workers (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) found that despite large diversity at the amino acid sequence level, certain subparts within Kabat CDRs adopt nearly identical peptide backbone conformations. These subparts are named are L1, L2, and L3 or H1, H2, and H3, where "L" and "H" refer to the light and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs . Other boundaries defining CDRs that overlap with Kabat CDRs have been described by Padlan (FASEB J.9:133-139(1995)) and MacCallum (J Mol Biol 262(5):732-45(1996)). Additionally, others The definition of CDR boundaries may not strictly follow one of the above systems but will still overlap with Kabat CDRs, although following specific residues or groups of residues or even entire CDRs does not significantly affect the prediction or experimental finding of antigen binding, they can be shortened or lengthened. In this application, the IMGT numbering system is used.

In this application, the term "FR" generally refers to the more highly conserved portions of antibody variable domains, which are known as framework regions. For example, the variable domains of native heavy and light chains may each comprise four FR regions, namely four in VH (H-FR1, H-FR2, H-FR3 and H-FR4), and four in VL. (L-FR1, L-FR2, L-FR3 and L-FR4). "Framework regions" generally refer to the portions of antibody variable regions recognized in the art that exist between the more divergent (i.e., hypervariable) CDRs. Such framework regions are typically referred to as frameworks 1 to 4 (FR1, FR2, FR3 and FR4) and provide a backbone for the presentation of six CDRs (three from the heavy chain and three from the light chain) in three dimensions to Forms an antigen-binding surface.

In this application, the term "homology" may generally be equated with sequence "identity." Homologous sequences may include amino acid sequences that may be at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence. . Typically, homologs will contain active sites, etc. that are identical to the subject amino acid sequence. Homology can be considered in terms of similarity (i.e. amino acid residues with similar chemical properties/functions) or in terms of Homology is expressed in terms of sequence identity. In this application, reference to an amino acid sequence or a nucleotide sequence having a percent identity with any one of the SEQ ID NOs refers to a sequence having said percent identity over the entire length of the mentioned SEQ ID NO. the sequence of.

To determine sequence identity, sequence alignment can be performed, which can be performed by various means known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software, etc. One skilled in the art will be able to determine appropriate parameters for alignment, including any algorithms required to achieve optimal alignment across the full-length sequences being compared.

In this application, the terms "upstream" and "downstream" are functionally defined and generally refer to the direction or polarity of the encoding nucleotide sequence chain. The "upstream" direction means that the nucleotide is located in the 5' direction of a given polynucleotide sequence, i.e., towards the starting nucleotide. With respect to amino acid sequences, the term "upstream" is interpreted to mean amino acids located in the N-terminal direction, ie towards the beginning of the polypeptide chain.

In this application, the term "isolated nucleic acid molecule" generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides or their analogues of any length, isolated from their natural environment or artificially synthesized .

In this application, the term "construct" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells. The constructs may include vectors primarily for insertion of DNA or RNA into a cell, vectors primarily for replication of DNA or RNA, and vectors primarily for expression of transcription and/or translation of DNA or RNA. The vectors also include vectors having a variety of the above-mentioned functions. The construct may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce the desired expression product by culturing a suitable host cell containing the vector.

In this application, the term "expression vector" generally refers to a plasmid, phage, virus or vector used to express a polypeptide from a polynucleotide sequence. An expression vector may contain a transcription unit that contains an assembly of (1) one or more genetic elements that have a regulatory role in the expression of the gene, such as a promoter or enhancer, and (2) a structure or sequence encoding a binding agent, which transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences. Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence that enables the host cell to secrete the translated protein out of the cell. Alternatively, when expressing a recombinant protein without a leader or transport sequence, the recombinant protein will include an amino-terminal methionyl residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide the final peptide product.

In this application, the term "viral vector" is used broadly to refer to nucleic acid molecules (e.g., transfer plasmids) or viral particles that mediate the transfer of nucleic acids, including virus-derived nucleic acid elements that generally facilitate the transfer or integration of nucleic acid molecules into the genome of a cell. . Viral particles typically include various viral components and sometimes host cell components in addition to nucleic acids. Viral vectors can refer to viruses or viral particles capable of transferring nucleic acids into cells, or to the transferred nucleic acids themselves.

In this application, the term "lentivirus" generally refers to the group (or genus) of complex retroviruses. Exemplary lentiviruses include but Not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); visna-maedivirus (VMV) virus; caprine arthritis-encephalitis virus (CAEV); equine infectious disease anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). In one embodiment, HIV-based vector backbones (ie, HIV cis-acting sequence elements) are preferred. In particular embodiments, lentiviruses are used to deliver CAR-containing polynucleotides to cells.

As used herein, the term "host cell" or "cell" generally refers to an individual cell that can or has contained a vector comprising an isolated nucleic acid molecule as described herein, or is capable of expressing an isolated antigen-binding fragment as described herein. lines or cell cultures. The host cells may include progeny of a single host cell. Due to natural, accidental or intentional mutations, the progeny cells may not necessarily be morphologically or genomically identical to the original parent cells, as long as they are able to express the isolated antigen-binding fragments described in this application. The host cells can be obtained by transfecting cells in vitro using the vectors described in this application. The host cell can be a prokaryotic cell (such as Escherichia coli) or a eukaryotic cell (such as yeast cell, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells or myeloma cells). For example, the host cell may be an E. coli cell. For example, the host cell may be a yeast cell. For example, the host cell may be a mammalian cell. For example, the mammalian cells may be CHO-K1 cells.

In this application, the term "T cell" or "T lymphocyte" may be any T cell, such as a cultured T cell, such as a primary T cell, or a T cell from a cultured T cell line, such as Jurkat, SupTI, etc., or T cells obtained from a mammal (preferably a primate species, including monkey, dog or human). If obtained from a mammal, the T cells can be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells can also be enriched or cultured. T cells can be obtained by maturing hematopoietic stem cells into T cells in vitro or in vivo. In an exemplary aspect, the T cells are human T cells. In an exemplary aspect, the T cells are T cells isolated from humans. T cells can be any type of T cell, including NKT cells, and can be of any developmental stage, including but not limited to CD4+/CD8+ double-positive T cells; CDA+ helper T cells; e.g., Th1 and Th2 cells, CD8+ T cells (e.g. Cytotoxic T cells); peripheral blood mononuclear cells (PBMC); peripheral blood leukocytes (PBL); tumor infiltrating cells (TIL); memory T cells; unprocessed T cells, etc. Preferably, the T cells are CD8+ T cells or CD4+ T cells. In some alternatives, the T cells are allogeneic (from a different donor of the same species) to the recipient cell or subject to whom the cells are to be received (e.g., the cells are in the form of a therapeutic composition); in some alternatives In one approach, the T cells are autologous (the donor and recipient are the same); in some alternative approaches, the T cells are syngeneic (the donor and recipient are different, but identical twins).

In this application, the term "immune effector cells" generally refers to immune cells that participate in immune responses and perform effector functions. For example, the exercise of effector functions may include clearing foreign antigens or promoting immune effector responses. Immune effector cells can Including plasma cells, T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells and bone marrow-derived phagocytes.

The immune effector cells of the present application may be autologous/autogeneic ("own") or non-autologous ("non-own", such as allogeneic, syngeneic or allogeneic). In this application, the term "autologous" generally refers to cells from the same subject. "Allogeneic" generally refers to cells of the same species but genetically different than the cells being compared. "Isogenic" generally refers to cells from a different subject that are genetically identical to the cells being compared. "Allogeneic" generally refers to cells that are of a different species than the cells to which they are compared. In some embodiments, the cells of the present application are autologous or allogeneic.

In this application, the term "modification" generally refers to altering the state or structure of a cell and/or an alteration of the state or structure of a cell. The change is usually compared with the state or structure of the corresponding cell without the modification. The change may include changes in the expression level or function of endogenous genes, such as down-regulation of the expression level of endogenous genes in cells through genetic engineering means, Up-regulation or non-expression, the genetic engineering means may include homologous recombination, CRISPR/Cas9 system gene editing, etc.; the changes may also include changes in cellular protein expression, structure or function, such as through the endogenous gene expression level or Changes in corresponding protein expression, structure or function achieved by changes in function, such as changes in protein expression, structure or function achieved by regulating protein translation and post-translational modification; the changes may also include the introduction of exogenous Genes, expression of foreign proteins, etc.

In this application, the term "nucleic acid" or "polynucleotide" or "nucleic acid molecule" generally refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and their polymers in single- or double-stranded form. Unless specifically limited, the term may include nucleic acids containing analogs of natural nucleotides that have similar binding properties to a reference nucleic acid (e.g., sequence information is shown) and in a manner similar to naturally occurring nucleotides. metabolism. Unless otherwise stated, the sequence of a nucleic acid may include variants thereof modified in a conservative manner, such as degenerate codon substitutions, alleles, orthologs, SNPs, and complementary sequences, as well as the sequences specifically indicated.

In this application, the term "expression" generally refers to the transcription and/or translation of a specific nucleotide sequence.

In this application, the terms "tumor" and "cancer" are used interchangeably and generally refer to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body through the bloodstream and lymphatic system. Examples of various cancers are described herein and include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, Lung cancer, etc. The term "cancer" or "tumor" includes premalignant as well as malignant cancers and tumors and also encompasses solid and non-solid tumors.

In this application, the term "human epidermal growth factor receptor 2 (Her2/ErbB2)", also known as Her2/Neu, ErbB-2, CD340 or p185, usually refers to a tyrosine kinase active transmembrane Glycoprotein, belonging to the EGFR receptor family. The amino acid sequence of the human HER2 protein can be found in UniProt/Swiss-Prot accession number P04626. In this application, the isolated antigen-binding fragment can bind to HER2 protein. In this application, the terms "HER2 protein", "HER2 antigen" and "HER2-Fc recombinant protein" are used interchangeably and include any variant or isoform thereof naturally expressed by a cell. Her2 is a tyrosine kinase receptor (RTK), belonging to the epidermal growth factor receptor (EGFR/ErbB) family, which consists of 1255 amino acids, including four extracellular domains (I, II, III and IV), a It has a transmembrane region, a domain with tyrosine kinase activity and a carboxyl-terminal tail containing tyrosine residues and anchoring sites for intracellular signaling molecules. The molecular weight is approximately 185kD. EGFR family members have similar structures. Extracellular domains I and III are involved in the binding of receptors to ligands, causing receptor conformational changes and thus receptor activation. Extracellular domains II and IV are involved in receptor dimerization. Her2 has a special open structure. It can activate itself without the participation of specific ligands and form homodimers or heterodimers with other receptors in the EGFR family. It is a heterodimer between family members. The preferred molecule for polymerization.

In this application, the term "Integrin", also known as integrin, is a transmembrane receptor that mediates the connection between a cell and its external environment (such as extracellular matrix, Extra Cellular Matrix) , a large family of cell surface receptors commonly found on the cell surface of vertebrates that are responsible for mediating cell-cell and cell-extracellular matrix (ECM) adhesion. There are at least 24 different integrins, each a heterodimer composed of alpha and beta subunits. Non-limiting examples of integrins include: α1β1, α2β1, α3β1, α4β1, α5β1, α6β1, α7β1, α8β1, α9β1, α10β1, α11β1, αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, αIIbβ3, α4β7, αEβ7, α6β4, αLβ2 , αMβ2, αXβ2, αDβ2, and combinations thereof. In some embodiments, the integrin is αvβ3 integrin, αIIbβ3 integrin, or αvβ6 integrin. Some integrins are thought to play an active role in promoting certain diseases, including cancer. For example, αvβ3 integrin has been implicated in promoting the aggressive phenotype of melanoma and glioblastoma. For example, αvβ6 is highly upregulated in oral squamous cell carcinoma (OSCC), pancreatic, ovarian, and colon cancer.

αvβ6 integrin has been identified as a receptor for foot-and-mouth disease virus (FMDV) in vitro by binding to the RGD motif in the viral capsid protein VP1. One mode of binding of FMDV to cells is through a small 31-amino-acid loop on its protein shell. This FMDV loop binds to αvβ6 with high selectivity and specificity. PCT Publication No. WO07/039728 describes the radiolabeled αvβ6 targeting peptide FMDV2 A20, consisting of the 20 core amino acids of the FMDV loop, which binds immobilized human αvβ6 with high specificity and selectivity in a competitive ELISA binding assay.

As used herein, the term "pharmaceutically acceptable" generally refers to a term that is commensurate with a reasonable benefit/risk ratio, suitable within the scope of reasonable medical judgment for use in contact with human and animal tissue without undue toxicity, irritation, those compounds, materials, compositions and/or dosage forms that may cause allergic reactions or other problems or complications.

As used herein, the term "pharmaceutically acceptable carrier" generally refers to any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption delaying agents, surfactants, fillers, disintegrating agents agent, adhesive, thinner Lubricants, glidants, pH adjusters, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, etc. compatible with drug administration. The use of such media and reagents for pharmaceutically active substances is well known in the art. The compositions may contain other active compounds that provide complementary, additional or enhanced therapeutic functions.

In this application, the term "effective amount" or "effective dose" generally refers to an amount sufficient to achieve, or at least partially achieve, the desired effect. A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is generally one that, when used alone or in combination with another therapeutic agent, promotes resolution of disease (either through a reduction in the severity of disease symptoms, the frequency of asymptomatic periods of disease An amount of any drug that is evidenced by an increase in the intensity and duration of the disease, or by the prevention of impairment or disability due to the disease.

In this application, the term "comprises" generally means including, encompassing, containing or encompassing. In some cases, it also means "for" or "composed of".

In this application, the term "about" generally refers to a variation within the range of 0.5% to 10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.

In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats or monkeys, and the like.

Detailed description of the invention

chimeric antigen receptor

In certain embodiments, the antigen-binding domain includes a ligand or functional fragment thereof, or an antibody or antigen-binding fragment thereof. For example, the first antigen-binding domain may be a ligand or a functional fragment thereof, and the second antigen-binding domain may be an antibody or an antigen-binding fragment thereof. For another example, the first antigen-binding domain and the second antigen-binding domain can both be ligands or functional fragments thereof. For another example, the first antigen-binding domain and the second antigen-binding domain may both be antibodies or antigen-binding fragments thereof.

For example, the first antigen binding domain may include an integrin ligand, an anti-integrin scFv antibody, or an anti-integrin VHH antibody.

In certain embodiments, the linker includes a peptide linker. For example, flexible and/or soluble peptide linkers, such as glycine- and serine-rich peptide linkers.

For example, linkers rich in glycine and serine (and/or threonine) include at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 % or 99% of such amino acids. In some embodiments, it includes at least or at least about 50%, 55%, 60%, 70%, or 75% glycine, serine, and/or threonine. In some embodiments, the linker consists essentially entirely of glycine, serine, and/or threonine. The length of the linker is generally between about 5 and about 50 amino acids, usually between 10 or about 10 and 30 or about 30 amino acids, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, and in some instances between 10 and 25 amino acids in length. Exemplary linkers include linkers having various numbers of repeats of the sequence GGGGS (G ₄ S; SEQ ID NO: 31), such as between 2, 3, 4, and 5 repeats of such sequence. Exemplary linkers include those having or consisting of the sequence shown in SEQ ID NO: 14 (GGGGSGGGGSGGGGSGGGGS).

In some embodiments, the linker has an amino acid sequence containing the sequence set forth in SEQ ID NO: 14. A fragment (eg, scFv) may include a VH region or a portion thereof, followed by a linker, followed by a VL region or a portion thereof. A fragment (eg, scFv) may include a VL region, or a portion thereof, followed by a linker, followed by a VH region, or a portion thereof.

In certain embodiments, the N-terminus of the first antigen-binding domain is directly or indirectly connected to the C-terminus of the second antigen-binding domain. For example, the first antigen-binding domain is located downstream of the second antigen-binding domain, and the N-terminus of the first antigen-binding domain and the C-terminus of the second antigen-binding domain can be connected through a peptide linker.

In certain embodiments, the C-terminus of the first antigen-binding domain is directly or indirectly connected to the N-terminus of the second antigen-binding domain. For example, the first antigen-binding domain is located upstream of the second antigen-binding domain, and the C-terminus of the first antigen-binding domain and the N-terminus of the second antigen-binding domain can be connected through a peptide linker.

For example, the first antigen-binding domain and the second antigen-binding domain may be connected in the following ways:

i) Second antigen binding domain VH - First antigen binding domain - Second antigen binding domain VL; or ii) Second antigen binding domain VL-first antigen-binding domain-second antigen-binding domain VH; where "-" represents a peptide linker or is absent.

i) first antigen binding domain VH - second antigen binding domain - first antigen binding domain VL; or ii) first antigen binding domain VL - second antigen binding domain - first antigen binding domain VH; where "-" represents Peptide linker or not present.

In certain embodiments, the two ends of the integrin ligand are respectively connected to the VH and VL of the anti-tumor antigen scFv antibody through linkers.

For example, from N-terminus to C-terminus, the extracellular antigen-binding domain may include the following structure:

i) anti-tumor antigen antibody VH-anti-tumor antigen antibody VL-integrin ligand;

ii) anti-tumor antigen antibody VL-anti-tumor antigen antibody VH-integrin ligand;

iii) Integrin ligand-anti-tumor antigen antibody VH-anti-tumor antigen antibody VL;

iv) Integrin ligand-anti-tumor antigen antibody VL-anti-tumor antigen antibody VH;

v) Anti-tumor antigen antibody VH-integrin ligand-anti-tumor antigen antibody VL; or

vi) Anti-tumor antigen antibody VL-integrin ligand-anti-tumor antigen antibody VH;

where "-" represents the peptide linker or its absence.

In certain embodiments, the extracellular antigen binding domain comprises an anti-integrin VHH antibody and an anti-tumor antigen scFv antibody.

In certain embodiments, the N-terminus of the anti-integrin VHH antibody is directly or indirectly linked to the C-terminus of the anti-tumor antigen scFv antibody.

In certain embodiments, the C-terminus of the anti-integrin VHH antibody is directly or indirectly linked to the N-terminus of the anti-tumor antigen scFv antibody.

In certain embodiments, the anti-integrin VHH antibody is located between the VH and VL of the anti-tumor antigen scFv antibody, and the anti-integrin VHH antibody is directly or indirectly connected to the VH and VL of the anti-tumor antigen scFv antibody, respectively. connect.

In certain embodiments, the anti-integrin VHH antibody and the anti-tumor antigen scFv antibody are linked by a linker.

In certain embodiments, the N-terminus of the anti-integrin VHH antibody is connected to the C-terminus of the anti-tumor antigen scFv antibody through a linker.

In certain embodiments, the C-terminus of the anti-integrin VHH antibody is connected to the N-terminus of the anti-tumor antigen scFv antibody through a linker.

In certain embodiments, both ends of the anti-integrin VHH antibody are respectively connected to the VH and VL of the anti-tumor antigen scFv antibody through linkers.

i) anti-tumor antigen antibody VH-anti-tumor antigen antibody VL-anti-integrin VHH antibody;

ii) anti-tumor antigen antibody VL-anti-tumor antigen antibody VH-anti-integrin VHH antibody;

iii) anti-integrin VHH antibody-anti-tumor antigen antibody VH-anti-tumor antigen antibody VL;

iv) Anti-integrin VHH antibody-anti-tumor antigen antibody VL-anti-tumor antigen antibody VH;

v) anti-tumor antigen antibody VH-anti-integrin VHH antibody-anti-tumor antigen antibody VL; or

vi) Anti-tumor antigen antibody VL-anti-integrin VHH antibody-anti-tumor antigen antibody VH;

where "-" represents the peptide linker or its absence.

In certain embodiments, the extracellular antigen binding domain comprises an integrin ligand and an anti-tumor antigen VHH antibody.

In certain embodiments, the N-terminus of the integrin ligand is directly or indirectly linked to the C-terminus of the anti-tumor antigen VHH antibody.

In certain embodiments, the C-terminus of the integrin ligand is directly or indirectly linked to the N-terminus of the anti-tumor antigen VHH antibody.

In certain embodiments, the N-terminus of the integrin ligand is connected to the C-terminus of the anti-tumor antigen VHH antibody through a linker.

In certain embodiments, the C-terminus of the integrin ligand is connected to the N-terminus of the anti-tumor antigen VHH antibody through a linker.

i) anti-tumor antigen antibody VHH-integrin ligand; or ii) integrin ligand-anti-tumor antigen antibody VHH; where "-" represents a peptide linker or is absent.

In certain embodiments, the extracellular antigen binding domain comprises an anti-integrin VHH antibody and an anti-tumor antigen VHH antibody.

In certain embodiments, the N-terminus of the anti-integrin VHH antibody is directly or indirectly linked to the C-terminus of the anti-tumor antigen VHH antibody.

In certain embodiments, the C-terminus of the anti-integrin VHH antibody is directly or indirectly linked to the N-terminus of the anti-tumor antigen VHH antibody.

In certain embodiments, the N-terminus of the anti-integrin VHH antibody and the C-terminus of the anti-tumor antigen VHH antibody are connected through a linker.

In certain embodiments, the C-terminus of the anti-integrin VHH antibody and the N-terminus of the anti-tumor antigen VHH antibody are connected through a linker.

i) anti-tumor antigen antibody VHH-anti-integrin VHH antibody; or ii) anti-integrin VHH antibody-anti-tumor antigen antibody VHH; where "-" represents a peptide linker or is absent.

In certain embodiments, the extracellular antigen binding domain comprises an anti-integrin scFv antibody and an anti-tumor antigen VHH antibody.

In certain embodiments, the N-terminus of the anti-integrin scFv antibody is directly or indirectly linked to the C-terminus of the anti-tumor antigen VHH antibody.

In certain embodiments, the C-terminus of the anti-integrin scFv antibody is directly or indirectly linked to the N-terminus of the anti-tumor antigen VHH antibody.

In certain embodiments, the anti-tumor antigen VHH antibody is located between the VH and VL of the anti-integrin scFv antibody, and the anti-tumor antigen VHH antibody is directly or indirectly connected to the VH and VL of the anti-integrin scFv antibody, respectively. connect.

In certain embodiments, the anti-tumor antigen VHH antibody and the anti-integrin scFv antibody are linked by a linker.

In certain embodiments, the N-terminus of the anti-tumor antigen VHH antibody is connected to the C-terminus of the anti-integrin scFv antibody through a linker.

In certain embodiments, the C-terminus of the anti-tumor antigen VHH antibody is connected to the N-terminus of the anti-integrin scFv antibody through a linker.

In certain embodiments, both ends of the anti-tumor antigen VHH antibody are respectively connected to the VH and VL of the anti-integrin scFv antibody through linkers.

i) anti-integrin antibody VH-anti-integrin antibody VL-anti-tumor antigen VHH antibody;

ii) anti-integrin antibody VL-anti-integrin antibody VH-anti-tumor antigen VHH antibody;

iii) anti-tumor antigen VHH antibody-anti-integrin antibody VH-anti-integrin antibody VL;

iv) anti-tumor antigen VHH antibody-anti-integrin antibody VL-anti-integrin antibody VH;

iii) anti-integrin antibody VH-anti-tumor antigen VHH antibody-anti-integrin antibody VL;

iv) anti-integrin antibody VL-anti-tumor antigen VHH antibody-anti-integrin antibody VH;

where "-" represents the peptide linker or its absence.

In certain embodiments, the anti-tumor antigen scFv antibody and the anti-integrin scFv antibody are linked by a linker.

In certain embodiments, the N-terminus of the anti-tumor antigen scFv antibody and the C-terminus of the anti-integrin scFv antibody are connected through a linker.

In certain embodiments, the C-terminus of the anti-tumor antigen scFv antibody and the N-terminus of the anti-integrin scFv antibody are connected through a linker.

In certain embodiments, both ends of the anti-tumor antigen scFv antibody are respectively connected to the VH and VL of the anti-integrin scFv antibody through linkers.

In certain embodiments, both ends of the anti-integrin scFv antibody are respectively connected to the VH and VL of the anti-tumor antigen scFv antibody through linkers.

i) anti-integrin antibody VH-anti-integrin antibody VL-anti-tumor antigen VH-anti-tumor antigen VL;

ii) anti-integrin antibody VL-anti-integrin antibody VH-anti-tumor antigen VH-anti-tumor antigen VL;

iii) anti-tumor antigen VH-anti-tumor antigen VL-anti-integrin antibody VH-anti-integrin antibody VL;

iv) anti-tumor antigen VH-anti-tumor antigen VL anti-integrin antibody VL-anti-integrin antibody VH;

iii) anti-integrin antibody VH-anti-tumor antigen VH-anti-tumor antigen VL-anti-integrin antibody VL; or

iv) anti-tumor antigen VH-anti-integrin antibody VH-anti-integrin antibody VL-anti-tumor antigen VL;

where "-" represents the peptide linker or its absence.

In certain embodiments, wherein the integrin is αvβ6.

In certain embodiments, wherein the αvβ6 ligand or functional _fragment thereof can _be _represented by the following formula _: B _m _RGDLX ₁ For amino acids, n is a number between 1 and 35, and m is a number between 1 and 35. Preferably, m is chosen such that B is long enough to promote a core of hydrophobic/non-covalent interactions. The exact nature of these residues depends on the overall design of the region. In particular, preference is given to hydrophobic interactions (from residues such as Val, Ile, Leu) and/or electrostatic interactions (using Asp, Glu, Lys and/or Arg and their counterparts at X1 and/or X2 material ion pair).

In certain embodiments, wherein the αvβ6 ligand or functional fragment thereof comprises or consists of an amino acid sequence selected from the group consisting of: NAVPNLRGDLQVLAQKVART (FMDV2 A20; SEQ ID NO: 4), NAVPNLRGDLQVLAQKVA G T(FMDV2 A20(R19G); SEQ ID NO:5), NAVPNLRGDLQVLAQ R VART(FMDV2 A20(K16R); SEQ ID NO:32), GFTTGRRGDLATIHGMNRPF(LAP A20; SEQ ID NO:33), YTASARGDLAHLTTTHARHL(FMDV1A20; SEQ ID NO:34), and combinations thereof.

In certain embodiments, wherein the tumor antigen is selected from: HER2, MUCl, ROR1, AFP, FAP, MAGA, MUC16, EphA2, ErbB, PSCA, IL13Rα2, EPCAM, EGFR, EGFRVIII, PSMA, GPC3, CEA, GD2, Mesothelin, PD-L1, CD133, AXL, DLL3, LMP1, MG7, PMEL, ROR2, VEGFR2, CD171, One or more of CLD18, FRα and cMet.

In certain embodiments, wherein the tumor antigen is HER2.

In certain embodiments, wherein the anti-HER2 antibody or antigen-binding fragment thereof comprises an antibody heavy chain variable region (VH), wherein the VH further comprises the amino acid sequence set forth in SEQ ID NO: 2 or is identical to SEQ ID NO :2 The sequence shown exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or Sequence with 99% sequence identity and retains the ability to bind to HER2.

In certain embodiments, wherein the anti-HER2 antibody or antigen-binding fragment thereof comprises an antibody heavy chain variable region (VL), wherein the VL further comprises the amino acid sequence set forth in SEQ ID NO: 1 or is identical to SEQ ID NO :1 The sequence shown exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or Sequence with 99% sequence identity and retains the ability to bind to HER2.

In certain embodiments, wherein the anti-HER2 scFv antibody comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 2 Or exhibit at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the sequence shown in SEQ ID NO:2 %, 98% or 99% sequence identity, the VL comprising the amino acid sequence shown in SEQ ID NO: 1 or exhibiting at least 85%, 86%, 87%, 88% with the sequence shown in SEQ ID NO: 1 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

For example, the anti-HER2 scFv antibody can comprise the amino acid sequence set forth in SEQ ID NO:3 or exhibit at least 85%, 86%, 87%, 88%, 89%, 90% of the sequence set forth in SEQ ID NO:3 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

In certain embodiments, from N-terminus to C-terminus, the extracellular antigen-binding domain sequentially includes αvβ6 ligand-anti-HER2 VH-anti-HER2 VL, or αvβ6 ligand-anti-HER2 VL-anti-HER2 VH.

For example, the extracellular antigen-binding domain may comprise an αvβ6 ligand or a functional fragment thereof and an anti-HER2 scFv antibody.

i) anti-HER2 antibody VH-anti-HER2 antibody VL-αvβ6 ligand;

ii) anti-HER2 antibody VL-anti-HER2 antibody VH-αvβ6 ligand;

iii) αvβ6 ligand-anti-HER2 antibody VH-anti-HER2 antibody VL;

iv) αvβ6 ligand-anti-HER2 antibody VL-anti-HER2 antibody VH;

v) Anti-HER2 antibody VH-αvβ6 ligand-anti-HER2 antibody VL; or

vi) Anti-HER2 antibody VL-αvβ6 ligand-anti-HER2 antibody VH;

where "-" represents the peptide linker or its absence.

In certain embodiments, wherein the second antigen binding domain includes an anti-HER2 VHH antibody.

For example, the extracellular antigen binding domain may comprise an αvβ6 ligand or a functional fragment thereof and an anti-HER2 VHH antibody.

i) anti-HER2 VHH antibody-αvβ6 ligand; or ii) αvβ6 ligand-anti-HER2 VHH antibody; where "-" represents a peptide linker or is absent.

In this application, the term "chimeric antigen receptor (CAR)" generally refers to a receptor that does not exist in nature and can confer immune effector cell specificity for a particular antigen.

In some embodiments, the CAR comprises: i) an extracellular antigen-binding domain; ii) a transmembrane domain; and iii) an intracellular signaling domain.

In the CAR, the (i) extracellular antigen-binding domain includes the above-described antibody of the present application or a functional fragment thereof, and it contains an antigen-binding region. Antibodies for CAR are preferably in the form of antibody fragments, more preferably in the form of Fab or scFv form, but not particularly limited thereto.

In the CAR, the (ii) transmembrane domain may be in a form linked to an extracellular domain, and may be derived from a natural or synthetic transmembrane domain. When derived from natural occurrence, it can be derived from a membrane-bound protein or a membrane-permeable protein, which can be derived from a variety of proteins (such as the alpha, beta or theta chain of the T cell receptor, CD28, CD3ε, CD45, CD4, A portion of the membrane permeable region of CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 or CD8, but not limited thereto). Sequences of such transmembrane domains can be obtained from literature known in the art for which portions of the membrane-permeable regions of membrane-permeable proteins are known.

In the CAR, the (iii) intracellular signal transduction domain may exist within the cell in a form linked to a transmembrane domain. The intracellular signal transduction domain of the present application is a region that generates or/and transmits signals that primarily cause cell activation when an antigen binds to the antigen-binding site of the CAR. The intracellular signal transduction domain is not particularly limited in type as long as it can transmit a signal that can cause activation of cells (especially immune cells) when an antigen binds to an antigen-binding site located outside the cell. Various types of intracellular signaling domains can be used, such as immunoreceptor tyrosine-based activation motifs or ITAMs, and ITAMs can include CD3θ (ξ, θ), TCRθ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b, CD278, CD66d, DAP10, DAP12, FcεRI (especially γ), and combinations thereof (one or more than two), but are not limited thereto.

In addition, depending on the cell type, the CAR of the present application may further include a costimulatory domain together with the intracellular signal transduction domain, but is not limited thereto. The costimulatory domain is a part included in the CAR of the present application, and plays a role in transmitting the maximum activation signal in addition to the primary signal to the corresponding cells through the intracellular signal transduction domain. It refers to the intracellular part of the CAR that contains the intracellular domain of the costimulatory molecule. That is, some immune cells (such as T lymphocytes and NK cells) require 2 signals (i.e., primary activation signal and costimulatory signal) to achieve maximum activation. The CAR may also optionally contain a costimulatory domain such that binding of the antigen to the extracellular domain results in the transmission of primary activating and costimulatory signals.

A costimulatory molecule is a cell surface molecule, which means a molecule necessary to elicit a sufficient response of an immune cell to an antigen, and its kind is not particularly limited as long as it is known in the art. For example, it may be selected from the group consisting of: ligands that specifically bind to MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins and cytokine receptors, integrins, SLAM proteins (Signal Transduction Lymphocytes Activating molecules), NK cell activation receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18, lymphocyte function related Antigen-1), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30 , NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1( CD226), SLAMF4 (CD244, 2B4), CD84, CD96, CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, CD83, PD-1, and combinations thereof. The costimulatory domain may be an intracellular portion of a molecule selected from the group consisting of such costimulatory molecules and combinations thereof (one or more than two).

The costimulatory domain can be linked to the N-terminus or C-terminus of the signal transduction domain and can be included in multiple signal transduction domains. For example, from N-terminus to C-terminus, the CAR sequentially includes: extracellular antigen-binding domain, transmembrane domain, intracellular costimulatory signal domain and intracellular signal transduction domain.

Each domain that makes up a CAR can be linked directly or via a polypeptide linker. Even if the CAR of the present application contains a linker, its length is not particularly limited if it is a linker that can induce T cell activation through the intracellular domain when the antigen binds to the antigen-binding domain located outside the cell.

For example, from N-terminus to C-terminus, the chimeric antigen receptor may include the following structure:

i) Anti-HER2 antibody VH-anti-HER2 antibody VL-αvβ6 ligand-IgG1 Fc(hinge)-CD28(TM/CD)-CD3ζ;

ii) Anti-HER2 antibody VL-anti-HER2 antibody VH-αvβ6 ligand-IgG1 Fc(hinge)-CD28(TM/CD)-CD3ζ;

iii) αvβ6 ligand-anti-HER2 antibody VH-anti-HER2 antibody VL-IgG1 Fc(hinge)-CD28(TM/CD)-CD3ζ;

iv) αvβ6 ligand-anti-HER2 antibody VL-anti-HER2 antibody VH-IgG1 Fc(hinge)-CD28(TM/CD)-CD3ζ;

v) Anti-HER2 antibody VH-αvβ6 ligand-anti-HER2 antibody VL-IgG1 Fc(hinge)-CD28(TM/CD)-CD3ζ;

vi) Anti-HER2 antibody VL-αvβ6 ligand-anti-HER2 antibody VH-IgG1 Fc(hinge)-CD28(TM/CD)-CD3ζ;

where "-" represents the peptide linker or its absence.

For another example, from N-terminus to C-terminus, the chimeric antigen receptor may include the following structure:

i) anti-HER2 VHH antibody-αvβ6 ligand-IgG1 Fc(hinge)-CD28(TM/CD)-CD3ζ; or ii) αvβ6 ligand-anti-HER2 VHH antibody--IgG1 Fc(hinge)-CD28(TM/CD )-CD3ζ;

where "-" represents the peptide linker or its absence.

i) CD8α-anti-HER2 antibody VH-anti-HER2 antibody VL-αvβ6 ligand-IgG1 Fc(hinge)-CD28(TM/CD)-CD3ζ;

ii) CD8α-anti-HER2 antibody VL-anti-HER2 antibody VH-αvβ6 ligand-IgG1 Fc(hinge)-CD28(TM/CD)-CD3ζ;

iii) CD8α-αvβ6 ligand-anti-HER2 antibody VH-anti-HER2 antibody VL-IgG1 Fc(hinge)-CD28(TM/CD)-CD3ζ;

iv) CD8α-αvβ6 ligand-anti-HER2 antibody VL-anti-HER2 antibody VH-IgG1 Fc(hinge)-CD28(TM/CD)-CD3ζ;

v) CD8α-anti-HER2 antibody VH-αvβ6 ligand-anti-HER2 antibody VL-IgG1 Fc(hinge)-CD28(TM/CD)-CD3ζ;

vi) CD8α-anti-HER2 antibody VL-αvβ6 ligand-anti-HER2 antibody VH-IgG1 Fc(hinge)-CD28(TM/CD)-CD3ζ;

where "-" represents the peptide linker or its absence.

In certain embodiments, the signal peptide fragment comprises the amino acid sequence shown in SEQ ID NO:7. For example, the chimeric antigen receptor may comprise the amino acid sequence shown in any one of SEQ ID NOs: 22-27.

Isolated nucleic acid molecules and constructs

On the other hand, the present application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric antigen receptor described in the present application. The base combination of the nucleic acid molecule is not particularly limited as long as it encodes a CAR protein, and can be produced by polynucleotide synthesis techniques known in the art. When an amino acid sequence is identified, techniques for generating a nucleotide sequence encoding the amino acid sequence based on codon information known in the art are well known in the art. The isolated nucleic acid molecules may be provided as single-stranded or double-stranded nucleic acid molecules containing all DNA, cDNA and RNA sequences.

In certain embodiments, wherein the integrin is αVβ6.

In certain embodiments, the αVβ6 ligand or functional fragment thereof comprises the amino acid sequence shown in any one of SEQ ID NOs: 4-5, 32-34.

In certain embodiments, wherein the tumor antigen is HER2.

For example, the isolated construct or constructs comprise a nucleic acid molecule encoding a first chimeric antigen receptor and a nucleic acid molecule encoding a second chimeric antigen receptor, wherein the first chimeric antigen receptor comprises a specific The second chimeric antigen receptor comprises an extracellular antigen-binding domain that specifically binds a tumor antigen.

For example, the isolated construct or constructs comprise a nucleic acid molecule encoding a first chimeric antigen receptor and a nucleic acid molecule encoding a second chimeric antigen receptor, wherein the first chimeric antigen receptor comprises a specific The second chimeric antigen receptor includes an extracellular antigen-binding domain that specifically binds to a tumor antigen.

For example, the isolated construct or constructs comprise a nucleic acid molecule encoding a first chimeric antigen receptor and a nucleic acid molecule encoding a second chimeric antigen receptor, wherein the first chimeric antigen receptor comprises a specific The second chimeric antigen receptor includes an extracellular antigen-binding domain that specifically binds to HER2.

In certain embodiments, wherein the anti-HER2 antibody or antigen-binding fragment thereof comprises an antibody heavy chain variable region (VH), wherein the VH further comprises the amino acid sequence set forth in SEQ ID NO: 2 or is identical to SEQ ID NO :2 The sequence shown exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or Sequences with 99% sequence identity.

In certain embodiments, wherein the anti-HER2 antibody or antigen-binding fragment thereof comprises an antibody heavy chain variable region (VL), wherein the VL further comprises the amino acid sequence set forth in SEQ ID NO: 1 or is identical to SEQ ID NO :2 The sequence shown exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or Sequences with 99% sequence identity.

For example, the isolated construct or constructs comprise a nucleic acid molecule encoding a first chimeric antigen receptor and a nucleic acid molecule encoding a second chimeric antigen receptor, wherein the first chimeric antigen receptor comprises a specific an extracellular antigen-binding domain that specifically binds to αVβ6, the second chimeric antigen receptor comprising an extracellular antigen-binding domain that specifically binds to HER2; wherein the extracellular antigen-binding domain that specifically binds to integrin includes αVβ6 A ligand or a functional fragment thereof, the extracellular antigen-binding domain that specifically binds a tumor antigen includes an anti-HER2 scFv antibody.

In some embodiments, the VH and the VL are connected through a linker.

In certain embodiments, wherein the anti-HER2 scFv antibody comprises the amino acid sequence set forth in SEQ ID NO:3 or shares at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, Sequences with 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

For example, the isolated construct or constructs comprise a nucleic acid molecule encoding a first chimeric antigen receptor and a nucleic acid molecule encoding a second chimeric antigen receptor, wherein the first chimeric antigen receptor comprises a specific an extracellular antigen-binding domain that specifically binds αVβ6, the second chimeric antigen receptor comprising an extracellular antigen-binding domain that specifically binds HER2; wherein the extracellular antigen-binding domain that specifically binds integrin comprises SEQ The amino acid sequence shown in any one of ID NO: 4-5, 32-34, the extracellular antigen-binding domain that specifically binds to tumor antigens includes the amino acid sequence shown in SEQ ID NO: 3.

In certain embodiments, wherein the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 10 or shares at least 85%, 86%, 87%, 88%, 89% with the sequence set forth in SEQ ID NO: 10 Sequences with %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

In certain embodiments, wherein the intracellular signal transduction domain comprises the amino acid sequence set forth in SEQ ID NO: 13 or shares at least 85%, 86%, 87%, Sequences with 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

In certain embodiments, the first chimeric antigen receptor and the second chimeric antigen receptor each further comprise an intracellular costimulatory signaling domain, the intracellular costimulatory signaling domain comprising: one or more proteins from the following group Intracellular costimulatory signaling domain: CD28, 4-1BB (CD137), CD27, CD2, CD7, CD8A, CD8B, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40, MyD88 and their variants.

In certain embodiments, wherein the intracellular costimulatory signaling domain comprises the amino acid sequence shown in any one of SEQ ID NO: 11-12 or is identical to the amino acid sequence shown in any one of SEQ ID NO: 11-12 The sequence exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity the sequence of.

In certain embodiments, the hinge region comprises the amino acid sequence shown in any one of SEQ ID NO:8-9 or exhibits at least 85%, Sequences with 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

In certain embodiments, from the N-terminus to the C-terminus, the first chimeric antigen receptor and the second chimeric antigen receptor each comprise an extracellular antigen-binding domain, a hinge region, a transmembrane domain, an intracellular cofactor, Stimulatory signaling domain and intracellular signaling domain.

In certain embodiments, from the N-terminus to the C-terminus, the hinge region, transmembrane domain, intracellular costimulatory signaling domain and intracellular signaling domain comprise the amino acid sequence shown in SEQ ID NO: 21 or Exhibit at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98% or 99% sequence identity.

In certain embodiments, from the N-terminus to the C-terminus, the second chimeric antigen receptor contains The extracellular antigen-binding domain, hinge region, transmembrane domain, intracellular costimulatory signaling domain and intracellular signaling domain of the antigen.

For example, the isolated construct or constructs contain a nucleic acid molecule encoding a first chimeric antigen receptor and a nucleic acid molecule encoding a second chimeric antigen receptor, from N-terminus to C-terminus, wherein the first chimeric antigen receptor The first chimeric antigen receptor of the chimeric antigen receptor sequentially includes an extracellular antigen-binding domain that specifically binds to integrins, a hinge region, a transmembrane domain, an intracellular costimulatory signaling domain, and an intracellular signaling structure. Domain, the second chimeric antigen receptor sequentially includes an extracellular antigen-binding domain that specifically binds a tumor antigen, a hinge region, a transmembrane domain, an intracellular costimulatory signaling domain, and an intracellular signaling domain.

For example, the isolated construct or constructs contain a nucleic acid molecule encoding a first chimeric antigen receptor and a nucleic acid molecule encoding a second chimeric antigen receptor, from N-terminus to C-terminus, wherein the first chimeric antigen receptor The chimeric antigen receptor includes an extracellular antigen-binding domain, a hinge region, a transmembrane domain, an intracellular costimulatory signaling domain and an intracellular signaling domain that specifically binds αVβ6, and the second chimeric antigen receptor It contains an extracellular antigen-binding domain, a hinge region, a transmembrane domain, an intracellular costimulatory signaling domain and an intracellular signaling domain that specifically binds to HER2.

For example, the isolated construct or constructs contain a nucleic acid molecule encoding a first chimeric antigen receptor and a nucleic acid molecule encoding a second chimeric antigen receptor, from N-terminus to C-terminus, wherein the first chimeric antigen receptor The chimeric antigen receptor includes an extracellular antigen-binding domain, a hinge region, a transmembrane domain, an intracellular costimulatory signaling domain and an intracellular signaling domain containing an αVβ6 ligand or a functional fragment thereof, and the second chimeric antigen receptor The combined antigen receptor includes an extracellular antigen-binding domain, a hinge region, a transmembrane domain, an intracellular costimulatory signaling domain and an intracellular signaling domain containing an anti-HER2 scFv antibody; wherein the specific binding integrin The extracellular antigen-binding domain includes the amino acid sequence shown in any one of SEQ ID NO:4-5, 32-34 or displays at least one amino acid sequence shown in any one of SEQ ID NO:4-5, 32-34. Sequences with 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, The extracellular antigen-binding domain that specifically binds to a tumor antigen comprises the amino acid sequence shown in SEQ ID NO: 3 or exhibits at least 85%, 86%, 87%, 88%, or Sequences with 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

For example, the isolated construct or constructs comprise a nucleic acid molecule encoding a first chimeric antigen receptor and a nucleic acid molecule encoding a second chimeric antigen receptor, the first chimeric antigen receptor and the second chimeric antigen receptor Chimeric antigen receptors can be combinations of:

First chimeric antigen receptor: αvβ6 ligand-IgG1 Fc(hinge)-CD28(TM/CD)-CD3ζ; and

Second chimeric antigen receptor: anti-HER2 antibody VH-anti-HER2 antibody VL-IgG1 Fc(hinge)-CD28(TM/CD)-CD3ζ or anti-HER2 antibody VL-anti-HER2 antibody VH-IgG1 Fc(hinge)-CD28 (TM/CD)-CD3ζ.

For example, the isolated construct or constructs contain a nucleic acid molecule encoding a first chimeric antigen receptor and a nucleic acid molecule encoding a second chimeric antigen receptor, from N-terminus to C-terminus, wherein the first chimeric antigen receptor The chimeric antigen receptor sequentially includes a signal peptide fragment, an extracellular antigen-binding domain that specifically binds to integrins, a hinge region, a transmembrane domain, an intracellular costimulatory signaling domain, and an intracellular signaling domain, wherein the third The two chimeric antigen receptors sequentially include a signal peptide fragment, an extracellular antigen-binding domain that specifically binds tumor antigens, a hinge region, a transmembrane domain, an intracellular costimulatory signaling domain, and an intracellular signaling domain.

For example, the isolated construct or constructs contain a nucleic acid molecule encoding a first chimeric antigen receptor and a nucleic acid molecule encoding a second chimeric antigen receptor, from N-terminus to C-terminus, wherein the first chimeric antigen receptor The chimeric antigen receptor includes a signal peptide fragment, an extracellular antigen-binding domain that specifically binds αVβ6, a hinge region, a transmembrane domain, an intracellular costimulatory signaling domain and an intracellular signaling domain, and the second chimeric antigen receptor The combined antigen receptor includes a signal peptide fragment, an extracellular antigen-binding domain that specifically binds to HER2, a hinge region, a transmembrane domain, an intracellular costimulatory signaling domain, and an intracellular signaling domain.

For example, the isolated construct or constructs contain a nucleic acid molecule encoding a first chimeric antigen receptor and a nucleic acid molecule encoding a second chimeric antigen receptor, from N-terminus to C-terminus, wherein the first chimeric antigen receptor The chimeric antigen receptor includes a signal peptide fragment, an extracellular antigen-binding domain containing αVβ6 ligand or a functional fragment thereof, a hinge region, a transmembrane domain, an intracellular costimulatory signaling domain and an intracellular signaling domain, so The second chimeric antigen receptor includes a signal peptide fragment, an extracellular antigen-binding domain containing an anti-HER2 scFv antibody, a hinge region, a transmembrane domain, an intracellular costimulatory signaling domain and an intracellular signaling domain; wherein The extracellular antigen-binding domain that specifically binds to integrins includes the amino acid sequence shown in any one of SEQ ID NO: 4-5, 32-34 or is the same as any of SEQ ID NO: 4-5, 32-34. One of the indicated sequences exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or Sequences with 99% sequence identity, the extracellular antigen-binding domain that specifically binds tumor antigens includes the amino acid sequence shown in SEQ ID NO:3 or exhibits at least 85% or 86% of the sequence identity with the sequence shown in SEQ ID NO:3 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

First chimeric antigen receptor: CD8α-αvβ6 ligand-IgG1 Fc(hinge)-CD28(TM/CD)-CD3ζ; and

Second chimeric antigen receptor: CD8α-anti-HER2 antibody VH-anti-HER2 antibody VL-IgG1 Fc(hinge)-CD28(TM/CD)-CD3ζ or CD8α-anti-HER2 antibody VL-anti-HER2 antibody VH-IgG1 Fc( hinge)-CD28(TM/CD)-CD3ζ.

In certain embodiments, the first chimeric antigen receptor comprises an amino acid sequence shown in any one of SEQ ID NO: 29-30 and a sequence representation shown in any one of SEQ ID NO: 29-30 Sequences with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity .

In certain embodiments, the second chimeric antigen receptor comprises an amino acid sequence shown in SEQ ID NO: 28 that is at least 85%, 86%, 87%, 88% identical to the sequence shown in SEQ ID NO: 28 , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

For example, the isolated construct or constructs comprise a nucleic acid molecule encoding a first chimeric antigen receptor and a nucleic acid molecule encoding a second chimeric antigen receptor, and the first chimeric antigen receptor may comprise SEQ. The amino acid sequence shown in any one of ID NO: 29-30, the second chimeric antigen receptor may include the amino acid sequence shown in SEQ ID NO: 28.

In certain embodiments, the construct includes an expression vector. In this application, "expression" generally means the production of a protein or nucleic acid in a cell.

In this application, an "expression vector" is a vector capable of expressing a protein or nucleic acid (RNA) of interest in a suitable host cell, and refers to a vector containing the necessary elements operably linked to allow the insertion of an expressible polynucleotide (gene). Adjustment element. "Operably linked" refers to a functional connection between a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein or RNA of interest to perform a general function, and means that a gene is expressed by being linked to an expression control sequence. Operable linkage to the expression vector can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes commonly known in the art. "Expression control sequence" refers to a DNA sequence that controls the expression of an operably linked polynucleotide sequence in a particular host cell. Such regulatory sequences include promoters for transcription, any operator sequences for regulating transcription, sequences encoding appropriate mRNA ribosome binding sites, sequences regulating termination of transcription and translation, initiation codons, termination Codons, polyadenylation signals, and enhancers. The promoter of the vector can be constitutive or inducible. Operably linked gene sequences and expression control sequences may be contained in a single expression vector containing a selectable marker and/or origin of replication for selection of host cells containing the vector. In addition, expression vectors containing A signal sequence or leader sequence for membrane targeting or secretion, and can be prepared in a variety of ways depending on the purpose.

The type of expression vector of the present application is not particularly limited as long as it is a commonly used vector in the field of cloning, and examples thereof include, but are not limited to, plasmid vectors, cosmid vectors, phage vectors, and viral vectors. Plasmids include plasmids derived from E. coli (pBR322, pBR325, pUC118 and pUC119, pET-22b(+)), plasmids derived from Bacillus subtilis (pUB110 and pTP5) and plasmids derived from yeast ( YEp13, YEp24 and YCp50). As the virus, animal viruses such as retrovirus, adenovirus or vaccinia virus, and insect viruses such as baculovirus can be used, and pcDNA can be used.

In the present application, the expression vector includes a polynucleotide encoding a first chimeric antigen receptor; a polynucleotide encoding a second chimeric antigen receptor may be provided in a form that is simultaneously included (inserted) in one vector, or may Provided by each being contained (inserted) in two (including different types) vectors.

cell

In certain embodiments, the cell comprises a polynucleotide encoding a chimeric antigen receptor; the chimeric antigen receptor comprises an extracellular antigen binding domain, wherein the extracellular antigen binding domain comprises a first antigen a binding domain and a second antigen-binding domain, wherein the first antigen-binding domain specifically binds integrin.

In certain embodiments, the cell contains a polynucleotide encoding a first chimeric antigen receptor; and a polynucleotide encoding a second chimeric antigen receptor may be simultaneously included (inserted) in one vector. A construct provided in a form is introduced, or a plurality of constructs provided in a form in which polynucleotides are separately contained (inserted) in each of 2 vectors can be introduced into one or more cells; wherein the first insert Synthetic antigen receptors specifically bind integrins.

In the present application, cells may include heterologous nucleic acid molecules (such as encoding the chimeric nucleic acid molecules of the present application) introduced into the cells by any means (such as electroporation, calphosphatase precipitation, microinjection, transformation, viral infection, etc.). Prokaryotic or eukaryotic cells containing polynucleotides containing antigen receptors).

In some embodiments, introduction of a heterologous nucleic acid molecule is accomplished by first stimulating a cell, such as by subjecting the cell to induction of, for example, proliferation, survival, and/or activation (e.g., as measured by expression of cytokines or activation markers). The activated cells are then transduced and expanded in culture to a number sufficient for clinical use.

Various methods for introducing genetically engineered components (eg, CARs) are well known and can be used with the provided methods and compositions. Exemplary methods include methods for transferring nucleic acid encoding a receptor, including via viruses (eg, retroviruses or lentiviruses), transduction, transposons, and electroporation.

In some embodiments, recombinant infectious viral particles (such as, for example, vectors derived from simian virus 40 (SV40), adenovirus, adeno-associated virus (AAV)) are used to transfer the recombinant polynucleotide into cells. In some embodiments, recombinant polynucleotides are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy April 2014 3rd. doi: 10.1038/gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol TherNucl Acids 2, e93; Park et al., Trends Biotechnol. November 29, 2011(11):550-557).

In some embodiments, recombinant polynucleotides are transferred into T cells via electroporation (see, e.g., Chicaybam et al., (2013) PLoS ONE 8(3):e60298 and Van Tedeloo et al., (2000) Gene Therapy7(16) :1431-1437). In some embodiments, the recombinant polynucleotide is transferred into T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4):427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2 , e74; and Huang et al. (2009) Methods Mol Biol 506:115-126). Other methods of introducing and expressing genetic material into immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.), protoplast fusion, cationic liposome-mediated Transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7:2031-2034 (1987)) .

The type of (host) cell of the present application is not particularly limited as long as it can be used to express the polynucleotide encoding the antibody or fragment thereof contained in the construct of the present application. Transformed cells (host cells) with constructs according to the present application can be prokaryotes (e.g. E. coli), eukaryotes (e.g. yeast or other fungi), plant cells (e.g. tobacco or tomato plant cells), animal cells (e.g. human cells, monkey cells, hamster cells, rat cells and mouse cells), insect cells or hybridomas derived therefrom. For example, they may be cells derived from mammals, including humans.

The cells are generally eukaryotic cells, such as mammalian cells, and often human cells. In some embodiments, the cells are derived from blood, bone marrow, lymph or lymphoid organs and are immune system cells, such as innate or adaptive immune cells, such as bone marrow or lymphocytes, including lymphocytes, typically T cells and/or NK cell. Other exemplary cells include stem cells, such as multipotent stem cells and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells are typically primary cells, such as those isolated directly from the individual and/or isolated and frozen from the individual. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as the full T cell population, CD4+ cells, CD8+ cells, and subsets thereof, such as those cells according to the following definitions: function, activation state , maturity, differentiation potential, expansion, recycling, location and/or persistence, antigen specificity, antigen receptor type, presence in specific organs or compartments, marker or cytokine secretion profile and/or degree of differentiation . With reference to the subject being treated, the cells may be allogeneic and/or autologous. Included among such methods are off-the-shelf methods. In some aspects, the As in off-the-shelf technology, the cells are pluripotent and/or multipotent cells, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, such methods include isolating cells from a subject, preparing, processing, culturing and/or engineering them as described herein, and reintroducing them to the same patient before or after cryopreservation middle.

In certain embodiments, the cells include immune effector cells. The type of immune cells (immune effector cells) is not particularly limited as long as they are cells known in the art to participate in human immune function.

In certain embodiments, the immune effector cells include T cells, B cells, NK (natural killer) cells, macrophages, NKT (natural killer T) cells, monocytes, dendritic cells, granulocytes, Lymphocytes, leukocytes, and/or peripheral blood mononuclear cells.

In some embodiments, the cells are T cells. The term "T cells" generally refers to lymphocytes that originate from the thymus and play a major role in cellular immunity. T cells include CD4 ⁺ T cells (helper T cells, TH cells), CD8 ⁺ T cells (cytotoxic T cells, CTL), memory T cells, regulatory T cells (Treg cells), and natural killer T cells. In this application, the T cells introduced into the CAR may preferably be CD8 ⁺ T cells, but are not limited thereto.

In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.

In certain embodiments, the cells include CAR-T cells or CAR-NK cells.

pharmaceutical composition

On the other hand, the present application provides a pharmaceutical composition, which contains the chimeric antigen receptor described in the present application, the nucleic acid molecule described in the present application, the construct described in the present application or the cell described in the present application, and optionally a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" refers to the ingredients of a pharmaceutical composition other than the active ingredients that are non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

In some aspects, the choice of vector is determined in part by the particular cell, nucleic acid molecule, and/or by the method of administration. Therefore, a variety of suitable vectors exist. For example, pharmaceutical compositions may contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. Preservatives or mixtures thereof are generally present in an amount of from about 0.0001% to about by weight of the total composition. Present in an amount of 2%. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosage and concentration used, and include (but are not limited to): buffers, such as phosphoric acid, citric acid and other organic acids; antioxidants, including ascorbic acid and methyl sulfide. Acid; preservatives (such as stearyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; parabens Esters, such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues ) polypeptide; protein, such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer, such as polyvinylpyrrolidone; amino acid, such as glycine, glutamine, asparagine, histidine, arginine or Lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions , such as sodium; metal complexes (eg, Zn-protein complexes); and/or nonionic surfactants, such as polyethylene glycol (PEG).

In some aspects, a buffering agent is included in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffers is used. Buffers or mixtures thereof are typically present in an amount from about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams &Wilkins; 21st Edition (May 1, 2005).

Pharmaceutical compositions of the present application may also contain more than one active ingredient suitable for the particular indication, disease or condition for which the cell is being treated (e.g., those having activities that are complementary to that of the cell), where each activity is mutually exclusive No adverse effects. Such active ingredients are suitably present in combination and amounts effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents.

Application is carried out using standard application techniques, formulations and/or devices. Formulations and devices for storing and administering the compositions, such as syringes and vials, are provided. Administration of cells can be autologous or allogeneic. For example, immunoreactive cells or progenitor cells can be obtained from one subject and administered to the same subject or to a different compatible subject. Immunoreactive cells or progeny thereof derived from peripheral blood (eg, derived in vivo, ex vivo, or ex vivo) can be administered via local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. When a therapeutic composition (eg, a pharmaceutical composition containing genetically modified immunoreactive cells) is administered, it will generally be formulated in a unit dose injectable form (solution, suspension, emulsion).

Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell population is administered parenterally. The term "parenteral" as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal, intracranial, intrathoracic and intraperitoneal administration. In some embodiments, the cell population is administered to the subject via intravenous, intraperitoneal, or subcutaneous injection using peripheral systemic delivery. use.

In some embodiments, the compositions are provided as sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which, in some aspects, can be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, particularly by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range to enhance longer contact time with specific tissues. Liquid or viscous compositions may include a carrier, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

Sterile injectable solutions may be prepared by incorporating the binding molecule into a solvent, such as mixed with a suitable carrier, diluent, or excipient, such as sterile water, physiological saline, dextrose, dextrose, or the like. preparation. The composition can also be lyophilized. Depending on the route of administration and the desired formulation, the compositions may contain auxiliary substances such as wetting, dispersing or emulsifying agents (e.g. methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavorings agents, pigments and the like. In some aspects, standard texts may be consulted for preparation of suitable formulations.

Various additives may be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents and buffers. Protection against microbial activity can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption such as aluminum monostearate and gelatin.

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibodies in the form of shaped articles such as films or microcapsules.

Formulations for in vivo administration are generally sterile. Sterility can be readily achieved by, for example, filtration through sterile filtration membranes.

Uses and methods

In certain embodiments, the tumor comprises a HER2-positive tumor.

On the other hand, the present application provides chimeric antigen receptors described in the present application, nucleic acid molecules described in the present application, and The use of the construct, the cells described in this application, or the pharmaceutical compositions described in this application in the preparation of medicines for preventing and/or treating diseases or conditions with abnormal expression of HER2.

In certain embodiments, the tumor comprises a HER2-positive tumor.

Non-limiting examples of different types of tumors suitable for treatment with cells or compositions of the present application include ovarian cancer, breast cancer, lung cancer, bladder cancer, thyroid cancer, liver cancer, pleural cancer, pancreatic cancer, cervical cancer, prostate cancer , testicular cancer, colon cancer, anal cancer, bile duct cancer, gastrointestinal carcinoid tumor, esophageal cancer, gallbladder cancer, rectal cancer, appendix cancer, small bowel cancer, stomach (stomach) cancer, kidney cancer (such as renal cell carcinoma) ), central nervous system cancer, skin cancer, choriocarcinoma, head and neck cancer, bone cancer, osteosarcoma, fibrosarcoma, neuroblastoma, glioma, melanoma, leukemia (such as acute lymphoblastic leukemia, chronic lymphocytic leukemia leukemia, acute myeloid leukemia, chronic myeloid leukemia, or hairy cell leukemia), lymphoma (such as non-Hodgkin lymphoma, Hodgkin lymphoma, B-cell lymphoma, or Burkitt's lymphoma), and multiple myeloma.

On the other hand, the present application provides a method for preventing and/or treating autoimmune diseases, which includes administering an effective amount of the chimeric antigen receptor described in the present application to a subject in need thereof. The nucleic acid molecules, the constructs described in this application, the cells described in this application, and/or the pharmaceutical compositions described in this application.

Autoimmune diseases generally include diseases or pathologies caused by immune responses (eg, autoantibody responses or cell-mediated responses) directed against self-tissue or tissue components. Examples of autoimmune diseases suitable for treatment using the cells or pharmaceutical compositions of the present application include, but are not limited to, organ-specific autoimmune diseases in which the autoimmune response is directed against a single tissue, such as type I diabetes, myasthenia gravis, vitiligo, Graves Stalin's disease, Hashimoto's disease, Addison's disease, autoimmune gastritis, and autoimmune hepatitis; and autoimmune responses Non-organ-specific autoimmunity directed against components present in several or many organs throughout the body Diseases such as systemic lupus erythematosus, progressive systemic sclerosis and variants, polymyositis, and dermatomyositis. Additional autoimmune diseases include, for example, pernicious anemia, primary biliary cirrhosis, autoimmune thrombocytopenia, Sjogren's syndrome, and multiple sclerosis.

On the other hand, the present application provides a method for preventing and/or treating diseases or conditions with abnormal expression of HER2, which includes administering an effective amount of the chimeric antigen receptor described in the present application to a subject in need thereof. The present application Described nucleic acid molecule, this The construct described in the application, the cell described in the application, and/or the pharmaceutical composition described in the application.

Methods for administering cells for adoptive cell therapy are known and can be used in conjunction with the provided methods and compositions. For example, adoptive T cell therapy is described in, for example, Gruenberg et al., U.S. Patent Application Publication No. 2003/0170238; Rosenberg, U.S. Patent No. 4,690,915; Rosenberg (2011) Nat Rev ClinOncol. 8(10):577- 85) in. See e.g. 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.

In some embodiments, cell therapy (e.g., adoptive cell therapy, such as adoptive T cell therapy) is performed by autologous transfer, wherein cells are isolated and/or otherwise prepared from the subject to receive the cell therapy, or from The cells are isolated and/or otherwise prepared from a sample derived from the subject. Thus, in some aspects, cells are derived from a subject (eg, a patient) in need of treatment, and the cells are administered to the same subject after isolation and processing.

In some embodiments, cell therapy (e.g., adoptive cell therapy, such as adoptive T cell therapy) is performed by allogeneic transfer from a subject other than the subject to receive or ultimately receive the cell therapy. A person (eg, a first subject) isolates and/or otherwise prepares the cells. In such embodiments, such cells are subsequently administered to a different subject of the same species (eg, 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 subtype as the first subject.

In some embodiments, the subject to which the cells, cell populations, or compositions are administered is a primate, such as a human. In some embodiments, the subject to which the cells, cell populations, or compositions are administered is a non-human primate. In some embodiments, the non-human primate is a monkey (eg, macaque) or ape. The subject can be male or female and can be a subject of any suitable age (including infant, juvenile, young adult, adult, and elderly). In some embodiments, the subject is a non-primate mammal, such as a rodent (eg, mouse, rat, etc.). In some examples, the patient or subject is a validated animal model for disease, adoptive cell therapy, and/or for assessing toxic outcomes, such as cytokine release syndrome (CRS).

The cells may be administered by any suitable means, such as by injection, such as intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, Intracameral injection, subconjunctival injection, subconjunctival injection, subtenon bulbar injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, it is administered by parenteral, intrapulmonary and intranasal administration, and if necessary for local treatment, by intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, intracranial, intrathoracic, or subcutaneous administration. Dosing and administration may depend in part on whether the administration is transient or long-term. Various dosing schedules include (But not limited to) single administration or multiple administrations at various time points, bolus administration, and pulse infusion.

For the prevention or treatment of disease, the appropriate dosage of the binding molecule, recombinant receptor or cell may depend on the type of disease to be treated, the type of binding molecule or recombinant receptor, the severity and course of the disease, the binding molecule or recombinant receptor Whether administration is for prophylactic or therapeutic purposes, prior therapies, patient clinical history and response to recombinant receptors or cells, and the judgment of the attending physician. In some embodiments, the compositions and molecules and cells are suitable for administration to a patient either once or over a series of treatments.

In some embodiments, toxicity and/or side effects of treatment can be monitored and used to adjust the dose and/or frequency of administration of the CAR, cells, and/or compositions. For example, adverse events and laboratory abnormalities can be monitored and used to adjust the dose and/or frequency of administration. Adverse events include infusion reactions, cytokine release syndrome (CRS), neurotoxicity, macrophage activation syndrome, and tumor lysis syndrome (TLS). Any one of these events may establish dose-limiting toxicity and warrant dose reduction and/or discontinuation of treatment. Other side effects or adverse events that may be used as a guide to establishing dosage and/or frequency of administration include non-hematologic adverse events, which include (but are not limited to) fatigue, pyrexia or febrile neutropenia, increased transaminases, persistent immobility Duration (e.g., less than or equal to 2 weeks or less than or equal to 7 days), headache, bone pain, hypotension, hypoxia, chills, diarrhea, nausea/vomiting, neurotoxicity (e.g., confusion, aphasia, seizures, convulsions , lethargy and/or altered state of consciousness), disseminated intravascular coagulation, other asymptomatic non-hematological clinical laboratory abnormalities such as electrolyte abnormalities. Other side effects or adverse events that may be used as a guide in establishing dosage and/or frequency of administration include hematologic adverse events, including (but not limited to) neutropenia, leukopenia, thrombocytopenia, animal and/or Or B cell hypoplasia and hypogammaglobinemia (hypogammaglobinemia).

In some embodiments, treatment according to provided methods can result in a lower rate of toxicity and/or a lower degree of toxicity, toxicity outcomes or symptoms, profiles, factors or properties that promote toxicity, such as symptoms or outcomes associated with or are indicative of symptoms or results of: cytokine release syndrome (CRS) or neurotoxicity, such as severe CRS or severe neurotoxicity, such as compared to administration of other therapies.

In some embodiments, for example, where the subject is a human, the dose includes more than about 1× ¹⁰ total CAR-expressing cells, T cells, or peripheral blood mononuclear cells (PBMC) and less than about 2×10 ⁹ total CAR-expressing cells, T cells or peripheral blood mononuclear cells (PBMC), such as in the range of about 2.5×10 ⁷ to about 1.2×10 ⁹ such cells, such as 2.5×10 ⁷ , 5×10 ⁷ , 1.5×10 ⁸ , 3×10 ^{8 , 4.5×10 8} ^, 8×10 ⁸ or 1.2×10 ⁹ total such cells, or a range between any two of the above values.

In some embodiments, the dose of genetically engineered cells includes between at or about 2.5 x ¹⁰ CAR-expressing T cells, total T cells, or total peripheral blood mononuclear cells (PBMC) and at or about 1.2 x 10 ⁹ CAR-expressing T cells, total T cells, or total PBMC, between at or about 5.0×10 ⁷ CAR-expressing T cells and at or about 4.5×10 ⁸ CAR-expressing T cells of T cells, total T cells, or total peripheral blood mononuclear cells (PBMC), between at or about 1.5 × 10 ⁸ CAR-expressing T cells and at or about 3.0 × 10 ⁸ CAR-expressing T cells, Between total T cells or total PBMC (endpoint values included).

In some embodiments, the number is with respect to the total number of CD3+ or CD8+, and in some cases also with respect to the CAR-expressing cells. In some embodiments, the dose comprises from about 2.5 x ¹⁰ to or to about 1.2 x ¹⁰ CD3 ⁺ or CD8 ⁺ total T cells or CD3 ⁺ CAR-expressing cells or CD8 ⁺ CAR-expressing cells, inclusive value). In some embodiments, the dose of T cells includes CD4 ⁺ T cells, CD8 ⁺ T cells, or CD4 ⁺ and CD8 ⁺ T cells.

In some embodiments, for example, where the subject is a human, the dose of CD8+ T cells, including where the dose includes CD4+ and CD8+ T cells, includes between at or about ¹ x 10 and between at or about 2×10 ⁹ total CAR-expressing CD8+ cells, for example, in the range from at or about 5×10 ⁷ to at or about 4.5×10 ⁸ such cells, for example at or about 2.5× 10 ⁷ , at or about 5×10 ⁷ , at or about 1.5×10 ⁸ , at or about 3×10 ⁸ , at or about 4.5×10 ⁸ , at or about 8×10 ⁸ , or at or about 1.2× 10 ⁹ total such cells, or a range between any two of the previous values.

In some embodiments, the dose of cells, e.g., CAR-T cells, is administered to the subject as a single dose or over a period of two weeks, one month, three months, six months, one year, or more. Apply only once within a period of time. In some embodiments, multiple doses are administered to the patient, and each dose or the total dose can be within any of the foregoing values.

Without intending to be limited by any theory, the following examples are only to illustrate the chimeric antigen receptor, preparation methods and uses of the present application, and are not intended to limit the scope of the invention of the present application.

Example

Example 1

Flow fluorescent antibodies of anti-HER2 and anti-ITGB6 were used to detect the expression of HER2 and ITGB6 (a subunit of αvβ6) on the surface of various tumor cell lines, respectively, and the mean fluorescence intensity (MFI) and the MFI of its own unstained cell lines were used. After quantification processing, the relative MFI of HER2 and ITGB6 was finally obtained.

The results (Table 1) show that multiple tumor cell lines express both HER2 and ITGB6 on the cell surface, suggesting that it is reasonable to target HER2 and integrin αvβ6 at the same time.

Table 1

Note: The expression strength of Her2 and ITGB6 is measured by the MFI value after normalization. For Her2, MFI>500
is +++; 50-500 is ++; 5-50 is +; <5 is -; for ITGB6: MFI>10 is +++; 5-10 is ++; 2-5 is +, <2 is -.

Example 2

As shown in Figure 1, different combinations of Her2 antigen sequences and αvβ6-recognizing sequences will be combined to construct a CAR with the function of recognizing dual targets.

The nucleotide sequences of anti-HER2 and FMDV2 A20 were obtained through gene synthesis. The nucleotide sequences encoding the above chimeric antigen receptors were further obtained through overlapping PCR and site-directed mutagenesis technology. The NcoI and XhoI enzyme cutting sites were used to obtain the nucleotide sequences. A series of nucleotide sequences encoding chimeric antigen receptors were cloned into the gamma-retroviral vector PSTITCH. The amino acid sequences of different fragments of chimeric antigen receptors are shown in Table 2.

Among them, the light chain region and heavy chain region of the single-chain variable fragment of anti-HER2 are respectively derived from the light chain variable region and heavy chain variable region of the Herceptin antibody. The FMDV2 A20 fragment can specifically bind to integrin αvβ6. FMDV2 C20 fragment replaces RGDL in the A20 short peptide with AAAA, destroying its ability to bind to integrin αvβ6. The hinge region is selected from the CH2/3 domain of the Fc segment of human IgG1. In order to avoid FcγR receptor interaction in the hinge region In combination, mutations were made in the PELLGG and ISR motifs of the sequence. The transmembrane region and intracellular co-stimulatory domain of the chimeric antigen receptor were selected from the transmembrane region and intracellular domain of CD28, in which the PYAPP domain was replaced with AYAAA. It can reduce the production of IL-2 by CAR-T cells and increase the resistance to Treg cells. The main signaling domain of the chimeric antigen receptor is selected from the intracellular domain of the CD3ζ chain.

Table 2

Example 3

The CAR molecules E007, E008 and E009 constructed in Implementation 2 were selected for most subsequent experiments. Each set of plasmids was transferred into HEK293T cells by transient transfection. After the cells were cultured overnight, the digested cells were harvested after trypsin treatment and mixed with anti-hIgG-PE antibody, HER2-FITC protein and αvβ6-PE protein respectively. After incubation at room temperature for half an hour, flow cytometry was performed.

The results (Figure 2) show that all three groups of CARs can be expressed on the surface of HEK293T cell membranes. Among them, E008 CAR can specifically recognize the two targets of HER2 and αvβ6, while E007 and E009 can only recognize the single target of HER2. Therefore, this application obtained a CAR molecule that can simultaneously recognize the dual targets of HER2 and αvβ6. In other experiments, it was also confirmed that E010 and E012 have the function of recognizing the dual targets of HER2 and αvβ6.

Example 4

Three sets of CARs, E007, E008 and E009, were transferred into T cells through gamma-retrovirus to prepare different CAR-T cells. Anti-hIgG-PE, an antibody specific to the CAR hinge region, was used to detect the presence of CAR on the T cell membrane. The results (Figure 3) show that all three groups of CARs can be expressed on the T cell membrane.

Example 5

The xCELLigence RTCA eSight system was used to monitor the killing ability of CAR-T cells against the tumor cell line MDA-MB-468 (HER2 ^- αvβ6 ⁺ ) in real time. As shown in Figure 4, when the effect-to-target ratio was 1:1, it was better than that without Compared with cells transfected with CAR-T, only E008 CAR-T cells had a killing effect on the MDA-MB-468 cell line, and neither E007 nor E009 CAR-T cells had an obvious killing effect on this tumor cell, indicating that E008 CAR-T The cells can also effectively kill αvβ6 single target cells.

CAR-T cells and MDA-MB-468 cell line were co-incubated at an effect-to-target ratio of 0.1:1 for 48 hours. After collecting the reaction supernatant, a commercial ELISA kit (BD, Human IFN-γ ELISA Set) was used to detect the γ-interferon content in the supernatant. The results (Figure 5) showed that compared with cells not transfected with CAR-T, E008 CAR-T cells can release higher levels of cytokines after encountering target cells, while E007 and E009 CAR-T cells only release low levels of cytokines. Cytokine release levels were consistent with cell killing assay results.

Example 6

The xCELLigence RTCA eSight system was used to monitor in real time the killing ability of CAR-T cells against the tumor cell line NCI-H358 (HER2+αvβ6+) that co-expresses HER2 and αvβ6. As shown in Figure 6, when the effect-to-target ratio is 1:1 , compared with cells not transfected with CAR-T, E008 showed high tumor cell killing ability, E009 had tumor cell killing ability but weaker than E008, so E008 recognized the dual target of HER2 and αvβ6 at the same time and therefore had better tumor killing ability.

CAR-T cells and NCI-H358 cell line were co-incubated at an effect-to-target ratio of 1:1 for 48 hours. After collecting the reaction supernatant, a commercial ELISA kit (BD, Human IFN-γ ELISA Set) was used to detect the content of γ-interferon in the supernatant. The results showed that compared with cells not transfected with CAR-T, E008 and E007 Both CAR-T cells and target cells can release higher levels of cytokines after co-incubation. The cytokine levels are consistent with the killing ability of the target cells. This further confirms the superiority of identifying dual targets of HER2 and αvβ6 at the same time, and the ability to identify dual targets. CAR T cells release more cytokines and better cell proliferation after antigen stimulation.

Example 7

As shown in Figure 8, to verify the in vivo effect of CAR-T cells, all experimental mice were immunodeficient B-NDG female mice aged 6-8 weeks. On Day 0, 1E+7 NCI-H358 tumor cells were resuspended with PBS. Suspended and inoculated into mice subcutaneously through subcutaneous tumor-bearing technology, when the tumor size was about 70 mm3, they were divided into 4 groups, including 3 mice in the Mock group and 6 mice in the other experimental groups. On Day 4, each Mice were injected with 1E+7 CAR-T positive cells into the tail vein (the Mock group was calculated based on 50% CAR-T positivity rate), and then the tumor volume was measured regularly with a vernier caliper. Tumor volume (mm3) = 1/2a*b2 , where a represents the length of the tumor and b represents the width of the tumor.

The results are shown in Figure 9A-9B. Dual-target recognition CAR-T cells can effectively inhibit tumor growth. At the same time, CAR-T cell infusion did not cause abnormal changes in the weight of mice. This preliminarily shows that dual-target recognition CAR-T cells are safe. of.

Example 8

The xCELLigence RTCA eSight system was used to monitor the killing ability of CAR-T cells against the tumor cell line NCI-H358 (HER2 ⁺ αvβ6 ⁺ ) in real time. Figure 10A shows that when the effect-to-target ratio is 3:1, compared with untransfected CAR- Compared with T cells, CAR-T cells with different sequence arrangements can effectively kill tumor cells.

The xCELLigence RTCA eSight system was used to monitor the killing ability of CAR-T cells against the tumor cell line MDA-MB-468 (HER2-αvβ6 ⁺ ) in real time. Figure 10B shows that when the effect-to-target ratio is 3:1, compared with untransfected Compared with CAR-T cells, CAR-T cells with different sequence combinations can effectively kill tumor cells.

Only E008, E010 and E012 CAR-T cells had a significant killing effect on the MDA-MB-468 cell line, while E007, E009, E011 and E013 CAR-T cells had no obvious killing effect on this tumor cell, indicating that E008, E010 and E012 CAR-T cells can also effectively kill αvβ6 single target cells.

Example 9

In order to detect the affinity of different CAR-T cells to the target, different groups of CAR-T cells with similar positive rates (except the NT group) and consistent cell numbers were incubated with different concentrations of HER2-FITC protein for half an hour, and then flowed The change of fluorescence intensity was detected by using the formula. The results (Figure 11) showed that compared with E007 CAR-T cells, the affinity of E008 and E009 CAR-T cells for HER2-FITC protein was significantly reduced, which suggested that E008 has a high ability to retain tumors. While having cell killing ability, it may reduce the ability to recognize target cells expressing low levels of Her2, thereby potentially increasing clinical safety.

Example 10

SKMEL28 tumor cells weakly express HER2 but do not express αvβ6, which can simulate to a certain extent the situation in which normal tissues in the body weakly express HER2 but do not express αvβ6. Figure 12 shows that when the effect-to-target ratio is 1:1, compared with not Compared with cells transfected with CAR-T, E007 CAR-T cells with high affinity for HER2 show a stronger killing effect on target cells, but E008 and E009 CAR-T cells have no obvious killing effect on target cells, which suggests that E008 and E009 CAR-T cells with low affinity for HER2 may have a higher safety profile in vivo.

Example 11

As shown in Figure 13, the CEA antigen sequence and the αvβ6 recognition sequence are combined to construct a CAR with the function of recognizing dual targets. Among them, the amino acid sequence of Y002 is shown in SEQ ID NO:56, and the amino acid sequence of Y007 is shown in SEQ ID NO:58.

The chimeric antigen receptor sequence containing anti-CEA scFv/FMDV2 A20 extracellular domain and anti-CEA scFv/FMDV2 C20 extracellular domain was obtained by overlapping PCR. The series of chimeric antigen receptor sequences were combined through NcoI and XhoI restriction sites. The antigen receptors were cloned into the gamma-retroviral vector PSTITCH respectively. The anti-CEA scFv sequence comes from the BW431/26 antibody.

Example 12

As shown in Figure 14, a CAR with the function of recognizing dual targets will be constructed by combining the HER2 antigen sequence and the sequence that recognizes αvβ6. Among them, the amino acid sequence of Y021 is shown in SEQ ID NO:63, and the amino acid sequence of Y022 is shown in SEQ ID NO:65.

The chimeric antigen receptor sequence containing anti-HER2 scFv/LAP A20 extracellular domain and anti-HER2 scFv/LAP C20 extracellular domain was obtained by overlapping PCR method. The series of chimeric antigen receptor sequences were sequenced through NcoI and XhoI restriction sites. zygogenic antigen The receptors were cloned into the gamma-retroviral vector PSTITCH. The anti-HER2 scFv sequence comes from the Herceptin antibody, LAP A20 comes from the LAP part of the TGF-β1 precursor, and the LAP C20 fragment replaces RGDL in the LAP A20 short peptide with AAAA, destroying its binding to integrin αvβ6. ability, its amino acid coding sequence is: GFTTGRAAAAAATIHGMNRPF (SEQ ID NO: 61), and the nucleotide sequence of LAP C20 is: gggttcactaccggccgcgcAGcCGcCgcGgccaccattcatggcatgaaccggcctttc (SEQ ID NO: 62).

Example 13

Y002 and Y007 CAR molecules were constructed by transfecting T cells with gamma-retrovirus to prepare different CAR-T cells. Anti-hIgG-PE, an antibody specific for the CAR hinge region, was used to detect the presence of CAR on the T cell membrane. Expression, while using CEA protein and αvβ6 protein to detect the specificity of CAR receptors respectively. The results (Figure 15) show that both Y002 and Y007 CAR can be expressed on the T cell membrane, among which Y002 can specifically bind to the two targets of CEA and αvβ6 , while Y007 only binds to the CEA target.

Example 14

The Y021 and Y022 CAR molecules were constructed by transfecting T cells with gamma-retrovirus to prepare different CAR-T cells. GFP was used to detect the positive rate of CAR-T cells, while HER2 protein and αvβ6 protein were used to detect CAR respectively. Regarding the specificity of the receptor, the results (Figure 16) show that Y021 can specifically bind to both HER2 and αvβ6 targets, while Y022 only binds to the HER2 target.

Example 15

After incubating the tumor cell lines SKOV3 and BxPC-3 with anti-CEA (APC) antibodies, flow cytometry was used to detect the CEA expression levels on the membrane surfaces of the two cell lines. The unstained cell lines were used as negative gate controls. The results (Figure 17) showed that the expression of CEA was basically undetectable on the SKOV3 cell membrane surface, while more than 99% of the cells on the BxPC-3 cell membrane surface expressed CEA antigen.

Example 16

The xCELLigence RTCA eSight system was used to monitor the killing ability of CAR-T cells against the tumor cell line SKOV3 (CEA-αvβ6+) in real time. Figure 18 shows that when the effect-to-target ratio is 1:1, compared with untransfected cells, Y007 CAR-T cells have a weak killing effect on the SKOV3 cell line. This may be due to the weak CEA expression on the surface of SKOV3 cells. Compared with Y007 CAR-T cells, Y002 CAR-T cells have a weak killing effect on the SKOV3 cell line. It is more obvious, indicating that this killing depends on the specific binding of FMDV2 A20 to αvβ6.

Example 17

CAR-T cells and SKOV3 cell lines were co-incubated at an effect-to-target ratio of 1:1 for 24 hours. After collecting the reaction supernatant, use a commercial ELISA kit (BD, Human IL-2 set) to detect the IL-2 content in the supernatant. The results (Figure 19) show that compared with cells not transfected with CAR-T, Y002 CAR-T cells can release higher levels of cytokines after encountering target cells. Y007 CAR-T cells only release low levels of cytokines. The cytokine release levels are consistent with the results of cell killing experiments.

Example 18

The xCELLigence RTCA eSight system was used to monitor the killing ability of CAR-T cells against the tumor cell line BxPC-3 (CEA+αvβ6+) in real time. Figure 20 shows that when the effect-to-target ratio is 1:1, compared with untransfected cells , both Y002 and Y007 CAR-T cells have a good killing effect on the tumor cell line BxPC-3. Compared with Y007 CAR-T cells, the killing trend of Y002 dual-target CAR-T cells on BxPC-3 is more obvious. This implies that dual targets promote the killing function of CAR-T cells.

Example 19

CAR-T cells and BxPC-3 cell lines were co-incubated at an effect-to-target ratio of 1:1 for 24 hours. After collecting the reaction supernatant, use a commercial ELISA kit (BD, Human IL-2 set) to detect the IL-2 content in the supernatant. The results (Figure 21) show that compared with cells not transfected with CAR-T, Y002 CAR-T cells can release higher levels of cytokines after encountering target cells. The level of cytokine release by Y007 CAR-T cells after encountering target cells is slightly lower than that of Y002 CAR-T cells. Cytokine release levels and cell killing The experimental results are consistent.

Example 20

The xCELLigence RTCA eSight system was used to monitor the killing ability of CAR-T cells against the tumor cell line MDA-MB-468 (HER2-αvβ6+) in real time. Figure 22 shows that when the effect-to-target ratio is 1:1, compared with untransfected cells In comparison, Y022 CAR-T cells have a weak killing effect on the MDA-MB-468 cell line. This may be due to the weak HER2 expression on the surface of the target cells. Compared with Y022 CAR-T cells, Y021 CAR-T The cell killing of the MDA-MB-468 cell line was more obvious, indicating that this killing relied on the specific binding of LAP A20 to αvβ6.

Example 21

CAR-T cells and MDA-MB-468 cell line were co-incubated at an effect-to-target ratio of 1:1 for 48 hours. After collecting the reaction supernatant, a commercial ELISA kit (BD, Human IFN-γset) was used to detect the IFN-γ content in the supernatant. The results (Figure 23) showed that compared with cells not transfected with CAR-T, Y021 CAR-T cells can release higher levels of cytokines after encountering target cells, while Y022 CAR-T cells only release low levels of cytokines. The factor release levels were consistent with the results of cell killing experiments.

Example 22

The xCELLigence RTCA eSight system was used to monitor the killing ability of CAR-T cells against the tumor cell line BxPC-3 (HER2+αvβ6+) in real time. Figure 24 shows that when the effect-to-target ratio is 1:1, compared with untransfected cells , Y021 and Y022 CAR-T cells both have good killing effects on the tumor cell line BxPC-3.

Example 23

CAR-T cells and BxPC-3 cell lines were co-incubated at an effect-to-target ratio of 1:1 for 48 hours. After collecting the reaction supernatant, a commercial ELISA kit (BD, Human IFN-γset) was used to detect the IFN-γ content in the supernatant. The results (Figure 25) showed that compared with cells not transfected with CAR-T, Y021 and Y022 CAR-T cells release high levels of cytokines after encountering target cell stimulation.

Claims

A chimeric antigen receptor comprising an extracellular antigen-binding domain, wherein the extracellular antigen-binding domain comprises a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain specifically binds integrins .
The chimeric antigen receptor according to claim 1, wherein the antigen-binding domain includes a ligand or a functional fragment thereof, or an antibody or an antigen-binding fragment thereof.
The chimeric antigen receptor according to any one of claims 1-2, wherein the first antigen-binding domain includes an integrin ligand or a functional fragment thereof, or an anti-integrin antibody or an antigen-binding fragment thereof.
The chimeric antigen receptor according to any one of claims 1-3, wherein the antibody or antigen-binding fragment thereof comprises a full-length antibody, Fab, single chain variable fragment (scFv), di-scFv or single domain Antibodies (VHH).
The chimeric antigen receptor according to any one of claims 1 to 4, wherein the first antigen binding domain is located upstream, downstream and/or in the middle of the second antigen binding domain.
The chimeric antigen receptor according to any one of claims 1 to 5, wherein the first antigen binding domain is directly or indirectly connected to the second antigen binding domain.
The chimeric antigen receptor of claim 6, wherein the indirect connection includes connection through a linker.
The chimeric antigen receptor of claim 7, wherein the linker comprises a peptide linker.
The chimeric antigen receptor according to any one of claims 1 to 8, wherein the N-terminus of the first antigen-binding domain is directly or indirectly connected to the C-terminus of the second antigen-binding domain.
The chimeric antigen receptor according to any one of claims 1 to 8, wherein the C-terminus of the first antigen-binding domain is directly or indirectly connected to the N-terminus of the second antigen-binding domain.
According to the chimeric antigen receptor according to any one of claims 1 to 8, when the second antigen binding domain is scFv, the first antigen binding domain is located between VH and VL of the second antigen binding domain, so The first antigen binding domain is directly or indirectly connected to VH and VL of the second antigen binding domain respectively.
According to the chimeric antigen receptor according to any one of claims 1 to 8, when the first antigen binding domain is scFv, the second antigen binding domain is located between VH and VL of the first antigen binding domain, so The second antigen binding domain is directly or indirectly connected to VH and VL of the first antigen binding domain respectively.
The chimeric antigen receptor according to any one of claims 1-12, wherein the extracellular antigen-binding domain comprises an integrin ligand and an anti-tumor antigen scFv antibody.
According to the chimeric antigen receptor of claim 13, the N-terminus of the integrin ligand is directly or indirectly connected to the C-terminus of the anti-tumor antigen scFv antibody.
According to the chimeric antigen receptor of claim 13, the C-terminus of the integrin ligand is directly or indirectly connected to the N-terminus of the anti-tumor antigen scFv antibody.
The chimeric antigen receptor according to claim 13, the integrin ligand is located between the VH and VL of the anti-tumor antigen scFv antibody, and the integrin ligand is respectively connected to the VH and VL of the anti-tumor antigen scFv antibody. VL is connected directly or indirectly.
According to the chimeric antigen receptor according to any one of claims 13-16, the integrin ligand and the anti-tumor antigen scFv antibody are connected through a linker.
According to the chimeric antigen receptor of claim 13, the N-terminus of the integrin ligand is connected to the C-terminus of the anti-tumor antigen scFv antibody through a linker.
According to the chimeric antigen receptor of claim 13, the C-terminus of the integrin ligand is connected to the N-terminus of the anti-tumor antigen scFv antibody through a linker.
According to the chimeric antigen receptor of claim 13, both ends of the integrin are respectively connected to the VH and VL of the anti-tumor antigen scFv antibody through linkers.
The chimeric antigen receptor according to any one of claims 1-20, wherein the integrin includes an integrin abnormally expressed on the surface of tumor cells.
The chimeric antigen receptor according to any one of claims 1-21, wherein the integrin is selected from: one or more of αIIβ3, α8β1, α5β1, αvβ1, αvβ3, αvβ5, αvβ6 and αvβ8.
The chimeric antigen receptor according to any one of claims 1-22, wherein the integrin is αvβ6.
The chimeric antigen receptor according to any one of claims 1-23, wherein the first antigen-binding domain includes an αvβ6 ligand or a functional fragment thereof, or an anti-αvβ6 antibody or an antigen-binding fragment thereof.
The chimeric antigen receptor according to claim 24, wherein the αvβ6 ligand or functional fragment thereof comprises the amino acid sequence shown in any one of SEQ ID NO: 4-5.
The chimeric antigen receptor according to any one of claims 1-25, wherein the second antigen-binding domain specifically binds a tumor antigen.
The chimeric antigen receptor according to claim 26, wherein the tumor antigen is selected from: HER2, MUCl, ROR1, AFP, FAP, MAGA, MUC16, EphA2, ErbB, PSCA, IL13Rα2, EPCAM, EGFR, EGFRVIII, PSMA , one or more of GPC3, CEA, GD2, Mesothelin, PD-L1, CD133, AXL, DLL3, LMP1, MG7, PMEL, ROR2, VEGFR2, CD171, CLD18, FRα and cMet.
The chimeric antigen receptor according to any one of claims 26-27, wherein the tumor antigen is HER2.
The chimeric antigen receptor according to any one of claims 1-28, wherein the second antigen-binding domain includes an anti-HER2 antibody or an antigen-binding fragment thereof.
The chimeric antigen receptor according to claim 29, wherein the anti-HER2 antibody or antigen-binding fragment thereof comprises an antibody heavy chain variable region (VH), wherein the VH further comprises the amino acid shown in SEQ ID NO: 2 sequence.
The chimeric antigen receptor according to any one of claims 29-30, wherein the anti-HER2 antibody or antigen-binding fragment thereof comprises an antibody heavy chain variable region (VL), wherein the VL further comprises SEQ ID NO. :The amino acid sequence shown in 1.
The chimeric antigen receptor of any one of claims 1-31, wherein the second antigen binding domain comprises an anti-HER2 scFv antibody.
The chimeric antigen receptor of claim 32, wherein the anti-HER2 scFv antibody comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), wherein the VH comprises SEQ ID NO: The amino acid sequence shown in 2, the VL includes the amino acid sequence shown in SEQ ID NO:1.
The chimeric antigen receptor according to claim 32, wherein the anti-HER2 scFv antibody comprises the amino acid sequence shown in SEQ ID NO: 3.
The chimeric antigen receptor according to any one of claims 29-34, wherein the N-terminus of the αvβ6 ligand is directly or indirectly connected to the C-terminus of the anti-HER2 scFv antibody.
The chimeric antigen receptor according to claim 35, from the N-terminus to the C-terminus, wherein the extracellular antigen-binding domain sequentially comprises anti-HER2 VH-anti-HER2 VL-αvβ6 ligand, or anti-HER2 VL-anti-HER2 VH-αvβ6 ligand.
According to the chimeric antigen receptor according to any one of claims 29-34, the C-terminus of the αvβ6 ligand is directly or indirectly connected to the N-terminus of the anti-HER2 scFv antibody.
The chimeric antigen receptor according to claim 37, from the N-terminus to the C-terminus, wherein the extracellular antigen-binding domain sequentially comprises αvβ6 ligand-anti-HER2 VH-anti-HER2 VL, or αvβ6 ligand-anti-HER2 VL-anti-HER2 VH.
The chimeric antigen receptor according to any one of claims 29-34, the αvβ6 ligand is located between the VH and VL of the anti-HER2 scFv antibody, and the second antigen-binding domain is respectively with the first antigen The VH and VL of the binding domain are connected directly or indirectly.
The chimeric antigen receptor according to claim 39, from the N-terminus to the C-terminus, wherein the extracellular antigen-binding domain sequentially includes anti-HER2 VH-αvβ6 ligand-anti-HER2 VL, or anti-HER2 VL-αvβ6 ligand. Body-anti-HER2 VH.
According to the chimeric antigen receptor according to any one of claims 29-40, the αvβ6 ligand is connected to the VH or VL of the anti-HER2 scFv antibody through a linker.
The chimeric antigen receptor according to any one of claims 17-41, wherein the linker comprises the amino acid sequence shown in SEQ ID NO: 14.
According to the chimeric antigen receptor of claims 1-42, the extracellular antigen-binding domain comprises the amino acid sequence shown in any one of SEQ ID NO: 15-20.
The chimeric antigen receptor according to any one of claims 1-43, said chimeric antigen receptor further comprising a transmembrane domain, said transmembrane domain comprising one or more selected from the group consisting of: Transmembrane domains of species proteins: CD8, CD28, CD3ε (CD3e), 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L(CD154), TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33 , CD37, CD64, SLAM and their variants.
The chimeric antigen receptor of claim 44, wherein the transmembrane domain comprises a transmembrane domain derived from CD28 or a variant thereof.
The chimeric antigen receptor according to any one of claims 44-45, wherein the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 10.
The chimeric antigen receptor according to any one of claims 1-46, said chimeric antigen receptor further comprising an intracellular signal transduction domain, said intracellular signal transduction domain comprising a protein selected from the group consisting of: Intracellular signaling domain of one or more proteins from the following group: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FceRIγ, FceRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A , simian immunodeficiency virus PBj14 Nef, DAP10, DAP-12 and domains containing at least one ITAM.
The chimeric antigen receptor of claim 47, wherein the intracellular signaling domain comprises a signaling domain derived from CD3ζ.
The chimeric antigen receptor according to any one of claims 47-48, wherein the intracellular signal transduction domain comprises the amino acid sequence shown in SEQ ID NO: 13.
The chimeric antigen receptor according to any one of claims 1-49, wherein the chimeric antigen receptor comprises an extracellular antigen-binding domain, a transmembrane domain and an intracellular signal transduction domain.
The chimeric antigen receptor according to any one of claims 1 to 50, wherein the chimeric antigen receptor further comprises an intracellular costimulatory signaling domain, and the intracellular costimulatory signaling domain comprises a source Intracellular costimulatory signaling domain of one or more proteins selected from the following group: CD28, 4-1BB (CD137), CD27, CD2, CD7, CD8A, CD8B, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40, MyD88 and their variants.
The chimeric antigen receptor of claim 51 , wherein the intracellular costimulatory signaling domain is derived from the costimulatory signaling domain of CD28 or a variant thereof.
The chimeric antigen receptor of any one of claims 51-52, wherein the intracellular costimulatory signaling domain comprises the amino acid sequence shown in SEQ ID NO: 11-12.
The chimeric antigen receptor according to any one of claims 1-53, wherein the chimeric antigen receptor further comprises a hinge region, the hinge region is located between the transmembrane domain and the extracellular antigen binding structure between domains, the hinge region comprising a hinge region derived from one or more proteins selected from the group consisting of: CD28, CD8, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8A, PD -1. ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT and their variants.
The chimeric antigen receptor according to claim 54, said hinge region comprising an IgG1 Fc domain or a variant thereof.
According to the chimeric antigen receptor according to any one of claims 54-55, the hinge region comprises the amino acid sequence shown in any one of SEQ ID NO: 8-9.
The chimeric antigen receptor according to any one of claims 1-56, from the N-terminus to the C-terminus, the chimeric antigen receptor includes an extracellular antigen-binding domain, a hinge region, a transmembrane domain, a cellular domain, and a transmembrane domain. Intrinsic costimulatory signaling domain and intracellular signaling domain.
The chimeric antigen receptor according to claim 57, from the N-terminus to the C-terminus, the hinge region, the transmembrane domain, the intracellular costimulatory signaling domain and the intracellular signaling domain of the chimeric antigen receptor Contains the amino acid sequence shown in SEQ ID NO:21.
The chimeric antigen receptor according to any one of claims 1 to 58, said chimeric antigen receptor further comprising a signal peptide fragment, the C-terminus of the signal peptide fragment being in contact with the extracellular antigen-binding domain. N-terminal connection.
The chimeric antigen receptor of claim 59, wherein the signal peptide fragment comprises a CD8α signal peptide fragment.
According to the chimeric antigen receptor according to any one of claims 59-60, the signal peptide fragment comprises the amino acid sequence shown in SEQ ID NO:7.
The chimeric antigen receptor according to any one of claims 1-61, said chimeric antigen receptor comprising the amino acid sequence shown in any one of SEQ ID NO: 22-27.
An isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric antigen receptor of any one of claims 1-62.
A construct comprising the nucleic acid molecule of claim 63.
Isolated one or more constructs comprising a nucleic acid molecule encoding a first chimeric antigen receptor and a second chimeric antibody encoding A nucleic acid molecule of a proreceptor, wherein the first chimeric antigen receptor comprises an extracellular antigen binding domain that specifically binds an integrin.
The construct of claim 65, wherein the extracellular antigen-binding domain that specifically binds integrin includes an integrin ligand or functional fragment thereof, or an anti-integrin antibody or antigen-binding fragment thereof.
The construct of claim 66, wherein the antibody or antigen-binding fragment thereof comprises a full-length antibody, Fab, single chain variable fragment (scFv), di-scFv or single domain antibody (VHH).
The construct of any one of claims 65-67, wherein the extracellular antigen-binding domain that specifically binds integrin includes an integrin ligand or a functional fragment thereof, an anti-integrin scFv antibody or an antigen-binding thereof Fragments, anti-integrin VHH antibodies or antigen-binding fragments thereof.
The construct of any one of claims 68, wherein the integrin comprises an integrin aberrantly expressed on the surface of tumor cells.
The construct of any one of claims 65-69, wherein the integrin is selected from: one or more of αIIβ3, α8β1, α5β1, αvβ1, αvβ3, αvβ5, αvβ6 and αvβ8.
The construct of any one of claims 65-70, wherein said integrin is αVβ6.
The construct of any one of claims 65-71, wherein the extracellular antigen-binding domain that specifically binds integrin includes an αVβ6 ligand or a functional fragment thereof, or an anti-αVβ6 antibody or an antigen-binding fragment thereof.
The construct according to any one of claims 65-72, wherein the αVβ6 ligand or functional fragment thereof comprises the amino acid sequence shown in any one of SEQ ID NO: 4-5.
The construct of any one of claims 65-73, wherein the second chimeric antigen receptor comprises an extracellular antigen binding domain that specifically binds a tumor antigen.
The construct of claim 74, wherein the extracellular antigen-binding domain that specifically binds a tumor antigen comprises a tumor antigen antibody or an antigen-binding fragment thereof.
The construct according to any one of claims 74-75, wherein the tumor antigen is selected from: HER2, MUCl, ROR1, AFP, FAP, MAGA, MUC16, EphA2, ErbB, PSCA, IL13Rα2, EPCAM, EGFR, One or more of EGFRVIII, PSMA, GPC3, CEA, GD2, Mesothelin, PD-L1, CD133, AXL, DLL3, LMP1, MG7, PMEL, ROR2, VEGFR2, CD171, CLD18, FRα and cMet.
The construct of any one of claims 74-76, wherein the tumor antigen is HER2.
The construct of claim 77, wherein the extracellular antigen-binding domain that specifically binds a tumor antigen comprises an anti-HER2 antibody or antigen-binding fragment thereof.
The construct of claim 78, wherein the anti-HER2 antibody or antigen-binding fragment thereof comprises an antibody heavy chain variable region (VH), wherein the VH further comprises the amino acid sequence shown in SEQ ID NO: 2.
The construct of any one of claims 78-79, wherein the anti-HER2 antibody or antigen-binding fragment thereof comprises an antibody heavy chain variable region (VL), wherein the VL further comprises SEQ ID NO: 1 The amino acid sequence shown.
The construct of any one of claims 78-80, wherein the extracellular antigen-binding domain that specifically binds a tumor antigen comprises an anti-HER2 scFv antibody.
The construct of any one of claims 78-81, wherein the anti-HER2 scFv antibody comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), wherein the VH comprises SEQ. The amino acid sequence shown in ID NO:2, the VL includes the amino acid sequence shown in SEQ ID NO:1.
The construct of claim 82, wherein said VH and said VL are connected by a linker.
The construct of claim 83, wherein the linker comprises the amino acid sequence shown in SEQ ID NO:14.
The construct of any one of claims 81-84, wherein the anti-HER2 scFv antibody comprises the amino acid sequence shown in SEQ ID NO:3.
The construct of any one of claims 65-84, each of the first chimeric antigen receptor and the second chimeric antigen receptor further comprises a transmembrane domain, the transmembrane domain comprising Transmembrane domain of one or more proteins from the following group: CD8, CD28, CD3ε (CD3e), 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ζ, CTLA-4, LAG- 3. CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L(CD154), TIM1, CD226, DR3, CD45, CD80, CD86 , CD9, CD16, CD22, CD33, CD37, CD64, SLAM and their variants.
The construct of claim 86, wherein the transmembrane domain comprises a transmembrane domain derived from CD28 or a variant thereof.
The construct according to any one of claims 86-87, wherein the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 10.
The construct of any one of claims 65-88, each of the first chimeric antigen receptor and the second chimeric antigen receptor further comprises an intracellular signal transduction domain, the intracellular signal transduction domain The conduction domain includes an intracellular signaling domain derived from one or more proteins selected from the group consisting of CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FceRIγ, FceRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, DAP10, DAP-12 and domains containing at least one ITAM.
The construct of claim 89, wherein the intracellular signaling domain comprises a signaling domain derived from CD3ζ.
The construct according to any one of claims 89-90, wherein the intracellular signal transduction domain comprises the amino acid sequence shown in SEQ ID NO: 13.
The construct according to any one of claims 65-91, the first chimeric antigen receptor and the second chimeric antigen receptor each comprise an extracellular antigen binding domain, a transmembrane domain and an intracellular signal transduction domain. guide domain.
The construct of any one of claims 65-92, each of the first chimeric antigen receptor and the second chimeric antigen receptor further comprises an intracellular costimulatory signaling domain, the intracellular co-stimulatory signaling domain The stimulatory signaling domain includes an intracellular costimulatory signaling domain derived from one or more proteins selected from the group consisting of: CD28, 4-1BB (CD137), CD27, CD2, CD7, CD8A, CD8B, OX40 , CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1 , LIGHT, JAML, CD244, CD100, ICOS, CD40, MyD88 and their variants.
The construct of claim 93, wherein the intracellular costimulatory signaling domain is derived from the costimulatory signaling domain of CD28 or a variant thereof.
The construct of any one of claims 93-94, wherein the intracellular costimulatory signaling domain comprises the amino acid sequence shown in SEQ ID NO: 11-12.
The construct according to any one of claims 65-95, each of the first chimeric antigen receptor and the second chimeric antigen receptor further comprises a hinge region, the hinge region is located in the transmembrane domain Between the extracellular antigen-binding domain and the hinge region, the hinge region includes a hinge region derived from one or more proteins selected from the following group: CD28, CD8, IgG1, IgG4, IgD, 4-1BB, CD4 , CD27, CD7, CD8A, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT and their variants.
The construct of claim 96, said hinge region comprising an IgG1 Fc domain or a variant thereof.
According to the construct of any one of claims 96-97, the hinge region comprises the amino acid sequence shown in any one of SEQ ID NO:8-9.
According to the construct according to any one of claims 65-98, from the N-terminus to the C-terminus, the first chimeric antigen receptor and the second chimeric antigen receptor each comprise an extracellular antigen binding domain, Hinge region, transmembrane domain, intracellular costimulatory signaling domain and intracellular signaling domain.
The construct according to claim 99, from the N-terminus to the C-terminus, the hinge region, transmembrane domain, intracellular costimulatory signaling domain and intracellular signaling domain comprise SEQ ID NO: 21 Amino acid sequence.
According to the construct according to any one of claims 65-100, from the N-terminus to the C-terminus, the first chimeric antigen The receptor sequentially contains an extracellular antigen-binding domain that specifically binds integrins, a hinge region, a transmembrane domain, an intracellular costimulatory signaling domain, and an intracellular signaling domain.
According to the construct according to any one of claims 65-101, from the N-terminus to the C-terminus, the second chimeric antigen receptor includes an extracellular antigen-binding domain that specifically binds a tumor antigen, a hinge region, transmembrane domain, intracellular costimulatory signaling domain, and intracellular signaling domain.
The construct according to any one of claims 65 to 102, each of the first chimeric antigen receptor and the second chimeric antigen receptor further comprises a signal peptide fragment, the C-terminus of the signal peptide fragment being connected to the The N-terminal linkage of the extracellular antigen-binding domain is described.
The construct of claim 103, the signal peptide fragment comprises a CD8α signal peptide fragment.
According to the construct of any one of claims 102-103, the signal peptide fragment comprises the amino acid sequence shown in SEQ ID NO:7.
According to the construct of any one of claims 65-105, the first chimeric antigen receptor comprises the amino acid sequence shown in any one of SEQ ID NO: 29-30.
According to the construct of any one of claims 65-106, the second chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 28.
The construct of any one of claims 65-107, wherein the nucleic acid molecule encoding the first chimeric antigen receptor and the nucleic acid molecule encoding the second chimeric antigen receptor are in the same construct.
The construct of any one of claims 65-107, wherein the nucleic acid molecule encoding a first chimeric antigen receptor and the nucleic acid molecule encoding a second chimeric antigen receptor are in separate constructs.
The construct of any one of claims 65-109, comprising an expression vector.
The construct of any one of claims 65-110, comprising a DNA vector, an RNA vector, a plasmid and/or a viral vector.
The construct of claim 111, said viral vector comprising a lentiviral vector, an adenoviral vector, an adeno-associated viral vector or a retroviral vector.
Cells comprising the nucleic acid molecule of any one of claims 63 or the construct of any one of claims 64-112, and/or expressing the chimera of any one of claims 1-62 Antigen receptors.
The cell of claim 113, wherein said cell comprises an immune effector cell.
The cell according to claim 114, the immune effector cells include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes , leukocytes and/or peripheral blood mononuclear cells.
The cell of any one of claims 113-115, comprising a mammalian cell.
The cell of any one of claims 113-116, comprising a human cell.
The cell of any one of claims 113-117, comprising autologous or non-autologous immune effector cells.
The cell of claims 114-118, wherein the immune cells comprise modified immune effector cells.
The cell of claim 119, wherein the modified immune cells comprise cells that reduce immune rejection resulting from allogeneic cell therapy.
The cell of any one of claims 113-120, wherein the cell comprises a CAR-T cell or a CAR-NK cell.
Pharmaceutical composition, comprising the chimeric antigen receptor according to any one of claims 1-62, the nucleic acid molecule according to any one of claims 63, and the construct according to any one of claims 64-112 The body or cell of any one of claims 113-121, and optionally a pharmaceutically acceptable carrier.
The chimeric antigen receptor according to any one of claims 1-62, the nucleic acid molecule according to claim 63, the construct according to any one of claims 64-112, any one of claims 113-121 The use of the cells described in claim 122 or the pharmaceutical composition described in claim 122 in the preparation of medicines for preventing and/or treating tumors or autoimmune diseases.
The use of claim 123, wherein the tumor comprises a solid tumor and/or a hematological tumor.
The use according to any one of claims 123-124, wherein the tumor includes breast cancer, gastric cancer, ovarian cancer, cervical cancer, urothelial cancer, esophageal cancer, bladder cancer, colorectal cancer, endometrial cancer , kidney cancer, lung cancer, pancreatic cancer, head and neck cancer, sarcoma, glioblastoma, prostate cancer and/or thyroid cancer.
The use of claims 123-125, wherein the tumor comprises a HER2-positive tumor.
The chimeric antigen receptor according to any one of claims 1-62, the nucleic acid molecule according to claim 63, the construct according to any one of claims 64-112, any one of claims 113-121 The use of the cells described in claim 122 or the pharmaceutical composition described in claim 122 in the preparation of medicines for preventing and/or treating diseases or conditions with abnormal expression of HER2.
The use according to claim 127, wherein the disease or condition in which HER2 expression is abnormal comprises a disease or condition in which HER2 expression is upregulated.
The use according to any one of claims 127-128, wherein the disease or disorder with abnormal expression of HER2 includes a tumor.
The use of claim 129, wherein the tumor comprises a HER2-positive tumor.
A method for preventing and/or treating tumors, comprising administering to a subject in need an effective amount of the chimeric antigen receptor of any one of claims 1-62, the nucleic acid molecule of claim 63, The construct of any one of claims 64-112, the cell of any one of claims 113-121, and/or the pharmaceutical composition of claim 122.
A method for preventing and/or treating autoimmune diseases, comprising administering to a subject in need an effective amount of the chimeric antigen receptor according to any one of claims 1-62, the nucleic acid according to claim 63 Molecule, construct according to any one of claims 64-112, cell according to any one of claims 113-121, and/or pharmaceutical composition according to claim 122.
A method for preventing and/or treating diseases or conditions with abnormal expression of HER2, comprising administering to a subject in need an effective amount of the chimeric antigen receptor according to any one of claims 1-62, claim 63 The nucleic acid molecule, the construct of any one of claims 64-112, the cell of any one of claims 113-121, and/or the pharmaceutical composition of claim 122.