WO2022257984A1

WO2022257984A1 - Preparation for enhanced chimeric antigen receptor (car) cells and use thereof

Info

Publication number: WO2022257984A1
Application number: PCT/CN2022/097680
Authority: WO
Inventors: 胡广; 郭继刚; 张荣胜; 井洋洋; 马方琳; 戴强; 杜楠; 谭涛超; 魏巧娥; 贾向印; 黄星星
Original assignee: 南京驯鹿医疗技术有限公司
Priority date: 2021-06-08
Filing date: 2022-06-08
Publication date: 2022-12-15
Also published as: CN117441020A

Abstract

Provided is an immune cell capable of expressing multiple components. The immune cell comprises two types of chimeric antigen receptors. The first type of chimeric antigen receptor can enhance the proliferation or activity of immune cells. The immune cell can also express competitive receptors with a missing function such as dNTGF-βRII, can solve the problem of immunosuppressive microenvironments existing in solid tumor tissues, and has a greatly improved ability to kill solid tumors. Meanwhile, the immune cell also expresses a tEGFR molecule, CAR-T cells in vivo can be effectively removed by means of injecting an antibody for EGFR, and a security risk caused by the CAR-T cells is safeguarded against.

Description

Preparation and application of an enhanced chimeric antigen receptor (CAR) cell

technical field

This application belongs to the technical field of cellular immunotherapy, and relates to a preparation method of enhanced chimeric antigen receptor (Chimeric Antigen Receptor, CAR) cells, including the simultaneous expression of two types of CAR and immunosuppressive receptors.

Background technique

Chimeric antigen receptor (Chimeric antigen receptor, CAR) T cell (CAR-T) immunotherapy uses the cell killing ability of T cells to specifically recognize the surface antigen of tumor cells by the single-chain antibody sequence on the synthesized CAR molecule to kill tumor cells. tumor cells. This therapy has shown significant efficacy in B-cell malignancies, including B-cell acute lymphoblastic leukemia (B-ALL) and B-cell non-Hodgkin's lymphoma (B-NHL) [1]. In August and October 2017, the FDA approved two CAR-T cell therapy drugs, Novartis’s Kymriah and Kite’s Yescarta, respectively. So far, the FDA has approved 4 CAR-T cell therapies.

As Novartis and Kite's cell therapy products Kymriah and Yescarta have been approved by the FDA based on phase II clinical data, the traditional drug development cycle has been greatly shortened, and their unprecedented efficacy has attracted the interest of a large number of companies and researchers. The number of clinical trials of CAR-T cell therapy has also increased rapidly, with the largest number of clinical trials in the United States and China.

For hematological tumors, in addition to the marketed CAR-T cell therapy products targeting B cell target CD19, CAR-T cell targets represented by CD20, CD22, and BCMA are used to treat B cell leukemia, B cell lymphoma, Multiple myeloma has also achieved remarkable curative effect[2-4]. In addition to B-cell tumors, research on T-cell tumors and myeloid leukemia is also in progress[5-6]. These results indicate that the use of CAR-T Cell technology platforms can treat a variety of tumors.

Hematological tumors account for only 10% of all tumor types, and the other 90% are solid tumors. There are also many reports on the research of CAR-T cells in the treatment of solid tumors. Due to the differences between solid tumors and hematological tumors, CAR-T cells face more challenges in the treatment of solid tumors. The reported clinical results show that CAR - T cells are less effective in solid tumors than in hematologic tumors.

Compared with hematological tumors, solid tumors have different basic characteristics[7]. First, solid tumors have a dense tissue structure, and there is a stroma layer composed of fibroblasts and collagen around the tumor, allowing CAR-T cells to enter the tumor tissue and tumor tissue. Cell contact is more difficult; tumor tissue is composed of a variety of cells, in addition to tumor cells, it also contains regulatory T cells Treg, myeloid suppressor cells MDSC, tumor-associated macrophages TAM, immature dendritic cells iDC, stromal fibroblasts Cell Fibroblastic cells and immunosuppressive cytokine TGFβ, etc. These factors together form a tumor immunosuppressive microenvironment, which makes tumor infiltrating T cells lose the ability to kill tumors; tumor cells in solid tumors are not completely the same tumor cells, and there is heterogeneity , CAR-T cell therapy targeting a single target may not be able to effectively kill all tumor cells; in addition, it is difficult to find tumor-specific antigens (TSA) as the target of CAR-T cells, and most of them currently use tumor-associated antigens (TAA) As the target of cell therapy, these TAAs are highly expressed on the surface of tumor cells and lowly expressed in other healthy tissues, which may cause toxic side effects of On target off tumor, all of which have brought challenges to the CAR-T cell therapy of solid tumors .

According to the characteristics of solid tumors, researchers have also proposed different strategies to overcome the obstacles faced by CAR-T cells in the treatment of solid tumors [8].

First of all, for the selection of targets, look for targets with high tumor-specific expression to reduce safety risks. At the same time, a dual-target logic method can also be used to design, and only tumor cells will activate T cells to play a killing function. For the heterogeneity of tumor cells, in addition to targeting a single tumor target, dual-target or multi-target CAR design can be used.

The second is to target the tumor immunosuppressive microenvironment. Gene editing methods can be used to knock out immune checkpoint molecules (such as PD1) in CAR-T cells, and CAR-T cells can also secrete antibody molecules that bind to immune checkpoints (such as PD1). PD1, PDL1, CTLA4, CD47, SIRPa), thereby blocking the activation and inhibition of immune cells by the tumor microenvironment; at the same time, it can also allow CAR-T cells to secrete cytokines (such as IL12) to enhance the killing effect of CAR-T cells.

In addition, related chemokine receptors can be expressed on the surface of CAR-T cells to enhance the tropism and infiltration ability of CAR-T cells to solid tumors and other methods to enhance the effect of CAR-T cells in treating solid tumors.

At present, there are many reports on CAR-T cell therapy for solid tumors [9], and a number of different targets have been verified for different solid tumor preclinical test data, and have shown significant efficacy. Since the preclinical test data are obtained based on tumor cell lines and mouse xenograft tumor models, in vitro studies based on tumor cell lines and mouse xenograft tumor models cannot truly reflect the real tumor conditions in patients. These preclinical data need to be further verified in clinical trials.

So far, there are 670 CAR-T cell therapy projects registered in ClinicalTrials.org, of which 167 are CAR-T cell therapy projects for solid tumors. Published clinical data of CAR-T cell therapy for solid tumors showed that all 6 patients treated with MSLN for ovarian cancer achieved stable disease [10], and 2 of 6 patients treated with pancreatic cancer achieved stable disease [11] ], a patient with mesothelioma achieved partial remission[12]; 9% partial remission was obtained in the treatment of bile duct and pancreatic cancer with HER2 targets, and 45% of the disease was stable[13]. The treatment of sarcoma and glioblastoma were respectively 24% had stable disease and 7% had a partial response [14]; targeting GD2 for neuroblastoma had a 27% complete response [15]; targeting PSMA for prostate cancer had a 40% partial response Relief [16].

These early clinical data show that CAR-T has a potential therapeutic effect in the treatment of solid tumors. Due to the small number of clinical cases, more clinical trials are still in progress. It is believed that with the update of clinical trial data, and the treatment of solid tumors With the improvement of the CAR-T cell design, the future CAR-T cell therapy of solid tumors will surely achieve a breakthrough.

Currently reported CAR-T cell therapy for solid tumors, clinical data show preliminary curative effect, but has not yet achieved a similar effect to the treatment of hematological tumors, and the reported existing technologies mostly use traditional second-generation CARs -T technology, or use enhanced CAR-T technology to overcome the tumor immunosuppressive microenvironment, or promote the infiltration ability of CAR-T cells into solid tumor tissues, but failed to solve the problem of CAR-T cells in solid tumor patients at the same time Amplifying and Overcoming Immunosuppressive Microenvironmental Issues in Solid Tumors.

Contents of the invention

In one aspect, the present application provides an immune cell expressing two types of chimeric antigen receptors, wherein the immune cell comprises T cells, and the immune cell expresses any one or more of the following components:

(a) a first class chimeric antigen receptor (CAR);

(b) a second class of chimeric antigen receptors (CARs);

(c) a loss-of-function immunosuppressive receptor; and

(d) A truncated form of EGFR molecule (tEGFR).

In certain embodiments, the expression of the first type of chimeric antigen receptor can enhance the function of immune cells, such as: CD19 CAR can improve the expansion ability of chimeric antigen receptor cells, CD5 CAR can Eliminate immune cells such as host T cells and Treg cells. Chimeric antigen receptors such as BCMA, CD20, CD22, CD5, and CD7 also have similar or broader functions to enhance CAR-T cells.

In certain embodiments, the second chimeric antigen receptor targets a target selected from the group consisting of: MSLN, HER2, GPC3, EGFRVIII, Claudin18.2, CD70, GD2, CEA, CS1, DLL3, EGFR, ErbB1, FAP, Folate Receptor, GPC1, gp100, MUC16, MUC1, NKG2D, PSCA, PSMA, ROR1, and VEGFR2.

On the other hand, in order to overcome the influence of the immunosuppressive microenvironment of solid tumors on the function of CAR-T cells, the present application also allows the above-mentioned CAR-T cells to express the signaling molecules involved in the tumor immunosuppressive microenvironment, wherein the signaling molecules are A functional immunosuppressive molecule receptor that can competitively bind to immunosuppressive molecules highly expressed in tumors. After the immunosuppressive molecule binds to the innately expressed immunosuppressive molecule receptor on the CAR-T, it can inhibit the killing function of immune cells on tumors, causing tumor proliferation and reducing the therapeutic effect of CAR-T. However, the modified immunosuppressive molecule receptor with loss of function can competitively bind to the immunosuppressive molecule in the tumor, but fails to induce the immunosuppressive effect after binding, and the immunosuppressive microenvironment of the solid tumor that CAR-T cells are subjected to is relieved or Elimination, the ability to kill solid tumors has been improved.

In certain embodiments, the engineered loss-of-function immunosuppressive molecule receptor comprises dnTGFβRII, a competitive receptor for TGFβ.

On the other hand, the immune cells also express tEGFR molecules, and the injection of EGFR antibodies can effectively eliminate CAR-T cells in the body, thereby ensuring the safety risks posed by CAR-T cells.

In some embodiments, because certain first-type chimeric antigen receptor T cells can lead to the loss of healthy B cells in the patient, which will cause humoral immune deficiency in the patient, in order to be able to control the survival of CAR-T cells in the body, this The applied CAR-T also expresses tEGFR molecules, which can be used as a safety switch for CAR-T cells. Through the injection of EGFR antibodies, CAR-T cells in the body can be effectively eliminated, thereby ensuring the safety risks posed by CAR-T cells.

In certain embodiments, the first type of chimeric antigen receptor (CAR) is a fully human CAR.

In certain embodiments, the first class of chimeric antigen receptors (CARs) is fully human CD19, CD20, CD22, CD5, CD7, and BCMA CARs.

In certain embodiments, the first class of chimeric antigen receptors (CARs) comprises a first binding domain, a first transmembrane domain, a first co-stimulatory domain, and an intracellular signaling domain, the The first binding domain comprises a combination of one or more antibodies or fragments thereof specifically binding to CD19, wherein each combination of said antibodies or fragments thereof comprises heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2) and heavy chain complementarity determining region 3 (HCDR3), the amino acid sequences of said HCDR1, HCDR2 and HCDR3 are respectively shown in SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49. .

In certain embodiments, the combination of antibodies or fragments thereof further comprises light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2) and light chain complementarity determining region 3 (LCDR3), said LCDR1 The amino acid sequences of , LCDR2, and LCDR3 are shown in SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, respectively.

In certain embodiments, the second class of chimeric antigen receptors (CARs) comprises a second binding domain, a second transmembrane domain, a second co-stimulatory domain, and an intracellular signaling domain.

In certain embodiments, said second binding domain comprises a combination of one or more antibodies or fragments thereof that specifically bind MSLN (mesothelin), wherein each of said antibodies comprises a heavy chain complementarity determining region 1 ( HCDR1), heavy chain complementarity determining region 2 (HCDR2) and heavy chain complementarity determining region 3 (HCDR3), the amino acid sequences of HCDR1, HCDR2 and HCDR3 are independently selected from the following combinations:

(1) HCDR1 such as the amino acid sequence of SEQ ID NO: 37, HCDR2 such as the amino acid sequence of SEQ ID NO: 38, and HCDR3 such as the amino acid sequence of SEQ ID NO: 39;

(2) HCDR1 such as the amino acid sequence of SEQ ID NO: 58, HCDR2 such as the amino acid sequence of SEQ ID NO: 59, and HCDR3 such as the amino acid sequence of SEQ ID NO: 60;

(3) HCDR1 such as the amino acid sequence of SEQ ID NO: 69, HCDR2 such as the amino acid sequence of SEQ ID NO: 70, and HCDR3 such as the amino acid sequence of SEQ ID NO: 71;

(4) HCDR1 such as the amino acid sequence of SEQ ID NO: 80, HCDR2 such as the amino acid sequence of SEQ ID NO: 81, and HCDR3 such as the amino acid sequence of SEQ ID NO: 82; and

(5) HCDR1 such as the amino acid sequence of SEQ ID NO: 91, HCDR2 such as the amino acid sequence of SEQ ID NO: 92, and HCDR3 such as the amino acid sequence of SEQ ID NO: 93.

In certain embodiments, the combination of antibodies or fragments thereof further comprises light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2) and light chain complementarity determining region 3 (LCDR3), said LCDR1 The amino acid sequences of LCDR2 and LCDR3 are independently selected from the following combinations:

(1) LCDR1 such as the amino acid sequence of SEQ ID NO: 40, LCDR2 such as the amino acid sequence of SEQ ID NO: 41, and LCDR3 such as the amino acid sequence of SEQ ID NO: 42;

(2) LCDR1 such as the amino acid sequence of SEQ ID NO: 61, LCDR2 such as the amino acid sequence of SEQ ID NO: 62, and LCDR3 such as the amino acid sequence of SEQ ID NO: 63;

(3) LCDR1 such as the amino acid sequence of SEQ ID NO: 72, LCDR2 such as the amino acid sequence of SEQ ID NO: 73, and LCDR3 such as the amino acid sequence of SEQ ID NO: 74; and

(4) LCDR1 such as the amino acid sequence of SEQ ID NO: 83, LCDR2 such as the amino acid sequence of SEQ ID NO: 84, and LCDR3 such as the amino acid sequence of SEQ ID NO: 85;

(5) LCDR1 such as the amino acid sequence of SEQ ID NO: 94, LCDR2 such as the amino acid sequence of SEQ ID NO: 95, and LCDR3 such as the amino acid sequence of SEQ ID NO: 96.

On the other hand, the application provides a chimeric antigen receptor (CAR) targeting MSLN (mesothelin), comprising an antigen-binding domain, a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain .

In certain embodiments, said antigen binding domain comprises a combination of one or more antibodies or fragments thereof that specifically bind MSLN (mesothelin), wherein each of said antibodies comprises a heavy chain complementarity determining region 1 (HCDR1 ), heavy chain complementarity determining region 2 (HCDR2) and heavy chain complementarity determining region 3 (HCDR3), the amino acid sequences of HCDR1, HCDR2 and HCDR3 are independently selected from the following combinations:

(1) HCDR1 such as the amino acid sequence of SEQ ID NO: 58, HCDR2 such as the amino acid sequence of SEQ ID NO: 59, and HCDR3 such as the amino acid sequence of SEQ ID NO: 60;

(2) HCDR1 such as the amino acid sequence of SEQ ID NO: 69, HCDR2 such as the amino acid sequence of SEQ ID NO: 70, and HCDR3 such as the amino acid sequence of SEQ ID NO: 71;

(3) HCDR1 such as the amino acid sequence of SEQ ID NO: 80, HCDR2 such as the amino acid sequence of SEQ ID NO: 81, and HCDR3 such as the amino acid sequence of SEQ ID NO: 82; and

(4) HCDR1 such as the amino acid sequence of SEQ ID NO: 91, HCDR2 such as the amino acid sequence of SEQ ID NO: 92, and HCDR3 such as the amino acid sequence of SEQ ID NO: 93.

In certain embodiments, the combination of antibodies or fragments thereof further comprises light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2) and light chain complementarity determining region 3 (LCDR3), said LCDR1 The amino acid sequences of , LCDR2, and LCDR3 are independently selected from the following combinations:

(1) LCDR1 such as the amino acid sequence of SEQ ID NO: 61, LCDR2 such as the amino acid sequence of SEQ ID NO: 62, and LCDR3 such as the amino acid sequence of SEQ ID NO: 63;

(2) LCDR1 such as the amino acid sequence of SEQ ID NO: 72, LCDR2 such as the amino acid sequence of SEQ ID NO: 73, and LCDR3 such as the amino acid sequence of SEQ ID NO: 74;

(3) LCDR1 such as the amino acid sequence of SEQ ID NO: 83, LCDR2 such as the amino acid sequence of SEQ ID NO: 84, and LCDR3 such as the amino acid sequence of SEQ ID NO: 85; and

(4) LCDR1 such as the amino acid sequence of SEQ ID NO: 94, LCDR2 such as the amino acid sequence of SEQ ID NO: 95, and LCDR3 such as the amino acid sequence of SEQ ID NO: 96.

In certain embodiments, the antigen binding domain comprises a heavy chain variable region, the amino acid sequence of which is shown in SEQ ID NO: 34, 44, 54, 65, 76 and 87.

In some embodiments, the combination of antibodies or fragments thereof further comprises a light chain variable region, the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 36, 46, 56, 67, 78 and 89 Show.

In certain embodiments, the combination of antibodies or fragments thereof comprises a light chain variable region and a heavy chain variable region connected by a linker.

In some embodiments, the amino acid sequence included in the sequence of the linker is shown in SEQ ID NO:9.

In certain embodiments, the combination of antibodies or fragments thereof is a single chain antibody or a single domain antibody.

In some embodiments, the amino acid sequence contained in the CD19 binding domain (scFv) is shown in SEQ ID No: 2; the amino acid sequence contained in the MSLN binding domain (scFv) is selected from any of the following: SEQ ID No: ID Nos: 5, 57, 68, 101, 79 and 90.

In some embodiments, the dnTGFβRII receptor comprises the amino acid sequence shown in SEQ ID No: 28 or a functional variant thereof.

In certain embodiments, it comprises a truncated form of EGFR molecule (tEGFR).

In some embodiments, the truncated EGFR molecule comprises the amino acid sequence shown in SEQ ID No: 27 or a functional variant thereof.

In some embodiments, the first type of chimeric antigen receptor is linked to the signaling molecule via a 2A peptide, and the second type of chimeric antigen receptor is linked to tEGFR via a 2A peptide.

In certain embodiments, the 2A peptide includes P2A, T2A, the P2A comprises the amino acid sequence shown in SEQ ID NO: 29 or a functional variant thereof, and the T2A comprises the amino acid sequence shown in SEQ ID NO: 30 or functional variants thereof.

In certain embodiments, the transmembrane domain comprises a polypeptide selected from the group consisting of α, β or ζ chains of T cell receptors, CD28, CD3e, CD45, CD4, CD5, CD8a, CD9, CD16, CD22 , MSLN, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

In some embodiments, the transmembrane domain comprises the amino acid sequence shown in SEQ ID No: 13 or a functional variant thereof.

In certain embodiments, the co-stimulatory domain comprises a polypeptide selected from the group consisting of CD28, 4-1BB, OX-40, and ICOS.

In some embodiments, the co-stimulatory domain comprises the amino acid sequence shown in SEQ ID No: 15 or a functional variant thereof.

In certain embodiments, the intracellular signaling domain comprises a signaling domain from CD3z.

In some embodiments, the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID No: 17 or a functional variant thereof.

In some embodiments, the CAR further comprises a hinge region connecting the antigen binding domain and the transmembrane domain.

In some embodiments, the hinge region comprises the amino acid sequence shown in SEQ ID No: 11 or a functional variant thereof.

In some embodiments, the CAR is also linked to a signal peptide.

In some embodiments, the signal peptide comprises the amino acid sequence shown in SEQ ID No: 8 or a functional variant thereof.

In some embodiments, the CAR comprises the amino acid sequences shown in SEQ ID Nos: 19, 32, 97, 98, 99 and 100 or functional variants thereof.

In certain embodiments, the CAR comprises SEQ ID No: 1, 3, 4, 6, 7, 10, 12, 14, 16, 18, 20, 21, 23, 24, 26, 31, 33, 35, 43, Nucleic acid sequences shown in 45, 53, 55, 64, 66, 75, 77, 86 and 88 or functional variants thereof.

On the other hand, the application provides a vector comprising SEQ ID No: 1, 3, 4, 6, 7, 10, 12, 14, 16, 18, 20, 21, 23, 24, 26, 31, 33, 35, 43, 45, 53, 55, 64, 66, 75, 77, 86 and 88 nucleic acid sequences or functional variants thereof.

In certain embodiments, the vector is selected from plasmids, retroviral vectors and lentiviral vectors.

In another aspect, the present application provides an immune effector cell, which comprises the CAR, the nucleic acid molecule, or the vector.

In certain embodiments, the immune effector cells are selected from T lymphocytes and natural killer (NK) cells.

In another aspect, the present application provides a method for preparing immune effector cells, which includes introducing the vector into the immune effector cells.

In another aspect, the present application provides a composition comprising the immune effector cells.

On the other hand, the present application provides the use of the CAR, nucleic acid molecule, carrier, and immune effector cell in the preparation of a drug, wherein the drug is used to treat diseases related to the expression of the second type of CAR targeting target. disease or condition.

In some embodiments, the use is that the disease or disorder associated with the expression of the second type of CAR targeting target is cancer or malignant tumor.

In another aspect, the present application provides a chimeric antigen receptor (CAR) targeting MSLN.

In certain embodiments, the CAR comprises an antigen-binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain, and the antigen-binding domain comprises one or more MSLN-specific binding The combination of antibodies or fragments thereof), wherein each of said antibodies comprises heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2) and heavy chain complementarity determining region 3 (HCDR3), said The amino acid sequences of HCDR1, HCDR2 and HCDR3 are independently selected from the following combinations:

In certain embodiments, the combined antigen-binding domain of the antibody or fragment thereof comprises a heavy chain variable region, the amino acid sequence of which is SEQ ID NO: 34, 44, 54, 65, 76 and 87 are shown.

In certain embodiments, the antigen-binding domain includes a heavy chain variable region, a linker, and a light chain variable region sequentially from the N-terminus to the C-terminus.

In some embodiments, the CD19 binding domain (ScFv) comprises the amino acid sequence shown in SEQ ID No: 2; wherein the amino acid sequence comprised by the MSLN binding domain (scFv) is selected from any of the following : SEQ ID Nos: 5, 57, 68, 101, 79 and 90.

In certain embodiments, the CAR is also linked to a truncated EGFR molecule (tEGFR) through a self-cleaving peptide.

In certain embodiments, the self-cleaving peptide comprises P2A, T2A or F2A.

In some embodiments, the EGFR molecule of the truncated form comprises the amino acid sequence shown in SEQ ID No: 27 or its functional variant; the P2A comprises the amino acid sequence shown in SEQ ID NO: 29 or its function Sexual variant; The T2A comprises the amino acid sequence shown in SEQ ID NO: 30 or a functional variant thereof.

In certain embodiments, the CAR, wherein the transmembrane domain comprises a polypeptide selected from the group consisting of α, β or ζ chains of T cell receptors, CD28, CD3e, CD45, CD4, CD5, CD8a , CD9, CD16, CD22, MSLN, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

In certain embodiments, the CAR further comprises a hinge region connecting the antigen binding domain and the transmembrane domain.

In some embodiments, the CAR is also linked to a signal peptide.

In certain embodiments, the CAR comprises the amino acid sequences shown in SEQ ID Nos: 32, 97, 98, 99 and 100 or functional variants thereof.

On the other hand, the present application also provides an isolated nucleic acid molecule encoding the CAR described in any one of claims 40-61.

In certain embodiments, the isolated nucleic acid molecule comprises SEQ ID No: 3, 4, 7, 10, 12, 14, 16, 18, 20, 21, 23, 33, 35, 53, 55, Nucleic acid sequences shown in 64, 66, 75, 77, 86 and 88 or functional variants thereof.

On the other hand, the present application also provides a vector comprising the above-mentioned nucleic acid molecule.

In certain embodiments, wherein the vector is selected from plasmids, retroviral vectors and lentiviral vectors.

In another aspect, the present application also provides an immune cell comprising the above-mentioned CAR, nucleic acid molecule, or vector.

In certain embodiments, wherein the immune cells are selected from T lymphocytes and natural killer (NK) cells.

On the other hand, the present application also provides a method for preparing immune cells, which includes introducing the above-mentioned carrier into the immune cells.

On the other hand, the present application also provides a pharmaceutical composition, which comprises the above-mentioned immune cells, and a pharmaceutically acceptable adjuvant.

On the other hand, the present application also provides the use of the CAR, the nucleic acid molecule, the vector, or the immune cell in the preparation of a drug, wherein the drug is used to treat the expression of MSLN related diseases or conditions.

In certain embodiments, the use, wherein the disease or disorder associated with MSLN expression is cancer or malignant tumor.

In certain embodiments, in the use, the tumor is preferably a solid tumor.

In another aspect, the present application provides a fully human antibody targeting MSLN or a single chain antibody or fragment thereof, wherein the light chain variable region of the fully human antibody includes LCDR1, LCDR2 and LCDR3, and the heavy chain variable region includes HCDR1, HCDR2 and HCDR3, wherein the LCDR1, LCDR2, LCDR3 and/or HCDR1, HCDR2, HCDR3 are selected from one of the following combinations:

(1) CDRs of #2 clone: the amino acid sequence of LCDR1 is shown in SEQ ID NO: 61;

The amino acid sequence of LCDR2 is shown in SEQ ID NO: 62;

The amino acid sequence of LCDR3 is shown in SEQ ID NO: 63;

The amino acid sequence of HCDR1 is shown in SEQ ID NO: 58;

The amino acid sequence of HCDR2 is shown in SEQ ID NO: 59;

The amino acid sequence of HCDR3 is shown in SEQ ID NO: 60;

(2) CDRs of #5 clone: the amino acid sequence of LCDR1 is shown in SEQ ID NO: 72;

The amino acid sequence of LCDR2 is shown in SEQ ID NO: 73;

The amino acid sequence of LCDR3 is shown in SEQ ID NO: 74;

The amino acid sequence of HCDR1 is shown in SEQ ID NO: 69;

The amino acid sequence of HCDR2 is shown in SEQ ID NO: 70;

The amino acid sequence of HCDR3 is shown in SEQ ID NO: 71;

(3) CDRs of #118 clone: the amino acid sequence of LCDR1 is shown in SEQ ID NO: 83;

The amino acid sequence of LCDR2 is shown in SEQ ID NO: 84;

The amino acid sequence of LCDR3 is shown in SEQ ID NO: 85;

The amino acid sequence of HCDR1 is shown in SEQ ID NO: 80;

The amino acid sequence of HCDR2 is shown in SEQ ID NO: 81;

The amino acid sequence of HCDR3 is shown in SEQ ID NO: 82; and

(4) CDRs of #119 clone: the amino acid sequence of LCDR1 is shown in SEQ ID NO: 94;

The amino acid sequence of LCDR2 is shown in SEQ ID NO: 95;

The amino acid sequence of LCDR3 is shown in SEQ ID NO: 96;

The amino acid sequence of HCDR1 is shown in SEQ ID NO: 91;

The amino acid sequence of HCDR2 is shown in SEQ ID NO: 92;

The amino acid sequence of HCDR3 is shown in SEQ ID NO:93.

In some embodiments, the amino acid sequence of the heavy chain variable region and/or the light chain variable region is selected from any combination of the following:

(1) The sequence shown in SEQ ID NO: 54 or its heavy chain variable region sequence with at least 90% sequence identity, and the sequence shown in SEQ ID NO: 56 or its light chain sequence with at least 90% sequence identity can be Variable region sequences (#2 clone);

(2) The sequence shown in SEQ ID NO: 65 or its heavy chain variable region sequence with at least 90% sequence identity, and the sequence shown in SEQ ID NO: 67 or its light chain sequence with at least 90% sequence identity can be Variable region sequences (#5 clone);

(3) The sequence shown in SEQ ID NO: 76 or its heavy chain variable region sequence with at least 90% sequence identity, and the sequence shown in SEQ ID NO: 78 or its light chain sequence with at least 90% sequence identity can be Variable region sequences (#118 clone);

(4) The sequence shown in SEQ ID NO: 87 or its heavy chain variable region sequence with at least 90% sequence identity, and the sequence shown in SEQ ID NO: 89 or its light chain sequence with at least 90% sequence identity can be Variable region sequences (#119 clone).

(1) The heavy chain variable region sequence shown in SEQ ID NO: 54, and the light chain variable region sequence shown in SEQ ID NO: 56 (#2 clone);

(2) The heavy chain variable region sequence shown in SEQ ID NO: 65, and the light chain variable region sequence shown in SEQ ID NO: 67 (#5 clone);

(3) The heavy chain variable region sequence shown in SEQ ID NO: 76, and the light chain variable region sequence shown in SEQ ID NO: 78 (#118 clone);

(4) The heavy chain variable region sequence shown in SEQ ID NO: 87, and the light chain variable region sequence shown in SEQ ID NO: 89 (#119 clone).

In some embodiments, the single-chain antibody targeting MSLN comprises the amino acid sequences shown in SEQ ID NO: 57, 68, 79, 90, 101.

On the other hand, the present application also includes isolated nucleic acid molecules encoding the above-mentioned fully human antibodies or single chain antibodies or fragments thereof.

On the other hand, the present application also includes an expression vector comprising the nucleic acid molecule described in the present application. In some embodiments, the vectors are plasmid, retroviral and lentiviral vectors.

On the other hand, the present application also includes a host cell comprising the expression vector described in the present application.

On the other hand, the present application also includes a pharmaceutical composition, which includes the fully human antibody or its single-chain antibody or fragment described in the present application, and a pharmaceutically acceptable carrier or diluent.

On the other hand, the present application also includes a method for treating a disease or disorder, by administering to a patient in need a therapeutically effective amount of the fully human antibody or its single-chain antibody or fragment described herein, or a host cell , or a pharmaceutical composition to eliminate, inhibit or reduce the activity of MSLN, thereby preventing, alleviating, improving or inhibiting diseases or conditions.

The present application also includes the use of the above-mentioned fully human antibody or its single-chain antibody or fragment or the above-mentioned host cell in the preparation of a medicament for eliminating, inhibiting or reducing the activity of MSLN, thereby preventing, alleviating, improving or inhibiting diseases or diseases.

The present application also includes the use of the above-mentioned fully human antibody or its single-chain antibody or fragment or the above-mentioned host cell as a drug or in treatment, for example, for eliminating, inhibiting or reducing the activity of MSLN, thereby preventing, alleviating, improving or inhibiting the disease or illness.

In some embodiments, the disease or condition is cancer.

In some embodiments, the cancer is a solid tumor.

In some embodiments, the cancer is selected from: mesothelioma, lung cancer, pancreatic cancer, breast cancer, ovarian cancer.

On the other hand, the present application also includes a kit for detecting MSLN protein in a sample. The kit includes the fully human antibody or its single-chain antibody or fragment described in this application. The detection can be in vitro or in vivo.

On the other hand, the present application also includes antibodies or fragments that compete for the same epitope with the fully human antibody or its single chain antibody or fragment described in the present application.

Description of drawings

Figure 1 is a design diagram of the enhanced anti-MSLN chimeric antigen receptor (Armored MSLN-CAR) that promotes the expansion of CAR-T cells in vivo and resists the tumor immunosuppressive microenvironment.

Fig. 2 is a map of the second generation MSLN-CAR lentiviral expression plasmid pCDH-MSLN-CAR (P01).

Figure 3 is a map of the lentiviral expression plasmid pCDH-MSLN-CAR-dnTGFβRII (P17) expressing MSLN-CAR and dnTGFβRII receptor.

Fig. 4 is a map of the lentiviral expression plasmid pCDH-CD19-CAR-tEGFR (P43) expressing CD19-CAR and tEGFR molecular switch.

Figure 5 is a graph showing the lentivirus packaging and titer detection of P01, P17, and P43 plasmids.

Figure 6 is a graph showing the positive rate detection of CD19-CAR, tEGFR, MSLN-CAR, and dnTGFβRII expression in CAR-T cells.

Figure 7 is a graph showing the ability of CAR-T cells to kill target cells in vitro.

Figure 8 is a diagram showing the secretion of IFNγ cytokines by CAR-T cells in vitro killing target cells.

Fig. 9 is a detection diagram of CD107a degranulation of CAR-T cells activated by target cells.

Fig. 10 is a detection diagram of CD107a degranulation of CAR-T cells activated by target cells.

Figure 11 is a graph showing the detection of the proliferation ability of CAR-T cells repeatedly stimulated by MSLN and CD19 dual target cell antigens.

Figure 12 is a graph showing that CD19-CAR promotes the expansion of enhanced CAR-T cells and improves the ability of CAR-T cells to kill tumor target cells.

Fig. 13 is a graph showing that dnTGFβRII receptor competitively inhibits the phosphorylation of SMAD2 protein in T cells by TGFβ-1 cytokine.

Fig. 14 is a diagram showing the inhibitory effect of dnTGFβRII receptor competitively resisting TGFβ-1 cytokine on T cells killing tumor cells.

Figure 15 is a graph showing the inhibition of ovarian cancer cell line SK-OV3 subcutaneous xenograft tumor growth by targeting MSLN-CAR-T cells.

Fig. 16 is a diagram of body weight changes in mice with subcutaneously transplanted tumors of the ovarian cancer cell line SK-OV3 treated with MSLN-CAR-T cells.

Figure 17 is a diagram of the in vivo expansion of MSLN-CAR-T cells in the SK-OV3 ovarian cancer cell line mouse subcutaneous xenograft tumor model.

Figure 18 is the results of the tested drugs acting on the SK-OV-3 xenograft tumor model; among them, Figure 18A is the tumor growth curve of the high-dose CAR-T group; Figure 18B is the tumor growth curve of the low-dose CAR-T group.

Figure 19 is the results of the tested drugs acting on the SK-OV-3 transplanted tumor model; among them, Figure 19A is the body weight change curve of the high-dose CAR-T group; Figure 19B is the body weight change curve of the low-dose CAR-T group; Figure 19C It is the relative body weight change curve of the high-dose CAR-T group; FIG. 19D is the relative body weight change curve of the low-dose CAR-T group.

Figure 20 shows the results of the enzyme-linked immunosorbent assay (ELISA) of part of the phage monoclonals panned and the target antigen and the control antigen, the negative control is the negative control of the phage, and the positive control 1 is the positive of the addition of the MSLN antibody MSLNAb (RD). control.

Figure 21 shows the results of flow cytometric analysis of the binding of some phage monoclonals to CHO-K1-MSLN and CHO-K1 cells, and the negative control is the negative control of phage.

Figures 22A, 22B, 22C, and 22D show the results of flow cytometric analysis (peak shape) of the binding of the screened phage monoclonal to various MSLN positive and negative cell lines, and the negative control is the negative control of phage.

Figures 23A, 23B, 23C, and 23D show the results of ELISA analysis of the screened phage monoclonals with a variety of different MSLN antigenic proteins and non-related antigens. Negative control is the negative control of phage, positive control 1 is the positive control of adding MSLN antibody MSLN Ab (RD), and positive control 2 is the positive control of adding MSLN Ab (Biolegend). Among them, the histograms corresponding to each test antibody and the control group indicate from left to right the reagents KACTUS-MSLN-Bio (Glu296-Gly588), KACTUS-MSLN-Bio (Glu296-Asn 494), ACRO-MSLN-cyno , ACRO-MSLN-mouse, KACTUS BAFFR-Bio, KACTUS CD5-Bio, SA test results.

Fig. 24A, 24B, 24C have shown the flow cytometric analysis result (peak figure) of the combination of the protein supernatant of recombinant expression and multiple different MSLN positive and negative cell lines, and negative control is not to add protein supernatant and only add two Antibiotic control.

Figures 25A, 25B, and 25C show the results of ELISA analysis of recombinantly expressed protein supernatants with a variety of different MSLN antigenic proteins and non-related antigens. The negative control is the control without protein supernatant and only the secondary antibody is added, the positive control 1 is the positive control with the MSLN antibody MSLN Ab (RD), and the positive control 3 is the positive control with the addition of HUYP218 (an antibody that is clinically positive for MSLN). Among them, the histograms corresponding to each test antibody and the control group indicate from left to right the reagents KACTUS-MSLN-Bio (Glu296-Gly588), KACTUS-MSLN-Bio (Glu296-Asn 494), ACRO-MSLN-cyno , ACRO-MSLN-mouse, KACTUS BAFFR-Bio, KACTUS CD5-Bio, SA test results.

Detailed ways

Definition of Terms

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art.

In this application, "antibody" refers to an immunoglobulin secreted by plasma cells (effector B cells) and used by the body's immune system to neutralize foreign substances (polypeptides, viruses, bacteria, etc.). This foreign substance is accordingly called an antigen. The basic structure of a classical antibody molecule is a 4-mer composed of 2 identical heavy chains and 2 identical light chains. According to the conservative difference in amino acid sequence, the heavy chain and light chain are divided into variable region (V) located at the amino terminal and constant region (C) located at the carboxy terminal. The variable region of the heavy chain (VH) and the variable region of the light chain (VL) interact to form the antigen binding site (Fv). In some cases, antibody can also be used to refer to antibody fragments that have antigen-binding ability, such as scFv, Fab, F(ab')2, and the like.

In this application, a "single chain fragment variable (scFv)" is composed of an antibody heavy chain variable region and a light chain variable region connected by a short peptide to form a peptide chain. Through correct folding, the variable regions from the heavy chain and the light chain interact through non-covalent bonds to form Fv segments, so scFv can better retain its affinity activity for antigens.

In this application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids other than the CDR region of a non-human antibody (such as a mouse antibody) are replaced with corresponding amino acids derived from human immunoglobulins. In the CDR regions, small additions, deletions, insertions, substitutions or modifications of amino acids may also be permissible so long as they still retain the ability of the antibody to bind a particular antigen. A humanized antibody optionally will comprise at least a portion of a human immunoglobulin constant region. A "humanized antibody" may retain antigen specificity similar to that of the original antibody. "Humanized" forms of non-human (eg, murine) antibodies may contain, at a minimum, chimeric antibodies of sequence derived from non-human immunoglobulin. In some cases, CDR region residues in a human immunoglobulin (recipient antibody) can be replaced with a non-human species (donor antibody) (such as mouse, rat) having the desired properties, affinity and/or capabilities. , rabbit or non-human primate) residue substitution in the CDR region. In certain instances, FR region residues of the human immunoglobulin may be replaced with corresponding non-human residues. In addition, humanized antibodies can contain amino acid modifications that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody properties, such as binding affinity.

In this application, the term "fully human antibody" generally refers to an antibody comprising only human immunoglobulin protein sequences. Fully human antibodies may contain murine sugar chains if they are produced in mice, in mouse cells, or in hybridomas derived from mouse cells. Similarly, a "murine antibody", "mouse antibody" or "rat antibody" refers to an antibody comprising only mouse or rat immunoglobulin sequences, respectively. Fully human antibodies can be produced in humans, in transgenic animals with human immunoglobulin germline sequences, by phage display or other molecular biology methods. Exemplary techniques that can be used to make antibodies are known in the art.

A "chimeric antigen receptor (CAR)", also known as chimeric T cell receptor, chimeric immune receptor, is a membrane protein receptor molecule engineered to confer desired specificity on immune effector cells , such as the ability to bind to a specific tumor antigen. Chimeric antigen receptors generally consist of an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In some instances, the antigen binding domain is a scFv sequence responsible for recognizing and binding a specific antigen. Intracellular signaling domains usually include immunoreceptor tyrosine activation motifs (ITAMs), such as signaling domains derived from CD3z molecules, which are responsible for activating immune effector cells and producing killing effects. In addition, chimeric antigen receptors may also include a signal peptide at the N-terminus responsible for the intracellular localization of the nascent protein, and a hinge region between the antigen-binding domain and the transmembrane domain. In addition to signaling domains, intracellular signaling domains may also include co-stimulatory domains derived from molecules such as 4-1BB or CD28.

In this application, the term "MSLN" is a tumor differentiation antigen. MSLN (Mesothelin), called mesothelin, is a cell surface glycoprotein with a molecular weight of 40kDa. The MSLN gene encodes a precursor protein, which produces two protein products, megakaryocyte-potentiating factor (MPF) and mesothelin, after proteolysis. Mesothelin can exert its own function by anchoring on the cell surface through glycosylphosphatidylinositol. Mesothelin exists in normal mesothelial cells, and its expression is low in normal tissues, but it is highly expressed in tumors such as mesothelioma, lung cancer, pancreatic cancer, breast cancer, and ovarian cancer. Therefore, mesothelin is a potential treatment for cancer. target.

In this application, the term "MSLN binding domain" generally refers to the extracellular domain of MSLN CAR, which can specifically bind to an antigen. For example, the extracellular binding domain of MSLN can be a receptor that can specifically bind to the MSLN polypeptide expressed on human cells, a chimeric antigen receptor that can specifically bind to the MSLN polypeptide expressed on human cells, an anti-MSLN antibody or its Antigen-binding fragments. The terms "binding domain", "extracellular domain", "extracellular binding domain", "antigen-specific binding domain" and "extracellular antigen-specific binding domain" are used interchangeably in this application used, and a CAR having the ability to specifically bind a target antigen of interest (eg, MSLN) is provided. The MSLN binding domain may be of natural, synthetic, semi-synthetic or recombinant origin.

In this application, the term "antibody" generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a specific antigen. For example, an antibody may comprise an immunoglobulin composed of at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, and includes any molecule comprising an antigen-binding portion thereof. The term "antibody" includes monoclonal antibodies, antibody fragments or antibody derivatives, including but not limited to human antibodies, humanized antibodies, chimeric antibodies, single domain antibodies (e.g., dAb, VH or VHH), single chain antibodies (e.g. , scFv). In the present application, a "fragment" of an antibody may refer to an antigen-binding fragment of an antibody, for example, Fab, Fab' and (Fab')2 fragments and the like. The term "antibody" also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives thereof. Each heavy chain can be composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain can be composed of a light chain variable region (VL) and a light chain constant region. The VH and VL regions can be further distinguished into hypervariable regions called complementarity determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs). Each VH and VL may consist of three CDR and four FR regions, which may be arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

In this application, the term "single-chain antibody" may also be referred to as scFv, which refers to an antibody consisting of one chain connected by a linker peptide between the variable region of the heavy chain and the variable region of the light chain. The term "single domain antibody" refers to an antibody formed solely from the variable domain of the heavy chain.

In this application, the term "transmembrane domain" (Transmembrane Domain) generally refers to the domain in CAR that passes through the cell membrane, which is connected to the intracellular signal transduction domain and plays a role in transmitting signals. In the present application, the transmembrane domain may be CD8a transmembrane domain.

In this application, the term "co-stimulatory domain" generally refers to an intracellular domain that can provide immune co-stimulatory molecules, which are cell surface molecules required for an effective response of lymphocytes to antigens. The costimulatory domain may include the costimulatory domain of CD28, and may also include the costimulatory domain of the TNF receptor family, such as the costimulatory domain of OX40 and 4-1BB.

In the present application, the term "hinge region" generally refers to the connecting region between the antigen binding region and the binding region of an immune cell Fc receptor (FcR). In the present application, the hinge region may be the CD8a hinge region.

In this application, the term "intracellular signaling domain" generally refers to the component of CAR located in intracellular signal transduction, which includes a signaling domain and a domain that specifically binds to the receptor component, for example: its It may be selected from CD3ζ intracellular domain, CD28 intracellular domain, CD28 intracellular domain, 4-1BB intracellular domain and OX40 intracellular domain.

In this application, the term "signal peptide" generally refers to a short (5-30 amino acids in length) peptide chain that directs the transfer of newly synthesized proteins to the secretory pathway.

In this application, the term "self-cleaving peptide" refers to self-cleaving 2A peptide, which can realize the function of cleaving protein through ribosome jumping instead of proteolysis, which may include but not limited to T2A, F2A and P2A, etc.

In this application, the term "marker detection signal" generally refers to a gene, protein or other molecule with known function or sequence that can function as a specific marker and emit a detectable signal. The label detection signal can be a fluorescent protein, such as: GFP, RFP, YFP and the like. The marker detection signal can be tEGFR. The terms "EGFRt" and "tEGFR" are used interchangeably in this application to refer to the gene encoding a truncated human epidermal growth factor receptor polypeptide that lacks the distal membrane EGF-binding domain and cytoplasmic signaling tail, but retains the Extracellular epitopes recognized by anti-EGFR antibodies. tEGFR can be used as a non-immunogenic selection tool as well as a tracking marker for functions in genetically modified cells. In this application, it can be used as a marker molecule for CAR-T cells, and can be used to clear CAR-T cells in the body if necessary. ADCC pathway (cetuximab mediated ADCC pathway) ( See US8802374B2), that is, it is used as a safety switch during clinical transformation.

In this application, "EGFR antibody" refers to the ability to induce antibody-dependent cell-mediated cytotoxicity (antibody dependent cell-mediated cytotoxicity), allowing immune cells to attack CAR-T cells with truncated epidermal growth factor receptor (EGFRt), Antibodies that assist in clearing CAR-T cells. The EGFR antibody can be used when patients have severe adverse reactions after infusion of CAR-T or other situations that require removal of CAR-T cells. It can assist in the removal of CAR-T cells and alleviate symptoms related to CAR-T treatment. The EGFR antibodies include, but are not limited to, cetuximab, panitumumab, necituzumab, and nimotuzumab.

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

In this application, the term "vector" generally refers to a nucleic acid delivery tool into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. The vector can be expressed by transforming, transducing or transfecting the host cell, so that the genetic material elements it carries can be expressed in the host cell. A vector may contain various elements that control expression. In addition, the vector may also contain an origin of replication. The vector may also include components that assist its entry into the cell.

In this application, the term "cell" generally refers to a single cell, cell line or cell culture that can be or has been the recipient of a subject's plasmid or vector, which includes the nucleic acid molecules described herein or the nucleic acid molecules described herein. the carrier described. Cells can include progeny of a single cell. Due to natural, accidental or deliberate mutations, the progeny may not necessarily be completely identical (either in the morphology of the total DNA complement or in the genome) to the original parent cell. Cells may include cells transfected in vitro with the vectors described herein.

In this application, the term "immunoconjugate" generally refers to the conjugation of the other agent (e.g., chemotherapeutics, radioactive elements, cytostatic and cytotoxic agents) to the antibody or antigen-binding fragment thereof (e.g. , a conjugate formed by covalently linking a linker molecule), the conjugate can be specifically bound to an antigen on a target cell by the antibody or antigen-binding fragment thereof, and the other agent can be delivered to the target cell (e.g. , tumor cells).

In this application, the term "pharmaceutical composition" generally refers to a composition for the prevention/treatment of a disease or condition. The pharmaceutical composition may comprise the isolated antigen binding protein described herein, the nucleic acid molecule described herein, the carrier described herein and/or the cell described herein, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical composition may also contain one or more (pharmaceutically effective) carriers and other suitable preparations. Acceptable ingredients of the compositions can be nontoxic to recipients at the dosages and concentrations employed. Pharmaceutical compositions of the present application include, but are not limited to, liquid, frozen and lyophilized compositions.

In this application, the term "pharmaceutically acceptable carrier" generally refers to a pharmaceutically acceptable carrier, excipient or stabilizer which is effective in the dosage and concentration employed to the cell or mammal to which it is exposed. non-toxic. A physiologically acceptable carrier may include suitable substances. Refers to the pharmaceutically acceptable carrier (carrier) and the carrier (vector) used to insert nucleic acid in genetic engineering are usually not the same substance.

In this application, the term "directly connected" may be opposed to the term "indirectly connected", and the term "directly connected" generally refers to a direct connection. For example, the direct connection may be a case where substances are directly connected without a spacer. The spacer may be a linker. For example, the linker can be a peptide linker. The term "indirectly linked" generally refers to the situation where substances are not directly linked. For example, the indirect connection may be through a spacer. For example, in the isolated antigen-binding protein described herein, the C-terminus of L-FR1 may be directly or indirectly linked to the N-terminus of LCDR1.

In this application, the term "comprises" generally refers to the meanings of including, encompassing, containing or encompassing. In some cases, it also means "for" and "consisting of".

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

In this application, the term "immunosuppressive receptor" generally refers to a receptor that binds to an immunosuppressive molecule highly expressed in tumors. After the immunosuppressive molecules are combined with the immunosuppressive receptors innately expressed on the CAR-T, it can inhibit the killing function of the immune cells on the tumor, causing tumor proliferation and reducing the therapeutic effect of CAR-T. However, the modified immunosuppressive molecule receptor with loss of function can competitively bind to the immunosuppressive molecule in the tumor, but fails to induce the immunosuppressive effect after binding, and the immunosuppressive microenvironment of the solid tumor that CAR-T cells are subjected to is relieved or Elimination, the ability to kill solid tumors has been improved.

Example

The following examples are used to further describe the present application, but these examples do not limit the scope of the present application.

The experimental methods not indicating specific conditions in the examples of the present application are generally in accordance with conventional conditions, or in accordance with the conditions suggested by raw material or commodity manufacturers. Reagents without specific sources indicated are conventional reagents purchased in the market.

Example 1: Enhanced anti-MSLN-CAR (Armored MSLN-CAR) design for promoting CAR-T cell expansion in vivo and resisting tumor immunosuppressive microenvironment

In order to enhance the curative effect of CAR-T cells in treating solid tumors, the enhanced MSLN-CAR-T design of this application is considered from two aspects. On the one hand, CD19-CAR molecules are used to promote the expansion of CAR-T cells in patients; On the one hand, the dnTGFβRII receptor without intracellular signal is used to resist the inhibitory effect of the immune microenvironment of tumor tissue on T cell function. In addition, for the safety of CAR-T cell application in vivo, tEGFR is used as CAR in the design of CAR - A molecular safety switch for T cells.

This application adopts lentivirus to infect T cells to construct enhanced MSLN-CAR-T cells. Due to the limitation of the packaging capacity of lentivirus, the design of this application’s enhanced MSLN-CAR-T cells is to co-infect T cells with two lentiviral expression vectors. One of the lentiviral expression vectors expressed CD19-CAR and tEGFR molecular safety switch, and the other lentiviral expression vector expressed MSLN-CAR and dnTGFβRII receptor. The schematic diagram of the design is shown in Figure 1, in which ① the MSLN-CAR molecule functions to recognize the MSLN antigen on the surface of tumor cells, activate T cells, and play the role of MSLN-CAR-T cells in killing MSLN-positive tumor cells. ②The role of CD19-CAR molecules is to recognize B cells in the patient's blood system, thereby stimulating and activating T cells, enabling CAR-T cells to achieve rapid expansion in the patient's body, and due to the persistence of B cells in the patient's body, it can promote CAR-T cells. Survival of T cells in the patient's body. ③The dnTGFβRII molecule acts as a TGFβRII receptor molecule without an intracellular domain, which can competitively resist the inhibitory effect of TGFβ-1 cytokines in the tumor tissue microenvironment on the function of CAR-T cells.

The CD19-targeting single-chain antibody scFv sequence in the CD19-CAR structure comes from the fully human anti-human CD19 single-chain antibody sequence (nucleotide sequence SEQ ID NO: 1, amino acid sequence SEQ ID NO: 2) obtained through internal screening of the company , the molecular switch tEGFR sequence is a human truncated EGFR receptor sequence (nucleotide sequence SEQ ID NO: 3) without an intracellular domain.

The scFv sequence of the single-chain antibody targeting MSLN in the MSLN-CAR structure refers to the humanized anti-MSLN single-chain antibody sequence huYP218 disclosed in the patent US2016/0229919A1 (nucleotide sequence SEQ ID NO 4, amino acid sequence SEQ ID NO: 5) , the dnTGFβRII receptor sequence without intracellular signal is a truncated human TGFβRII receptor sequence (SEQ ID NO: 6).

The signal peptide sequence used by the chimeric antigen receptor CAR is CD8a SP (nucleotide sequence SEQ ID NO: 7, amino acid sequence SEQ ID NO: 8), and the Linker sequence connecting the heavy chain and light chain of the single-chain antibody is (G4S) 3 (amino acid sequence SEQ ID NO: 9), the hinge region is CD8hinge (nucleotide sequence SEQ ID NO: 10, amino acid sequence SEQ ID NO: 11), and the transmembrane region is CD8TM (nucleotide sequence SEQ ID NO: 12 , amino acid sequence SEQ ID NO: 13), the second signal (co-stimulatory signal domain) is 41BB (nucleotide sequence SEQ ID NO: 14, amino acid sequence SEQ ID NO: 15), the first signal (intracellular signal transduction domain) is CD3z (nucleotide sequence SEQ ID NO: 16, amino acid sequence SEQ ID NO: 17).

Example 2: Design and construction of the third generation lentiviral packaging system and lentiviral vector

This application uses the third-generation lentiviral packaging system, including expression plasmids, envelope plasmids, and a four-plasmid system of packaging plasmids.

The four plasmids of the lentiviral packaging system were purchased from YouBio. The backbone plasmid of the expression plasmid is pCDH-EF1-MCS-T2A-Puro (Catalog#: VT1482), and the envelope plasmid is pMD2.G (Catalog#: VT1443) The packaging plasmids are pMDLgpRRE (Catalog#: VT1449) and pRSV-Rev (Catalog#: VT1445), respectively. The construction method of the lentiviral expression plasmid designed in this application is as follows:

1. Constructed the second-generation MSLN-CAR lentiviral expression plasmid pCDH-MSLN-CAR (P01) used as a control: the MSLN-CAR sequence (nucleotide sequence SEQ ID NO: 18, amino acid sequence SEQ ID NO: 19) Gene synthesis was inserted into the multiple cloning site of the pCDH-EF1-MCS-T2A-Puro plasmid, and the T2A-Puro sequence was removed to transform into pCDH-MSLN-CAR (P01) (nucleotide sequence SEQ ID NO: 20); lentivirus The map of the expression plasmid is shown in Figure 2.

2. Constructed the lentiviral expression plasmid pCDH-MSLN-CAR-dnTGFβRII (P17) expressing MSLN-CAR and dnTGFβRII receptor: MSLN-CAR-P2A-dnTGFβRII sequence (nucleotide sequence SEQ ID NO: 21, amino acid sequence SEQ ID NO: 22) Gene synthesis was inserted into the multiple cloning site of the pCDH-EF1-MCS-T2A-Puro plasmid, and the T2A-Puro sequence was removed to transform into pCDH-MSLN-CAR-dnTGFβRII (P17) (nucleotide sequence SEQ ID NO: 23); the map of the lentiviral expression plasmid is shown in Figure 3.

3. Constructed the lentiviral expression plasmid pCDH-CD19-CAR-tEGFR (P43) expressing CD19-CAR and tEGFR molecular switch: the CD19-CAR-T2A-tEGFR sequence (nucleotide sequence SEQ ID NO: 24, amino acid sequence SEQ ID NO: 25) Gene synthesis was inserted into the multiple cloning site of the pCDH-EF1-MCS-T2A-Puro plasmid, and the T2A-Puro sequence was removed to transform into pCDH-CD19-CAR-tEGFR(P43) (nucleotide sequence SEQ ID NO: 26); the map of the lentiviral expression plasmid is shown in Figure 4.

Packaging and virus titer detection of embodiment 3 lentiviral vectors

Lentiviral vector packaging method

239T cells were inoculated in culture dishes of appropriate specifications 24 hours in advance. The cell culture medium was DMEM + 10% FBS without antibiotics. The inoculation cell density was 70%-80%, so that the cell density at the time of infection was above 90%.

The next day, use Lipo3000 reagent for transfection, and configure A and B solutions according to the reagent instructions:

Liquid A: Dissolve four plasmids in serum-free Optim-MEM medium (four-plasmid system), calculate the volume of the required plasmids (pMD2.G, pMDLg-RRE, pRSV-Rev, Transfer vector) according to the table below, and then add P3000 for mixing.

Solution B: Dissolve Lipo3000 in an equal volume of serum-free DMEM medium (mix gently and let stand for 5 minutes).

Table 1. Lentivirus packaging system (15cm dish)

components

Usage amount

pRSV-RevpRSV-Rev	5.5μg5.5μg
pMDlg-pRREpMDlg-pRRE	11.7μg11.7μg
pMD2.GpMD2.G	7.8μg7.8μg
pCDH Transfer vectorpCDH Transfer vector	12.5μg12.5μg
PAdVantage vectorPAdVantage vector	2.5μg2.5μg
TotalTotal	37.5μL37.5μL
P3000P3000	75μL75μL
lip3000lip3000	88.9μL88.9μL
MediumMedium	4.5ml+4.5ml4.5ml+4.5ml

Add solution A to solution B dropwise, mix gently, and incubate at room temperature for 20 minutes. The transfection complexes were evenly added to the medium in the dish. After 6 h, the transfected cells were replaced with DMEM medium containing 10% FBS. Incubate at 37°C for 24-48h, collect the virus supernatant, centrifuge at 1500g, 4°C for 5min to remove cell debris.

Concentration and purification method of lentivirus

Use the Lenti-X ^TM Concentrator reagent to concentrate the collected lentivirus supernatant. The specific steps are as follows:

1. Add 1/3 of the total volume of Lenti-X ^TM Concentrator; 2. Mix well and incubate at 4°C for more than 2 hours; 3.4°C, 1500g, centrifuge for 45min; 4. Discard the supernatant and add an appropriate volume of T cells The medium (X-VIVO 15) dissolves the lentiviral pellet, and freezes it at -80°C.

Lentivirus titer determination method

The titer of lentivirus was determined by flow cytometry, the specific method is as follows:

(1) Plate (293CT cells) 24 hours in advance (1-2E+05 cells per well) (day1); (2) Digest one well of 293CT cells and add different volumes of virus (0.1/0.5/1ul) (day2 ); (3) Cell replacement (day3); (4) Flow cytometric detection of CAR+ (day4); (5) Titer calculation formula: virus titer (/ml) = positive rate × cell number / virus volume μL

pCDH-MSLN-CAR(P01), pCDH-MSLN-CAR-dnTGFβRII(P17), pCDH-CD19-CAR-tEGFR(P43) were packaged with lentivirus, and the virus titer was detected. The experimental results are shown in Figure 6 , P01, P17, and P43 lentivirus titers were 4.5E+08TU/ml, 3.4E+08TU/ml, and 5.6E+08TU/ml, respectively.

Example 4: Preparation of enhanced CAR-T cells that promote in vivo expansion of CAR-T cells and resist tumor immunosuppressive microenvironment, CAR ⁺ positive rate detection

First, collect peripheral blood mononuclear lymphocytes from healthy donors, or purchase PBMCs from healthy donors, and use the T cell isolation kit EasySep ^TM Human T Cell Isolation Kit (Stem cell company, catalog number: 17951) to extract PBMCs T cells were further sorted. The obtained T cells were cultured using X-VIVO15 (Lonza, 04-418Q) medium, while adding recombinant human IL2 cytokine (R&D, product number: 202-IL-050) at a final concentration of 100 IU/ml, and followed by Add CD3/CD28 magnetic beads (Dynabeads ^TM Human T-Activator CD3/CD28, GIBCO, catalog number: 11132D) to stimulate T cells at a ratio of 1:1 to activate T cells. The cell density is 1×10 ⁶ cells/mL. , cultured under 5% CO ₂ conditions.

After T cell stimulation and activation for 24 hours, lentivirus infection was performed. Gently pipette the cells to separate them, take a small amount of cells and count them; centrifuge at 500g for 5 minutes, carefully absorb the supernatant and discard; resuspend the T cells with CART cell complete medium, adjust the cell density to >2E+06cells/ml, and take the required amount of Put the cells into the cell culture plate, mix and add a certain volume of two virus concentrates according to the titer and MOI of the virus concentrate, add complete medium to adjust the final cell density to 2E+06cells/ml; start the centrifuge in advance to preheat To 32°C, put the cell culture plate into the centrifuge and balance it, and centrifuge at 2000g at 32°C for 1h; after the centrifugation, add complete medium at a volume ratio of 1:1, and place it in a constant temperature incubator at 37°C for overnight cultivation; the next day Discard the supernatant by centrifugation, resuspend with complete medium (density about 3E+05c ~ 5E+05cells/ml), and place in a constant temperature incubator at 37°C to continue culturing.

After 96 hours of virus infection, collect the cell suspension, centrifuge at 300g for 5min, discard the supernatant, resuspend with 1-1.5ml of complete medium and blow gently to separate the cells from the beads, transfer to a 1.5-2ml centrifuge tube, and place in DynaMag ^TM - Let stand in 2 for 1 minute, carefully pipette the cell suspension with a 1ml tip into a 1.5ml EP tube or a 15ml centrifuge tube (depending on the number of initial cells cultured), and remove the magnetic beads. Take a small amount of cells for counting, adjust the cell density to 5E+05cells/ml with complete medium, transfer to a suitable culture bottle or dish, and place in a constant temperature incubator at 37°C to continue culturing.

Cell growth was monitored every day to keep the cell density at 3×10 ⁵ cells/-l-2×10 ⁶ cells/ml during culture. About 7 days after T cells are infected with lentiviral vector, the positive rate of CD19-CAR, tEGFR, MSLN-CAR, and dnTGFβRII expression can be detected, and the dnTGFβRII-CD19-MSLN infected by tEGFR-CD19-CAR and dnTGFβRII-MSLN-CAR double virus can be obtained - CAR-T cells.

The positive rate detection of CAR-T cells is shown in Figure 6. Among them, UTD (Untransduced T Cell untransfected cells) cells were not infected control cells, MSLN-CAR, CD19-CAR, tEGFR, dnTGFβRII expression were all negative; pCDH-MSLN-CAR (P01) lentivirus infection obtained In MSLN-CAR-T (P01) cells, the positive rate of MSLN-CAR is about 70%, and the expression of CD19-CAR, tEGFR, and dnTGFβRII is negative; the dnTGFβRII-MSLN- In CAR-T cells (P17), the positive rate of MSLN-CAR is about 70%, the positive rate of dnTGFβRII expression is about 27.6%, and the expression of CD19-CAR and tEGFR is negative; pCDH-CD19-CAR-tEGFR (P43) lentivirus infection obtained CD19-CAR-T cells (P43), CD19-CAR positive rate is about 21.6%, tEGFR positive rate is 63.5%, MSLN-CAR, dnTGFβRII expression is negative; pCDH-MSLN-CAR(P01)+pCDH-CD19- CAR-tEGFR (P43) double virus infected cells CD19-MSLN-CAR-T cells (P01+P43), MSLN-CAR positive rate is about 60%, CD19-CAR positive rate is about 12.24%, tEGFR positive rate is 36.84 %, the expression of dnTGFβRII was negative, and the double positive rate of MSLN-CAR and CD19-CAR was 27.5% (the infection efficiency of pCDH-CD19-CAR-tEGFR was indicated by tEGFR);

pCDH-MSLN-CAR-dnTGFβRII(P17)+pCDH-CD19-CAR-tEGFR(P43) double virus infected cells dnTGFβRII-CD19-MSLN-CAR-T cells (P17+P43), the MSLN-CAR positive rate was about 58% %, dnTGFβRII positive rate was 20.7%, CD19-CAR positive rate was about 9.9%, tEGFR positive rate was 36.08%, MSLN-CAR, CD19-CAR double positive rate was 26.7% (indicated by tEGFR pCDH-CD19-CAR-tEGFR infection efficiency).

Example 5: Detection of the killing ability of CAR-T cells on target cells in vitro

(1) Experimental materials:

Luciferase Assay (Promega, catalog number: E2520), X-VIVO 15 Medium (Lonza, catalog number: 04-418Q), 96-well flat-bottom white plate without cover (Greiner, catalog number: 655075), 96-well flat-bottomed transparent plate with cover (Greiner, catalog number: 655180), PerkinElmer EnVision 2103 Multilabel Reader

(2) Samples to be tested:

Negative control UTD cells not infected with virus, MSLN-CAR-T cells (P01), dnTGFβRII-MSLN-CAR-T cells (P17), CD19-CAR-T cells (P43), P01, P43 double virus infection CD19-MSLN -CAR-T cells (P01+P43), dnTGFβRII-CD19-MSLN-CAR-T cells (P17+P43) infected by P17, P43 double virus

(3) Experimental method:

Select the target cell SKOV-3C16-fluc, after digestion, resuspend in X-VIVO 15 (without IL2, no serum), adjust to a density of 1E+5/ml, and spread 100 μL/well on 96 in the orifice plate.

Aspirate CAR-T cells, centrifuge at 300g for 5 minutes, resuspend in X-VIVO 15, adjust the density to 1E+6/ml and then dilute to CAR+ density of 5E+5/ml, 2.5E+5 /ml, 1.25E+5/ml, 0.625E+5/ml, 0.3125E+5/ml, the immediate effect target ratio is 10, 5, 2.5, 1.25, 0.625, 0.3125, and the adjusted CAR-T Spread 100 μL/well of cells in a 96-well plate co-cultured with target cells, mix well, and set up a spontaneous group of effector cells, spread the suspension and X-VIVO 15 in a 96-well plate at a volume ratio of 1:1 , in preparation for the detection of cytokines.

Place in a CO ₂ incubator for 17-19 hours, centrifuge at 500g for 5 minutes, absorb the supernatant at 100 μL/well (be careful not to suck the cells), place it in another clean 96-well plate, mark it, and store it in a -20°C refrigerator , for cytokine detection.

100 μL/well The

Add Reagent into the cell suspension, operate in the dark, and let it stand for 5-10 minutes, then test the expression of Luciferase on the computer.

The formula for calculating the percentage of CAR-T cells killing target cells is:

Killing rate (Lysis%)=(pure Target-value-experimental group reading value)/pure Target reading value

(4) Experimental results

As shown in Figure 7, it can be seen from the experimental results that MSLN-CAR-T cells (P01), dnTGFβRII-MSLN-CAR-T cells (P17), P01, P43 double virus infection of CD19-MSLN-CAR-T cells (P01 +P43), P17, P43 double virus-infected dnTGFβRII-CD19-MSLN-CAR-T cells (P17+P43) had significant tumor killing ability against MSLN-positive ovarian cancer cell lines SK-OV3 and OVCAR3, while the negative control UTD cells and CD19-CAR-T cells (P43) had no killing effect on SK-OV3 and OVCAR3 (Fig. 7a, 7b).

For the CD19-positive NALM-6-977 tumor cell line overexpressing MSLN antigen, CAR-T cells in P01, P17, P43, P01+P43, P17+P43 groups all had significant tumor killing ability, while the negative control UTD cells did not Killing effect (Fig. 7c).

For the CD19-positive Raji tumor cell line, only CD19-CAR-expressing CAR-T cells P43, P01+P43, and P17+P43 have killing ability, while UTD, P01, and P17 have no killing effect (Figure 7d). For the CD19KO-Raji cell line knocked out of CD19, none of the groups had the effect of killing CD19KO-Raji tumor cells (Fig. 7e).

Example 6: IFNγ cytokine secretion of CAR-T cells killing target cells in vitro

Take the cell killing ability test, that is, the supernatant after the co-incubation of CAR-T cells and target cells in each group in Test Example 5 (E:T=5:1); use Cisbio Human IFNγKits (product number: 62HIFNGPEG) to detect the supernatant The concentration of IFN-γ in the medium, the specific experimental steps are as follows:

(1) Before the experiment, the reagents should be kept at room temperature for at least 30 minutes.

(2) Sample preparation: After centrifuging each group of samples at high speed, suck out the supernatant and transfer it to a clean EP tube to ensure the removal of residual cell debris, and dilute the original solution 5 times with diluent or cell culture medium (X-VIVO 15) After that, it is ready for use.

(3) Standard product configuration: prepare a standard bottle with distilled water to make the volume conform to the volume on the bottle label, and dilute the melted standard stock solution 3 times with cell culture medium (X-VIVO 15). Take 60 μL of the stock solution, add it to 120 μL of cell culture medium, and mix gently to prepare a standard curve for the standard substance Std7 (4000 pg/mL) with a high-concentration standard substance (Std 7), and serially dilute as follows: from Std 6 to Std 0, each Take 110 μL of diluent or cell culture medium from the bottle, add 100 μL of standard product to 110 μL of dilute solution or cell culture medium, mix gently, repeat serial dilution to make standard products, and mark them as std6, std5, std4, std3, std2, std1, Std 0 (negative control) is the diluent or culture medium.

(4) INFγantibodies working solution configuration: Use

detection buffer#

3, 20 times to dilute 20X stock solution (INFγEu Cryptate antibody), use

detection buffer#

3, 20 times to dilute 20X stock solution (INFγd2 antibody), before the experiment, press 1:1 Pre-mix two ready-to-use diluted antibody solutions to become INFγantibodies working solution.

(5) Detection steps: Take 16 μL/well of standard and sample and add to HTRF 96 well low volume plates (set up 2-3 duplicate wells); add 4 μL of pre-mixed INFγantibodies working solution to all wells; seal the plate and incubate at room temperature for 3 hours at

Plates were read with a PerkinElmer EnVision 2103 Multilabel Reader under the program.

The experimental results obtained are shown in Figure 8. In the CAR-T cell alone group and in the co-incubation group with MSLN-negative K562 target cells, the levels of IFNγ were very low. In the co-incubation group with MSLN-positive target cells OVCAR3, MSLN-CAR-T cells (G2), dnTGFβRII-MSLN-CAR-T cells (G3), double virus-infected cells dnTGFβRII-CD19-MSLN-CAR-T (G4), CD19-MSLN-CAR-T (G6) infected with double viruses had significant secretion of IFNγ cytokines, while negative control UTD (G1), and CD19-CAR-T (G5) groups had lower levels of IFNγ cytokine secretion. Low. In co-incubation experiments with CD19-positive NALM-6-977 tumor cells overexpressing MSLN, MSLN-CAR-T cells (G2), dnTGFβRII-MSLN-CAR-T cells (G3), dnTGFβRII-CD19-MSLN-CAR -T (G4), CD19-CAR-T (G5), and CD19-MSLN-CAR-T (G6) all had significant IFNγ cytokine secretion, and the UTD group had no IFNγ cytokine secretion.

Example 7: Detection of CD107a degranulation activated by CAR-T cells

(1) Experimental materials

PE Mouse Anti-Human CD107a (BD, 555801), PE Mouse IgG1, κIsotype Control (BD, 555749), FITC-Labeled Human CD19 (20-291) Protein, His Tag (ACRO, CD9-HF2H2), Biotinylated Human Mesothelin/ MSLN (296-580), Fc Tag (ACRO, MSN-H826x), Brilliant Violet 421 ^TM Streptavidin (Biolegend, 405225), X-VIVO ^TM 15 cell culture medium (Lonza, 04-418Q), DPBS (Gibco, 14190- 144), 96-well flat bottom plate (Costar), Stain Buffer (BD, 554657), Monensin (BD, 554724), flow cytometer (Beckman), cell culture incubator (Thermo), high-speed centrifuge (Thermo)

(2) Sample to be tested

(3) Experimental method

Tumor cell lines K562, SK-OV3, and OVCAR3 were resuspended in X-VIVO ^TM 15, adjusted to a density of 1e+06/mL, and 100 μL per well was added to a 96-well flat-bottom plate.

CAR-T cells were resuspended with X-VIVO ^TM 15, adjusted to a density of 2e+06/mL, 100 μL per well were added to the corresponding tumor cells, and each group had two replicates.

Add 5 μL PE Mouse Anti-Human CD107a antibody to each well and incubate for 1 hour. At the same time, set the isotype group and add 5 μL PE Mouse IgG1, κIsotype Control.

Dilute the Golgi inhibitor Monensin at 1:50, add 10 μL Monensin to each well and incubate for 4 hours, centrifuge at 500 g for 5 minutes, and discard the supernatant.

Resuspend in 100 μL Stain Buffer, add 3 μL biotin-labeled MSLN protein and 3 μL FITC-Labeled Human CD19 to each well, mix well, and incubate at 4°C for 30 min.

Add 200μL DPBS to each well, mix well, centrifuge at 500g for 5min, and discard the supernatant.

Resuspend in 100 μL Stain Buffer, add 1 μL 500-fold diluted Brilliant Violet 421 ^TM Streptavidin to each well, mix well, and incubate at 4°C for 30 min.

Add 200μL DPBS to each well, mix well, centrifuge at 500g for 5min, discard the supernatant

Resuspend in 100μL Stain Buffer and test on the machine.

(4) Experimental results

The experimental results are shown in Figures 9 and 10. After co-incubation with MSLN-expression-negative tumor cell line K562, the expression ratio of CD107a in MSLN-CAR-positive cells was low, while after co-incubation with MSLN-expression-positive ovarian cancer cell lines SK-OV3, OVCAR3, MSLN-CAR- T cells (P01), dnTGFβRII-MSLN-CAR-T cells (P17), P01, P43 double virus infection of CD19-MSLN-CAR-T cells (P01+P43), P17, P43 double virus infection of dnTGFβRII-CD19-MSLN - The expression ratio of CD107a in MSLN-CAR positive cells in the CAR-T cell (P17+P43) group was significantly increased, while the expression ratio of CD107a in the negative control UTD group, CD19-CAR-T cell (P43) group was lower.

Example 8: Detection of the proliferation ability of CAR-T cells repeatedly stimulated by double target cell antigens

(1) Experimental materials

X-VIVO

^TM 15 medium (Lonza company, product number: BEBP04-744Q), Mc'oy's 5A (Modified) medium (Gibco company, product number: 16600082), Mitomycin C (Selleck company, product number: S8146), APC Streptavidin (BD Biosciences, catalog number: 554067), Stain Buffer (BD Biosciences, catalog number: 554657), Biotinylated Human Mesothelin, Fc Tag (Acrobiosystems, MSN-H826x), SK-OV-3 cell line (Shanghai Gaining Biotechnology Co., Ltd. , Cat. No.: CM-H143), Raji cell line (ATCC, Cat. No.: CCL-86), fluorescence cytometer (Shanghai Ruiyu Biotechnology Co., Ltd.)

(2) Sample to be tested

(3) Experimental method

SK-OV3 cells were digested, Raji cells were drawn, counted, and the cells were inoculated in a 12-well plate, with 3×10 ⁵ cells in each well, and the total volume of the medium was 1 mL/well, and cultured for 24 hours.

Dilute Mitomycin C with complete medium, shake and mix, add the diluted Mitomycin C to the prepared cells, the final concentration is 30mg/mL, and incubate for 3h.

T cells were collected, counted, and UTD was used to adjust the positive rate of CAR-T cells. Centrifuge at 300×g for 5 minutes. Discard the supernatant, wash the T cells with 1×PBS, and centrifuge at 300×g for 5 minutes. Resuspend T cells with X-VIVO medium, adjust the density to 5×10 ⁵ cells/mL, and set aside.

Discard the SK-OV3+Raji cell culture medium, wash the cells with 1×PBS, and repeat twice.

T cells were co-cultured with SK-OV3+Raji cells (this time point was recorded as Day 1), and T cells were seeded into well plates at a volume of 2 mL/well.

After 48 hours of co-cultivation (this time point was recorded as Day 3), the number of CAR-T cells was counted, and the positive rate of CAR-T cells was detected by flow cytometry. 1×10 ⁶ cells were taken for the next round of antigen stimulation experiments. Repeat the stimulation for three rounds.

The formula for calculating the expansion ratio of CAR-T cells is as follows:

(4) Experimental results

The experimental results are shown in Figure 11. When CAR-T cells were cultured alone, there was no tumor cell stimulation, and on D3 days after one round of stimulation, there was no expansion of CAR-T cells. In the case of co-culture with the tumor cell line SK-OV3+Raji, the negative control group had no expansion of UTD cells, MSLN-CAR-T cells (P01), dnTGFβRII-MSLN-CAR-T cells (P17), CD19-CAR- T cells (P43), P01, P43 double virus infection of CD19-MSLN-CAR-T cells (P01+P43), P17, P43 double virus infection of dnTGFβRII-CD19-MSLN-CAR-T cells (P17+P43) significantly Expansion, in which CAR-T cells with CD19-CAR and MSLN-CAR double CAR molecules P17+P43, P01+P43 have significantly higher expansion folds than CAR-T cells with only MSLN-CAR or CD19-CAR molecules.

Example 9: CD19-CAR promotes the expansion of enhanced CAR-T cells and increases anti-tumor ability

In order to evaluate the expansion advantage of enhanced MSLN-CAR-T cells with CD19-CAR molecules in the human body and the ability to kill target cells, each group of CAR-T cells (UTD cells, MSLN-CAR-T cells, dnTGFβRII-MSLN-CAR-T cells, CD19-CAR-T cells, double virus infected CD19-MSLN-CAR-T cells, P17, P43 double virus infected dnTGFβRII-CD19-MSLN-CAR-T cells) were first positive for CD19 Target cells NALM-6 were co-incubated for 4 hours, and then incubated with MSLN-positive target cells OVCAR3 for 7 hours to detect the killing ability of CAR-T cells in each group on OVCAR3 target cells. The experimental method is shown in Example 5.

The experimental results are shown in Figure 12. It can be seen that the CAR-T cell group with CD19-CAR molecules after incubation with NALM-6 for 4 hours: dnTGFβRII-CD19-MSLN-CAR-T, CD19-MSLN-CAR-T The killing ability of OVCAR3 target cells is significantly higher than that of CAR-T cell groups without CD19-CAR molecules: MSLN-CAR-T, dnTGFβRII-MSLN-CAR-T. The experimental results prove that in the presence of CD19-positive target cells, CD19-CAR molecules can promote the expansion of CAR-T cells, thereby increasing the killing effect of CAR-T on MSLN-positive target cells.

Example 10: dnTGFβRII blocks TGFβ-1 cytokine signaling in T cells

(1) Experimental materials

Human TGF-Beta1/TGFB1 Protein, Tag Free (ACRO, TG1-H4212), Phospho-SMAD2 (Ser465/Ser467) (E8F3R) Rabbit mAb (CST, 18338), PE anti-human TGF-β Receptor II Antibody (Biolegend, 399704 ), PE Rat IgG2b, κIsotype Ctrl Antibody (Biolegend, 400607), Alexa

647 Donkey anti-rabbit IgG (minimal x-reactivity) Antibody (Biolegend, 406414), Invitrogen ^™ eBioscience ^™ Fixation/Permeabilization Concentrate (Invitrogen, 501129082), Invitrogen ^™ UltraPure ^™ DNase/RNase-Free Distilled Water (Invitrogen, 15, 1097) Acetic acid (Sanko, A501931-0500).

(2) Sample to be tested

(3) Experimental method

Take 2e+06 CAR-T cells, divide them into two, centrifuge at 300g for 5min, and discard the supernatant. One was resuspended in X-VIVO ^TM 15, and the other was resuspended in X-VIVO ^TM 15 plus 5ng/mL TGF-Beta1, incubated in a cell culture incubator for 40 minutes, centrifuged at 300g for 5 minutes, and discarded the supernatant. Resuspend with 100 μL Stain Buffer, add 5 μL PE anti-human TGF-β Receptor II Antibody to each well, set the isotype group at the same time and add 5 μL PE Rat IgG2b, κIsotype Ctrl Antibody, and incubate at 4°C for 30 min. Add 200 μL DPBS to each well, mix well, centrifuge at 500 g for 5 min, discard the supernatant, mix with 200 μL Fixation buffer, and incubate at room temperature for 1 h in the dark. 500g, centrifuge for 5min, discard the supernatant.

Dilute 10X Permeabilization buffer to 1X with water, resuspend cells in 300μL 1X Permeabilization buffer, centrifuge at 500g for 5min, discard supernatant.

Dilute Phospho-SMAD2(Ser465/Ser467)(E8F3R) Rabbit mAb with 1X Permeabilization buffer at a ratio of 1:400, add 100 μL per well to the corresponding cells, resuspend, and incubate at room temperature for 1 h in the dark.

Add 200μL DPBS to each well, mix well, centrifuge at 500g for 5min, and discard the supernatant. Dilute Alexa with 1X Permeabilization buffer at a ratio of 1:4000

647 Donkey anti-rabbit IgG, add 100 μL per well to the corresponding cells, resuspend, and incubate at room temperature for 1 hour in the dark. Add 200μL DPBS to each well, mix well, centrifuge at 500g for 5min, and discard the supernatant. Resuspend in 100μL 1X Permeabilization buffer, and test on the machine.

(4) Experimental results

The experimental results are shown in Figure 13. After adding 5ng/mL TGFβ-1 cytokine to the CAR-T medium of each group, the negative control UTD cells, MSLN-CAR-T cells (P01), CD19-CAR-T Cells (P43), P01, P43 double virus infection of CD19-MSLN-CAR-T cells (P01+P43) increased SMAD2 protein phosphorylation level, while the CAR-T cell group with dnTGFβRII receptor: dnTGFβRII-MSLN- CAR-T cells (P17), P17, P43 double virus infection dnTGFβRII-CD19-MSLN-CAR-T cells (P17+P43) corresponding SMAD2 protein phosphorylation levels were lower, indicating dnTGFβRII receptor competitive resistance Inhibitory effect of TGFβ-1 cytokine on T cell function.

Example 11: dnTGFβRII resists the inhibitory effect of TGFβ-1 cytokine on the ability of T cells to kill tumor target cells

(1) Experimental materials

ONE-Glo ^TM Luciferase Assay System (Promeg, 72056), dissolve the substrate according to the instructions, aliquot 10 mL in each tube, and freeze at -20°C; Trypsin-EDTA (0.25%), phenol red (Thermo, 25200114) 96-well enzyme label Plate (Costar, 3922), microplate reader (Perkin Elmer)

(2) Sample to be tested

MSLN-CAR-T cells (P01), dnTGFβRII-MSLN-CAR-T cells (P17), P01, P43 double virus infection of CD19-MSLN-CAR-T cells (P01+P43), P17, P43 double virus infection of dnTGFβRII -CD19-MSLN-CAR-T cells (P17+P43)

(3) Experimental method

Add 125 μL of X-VIVO ^TM 15 or X-VIVO ^TM 15 plus 2 ng/mL TGFβ-1 in a 96-well plate. Take 2e+06 CAR-T cells, divide them into two, centrifuge at 300g for 5min, and discard the supernatant. One part was resuspended with 1mL X-VIVO ^TM 15 to a density of 1e+06/mL, and one part was resuspended with 1mL X-VIVO ^TM 15 plus 2ng/mL TGF-Beta1 to a density of 1e+06/mL. Take 250 μL each into the corresponding empty wells of the 96-well plate, and repeat 3 times in each group. Take 125 μL from the 250 μL cell solution into the 125 μL culture medium well, mix well, then take 125 μL into the 125 μL culture medium well, and repeat this 5 times . Take 100 μL of the above-mentioned serially diluted cells into a new marked 96-well plate, while retaining 3 wells and only add 100 μL of the corresponding culture solution, and incubate in the cell culture incubator for 24 hours.

The next day, target cells SKOV3-C16-F were digested, centrifuged at 300 g for 5 min, and the supernatant was discarded. Resuspend with X-VIVO ^TM 15 to adjust the density to 1e+05/mL, add 100 μL per well to the corresponding 96-well plate, and incubate in the cell culture incubator for 18 hours. Centrifuge at 500g for 10min, discard 100μL supernatant, add 100μL substrate, mix well, incubate at room temperature for 15min, take 100μL into a 96-well microplate, use a microplate reader to read the fluorescence value and analyze the experimental results

(4) Experimental results

The experimental results are shown in Figure 14. Each group of CAR-T cells treated with TGFβ-1 cytokines was co-incubated with target cells to evaluate the effect of TGFβ-1 cytokines on the ability of CAR-T cells to kill tumor cells.

MSLN-CAR-T cells (P01), P01, P43 double virus infection CD19-MSLN-CAR-T cells (P01+P43) group were treated with TGFβ-1 cytokines to significantly weaken the killing ability of target cells. And the CAR-T cell group with dnTGFβRII receptor: dnTGFβRII-MSLN-CAR-T cells (P17), P17, P43 double virus infected dnTGFβRII-CD19-MSLN-CAR-T cells (P17+P43), after TGFβ -1 cytokine treatment has almost no effect on the killing ability of target cells.

Example 12: In vivo drug efficacy evaluation of CAR-T cell mouse ovarian cancer subcutaneous xenograft model

In order to evaluate the therapeutic effect of enhanced CAR-T cells on the xenograft tumor model in mice, a subcutaneous xenograft tumor model of ovarian cancer cell line was constructed, and severe immunodeficiency NCG mice (purchased by Jizui Pharmaco), female, 6- 8 weeks old, feeding environment: SPF grade. After one week of adaptive feeding, the mice were randomly divided into 4 groups with 6 mice in each group. Each mouse was subcutaneously inoculated with 5×10 ⁶ SK-OV3 tumor cells in the right scapula to establish the model,

When the average tumor volume grew to 150cm ³ , they were randomly divided into groups, and each mouse was infused with 2×10 ⁷ CART cells, and the body weight and tumor size of the mice were recorded 2 or 3 times a week, and the effects of different CARTs on SK-OV3 in vivo were compared. Killing of tumor cells.

After the mice were modeled and CAR-T cells were reinfused, the results of tumor size and mouse weight changes are shown in Figure 15 and Figure 16. It can be seen from Figure 15 that the tumor volume of the control group mice injected with UTD continued to grow, and the mice injected with MSLN-CAR-T (P01) group, dnTGFβRII-MSLN-CAR-T (P17) group, dnTGFβRII-CD19-MSLN -The tumor volume of mice in the CAR-T (P17+P43) group began to decrease one week after CAR-T cell injection, and the tumor disappeared three weeks later, indicating that CAR-T cells targeting MSLN can be effective in mouse xenograft models Kill SK-OV3 tumor cells. Figure 16 shows that the weight of the mice in each group injected with CAR-T cells did not decrease, and the mental state and hair state of the mice were normal, indicating that the injected MSLN-CAR-T had no obvious side effects on the mice.

Example 13: Pharmacokinetic detection of CAR-T cells in mice with xenografted tumors

Proliferation analysis of CAR-T cells in mice with transplanted tumors, using NCG mice (purchased by Jizui Pharmaceutical Co., Ltd.), female, 6-8 weeks, feeding environment: SPF grade. After one week of adaptive feeding, the mice were randomly divided into 4 groups with 6 mice in each group. Each mouse was subcutaneously inoculated with 5×10 ⁶ SK-OV3 tumor cells in the right scapula to establish a model. After the average tumor volume grew to 150 cm ³ , they were randomly divided into groups, and each mouse was reinfused with 2×10 ⁷ CART cells. After the reinfusion of CART cells, on the 1st, 7th, 14th, and 21st days, the blood of the mice was collected from the orbital vein, and quantitative PCR was used to detect the copy number of MSLN-CAR molecules in the peripheral blood of the CART cells in the mice to reflect the presence of CAR-T cells in small The number of amplification in mice after being specifically stimulated by SK-OV3 tumor cells in mice. As shown in Figure 17, there was no significant expansion of UTD cells in the control group after reinfusion, MSLN-CAR-T (P01) group, dnTGFβRII-MSLN-CAR-T (P17) group, dnTGFβRII-CD19-MSLN-CAR-T ( The CAR-T cells in the P17+P43) group expanded significantly on the 7th day after reinfusion, and the CAR-T cells returned to the basic level on the 14th day.

Example 14: Establishment of human SK-OV-3 cell line NCG mouse subcutaneous xenograft tumor model and pharmacodynamics of CAR T cells in this model

1. Purpose of the experiment

By inoculating human ovarian adenocarcinoma cells (SK-OV-3 cells) subcutaneously in NCG mice, the pharmacodynamic evaluation of the anti-tumor effect of CAR-T cell drugs was performed on this tumor model.

2. Experimental materials

2.1 Cell lines

SK-OV-3 cells are from Shanghai Reindeer Biotechnology Co., Ltd., and the culture conditions are Mc'oy's 5a medium plus 10% heat-inactivated fetal bovine serum (FBS), placed in a 37°C, 5% CO ₂ incubator cultivated in. Cells in logarithmic growth phase will be used for subcutaneous tumor inoculation in NCG mice.

2.2 Experimental animals

2.2.1 Source of experimental animals

162 NCG mice, female, weighing about 18-24 g, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. Breeding environment: SPF grade. Adaptive feeding was carried out for at least 3 days before the formal experiment.

2.2.2 Experimental Animal Feeding

All the experimental mice were kept in an IVC constant temperature and pressure system in an SPF grade animal room, where the temperature was 20-26° C., the humidity was 40-70%, and the light cycle was 12 hours bright and 12 hours dark. No more than 6 mice were raised in each cage box, and the size of the cage box was 325mm×210mm×180mm. The litter used in the cage box was autoclaved corncobs, which were replaced twice a week. During the whole experiment, all the experimental mice were free to eat and drink, the feed was sterilized by Co60 irradiation, the drinking water was sterilized by high pressure, and the feed and drinking water were kept in sufficient supply. All personnel entering and leaving the animal breeding room or experimental operators should wear sterile lab coats, disposable medical masks and gloves. Each breeding cage has a corresponding clear and detailed label, which includes: IACUC approval number LDIACUC006, number of animals, sex, strain, date of receipt, project number, group, current experimental stage, and the person in charge of the experiment. All experimental animals will be acclimatized for at least 3 days prior to experimental use.

2.3 Test drugs

The tested drugs include UTD cells, P43 cells, P01 cells, P17 cells, P01+P43 cells, P17+P43 cells, C1+P43 cells and C2+P43 cells. Wherein, UTD cells, P43 cells, P01 cells, P17 cells, P01+P43 cells, P17+P43 cells are the same as those described in Example 4, and C1+P43 cells were obtained using pCDH-MSLN-CAR-dnTGFβRII(C1)+pCDH -CD19-CAR-tEGFR(P43) double virus infected cells dnTGFβRII-CD19-MSLN-CAR-T cells (C1+P43), C2+P43 cells were pCDH-MSLN-CAR-dnTGFβRII(C2)+pCDH-CD19 -CAR-tEGFR(P43) double virus infected cells dnTGFβRII-CD19-MSLN-CAR-T cells (C2+P43). The pCDH-CD19-CAR-tEGFR (P43) virus used for the infection of C1+P43 cells and C2+P43 cells is the same as that described in Example 2 and Example 4, and the basic structures of C1 and C2 are the same as in Example 2 and Example 4 The lentivirus P17 recorded in is the same, the only difference is that the scFv sequence of the single-chain antibody targeting MSLN in the C1 structure is shown in SEQ ID NO: 101, and the scFv sequence of the single-chain antibody targeting MSLN in the C2 structure is shown in SEQ ID NO: 90 shown.

3. Experimental steps and methods

3.1 Culture of SK-OV-3 tumor cells

SK-OV-3 cells were cultured in Mc'oy'5A medium with 10% heat-inactivated fetal bovine serum (FBS), placed in a 37°C, 5% CO ₂ incubator. Cells in logarithmic growth phase will be used for subcutaneous tumor inoculation in NCG mice.

3.2 SK-OV-3 cell inoculation, grouping and administration

The SK-OV-3 cells in the logarithmic growth phase were harvested, the inoculated amount was 8×106/mouse, the inoculated cell volume was 0.2ml/mouse (containing 50% Matrigel), and inoculated subcutaneously on the right side of NCG mice. After the tumor volume of the mice grew to 150-200 mm ³ , 108 mice were selected according to the tumor volume and divided into 18 groups randomly, with 6 mice in each group. The day of group administration was Day 0 (D0). On D0, in the low-dose group (G11-G18), before CAR-T injection, 1×10 ⁶ NALM6 cells were injected into the tail vein (after being treated with MMC for 3 h), and the same treated NALM6 cells were injected on D7, D14, and D21. cell. The detailed administration method, dosage and route of administration are shown in Table 2.

Table 2. Grouping and administration

High dose administration group:

Note: N: number of animals used; i.v.: tail vein injection; i.p: intraperitoneal injection

Dosing volume: Adjust the dosing volume to 200 μL according to the body weight of the tumor-bearing mice.

Low dose administration group:

Note: N: number of animals used; i.v.: administration by tail vein injection

Day0: Administration on day 0

3.3 Experimental observation

Throughout the experiment, the use and observation of experimental animals were carried out in accordance with the relevant regulations of AAALAC animal use and management. After the experimental animals were inoculated with tumor tissues, they were observed every day, and their morbidity and death were recorded. During routine experiments, all experimental animals were monitored and recorded for behavior, food intake, water intake, body weight change, hair luster and other abnormalities.

3.4 Evaluation Indicators

It is mainly to detect the tumor growth curve of SK-OV-3 cells on NCG mice and the growth inhibitory effect or complete cure ability of CAR-T on SK-OV-3 cell line tumor models.

3.4.1 Measurement of tumor volume and body weight of tumor-bearing mice: measure twice a week with a vernier caliper, the formula for calculating tumor volume is V=0.5a×b2, where a and b represent the long diameter and wide diameter of the tumor, respectively;

3.4.2 Tumor growth inhibition rate TGI (%) = 100-(delta T/delta C) × 100 = 100-(Ti-T0)/(Vi-V0) × 100%, Ti is given to the CAR-T cell group The average tumor volume after drug administration, T0 is the average tumor volume of the CAR-T cell group at the first administration, Vi is the average tumor volume of the vehicle control group after administration, and V0 is the average tumor volume of the vehicle control group at the first administration T/C%=TRTV/CRTV×100%, RTV=Vt/V0, T/C% is the tumor relative growth rate, and T and C are respectively the tumor volume of administration group and matched group at a specific time point ( TV).

3.4.3 The body weight of all tumor-bearing mice was measured twice a week. The body weight change uses the formula RCBW%=(BWi-BW0)/BW0×100%, BWi is the current body weight of the mouse, and BW0 is the mouse body weight on the day of grouping;

3.5 Sample collection

On D1, D7, D14, D21, and D28 days after CAR-T injection in the drug effect group, 100 μL of blood was collected from the orbit using EDTA anticoagulant tubes.

3.6 Data Analysis

All the data are represented by Mean±SEM, and One-Way ANOVA is used to compare whether there is a significant difference in the tumor volume between the treatment group and the control group. All data were analyzed with Graphpad, and P<0.05 was considered to have a significant difference.

4. Experimental results

On the 22nd day of administration, some mice were euthanized because the tumor volume exceeded 2000mm ³ , and all statistical data were cut off on the 22nd day of administration.

On the 22nd day after administration, the average tumor volume of PBS control group was 1861.70±178.19mm ³ , HD UTD, 2×107/mouse administration group, HD P43, 2×107/mouse administration group, HD P01, 2×107 administration group /mouse administration group, HD P17, 2×107/mouse administration group, HD P01+P43, 2×107/mouse administration group, HD P17+P43, 2×107/mouse, iv administration group, HD C1 +P43, 2×107/mouse administration group, HD C2+P43, 2×107/mouse administration group, HD P17+P43, 2×107/mouse, ip administration group, LD UTD, 5×10 ⁶ / mouse administration group, LD P43, 5×10 ⁶ /mouse administration group, LD P01, 5×10 ⁶ /mouse administration group, LD P17, 5×10 ⁶ /mouse administration group, LD P01+P43, 5 The mean tumor volumes of the ×10 ⁶ /mouse administration group, LD C1+P43, 5×10 ⁶ /mouse administration group and LDC2+P43, 5×10 ⁶ /mouse administration group were 1761.94±157.75mm ³ , 1886.56±207.88mm ³ , 45.76±25.15mm ³ , 10.03±6.39mm ³ , 22.30±7.08mm ³ , 38.34±26.38mm ³ , 1227.07±96.91mm ³ , 51.69±38.08mm 35 , 42.39±3.2mm ³ , 71mm 35 , 42.39±3.2mm 3 , 71mm ³ 131.59mm ³ , 2036.84±217.75mm ³ , 1522.94±191.47mm ³ , 776.38±234.11mm ³ , 412.03±202.90mm ³ , 195.75±54.39mm ³ , 1523.93±119.21mm ³ and ^2131.46mm Compared with the control group, the tumor growth inhibition rate (TGI) was 6.41%, -1.61%, 185.29%, 196.78%, 192.85%, 187.66%, 40.95%, 183.33%, 186.40%, -16.28%, -11.28% , 21.91%, 70.05%, 93.53%, 137.24%, 21.82% and 129.43%.

Compared with the PBS control group (day 22), HD P01, 2×10 ⁷ /mouse administration group, HD P17, 2×10 ⁷ /mouse administration group, HD P01+P43, 2×10 ⁷ /mouse administration group drug group, HD P17+P43, 2×10 ⁷ /mouse, iv administration group, HD C1+P43, 2×10 ⁷ /mouse administration group, HD C2+P43, 2×10 ⁷ /mouse administration group, HD P17+P43, 2×10 ⁷ /mouse, ip administration group, LD P17, 5×10 ⁶ /mouse administration group, LD P01+P43, 5×10 ⁶ /mouse administration group, and LD C2+P43 , the tumor volume of the 5×10 ⁶ /mouse administration group was significantly reduced (P<0.001), indicating that CAR-T has a very significant tumor inhibitory effect in mice, and has a long-lasting tumor inhibitory effect.

And on the 36th day after administration, the experiment ended, HD P17, 2×10 ⁷ /mouse administration group, HD P01+P43, 2×10 ⁷ /mouse administration group, HD P17+P43, 2×10 ⁷ /mouse administration group , the tumors in the ip administration group all subsided, showing a very significant tumor-suppressing effect.

During this experiment, 70# mice in the LDP43, 5×10 ⁶ /mouse administration group and 102# mice in the LDC1+P43, 5×10 ⁶ /mouse administration group died abnormally on D27, which may be due to Influenced by individual differences, the body weight and preclinical behavior of the mice in the remaining groups did not have significant abnormal changes, indicating that the tumor-bearing mice had good tolerance to each test drug at the test dose (see Figures 18 and 19 ).

5. Experiment summary and discussion

The main purpose of this experiment is to establish a CDX tumor model by inoculating SK-OV-3 cell line subcutaneously in NCG mice, and to evaluate the pharmacodynamics of the anti-tumor effect of CAR-T cells on this tumor model.

The results of pharmacodynamic experiments showed that compared with the control group, HD P01, 2×10 ⁷ /mouse administration group, HD P17, 2×10 ⁷ /mouse administration group, HD P01+P43, 2×10 ⁷ /mouse administration group Administration group, HD P17+P43, 2×10 ⁷ /mouse, iv administration group, HD C1+P43, 2×10 ⁷ /mouse administration group, HD C2+P43, 2×10 ⁷ /mouse administration group , HD P17+P43, 2×10 ⁷ /mouse, ip administration group, LD P17, 5×10 ⁶ /mouse administration group, LD P01+P43, 5×10 ⁶ /mouse administration group, LD P17+P43 , 5×10 ⁶ /mouse administration group and LDC2+P43, the tumor volume of 5×10 ⁶ /mouse administration group was significantly reduced (P<0.001), indicating that CAR-T has a very significant role in mice Anti-tumor effect, and has a long-term anti-tumor effect.

At the same time, the body weight and preclinical behavior of the mice in each group showed no obvious abnormal changes, indicating that the tumor-bearing mice had good tolerance to each test drug at the test dose.

Example 15 MSLN antibody enrichment and screening using phage antibody library

Using appropriate negative panning and positive panning strategies to enrich the specific antibody clones targeting MSLN protein from the phage antibody library.

The phage antibody library used was constructed by the applicant, including natural library, semi-synthetic library and single domain library. The semi-synthetic phage antibody library, used together with the natural library, solves the problem that the natural library may lack MSLN high-affinity antibody clones. The single-domain phage antibody library is an antibody library composed only of the variable region amino acids of heavy chain antibodies. Its molecular weight is only 12-15kDa, but it has similar or higher specificity and affinity than traditional antibodies.

Using different antibody libraries, through 3 rounds of protein panning, a significant increase in recovery was observed in each panning, demonstrating the effective enrichment of antibody clones, followed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS) screening of specific clones from the enriched phage pool.

The purpose and principle of screening: The phage pool enriched by the affinity panning step contains phage antibodies of various properties: specific clones, non-specific clones and negative clones. In order to obtain specific clones, we need to isolate monoclonals from them, package them into monoclonal phages, and conduct preliminary screening on a large number of monoclonals by enzyme-linked immunoassay (ELISA) and flow cytometry (FACS), and select them to simultaneously specifically bind Monoclonal MSLN protein and MSLN positive cell line CHO-K1-MSLN. The specific monoclonal is further determined by DNA sequencing to determine the unique antibody sequence contained therein.

In the ELISA primary screening, through the combination of streptavidin and biotin Biotin, the biotinylated target protein KACTUS-MSLN-Bio (Glu296-Gly588), KACTUS-MSLN-Bio (Glu296-Asn 494) in The conformation of the antigen in the reaction solution is closer to the natural state. Those that only bind to KACTUS-MSLN-Bio (Glu296-Gly588) but not Streptavidin are identified as specific clones, and some of these specific clones bind to KACTUS-MSLN-Bio (Glu296-Asn 494). The FACS primary screening was carried out using the positive cell line CHO-K1-MSLN with high expression of MSLN and the cell line CHO-K1 negative for MSLN, and those that only combined with CHO-K1-MSLN cells but not with CHO-K1 cells were identified as specific clones. Through ELISA and FACS two primary screenings, we can obtain candidate antibodies that can not only bind to the recombinantly expressed KACTUS-MSLN-Bio (Glu296-Gly588) protein, but also recognize the natural state MSLN molecules on the cell surface for subsequent further screening.

Brief steps of ELISA experiment:

1) Cultivate and package monoclonal phage in a deep-well 96-well plate;

2) Dilute Streptavidin to 2 μg/mL with PBS, add 100 μL/well to a high-binding microtiter plate, and bind at room temperature for 2 hours;

3) Discard the coating solution, add 250 μL of blocking solution to each well, and block overnight at 4°C;

4) Wash the plate twice with 250 μL of washing solution;

5) Dilute the biotin-labeled target protein to 2 μg/mL with PBS, add 100 μg/well to the microtiter plate pre-coated with Streptavidin, and combine at room temperature for 0.5 h;

6) Wash the plate twice with 250 μL of washing solution;

7) Add 100 μL of the phage supernatant cultured in step 1) to the wells coated with the target antigen, and bind at 4°C for 2 hours;

8) Wash the plate 4 times with 250 μL of washing solution;

9) Add mouse anti M13 primary antibody diluted 1:2000, 100 μL/well, and incubate at room temperature for 45 minutes;

10) Wash the plate 4 times with 250 μL washing solution;

11) Add 1:2000 diluted HRP Donkey anti-mouse IgG secondary antibody, 100 μL/well, incubate at room temperature for 45 minutes;

12) Wash the plate 6 times with 250 μL rinse solution;

13) Add 100 μL TMB chromogenic substrate, and develop color for 5 to 10 minutes;

14) Add 100 μL of 2M H ₂ SO ₄ to terminate the reaction, and read the result on a microplate reader.

Brief steps of FACS primary screening experiment:

1) Cultivate and package monoclonal phage in a deep-well 96-well plate;

2) Wash CHO-K1-MSLN and CHO-K1 cells twice with PBS, resuspend in PBS to a concentration of 1x10 ⁷ /mL, and dispense 50 μL into 96-well U-bottom well plates;

3) Add 50 μL of packaged monoclonal phage to each well, mix well, and combine at 4°C for 1 hour;

4) Wash twice with 180 μL PBS;

5) Add mouse anti M13 primary antibody diluted 1:2000, 100 μL/well, mix well by pipetting, and incubate at 4°C for 30 minutes;

6) Wash once with 180 μL PBS;

7) Add FITC horse anti mouse-IgG (H+L) secondary antibody diluted 1:300, 100 μL/well, mix well by pipetting, and incubate at 4°C for 30 minutes;

8) Wash twice with 180 μL PBS; finally resuspend the cells with 100 μL PBS;

9) Detect the fluorescence intensity of the FITC channel of the sample on the flow cytometer, and analyze the results.

Main materials and reagents:

Helper phage KO7, Thermo/Invitrogen, 18311019

Streptavidin (SA), Pierce, 21125

KACTUS-MSLN-Bio(Glu296-Gly588), Biotinylated Human Mesothelin, Avitag ^™ , His Tag, KACTUS, MSL-HM280B

KACTUS-MSLN-Bio(Glu296-Asn494), Biotinylated truncation Human Mesothelin, Avitag ^TM , His Tag, KACTUS

High binding ELSIA plate, Costar, #3590

Corning 96 Well Clear Round Bottom TC-Treated Microplate, Costar, #3799

Blocking solution: PBS+3%BSA

Rinse solution: PBS+0.1% Tween20

Soluble one-component TMB substrate solution, Tiangen, PA-107-02

Anti-M13 Bacteriophage Coat Protein g8p antibody, abcam, ab9225

HRP Goat anti-mouse IgG (minimal x-reactivity) Antibody, Biolegend, 405306

HRP Goat anti-rat IgG (minimal x-reactivity) Antibody, Biolegend, 405405

HRP Donkey anti-human IgG (minimal x-reactivity) Antibody, Biolegend, 410902

FITC horse anti mouse-IgG(H+L), Vector, FI2000

MSLN Ab (Biolegend), pufified anti human Mesothelin, Biolegend, 530101

MSLN Ab(RD), PE anti-human Mesothelin, Rat IgG, R&D, FAB3265P

Experimental results:

Monoclonals were randomly selected from the enriched phage antibody pool, packaged into phages, and the binding of monoclonal phages to KACTUS-MSLN-Bio protein, KACTUS-MSLN-Bio (Glu296-Asn 494) protein and SA protein was detected by phage ELISA , found only combined with KACTUS-MSLN-Bio (Glu296-Gly588), not combined with KACTUS-MSLN-Bio (Glu296-Asn 494), or combined with KACTUS-MSLN-Bio (Glu296-Gly588), KACTUS-MSLN-Bio ( Glu296-Asn 494) specific phage antibody clones combined simultaneously. The ELISA results of some clones are shown in Figure 20. Wherein the negative control is the negative control of phage, the positive control 1 is the positive control of adding MSLN antibody MSLN Ab (RD), and the positive control 2 is the positive control of adding MSLNAb (Biolegend); it can be seen from the figure that the clones of G1～G9 are compatible with the target The antigen KACTUS-MSLN-Bio (Glu296-Gly588) combined well, and did not bind to the control antigen Streptavidin (SA), with good specificity, among which G2, G4～G7 also combined with KACTUS-MSLN-Bio (Glu296- Asn 494), indicating that these clones can bind to different regions of the MSLN antigen with good specificity. The G10 clone does not bind to the target antigen KACTUS-MSLN-Bio (Glu296-Gly588), KACTUS-MSLN-Bio (Glu296-Asn 494) and streptavidin (SA), and is a negative clone.

The FACS primary screening results of some clones are shown in Figure 21. The negative control is the negative control of phage; it can be seen from the figure that the clones G1-G9 combined with the MSLN-positive cell line CHO-K1-MSLN, but not with the MSLN-negative cell line CHO-K1, are specific clones, and the G10 clone is negative Cloning (does not bind to either cell).

Through ELISA detection and FACS primary screening, we obtained a total of 359 specific clones.

Example 16. Identification of Monoclonal Specificity by FACS Using Multiple Cell Lines

The purpose and principle of the experiment: the antibody used for treatment must have very good target specificity, and only bind the target antigen, not any unrelated antigen; on the other hand, the amino acid sequence of the same antigen on different cell lines will be different (Isomers or mutants) or binding ligands are different, and it is also necessary to investigate whether our antibodies can bind to cells positive for various target proteins. In order to further analyze the specificity and universality of these monoclonals and find the best candidate clones, we further evaluated the specificity of the primary screening clones by flow cytometry. In this experiment, we used a variety of MSLN-positive cell lines and a variety of MSLN-negative cell lines to react with these monoclonal phage antibodies, and analyzed whether these clones could bind to the MSLN antigen on the cell lines and whether they interacted with other cells that did not express Cell lines with MSLN have any non-specific binding. Through this experiment, we obtained several clones with excellent specificity.

Experimental method: the same as the FACS primary screening;

Main samples and reagents:

CHO-K1-MSLN, OVCAR-3, ASPC-1 cell lines, MSLN positive cell lines (+);

CHO-K1, Nalm6 cell line, MSLN negative cell line (-);

The rest of the reagents were the same as the FACS primary screening.

Experimental results:

Antibodies used for therapy must have very good target specificity. In order to further analyze the specificity of these monoclonal antibodies, we identified the only clone obtained in Example 2 on more antigens and cell lines using ELISA and flow cytometry. The results are shown in Figure 22A, 22B, 22C, 22D, the negative control is negative phage antibody clone, #2, #3, #5, #7, #8, #12, #14, #24, #26 can be seen , #28, #29 (Fig. 22A), #30, #33, #34, #39, #40, #43, #46, #55, #75, #76, #77 (Fig. 22B), #84 , #85, #86, #87, #90, #91, #93, #96, #97, #107, #110 (Figure 22C), #116, #118, #119, #120, #121, #122 (Fig. 22D) clone combined with the MSLN positive cell line CHO-K1-MSLN, and did not combine with the MSLN negative cell line, wherein #24, #26, #28, #29 (Fig. 22A), #30, #43 (Fig. 22B), #86, #96 (Fig. 22C), #118-122 (Fig. 22D) No. clones also combined with MSLN positive cell line OVCAR-3, #12, #14, #24, #26, #28, #29 (Fig. 22A), #30, #33, #34, #39, #40, #43, #46, #55, #75, #76, #77 (Fig. 22B), #84, #85, #86, #87, #91, #93, #97, #107, #110 (Fig. 22C), #119, #121, #122 (Fig. 22D) clones were also compatible with the MSLN positive cell line ASPC- 1 binding, good specificity.

Example 17. Identification of Monoclonal Specificity by ELISA Using Different Antigens

Purpose and principle of the experiment: Antibodies used for treatment must have very good target specificity and only bind to the target antigen, not to any unrelated antigen. In order to further analyze the specificity and universality of these monoclonals and find the best candidate clones, we further evaluated the specificity of the primary screening clones by enzyme-linked immunoassay (ELISA). In this experiment, we used MSLN antigens purchased from different companies and a variety of MSLN-unrelated antigens to react with these monoclonal phage antibodies, and analyzed whether these clones could bind to different MSLN antigens and whether they could bind to other MSLN-unrelated antigens Any non-specific binding. Through this experiment, we obtained several clones with excellent specificity.

Experimental method: the same as the ELISA primary screening;

The main samples and reagents are listed in Table 3:

table 3

The rest of the reagents are the same as the ELISA primary screening.

Experimental results:

Antibodies used for therapy must have very good target specificity. In order to further analyze the specificity of these monoclonal antibodies, we identified multiple clones obtained in Example 2 on various antigens using enzyme-linked immunosorbent assay (ELISA). The results are shown in Figure 23A, 23B, 23C, 23D, the negative control is the negative control of phage, the positive control 1 is the positive control of adding MSLN antibody MSLN Ab (RD), and the positive control 2 is the positive control of adding MSLN Ab (Biolegend) ;As can be seen from the figure, #2, #3, #5, #7, #8, #12, #14, #24, #26, #28, #29 (Fig. 23A), #30, #33, # 34, #39, #40, #43, #46, #55, #75, #76, #77 (Fig. 23B), #84, #85, #86, #87, #90, #91, #93 , #96, #97, #107, #110 (Figure 23C), #116, #118, #119, #120, #121, #122 (Figure 23D) clones and KACTUS-MSLN-Bio (Glu296-Gly588 ) protein has strong binding, and does not bind to non-related antigens KACTUS BAFFR-Bio, KACTUS CD5-Bio and SA, wherein #24, #26, #28, #29 (Figure 23A), #30, #33, # 34, #39, #40, #43, #46, #55, #75, #76, #77 (Fig. 23B), #84, #85, #86, #87, #90, #91, #93 , #96, #97, #107, #110 (Fig. 23C) clones combined with KACTUS-MSLN-Bio (Glu296-Asn 494) protein, indicating that these clones can bind to different regions of MSLN antigen with good specificity; in addition # 2, #24, #26, #28, #29 (Fig. 23A), #30, #33, #39, #46, #75, #76, #77 (Fig. 23B), #84, #85, # 86, #87, #91, #93, #97, #107, #110 (Figure 23C), #116, #119, #122 (Figure 23D) clones combined with ACRO-MSLN-cyno, monkey cross, #28, #29 (Fig. 23A), #30, #39, #46, #75, #76, #77 (Fig. 23B), #84, #85, #86, #91, #93, #97, Clones #107 and #110 (Fig. 23C) combined with ACRO-MSLN-mouse, and there was a mouse cross.

Example 18. Identification of antibody specificity by FACS using multiple cell lines after recombinant expression

The purpose and principle of the experiment: the antibody used for treatment must have very good target specificity, and only bind the target antigen, not any unrelated antigen; on the other hand, the amino acid sequence of the same antigen on different cell lines will be different (Isomers or mutants) or binding ligands are different, and it is also necessary to investigate whether our antibodies can bind to cells positive for various target proteins. In order to further analyze the specificity and universality of these monoclonals and find the best candidate clones, we expressed them through recombinant expression, that is, constructing the specific sequence obtained after identification at the phage level on an expression vector, and electrotransferring into cells After culturing, the supernatant expressed by the cells was collected, and the specificity of the clones was further evaluated at the protein level by flow cytometry. In this experiment, we used a variety of MSLN-positive cell lines and a variety of MSLN-negative cell lines to react with these recombinantly expressed antibodies, and analyzed whether these clones could bind to the MSLN antigen on the cell lines and whether they interacted with other cells that did not express Cell lines with MSLN have any non-specific binding. Through this experiment, we obtained several clones with excellent specificity.

Brief steps of FACS experiment:

1) Prepare the supernatant of the recombinantly expressed protein to be identified;

2) The cells to be used were washed twice with PBS, resuspended in PBS containing 5% FBS to a concentration of 5x10 ⁶ /mL, and dispensed into 96-well U-bottom well plates in 100 μL;

3) Place at 4°C and seal for 30 minutes;

4) Add protein supernatant and incubate at 4°C for 1 hour;

5) Wash twice with 180 μL PBS;

6) Add Anti human IgG (647-conjugated) secondary antibody diluted 1:300, 100 μL/well, mix by pipetting, and incubate at 4°C for 30 minutes;

7) Wash twice with 180 μL PBS; finally resuspend the cells with 100 μL PBS;

8) Detect the fluorescence intensity of the APC channel of the sample on the flow cytometer, and analyze the results.

Main samples and reagents:

CHO-K1-MSLN, Nalm6-977, OVCAR-3, ASPC-1 cell lines, MSLN positive cell lines (+);

CHO-K1, JURKAT cell line, MSLN negative cell line (-);

Anti human IgG (647-conjugated) secondary antibody, Jackson, Cat. No.: 146154

Experimental results:

Antibodies used for therapy must have very good target specificity. In order to further analyze the specificity of these monoclonal antibodies, we recombinantly expressed the specific clones obtained in Examples 4 and 5 into protein supernatants and performed enzyme-linked immunosorbent adsorption and flow cytometry on more antigens and cell lines. Identification. The results are shown in Figures 24A, 24B, and 24C. The negative control is the control without adding protein supernatant and only adding secondary antibody. You can see #2, #3, #5, #7, #8, #12, #14 , #24, #26, #28, #29, #30, #33 (Fig. 24A), #55, #75, #76, #77, #84, #85, #86, #87 (Fig. 24B) , #90, #91, #93, #96, #116, #119 (Fig. 24C) clones combined with the two MSLN positive cell lines CHO-K1-MSLN and Nalm6-977, the median fluorescence intensity (MFI) All are relatively strong, do not bind to MSLN-negative cell lines, MFI is low, specificity is good, and they are specific clones. Among these specific clones, #5, #8, #14, #24, #26, #28, #29 , #30, #33 (Fig. 24A), #55, #76, #77, #84, #86, #87 (Fig. 24B), #90, #91, #93, #96, #116, #119 (Fig. 24C) No. clone also combined with MSLN positive cell line OVCAR-3; #3, #14, #24, #26, #28, #29, #30, #33 (Fig. 24A), #55, #76 , #77, #84, #85, #86, #87 (Fig. 24B), #90, #91, #93, #96, #116, #119 (Fig. 24C) clones were also compatible with the MSLN positive cell line ASPC -1 combination; additional #34, #39, #40, #43, #46 (FIG. 24B), #97, #107, #110, #118, #121, #120, #122 (FIG. 24C) clones It has a weak combination with MSLN-negative cell lines and is a non-specific antibody, which does not meet the experimental needs.

Example 19 Identification of antibody specificity by ELISA using different antigens after recombinant expression

Purpose and principle of the experiment: Antibodies used for treatment must have very good target specificity and only bind to the target antigen, not to any unrelated antigen. In order to further analyze the specificity and universality of these monoclonals and find the best candidate clones, we expressed them through recombinant expression, that is, constructing the specific sequence obtained after identification at the phage level on an expression vector, and electrotransferring into cells After culturing, the supernatant expressed by the cells was collected, and the specificity of the clone was further evaluated at the protein level by enzyme-linked immunoassay (ELISA). In this experiment, we used MSLN antigens purchased from different companies and various MSLN-unrelated antigens to react with recombinantly expressed antibodies, and analyzed whether these clones could bind to different MSLN antigens and whether they could bind to other MSLN-unrelated antigens. Any non-specific binding. Through this experiment, we obtained several clones with excellent specificity.

Brief steps of ELISA experiment:

4) Wash the plate twice with 250 μL washing solution;

6) Wash the plate twice with 250 μL of washing solution;

7) Add 100 μL of the protein supernatant prepared in step 1) to the wells coated with the target antigen, and bind at 4°C for 45 minutes;

8) Wash the plate 4 times with 250 μL of washing solution;

9) Add anti human-HRP secondary antibody diluted 1:2000, 100 μL/well, and incubate at room temperature for 45 minutes;

10) Wash the plate 6 times with 250 μL of washing solution;

11) Add 100 μL TMB chromogenic substrate, and develop color for 5 to 10 minutes;

12) Add 100 μL of 2M H ₂ SO ₄ to terminate the reaction, and read the result on a microplate reader.

The main samples and reagents are shown in Table 4:

Table 4

The rest of the reagents are the same as the ELISA primary screening.

Experimental results:

Antibodies used for therapy must have very good target specificity. In order to further analyze the specificity of these monoclonal antibodies, we recombinantly expressed the specific clones obtained in Examples 4 and 5 into protein supernatants and identified them by enzyme-linked immunosorbent assay (ELISA). The results are shown in Figures 25A, 25B, and 25C. The secondary antibody control is the control without protein supernatant and only the secondary antibody is added. The positive control 1 is the positive control with the MSLN antibody MSLN Ab (RD), and the positive control 3 is the positive control with the addition of HUYP218 ( MSLN clinically positive antibody) positive control; As can be seen from the figure, #2, #3, #5, #7, #8, #12, #14, #24, #26, #28, #29, #30, #33 (Fig. 25A), #34, #39, #40, #43, #46, #55, #75, #76, #77, #84, #85, #86, #87 (Fig. 25B), #90, #91, #93, #96, #107, #110, #116, #118, #119, #120, #121, #122 (Figure 25C) clones and KACTUS-MSLN-Bio (Glu296- Gly588) protein has varying degrees of binding, and does not bind to non-related antigens KACTUS BAFFR-Bio, KACTUS IL10-Bio and SA, of which #3, #14, #24, #26, #28, #29, #30, #33 (Fig. 25A), #34, #39, #40, #43, #46, #55, #75, #76, #77, #84, #85, #86, #87 (Fig. 25B), #90, #91, #93, #96, #107, #110 (Fig. 25C) clones have varying degrees of binding to the KACTUS-MSLN-Bio (Glu296-Asn 494) protein, indicating that these clones can bind to the MSLN antigen Different regions with good specificity; additional #2, #12, #14, #24, #26, #28, #29, #30, #33 (Figure 25A), #34, #39, #40, #46 , #75, #76, #77, #84, #85, #86, #87 (Fig. 25B), #91, #93, #107, #110, #116, #119, #122 (Fig. 25C) Clones #28, #29, #30, #33 (Figure 25A), #34, #39, #43, #46, #76, #77, #84 combined with ACRO-MSLN-cyno, and monkey crosses were present , #85, #86 (FIG. 25B), #91, #93, #107 (FIG. 25C) clones were combined with ACRO-MSLN-mouse, and there was a mouse cross. At the same time, clone #97 (Fig. 25C) had weak non-specific binding to MSLN irrelevant antigens, which did not meet the experimental requirements.

The present invention uses fully human phages for antibody screening to directly obtain fully human monoclonal antibodies. Compared with traditional hybridoma technology, it saves the difficult step of humanizing mouse antibodies, and fully human antibodies have lower immunogenicity than humanized mouse antibodies, and are used in antibody drugs, detection reagents, etc. Has the potential to be better.

The affinity determination of embodiment 20 antibody

Experiment purpose and principle:

The affinity between antibody and antigen may have an important impact on the killing effect and duration of CAR-T or antibody drugs in patients. In order to determine this important property, we used the molecular interaction technology (BLI) of Sartorius to analyze the Clones #2, #5, #118 and #119 were screened in Example 16-19, and the reference antibody huYP218 was tested for affinity.

The biomembrane interferometry technology used in the system is a label-free technology that provides high-throughput biomolecular interaction information in real time. The instrument emits white light to the surface of the sensor and collects the reflected light. The reflected light spectrum of different frequencies is affected by the thickness of the optical film layer of the biosensor. Some frequencies of reflected light form constructive interference (blue), while others are destructive. Interference (red). These interferences are detected by the spectrometer and form an interference spectrum, which is displayed by the phase shift intensity (nm) of the interference spectrum. Therefore, once the number of molecules bound to the sensor surface increases or decreases, the spectrometer will detect the shift of the interference spectrum in real time, and this shift directly reflects the thickness of the biofilm on the sensor surface, from which the biomolecular interaction information can be obtained. High-quality data, so as to determine the kinetic parameters of biomolecular interactions (Kon, Kdis and KD), provide important information for the research and development process.

Brief experimental steps:

1) Dilute the anti-MSLN IgG (constructed by fusing the MSLN scFv sequence with human IgG4Fc) to 10 μg/mL with loading buffer (1×PBS, pH 7.2, 0.1% BSA and 0.02% Tween 20), and in the biosensor On the sample.

2) After a 300s equilibration period, monitor the binding kinetics of MSLN antibody and MSLN antigen multi-concentration (see Table 5). Binding for 60 s and dissociation for 60 s were performed in parallel at each concentration.

Table 5 Detection conditions

Note: mAb 02 is obtained by fusing the #2 cloned scFv sequence with human IgG4Fc, similar to mAb 05, mAb 118, and mAb 119.

3) Wash 3 times with 10mM Glycine-HCl, pH1.5 to regenerate the chip.

4) Binding constants were analyzed by using a 1:1 binding site model (BLI analysis software V11.0).

Experimental results:

Affinity refers to the strength of the binding of a single molecule to its ligand and is usually measured and reported by the equilibrium dissociation constant (KD), which can be used to assess and rank the strength of the interaction between two molecules. The binding of an antibody to its antigen is a reversible process, and the rate of the binding reaction is proportional to the concentration of the reactant. The smaller the KD value, the greater the affinity of the antibody for its target. As shown in Table 6: #2, #5, #118 and #119 clones can all bind to the MSLN antigen, and #119 clone has a slightly higher affinity than other clones.

Table 6 affinity determination results

LigandLigand	KD(M)KD(M)	kon(1/Ms)kon(1/Ms)	kdis(1/s)kdis(1/s)	R^2R^2
huYP218huYP218	2.36E-092.36E-09	2.97E+052.97E+05	7.00E-047.00E-04	0.99830.9983
mAb 02mAb 02	3.19E-073.19E-07	2.69E+052.69E+05	8.90E-048.90E-04	0.99410.9941
mAb 05 mAb 05	2.51E-072.51E-07	4.52E+054.52E+05	1.13E-011.13E-01	0.99710.9971
mAb 118 mAb 118	1.15E-061.15E-06	2.83E+052.83E+05	3.27E-013.27E-01	0.99330.9933
mAb 119 mAb 119	4.32E-094.32E-09	2.26E+052.26E+05	9.76E-049.76E-04	0.99910.9991

references

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Claims

An immune cell expressing any one or more of the following components:

(a) a first class chimeric antigen receptor (CAR);

(b) a second class of chimeric antigen receptors (CAR);

(c) loss-of-function immunosuppressive receptors; and

(d) A truncated form of the EGFR molecule (tEGFR).
The immune cell according to claim 1, wherein the first type of CAR is a fully human CAR.
The immune cell according to claim 1 or 2, wherein the first type of CAR is selected from CD19, CD20, CD22, CD5, CD7 and BCMA CAR.
The immune cell according to any one of claims 1-3, wherein the second type of CAR is different from the first type of CAR, and is selected from any one or more of the following targets MSLN, HER2, GPC3, EGFRVIII, Claudin18. 2. CARs of CD70, GD2, CEA, CS1, DLL3, EGFR, ErbB1, FAP, Folate Receptor, GPC1, gp100, MUC16, MUC1, NKG2D, PSCA, PSMA, ROR1 or VEGFR2.
The immune cell according to any one of claims 1-4, wherein the functionally deficient immunosuppressive receptors comprise dnTGFβRII, dnPD1.
The immune cell according to any one of claims 1-5, wherein the first type of CAR comprises a first binding domain, a first transmembrane domain, a first co-stimulatory domain, and a first intracellular signaling structure domain, the first binding domain comprises one or more first antibodies or fragments thereof that specifically bind to CD19, wherein the first antibodies or fragments thereof comprise a first heavy chain complementarity determining region 1 (HCDR1), a first Heavy chain complementarity determining region 2 (HCDR2) and first heavy chain complementarity determining region 3 (HCDR3), the amino acid sequences of HCDR1, HCDR2, and HCDR3 are respectively as SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO :49.
The immune cell according to claims 1-6, wherein the first antibody or fragment thereof further comprises a first light chain complementarity determining region 1 (LCDR1), a first light chain complementarity determining region 2 (LCDR2) and a first light chain complementarity determining region Chain complementarity determining region 3 (LCDR3), the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, respectively.
The immune cell according to any one of claims 1-7, wherein the second type of CAR comprises a second binding domain, a second transmembrane domain, a second co-stimulatory domain, and a second intracellular signaling structure domain, the second binding domain comprises one or more second antibodies or fragments thereof that specifically bind MSLN (mesothelin), wherein the antibodies or fragments thereof comprise a second heavy chain complementarity determining region 1 (HCDR1) , the second heavy chain complementarity determining region 2 (HCDR2) and the second heavy chain complementarity determining region 3 (HCDR3), the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are independently selected from the following combinations:

(1) HCDR1 such as the amino acid sequence of SEQ ID NO: 37, HCDR2 such as the amino acid sequence of SEQ ID NO: 38, and HCDR3 such as the amino acid sequence of SEQ ID NO: 39;

(2) HCDR1 such as the amino acid sequence of SEQ ID NO: 58, HCDR2 such as the amino acid sequence of SEQ ID NO: 59, and HCDR3 such as the amino acid sequence of SEQ ID NO: 60;

(3) HCDR1 such as the amino acid sequence of SEQ ID NO: 69, HCDR2 such as the amino acid sequence of SEQ ID NO: 70, and HCDR3 such as the amino acid sequence of SEQ ID NO: 71;

(4) HCDR1 such as the amino acid sequence of SEQ ID NO: 80, HCDR2 such as the amino acid sequence of SEQ ID NO: 81, and HCDR3 such as the amino acid sequence of SEQ ID NO: 82; and

(5) HCDR1 such as the amino acid sequence of SEQ ID NO: 91, HCDR2 such as the amino acid sequence of SEQ ID NO: 92, and HCDR3 such as the amino acid sequence of SEQ ID NO: 93.
The immune cell according to any one of claims 1-8, wherein the second antibody or fragment thereof specifically binding to MSLN (mesothelin) further comprises a second light chain complementarity determining region 1 (LCDR1), a second Light chain complementarity determining region 2 (LCDR2) and second light chain complementarity determining region 3 (LCDR3), the amino acid sequences of LCDR1, LCDR2 and LCDR3 are independently selected from the following combinations:

(1) LCDR1 such as the amino acid sequence of SEQ ID NO: 40, LCDR2 such as the amino acid sequence of SEQ ID NO: 41, and LCDR3 such as the amino acid sequence of SEQ ID NO: 42;

(2) LCDR1 such as the amino acid sequence of SEQ ID NO: 61, LCDR2 such as the amino acid sequence of SEQ ID NO: 62, and LCDR3 such as the amino acid sequence of SEQ ID NO: 63;

(3) LCDR1 such as the amino acid sequence of SEQ ID NO: 72, LCDR2 such as the amino acid sequence of SEQ ID NO: 73, and LCDR3 such as the amino acid sequence of SEQ ID NO: 74;

(4) LCDR1 such as the amino acid sequence of SEQ ID NO: 83, LCDR2 such as the amino acid sequence of SEQ ID NO: 84, and LCDR3 such as the amino acid sequence of SEQ ID NO: 85; and

(5) LCDR1 such as the amino acid sequence of SEQ ID NO: 94, LCDR2 such as the amino acid sequence of SEQ ID NO: 95, and LCDR3 such as the amino acid sequence of SEQ ID NO: 96.
The immune cell according to any one of claims 1-9, wherein the first antigen-binding domain comprises a first heavy chain variable region (VH), which comprises the amino acid sequence shown in SEQ ID NO:44.
The immune cell according to any one of claims 1-10, wherein the second antigen binding domain comprises a second heavy chain variable region (VH) comprising SEQ ID NO: 34, 54, 65, 76 and the amino acid sequence shown in 87.
The immune cell according to any one of claims 1-11, wherein the first antigen-binding domain comprises a first light chain variable region (VL), which comprises the amino acid sequence shown in SEQ ID NO:46.
The immune cell according to any one of claims 1-12, wherein the second antigen binding domain comprises a second light chain variable region (VL) comprising SEQ ID NO: 36, 56, 67, 78 and the amino acid sequence shown in 89.
The immune cell according to any one of claims 1-13, wherein the first light chain variable region and the first heavy chain variable region are connected by a first linker, and/or the second light chain variable region region and the second heavy chain variable region are connected by a second linker.
The immune cell according to any one of claims 1-14, wherein the sequence of the first and/or second linker comprises the amino acid sequence shown in SEQ ID NO:9.
The immune cell according to any one of claims 1-15, wherein the combination of the first and/or second antibodies or fragments thereof is a single chain antibody or a single domain antibody.
The immune cell according to any one of claims 1-16, wherein the CD19 antigen-binding domain comprises scFv, and the amino acid sequence contained in the scFv is SEQ ID No:2.
The immune cell according to any one of claims 1-17, wherein the MSLN binding domain comprises scFv, the scFv comprising an amino acid sequence selected from any of the following: SEQ ID No: 5, 57, 68, 101 , 79 and 90.
The immune cell according to any one of claims 1-18, wherein the dnTGFβRII receptor comprises the amino acid sequence shown in SEQ ID No: 28 or a functional variant thereof.
The immune cell according to any one of claims 1-19, wherein the truncated EGFR molecule comprises the amino acid sequence shown in SEQ ID No: 27 or a functional variant thereof.
The immune cell according to any one of claims 1-20, wherein the first type of CAR and the immunosuppressive receptor are co-expressed in the immune cell through the action of 2A peptide, and the second type of CAR is co-expressed with the immune cell The tEGFR is co-expressed in the immune cells by the action of 2A peptide.
According to the immune cell according to any one of claims 1-21, the 2A peptide is selected from P2A, T2A or F2A, and the P2A comprises the amino acid sequence shown in SEQ ID NO: 29 or a functional variant thereof, the T2A comprises the amino acid sequence shown in SEQ ID NO: 30 or a functional variant thereof.
The immune cell according to any one of claims 1-22, wherein the first and/or second transmembrane domains comprise polypeptides selected from the following proteins: α, β or ζ chains of T cell receptors, CD28, CD3e, CD45, CD4, CD5, CD8a, CD9, CD16, CD22, MSLN, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.
The immune cell according to any one of claims 1-23, wherein the first and/or second transmembrane domains comprise the amino acid sequence shown in SEQ ID No: 13 or a functional variant thereof.
The immune cell according to any one of claims 1-24, wherein the first and/or second co-stimulatory domain comprises a polypeptide selected from the group consisting of CD28, 4-1BB, OX-40 and ICOS.
The immune cell according to any one of claims 1-25, wherein the first and/or second co-stimulatory domain comprises the amino acid sequence shown in SEQ ID No: 15 or a functional variant thereof.
The immune cell according to any one of claims 1-26, wherein the first and/or second intracellular signaling domain comprises a signaling domain from CD3z.
The immune cell according to any one of claims 1-27, wherein the first and/or second intracellular signaling domain comprises the amino acid sequence shown in SEQ ID No: 17 or a functional variant thereof.
The immune cell according to any one of claims 1-28, wherein the first and/or second CAR further comprises a hinge region connecting the first antigen-binding domain and the first span a membrane domain or said second antigen binding domain and said second transmembrane domain.
The immune cell according to any one of claims 1-29, wherein the hinge region comprises the amino acid sequence shown in SEQ ID No: 11 or a functional variant thereof.
The immune cell according to any one of claims 1-30, wherein the first and/or second CAR is further linked to a signal peptide.
The immune cell according to any one of claims 1-31, wherein the signal peptide comprises the amino acid sequence shown in SEQ ID No: 8 or a functional variant thereof.
The immune cell according to any one of claims 1-32, wherein the first type of CAR comprises the amino acid sequence shown in SEQ ID No: 19 or a functional variant thereof, and/or the second type of CAR Comprising the amino acid sequences shown in SEQ ID Nos: 32, 97, 98, 99 and 100 or functional variants thereof.
An isolated nucleic acid molecule encoding any component or combination thereof expressed on the immune cell of any one of claims 1-33.
The nucleic acid molecule of separation according to claim 34, which comprises SEQ ID No: 1, 3, 4, 6, 7, 10, 12, 14, 16, 18, 20, 21, 23, 24, 26, 31, Nucleic acid sequences shown in 33, 35, 43, 45, 53, 55, 64, 66, 75, 77, 86 and 88 or functional variants thereof.
A vector comprising the nucleic acid molecule of claim 34 or 35.
The vector according to claim 36, wherein said vector is selected from the group consisting of plasmids, retroviral vectors and lentiviral vectors.
The immune cell according to claims 1-33, wherein said immune cell is selected from T lymphocytes and natural killer (NK) cells.
A method for preparing immune effector cells, comprising introducing the carrier of claim 36 into immune cells.
A pharmaceutical composition comprising the immune cell according to any one of claims 1-33.
The use of the immune cell according to any one of claims 1-33, or the nucleic acid molecule according to claim 34 or 35, or the carrier according to claim 36 or 37 in the preparation of a medicine, wherein the medicine is used For the treatment of diseases or diseases associated with the expression of the second type of CAR targeting target antigen.
The use according to claim 41, wherein the disease or disease related to the expression of the second type of CAR targeting target antigen is cancer or malignant tumor.
The use according to claim 42, the tumor is a solid tumor.
A CAR targeting MSLN (mesothelin), comprising an antigen binding domain, a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain, the antigen binding domain comprising one or more specific binding Combinations of antibodies to MSLN (mesothelin) or fragments thereof, wherein each of said antibodies comprises heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2) and heavy chain complementarity determining region 3 (HCDR3) , the amino acid sequences of HCDR1, HCDR2 and HCDR3 are independently selected from the following combinations:

(1) HCDR1 such as the amino acid sequence of SEQ ID NO: 58, HCDR2 such as the amino acid sequence of SEQ ID NO: 59, and HCDR3 such as the amino acid sequence of SEQ ID NO: 60;

(2) HCDR1 such as the amino acid sequence of SEQ ID NO: 69, HCDR2 such as the amino acid sequence of SEQ ID NO: 70, and HCDR3 such as the amino acid sequence of SEQ ID NO: 71;

(3) HCDR1 such as the amino acid sequence of SEQ ID NO: 80, HCDR2 such as the amino acid sequence of SEQ ID NO: 81, and HCDR3 such as the amino acid sequence of SEQ ID NO: 82; and

(4) HCDR1 such as the amino acid sequence of SEQ ID NO: 91, HCDR2 such as the amino acid sequence of SEQ ID NO: 92, and HCDR3 such as the amino acid sequence of SEQ ID NO: 93.
The CAR according to claim 44, wherein the combination of the antibody or fragment thereof further comprises light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2) and light chain complementarity determining region 3 (LCDR3), The amino acid sequences of LCDR1, LCDR2, and LCDR3 are independently selected from the following combinations:

(1) LCDR1 such as the amino acid sequence of SEQ ID NO: 61, LCDR2 such as the amino acid sequence of SEQ ID NO: 62, and LCDR3 such as the amino acid sequence of SEQ ID NO: 63;

(2) LCDR1 such as the amino acid sequence of SEQ ID NO: 72, LCDR2 such as the amino acid sequence of SEQ ID NO: 73, and LCDR3 such as the amino acid sequence of SEQ ID NO: 74;

(3) LCDR1 such as the amino acid sequence of SEQ ID NO: 83, LCDR2 such as the amino acid sequence of SEQ ID NO: 84, and LCDR3 such as the amino acid sequence of SEQ ID NO: 85; and

(4) LCDR1 such as the amino acid sequence of SEQ ID NO: 94, LCDR2 such as the amino acid sequence of SEQ ID NO: 95, and LCDR3 such as the amino acid sequence of SEQ ID NO: 96.
The CAR according to any one of claims 44-45, wherein the combination of the antibody or its fragments comprises a heavy chain variable region, the amino acid sequence of the heavy chain variable region is as SEQ ID NO: 34, 54, 65 , 76 and 87.
According to the CAR according to any one of claims 44-46, wherein the combination of the antibody or its fragments further comprises a light chain variable region, the amino acid sequence of the light chain variable region is as SEQ ID NO: 36, 56, 67, 78 and 89.
According to the CAR according to any one of claims 44-47, the light chain variable region and the heavy chain variable region contained in the combination of the antibodies or fragments thereof are connected by a linker.
According to the CAR according to any one of claims 44-48, the amino acid sequence contained in the sequence of the linker is shown in SEQ ID NO: 9.
The CAR according to any one of claims 44-49, wherein the combination of antibodies or fragments thereof is a single chain antibody or a single domain antibody.
The CAR according to any one of claims 44-50, wherein the amino acid sequence comprised by the MSLN binding domain (scFv) is selected from any of the following: SEQ ID No: 5, 57, 68, 101, 79 and 90.
The CAR according to any one of claims 44-51, wherein the CAR is also connected to a truncated EGFR molecule (tEGFR) through a self-cleaving peptide.
The CAR of claim 52, wherein the self-cleaving peptide comprises P2A, T2A or F2A.
The CAR according to claim 52 or 53, wherein the truncated EGFR molecule comprises the amino acid sequence shown in SEQ ID No: 27 or a functional variant thereof; the P2A comprises the amino acid sequence shown in SEQ ID NO: 29 An amino acid sequence or a functional variant thereof; the T2A comprises the amino acid sequence shown in SEQ ID NO: 30 or a functional variant thereof.
The CAR according to any one of claims 44-54, wherein the transmembrane domain comprises a polypeptide selected from the following proteins: α, β or ζ chain of T cell receptor, CD28, CD3e, CD45, CD4, CD5, CD8a, CD9, CD16, CD22, MSLN, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.
The CAR according to any one of claims 44-55, wherein the transmembrane domain comprises the amino acid sequence shown in SEQ ID No: 13 or a functional variant thereof.
The CAR according to any one of claims 44-56, wherein the co-stimulatory domain comprises a polypeptide selected from the following proteins: CD28, 4-1BB, OX-40 and ICOS.
The CAR according to any one of claims 44-57, wherein the co-stimulatory domain comprises the amino acid sequence shown in SEQ ID No: 15 or a functional variant thereof.
The CAR according to any one of claims 44-58, wherein the intracellular signaling domain comprises a signaling domain from CD3z.
The CAR according to any one of claims 44-59, wherein the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID No: 17 or a functional variant thereof.
The CAR according to any one of claims 44-60, wherein the CAR further comprises a hinge region connecting the antigen-binding domain and the transmembrane domain.
The CAR according to any one of claims 44-61, wherein the hinge region comprises the amino acid sequence shown in SEQ ID No: 11 or a functional variant thereof.
The CAR according to any one of claims 44-62, wherein the CAR is further linked to a signal peptide.
The CAR according to any one of claims 44-63, wherein the signal peptide comprises the amino acid sequence shown in SEQ ID No: 8 or a functional variant thereof.
The CAR according to any one of claims 44-64, comprising the amino acid sequence shown in SEQ ID No: 32, 97, 98, 99 and 100 or a functional variant thereof.
An isolated nucleic acid molecule encoding the CAR of any one of claims 44-65.
The isolated nucleic acid molecule of claim 66 comprising SEQ ID No: 3, 4, 7, 10, 12, 14, 16, 18, 20, 21, 23, 33, 35, 53, 55, 64, Nucleic acid sequences shown in 66, 75, 77, 86 and 88 or functional variants thereof.
A vector comprising the nucleic acid molecule of claim 66 or 67.
The vector according to claim 68, wherein said vector is selected from the group consisting of plasmids, retroviral vectors and lentiviral vectors.
An immune cell comprising the CAR according to claims 44-65, the nucleic acid molecule according to claim 66 or 67, or the carrier according to claim 68 or 69.
The immune cell according to claim 70, wherein said immune cell is selected from T lymphocytes and natural killer (NK) cells.
A method for preparing immune cells, comprising introducing the carrier according to claim 68 or 69 into the immune cells.
A pharmaceutical composition comprising the immune cell according to claim 70 or 71, and a pharmaceutically acceptable adjuvant.
The use of the CAR described in claims 44-65, the nucleic acid molecule described in claim 66 or 67, the carrier described in claim 68 or 69, or the immune cell described in claim 70 or 71 in the preparation of medicines , wherein the medicament is for the treatment of a disease or condition associated with MSLN expression.
The use according to claim 74, wherein the disease or condition associated with MSLN expression is cancer or malignant tumor.
The use according to claim 75, the tumor is a solid tumor.