Modified immune cell and use thereof

Abstract

A modified immune cell, characterized in that the modified immune cell can secrete two binding molecules, wherein the first binding molecule is a VEGF binding molecule or a VEGFR binding molecule, and the second binding molecule is a PDL1 binding molecule or a PD-1 binding molecule. The immune cell can quickly infiltrate a tumor microenvironment, proliferate rapidly and release a large number of cytokines, and thus has a good anti-tumor effect.

Description

Modified immune cells and their uses Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a modified immune cell, characterized in that it can secrete two binding molecules, wherein one binding molecule is a VEGF binding molecule or a VEGFR binding molecule, and the other binding molecule is a PDL1 binding molecule or a PD-1 binding molecule. The present application also relates to the use of the immune cell. Background Art

[0002] Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T) involves genetically modifying T cells to express chimeric antigen receptors (CARs) that recognize tumor cell surface antigens, thereby enhancing T cell-specific tumor killing. CARs typically consist of a single-chain variable fragment (scFv) that specifically recognizes a tumor-associated antigen (TAA), a hinge region, a transmembrane region, and an intracellular signaling domain.

[0003] The programmed cell death-1 (PD-1) / programmed cell death-ligand 1 (PDL1) immunosuppressive signaling pathway inhibits T cell proliferation, reduces effector T cell function, induces immune tolerance, and promotes T cell apoptosis, severely impairing the anti-tumor efficacy of tumor-infiltrating T cells. Numerous marketed PD-1 and PDL1 antibody drugs have demonstrated significant efficacy across a wide range of tumor indications, a significant advancement in the biopharmaceutical field. This has also prompted increased focus within the industry on the development of drugs that relieve immunosuppression within the tumor immune microenvironment and restore "immune normalization."

[0004] Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), is a highly specific vascular endothelial growth factor that promotes vascular endothelial cell growth, including increased vascular permeability, extracellular matrix degeneration, endothelial cell migration, proliferation, and angiogenesis. VEGFR (vascular endothelial growth factor receptor) is a key transmembrane tyrosine kinase receptor that regulates angiogenesis and consists of three major isoforms: VEGFR-1 (Flt-1), VEGFR-2 (KDR / Flk-1), and VEGFR-3 (Flt-4). Its extracellular domain binds VEGF ligands (such as VEGF-A and VEGF-C), while its intracellular domain activates downstream signaling pathways (such as PI3K-AKT and RAS-MAPK) through kinase activity, promoting endothelial cell proliferation, migration, and increased vascular permeability. Antibodies or inhibitors targeting VEGF / VEGFR can inhibit tumor growth and metastasis by blocking signal transduction, making it an important target for anti-angiogenic therapy.

[0005] In recent years, CAR-T immunotherapy has shown significant therapeutic effects in the treatment of acute leukemia and non-Hodgkin's lymphoma, but the effect of CAR-expressing immune cells in solid tumors is limited. The main reasons include the limited infiltration of CAR-T in the tumor site and the restriction of CAR-T amplification in the tumor site by the tumor microenvironment, such as upregulation of immune checkpoints.

[0006] Currently, CAR-T immunotherapy urgently needs to be further optimized to enhance the expansion and infiltration ability of CAR-T in tumor sites. Summary of the Invention

[0007] The present application provides a modified immune cell and a method for promoting immune cell infiltration into the tumor microenvironment and effective expansion. The immune cell provided in the present application can secrete two binding molecules, wherein one binding molecule is a VEGF binding molecule or a VEGFR binding molecule, and the other binding molecule is a PDL1 binding molecule or a PD-1 binding molecule; the immune cell has one or more of the following advantages: (1) it can release more cytokines (including IL-2 and IFN-γ), (2) it can increase the number of CAR-T cells infiltrating in the body, (3) it can reduce the exhaustion of CAR-T cells in the body, and (4) it can significantly inhibit tumor growth under the premise of safety, and synergistically exert anti-tumor effects.

[0008] At the same time, the immune cells provided in the present application have similar advantages and effects on different targets. Using immune cells to secrete two binding molecules, one of which is a VEGF binding molecule or a VEGFR binding molecule, and the other is a PDL1 binding molecule or a PD-1 binding molecule, has a better effect than intraperitoneal injection of a combination of VEGF antibodies and PDL1 antibodies.

[0009] On the one hand, the present application provides a modified immune cell characterized in that it can secrete two binding molecules, wherein the first binding molecule can be a VEGF binding molecule or a VEGFR binding molecule, and the second binding molecule can be a PDL1 binding molecule or a PD-1 binding molecule.

[0010] In certain embodiments, the first binding molecule may be a VEGF binding molecule, and the second binding molecule may be a PDL1 binding molecule.

[0011] In certain embodiments, the first binding molecule may be a VEGF binding molecule, and the second binding molecule may be a PD-1 binding molecule.

[0012] In certain embodiments, the first binding molecule may be a VEGFR binding molecule, and the second binding molecule may be a PDL1 antibody.

[0013] In certain embodiments, the first binding molecule may be a VEGFR antibody, and the second binding molecule may be a PD-1 antibody.

[0014] In certain embodiments, the binding molecule can be selected from the group consisting of an antibody or an antigen-binding fragment thereof, a fusion protein, a polypeptide, a truncation, and a genetically engineered scaffold protein.

[0015] In certain embodiments, the antibody is selected from the group consisting of a monoclonal antibody, a single-chain antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

[0016] In certain embodiments, the antigen binding protein is selected from the group consisting of Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv, VHH and dAb.

[0017] In certain embodiments, the binding molecule comprises a scFv.

[0018] In certain embodiments, the binding molecule comprises a VHH.

[0019] In certain embodiments, the two binding molecules secreted by the immune cells may be secreted in the form of monoclonal antibodies.

[0020] In certain embodiments, the two binding molecules secreted by the immune cells may be secreted in the form of a bispecific antibody.

[0021] In certain embodiments, the immune cells may be derived from iPSCs.

[0022] In certain embodiments, the immune cells may be one or more cells selected from the group consisting of NK cells, NKT cells, macrophages, and T cells.

[0023] In certain embodiments, the immune cells may be T cells.

[0024] In certain embodiments, the immune cells may express a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0025] In certain embodiments, the CAR may comprise a targeting moiety.

[0026] In certain embodiments, the targeting moiety can be a scFv.

[0027] In certain embodiments, the targeting moiety may be a VHH.

[0028] In certain embodiments, the targeting moiety specifically binds to and / or recognizes a tumor antigen, and the antigen is selected from one or more antigens in the following group: CD19, BCMA, HER2, Mesothelin, GPC3, Muc 1, ROR1 and CDH17.

[0029] In certain embodiments, the targeting moiety specifically binds to and / or recognizes GPC3.

[0030] In certain embodiments, the targeting moiety specifically binds to and / or recognizes ROR1.

[0031] In certain embodiments, the targeting moiety specifically binds to and / or recognizes CDH17.

[0032] In certain embodiments, the CAR may comprise a signal peptide.

[0033] In certain embodiments, the peptide may be a signal peptide derived from CD8 protein.

[0034] In certain embodiments, the CAR may comprise a hinge region comprising a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

[0035] In certain embodiments, the hinge region may be a hinge region derived from CD8.

[0036] In certain embodiments, the CAR may comprise a transmembrane region comprising a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

[0037] In certain embodiments, the transmembrane region may be a transmembrane region derived from CD8.

[0038] In certain embodiments, the CAR may comprise a costimulatory domain comprising a costimulatory domain derived from one or more proteins selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, 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, a ligand of CD83, CD40, and MyD88.

[0039] In certain embodiments, the costimulatory domain may be a costimulatory domain derived from 4-1BB.

[0040] In certain embodiments, the CAR may comprise an intracellular signaling domain comprising an intracellular signaling domain derived from one or more proteins selected from the group consisting of CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM.

[0041] In certain embodiments, the intracellular signaling domain may be an intracellular signaling domain derived from CD3ζ.

[0042] In certain embodiments, the CAR comprises a leader peptide.

[0043] In certain embodiments, the leader peptide is an 1gκ leader peptide.

[0044] In certain embodiments, the CAR may comprise a cleavage peptide comprising one or more cleavage peptides selected from the group consisting of T2A, P2A, E2A, and F2A.

[0045] In certain embodiments, the cleavage peptide may be T2A.

[0046] In certain embodiments, the CAR can be linked to a first domain and a second domain that express a secreted form of an antibody, wherein the first domain can express a VEGF binding molecule or a VEGFR binding molecule, and the second domain can express a PDL1 binding molecule or a PD-1 binding molecule.

[0047] In certain embodiments, the first domain and the second domain are connected by one or more linkers.

[0048] In certain embodiments, the linker may comprise an amino acid sequence of (GGGGS)n, wherein n is any positive integer between 1 and 10.

[0049] In certain embodiments, the linker may comprise an amino acid sequence of (GGS)n, wherein n is any positive integer between 1 and 10.

[0050] In certain embodiments, the linker may be (GGS)5.

[0051] In certain embodiments, the linker may be (GGGGS)3.

[0052] In certain embodiments, the first domain and the second domain are connected by a cleavage peptide.

[0053] In certain embodiments, the cleavage peptide may be one or more cleavage peptides selected from the group consisting of T2A, P2A, E2A, and F2A.

[0054] In certain embodiments, the cleavage peptide is T2A.

[0055] In certain embodiments, the CAR may comprise a CD8 signal peptide, a GPC3 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a first domain, a (GGGGS)3 linker, and a second domain, which are sequentially connected.

[0056] In certain embodiments, the CAR may comprise a CD8 signal peptide, a GPC3 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a first domain, a T2A cleavage peptide, and a second domain connected in sequence.

[0057] In certain embodiments, the CAR may comprise a CD8 signal peptide, a GPC3 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a second domain, a (GGGGS)3 linker, and a first domain, which are sequentially connected.

[0058] In certain embodiments, the CAR may comprise a CD8 signal peptide, a GPC3 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a second domain, a T2A cleavage peptide, and a first domain, which are sequentially connected.

[0059] In certain embodiments, the CAR may comprise a CD8 signal peptide, a ROR1 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a first domain, a (GGGGS)3 linker, and a second domain, which are sequentially connected.

[0060] In certain embodiments, the CAR may comprise a CD8 signal peptide, a ROR1 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a first domain, a T2A cleavage peptide, and a second domain connected in sequence.

[0061] In certain embodiments, the CAR may comprise a CD8 signal peptide, a ROR1 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a second domain, a (GGGGS)3 linker, and a first domain connected in sequence.

[0062] In certain embodiments, the CAR may comprise a CD8 signal peptide, a ROR1 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a second domain, a T2A cleavage peptide, and a first domain, which are sequentially connected.

[0063] In certain embodiments, the CAR may comprise a CD8 signal peptide, a CDH17 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a first domain, a (GGGGS)3 linker, and a second domain, which are sequentially connected.

[0064] In certain embodiments, the CAR may comprise a CD8 signal peptide, a CDH17 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a first domain, a T2A cleavage peptide, and a second domain connected in sequence.

[0065] In certain embodiments, the CAR may comprise a CD8 signal peptide, a CDH17 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a second domain, a (GGGGS)3 linker, and a first domain connected in sequence.

[0066] In certain embodiments, the CAR may comprise a CD8 signal peptide, a CDH17 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a second domain, a T2A cleavage peptide, and a first domain, which are sequentially connected.

[0067] In certain embodiments, the immune cell may comprise the nucleic acid sequences shown in SEQ ID NO:1, SEQ ID NO:52 and SEQ ID NO:54.

[0068] In certain embodiments, the immune cells may express an amino acid sequence comprising SEQ ID NO:6, SEQ ID NO:53, and SEQ ID NO:55.

[0069] On the other hand, the present application also provides one or more isolated nucleic acid molecules, which can encode the CAR described in the present application.

[0070] In certain embodiments, the nucleic acid molecule may comprise the nucleic acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 52, and SEQ ID NO: 54.

[0071] In certain embodiments, the nucleic acid molecule may further comprise a promoter.

[0072] In certain embodiments, the promoter may be the EF-1α promoter.

[0073] On the other hand, the present application also provides a vector comprising the nucleic acid molecule described in the present application.

[0074] In certain embodiments, the vector may be a viral vector.

[0075] In certain embodiments, the vector may be a lentiviral vector.

[0076] On the other hand, the present application also provides a pharmaceutical composition comprising the immune cells described in the present application, the nucleic acid molecules described in the present application, and / or the vector described in the present application, and optionally a pharmaceutically acceptable carrier.

[0077] On the other hand, the present application also provides a method for preparing modified immune cells, which comprises introducing the vector described in the present application into immune effector cells.

[0078] On the other hand, the present application also provides the use of the immune cells, the nucleic acid molecules, the vectors, and / or the pharmaceutical compositions described in the present application in the preparation of drugs for preventing, treating and / or alleviating diseases and / or conditions.

[0079] On the other hand, the present application also provides the immune cells, the nucleic acid molecules, the vectors, and / or the pharmaceutical compositions described in the present application, which are used to prevent, treat and / or alleviate diseases and / or conditions.

[0080] On the other hand, the present application also provides a method for preventing, treating and / or alleviating a disease and / or condition, which comprises administering the cells described herein and / or the pharmaceutical composition described herein to a subject in need thereof.

[0081] On the other hand, the present application also provides a method for promoting the infiltration of immune cells into the tumor microenvironment, which comprises the following steps: causing the immune cells to secrete two binding molecules, wherein one binding molecule is a VEGF binding molecule or a VEGFR binding molecule, and the other binding molecule is a PDL1 binding molecule or a PD-1 binding molecule.

[0082] On the other hand, the present application also provides a method for enhancing the release of cytokines by immune cells, which comprises the following steps: causing the immune cells to secrete two binding molecules, wherein one binding molecule is a VEGF binding molecule or a VEGFR binding molecule, and the other binding molecule is a PDL1 binding molecule or a PD-1 binding molecule.

[0083] In certain embodiments, the cytokines comprise interleukins, interferons, and / or tumor necrosis factors.

[0084] In certain embodiments, the cytokine comprises IL-2 and / or IFN-γ.

[0085] In certain embodiments, the method further comprises the step of deriving the immune cells from iPSCs.

[0086] In certain embodiments, the immune cells in the method are one or more cells selected from the group consisting of NK cells, NKT cells, macrophages, and T cells.

[0087] In certain embodiments, the immune cells in the method are T cells.

[0088] According to common knowledge in the art, for antibodies without human-mouse cross-binding activity, alternative in vivo mouse experiments are often performed using antibodies with mouse binding activity against the same target to help verify the biological function and pharmacological effects of the target in animal models. For example, the article published in 2009, Krupitskaya Y, Wakelee HA. Ramucirumab, a fully human mAb to the transmembrane signaling tyrosine kinase VEGFR-2 for the potential treatment of cancer. Curr Opin Investig Drugs. 2009 Jun; 10(6): 597-605. PMID: 19513949. Based on the experiments in the article, an antibody with mouse binding activity against the same target (DC-101) can be used to provide important information about the biological function and pharmacological effects of the target in mouse models; based on the preliminary evidence provided by the mouse model, antibodies active against human VEGFR-2 (such as Ramucirumab) are developed to ensure that they can be translated into human treatment.

[0089] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] The features and advantages of the invention of this application can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0091] Figure 1 shows a schematic diagram of the CAR structure and mechanism of action.

[0092] Figure 2 shows a schematic diagram of the CAR vector sequence combination.

[0093] Figure 3A shows the results of IFN-γ release detection after GPC3-targeted CAR-T cells were co-incubated with target cells HuH-7 for 24 hours.

[0094] Figure 3B shows the results of IL-2 release detection after GPC3-targeted CAR-T cells were co-incubated with target HuH-7 cells for 24 hours.

[0095] Figure 4A shows the VEGF antibody concentration in the supernatant of CAR-T cells after co-incubation of GPC3-targeted CAR-T with target HuH-7 cells.

[0096] Figure 4B shows the results of the detection of PDL1 antibody concentration in the supernatant of CAR-T cells after co-incubation of GPC3-targeted CAR-T with target cells HuH-7.

[0097] Figure 5A shows the tumor growth curve after GPC3-targeted CAR-T (or T) infusion (n=6, Mean±SEM).

[0098] Figure 5B shows the in vivo expansion change curve after GPC3-targeted CAR-T (or T) infusion.

[0099] Figure 5C shows the results of the detection of changes in the secretion level of cytokine IFN-γ in the peripheral blood of mice after the reinfusion of GPC3-targeted CAR-T (or T).

[0100] Figure 5D shows the results of the weight change test of mice after the infusion of targeted GPC3 CAR-T (or T).

[0101] Figure 5E shows the percentage of GPC3-targeted CAR-T (or T) cells infiltrating into tumor tissue.

[0102] Figure 5F shows the results of immunofluorescence staining of tumor tissue blood vessels and CAR-T infiltration after G3 CAR-T and G3-aVEGF-2A-aPDL1 CAR-T reinfusion.

[0103] Figure 5G shows the results of quantitative immunofluorescence detection of vascular distribution in tumor tissue after reinfusion of G3 CAR-T and G3-aVEGF-2A-aPDL1 CAR-T.

[0104] Figure 5H shows the results of quantitative immunofluorescence detection of CAR-T infiltration in tumor tissue after reinfusion of G3 CAR-T and G3-aVEGF-2A-aPDL1 CAR-T.

[0105] Figure 6 shows the in vivo anti-tumor effect test results comparing G3 CAR-T cells that simultaneously secrete aVEGF scFv and aPDL1 scFv (i.e., G3-aVEGF-2A-aPDL1) and G3 CAR-T cells that secrete VEGF and PDL1 dual antibodies (i.e., G3-aVEGF-linker-aPDL1 CAR-T).

[0106] Figure 7A shows the tumor growth curve after GPC3-targeted CAR-T (or T) infusion (n=5, Mean±SEM).

[0107] Figure 7B shows the in vivo expansion change curve after GPC3-targeted CAR-T (or T) infusion.

[0108] Figure 7C shows the changes in cytokine IFN-γ secretion in the peripheral blood of mice after GPC3-targeted CAR-T (or T) infusion.

[0109] Figure 7D shows the results of flow cytometric detection of GPC3-targeted CAR-T (or T) cell exhaustion markers in mouse peripheral blood.

[0110] Figure 8A shows the infiltration ratio of GPC3-targeted CAR-T (or T) cells in tumor tissues.

[0111] Figure 8B shows the number of GPC3-targeted CAR-T (or T) cells infiltrating tumor tissues.

[0112] Figure 8C shows the ratio of GPC3-targeted CAR-T (or T) cell exhaustion markers infiltrating mouse tumor tissue.

[0113] Figure 8D shows the multiples of peripheral blood VEGF antibody concentration relative to the corresponding antibody concentration in the tumor microenvironment.

[0114] Figure 8E shows the multiples of the PDL1 antibody concentration in peripheral blood relative to the corresponding antibody concentration in the tumor microenvironment.

[0115] Figure 9A shows the curve of tumor volume change after ROR1-targeted CAR-T (or T) reinfusion.

[0116] Figure 9B shows the expansion curve of ROR1-targeted CAR-T (or T) in the peripheral blood of mice after reinfusion.

[0117] Figure 9C shows the changes in cytokine IFN-γ secretion in the peripheral blood of mice after ROR1-targeted CAR-T (or T) infusion.

[0118] Figure 9D shows the changes in mouse body weight after ROR1-targeted CAR-T (or T) transfusion.

[0119] Figure 9E shows the tumor infiltration ratio of ROR1-targeted CAR-T (or T).

[0120] Figure 10A shows the effects of different secretory antibody combinations on tumor inhibition in the GPC3 target liver cancer model.

[0121] Figure 10B shows the effects of different secretory antibody combinations on T cell infiltration in tumors in the GPC3-targeted liver cancer model.

[0122] Figure 10C shows the effects of different secretory antibody combinations on tumor inhibition in a CDH17-targeted colon cancer model. DETAILED DESCRIPTION

[0123] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0124] Definition of terms

[0125] In this application, the term "binding molecule" generally refers to a natural or artificially designed molecule that can specifically recognize or bind to a target molecule, and the binding molecule has a blocking effect on the target molecule. The biological molecule can be an antibody or antigen-binding fragment thereof, a fusion protein, a polypeptide, a truncation, a genetically engineered scaffold protein (an artificially designed protein not derived from an antibody), an aptamer, a small molecule inhibitor, or a natural ligand analog.

[0126] In this application, the term "immune cell" generally refers to cells involved in or associated with an immune response. The immune cell may include lymphocytes and various phagocytes. The immune cell may also include natural and genetically modified immune cells. In this application, the genetically modified immune cell may express a chimeric antigen receptor (CAR). In this application, the immune cell may include a T cell.

[0127] In this application, the term "modified" generally refers to an alteration or modification made to a cell. For example, the genetic modification can be a modification at the gene level, transcription level, and / or translation level. For another example, the genetic modification can include a change in any genetic characteristic of an organism (including its tissues, cells, DNA, mRNA, or proteins and fragments thereof). The genetic modification can include causing the organism to express a specific protein or fragment thereof. For example, the modification can include causing the organism to contain a vector capable of expressing the specific protein or fragment thereof.

[0128] In this application, the term "PD-1" generally refers to programmed cell death 1, also known as "programmed cell death 1", "CD279", "cluster of differentiation 279", "PD1", "PDCD1". PD-1 is generally expressed on T cells, B cells, natural killer T cells, activated monocytes and dendritic cells (DC), and is involved in apoptosis. PD-1 generally comprises an extracellular IgV domain, a transmembrane region and an intracellular domain. PD-1 can bind to two ligands, PDL1 and PD-L2. The "PD-1" includes any natural PD-1 of any vertebrate origin, including mammals, such as primates (e.g., humans and crab-eating macaques) and rodents (e.g., mice and rats). The term covers "full length", unprocessed PD-1 as well as any form of PD-1 produced by cell processing. PD-1 may exist as a transmembrane protein or as a soluble protein. “PD-1” includes intact PD-1 and fragments thereof, as well as functional variants, isoforms, species homologs, derivatives, analogs of PD-1, and analogs having at least one common epitope with PD-1.

[0129] In this application, the term "PD-L1" or "PDL1" generally refers to programmed cell death 1 ligand 1, also known as B7 homolog 1, B7-H1, cluster of differentiation 274, (3)274 or CD274, which downregulates T cell activation and cytokine secretion after binding to PD-1. "PDL1" includes any native PDL1 of any vertebrate origin, including mammals, such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats). The term covers "full-length", unprocessed PD-L1 as well as any form of PDL1 produced by cell processing. PDL1 can exist as a transmembrane protein or as a soluble protein. "PDL1" includes complete PDL1 and fragments thereof, and also includes functional variants, isoforms, species homologs, derivatives, analogs, and analogs having at least one common epitope with PDL1. The basic structure of PDL1 includes four domains: an extracellular Ig-like V-type domain and an Ig-like C2-type domain, a transmembrane domain, and a cytoplasmic domain.

[0130] In this application, the term "VEGF" or "VEGF-A" generally refers to human vascular endothelial growth factor and the related 121, 145, 189 and 206 amino acid human vascular endothelial growth factors, as well as their naturally occurring allelic and processed forms. VEGF-A is part of a gene family that includes VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and PlGF. VEGF-A primarily binds to two high-affinity receptor tyrosine kinases, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1 / KDR), the latter being the primary transmitter of VEGF-A's mitogenic signal in vascular endothelial cells. In addition, neuropilin-1 has been identified as a heparin-binding VEGF-A isoform of the receptor and may play a role in vascular development. The term "VEGF" or "VEGF-A" also refers to VEGF from non-human species such as mouse, rat or primate.

[0131] In this application, the term "VEGFR" generally refers to the vascular endothelial growth factor receptor, whose family members include VEGFR-1, VEGFR-2, and VEGFR-3. It is a receptor tyrosine kinase. This protein binds to vascular endothelial growth factor (VEGF) to promote the proliferation, survival, migration and differentiation of endothelial cells, and plays an important role in angiogenesis, vascular development, vascular permeability, embryonic vascular development, cell migration and the regulation of cancer cells.

[0132] Throughout this application, the terms "polypeptide molecule," "polypeptide," and "peptide" are used interchangeably and generally refer to a polymer of amino acid residues. The term "fusion protein" generally refers to a polypeptide having at least two covalently linked moieties. Each moiety can be a polypeptide with a distinct property. This property can be a biological property, such as in vitro or in vivo activity. It can also be a simple chemical or physical property, such as binding to a target molecule or catalysis of a reaction. The two moieties can be directly linked by a single peptide bond or through a peptide linker.

[0133] In this application, the term "fusion protein" generally refers to a macromolecule formed by chemical and / or genetic fusion of two or more distinct protein molecules. A fusion protein can be the expression product of two or more genes recombined through DNA recombination technology, or a group of proteins that mediate the fusion of two or more cell membranes. In this application, a fusion protein can be a protein molecule containing these two domains, which is formed by genetically linking a target gene with an immunoglobulin Fc region or its variant fragment gene, and expressed in eukaryotic or prokaryotic cells.

[0134] In this application, the term "truncated protein" generally refers to a truncated protein, which is a variant generated by deleting specific regions of the native protein (such as transmembrane domains, non-functional domains), while retaining key functional structures (such as ligand binding domains). The truncated protein can be obtained by proteolysis or manipulation of the structural gene to eliminate the N- or C-terminal portion of the protein. Alternatively, the truncated protein can be obtained by prematurely terminating translation by causing a stop codon in the structural gene due to a nonsense mutation.

[0135] In this application, the term "genetically engineered scaffold protein" generally refers to an artificial protein that is modified by directed evolution or rational design based on a natural protein (non-antibody source).

[0136] In this application, the term "T cell receptor" (TCR) generally refers to the T cell antigen receptor, which is a molecular structure that T cells specifically recognize and bind to antigen peptide-MHC molecules. It is usually present on the T cell surface in the form of a complex with the CD3 molecule. The TCR of most T cells is composed of α and β peptide chains, while the TCR of a few T cells is composed of γ and δ peptide chains.

[0137] In this application, the term "monoclonal antibody" generally refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence, except for possible naturally occurring mutations that may be present in small amounts. In this application, the "monoclonal antibody" may be an antibody against PDL1. In this application, the "monoclonal antibody" may be an antibody against VEGF.

[0138] In this application, the term "bispecific antibody" generally refers to an antibody that has two different antigen binding sites within a single antibody molecule. In this application, the "bispecific antibody" can be a PDL1 antibody and a VEGF antibody.

[0139] In this application, the term "T cell", also known as "T lymphocyte", is a subtype of white blood cells that plays a central role in cell-mediated immunity. T cells can be distinguished from other lymphocytes such as B cells and natural killer cells by the presence of T cell receptors on their cell surface.

[0140] In this application, the term "chimeric antigen receptor" is generally referred to as "CAR", which generally refers to a fusion protein comprising an extracellular domain capable of binding to an antigen and at least one intracellular domain. In this application, the CAR may include an intracellular domain, and the intracellular domain includes a signaling domain and / or a costimulatory domain. In this application, a group of polypeptides of CAR may be located in the same polypeptide chain (for example, comprising a chimeric fusion protein), or may be discontinuous with each other, for example, they may be located in different polypeptide chains. In this application, the signal involved in induction can be transduced into the cytoplasm of T cells via the CD3ζ chain. In this application, the intracellular domain may include a primary signaling binding domain (for example, the main signaling domain of CD3ζ). In one aspect, the cytoplasmic signaling domain may also include one or more costimulatory domains derived from at least one costimulatory molecule. In this application, CAR may include a chimeric fusion protein, for example, containing an optional leader sequence at the amino terminus (N-ter).

[0141] In this application, the term "signaling domain" generally refers to a domain located inside a cell that can transduce a signal. In this application, the intracellular signaling domain can conduct a signal into the cell. For example, the intracellular signaling domain is the intracellular signaling domain of the chimeric antigen receptor. In this application, the signaling domain may comprise a portion selected from the group consisting of the signaling domains of CD3ζ, CD3δ, and CD3ε.

[0142] In this application, the term "costimulatory domain" generally refers to a domain in CAR that passes through the cell membrane and is connected to an intracellular signal transduction domain to transmit a signal. In this application, the costimulatory domain may include a portion selected from the group consisting of CD27, CD28, and 4-1BB costimulatory domains.

[0143] In this application, the term "hinge region" generally refers to the connecting region between the antigen binding region and the immune cell Fc receptor (FcR) binding region. For example, the hinge region can be the region between the heavy chain CH1 and CH2 functional regions of an immunoglobulin. In this application, the hinge region can be a region located between the scFv and the T cell membrane. The hinge region can be derived from IgG1 or IgG4, or from IgD or CD8. In this application, the hinge region can include a portion selected from the group consisting of the hinge region of IgG4, the hinge region of IgG1, and the hinge region of CD8.

[0144] In this application, the term "transmembrane region" generally refers to the transmembrane region connecting the extracellular antigen binding domain and the intracellular signaling domain, which is generally composed of a dimeric membrane protein, mainly including CD3ζ, CD4, CD8, CD28, etc., which can anchor the CAR structure to the T cell membrane. Different designs of the transmembrane region can affect the expression of the introduced CAR gene. In this application, the transmembrane region may include a portion selected from the following groups: the transmembrane region of CD8, the transmembrane region of CD28, and the transmembrane region of CD24.

[0145] In the present application, the term "single-chain antibody" (scFv) generally refers to an antibody formed by connecting the heavy chain variable region and the light chain variable region via a linker. In the present application, the linker can be a connecting peptide.

[0146] In this application, the terms "VHH" and "Nanobody" are used interchangeably and generally refer to antibodies that contain the variable antigen-binding domain of a heavy chain antibody.

[0147] In the present application, the term "tumor antigen" generally refers to an antigenic substance in or produced by a tumor cell that may have the ability to trigger an immune response in a host. For example, a tumor antigen can be a protein, polypeptide, peptide, or fragment thereof that constitutes a part of a tumor cell and can induce tumor-specific cytotoxic T lymphocytes. In some embodiments, the term "tumor antigen" can also refer to a biomolecule (e.g., protein, carbohydrate, glycoprotein, etc.) that is specifically or preferentially or differentially expressed and / or associated with a cancer cell on a cancer cell, thereby providing a preferential or specific target for cancer. For example, preferential expression can be conventional preferential expression, or preferential expression (e.g., in a specific organ or tissue) within a specific region of an organism, compared to any other cell in the organism. In the present application, the tumor antigen can include B lymphocyte surface antigens, TNF family members, HER family members, and GPC family members.

[0148] In this application, the term "B lymphocyte surface antigen" generally refers to an antigen located on the surface of a B lymphocyte produced by a B lymphocyte at different stages. For example, the B lymphocyte surface antigen may include CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, and CD40. In this application, the B lymphocyte surface antigen may include CD19.

[0149] In this application, the term "CD19" generally refers to the cluster of differentiation 19 protein, which is an antigenic determinant that can be detected on leukemic precursor cells. The accession number of human CD19 in UniProt / Swiss-Prot is P15391, and the accession number of the nucleotide sequence encoding human CD19 in GenBank is NM_001178098. In this application, CD19 can also include proteins containing mutations or functional fragments thereof, such as point mutations, fragments, insertions, deletions and splice variants of full-length wild-type CD19.

[0150] In the present application, the term "TNF family member" generally refers to a member belonging to the TNF (tumor necrosis factor) family. TNF family members may include CD40LG (TNFSF5), CD70 (TNFSF7), EDA, FASLG (TNFSF6), LTA (TNFSF1), LTB (TNFSF3), TNFSF4 (OX40L), TNFSF8 (CD153), TNFSF9 (4-1BB), TNFSF10 (TRAIL), TNFSF11 (RANKL), TNFSF12 (TWEAK), TNFSF13, TNFSF13B, TNFSF14, TNFSF15, TNFSF17 (BCMA) and TNFSF18. In the present application, the TNF family member may include BCMA and 4-1BB.

[0151] In this application, the term "BCMA" generally refers to B cell maturation antigen (BCMA, CD269). BCMA is a member of the tumor necrosis factor receptor (TNF) superfamily that binds to B cell activating factor (BAFF) and proliferation-inducing ligand (APRIL). BCMA is commonly found on the surface of plasma cells in patients with multiple myeloma.

[0152] The term "CD137 protein," also known as 4-1BB or TNFRS9, generally refers to a transmembrane protein of the tumor necrosis factor receptor superfamily (TNFRS). It is an activation-induced co-stimulatory molecule and an important regulator of the immune response. Studies have shown that CD137 agonistic monoclonal antibodies increase the expression of co-stimulatory molecules in many models and significantly enhance cytolytic T lymphocyte responses, exerting anti-tumor effects.

[0153] In this application, the term "HER family member" generally refers to a member belonging to the HER (human epidermal growth factor receptor) family. HER family members may include EGFR (ErbB-1), HER2 / c-neu (ErbB-2), Her3 (ErbB-3), and Her4 (ErbB-4). In this application, the HER family member may include HER2.

[0154] In this application, the term "HER2" generally refers to the human HER2 protein, which is a member of the HER family. For example, see Semba et al., PNAS (USA) 82: 6497-6501 (1985) and Yamamoto et al., Nature 319: 230-234 (1986). The GenBank accession number for human HER2 may include XP_024306409.1.

[0155] In this application, the term "GPC family member" generally refers to glypican. Six glypicans have been identified in mammals, namely GPC1 to GPC6. Glypicans are abnormally expressed in cancers, including human hepatocellular carcinoma, ovarian cancer, mesothelioma, pancreatic cancer, glioma, and breast cancer. In this application, the GPC family member may include GPC3.

[0156] As used herein, the term "GPC3" generally refers to the protein encoded by glypican 3 (NCBI database gene ID: 2719), an early marker for liver cancer. GPC3 is highly expressed in hepatocellular carcinoma and has been detected in tissue from patients with early-stage hepatocellular carcinoma. The GenBank accession number for human GPC3 is AAB87062.1.

[0157] In this application, the term "Mesothelin" (MSLN) generally refers to a tumor differentiation antigen that is normally present on mesothelial cells lining the pleura, peritoneum, and pericardium. It is highly expressed in cancers including malignant mesothelioma, pancreatic cancer, ovarian cancer, and lung adenocarcinoma.

[0158] In this application, the term "CDH17" is also referred to as "cadherin 17," "liver-intestinal cadherin," "LI-cadherin," and "intestinal peptide-related transporter HPT-1." The term encompasses "full-length," unprocessed CDH17, as well as any form of CDH17 produced by cellular processing. In this application, the term "CDH17" includes full-length wild-type CDH17 and mutants, fragments, variants, isoforms, and homologs thereof. For example, the CDH17 may include human CDH17. For example, the sequence of human CDH17 can be found in Genbank Accession No. NM004063.

[0159] In this application, the term "cytokine" generally refers to a class of small molecule proteins with a wide range of biological activities that are synthesized and secreted by immune cells (such as monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.) upon stimulation. The cytokine may have multiple functions such as regulating innate immunity and adaptive immunity, hematopoiesis, cell growth, APSC multipotent cells, and damaged tissue repair. In this application, the cytokine may include interleukins, interferons, tumor necrosis factor superfamily, colony stimulating factors, chemokines, and growth factors. For example, the cytokine may be an interleukin.

[0160] In the present application, the term "interleukin" generally refers to a secretory protein or signaling molecule that can promote the development and differentiation of T and / or B lymphocytes and / or hematopoietic cells. Interleukins can be synthesized by auxiliary CD4 T lymphocytes, as well as by monocytes, macrophages and endothelial cells. In the present application, the term "interleukin" can include full-length interleukins or fragments (such as truncated forms) or variants thereof, which substantially maintain the biological activity of the corresponding wild-type interleukin (for example, having a biological activity of at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or even at least 100% of the biological activity of the corresponding wild-type interleukin). The interleukin used herein can be from any mammalian species. In certain embodiments, the interleukin is from a species selected from humans, horses, cattle, mice, pigs, rabbits, cats, dogs, rats, goats, sheep and non-human primates. In certain embodiments, the interleukin can be a mutant form.

[0161] In this application, the term "retroviral vector" generally refers to an RNA virus that can reverse transcribe into a complementary DNA strand within infected cells. This single DNA strand serves as a template for synthesizing a second DNA strand, which is then incorporated into the cell's genomic DNA. These retroviral vectors utilize host cell enzymes to transcribe and replicate RNA into proteins, which are then packaged and released from the cell as infectious viruses. These retroviral vectors have high transduction efficiencies, effectively increasing gene delivery rates.

[0162] In this application, the term "lentiviral vector" refers to a gene therapy vector developed based on HIV-1 (human immunodeficiency virus type 1). These lentiviral vectors are capable of infecting both dividing and non-dividing cells. They can effectively infect nearly all mammalian cells, including neurons and hepatocytes, with high infection efficiency. Lentiviruses can effectively integrate exogenous genes into host chromosomes, thereby achieving persistent expression.

[0163] In the present application, the term "pharmaceutically acceptable carrier" may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes and / or nonionic surfactants, etc.

[0164] In this application, the term "nucleic acid molecule" includes DNA molecules and RNA molecules. A nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA. The term "promoter" generally refers to a DNA sequence that can regulate the expression of a selected DNA sequence operably linked to the promoter, thereby affecting the expression of the selected DNA sequence in a cell. For example, the nucleic acid molecule can encode the antigen-binding protein and / or the chimeric antigen receptor. For example, the nucleic acid molecule can include a promoter. For example, the promoter can be a constitutive promoter. For example, the promoter can be the EF1α promoter.

[0165] In this application, the term "vector" generally refers to a molecule to which one or more nucleic acid molecules of the present application can be attached. For example, the vector can be a viral vector. For example, the vector can be a lentiviral vector.

[0166] In this application, the term "antibody" generally refers to an immunoglobulin or its fragment or derivative thereof, encompassing any polypeptide comprising an antigen binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated and transplanted antibodies. Unless otherwise modified by the term "complete", as in "complete antibody", for the purposes of the present invention, the term "antibody" also includes antibody fragments, such as Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv (bispecific single-chain antibody), VHH and dAb and other antibody fragments that retain antigen binding function. Typically, such fragments should include an antigen binding domain.

[0167] In this application, the term "antigen-binding fragment" generally refers to one or more fragments that have the ability to specifically bind to an antigen. In this application, the antigen-binding fragment can include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

[0168] As used herein, the term "diagnosis" generally refers to the detection of a disease or condition, or the determination of the state or extent of a disease or condition, and also includes the detection of the cause of a disease or condition, the determination of the therapeutic effect of a drug therapy, or the prediction of a response pattern to a drug therapy or a xenobiotic. The diagnostic methods described herein can be used independently or in combination with other diagnostic and / or staging methods known in the medical field for a particular disease or condition.

[0169] As used herein, the term "prevent" generally refers to methods used to prevent, inhibit, or reduce the likelihood of the occurrence or recurrence of a disease or condition (e.g., cancer). It also refers to delaying the onset or recurrence of a disease or condition or delaying the onset or recurrence of symptoms of a disease or condition. "Prevent" and similar terms also include reducing the intensity, effects, symptoms, and / or burden of a disease or condition prior to its onset or recurrence.

[0170] As used herein, the term "treat" generally refers to any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may even include a minimal reduction in one or more measurable markers of the disease or condition being treated (e.g., cancer). Treatment may optionally involve a reduction or improvement in the symptoms of a disease or condition, or a delay in the progression of a disease or condition. "Treatment" does not necessarily indicate complete eradication or cure of a disease or condition or its associated symptoms.

[0171] In this application, the term "proliferation" generally refers to the generation of multiple individual cells by division of a starting cell. The multiple individual cells can be of the same type or of different types. The starting cells used for proliferation need not be the same as the cells produced by proliferation. For example, the proliferated cells can be generated from the growth and differentiation of a starting cell population.

[0172] In this application, the term "differentiation" generally refers to the process by which a non-specific or less specific cell acquires specific cell characteristics. A differentiated or differentiation-induced cell is a cell that occupies a more specific position in a cell lineage.

[0173] In this application, the term "ex vivo" generally refers to operations performed on cells, tissues and / or organs that have been removed from an organism. In some embodiments, the cells, tissues and / or organs can be returned to the organism or introduced into another organism by certain methods.

[0174] As used herein, the term "monoclonal antibody" generally refers to an antibody that is capable of specifically binding to one antigen or epitope.

[0175] In this application, the term "bispecific antibody" generally refers to an antibody that can specifically bind to two antigens or epitopes.

[0176] In this application, the term "in vitro" generally refers to removing or releasing a part of an organism from the organism.

[0177] In this application, the term "include" generally means to include, encompass, contain or encompass. In some cases, it also means "to be", "to be composed of..."

[0178] In this application, the term "and / or" should be understood to mean any one, two, more than one or any combination of the alternatives.

[0179] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0180] Detailed Description of the Invention

[0181] Chimeric Antigen Receptor (CAR)

[0182] On the one hand, the present application provides a chimeric antigen receptor (CAR) that can express two binding molecules in a secretory form, wherein the first binding molecule is a VEGF binding molecule or a VEGFR binding molecule, and the second binding molecule is a PDL1 binding molecule or a PD-1 binding molecule.

[0183] In the present application, the CAR is connected to express a first domain and a second domain of a secretory binding molecule, wherein the first domain expresses a VEGF binding molecule or a VEGFR binding molecule, and the second domain expresses a PDL1 binding molecule or a PD-1 binding molecule.

[0184] In the present application, the CAR and the first domain or the second structure can be directly connected, can be connected by a connector, or can be linked by a cleavage peptide. In the present application, the first domain and the second domain can be directly connected, can be connected by a connector, or can be linked by a cleavage peptide.

[0185] For example, the linker may comprise an amino acid sequence of (GGGGS)n, wherein n is any positive integer between 1 and 10. For example, the linker may comprise an amino acid sequence of (GGS)n, wherein n is any positive integer between 1 and 10. For example, the linker may comprise an amino acid sequence of (GGGGS)3, or may comprise an amino acid sequence of (GGS)5. For example, the cleavage peptide comprises one or more cleavage peptides selected from the group consisting of T2A, P2A, E2A, and F2A. For example, the cleavage peptide is T2A.

[0186] In the present application, the secreted binding molecule can be a natural or artificially designed molecule that specifically recognizes or binds to VEGF / VEGFR / PD-1 / PDL1, which can be referred to as a VEGF binding molecule, a VEGFR binding molecule, a PD-1 binding molecule, and a PDL1 binding molecule in the present application. The binding molecule can be an antibody or an antigen-binding fragment thereof, a fusion protein, a polypeptide, a truncation, a genetically engineered scaffold protein (an artificially designed protein of non-antibody origin), a nucleic acid aptamer, a small molecule inhibitor, or a natural ligand analog. The antibody can be selected from the group consisting of a monoclonal antibody, a single-chain antibody, a chimeric antibody, a humanized antibody, and a fully human antibody. The antigen-binding protein can be selected from the group consisting of Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv, VHH, and dAb.

[0187] In the present application, the VEGF binding molecule can be 2-11 antibody, bevacizumab, ranibizumab, brolucizumab, aflibercept, conbercept. In the present application, the VEGFR binding molecule can be DC101, ramucirumab. In the present application, the PD-1 binding molecule can be nivolumab, pembrolizumab, etc. In the present application, the PDL1 binding molecule can be YN035, atezolizumab, etc.

[0188] In the present application, the CAR may comprise a signal peptide. For example, the signal peptide may be a signal peptide derived from a CD8 protein. For example, the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 19.

[0189] In the present application, the CAR may comprise an intracellular domain, which may include a signaling domain and / or a co-stimulatory domain.

[0190] For example, the signaling domain can comprise a portion selected from the group consisting of CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM. For example, the signaling domain is the signaling domain of CD3ζ. For example, the signaling domain can comprise the amino acid sequence of SEQ ID NO:24 or an amino acid sequence having at least 80% homology thereto, and the nucleic acid molecule encoding the signaling domain can comprise the nucleic acid sequence of SEQ ID NO:24 or a nucleic acid sequence having at least 80% homology thereto.

[0191] For example, the costimulatory domain can include a portion selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, 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, a ligand of CD83, CD40, and MyD88. For example, the costimulatory domain is the costimulatory domain of 4-1BB. For example, the costimulatory domain can include the amino acid sequence shown in SEQ ID NO: 23 or an amino acid sequence having at least 80% homology thereto.

[0192] In the present application, the CAR may comprise a hinge region. For example, the hinge region may comprise a portion selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT. For example, the hinge region is the hinge region of CD8. For example, the hinge region may comprise the amino acid sequence shown in SEQ ID NO: 21 or an amino acid sequence having at least 80% homology thereto.

[0193] In the present application, the CAR may include a transmembrane region. For example, the transmembrane region may include a portion selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM. For example, the transmembrane region is the transmembrane region of CD8. For example, the transmembrane region may comprise the amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence having at least 80% homology thereto.

[0194] In the present application, the CAR may comprise a targeting moiety. In the present application, the targeting moiety may comprise an antibody or an antigen binding fragment. The antigen binding fragment may be selected from the group consisting of Fab, Fab', F(ab)2, F(ab')2, Fv, and ScFv fragments. For example, the targeting moiety may be ScFv.

[0195] In the present application, the targeting moiety can specifically bind to and / or recognize a tumor antigen. For example, the targeting moiety can specifically bind to and / or recognize a target selected from the group consisting of: a B lymphocyte surface antigen, a TNF family member, a HER family member, and a GPC family member. For example, the targeting moiety can specifically bind to and / or recognize a target selected from the group consisting of: CD19, BCMA, HER2, Mesothelin, GPC3, Muc 1, ROR 1, and CDH17.

[0196] In the present application, the targeting moiety is an antibody or antigen-binding fragment thereof that specifically binds to and / or recognizes GPC3, and the targeting moiety may comprise the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 80% homology thereto. In the present application, the nucleic acid molecule encoding the targeting moiety may comprise the nucleic acid sequence of SEQ ID NO: 41, or a nucleic acid sequence having at least 80% homology thereto.

[0197] In the present application, the targeting moiety is an antibody or antigen-binding fragment thereof that specifically binds to and / or recognizes ROR1, and the targeting moiety may comprise the amino acid sequence shown in SEQ ID NO: 18 or an amino acid sequence having at least 80% homology thereto. In the present application, the nucleic acid molecule encoding the targeting moiety may comprise the nucleic acid sequence shown in SEQ ID NO: 17 or a nucleic acid sequence having at least 80% homology thereto.

[0198] In the present application, the targeting moiety is an antibody or antigen-binding fragment thereof that specifically binds to and / or recognizes CDH17, and the targeting moiety may comprise the amino acid sequence shown in SEQ ID NO: 47 or an amino acid sequence having at least 80% homology thereto. In the present application, the nucleic acid molecule encoding the targeting moiety may comprise the nucleic acid sequence shown in SEQ ID NO: 46 or a nucleic acid sequence having at least 80% homology thereto.

[0199] The proteins, polypeptides, amino acid sequences, and / or nucleic acid sequences referred to in this application should also be understood to include at least the following: variants or homologs having the same or similar functions as the proteins or polypeptides, and / or nucleic acid sequences encoding variants or homologs having the same or similar functions as the proteins or polypeptides.

[0200] In the present application, the variant may be a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody or fragment thereof that specifically binds to a GPC3 protein, or an antibody or fragment thereof that specifically binds to a ROR1 protein, or an antibody or fragment thereof that specifically binds to a CDH17 protein). For example, the functional variant may comprise a protein or polypeptide having an amino acid change by at least 1, e.g., 1-30, 1-20, or 1-10, or for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, and / or insertions. The functional variant may substantially retain the biological properties of the protein or polypeptide before the change (e.g., substitution, deletion, or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding ability, or the biological function of the low-density lipoprotein receptor-related protein or fragment thereof) of the protein or polypeptide before the change. For example, the substitution may be a conservative substitution.

[0201] In the present application, the homolog can be a protein or polypeptide having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody or fragment thereof that specifically binds to a GPC3 protein, or an antibody or fragment thereof that specifically binds to a ROR1 protein, or an antibody or fragment thereof that specifically binds to a CDH17 protein).

[0202] In the present application, the CAR may further comprise a leader peptide. For example, the leader peptide may be an lgκ leader peptide. For example, the leader peptide may comprise the amino acid sequence shown in SEQ ID NO: 13. For example, the leader peptide may comprise the nucleic acid sequence shown in SEQ ID NO: 11 and SEQ ID NO: 12.

[0203] In the present application, the CAR may further comprise a cleavage peptide comprising one or more cleavage peptides in the following group: T2A, P2A, E2A or F2A. For example, the cleavage peptide may be T2A. For example, the cleavage peptide may comprise a nucleic acid sequence as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.

[0204] For example, the CAR comprises a CD8 signal peptide, a GPC3 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a first domain, a (GGGGS)3 linker, and a second domain connected in sequence.

[0205] For example, the CAR comprises a CD8 signal peptide, a GPC3 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a first domain, a T2A cleavage peptide, and a second domain connected in sequence.

[0206] For example, the CAR comprises a CD8 signal peptide, a GPC3 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a second domain, a (GGGGS)3 linker and a first domain connected in sequence.

[0207] For example, the CAR comprises a CD8 signal peptide, a GPC3 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a second domain, a T2A cleavage peptide and a first domain connected in sequence.

[0208] For example, the CAR comprises a CD8 signal peptide, a ROR1 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a first domain, a (GGGGS)3 linker and a second domain connected in sequence.

[0209] For example, the CAR comprises a CD8 signal peptide, a ROR1 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a first domain, a T2A cleavage peptide, and a second domain connected in sequence.

[0210] For example, the CAR comprises a CD8 signal peptide, a ROR1 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a second domain, a (GGGGS)3 linker and a first domain connected in sequence.

[0211] For example, the CAR comprises a CD8 signal peptide, a ROR1 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a second domain, a T2A cleavage peptide and a first domain, which are sequentially connected.

[0212] For example, the CAR comprises a CD8 signal peptide, a CDH17 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a first domain, a (GGGGS)3 linker and a second domain connected in sequence.

[0213] For example, the CAR comprises a CD8 signal peptide, a CDH17 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a first domain, a T2A cleavage peptide, and a second domain connected in sequence.

[0214] For example, the CAR comprises a CD8 signal peptide, a CDH17 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a second domain, a (GGGGS)3 linker and a first domain connected in sequence.

[0215] For example, the CAR comprises a CD8 signal peptide, a CDH17 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a second domain, a T2A cleavage peptide and a first domain connected in sequence.

[0216] In the present application, the immune cells comprise the nucleic acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 52 and SEQ ID NO: 54.

[0217] In the present application, the immune cells may express the amino acid sequences shown in SEQ ID NO:6, SEQ ID NO:53 and SEQ ID NO:55.

[0218] Modified immune cells

[0219] On the other hand, the present application provides a modified immune cell characterized in that it can secrete two binding molecules, wherein the first binding molecule is a VEGF binding molecule or a VEGFR binding molecule, and the second binding molecule is a PDL1 binding molecule or a PD-1 binding molecule.

[0220] In the present application, the immune cells can secrete two binding molecules simultaneously.

[0221] For example, the first binding molecule is a VEGF binding molecule, and the second binding molecule is a PDL1 binding molecule. For example, the first binding molecule is a VEGF binding molecule, and the second binding molecule is a PD-1 binding molecule. For example, the first binding molecule is a VEGFR binding molecule, and the second binding molecule is a PDL1 binding molecule. For example, the first binding molecule is a VEGFR binding molecule, and the second binding molecule is a PD-1 binding molecule.

[0222] For example, the VEGF-binding molecule or VEGFR-binding molecule is a scFv. For example, the VEGF-binding molecule or VEGFR-binding molecule is a VHH. For example, the PDL1-binding molecule or PD-1-binding molecule is a scFv. For example, the PDL1-binding molecule or PD-1-binding molecule is a VHH.

[0223] For example, both binding molecules are secreted as monoclonal antibodies. For example, one of the two binding molecules is secreted as a monoclonal antibody, while both binding molecules are secreted as bispecific antibodies. For example, both binding molecules are secreted as bispecific antibodies. For example, one or more of the two binding molecules are secreted as a fusion protein, polypeptide, truncation, genetically engineered scaffold protein, etc.

[0224] In the present application, the modified immune cells may comprise lymphocytes. In the present application, the modified immune cells comprise modified T cells.

[0225] In the present application, the modified immune cell expresses a chimeric antigen receptor (CAR). For example, the CAR can be the CAR described in the detailed description of the invention of the present application. For example, the CAR connects a first domain and a second domain expressing a secreted form of a binding molecule, wherein the first domain expresses a VEGF binding molecule or a VEGFR binding molecule, and the second domain expresses a PDL1 binding molecule or a PD-1 binding molecule.

[0226] In the present application, the modified immune cell may include a vector capable of expressing the chimeric antigen receptor (CAR). The vector may include a nucleotide molecule encoding the chimeric antigen receptor (CAR). For example, the vector may be selected from the group consisting of a retroviral vector, a lentiviral vector, and / or a transposon plasmid.

[0227] Nucleic acid molecules, vectors, and pharmaceutical compositions

[0228] On the other hand, the present application provides a nucleic acid molecule encoding the CAR described in the present application.

[0229] In the present application, the nucleic acid molecule further comprises a promoter. For example, the promoter is the EF-1α promoter.

[0230] In another aspect, the present application provides a vector comprising the nucleic acid molecule described in the present application.

[0231] In the present application, the vector may be a viral vector. For example, the vector is a lentiviral vector.

[0232] On the other hand, the present application provides a pharmaceutical composition comprising the immune cells described herein, the nucleic acid molecules described herein, and / or the vector described herein, and optionally a pharmaceutically acceptable carrier.

[0233] In the present application, the pharmaceutically acceptable carrier may include a buffer, an antioxidant, a preservative, a low molecular weight polypeptide, a protein, a hydrophilic polymer, an amino acid, a sugar, a chelating agent, a counter ion, a metal complex and / or a nonionic surfactant, etc.

[0234] The compositions described herein may comprise a therapeutically effective amount of the antibody or antigen-binding fragment thereof. The therapeutically effective amount is the dosage required to prevent and / or treat (at least partially treat) a condition or disorder (e.g., cancer) and / or any complications thereof in a subject suffering from or at risk of developing the condition.

[0235] Methods and uses

[0236] In another aspect, the present application provides a method for preparing modified immune cells, comprising introducing the vector described herein into immune effector cells.

[0237] On the other hand, the present application provides a use of the immune cells described in the present application, the nucleic acid molecules described in the present application, the vectors described in the present application, and / or the pharmaceutical compositions described in the present application in the preparation of a drug for preventing, treating and / or alleviating diseases and / or conditions.

[0238] On the other hand, the present application provides an immune cell described in the present application, a nucleic acid molecule described in the present application, a vector described in the present application, and / or a pharmaceutical composition described in the present application, which is used to prevent, treat and / or alleviate diseases and / or conditions.

[0239] On the other hand, the present application provides a method for preventing, treating and / or alleviating a disease and / or condition, which comprises administering the cells described herein and / or the pharmaceutical composition described herein to a subject in need thereof.

[0240] On the other hand, the present application provides a method for promoting the infiltration of immune cells into the tumor microenvironment, comprising the following steps: causing the immune cells to secrete two binding molecules, wherein one binding molecule is a VEGF binding molecule or a VEGFR binding molecule, and the other binding molecule is a PDL1 binding molecule or a PD-1 binding molecule.

[0241] On the other hand, the present application provides a method for enhancing the release of cytokines by immune cells, comprising the following steps: causing the immune cells to secrete two binding molecules, wherein one binding molecule is a VEGF binding molecule or a VEGFR binding molecule, and the other binding molecule is a PDL1 binding molecule or a PD-1 binding molecule.

[0242] In the present application, the cytokines include interleukins, interferons and / or tumor necrosis factors. For example, the cytokines include IL-2 and / or IFN-γ.

[0243] Without intending to be bound by any theory, the following examples are merely intended to illustrate the modified immune cells, preparation methods, and uses of the present application, and are not intended to limit the scope of the present invention.

[0244] Example

[0245] Example 1 Construction of Lentiviral Vector, Viral Packaging and Titer Detection

[0246] Taking GPC3 CAR-T targeted as an example, the immune cells were chemically synthesized to contain a CAR structural DNA fragment (referred to as G3) containing anti-GPC3-4-1BB-CD3zeta, anti-VEGFA (Bevacizumab) scFv (referred to as aVEGF), and anti-PDL1 (YN035) scFv (referred to as aPDL1) fragment. After combining the fragments, they were constructed into the lentiviral vector pCore (LV100A, System Biosciences) by the Gibson Assembly method to obtain different GPC3-targeted CAR lentiviral expression vectors (CAR structure is shown in Figure 2), namely pCore-G3, pCore-G3-aVEGF, pCore-G3-aPDL1, pCore-G3-aVEGF-2A-aPDL1, and pCore-G3-aVEGF-G4S linker-aPDL1.

[0247] The G3 CAR sequence is constructed by splicing the leader sequence CD8 leader peptide, anti-GPC3 ScFv, CD8 hinge region and transmembrane region, 4-1BB, and CD3zeta sequentially from the 5' end to the 3' end. The G3 CAR nucleotide sequence is shown in SEQ ID NO: 1; the amino acid sequence is shown in SEQ ID NO: 6; the CD8 leader peptide amino acid sequence is shown in SEQ ID NO: 19; the anti-GPC3 ScFv amino acid sequence is shown in SEQ ID NO: 20; the CD8 hinge region amino acid sequence is shown in SEQ ID NO: 21; the CD8 transmembrane region amino acid sequence is shown in SEQ ID NO: 22; the 4-1BB amino acid sequence is shown in SEQ ID NO: 23; and the CD3zeta amino acid sequence is shown in SEQ ID NO: 24.

[0248] The G3-aVEGF CAR sequence is constructed by sequentially splicing the CD8 leader peptide, anti-GPC3 scFv, CD8 hinge and transmembrane regions, 4-1BB, CD3zeta, 2A, IgK leader peptide, aVEGF scFv, and human IgG1 Fc from the 5' end to the 3' end. The aVEGF secretion signal peptide uses the IgK leader peptide, the nucleotide sequence of which is shown in SEQ ID NO:11, and the amino acid sequence of the IgK leader peptide is shown in SEQ ID NO:13. The aVEGF scFv nucleotide sequence is shown in SEQ ID NO:2, and the amino acid sequence is shown in SEQ ID NO:7. The human IgG1 Fc nucleotide sequence is shown in SEQ ID NO:35, and the amino acid sequence is shown in SEQ ID NO:36.

[0249] The G3-aPDL1 CAR sequence is constructed by sequentially splicing the CD8 leader peptide, anti-GPC3 ScFv, CD8 hinge and transmembrane regions, 4-1BB, CD3zeta, 2A, IgK leader peptide, aPDL1 scFv, and 6*His from the 5' end to the 3' end. The aPDL1 secretion signal peptide uses the IgK leader peptide, the nucleotide sequence of which is shown in SEQ ID NO:12 and the amino acid sequence is shown in SEQ ID NO:13; the aPDL1 nucleotide sequence is shown in SEQ ID NO:3 and the amino acid sequence is shown in SEQ ID NO:8; the 6*his nucleotide sequence is shown in SEQ ID NO:37 and the amino acid sequence is shown in SEQ ID NO:38.

[0250] The G3-aVEGF-2A-aPDL1 CAR sequence is constructed by splicing the leader sequence CD8 SP, anti-GPC3 ScFv, CD8 hinge and transmembrane regions, 4-1BB, CD3zeta, 2A, aVEGF-2A-aPDL1 (IgK leader peptide, aVEGF scFv, human IgG1 Fc, T2A, IgK leader peptide, aPDL1 scFv, 6*His) from the 5' end to the 3' end. The nucleotide sequence of aVEGF (Bevacizumab) -2A-aPDL1 (YN035) is shown in SEQ ID NO: 4, and the amino acid sequence is shown in SEQ ID NO: 9.

[0251] The G3-aVEGF-G4S linker-aPDL1 sequence is constructed by sequentially splicing the leader sequence CD8 SP, anti-GPC3 ScFv, CD8 hinge and transmembrane regions, 4-1BB, CD3zeta, 2A, IgK leader peptide, aVEGF-G4S linker-aPDL1, and 6*His from the 5' end to the 3' end. The nucleotide sequence of the aVEGF (Bevacizumab)-G4S linker-aPDL1 (YN035) dual antibody is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 10.

[0252] The 2A sequence is T2A, and the nucleotide sequence is shown in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.

[0253] After vector construction, virus packaging, virus harvesting and titer detection were performed. The lentiviral packaging plasmid mixture containing pMDLg.pRRE (Addgene#12251), pRSV-REV (Addgene#12253) and pMD2.G (Addgene#12259) was pre-mixed with polyetherimide (PEI) at a mass / volume ratio of 1:3 and then incubated at 25°C for 15 minutes. The transfection mixture was then added to adherent 293-T cells. After that, the cells were incubated at 37°C in a cell culture incubator with 5% CO2 for 16 hours and then fresh medium was replaced. After 48 hours, the virus was harvested, the supernatant was collected by centrifugation at room temperature, filtered through a 0.45μm PES filter, aliquoted and stored at -80°C. The virus titer was determined by titration of the transduced 293T cell line.

[0254] Example 2 Preparation of GPC3-targeted CAR-T cells: CAR viral transduction and CAR-T amplification

[0255] CAR-T cells generated by PBMC-derived T cells were transduced with G3, G3-aVEGF, G3-aPDL1, G3-aVEGF-2A-aPDL1, and G3-aVEGF-G4S linker-aPDL1 lentiviruses as experimental groups, and T cells were used as negative controls. The specific experiments are briefly described as follows:

[0256] Day 0: PBMC-derived T cells were reconstituted and resuspended in X-Vivo-15 + 5% FBS + 10 ng / mL IL-7 + 20 ng / mL IL-21 (complete medium). CD3 / CD28 Dynabeads activation beads were added at a ratio of 1:3 and activated for 24 hours.

[0257] Day 1 transduction: Add the corresponding volume of lentivirus at an MOI of 3, add polybrene at a dilution of 1:2000, centrifuge at 1000g for 1 hour, and then place in a 37°C, 5% CO2 incubator for 24 hours;

[0258] Day 2: Remove the virus, remove the cells, centrifuge and discard the supernatant, then use complete culture medium to press 0.7×10 6 After resuspending at a density of cells / mL, the cells were placed in a 37°C, 5% CO2 incubator for static culture. The cells were counted every 1-2 days and the complete culture medium was added to the cell density to 0.7×10 6 cells / mL and continue culturing;

[0259] Day 9: After counting the cells, take 3×10 5 The cells were tested for CAR positive rate; after the CAR positive rate was determined, Mock T cells were used to adjust the CAR positive rate to the same level. A portion was frozen for in vivo animal experiments, and the other portion was adjusted to a cell density of 1.0×10 6 cells / mL and continue to culture;

[0260] Day 10-13: Perform in vitro functional assays, including but not limited to flow cytometry, cytokine secretion, cytotoxicity studies, or target cell-specific repeated stimulation experiments.

[0261] Example 3 Targeting GPC3 CAR-T Cell IL-2 and IFN-γ Cytokine Release

[0262] The CAR-T cells prepared in Example 2 were used to analyze the ability of G3, G3-aVEGF, G3-aPDL1, G3-aVEGF-2A-aPDL1, and G3-aVEGF-G4S linker-aPDL1 CAR T cells to produce IFNγ and IL-2, while Mock T was used as a negative control. Different groups of CAR T cells were mixed with target cells Huh7 at an effector-target ratio of 1:1 (2×10 5 CAR-T: 2×10 5 Huh7=1:1) for 24 hours, and the supernatant was taken and passed through BD TM Cytometric Bead Array(CBA)Human IL-2Flex Set and BD TM The Cytometric Bead Array (CBA) Human IFN-γFlex Set was used to measure the concentrations of IFNγ and IL-2 produced in the culture supernatant according to the instructions.

[0263] Experimental results:

[0264] As shown in Figures 3A and 3B, the concentrations of IL-2 and IFNγ secreted by CAR T cells increased significantly after activation with GPC3-based CAR. There were no significant differences among the CAR-T groups.

[0265] Example 4 Detection of aVEGF and aPDL1 levels secreted by GPC3-targeted CAR-T cells

[0266] The CAR-T cells prepared in Example 2 were used, and the Mock T group and the group without target cells were used as negative controls. The CAR-T cells were mixed with the target cells Huh7 at an effector-target ratio of 1:1 (2×10 5 CAR-T: 2×10 5 The cells were co-cultured with G3-aVEGF, G3-aPDL1, G3-aVEGF-2A-aPDL1, and G3-aVEGF-G4S linker-aPDL1 CAR T cells for 120 h. The supernatant was then collected and analyzed for aVEGF and aPDL1 secretion levels by ELISA.

[0267] The specific process is to first coat a 96-well ELISA plate with 1 μg / mL PDL1 antigen and 1 μg / mL VEGF antigen, 100 μL per well, seal with tin foil to protect from light, and incubate overnight at 4°C. The next morning, discard the antigen solution in the plate and wash seven times with a plate washer. Then, add 100 μL of standard solution and 100 μL of the test sample to each well and incubate at 4°C for 1 hour. After incubation, discard the primary antibody solution in the plate and wash seven times with a plate washer. Then, add HRP-labeled secondary antibody solution to each well and incubate at 4°C for 1 hour. After incubation, discard the primary antibody solution in the plate and wash seven times with a plate washer. Add 200 μL of TMB to each well. After color development, add 50 μL of stop solution to each well and gently shake to mix. Immediately place the plate on a Bio Tek microplate reader and read at 450 nm.

[0268] Experimental results:

[0269] As shown in Figures 4A and 4B, no antibodies were detected in Mock T and G3 under stimulation of target cells Huh7, indicating the specificity of this ELISA method; 200 ng / mL aVEGF was detected in G3-aVEGF, G3-aVEGF-2A-aPDL1, and G3-aVEGF-G4S linker-aPDL1 under stimulation of target cells Huh7; 150 ng / mL aPDL1 was detected in G3-aPDL1, G3-aVEGF-2A-aPDL1, and G3-aVEGF-G4S linker-aPDL1 under stimulation of target cells Huh7, indicating that the constructed CAR-T cells secreting different antibodies can successfully secrete stable and similar amounts of antibodies under stimulation of target cells.

[0270] Example 5: Simultaneous secretion of aVEGF and aPDL1 can significantly enhance the anti-tumor effect of GPC3-targeted CAR-T in vivo

[0271] (1) The anti-tumor efficacy of Mock T, G3, G3-aVEGF, G3-aPDL1, and G3-aVEGF-2A-aPDL1 cells was evaluated in a B-NDG mouse (Biocytogenetic immunodeficient mouse) xenograft model. Huh7 target cells were injected 12 days in advance at the appropriate dose (3×10 6 The tumor size was measured using a digital caliper two to three times a week. The tumor volume was calculated according to the following formula: Tumor volume = ((length) × (width) 2 ) / 2. The average tumor size of the mice was about 80mm 3 The mice were divided into five groups: control group Mock T, G3, G3-aVEGF, G3-aPDL1, and G3-aVEGF-2A-aPDL1, with 6 mice in each group. Each group of mice was injected with 2.5×10 6Frozen control Mock T cells or G3, G3-aVEGF, G3-aPDL1, G3-aVEGF-2A-aPDL1 CAR-T cells derived from Example 2, wherein the total number of T cells in the control group was consistent with the total number of CAR-T cells, and the CAR positive rate in the other experimental groups was consistent. Afterwards, the mice were weighed 3 times a week, the tumors were measured 3 times with a digital caliper, and the coat color, excrement, food intake, body movement, and death of the mice were observed, and tumor inhibition curves and mouse survival curves were drawn. In accordance with the relevant laws and regulations on experimental animal ethics and various regulations, mice treated with Mock T and G3, G3-aVEGF, and G3-aPDL1 CAR-T cells were sacrificed on the 14th day. Mice treated with G3-aVEGF-2A-aPDL1 CAR T cells were recorded until the 26th day. Considering that aVEGF antibodies used for human therapeutics, such as aVEGF (Bevacizumab), do not have cross-reactivity with mouse VEGF, aVEGF was used as a surrogate antibody scFv in this animal experiment, namely aVEGF(2-11) scFv, whose nucleotide sequence is shown in SEQ ID NO:42 and amino acid sequence is shown in SEQ ID NO:43.

[0272] Experimental results:

[0273] As shown in Figure 5A, compared with the Mock T cell group, on day 14, tumors in the G3, G3-aVEGF, G3-aPDL1, and G3-aVEGF-2A-aPDL1 groups were observed to be significantly inhibited, and only the G3-aVEGF-2A-aPDL1 group showed significant tumor suppression (P < 0.001). This indicates that G3-aVEGF-2A-aPDL1 CAR T cells can inhibit the growth of CAR T cells at a lower dose (2.5 × 10 6 This indicates that under the same conditions, G3 CAR-T cells secreting aVEGF or aPDL1 antibody alone cannot significantly enhance the tumor suppression function of CAR-T cells. G3 CAR-T cells secreting both aVEGF and aPDL1 antibodies have significant synergistic anti-tumor therapeutic effects.

[0274] (2) On days 7, 13, 20, and 26 after adoptive transfer, the proportion of human CD3-positive T cells in the blood of all remaining mice in different groups was detected by FACS.

[0275] Experimental results:

[0276] As shown in Figure 5B, the G3-aVEGF-2A-aPDL1 CAR-T cell group was at 2.5×10 6The T cells showed significant expansion on day 20 under different doses and then decreased, while no obvious expansion of T cells occurred in mice treated with G3, G3-aVEGF, G3-aPDL1 CAR-T cells and Mock T cells throughout the treatment process.

[0277] (3) On the 7th, 13th, and 20th days after CAR-T transfusion, blood was collected by orbital blood sampling to detect the secretion of cytokine IFN-γ.

[0278] Experimental results:

[0279] As shown in Figures 5A and 5C, the G3-aVEGF-2A-aPDL1 CAR-T cell group was at 2.5×10 6 The mice treated with G3, G3-aPDL1, G3-aVEGF, and Mock T showed significant IFN-γ secretion on day 7, which then gradually increased on day 13 and decreased on day 20 as the tumor shrank ( Figure 5A ), while no obvious IFN-γ secretion was observed in mice treated with G3, G3-aPDL1, G3-aVEGF, and Mock T during the entire treatment process ( Figure 5C ).

[0280] IFN-γ secretion indicates the level of proliferation and killing of CAR-T cells after they infiltrate the tumor site and encounter tumor antigen stimulation. The G3-aVEGF-2A-aPDL1 CAR-T cell group showed obvious IFN-γ secretion and peak appearance, indicating that the combination of G3-aVEGF-2A-aPDL1 CAR-T cells has a significant synergistic effect of entering the tumor suppressive microenvironment, proliferating, and killing target cells.

[0281] (4) The body weight of each group of mice was monitored after the CAR-T transfusion process.

[0282] Experimental results:

[0283] As shown in Figure 5D, there was no significant decrease or fluctuation in the overall weight of the mice, indicating that all groups of CAR-T had high safety.

[0284] (5) Further explore the anti-tumor mechanism of G3-aVEGF-2A-aPDL1 CAR-T and perform flow cytometry analysis on the number and phenotype of intratumoral CAR-T cell infiltration.

[0285] The specific operation is as follows: prepare tissue digestion solution: PBS + 0.5 mg / mL collagenase IV + 0.10.5 mg / mL DNase I; take 250 mg of tumor tissue, add 1 mL of the above tissue digestion solution, and digest at 37°C for 1 hour; place a 70 μm cell sieve on a six-well plate, filter the digested tissue suspension, rinse with PBS three times, each time with 1 mL of PBS; centrifuge at 500g for 5 minutes, add 1 mL of red blood cell lysis solution, and lyse at room temperature for 10 minutes; centrifuge at 500g for 5 minutes, add 1 mL of PBS, resuspend, count, and take 3x10 5 Prepare two aliquots of cells, add the corresponding isotype antibody to one tube, add the antibody according to Table 1 to the other tube, incubate at 4°C for 30 minutes, add 1 mL of PBS, centrifuge at 500g for 5 minutes, add 50 μL of PBS, resuspend in 50 μL of Biolegend Count Beads, and load onto the analyzer.

[0286] Table 1: Detection of CAR-T cell typing, memory, and exhaustion markers in mice

[0287] Experimental results:

[0288] As shown in Table 2 and Figure 5E , the number of G3-aVEGF-2A-aPDL1 CAR-T infiltrates was higher, reaching 11,819 CAR-T cells / mg tumor. Flow cytometry analysis also revealed that the infiltration ratio of G3-aVEGF-2A-aPDL1 CAR-T was as high as 35% ( Figure 5E ), while that of PD1+CAR-T was only about 4.87%.

[0289] The above results further demonstrate that G3-aVEGF-2A-aPDL1 CAR-T secretes aVEGF and aPDL1 antibodies, which greatly improves the tumor suppressive microenvironment, significantly promotes CAR-T cell infiltration, reduces cell exhaustion, and ultimately leads to a significant improvement in the anti-tumor effect (Figure 5A).

[0290] Table 2. Absolute counts of CAR-T cell infiltration in tumor tissue

[0291] (6) Further analyze whether the secretion of aVEGF improves the vascular status of tumor tissue.

[0292] Experimental results:

[0293] As shown in Figures 5F, 5G, and 5H, tumor tissues were subjected to immunofluorescence staining (Figure 5F). Compared with G3 CAR-T, G3-aVEGF-2A-aPDL1 CAR-T showed reduced angiogenesis (Figure 5G) and a significantly increased proportion of CD3+ T cell infiltration (Figure 5H).

[0294] Example 6 Comparison of the in vivo anti-tumor effects of GPC3-targeted CAR-T cells that simultaneously secrete aVEGF and aPDL1 (i.e., G3-aVEGF-2A-aPDL1) and GPC3-targeted CAR-T cells that secrete aVEGF-aPDL1 dual antibodies (i.e., G3-aVEGF-linker-aPDL1CAR-T)

[0295] To further verify and compare the in vivo anti-tumor effects of G3 CAR-T cells that simultaneously secrete aVEGF and aPDL1 (i.e., G3-aVEGF-2A-aPDL1) and G3 CAR-T cells that secrete aVEGF-aPDL1 dual antibodies (i.e., G3-aVEGF-linker-aPDL1 CAR-T), a subcutaneous xenograft model was selected. 6-7 week-old B-NDG mice were subcutaneously inoculated with Huh7 cells (3×10 6 cells / mouse). When the average tumor volume approaches 80 mm 3 At 12 days after xenograft inoculation, the mice were randomly divided into 3 groups and injected with 1.0×10 7 CAR+ viable cells, including G3-aVEGF-2A-aPDL1 CAR-T cells, G3-aVEGF-linker-aPDL1 CAR-T cells, and Mock T (the same total T cell number as the CAR-T group). Tumor size was measured with a digital caliper three times a week. Tumor volume was calculated according to the following formula: Tumor volume = ((length) × (width) 2 ) / 2.

[0296] Considering that aVEGF antibodies used for human therapeutics, such as aVEGF (Bevacizumab), do not have cross-reactivity with mouse VEGF, aVEGF was used as a surrogate antibody scFv in this animal experiment, namely aVEGF(2-11) scFv, whose nucleotide sequence is shown in SEQ ID NO:42 and amino acid sequence is shown in SEQ ID NO:43.

[0297] In accordance with the relevant laws and regulations on experimental animal ethics, mice treated with mock T cells were killed on the 17th day.

[0298] Experimental results:

[0299] As shown in Figure 6, compared with the mock T cell group, tumor size in mice adoptively transferred with G3-aVEGF-2A-aPDL1 CAR-T cells and G3-aVEGF-linker-aPDL1 CAR-T cells was reduced by 87.6% and 37.8%, respectively, on day 17. This indicates that G3 CAR-T cells secreting different forms of aVEGF and aPDL1 antibodies (aVEGF, aPDL1, and VEGF-PDL1 dual antibodies) can enhance the anti-tumor effect of CAR-T. Relatively speaking, G3-aVEGF-2A-aPDL1 CAR-T cells have a faster anti-tumor onset time than G3-aVEGF-linker-aPDL1 CAR-T cells, indicating that the simultaneous secretion of aVEGF and aPDL1 antibodies has certain advantages.

[0300] The above experimental results show that as long as CAR-T cells secrete aVEGF and aPDL1 antibodies at the same time, they can achieve anti-tumor effects, and the secretion form is not limited to the simultaneous secretion of aVEGF and aPDL1 antibodies alone, or the secretion of aVEGF-aPDL1 dual antibodies.

[0301] Example 7 Comparison of the in vivo anti-tumor effects of GPC3-targeted CAR-T combined with intraperitoneal injection of VEGF antibody and PDL1 antibody and the triple drug combination G3-aVEGF-2A-aPDL1

[0302] (1) Verify and compare the in vivo anti-tumor effects of G3 CAR-T combined with intraperitoneal injection of aVEGF antibody (alternative antibody, i.e., 2-11 antibody) and aPDL1 antibody (PDL1 Ab, YN035) and the three-drug combination G3-aVEGF-2A-aPDL1. Similarly, considering that aVEGF antibodies used for human treatment such as aVEGF (Bevacizumab) have no cross-reactivity with mouse VEGF, aVEGF is an alternative antibody scFv, i.e., aVEGF (2-11) scFv, whose nucleotide sequence is shown in SEQ ID NO: 42 and amino acid sequence is shown in SEQ ID NO: 43.

[0303] The in vivo anti-tumor efficacy of Mock T, Mock T+aVEGF antibody+aPDL1 antibody, G3, G3+aVEGF antibody+aPDL1 antibody, and G3-aVEGF-2A-aPDL1 cells was evaluated in a B-NDG mouse (Biocytogenin immunodeficient mouse) xenograft model. Huh7 target cells were inoculated 12 days in advance at the appropriate dose (3x10 6 The tumor size was measured using a digital caliper two to three times a week. The tumor volume was calculated according to the following formula: Tumor volume = ((length) × (width) 2) / 2. The average tumor size of the mice was about 80mm 3 The mice were randomly divided into 5 groups, namely Mock T, Mock T+aVEGF antibody+aPDL1 antibody, G3, G3+aVEGF antibody+aPDL1 antibody, and G3-aVEGF antibody-2A-PDL1 antibody, with 5 mice in each group. 3x10 6 Cryopreserved CAR-T cells or control Mock T cells were used for the corresponding groups. The total number of Mock T cells in the control group was consistent with the total number of cells in the other experimental groups, and the CAR positivity rates in the other experimental groups were consistent. The Mock T+aVEGF antibody+aPDL1 antibodyb and G3 CAR-T+aVEGF antibody+aPDL1 antibody groups received intraperitoneal injections of 2.5 mg / kg of aVEGF antibody and 10 mg / kg of aPDL1 antibody twice weekly.

[0304] After that, the mice were weighed three times a week, and the tumors were measured three times with a digital caliper. The coat color, excrement, food and water intake, body movement, and death of the mice were observed. Tumor inhibition curves and mouse survival curves were drawn. In accordance with the relevant laws and regulations on experimental animal ethics, mice treated with Mock T and G3 cells were killed on the 16th and 18th days (over 2000 mm 3 The mice treated with Mock T+aVEGF antibody+aPDL1 antibody, G3+aVEGF antibody (+aPDL1 antibody, G3-aVEGF antibody-2A-PDL1 antibody cells) were recorded. On the 25th day, the mice treated with Mock T combined with aVEGF antibody and aPDL1 antibody were sacrificed (more than 2000 mm 3 ), mice treated with G3+aVEGF antibody+aPDL1 antibody, G3-aVEGF antibody-2A-aPDL1 antibody cells were recorded until day 30.

[0305] Experimental results:

[0306] As shown in Figure 7A, the G3-aVEGF-2A-aPDL1 group showed a significant inflection point starting on day 11, whereas this inflection point did not appear in the other groups. This suggests that the G3-aVEGF-2A-aPDL1 group exhibited a significant in vivo expansion advantage over the other groups, particularly the G3+aVEGF+aPDL1 group. Further comparison revealed no significant anti-tumor effect differences between the Mock T+aVEGF+aPDL1 group and the G3+aVEGF+aPDL1 group before day 16. Although tumor size was significantly reduced relative to that of the Mock T group, suggesting that the aVEGF+aPDL1 group had a certain inhibitory effect on the tumor itself, unlike the G3-aVEGF-2A-aPDL1 group, which exhibited a significant synergistic effect on day 11, intraperitoneal administration of the aVEGF+aPDL1 group did not significantly enhance CAR-T function before day 16, demonstrating the unique synergistic advantage of CAR-T secretion of aVEGF and aPDL1 antibodies. During long-term observation up to the 30th day, the tumors in the G3-aVEGF-2A-aPDL1 group continued to shrink, while the tumors in the other groups of mice were either eliminated because they exceeded the ethical limit, or their tumors continued to grow slowly, further illustrating the unexpected special advantages of the G3-aVEGF-2A-aPDL1 group in secreting aVEGF antibodies and aPDL1 antibodies over G3-CAR-T combined with intraperitoneal injection of aVEGF antibodies and aPDL1 antibodies.

[0307] (2) Compare the effects of G3 CAR-T combined with intraperitoneal injection of aVEGF and aPDL1 antibodies and G3 CAR-T simultaneously secreting aVEGF and aPDL1 antibodies on CAR-T in vivo expansion.

[0308] On days 7, 14, 21, and 28 after adoptive transfer, the proportion of human CD3-positive T cells in the mouse blood was detected by FACS.

[0309] Experimental results:

[0310] As shown in Figure 7B, the G3-aVEGF-2A-aPDL1 CAR-T cell group showed significant expansion on day 21, which then decreased as the tumor shrank. Throughout the treatment process, mice treated with all other groups showed no significant expansion or only minimal expansion, further demonstrating that the G3 CAR-T cells secreting aVEGF and aPDL1 antibody to promote CAR-T cell expansion in vivo has a unique advantage that is completely different from the G3 CAR-T cells combined with aVEGF and aPDL1 antibodies.

[0311] At the same time, on days 7, 14, 21, and 28 after CAR-T transfusion, blood was collected by orbital blood sampling to detect the secretion of cytokine IFN-γ.

[0312] Experimental results:

[0313] As shown in Figure 7C, the G3-aVEGF-2A-aPDL1 CAR-T cell group began to secrete higher IFN-γ on the 7th day, reached a peak on the 14th day, and then began to decline, which also showed a corresponding relationship with the growth curve of the tumor in this group in Figure 7A, indicating that G3 CAR-T secretes aVEGF and aPDL1 antibody-specific in vivo CAR-T expansion and tumor killing.

[0314] Although the Mock T+aVEGF+aPDL1 antibody group showed some IFN-γ secretion on days 21 and 28, the T cells in this group lacked specific cytotoxicity, and the rise in IFN-γ occurred not in the early stages (day 7) but in the late stages (day 21 and beyond), when GVHD is potentially developing, indicating that this group developed some GVHD. No significant IFN-γ secretion was observed in the other groups throughout the treatment process. IFN-γ secretion indicates the level of cytotoxicity of CAR-T cells upon antigen stimulation. The G3-aVEGF-2A-aPDL1 CAR-T cell group was the first to show a peak in IFN-γ secretion, indicating that the G3-aVEGF-2A-aPDL1 CAR-T cells are more likely to penetrate the tumor's suppressive microenvironment, undergo extensive proliferation, and kill target cells. This further demonstrates the unique advantages of the G3-aVEGF-2A-aPDL1 CAR-T group, which secretes both aVEGF and aPDL1 antibodies.

[0315] (3) Compare the effects of G3 CAR-T combined with intraperitoneal injection of aVEGF and aPDL1 antibodies and G3 CAR-T simultaneously secreting aVEGF and aPDL1 antibodies on tumor killing and CAR-T exhaustion phenotype in vivo. On the 21st day after adoptive transfer, the phenotype of human CD3-positive T cells in the peripheral blood of mice was detected by FACS.

[0316] Experimental results:

[0317] As shown in Figure 7D, CD39 was undetectable in all groups, possibly related to the donor-derived T cells themselves. Compared with the other groups, the Mock T+aVEGF+aPDL1 group showed higher T cell PD1 expression, indicating that GVHD causes T cell exhaustion and that aVEGF and aPDL1 antibodies have no effect on alleviating T cell fatigue in attacking normal tissues. PD1 expression was low in both the G3+aVEGF+aPDL1 and G3-aVEGF-2A-aPDL1 CAR-T groups, indicating that the simultaneous secretion of aVEGF and aPDL1 by G3 CAR-T cells can reduce CAR-T exhaustion in vivo.

[0318] (4) Verify and compare the effects of G3 CAR-T combined with aVEGF and aPDL1 (YN035) antibodies and G3 CAR-T simultaneously secreting aVEGF and aPDL1 antibodies on the CAR-T tumor microenvironment infiltration ability.

[0319] 24 days after CAR-T infusion, tumor tissue was punctured and analyzed by flow cytometry for the number and phenotype of intratumoral CAR-T cell infiltration. See Example 5 for details.

[0320] Experimental results:

[0321] As shown in Figures 8A and 8B , flow cytometry was used to detect the infiltration ratio of CAR-T cells. The infiltration ratio of G3-aVEGF-2A-aPDL1 CAR-T reached 48.8% ( Figure 8A ), and the absolute count reached 3.73×10 6 CAR-T cells / μg tumor tissue (Figure 8B), further demonstrating that G3-aVEGF-2A-aPDL1 CAR-T cells secrete aVEGF and aPDL1 antibodies, greatly improving the tumor suppressive microenvironment and significantly promoting CAR-T cell infiltration. No significant synergistic anti-tumor effect was observed when G3-CAR-T cells were combined with aVEGF and aPDL1 antibodies, whereas G3-CAR-T cells secreting both aVEGF and aPDL1 antibodies exhibited significant synergistic anti-tumor advantages.

[0322] In addition, it was found that the degree of exhaustion of G3-aVEGF-2A-aPDL1 CAR-T infiltrating into the tumor was lower than that of G3 CAR-T combined with aVEGF antibody and aPDL1 antibody (Figure 8C), further indicating that CAR-T has strong anti-tumor ability and lower exhaustion degree due to its self-secreting VEGF antibody and PDL1 antibody.

[0323] (5) Analyze the differences in the antibody levels in peripheral blood and tumor microenvironment between the G3 CAR-T combined with aVEGF and aPDL1 antibody group and the G3 CAR-T simultaneously secreting aVEGF and aPDL1 group.

[0324] The PDL1 antibody and VEGF antibody in the tumors of the peritoneal combined antibody group and the secretory antibody group were relatively defined as 1, and then the relative concentrations of the corresponding peripheral blood antibodies of the peritoneal combined antibody group and the secretory antibody group were clarified.

[0325] Experimental results:

[0326] As shown in Figures 8D and 8E, in the G3 CAR-T group combined with aVEGF antibody and aPDL1 antibody, the peripheral blood VEGF antibody and PDL1 antibody concentrations were much higher than the antibody concentrations corresponding to the tumor microenvironment (83.21 times and 62.8 times, respectively); in the G3 CAR-T group that simultaneously secreted aVEGF scFv and aPDL1 scFv, the peripheral blood VEGF antibody and PDL1 antibody concentrations were slightly higher than the antibody concentrations corresponding to the tumor microenvironment (9.81 times and 1.98 times, respectively) (Figures 8D and 8E).

[0327] These experimental results demonstrate that G3 CAR-T cells simultaneously secreting aVEGF and aPDL1 antibodies not only exert a highly effective anti-tumor effect, but also minimize potential peripheral side effects, such as the potential bleeding tendency associated with VEGF antibodies. This further demonstrates the unique advantages of G3 CAR-T cells that simultaneously secrete aVEGF and aPDL1 antibodies.

[0328] In summary, the above experimental results all indicate that G3 CAR-T cells that simultaneously secrete aVEGF and aPDL1 antibodies have a more effective anti-tumor effect than G3 CAR-T cells that combine aVEGF and aPDL1 antibodies.

[0329] Example 8 Targeting ROR1 CAR-T can simultaneously secrete aVEGF and aPDL1 to significantly enhance its anti-tumor effect in vivo

[0330] To further verify that the simultaneous secretion of aVEGF and aPDL1 antibodies by CAR-T cells can greatly enhance the in vivo anti-tumor ability of other CAR-T cells, the ROR1 target was selected, and a lentiviral plasmid targeting ROR1-4-1BB-CD3z-aVEGF-2A-aPDL1 (ROR1 CAR-T-aVEGF-2A-aPDL1) was constructed (the nucleotide sequence of the ROR1-targeting scFV is shown in SEQ ID NO: 17, and the amino acid sequence is shown in SEQ ID NO: 18). The ROR1-4-1BB-CD3zeta (ROR1 CAR-T) plasmid was used as a control. Virus packaging and titer testing were performed according to Example 1, and CAR-T cell preparation was performed according to Example 2. The prepared CAR-T cells were frozen and subjected to in vivo efficacy analysis. Similarly, considering that aVEGF antibodies used for human therapeutics, such as aVEGF (Bevacizumab), have no cross-reactivity with mouse VEGF, aVEGF is a surrogate antibody scFv, namely aVEGF(2-11)scFv, whose nucleotide sequence is shown in SEQ ID NO:42 and amino acid sequence is shown in SEQ ID NO:43.

[0331] The in vivo anti-tumor efficacy of Mock T, ROR1 CAR-T, and ROR1 CAR-T-aVEGF-2A-aPDL1 cells was evaluated in a B-NDG mouse (Biocytogenetic immunodeficient mouse) xenograft model of ovarian cancer. SKOV3 target cells were injected 20 days in advance at the appropriate dose (3×10 6 The tumor size was measured using a digital caliper two to three times a week. The tumor volume was calculated according to the following formula: Tumor volume = ((length) × (width) 2 ) / 2. The average tumor size of the mice was about 80mm 3 The mice were divided into three groups: Mock T, ROR1 CAR-T, and ROR1 CAR-T-aVEGF-2A-aPDL1, with 6-7 mice in each group. Each group of mice was injected with 5×10 6 The total number of T cells in the control group was consistent with the total number of CAR-T cells, and the CAR positive rates in the other experimental groups were consistent.

[0332] (1) The mice were weighed three times a week, and the tumors were measured three times with a digital caliper. The coat color, excrement, food and water intake, body movement, and death of the mice were observed. The tumor inhibition curve and mouse survival curve were drawn.

[0333] Experimental results:

[0334] As shown in Figure 9A, compared with the ROR1 CAR-T cell group, the tumor size of mice adoptively transferred with ROR1 CAR-T-aVEGF-2A-aPDL1 cells was significantly reduced on day 26, with a highly significant difference (P<0.0001). This indicates that ROR1 CAR-T-aVEGF-2A-aPDL1 cells, which simultaneously secrete aVEGF and aPDL1 antibodies, also have significant synergistic anti-tumor effects in the ovarian cancer tumor-bearing model.

[0335] (2) On the 7th and 14th day after adoptive transfer, the proportion of human CD3-positive T cells in the blood of all remaining mice in different groups was detected by FACS.

[0336] Experimental results:

[0337] As shown in Figure 9B, the ROR1 CAR-T-aVEGF-2A-aPDL1 cell group was 5×10 6 At the same dose, significant expansion was observed on day 7, followed by a decrease, while no significant expansion of T cells occurred in mice treated with ROR1 CAR-T cells or Mock T cells throughout the treatment process.

[0338] (3) On the 7th and 14th days after CAR-T transfusion, blood was collected by orbital blood sampling to detect the secretion of cytokine IFN-γ.

[0339] Experimental results:

[0340] As shown in Figure 9C, the ROR1-aVEGF-2A-aPDL1 CAR-T cell group exhibited significant IFN-γ secretion on day 7, which decreased as the tumor shrank. Throughout the treatment process, IFN-γ secretion was limited in the ROR1 CAR-T cell group. IFN-γ secretion indicates the level of proliferation and killing of CAR-T cells after infiltrating the tumor site and encountering tumor antigen stimulation. The ROR1-CAR-T-aVEGF-2A-aPDL1 cell group showed significant IFN-γ secretion and peaked, indicating that ROR1-aVEGF-2A-aPDL1 CAR-T cells simultaneously secrete aVEGF and aPDL1 antibodies, promoting CAR-T cells to enter the tumor suppressive microenvironment, proliferate, and kill target cells.

[0341] (4) The body weight of each group of mice was monitored after the CAR-T transfusion process.

[0342] Experimental results:

[0343] As shown in Figure 9D, there was no significant decrease or fluctuation in the overall weight of the mice, indicating that all groups of CAR-T had high safety.

[0344] (5) On the 26th day of CAR-T treatment, tumor tissues were collected from 3 mice in each group, and flow cytometry detection of CAR-T infiltrating tumors was performed according to Example 5.

[0345] Experimental results:

[0346] As shown in Figure 9E , the proportion of CAR-T cells infiltrating into the tumor in the ROR1-CAR-T-aVEGF-2A-aPDL1 cell group was significantly higher than that in the ROR1 CAR-T cell group (p<0.001), indicating that ROR1-aVEGF-2A-aPDL1 CAR-T cells simultaneously secrete aVEGF and aPDL1 antibodies, promoting the infiltration of CAR-T cells into the tumor microenvironment.

[0347] The above experimental results show that the simultaneous secretion of aVEGF and aPDL1 by CAR-T cells with different targets can enhance the anti-tumor ability of CAR-T cells.

[0348] Example 9 Targeting GPC3 CAR-T cells that simultaneously secrete VEGF / VEGFR inhibitors and PD1 / PDL1 immune checkpoint inhibitors can significantly enhance the anti-tumor effect of targeted GPC3 CAR-T cells in vivo

[0349] To further confirm that CAR-T cells simultaneously secreting VEGF-VEGFR2 pathway inhibitors and PD1-PDL1 pathway inhibitors can achieve in vivo anti-tumor synergistic effects similar to aVEGF&aPDL1 and that this trend is not target-restricted, plasmid construction was performed according to the method in Example 1. In addition to preparing G3 CAR-T vectors targeting GPC3, G3 CAR-aVEGF-2A-aPDL1 vectors based on two different aPDL1 antibody sequences, G3 CAR-aVEGF-2A-aPD1 vectors based on two different aPD1 antibody sequences, G3 CAR-aVEGFR2-2A-aPDL1 vectors, and G3 CAR-aCTLA4-2A-aPDL1 vectors with different mechanisms, CDH17 CAR-T vectors targeting CDH17, CDH17 CAR-aVEGF-2A-aPDL1 vectors, CDH17 CAR-aVEGF-2A-aPD1 vectors, CDH17 CAR-aVEGFR2-2A-aPDL1 vectors, and CDH17CAR-aCTLA4-2A-aPDL1 vectors were also constructed.

[0350] Considering that human therapeutic aVEGF antibodies such as aVEGF (Bevacizumab) have no cross-reactivity with mouse VEGF, the aVEGF scFV in the G3 CAR-aVEGF-2A-aPDL1 vector uses the 2-11 anti-mouse VEGF scFv (nucleotide sequence shown in SEQ ID NO.42, amino acid sequence shown in SEQ ID NO:43), and the aPDL1 scFV uses the YN035scFV (nucleotide sequence shown in SEQ ID NO:3, amino acid sequence shown in SEQ ID NO:8) or the atezolizumab scFV (nucleotide sequence shown in SEQ ID NO:29, amino acid sequence shown in SEQ ID NO:30);

[0351] The aVEGF scFV in the G3 CAR-aVEGF-2A-aPD1 vector is 2-11 anti-mouse VEGF scFv (nucleotide sequence shown in SEQ ID NO:42, amino acid sequence shown in SEQ ID NO:43), and the aPD1 scFV is nivolumab scFV (nucleotide sequence shown in SEQ ID NO:25, amino acid sequence shown in SEQ ID NO:26) or pembrolizumab scFV (nucleotide sequence shown in SEQ ID NO:33, amino acid sequence shown in SEQ ID NO:34);

[0352] Considering that human therapeutic aVEGFR2 antibodies such as aVEGFR2 (Ramucirumab) have no cross-reactivity with mouse VEGFR2, the aVEGFR2 scFV in the G3 CAR-aVEGFR2-2A-aPDL1 vector uses the DC101 anti-mouse VEGFR2 scFv (nucleotide sequence shown in SEQ ID NO:44, amino acid sequence shown in SEQ ID NO:45), and the aPDL1 scFV uses the YN035 scFV (nucleotide sequence shown in SEQ ID NO:3, amino acid sequence shown in SEQ ID NO:8);

[0353] The aCTLA4 scFV in the G3 CAR-aCTLA4-2A-aPDL1 vector uses tremelimumab / tremelimumab scFv (nucleotide sequence shown in SEQ ID NO: 31, amino acid sequence shown in SEQ ID NO: 32), and the aPDL1 scFV uses YN035 scFV (nucleotide sequence shown in SEQ ID NO: 3, amino acid sequence shown in SEQ ID NO: 8).

[0354] The CDH17 CAR-T vector core sequence is constructed by sequentially splicing the CD8 SP leader sequence, aCDH17 ScFv, the CD8 hinge and transmembrane regions, 4-1BB, and CD3zeta from the 5' end to the 3' end. The nucleotide sequence of the CDH17-targeting scFv is shown in SEQ ID NO:46, and the amino acid sequence is shown in SEQ ID NO:47.

[0355] The aVEGF scFV in the CDH17 CAR-aVEGF-2A-aPDL1 vector uses the 2-11 anti-mouse VEGF scFv (nucleotide sequence shown in SEQ ID NO:42, amino acid sequence shown in SEQ ID NO:43), and the aPDL1 scFV uses the atezolizumab scFV (nucleotide sequence shown in SEQ ID NO:29, amino acid sequence shown in SEQ ID NO:30);

[0356] The aVEGF scFV in the CDH17 CAR-aVEGF-2A-aPD1 vector uses the 2-11 anti-mouse VEGF scFv (nucleotide sequence shown in SEQ ID NO:42, amino acid sequence shown in SEQ ID NO:43), and the aPD1 scFV uses the pembrolizumab scFV (nucleotide sequence shown in SEQ ID NO:33, amino acid sequence shown in SEQ ID NO:34);

[0357] The aVEGFR2 scFV in the CDH17 CAR-aVEGFR2-2A-aPDL1 vector uses the DC101 anti-mouse VEGFR2 scFv (nucleotide sequence shown in SEQ ID NO:44, amino acid sequence shown in SEQ ID NO:45), and the aPDL1 scFV uses the YN035 scFV (nucleotide sequence shown in SEQ ID NO:3, amino acid sequence shown in SEQ ID NO:8).

[0358] The aCTLA4 scFV in the CDH17 CAR-aCTLA4-2A-aPDL1 vector uses tremelimumab / tremelimumab scFv (nucleotide sequence shown in SEQ ID NO: 31, amino acid sequence shown in SEQ ID NO: 32), and the aPDL1 scFV uses YN035 scFV (nucleotide sequence shown in SEQ ID NO: 3, amino acid sequence shown in SEQ ID NO: 8).

[0359] After the above plasmid construction was completed, virus packaging was performed, and CAR-T cells were prepared and cryopreserved according to the method of Example 2. The GPC3-targeted CAR-T cells, including G3CAR-T, were reinfused into the B-NDG (purchased from Biocytogen) mouse xenograft subcutaneous liver cancer cell huh7 tumor model to confirm the in vivo anti-tumor efficacy. Huh7 target cells were injected 12 days in advance at the appropriate dose (3×10 6 The tumor size was measured using a digital caliper two to three times a week. The tumor volume was calculated according to the following formula: Tumor volume = ((length) × (width) 2 ) / 2. The average tumor size of the mice was about 80mm 3 The mice were divided into 5 groups, with 5 mice in each group, and each group of mice was injected with 3×10 6The mice were weighed weekly, tumors were measured with a digital caliper, and the coat color, excrement, food and water intake, body movement, and death of the mice were observed. The tumor inhibition curve was then drawn. On D25, 2-3 representative mice in each group were collected for intratumoral CD3 + T cell detection.

[0360] In the same manner, 5×10 6 Each CDH17-targeted CAR-T cell was cryopreserved. The mice were weighed weekly, tumors were measured with a digital caliper, and their coat color, excrement, food and water intake, body movements, and mortality were observed. Tumor inhibition curves were then plotted.

[0361] The experimental results showed that the G3 CAR-aVEGF-2A-aPDL1 CAR-T, G3 CAR-aVEGF-2A-aPD1 CAR-T, and G3CAR-aVEGFR2-2A-aPDL1 CAR-T groups began to inhibit tumor growth on day 12, and the anti-tumor effects became more significant over time and were significantly better than the G3 CAR-T group. The G3 CAR-aCTLA4-2A-aPDL1 CAR-T group was only slightly better than the G3 CAR-T group but significantly inferior to the other groups. There were no significant differences between the two aPD1 antibody sequence groups, nor between the two aPDL1 antibody sequence groups (Figure 10A). Except for the G3 CAR-aCTLA4-2A-aPDL1 CAR-T group, the CD3+ T cell infiltration in the tumors of the other groups was significantly higher than that of the G3 CAR-T group (Figure 10B).

[0362] A similar trend was observed in the CDH17-targeted human colon cancer cell COLO205 tumor inhibition model (Figure 10C). The anti-tumor effects of the CDH17 CAR-aVEGF-2A-aPDL1 CAR-T, CDH17 CAR-aVEGF-2A-aPD1 CAR-T, and CDH17 CAR-aVEGFR2-2A-aPDL1 CAR-T groups were significantly better than those of the CDH17 CAR-T group. Although the CDH17 CAR-aCTLA4-2A-aPDL1 CAR-T group was slightly better than the CDH17 CAR-T group, it was significantly inferior to the other groups.

[0363] From the above examples, it can be seen that the simultaneous secretion of VEGF / VEGFR2 pathway and PD1 / PDL1 pathway inhibitory molecules can effectively improve the therapeutic effect of CAR-T with different targets. This degree of improvement cannot be simply achieved by combining CAR-T with antibody drugs; the effective treatment of diseases by combining antibodies of multiple pathways does not mean that the same degree of improvement in efficacy can be achieved by CAR-T secreting related antibodies as by CAR-T simultaneously secreting VEGF / VEGFR2 pathway antibodies and PD1 / PDL1 pathway antibodies. In other words, those skilled in the art cannot infer whether the combination of CAR-T to secrete the antibody has a good technical effect based on the technical effect of the antibody combination. A large number of experiments are needed to verify this before a conclusion can be reached.

[0364] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various changes to the embodiments currently listed herein will be apparent to those skilled in the art and are intended to fall within the scope of the appended claims and their equivalents.

Claims

1. A modified immune cell, characterized in that Two binding molecules may be secreted, wherein the first binding molecule is a VEGF binding molecule or a VEGFR binding molecule, and the second binding molecule is a PDL1 binding molecule or a PD-1 binding molecule.

2. The immune cell according to claim 1, wherein The first binding molecule is a VEGF binding molecule, and the second binding molecule is a PDL1 binding molecule.

3. The immune cell according to claim 1, wherein The first binding molecule is a VEGF binding molecule, and the second binding molecule is a PD-1 binding molecule.

4. The immune cell according to claim 1, wherein The first binding molecule is a VEGFR binding molecule, and the second binding molecule is a PDL1 binding molecule.

5. The immune cell according to claim 1, wherein The first binding molecule is a VEGFR binding molecule, and the second binding molecule is a PD-1 binding molecule.

6. The immune cell according to any one of claims 1 to 5, wherein the binding molecule is selected from the group consisting of an antibody or an antigen-binding fragment thereof, a fusion protein, a polypeptide, a truncation, and a genetically engineered scaffold protein.

7. The immune cell according to claim 6, wherein the antibody is selected from the group consisting of a monoclonal antibody, a single-chain antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

8. The immune cell of claim 6, wherein the antigen binding is selected from the group consisting of Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv, VHH and dAb.

9. The immune cell of any one of claims 1-8, wherein the binding molecule comprises a scFv.

10. The immune cell of any one of claims 1-8, wherein the binding molecule comprises a VHH. The immune cell according to any one of claims 1 to 10 , wherein the two binding molecules secreted by the immune cell are secreted in the form of monoclonal antibodies.

12. The immune cell according to any one of claims 1 to 11, wherein the two binding molecules secreted by the immune cell are secreted in the form of bispecific antibodies.

13. The immune cell according to any one of claims 1-12, wherein the immune cell is derived from iPSC.

14. The immune cell according to any one of claims 1 to 12, wherein the immune cell is one or more cells selected from the group consisting of NK cells, NKT cells, macrophages, and T cells.

15. The immune cell according to any one of claims 1 to 14, wherein the immune cell is a T cell.

16. The immune cell of any one of claims 1-15, wherein the immune cell expresses a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

17. The immune cell of claim 16, wherein the CAR comprises a targeting moiety.

18. The immune cell according to claim 17, wherein the targeting moiety is scFv or VHH.

19. The immune cell according to any one of claims 17-18, wherein the targeting portion specifically binds to and / or recognizes a tumor antigen, and the antigen is selected from one or more antigens in the following group: CD19, BCMA, HER2, Mesothelin, GPC3, Muc 1, ROR1 and CDH17.

20. The immune cell according to any one of claims 17-19, wherein the targeting moiety specifically binds to and / or recognizes GPC3.

21. The immune cell of any one of claims 17-19, wherein the targeting moiety specifically binds to and / or recognizes ROR1.

22. The immune cell of any one of claims 17-19, wherein the targeting moiety specifically binds to and / or recognizes CDH17.

23. The immune cell of any one of claims 16-22, wherein the CAR comprises a signal peptide. The immune cell according to claim 23 , wherein the signal peptide is a signal peptide derived from CD8 protein.

25. The immune cell of any one of claims 16-24, wherein the CAR comprises a hinge region comprising a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT. The immune cell according to claim 25 , wherein the hinge region is a hinge region derived from CD8.

27. The immune cell of any one of claims 16-26, wherein the CAR comprises a transmembrane region comprising a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM. The immune cell according to claim 27 , wherein the transmembrane region is a transmembrane region derived from CD8.

29. The immune cell of any one of claims 16-28, wherein the CAR comprises a costimulatory domain comprising a costimulatory domain derived from one or more proteins selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, 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, a ligand of CD83, CD40, and MyD88.

30. The immune cell of claim 29, wherein the costimulatory domain is a costimulatory domain derived from 4-1BB.

31. The immune cell of any one of claims 16-30, wherein the CAR comprises an intracellular signaling domain comprising an intracellular signaling domain derived from one or more proteins selected from the group consisting of CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM.

32. The immune cell of claim 31, wherein the intracellular signaling domain is an intracellular signaling domain derived from CD3ζ.

33. The immune cell of any one of claims 16-32, wherein the CAR comprises a leader peptide. The immune cell according to claim 33 , wherein the leader peptide is an 1gκ leader peptide.

35. The immune cell according to any one of claims 16-34, wherein the CAR comprises a cleavage peptide, and the cleavage peptide is selected from one or more cleavage peptides in the following group: T2A, P2A, E2A and F2A. The immune cell according to claim 35 , wherein the cleavage peptide is T2A.

37. An immune cell according to any one of claims 16-36, wherein the CAR is connected to express a first domain and a second domain of a secreted form of a binding molecule, wherein the first domain expresses a VEGF binding molecule or a VEGFR binding molecule, and the second domain expresses a PDL1 binding molecule or a PD-1 binding molecule.

38. The immune cell of claim 37, wherein the first domain and the second domain are connected by one or more linkers.

39. The immune cell according to claim 38, wherein the linker is selected from (GGS)n and / or (GGGGS)n, wherein n is any positive integer between 1 and 10.

40. The immune cell of any one of claims 38-39, wherein the linker is (GGS)5 (SEQ ID NO: 39).

41. The immune cell of any one of claims 38-40, wherein the linker is (GGGGS)3 (SEQ ID NO: 40).

42. The immune cell of any one of claim 41, wherein the first domain and the second domain are connected by a cleavage peptide.

43. The immune cell according to claim 42, wherein the cleavage peptide is one or more cleavage peptides selected from the group consisting of T2A, P2A, E2A and F2A.

44. The immune cell according to any one of claims 42-43, wherein the cleavage peptide is T2A.

45. The immune cell according to any one of claims 16-44, wherein the CAR comprises a CD8 signal peptide, a GPC3 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a first domain, a (GGGGS)3 linker, and a second domain connected in sequence.

46. ​​The immune cell according to any one of claims 16-44, wherein the CAR comprises a CD8 signal peptide, a GPC3 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a first domain, a T2A cleavage peptide, and a second domain connected in sequence.

47. The immune cell of any one of claims 16-44, wherein the CAR comprises a CD8 signal peptide, a GPC3 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a second domain, a (GGGGS)3 linker, and a first domain connected in sequence.

48. The immune cell of any one of claims 16-44, wherein the CAR comprises a CD8 signal peptide, a GPC3 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a second domain, a T2A cleavage peptide, and a first domain connected in sequence.

49. The immune cell according to any one of claims 16-44, wherein the CAR comprises a CD8 signal peptide, a ROR1 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a first domain, a (GGGGS)3 linker, and a second domain connected in sequence.

50. The immune cell according to any one of claims 16-44, wherein the CAR comprises a CD8 signal peptide, a ROR1 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a first domain, a T2A cleavage peptide, and a second domain connected in sequence.

51. The immune cell of any one of claims 16-44, wherein the CAR comprises a CD8 signal peptide, a ROR1 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a second domain, a (GGGGS)3 linker, and a first domain connected in sequence.

52. The immune cell according to any one of claims 16-44, wherein the CAR comprises a CD8 signal peptide, a ROR1 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a second domain, a T2A cleavage peptide, and a first domain connected in sequence.

53. The immune cell according to any one of claims 16-44, wherein the CAR comprises a CD8 signal peptide, a CDH17 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a first domain, a (GGGGS)3 linker, and a second domain connected in sequence.

54. The immune cell according to any one of claims 16-44, wherein the CAR comprises a CD8 signal peptide, a CDH17 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a first domain, a T2A cleavage peptide, and a second domain connected in sequence.

55. The immune cell of any one of claims 16-44, wherein the CAR comprises a CD8 signal peptide, a CDH17 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a second domain, a (GGGGS)3 linker, and a first domain connected in sequence.

56. The immune cell of any one of claims 16-44, wherein the CAR comprises a CD8 signal peptide, a CDH17 antibody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a T2A cleavage peptide, a second domain, a T2A cleavage peptide, and a first domain connected in sequence.

57. The immune cell according to any one of claims 16-56, comprising the nucleic acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 52 and SEQ ID NO:

54.

58. The immune cell according to any one of claims 16-56, wherein the immune cell expresses the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:53 and SEQ ID NO:

55.

59. One or more isolated nucleic acid molecules encoding the CAR of any one of claims 16-58.

60. The nucleic acid molecule according to claim 59, comprising the nucleic acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 52 and SEQ ID NO:

54.

61. The nucleic acid molecule of any one of claims 59-60, further comprising a promoter.

62. The nucleic acid molecule of claim 61, wherein the promoter is the EF-1α promoter.

63. A vector comprising the nucleic acid molecule of any one of claims 59-62.

64. The vector according to claim 63, which is a viral vector.

65. The vector according to claim 64, which is a lentiviral vector.

66. A pharmaceutical composition comprising the immune cell of any one of claims 1-58, the nucleic acid molecule of any one of claims 59-62, and / or the vector of any one of claims 63-65, and optionally a pharmaceutically acceptable carrier.

67. A method for preparing a modified immune cell, comprising introducing the vector of any one of claims 63-65 into an immune effector cell.

68. Use of the immune cell of any one of claims 1-58, the nucleic acid molecule of any one of claims 59-62, the vector of any one of claims 63-65, and / or the pharmaceutical composition of claim 66 in the preparation of a medicament for preventing, treating and / or alleviating a disease and / or condition.

69. The immune cell of any one of claims 1-58, the nucleic acid molecule of any one of claims 59-62, the vector of any one of claims 63-65, and / or the pharmaceutical composition of claim 71, for use in preventing, treating and / or alleviating a disease and / or condition.

70. A method for preventing, treating and / or alleviating a disease and / or condition, the method comprising administering the cell of any one of claims 1-58, and / or the pharmaceutical composition of claim 66, to a subject in need thereof.

71. A method for promoting immune cell infiltration into the tumor microenvironment, comprising the following steps: causing the immune cells to secrete two binding molecules, wherein: One binding molecule is a VEGF binding molecule or a VEGFR binding molecule, and the other binding molecule is a PDL1 binding molecule or a PD-1 binding molecule.

72. A method for enhancing the release of cytokines from immune cells, comprising the following steps: causing the immune cells to secrete two binding molecules, wherein: One binding molecule is a VEGF binding molecule or a VEGFR binding molecule, and the other binding molecule is a PDL1 binding molecule or a PD-1 binding molecule.

73. The method of claim 71 or 72, wherein the immune cells are derived from iPSCs.

74. The method of any one of claims 71-73, wherein the immune cells are selected from one or more cells in the group consisting of NK cells, NKT cells, macrophages, and T cells.

75. The method of any one of claims 71-74, wherein the immune cell is a T cell.

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