CN117441020A

CN117441020A - Preparation and application of enhanced Chimeric Antigen Receptor (CAR) cells

Info

Publication number: CN117441020A
Application number: CN202280040417.3A
Authority: CN
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: Nanjing Reindeer Biotechnology Co ltd
Current assignee: Nanjing Reindeer Biotechnology Co ltd
Priority date: 2021-06-08
Filing date: 2022-06-08
Publication date: 2024-01-23
Also published as: WO2022257984A1

Abstract

The immune cells can express various components including immune cells, wherein the immune cells comprise two chimeric antigen receptors, the first chimeric antigen receptor can enhance proliferation or activity of the immune cells, and the immune cells can simultaneously express competitive receptors with a missing function, such as dnTGF beta RII, so that the immune suppression microenvironment problem of solid tumor tissues can be overcome, and the capability of killing the solid tumor is improved. Meanwhile, the immune cells also express tEGFR molecules, and the CAR-T cells in the body can be effectively cleared by injecting antibodies of EGFR, so that the safety risk brought by the CAR-T cells is further ensured.

Description

Preparation and application of enhanced Chimeric Antigen Receptor (CAR) cells

Technical Field

The application belongs to the technical field of cellular immunotherapy, and relates to a preparation method of an enhanced chimeric antigen receptor (Chimeric Antigen Receptor, CAR) cell, which comprises the simultaneous expression of two types of CARs and an immunosuppression receptor.

Background

Chimeric antigen receptor (Chimeric antigen receptor, CAR) T cell (CAR-T) immunotherapy is the use of the cell killing capacity of T cells to kill tumor cells by specific recognition of tumor cell surface antigens by single chain antibody sequences on the synthesized CAR molecules. The therapy shows remarkable curative effects in B cell malignant tumors including B cell acute lymphoblastic leukemia (B-ALL), and B cell non-Hodgkin lymphoma (B-NHL) [1]. Two types of CAR-T cell therapeutics, kymeria from nova and yescanta from Kite, were approved by the FDA at 8 and 10 2017, respectively, and 4 CAR-T cell therapies have been approved by the FDA to date.

With the FDA approval of the cell therapy products kymrih and yescata of nova and Kite on the market by virtue of the second-phase clinical data, the period of drug development in the traditional sense is greatly shortened, and the interest of a large number of companies and researchers is attracted by virtue of the unprecedented efficacy, the number of clinical trials of CAR-T cell therapy is also rapidly increasing, with the greatest number of clinical trials in the united states and china.

In addition to the marketed CAR-T cell therapy products against B cell target CD19, CAR-T cell targets represented by CD20, CD22, BCMA have also achieved significant efficacy against hematological tumors [2-4], in addition to B cell tumors, studies against T cell tumors, myeloid leukemia are underway [5-6], these results all demonstrate that multiple tumors can be treated using CAR-T cell technology platform.

Of all tumor types, hematological tumors account for only 10%, with the other 90% being solid tumors. There are also many reports on the study of CAR-T cells in solid tumor treatment, and CAR-T cells face more challenges in solid tumor treatment due to the difference between solid tumors and hematological tumors, and the clinical results reported at present show that the therapeutic effect of CAR-T cells in solid tumors is poorer than that of hematological tumors.

Compared with the tumor of the blood system, the basic characteristics of the solid tumor are different [7], the solid tumor has a compact tissue structure, and a matrix layer formed by fibroblasts and collagen is arranged around the tumor, so that the contact difficulty of CAR-T cells entering the tumor tissue and the tumor cells is increased; the tumor tissue consists of a plurality of cells, and besides the tumor cells, the tumor tissue also contains regulatory T cell Treg, myeloid suppressor cell MDSC, tumor-related giant macrophage thin TAM, immature dendritic cell iDC, stromal fibroblast and immunosuppressive cytokine TGFbeta, and the factors form a tumor immunosuppression microenvironment together, so that tumor infiltrating T cells lose the capability of killing tumors; the tumor cells of solid tumors are not identical tumor cells, and there is heterogeneity that CAR-T cell therapy against a single target may not be effective in killing all tumor cells; in addition, it is difficult to find tumor-specific antigens (TSAs) as targets of CAR-T cells, and currently, tumor-associated antigens (TAAs) are mostly adopted as targets of cell therapy, and these TAAs are highly expressed on the surface of tumor cells, and simultaneously, are lowly expressed in other healthy tissues, which may cause toxic and side effects of On target off tumor, and all of these bring challenges to CAR-T cell therapy of solid tumors.

Different strategies have also been proposed by researchers against the characteristics of solid tumors to overcome the obstacles CAR-T cells face in solid tumor treatment [8].

Firstly, a target point with high tumor specificity expression is searched for aiming at the selection of the target point, so that the safety risk is reduced, and meanwhile, the design of a double-target logic method can be adopted, and only aiming at tumor cells, the T cells can be activated to play a killing function. For the heterogeneity characteristics of tumor cells, in addition to targeting a single tumor target, a dual-target or multi-target CAR design can be employed.

Secondly, aiming at tumor immunosuppression microenvironment, an immune checkpoint molecule (such as PD 1) can be knocked out in the CAR-T cell by using a gene editing method, and the CAR-T cell can also secrete antibody molecules (such as PD1, PDL1, CTLA4, CD47 and SIRPa) combined with the immune checkpoint, so that activation inhibition effect of the tumor microenvironment on immune cells is blocked; meanwhile, the cell factor (such as IL 12) secreted by the CAR-T cells can enhance the killing effect of the CAR-T cells.

In addition, the method of expressing the related chemokine receptor on the surface of the CAR-T cell to enhance the chemotaxis and infiltration capacity of the CAR-T cell to the solid tumor and the like can enhance the effect of the CAR-T cell on treating the solid tumor.

There are many reports of CAR-T cell therapies for solid tumors at present [9], and preclinical test data for different solid tumors have validated a number of different targets and show significant efficacy. Since preclinical test data are obtained based on tumor cell lines and mouse-based animal models of transplanted tumors, neither in vitro studies based on tumor cell lines nor in mice-based animal models of transplanted tumors truly reflect the actual tumor condition in patients, and these preclinical data need to be further validated in clinical trials.

There are 670 CAR-T cell therapy programs registered in clinical trimals. Org to date, of which 167 are CAR-T cell therapy programs directed to solid tumors. Clinical data of published solid tumor CAR-T cell treatment show that 6 patients treated with ovarian cancer by taking MSLN as a target obtain complete disease stabilization [10], 2 patients treated with pancreatic cancer obtain disease stabilization [11], and one patient treated with mesothelioma obtains partial relief [12]; treatment of bile duct and pancreatic cancer with HER2 target yielded 9% partial remission, 45% disease stability [13], treatment of sarcoma and glioblastoma with 24% disease stability and 7% partial remission [14], respectively; treatment of neuroblastoma with GD2 as target yielded 27% complete remission [15]; treatment of prostate cancer with PSMA as a target achieved 40% partial remission [16].

These early clinical data show potential therapeutic effects of CAR-T in solid tumor treatment, as fewer of these clinical cases, more clinical trials are underway, and it is believed that future CAR-T cell treatment of solid tumors must achieve breakthrough progress as the clinical trial data is updated, as well as improvements in CAR-T cell design for solid tumor treatment.

The reported clinical data of the CAR-T cell treatment aiming at the solid tumor show preliminary curative effect, but the similar effect of treating the blood tumor cannot be achieved, and the reported prior art adopts the traditional second-generation CAR-T technology or adopts the enhanced CAR-T technology to overcome the tumor immunosuppression microenvironment or promote the infiltration capacity of the CAR-T cell to the solid tumor tissue, but the problem of the amplification of the CAR-T cell in the solid tumor patient and the problem of the solid tumor immunosuppression microenvironment cannot be solved at the same time.

Disclosure of Invention

In one aspect, the present application provides an immune cell that can express two classes of chimeric antigen receptors, wherein the immune cell comprises a T cell that expresses any one or more of the following components:

(a) A first class of Chimeric Antigen Receptors (CARs);

(b) A second class of Chimeric Antigen Receptors (CARs);

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

(d) Truncated forms of EGFR molecules (tgfr).

In certain embodiments, expression of the first class of chimeric antigen receptors can enhance a function of an immune cell, including, for example: CD19 CARs can increase the expansion capacity of chimeric antigen receptor cells, and CD5 CARs can clear immune cells such as host T cells, treg cells, and the like. Chimeric antigen receptors such as BCMA, CD20, CD22, CD5, CD7 and the like also possess similar or broader functions to enhance CAR-T cells.

In certain embodiments, the second class of chimeric antigen receptors targets a target selected from the group consisting of: MSLN, HER2, GPC3, egfrvlll, claudin18.2, CD70, GD2, CEA, CS1, DLL3, EGFR, erbB1, FAP, fonate Receptor, GPC1, gp100, MUC16, MUC1, NKG2D, PSCA, PSMA, ROR1, and VEGFR2.

On the other hand, in order to overcome the influence of the solid tumor immunosuppressive microenvironment on the function of the CAR-T cells, the application also enables the CAR-T cells to express a signal molecule related to the tumor immunosuppressive microenvironment, wherein the signal molecule is a nonfunctional immunosuppressive molecule receptor and can competitively bind with an immunosuppressive molecule highly expressed in tumors. After the immunosuppressive molecule is combined with an immunosuppressive molecule receptor expressed on the CAR-T, the immunosuppressive molecule can inhibit the killing function of immune cells on tumors, so that tumor proliferation is caused, and the therapeutic effect of the CAR-T is reduced. The modified immunosuppression molecule receptor with the lost function can competitively bind with immunosuppression molecules in tumors, but cannot cause immunosuppression effect after binding, so that the immunosuppression microenvironment of the solid tumor suffered by the CAR-T cells is relieved or eliminated, and the capability of killing the solid tumor is improved.

In certain embodiments, the engineered loss-of-function immunosuppressive molecule receptor comprises the competitive receptor dntgfbetarii for tgfbeta.

On the other hand, the immune cells express tEGFR molecules at the same time, and the CAR-T cells in the body can be effectively cleared by injecting the EGFR antibody, so that the safety risk brought by the CAR-T cells is further ensured.

In some embodiments, because some first-class chimeric antigen receptor T cells may cause a loss of healthy B cells in a patient, which may cause a humoral immune deficiency in the patient, in order to control the persistence of CAR-T cells in the body, the CAR-T of the present application expresses tgfr molecules simultaneously, and may be used as a safety switch for CAR-T cells, and by injecting antibodies to EGFR, CAR-T cells in the body may be effectively cleared, thereby guaranteeing the safety risk brought by CAR-T cells.

In certain embodiments, the first class of Chimeric Antigen Receptor (CAR) is a fully human CAR.

In certain embodiments, the first class of Chimeric Antigen Receptor (CAR) is a fully human CD19, CD20, CD22, CD5, CD7, and BCMA CAR.

In certain embodiments, the first class of Chimeric Antigen Receptor (CAR) comprises a first binding domain comprising one or more antibodies or a combination of fragments thereof that specifically bind CD19, wherein each of the antibodies or combination of fragments thereof comprises a heavy chain complementarity determining region 1 (HCDR 1), a heavy chain complementarity determining region 2 (HCDR 2), and a heavy chain complementarity determining region 3 (HCDR 3), the amino acid sequences of HCDR1, HCDR2, and HCDR3 being as set forth in SEQ ID NOs: 47. SEQ ID NO:48 and SEQ ID NO: shown at 49. .

In certain embodiments, the combination of antibodies or fragments thereof further comprises light chain complementarity determining region 1 (LCDR 1), light chain complementarity determining region 2 (LCDR 2), and light chain complementarity determining region 3 (LCDR 3), the amino acid sequences of LCDR1, LCDR2, and LCDR3 being as set forth in SEQ ID NOs: 50. SEQ ID NO:51 and SEQ ID NO: 52.

In certain embodiments, the second class of Chimeric Antigen Receptor (CAR) comprises a second binding domain, a second transmembrane domain, a second costimulatory domain, and an intracellular signaling domain.

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

(1) As set forth in SEQ ID NO:37, and HCDR1 of the amino acid sequence of SEQ ID NO:38, and HCDR2 of the amino acid sequence of SEQ ID NO:39, HCDR3 of the amino acid sequence;

(2) As set forth in SEQ ID NO:58, and HCDR1 of the amino acid sequence of SEQ ID NO:59, and HCDR2 of the amino acid sequence of SEQ ID NO:60, HCDR3 of the amino acid sequence of seq id no;

(3) As set forth in SEQ ID NO:69, and HCDR1 of the amino acid sequence of SEQ ID NO:70, and HCDR2 of the amino acid sequence of SEQ ID NO:71, HCDR3 of the amino acid sequence;

(4) As set forth in SEQ ID NO:80, and HCDR1 of the amino acid sequence of SEQ ID NO:81, and HCDR2 of the amino acid sequence of SEQ ID NO:82, HCDR3 of the amino acid sequence; and

(5) As set forth in SEQ ID NO:91, HCDR1 of the amino acid sequence of SEQ ID NO:92, and HCDR2 of the amino acid sequence of SEQ ID NO:93, and HCDR3 of the amino acid sequence of seq id no.

In certain embodiments, the combination of antibodies or fragments thereof further comprises light chain complementarity determining region 1 (LCDR 1), light chain complementarity determining region 2 (LCDR 2), and light chain complementarity determining region 3 (LCDR 3), the amino acid sequences of LCDR1, LCDR2, and LCDR3 being independently selected from the group consisting of:

(1) As set forth in SEQ ID NO:40, as set forth in SEQ ID NO:41, and LCDR2 as set forth in SEQ ID NO:42, LCDR3 of the amino acid sequence;

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

(3) As set forth in SEQ ID NO:72, as set forth in SEQ ID NO:73, and LCDR2 as set forth in SEQ ID NO:74, LCDR3 of the amino acid sequence; and

(4) As set forth in SEQ ID NO:83, as set forth in SEQ ID NO:84, and LCDR2 as set forth in SEQ ID NO:85, LCDR3 of the amino acid sequence;

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

In another aspect, the present application provides a Chimeric Antigen Receptor (CAR) targeting MSLN (mesothelin) comprising an antigen binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

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

(1) As set forth in SEQ ID NO:58, and HCDR1 of the amino acid sequence of SEQ ID NO:59, and HCDR2 of the amino acid sequence of SEQ ID NO:60, HCDR3 of the amino acid sequence of seq id no;

(2) As set forth in SEQ ID NO:69, and HCDR1 of the amino acid sequence of SEQ ID NO:70, and HCDR2 of the amino acid sequence of SEQ ID NO:71, HCDR3 of the amino acid sequence;

(3) As set forth in SEQ ID NO:80, and HCDR1 of the amino acid sequence of SEQ ID NO:81, and HCDR2 of the amino acid sequence of SEQ ID NO:82, HCDR3 of the amino acid sequence; and

(4) As set forth in SEQ ID NO:91, HCDR1 of the amino acid sequence of SEQ ID NO:92, and HCDR2 of the amino acid sequence of SEQ ID NO:93, and HCDR3 of the amino acid sequence of seq id no.

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

(2) As set forth in SEQ ID NO:72, as set forth in SEQ ID NO:73, and LCDR2 as set forth in SEQ ID NO:74, LCDR3 of the amino acid sequence;

(3) As set forth in SEQ ID NO:83, as set forth in SEQ ID NO:84, and LCDR2 as set forth in SEQ ID NO:85, LCDR3 of the amino acid sequence; and

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

In certain embodiments, the antigen binding domain comprises a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 34. 44, 54, 65, 76 and 87.

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

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

In certain embodiments, the linker sequence comprises an amino acid sequence of SEQ ID NO: shown at 9.

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

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

In certain embodiments, the dntgfbetarii receptor comprises SEQ ID No:28 or a functional variant thereof.

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

In certain embodiments, the truncated form of the EGFR molecule comprises SEQ ID No:27 or a functional variant thereof.

In certain embodiments, the first class of chimeric antigen receptor is linked to each other via a 2A peptide and the signal molecule, and the second class of chimeric antigen receptor is linked to tgfr via a 2A peptide.

In certain embodiments, the 2A peptide comprises P2A, T a, the P2A comprising the amino acid sequence of SEQ ID NO:29 or a functional variant thereof, said T2A comprising the amino acid sequence set forth in SEQ ID NO:30 or a functional variant thereof.

In certain embodiments, the transmembrane domain comprises a polypeptide selected from the group consisting of: the 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.

In certain embodiments, the transmembrane domain comprises SEQ ID No:13 or a functional variant thereof.

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

In certain embodiments, the co-stimulatory domain comprises SEQ ID No:15 or a functional variant thereof.

In certain embodiments, the intracellular signaling domain comprises a signaling domain from CD3 z.

In certain embodiments, the intracellular signaling domain comprises SEQ ID No:17 or a functional variant thereof.

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

In certain embodiments, the hinge region comprises SEQ ID No:11 or a functional variant thereof.

In certain embodiments, the CAR is further linked to a signal peptide.

In certain embodiments, the signal peptide comprises SEQ ID No:8 or a functional variant thereof.

In certain embodiments, the CAR comprises SEQ ID No: 19. 32, 97, 98, 99 and 100 or a functional variant 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, 45, 53, 55, 64, 66, 75, 77, 86 and 88, or a functional variant thereof.

In another aspect, the present 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, or a functional variant thereof.

In certain embodiments, the vector is selected from the group consisting of a plasmid, a retroviral vector, and a lentiviral vector.

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

In certain embodiments, the immune effector cell is selected from the group consisting of a T lymphocyte and a Natural Killer (NK) cell.

In another aspect, the present application provides a method of preparing an immune effector cell comprising introducing the vector into an immune effector cell.

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

In another aspect, the application provides the use of the CAR, nucleic acid molecule, vector, immune effector cell in the manufacture of a medicament for treating a disease or disorder associated with the expression of a second class of CAR-targeted targets.

In certain embodiments, the use is that the disease or disorder associated with the expression of the second class of CAR-targeted targets is cancer or malignancy.

In another aspect, the present application provides a Chimeric Antigen Receptor (CAR) that targets MSLN.

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

In certain embodiments, the combined antigen binding domain of the antibody or fragment thereof comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 34. 44, 54, 65, 76 and 87.

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

In certain embodiments, the CD19 binding domain (ScFv) comprises the amino acid sequence set forth in SEQ ID No:2, and a polypeptide sequence represented by the following formula (2); wherein the MSLN binding domain (scFv) comprises an amino acid sequence selected from any one of the following: SEQ ID No: 5. 57, 68, 101, 79 and 90.

In certain embodiments, the CAR is further linked to a truncated form of EGFR molecule (tgfr) by a self-cleaving peptide.

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

In certain embodiments, the truncated form of the EGFR molecule comprises SEQ ID No:27 or a functional variant thereof; the P2A comprises SEQ ID NO:29 or a functional variant thereof; the T2A comprises 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: the 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.

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

In certain embodiments, the CAR is further linked to a signal peptide.

In certain embodiments, the CAR comprises SEQ ID No: 32. 97, 98, 99 and 100 or a functional variant thereof.

In another aspect, the present application also provides an isolated nucleic acid molecule encoding the CAR of any one of claims 40-61.

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

In another aspect, the present application also provides a vector comprising the nucleic acid molecule described above.

In certain embodiments, wherein the vector is selected from the group consisting of a plasmid, a retroviral vector, and a lentiviral vector.

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

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

In another aspect, the present application also provides a method of preparing an immune cell comprising introducing into an immune cell the vector described above.

In another aspect, the present application also provides a pharmaceutical composition comprising the immune cell described above, and a pharmaceutically acceptable adjuvant.

In another aspect, the present application also provides the use of the CAR, the nucleic acid molecule, the vector, or the immune cell in the manufacture of a medicament for treating a disease or disorder associated with MSLN expression.

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

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

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

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

the amino acid sequence of LCDR2 is SEQ ID NO: indicated at 62;

the amino acid sequence of LCDR3 is SEQ ID NO: indicated at 63;

the amino acid sequence of HCDR1 is SEQ ID NO: indicated at 58;

the amino acid sequence of HCDR2 is SEQ ID NO: 59;

the amino acid sequence of HCDR3 is SEQ ID NO: shown at 60;

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

the amino acid sequence of LCDR2 is SEQ ID NO: shown at 73;

the amino acid sequence of LCDR3 is SEQ ID NO: shown at 74;

the amino acid sequence of HCDR1 is SEQ ID NO: 69;

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

the amino acid sequence of HCDR3 is SEQ ID NO: shown at 71;

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

the amino acid sequence of LCDR2 is SEQ ID NO: shown at 84;

the amino acid sequence of LCDR3 is SEQ ID NO: indicated at 85;

the amino acid sequence of HCDR1 is SEQ ID NO: 80;

the amino acid sequence of HCDR2 is SEQ ID NO: shown at 81;

the amino acid sequence of HCDR3 is SEQ ID NO: 82; and

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

the amino acid sequence of LCDR2 is SEQ ID NO: 95;

the amino acid sequence of LCDR3 is SEQ ID NO: 96;

the amino acid sequence of HCDR1 is SEQ ID NO: 91;

the amino acid sequence of HCDR2 is SEQ ID NO: 92;

the amino acid sequence of HCDR3 is 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 one of the following combinations:

(1) SEQ ID NO:54 or a heavy chain variable region sequence having at least 90% sequence identity thereto, and SEQ ID NO:56 or a light chain variable region sequence (# 2 clone) having at least 90% sequence identity thereto;

(2) SEQ ID NO:65 or a heavy chain variable region sequence having at least 90% sequence identity thereto, and SEQ ID NO:67 or a light chain variable region sequence (# 5 clone) having at least 90% sequence identity thereto;

(3) SEQ ID NO:76 or a heavy chain variable region sequence having at least 90% sequence identity thereto, and SEQ ID NO:78 or a light chain variable region sequence having at least 90% sequence identity thereto (# 118 clone);

(4) SEQ ID NO:87 or a heavy chain variable region sequence having at least 90% sequence identity thereto, and SEQ ID NO:89 or a light chain variable region sequence having at least 90% sequence identity thereto (# 119 clone).

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

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

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

(4) SEQ ID NO:87, and SEQ ID NO:89, a light chain variable region sequence (# 119 clone).

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

In another aspect, the present application also includes isolated nucleic acid molecules encoding the fully human antibodies or single chain antibodies or fragments thereof described above.

In another aspect, the present application also includes an expression vector comprising a nucleic acid molecule as described herein. In some embodiments, the vector is a plasmid, retrovirus, or lentiviral vector.

In another aspect, the present application also includes a host cell comprising an expression vector as described herein.

In another aspect, the present application also includes a pharmaceutical composition comprising a fully human antibody or single chain antibody or fragment thereof described herein, and a pharmaceutically acceptable carrier or diluent.

In another aspect, the present application also includes a method of treating a disease or disorder by administering to a patient in need thereof a therapeutically effective amount of a fully human antibody or single chain antibody or fragment thereof, or a host cell, or a pharmaceutical composition described herein to eliminate, inhibit or reduce MSLN activity, thereby preventing, alleviating, ameliorating or inhibiting the disease or disorder.

The present application also includes the use of the fully human antibodies or single chain antibodies or fragments thereof described above or the host cells described above in the manufacture of a medicament for the elimination, inhibition or reduction of MSLN activity, thereby preventing, alleviating, ameliorating or inhibiting a disease or disorder.

The present application also includes the use of the fully human antibodies described above or single chain antibodies or fragments thereof or the host cells described above as a medicament or in therapy, e.g., for the elimination, inhibition or reduction of MSLN activity, thereby preventing, alleviating, ameliorating or inhibiting a disease or disorder.

In some embodiments, the disease or disorder 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.

In another aspect, the present application also includes a kit for detecting MSLN protein in a sample. The kit comprises the fully human antibody or the single chain antibody or the fragment thereof. The detection may be in vitro or in vivo.

In another aspect, the present application also includes antibodies or fragments that compete for the same epitope as the fully human antibodies or single chain antibodies or fragments thereof described herein.

Drawings

FIG. 1 is a diagram of an enhanced anti-MSLN chimeric antigen receptor (Armored MSLN-CAR) design that facilitates in vivo expansion of CAR-T cells and against a tumor immunosuppressive microenvironment.

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

FIG. 3 is a map of lentiviral expression plasmid pCDH-MSLN-CAR-dnTGF-beta RII (P17) expressing MSLN-CAR and dnTGF-beta RII receptor.

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

FIG. 5 shows the P01, P17 and P43 plasmid lentivirus packages and titer assays.

FIG. 6 is a graph of the positive rate detection of expression of the dnTGF-beta RII by a CAR-T cell CD19-CAR, tEGFR, MSLN-CAR.

FIG. 7 is a graph of the in vitro killing ability of CAR-T cells against target cells.

FIG. 8 is a graph of IFN gamma cytokine secretion from CAR-T cells in vitro killing target cells.

FIG. 9 is a graph of a detection of CD107a degranulation of CAR-T cells by target cells.

FIG. 10 is a graph of a detection of CD107a degranulation of CAR-T cells by target cells.

FIG. 11 is a graph of MSLN, CD19 dual target cell antigen repeat stimulation of CAR-T cell proliferation potency assay.

Figure 12 is a graph of CD19-CAR promoting expansion of enhanced CAR-T cells, improving CAR-T cell killing tumor target cells.

FIG. 13 is a graph showing that the dnTGF-beta RII receptor competitively inhibits the phosphorylation of the TGF-beta-1 cytokine by SMAD2 protein in T cells.

FIG. 14 is a graph showing the inhibition of T cell killing tumor cells by dnTGF-beta RII receptor against TGF-beta-1 cytokine.

FIG. 15 is a graph of inhibition of ovarian cancer cell line SK-OV3 subcutaneous engraftment tumor growth by targeted MSLN-CAR-T cells.

Figure 16 is a graph of body weight change in mice with ovarian cancer cell line SK-OV3 subcutaneously transplanted tumors targeted to MSLN-CAR-T cells.

FIG. 17 is an in vivo expansion of MSLN-CAR-T cells in a SK-OV3 ovarian cancer cell line mouse subcutaneous engraftment tumor model.

FIG. 18 is a graph showing the results of a test drug acting on SK-OV-3 transplant tumor models; wherein, figure 18A is a high dose CAR-T group tumor growth curve; fig. 18B is a low dose CAR-T group tumor growth curve.

FIG. 19 is a graph showing the results of a test drug acting on SK-OV-3 transplant tumor models; wherein, figure 19A is a high dose CAR-T group body weight change curve; FIG. 19B is a graph of low dose CAR-T group body weight change; FIG. 19C is a graph of the high dose CAR-T group versus body weight change; figure 19D is a graph of low dose CAR-T group relative body weight change.

FIG. 20 shows the results of an enzyme-linked immunosorbent assay (ELISA) of a portion of the phage monoclonal antibodies selected with the target antigen and the control antigen, the negative control being the negative control of the phage, and the positive control 1 being the positive control to which the MSLN antibody MSLNAb (RD) was added.

FIG. 21 shows the results of flow cytometric analysis of binding of a portion of phage monoclonal to CHO-K1-MSLN and CHO-K1 cells, with the negative control being that of phage.

FIGS. 22A, 22B, 22C, 22D show the results of flow cytometric analysis (peak plots) of binding of selected phage monoclonal to a variety of different MSLN positive and negative cell lines, the negative control being that of phage.

FIGS. 23A, 23B, 23C, 23D show ELISA assay results of the selected phage monoclonal and a plurality of different MSLN antigen proteins and non-associated antigens. The negative control was phage negative control, positive control 1 was positive control added with MSLN antibody MSLN Ab (RD), positive control 2 was positive control added with MSLN Ab (bioleged). Wherein, the bar graph corresponding to each test antibody and the control group shows the test results of the reagents KACTUS-MSLN-Bio (Glu 296-Gly 588), KACTUS-MSLN-Bio (Glu 296-Asn 494), ACRO-MSLN-cyno, ACRO-MSLN-mouse, KACTUS BAFFR-Bio, KACTUS CD5-Bio, SA in order from left to right.

FIGS. 24A, 24B, 24C show results of flow cytometric analysis (peak plots) of binding of recombinantly expressed protein supernatants to a variety of different MSLN positive and negative cell lines, the negative control being a control without protein supernatant plus only secondary antibody.

FIGS. 25A, 25B, and 25C show ELISA assay results of recombinantly expressed protein supernatants with a plurality of different MSLN antigen proteins and non-associated antigens. The negative control was a control without protein supernatant plus only secondary antibody, positive control 1 was a positive control plus MSLN antibody MSLN Ab (RD), positive control 3 was a positive control plus HUYP218 (MSLN clinically positive antibody). Wherein, the bar graph corresponding to each test antibody and the control group shows the test results of the reagents KACTUS-MSLN-Bio (Glu 296-Gly 588), KACTUS-MSLN-Bio (Glu 296-Asn 494), ACRO-MSLN-cyno, ACRO-MSLN-mouse, KACTUS BAFFR-Bio, KACTUS CD5-Bio, SA in order from left to right.

Detailed Description

Definition of terms

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

In this application, an "antibody" refers to an immunoglobulin secreted by plasma cells (effector B cells) that is used by the body's immune system to neutralize foreign substances (polypeptides, viruses, bacteria, etc.). The foreign substance is correspondingly referred to as an antigen. The basic structure of classical antibody molecules is a 4-mer consisting of 2 identical heavy chains and 2 identical light chains. Heavy and light chains are divided into a variable region (V) at the amino terminus and a constant region (C) at the carboxy terminus according to the conservative differences in amino acid sequences. The heavy chain variable region (VH) and the light chain variable region (VL) interact to form an antigen binding site (Fv). In some cases, antibodies may also be used to refer to antibody fragments having antigen binding capacity, such as scFv, fab, F (ab') 2 and the like.

In the present application, the "single chain antibody (single chain fragment variable, scFv)" is composed of an antibody heavy chain variable region and a light chain variable region linked by a short peptide into one peptide chain. By correct folding, the variable regions from the heavy and light chains interact through non-covalent bonds to form Fv fragments, so that scfvs can better retain their affinity for antigen.

In the present application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR regions of a non-human antibody (e.g., a mouse antibody) are replaced with the corresponding amino acids derived from a human immunoglobulin. Small additions, deletions, insertions, substitutions or modifications of amino acids in the CDR regions may also be permissible, provided that they still retain the ability of the antibody to bind to a particular antigen. The humanized antibody may optionally 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. A "humanized" form of a non-human (e.g., murine) antibody may minimally comprise chimeric antibodies derived from sequences of non-human immunoglobulins. In some cases, CDR region residues in a human immunoglobulin (recipient antibody) may be replaced with CDR region residues of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired properties, affinity and/or capability. In some cases, the FR region residues of the human immunoglobulin may be replaced with corresponding non-human residues. In addition, the humanized antibody may comprise amino acid modifications that are not in the recipient antibody or in the donor antibody. These modifications may be made to further improve the properties of the antibody, such as binding affinity.

In the present application, the term "fully human antibody" generally refers to an antibody comprising only human immunoglobulin protein sequences. If it is produced in a mouse, in a mouse cell or in a hybridoma derived from a mouse cell, the fully human antibody may contain a murine sugar chain. Similarly, "murine antibody", "mouse antibody" or "rat antibody" refer to antibodies comprising only mouse or rat immunoglobulin sequences, respectively. Fully human antibodies can be produced in humans by phage display or other molecular biological methods in transgenic animals with human immunoglobulin germline sequences. Exemplary techniques useful for making antibodies are known in the art.

A "Chimeric Antigen Receptor (CAR)", also known as a chimeric T cell receptor, chimeric immune receptor, is an engineered membrane protein receptor molecule that can confer desired specificity to immune effector cells, such as the ability to bind to a particular tumor antigen. Chimeric antigen receptors are generally composed of an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain. In some cases, the antigen binding domain is a scFv sequence responsible for recognizing and binding to a specific antigen. Intracellular signaling domains typically include an immune receptor tyrosine activation motif (ITAM), such as a signaling domain derived from a CD3z molecule, responsible for activating immune effector cells and producing killing. In addition, chimeric antigen receptors may also include a signal peptide at the amino terminus that is responsible for intracellular localization of the nascent protein, as well as a hinge region between the antigen binding domain and the transmembrane domain. In addition to the signaling domain, the intracellular signaling domain may also include a costimulatory domain derived from, for example, a 4-1BB or CD28 molecule.

In the present application, the term "MSLN" is a tumor differentiation antigen. MSLN (Mesothelin), called mesothelin, is a cell surface glycoprotein with a molecular weight of 40 kDa. The MSLN gene encodes a precursor protein that is proteolytically produced to yield both Megakaryocyte Potentiator (MPF) and mesothelin protein products. Mesothelin can be anchored to the cell surface by glycosyl phosphatidylinositol to perform its own function. Mesothelin exists in normal mesothelium cells and is low in expression in normal tissues, but is high in expression in tumors such as mesothelioma, lung cancer, pancreatic cancer, breast cancer, ovarian cancer and the like, so that the mesothelin is a potential target for treating the cancer.

In the present application, the term "MSLN binding domain" refers generally to the extracellular domain of a MSLN CAR, which domain can specifically bind to an antigen. For example, the MSLN extracellular binding domain may be a receptor capable of specifically binding to an MSLN polypeptide expressed on a human cell, a chimeric antigen receptor capable of specifically binding to an MSLN polypeptide expressed on a human cell, an anti-MSLN antibody, or an antigen binding fragment thereof. The terms "binding domain," "extracellular binding domain," "antigen-specific binding domain," and "extracellular antigen-specific binding domain" are used interchangeably herein and provide a CAR that has the ability to specifically bind a target antigen of interest (e.g., MSLN). The MSLN binding domain may be of natural, synthetic, semisynthetic or recombinant origin.

In the present application, the term "antibody" generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a particular antigen. For example, an antibody may comprise an immunoglobulin of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and include 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., dabs, VH, or VHH), single chain antibodies (e.g., scFv). In this application, a "fragment" of an antibody may refer to an antigen binding fragment of an antibody, e.g., 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, non-glycosylated antibodies, as well as any of the antigen-binding antibody fragments and derivatives thereof described. Each heavy chain may be composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain may be composed of a light chain variable region (VL) and a light chain constant region. VH and VL regions can be further distinguished as hypervariable regions called Complementarity Determining Regions (CDRs) interspersed with regions that are more conserved, called Framework Regions (FR). Each VH and VL may be composed of three CDRs and four FR regions, which may be arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody may mediate binding of the immunoglobulin to host tissues or factors including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

In the present application, the term "single chain antibody" may also be referred to as scFv, and refers to an antibody composed of one chain formed by connecting a heavy chain variable region and a light chain variable region through a connecting peptide. The term "single domain antibody" refers to antibodies formed solely from the heavy chain variable region.

In this application, the term "transmembrane domain" (Transmembrane Domain) generally refers to the domain of a CAR that passes through the cell membrane, which is linked to an intracellular signaling domain, and serves to transmit signals. In this application, the transmembrane domain may be a CD8a transmembrane domain.

In the present application, the term "costimulatory domain" generally refers to an intracellular domain that can provide an immune costimulatory molecule, which is a cell surface molecule required for the efficient response of lymphocytes to an antigen. The co-stimulatory domain may include the co-stimulatory domain of CD28, and may also include the co-stimulatory domain of the TNF receptor family, such as the co-stimulatory domains of OX40 and 4-1 BB.

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

In this application, the term "intracellular signaling domain" generally refers to a component of CAR that is located in intracellular signaling, comprising a signaling domain and a domain that specifically binds to the receptor component, for example: it may be selected from the group consisting of CD3 zeta intracellular domain, CD28 intracellular domain, 4-1BB intracellular domain and OX40 intracellular domain.

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

In the present application, the term "self-cleaving peptide" refers to a self-cleaving 2A peptide that can be proteolytically cleaved via ribosome hopping rather than protease hydrolysis to perform the function of a cleaving protein, which can include, but is not limited to, T2A, F2A, P2A, and the like.

In this application, the term "marker detection signal" generally refers to a gene, protein or other molecule of known function or sequence that is capable of performing a specific labeling function, signaling that can be detected. The label detection signal may be a fluorescent protein, such as: GFP, RFP, YFP, and the like. The marker detection signal may be tgfr. The terms "EGFRt" and "tgfr" are used interchangeably herein to refer to a 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 epitope recognized by the anti-EGFR antibody. tEGFR can be used as a non-immunogenic selection tool and tracking marker for genetically modified cell function. In this application, it can be used as a marker molecule for CAR-T cells for clearing CAR-T cell EGFR antibodies (e.g. cetuximab) mediated ADCC pathway (cetuximab mediated ADCC pathway) in vivo if necessary (see US8802374B 2), i.e. as a safety switch at the time of clinical transformation.

In this application, an "EGFR antibody" refers to an antibody that is capable of eliciting an antibody-dependent cellular cytotoxicity effect (antibody dependent cell-mediated cytotoxicity) that causes immune cells to attack CAR-T cells having truncated epidermal growth factor receptor (EGFRt) to assist in the clearance of the CAR-T cells. The EGFR antibodies can be used when serious adverse reactions occur after infusion of CAR-T by a patient or other conditions requiring clearance of CAR-T cells, which can assist in clearance of CAR-T cells, alleviating symptoms associated with CAR-T treatment. The EGFR antibodies include, but are not limited to, cetuximab, panitumumab, cetuximab and nituzumab.

In this application, the term "nucleic acid molecule" generally refers to an isolated form of a nucleotide, deoxyribonucleotide or ribonucleotide of any length, or an analogue isolated from its natural environment or synthesized synthetically.

In the present application, the term "vector" generally refers to a nucleic acid vector into which a polynucleotide encoding a protein can be inserted and the protein expressed. The vector may be expressed by transforming, transducing or transfecting a host cell such that the genetic element carried thereby is expressed within the host cell. One vector may contain a variety of elements that control expression. In addition, the vector may also contain a replication origin. The carrier may also include components that assist it in entering the cell.

In the present application, the term "cell" generally refers to a single cell, cell line or cell culture that may or may not be a recipient of a subject plasmid or vector, which includes a nucleic acid molecule as described herein or a vector as described herein. Cells may include progeny of a single cell. The offspring may not necessarily be identical to the original parent cell (either in the form of the total DNA complement or in the genome) due to natural, accidental or deliberate mutation. Cells may include cells transfected in vitro with the vectors described herein.

In this application, the term "immunoconjugate" generally refers to a conjugate formed by conjugation (e.g., covalent attachment via a linker molecule) of the other agent (e.g., a chemotherapeutic agent, a radioactive element, a cytostatic agent, and a cytotoxic agent) to the antibody or antigen-binding fragment thereof, which conjugate can specifically bind to an antigen on a target cell through the antibody or antigen-binding fragment thereof, delivering the other agent to the target cell (e.g., a tumor cell).

In the present application, the term "pharmaceutical composition" generally refers to a composition for the prevention/treatment of a disease or disorder. The pharmaceutical composition may comprise an isolated antigen binding protein described herein, a nucleic acid molecule described herein, a vector described herein and/or a cell described herein, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical compositions may also comprise one or more suitable formulations such as (pharmaceutically effective) carriers. The acceptable ingredients of the composition may be non-toxic to the recipient 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 that is non-toxic to the cells or mammals to which it is exposed at the dosages and concentrations employed. The physiologically acceptable carrier may include a suitable substance. Refers to a pharmaceutically acceptable carrier (carrier) that is not typically the same substance as the vector (vector) used to insert the nucleic acid in genetic engineering.

In this application, the term "directly connected" may be opposed to the term "indirectly connected," which generally refers to a direct connection. For example, the direct linkage may be where there is no spacer between the substances. The spacer may be a linker. For example, the linker may be a peptide linker. The term "indirect linkage" generally refers to the situation where the materials are not directly linked. For example, the indirect connection may be the case where the connection is through a spacer. For example, in the isolated antigen binding proteins described herein, the C-terminus of the L-FR1 and the N-terminus of the LCDR1 can be directly or indirectly linked.

In this application, the term "comprising" generally refers to the meaning of including, generalizing, containing or comprising. In some cases, the meaning of "as", "consisting of.

In this application, the term "about" generally means ranging from 0.5% to 10% above or below the specified value, e.g., ranging from 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% above or below the specified value.

In this application, the term "immunosuppressive receptor" generally refers to a receptor that binds to an immunosuppressive molecule that is highly expressed in a tumor. After the immunosuppressive molecule is combined with an immunosuppressant receptor expressed on the CAR-T, the immunosuppressant molecule can inhibit the killing function of immune cells on tumors, so that tumor proliferation is caused, and the treatment effect of the CAR-T is reduced. The modified immunosuppression molecule receptor with the lost function can competitively bind with immunosuppression molecules in tumors, but cannot cause immunosuppression effect after binding, so that the immunosuppression microenvironment of the solid tumor suffered by the CAR-T cells is relieved or eliminated, and the capability of killing the solid tumor is improved.

Examples

The present application is further described below in conjunction with specific examples, which are not intended to limit the scope of the present application.

The experimental methods for which specific conditions are not noted in the examples of the present application are generally conducted under conventional conditions or under conditions recommended by the manufacturer of the raw materials or goods. The reagents of specific origin are not noted and are commercially available conventional reagents.

Example 1: enhanced anti-MSLN-CAR (Armored MSLN-CAR) design to promote in vivo expansion of CAR-T cells and to combat tumor immunosuppression microenvironment

To enhance the efficacy of CAR-T cell therapy for solid tumors, the enhanced MSLN-CAR-T design of the present application, on the one hand, uses CD19-CAR molecules to promote expansion of CAR-T cells in a patient; on the other hand, dntgfbetarii receptor without intracellular signal is used to combat the inhibitory effect of immune microenvironment of tumor tissue on T cell function, and additionally, for the safety of CAR-T cell application in vivo, the design of CAR has tEGFR as a molecular safety switch for CAR-T cells.

The design of the enhanced MSLN-CAR-T cell is obtained by jointly infecting the T cell with two lentiviral expression vectors, wherein one lentiviral expression vector expresses a CD19-CAR and a tEGFR molecular safety switch, and the other lentiviral expression vector expresses MSLN-CAR and a dnTGF beta RII receptor. The schematic of the design is shown in FIG. 1, wherein (1) MSLN-CAR molecules act as MSLN antigens recognizing the surface of tumor cells, activating T cells, and exerting the effect of MSLN-CAR-T cells killing MSLN positive tumor cells. (2) The CD19-CAR molecule acts to recognize B cells in the patient's blood system, thereby stimulating activation of T cells, enabling the CAR-T cells to reach the ability to expand rapidly in the patient, and may promote the ability of the CAR-T cells to survive in the patient due to the sustained presence of B cells in the patient. (3) The dnTGF beta RII molecule acts as a TGF beta RII receptor molecule without intracellular domain, and can competitively resist the inhibition of TGF beta-1 cytokines in tumor tissue microenvironment to CAR-T cell functions.

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

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

The chimeric antigen receptor CAR uses a signal peptide sequence of CD8 aSP (nucleotide sequence SEQ ID NO:7, amino acid sequence SEQ ID NO: 8), a Linker sequence linking the heavy and light chains of the single chain antibody of (G4S) 3 (amino acid sequence SEQ ID NO: 9), a hinge region of CD8hinge (nucleotide sequence SEQ ID NO:10, amino acid sequence SEQ ID NO: 11), a transmembrane region of CD8TM (nucleotide sequence SEQ ID NO:12, amino acid sequence SEQ ID NO: 13), a second signal (co-stimulatory signaling domain) of 41BB (nucleotide sequence SEQ ID NO:14, amino acid sequence SEQ ID NO: 15), and a first signal (intracellular signaling domain) of CD3z (nucleotide sequence SEQ ID NO:16, amino acid sequence SEQ ID NO: 17).

Example 2: third generation lentivirus packaging system and lentivirus vector design construction

The present application uses a third generation lentiviral packaging system, including a four plasmid system that expresses plasmids, envelope plasmids, and packages plasmids.

4 plasmids of the lentiviral packaging system are purchased from YouBio Youbao organisms, a skeleton plasmid of an expression plasmid is pCDH-EF1-MCS-T2A-Puro (catalyst#: VT 1482), an envelope plasmid is pMD2.G (catalyst#: VT 1443), packaging plasmids are pMDLgpRRE (catalyst#: VT 1449) and pRSV-Rev (catalyst#: VT 1445), and the construction method of the lentiviral expression plasmid designed by the application is as follows:

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

2. Lentiviral expression plasmid pCDH-MSLN-CAR-dntgfbrii (P17) expressing MSLN-CAR and dntgfbrii receptor was constructed: MSLN-CAR-P2A-dnTGF beta RII sequence (nucleotide sequence SEQ ID NO:21, amino acid sequence SEQ ID NO: 22) is synthesized and inserted into the multiple cloning site of pCDH-EF1-MCS-T2A-Puro plasmid, and T2A-Puro sequence is removed to reconstruct pCDH-MSLN-CAR-dnTGF beta RII (P17) (nucleotide sequence SEQ ID NO: 23); the map of the lentiviral expression plasmid is shown in FIG. 3.

3. Lentiviral expression plasmid pCDH-CD 19-CAR-tgfr (P43) expressing the CD19-CAR and tgfr molecular switches was constructed: the CD19-CAR-T2A-tEGFR sequence (nucleotide sequence SEQ ID NO:24, amino acid sequence SEQ ID NO: 25) is synthesized and inserted into the multiple cloning site of the pCDH-EF1-MCS-T2A-Puro plasmid, and the T2A-Puro sequence is removed to reconstruct the pCDH-CD19-CAR-tEGFR (P43) (nucleotide sequence SEQ ID NO: 26); the map of the lentiviral expression plasmid is shown in FIG. 4.

EXAMPLE 3 packaging of lentiviral vectors and detection of viral titre

Lentiviral vector packaging method

239T cells are inoculated in a culture dish with proper specification 24h in advance, the cell culture solution is DMEM+10% FBS without antibiotics, and the inoculated cell density is 70% -80%, so that the cell density is more than 90% during infection.

The following day of transfection with Lipo3000 reagent, A, B solution was prepared according to the reagent instructions:

and (3) solution A: the four plasmids were dissolved in serum-free Optim-MEM medium (four plasmid system), and the volumes of the desired plasmids (pMD 2.G, pMDLg-RRE, pRSV-Rev, transfer vector) were calculated as shown in the following table, and then added to P3000 for mixing.

And (2) liquid B: lipo3000 was dissolved in an equal volume of serum-free DMEM medium (gently mixed and allowed to stand for 5 min).

TABLE 1 lentiviral packaging System (15 cm dish)

Component (A)

Usage amount

pRSV-Rev	5.5μg
pMDlg-pRRE	11.7μg
pMD2.G	7.8μg
pCDH Transfer vector	12.5μg
PAdVantage vector	2.5μg
Total	37.5μL
P3000	75μL
lip3000	88.9μL
Medium	4.5ml+4.5ml

The solution A is added into the solution B drop by drop, mixed gently and incubated for 20min at room temperature. The transfection complexes were added on average to the medium in the petri dishes. Transfected cells were plated after 6h with DMEM medium containing 10% fbs. Incubating at 37 ℃ for 24-48h, collecting virus supernatant, centrifuging at 1500g at 4 ℃ for 5min, and removing cell debris.

Method for concentrating and purifying slow virus

Using Lenti-X ^TM The concentration of lentivirus from collected lentivirus supernatants by the Concentrator reagent was performed as follows:

1. adding 1/3 of the total volume of Lenti-X ^TM A Concentrator;2. fully and uniformly mixing, and then placing the mixture into a temperature of 4 ℃ for incubation for more than 2 hours; centrifuging at 3.4 ℃ for 45min at 1500 g; 4. removing supernatant, adding appropriate volume of T cell culture medium (X-VIVO 15) to dissolve lentiviral precipitate, and sub-packaging at-80deg.C for freezing.

Lentivirus titer assay

Lentivirus titer was determined by flow cytometry, as follows:

(1) Plating (293 CT cells) 24h ahead (1-2E+05 cells per well) (day 1); (2) 293CT cells from one well were digested and different volumes of virus (0.1/0.5/1 ul) were added (day 2); (3) cell exchange fluid (day 3); (4) detecting car+ (day 4) in a stream; (5) a titer calculation formula: viral titer (/ ml) =positive rate x number of cells/viral volume μl

The pCDH-MSLN-CAR (P01), pCDH-MSLN-CAR-dnTGF beta RII (P17), pCDH-CD19-CAR-tEGFR (P43) were lentivirally packaged and tested for viral titres, and the experimental results were shown in FIG. 6, with P01, P17 and P43 lentiviruses having titres of 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, peripheral blood mononuclear lymphocytes were collected from healthy donors or PBMC from healthy donors were purchased from suppliers and isolated using the T cell isolation kit EasySep ^TM Human T Cell Isolation Kit (Stem cell Co., ltd.: 17951) T cells were further sorted from PBMC. The obtained T cells were cultured in X-VIVO15 (Lonza, 04-418Q) medium, while adding recombinant human IL2 cytokine (R) at a final concentration of 100IU/ml&D, cargo number: 202-IL-050) and magnetic beads (Dynabeads) of CD3/CD28 were added in a 1:1 ratio to T cells ^TM Human T-actioner CD3/CD28, GIBCO, cat: 11132D) Stimulation of activated T cells at a cell density of 1X 10 ⁶ Individual/mL, at 37 ℃,5% CO ₂ Culturing under the condition.

After 24 hours of T cell stimulation activation, lentiviral infection was performed. Lightly blowing the cells to disperse, and taking a small number of cells for counting; centrifuging 500g for 5min, carefully sucking the supernatant; resuspension T cells with CART cell complete culture medium, adjusting cell density to be more than 2E+06cells/ml, taking required amount of cells into a cell culture plate, adding two virus concentrates with a certain volume according to the virus concentrate liquid drop degree and MOI, adding complete culture medium to adjust the cell final density to be 2E+06cells/ml; starting the centrifuge in advance to preheat to 32 ℃, putting the cell culture plate into the centrifuge and balancing, and centrifuging for 1h at 2000g at 32 ℃; after centrifugation, adding a complete culture medium according to the volume ratio of 1:1, and placing the culture medium in a constant temperature incubator at 37 ℃ for culture overnight; the next day, the supernatant was centrifuged off, resuspended in complete medium (density about 3E+05c.about.5E+05cells/ml), and then placed in a constant temperature incubator at 37℃for further culture.

The cell suspension was collected 96 hours after virus infection, centrifuged at 300g for 5min, and the supernatant was discarded, resuspended in 1-1.5ml complete medium and gently beatenCells were separated from beads and transferred to 1.5-2ml centrifuge tubes in DynaMag ^TM 2, standing for 1 min, the 1ml gun head carefully aspirates the cell suspension into a 1.5ml EP tube or 15ml centrifuge tube (depending on the number of starting cells cultured), and removing the magnetic beads. A small amount of cell count was taken, the cell density was adjusted to 5E+05cells/ml with complete medium, transferred to a suitable flask or dish, and placed in a constant temperature incubator at 37℃for continuous culture.

The growth of cells was monitored daily to maintain a cell density of 3X 10 during culture ⁵ cells/-l-2×10 ⁶ cells/ml. The positive rate of expression of CD19-CAR, tEGFR, MSLN-CAR and dnTGF beta RII can be detected about 7 days after T cells are infected with lentiviral vectors, and the dnTGF beta RII-CD19-MSLN-CAR-T cells infected with the double viruses tEGFR-CD19-CAR and dnTGF beta RII-MSLN-CAR are obtained.

The CAR-T cell positive rate assay is shown in FIG. 6. Wherein UTD (Untransduced T Cell untransfected cells) cells are null and negative for infected control cells, MSLN-CAR, CD19-CAR, tEGFR, dnTGF βrii expression; pCDH-MSLN-CAR (P01) lentiviral infected MSLN-CAR-T (P01) cells obtained with a positive MSLN-CAR rate of about 70% and negative CD19-CAR, tEGFR, dnTGF βrii expression; dnTGF beta RII-MSLN-CAR-T cells (P17) obtained by lentiviral infection of pCDH-MSLN-CAR-dnTGF beta RII (P17) have a MSLN-CAR positive rate of about 70%, a dnTGF beta RII expression positive rate of about 27.6%, and CD19-CAR, tEGFR expression is negative; pCDH-CD 19-CAR-tggfr (P43) lentiviral infected CD19-CAR-T cells (P43), CD19-CAR positive rate of about 21.6%, tggfr positive rate of 63.5%, MSLN-CAR, dntgfβrii expression negative; pCDH-MSLN-CAR (P01) +pcdh-CD 19-CAR-tggfr (P43) double virally infected cells CD19-MSLN-CAR-T cells (p01+p43), MSLN-CAR positive rate of about 60%, CD19-CAR positive rate of about 12.24%, tggfr positive rate of 36.84%, dntgfbetarii expression negative, MSLN-CAR, CD19-CAR double positive rate of 27.5% (pCDH-CD 19-CAR-tggfr infection efficiency indicated with tggfr);

pCDH-MSLN-CAR-dntgfbrii (P17) +pcdh-CD 19-CAR-tggfri (P43) double virus infected cells dntgfbrii-CD 19-MSLN-CAR-T cells (p17+p43), MSLN-CAR positive rate of about 58%, dntgfbrii positive rate of 20.7%, CD19-CAR positive rate of about 9.9%, tggfr positive rate of 36.08%, MSLN-CAR, CD19-CAR double positive rate of 26.7% (pCDH-CD 19-CAR-tggfr infection efficiency indicated by tggfr).

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

(1) Experimental materials:

luciferase Assay (Promega, cat# E2520), X-VIVO 15 Medium (Lonza, cat# 04-418Q), 96-well flat-bottomed white plates without lids (Greiner, cat# 655075), 96-well flat-bottomed transparent lids (Greiner, cat# 655180), perkinElmer EnVision 2103 Multilabel Reader

(2) Sample to be measured:

negative control UTD cells without virus infection, MSLN-CAR-T cells (P01), dnTGF beta RII-MSLN-CAR-T cells (P17), CD19-CAR-T cells (P43), P01, P43 double virus infected CD19-MSLN-CAR-T cells (P01+P43), P17, P43 double virus infected dnTGF beta RII-CD19-MSLN-CAR-T cells (P17+P43)

(3) The experimental method comprises the following steps:

target cells SKOV-3C16-fluc were selected, and after digestion, X-VIVO 15 (without IL2, serum free) was resuspended to a density of 1E+5/ml and 100. Mu.L/well plated in 96 well plates requiring co-culture and targeting.

Absorbing CAR-T cells, centrifuging for 5min at 300g, re-suspending X-VIVO 15, calculating according to CAR+, adjusting the density to 1E+6/ml, diluting the density to be 5E+5/ml, 2.5E+5/ml, 1.25E+5/ml, 0.625E+5/ml and 0.3125E+5/ml, namely, the effective target ratios of 10, 5, 2.5, 1.25, 0.625 and 0.3125, respectively paving 100 mu L/hole of CAR-T cells with the adjusted density into a 96-hole plate co-cultured with target cells, uniformly mixing, additionally arranging an effector cell spontaneous group, paving the suspension and X-VIVO 15 into the 96-hole plate according to the volume ratio of 1:1, and preparing for detecting cytokines.

Placed in CO ₂ The incubator was incubated for 17-19h, centrifuged at 500g for 5min, and the supernatant was aspirated at 100. Mu.L/well (note that cells were not aspirated as much as possible),placing in another clean 96-well plate, marking, and storing in refrigerator at-20deg.C for cytokine detection.

100. Mu.L/well TheReagent is added into the cell suspension, the operation is carried out in a dark place, and after the reaction is placed for 5 to 10 minutes, the expression of luciferases is detected by an upper machine.

The calculation formula of the percentage of the CAR-T cells killing target cells is as follows:

killing rate (Lysis%) = (simple Target-value-experimental group read value)/simple Target read value

(4) Experimental results

As shown in fig. 7, it can be seen from the experimental results that MSLN-CAR-T cells (P01), dntgfbetarii-MSLN-CAR-T cells (P17), P01, P43 double virus infected CD19-MSLN-CAR-T cells (p01+p43), P17, P43 double virus infected dntgfbetarii-CD 19-MSLN-CAR-T cells (p17+p43) had significant tumor killing ability against MSLN expressing positive ovarian cancer cell line SK-OV3, OVCAR3, whereas negative control UTD cells and CD19-CAR-T cells (P43) had no killing effect on SK-OV3, OVCAR3 (fig. 7a, 7 b).

The positive NALM-6-977 tumor cell line, P01, P17, P43, P01+P43, P17+P43 group CAR-T cells, had significant tumor killing capacity against CD19 overexpressing MSLN antigen, while negative control UTD cells had no killing effect (FIG. 7 c).

For a Raji tumor cell line positive for CD19 expression, only CAR-T cells P43, p01+p43, p17+p43 expressing CD19-CAR had killing ability, UTD, P01, P17 had no killing effect (fig. 7 d). For CD19KO-Raji cell lines with CD19 knockdown, none of the groups had the effect of killing CD19KO-Raji tumor cells (FIG. 7 e).

Example 6: in vitro killing of target cells by CAR-T cells IFN gamma cytokine secretion

Taking cell killing capacity test, namely supernatant after each group of CAR-T cells and target cells are incubated together (E: T=5:1) in test example 5; the concentration of IFN-gamma in the supernatant was measured using Cisbio Human IFN gamma kit (cat# 62 HIFUNGPEG) and the following procedure was followed:

(1) The reagents were left at room temperature for at least 30min before the experiment.

(2) Sample preparation: after high speed centrifugation of each set of samples, the supernatant was aspirated and transferred to a clean EP tube to ensure removal of residual cell debris, and the stock was diluted 5-fold with diluent or cell culture medium (X-VIVO 15) for use.

(3) Standard substance configuration: the standard flask was prepared with distilled water to a volume corresponding to the volume of the flask, and the thawed standard stock solution was diluted 3-fold with a cell culture medium (X-VIVO 15). 60. Mu.L of stock solution was added to 120. Mu.L of cell culture medium, gently mixed to give standard Std7 (4000 pg/mL), and a standard curve was prepared with high concentration standard (Std 7) and serially diluted as follows: from Std6 to Std 0, 110. Mu.L of the diluent or cell culture medium was taken per bottle, 100. Mu.L of the standard was added to 110. Mu.L of the diluent or cell culture medium, gently mixed, and serial dilutions were repeated to prepare standards labeled Std6, std5, std4, std3, std2, std1, std 0 (negative control) as the diluent or medium, respectively.

(4) Infγ antibodies working solution configuration: the detection buffer #3 was used to dilute 20X stock solution (INFγ Eu Cryptate antibody) 20 times, the detection buffer #3 was used to dilute 20X stock solution (INFγd2 anti) 20 times, and two ready-to-use diluted antibody solutions were mixed 1:1 to obtain INFγ antibodies working solution prior to the experiment.

(5) The detection step comprises: 16. Mu.L/well of standard and sample were added to HTRF 96 well low volume plates (2-3 wells were set); 4. Mu.L of premixed INFγ antibodies working solution was added to all wells; sealing plate, incubating for 3 hours at room temperature, at PerkinElmer EnVision 2103 under program 2103 Multilabel Reader reads the plate.

The results of the experiments obtained are shown in FIG. 8, where the IFNγ levels were low in the CAR-T cell alone group and in the co-incubated group with MSLN-negative K562 target cells, respectively. In the co-incubation group with target cells OVCAR3 positive for MSLN expression, MSLN-CAR-T cells (G2), dntgfbrii-MSLN-CAR-T cells (G3), double virus infected cells dntgfbrii-CD 19-MSLN-CAR-T (G4), double virus infected CD19-MSLN-CAR-T (G6) had significant ifnγ cytokine secretion, whereas negative controls UTD (G1), and CD19-CAR-T (G5) group ifnγ cytokine secretion levels were lower. In the co-incubation experiments with MSLN-expressing CD19 positive NALM-6-977 tumor cells, MSLN-CAR-T cells (G2), dnTGF beta RII-MSLN-CAR-T cells (G3), dnTGF beta RII-CD19-MSLN-CAR-T (G4), CD19-CAR-T (G5), CD19-MSLN-CAR-T (G6) all had significant secretion of IFNγ cytokines, and UTD groups did not have secretion of IFNγ cytokines.

Example 7: CAR-T cell activated CD107a degranulation assay

(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 broth (Lonza, 04-418Q), DPBS (Gibco, 14190-144), 96 well Flat bottom plate (Costar), stain Buffer (BD, 554657), monensin (BD, 554724), flow cytometer (Beckman), cell incubator (Thermo), high speed centrifuge (Thermo)

(2) Sample to be measured

(3) Experimental method

Tumor cell lines K562, SK-OV3 and OVCAR3 were treated with X-VIVO respectively ^TM 15 resuspension, adjust density to 1e+06/mL, add 100. Mu.L per well to 96 well flat bottom plate.

X-VIVO for CAR-T cells ^TM 15 re-suspension, adjusting the density to 2e+06/mL, and adding 100 mu L of each well to corresponding tumor cells respectivelyTwo replicates per group.

mu.L PE Mouse Anti-Human CD107a antibody was added to each well and incubated for 1h, while the Isotype set was set and 5. Mu.L PE Mouse IgG1,. Kappa.isotype Control was added.

The golgi inhibitor Monensin was diluted 1:50, 10. Mu.L Monensin was added to each well and incubated for 4h,500g, centrifuged for 5min, and the supernatant discarded.

Resuspension with 100. Mu.L of Stain Buffer, 3. Mu.L of biotin-Labeled MSLN protein was added to each well, 3. Mu.L of FITC-Labeled Human CD19, mixed well, and incubated at 4℃for 30min.

200 mu L of DPBS is added to each well, mixed well, 500g and centrifuged for 5min, and the supernatant is discarded.

100. Mu.L of Stain Buffer was resuspended with 1. Mu.L of 500-fold dilution Brilliant Violet 421 per well ^TM Strepitavidins were mixed and incubated at 4℃for 30min.

200 mu L of DPBS is added into each hole, evenly mixed, 500g is centrifuged for 5min, and the supernatant is discarded

100 mu L of Stain Buffer is resuspended and checked on the machine.

(4) Experimental results

The experimental results are shown in fig. 9 and 10. The proportion of CD107a in MSLN-CAR positive cells after co-incubation with MSLN-expressing negative tumor cell line K562 was lower, whereas the proportion of CD107a in MSLN-CAR positive cells in MSLN-expressing positive ovarian cancer tumor cell line SK-OV3, OVCAR3 was significantly increased after co-incubation with MSLN-expressing positive tumor cell line SK-OV3, MSLN-CAR-T cell (P01), dntgfbetarii-MSLN-CAR-T cell (P17), P01, P43 double virus infected CD19-MSLN-CAR-T cell (p01+p43), P17, P43 double virus infected dntgfbetarii-CD 19-MSLN-CAR-T cell (p17+p43) group, whereas the proportion of CD107a in the negative control UTD group, CD19-CAR-T cell (P43) group was lower.

Example 8: dual target cell antigen repeat stimulation CAR-T cell proliferation capability assay

(1) Experimental materials

X-VIVO ^TM 15 Medium (Lonza, cat# BEBP 04-744Q), mc 'oy' S5A (Modified) medium (Gibco, cat# 16600082), mitomycin C (Selleck, cat# S8146), APC strepitavidin (BD Biosciences, cat# 554067),stain Buffer (BD Biosciences, cat# 554657), biotinylated Human Mesothelin, fc Tag (Acrobiosystems, MSN-H826 x), SK-OV-3 cell line (Shanghai Gaining Biotech Co., ltd., cat# CM-H143), raji cell line (ATCC, cat# CCL-86), fluorescent cytometer (Shanghai Rui Yu Biotech Co., ltd.)

(2) Sample to be measured

(3) Experimental method

Digesting SK-OV3 cells, sucking Raji cells, counting, inoculating cells into 12-well plate, and inoculating 3×10 cells in each well ⁵ The cells were cultured for 24 hours in a total volume of 1 mL/well.

The Mitomycin C is diluted by using a complete culture medium, mixed by shaking, and the diluted Mitomycin C is added into the prepared cells, and the final concentration is 30mg/mL, and incubated for 3 hours.

T cells were collected, counted, and CAR-T cell positive rates were adjusted to be consistent using UTD. Centrifuge at 300 Xg for 5min. The supernatant was discarded, and T cells were washed with 1 XPBS and centrifuged at 300 Xg for 5min. Resuspension of T cells with X-VIVO medium, density was adjusted to 5X 10 ⁵ And (3) one/mL for later use.

The SK-OV3+ Raji cell culture medium was discarded, and cells were washed with 1 XPBS and repeated twice.

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

Co-culturing for 48h (this time point is marked as Day 3), counting the number of CAR-T cells, detecting the positive rate of the CAR-T cells in a flow mode, and taking 1 multiplied by 10 ⁶ Individual cells were used for the next round of antigen stimulation experiments. The stimulation was repeated three times.

The CAR-T cell expansion fold calculation formula is as follows:

(4) Experimental results

The experimental results are shown in figure 11, where CAR-T cells were cultured alone without tumor cell stimulation, and where CAR-T cells were not expanded D3 days after one round of stimulation. In the case of co-culture with tumor cell line SK-ov3+raji, negative control group UTD cells were not expanded, MSLN-CAR-T cells (P01), dntgfbetarii-MSLN-CAR-T cells (P17), CD19-CAR-T cells (P43), P01, P43 double virus infected CD19-MSLN-CAR-T cells (p01+p43), P17, P43 double virus infected dntgfbetarii-CD 19-MSLN-CAR-T cells (p17+p43) were significantly expanded, with CAR-T cells p17+p43 with CD19-CAR, MSLN-CAR double CAR molecules, the fold expansion of p01+p43 was significantly higher than CAR-T cells with MSLN-CAR or CD19-CAR molecules alone.

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

To assess the predominance of expansion of enhanced MSLN-CAR-T cells with CD19-CAR molecules in humans, and the killing ability against target cells, each group of CAR-T cells (UTD cells, MSLN-CAR-T cells, dntgfbetarii-MSLN-CAR-T cells, CD19-CAR-T cells, double virus infected CD19-MSLN-CAR-T cells, P17, P43 double virus infected dntgfbetarii-CD 19-MSLN-CAR-T cells) was incubated with CD19 positive target cells NALM-6 for 4 hours followed by incubation with MSLN positive target cells OVCAR3 for 7 hours, and the killing ability of each group of CAR-T cells against OVCAR3 target cells was examined as described in example 5.

The experimental results are shown in FIG. 12, and it can be seen that the set of CAR-T cells bearing the CD19-CAR molecule after 4 hours of incubation with NALM-6: dntgfbetarii-CD 19-MSLN-CAR-T, has significantly higher killing capacity against OVCAR3 target cells than the CAR-T cell group without CD19-CAR molecule: MSLN-CAR-T, dnTGFβRII-MSLN-CAR-T. Experimental results prove that in the presence of the CD19 positive target cells, the CD19-CAR molecules can promote the expansion of the CAR-T cells, so that the killing effect of the CAR-T on the bar MSLN positive target cells is improved.

Example 10: dnTGF beta RII blocks the signaling of TGF beta-1 cytokines 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-βReceptor 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 ^TM eBioscience ^TM Fixation/Permeabilization Concentrate(Invitrogen，501129082)，Invitrogen ^TM UltraPure ^TM DNase/RNase-Free Distilled Water (Invitrogen, 10977015), acetic acid (Protect, A501931-0500).

(2) Sample to be measured

(3) Experimental method

Each CAR-T cell of 2e+06 was split equally into two parts, centrifuged at 300g for 5min and the supernatant discarded. One portion is used with X-VIVO ^TM 15 resuspension, one portion with X-VIVO ^TM 15, 5ng/mL TGF-Beta1 was added and resuspended, the cells were incubated in a cell incubator for 40min, centrifuged at 300g for 5min, and the supernatant was discarded. The wells were resuspended in 100. Mu.L of Stain Buffer, 5. Mu.L of PE anti-human TGF-. Beta. Receptor II Antibody were added to each well, the isotype set was set up and 5. Mu.L of PE Rat IgG2b,. Kappa. Isotype Ctrl Antibody were added and incubated for 30min at 4 ℃. 200 mu L of DPBS is added into each hole, the mixture is uniformly mixed, 500g is centrifuged for 5min, the supernatant is discarded, the mixture is uniformly mixed with 200 mu L Fixation buffer, and the mixture is incubated for 1h at room temperature in a dark place.500g, centrifuging for 5min, and discarding the supernatant.

10X Permeabilization buffer the cells were resuspended at 1X, 300. Mu.L 1X Permeabilization buffer with water, 500g, centrifuged for 5min and the supernatant discarded.

The Phospho-SMAD2 (Ser 465/Ser 467) (E8F 3R) Rabbit mAb was diluted 1:400 with 1X Permeabilization buffer, 100. Mu.L per well was added to the corresponding cells, resuspended, and incubated for 1h at room temperature in the absence of light.

200 mu L of DPBS is added to each well, mixed well, 500g and centrifuged for 5min, and the supernatant is discarded. Alexa was diluted 1:4000 with 1X Permeabilization buffer647 Donkey anti-rabit IgG was added to the corresponding cells at 100. Mu.L per well, resuspended, and incubated for 1h at room temperature in the absence of light. 200 mu L of DPBS is added to each well, mixed well, 500g and centrifuged for 5min, and the supernatant is discarded. 100 μL of 1X Permeabilization buffer are resuspended and checked on the machine.

(4) Experimental results

As shown in fig. 13, following 5ng/mL tgfβ -1 cytokine treatment in each group of CAR-T media, negative control UTD cells, MSLN-CAR-T cells (P01), CD19-CAR-T cells (P43), P01, P43 double virus infected CD19-MSLN-CAR-T cells (p01+p43) had elevated SMAD2 protein phosphorylation levels, whereas CAR-T cell groups with dntgfβrii receptor: the corresponding increase in the phosphorylation level of SMAD2 protein of dnTGF beta RII-MSLN-CAR-T cells (P17), P17, P43 double virus infected dnTGF beta RII-CD19-MSLN-CAR-T cells (P17 + P43) is lower, indicating that the dnTGF beta RII receptor competitively resists the inhibition of T cell function by TGF beta-1 cytokines.

Example 11: inhibition of T cell killing tumor target cell by dnTGF beta RII against TGF beta-1 cytokine

(1) Experimental materials

ONE-Glo ^TM Luciferase Assay System (Promeg, 72056), dissolve substrate, 10mL per tube, frozen at-20 ℃; trypsin-EDTA (0.25%), phenol red (Thermo, 25200114) 96-well ELISA plate (Costar, 3922),enzyme label instrument (Perkin Elmer)

(2) Sample to be measured

MSLN-CAR-T cell (P01), dnTGFβRII-MSLN-CAR-T cell (P17), P01, P43 double virus infected CD19-MSLN-CAR-T cell (P01+P43), P17, P43 double virus infected dnTGFβRII-CD19-MSLN-CAR-T cell (P17+P43)

(3) Experimental method

125. Mu.L of X-VIVO was added to a 96-well plate ^TM 15 or X-VIVO ^TM 15 plus 2ng/mL TGF beta-1. Each CAR-T cell of 2e+06 was split equally into two parts, centrifuged at 300g for 5min and the supernatant discarded. One portion was taken with 1mL of X-VIVO ^TM 15 to a density of 1e+06/mL, one portion was resuspended in 1mL X-VIVO ^TM 15 plus 2ng/mL TGF-Beta1 was resuspended to a density of 1e+06/mL. Each 250. Mu.L of the culture medium was taken into the corresponding 96-well plate well, 3 replicates of each group, 125. Mu.L of the culture medium was taken out of 250. Mu.L of the cell liquid into 125. Mu.L of the culture medium well, and the mixture was mixed well, and 125. Mu.L of the culture medium was taken out of 125. Mu.L of the culture medium well, and the above-mentioned procedures were repeated 5 times. 100. Mu.L of the above-described gradient diluted cells were taken into a new 96-well plate labeled, while 3 wells were kept and only 100. Mu.L of the corresponding culture medium was added, and incubated in a cell incubator for 24 hours.

The following day the target cells SKOV3-C16-F were digested, centrifuged at 300g for 5min and the supernatant discarded. With X-VIVO ^TM 15 resuspension was adjusted to 1e+05/mL, 100. Mu.L per well was added to the corresponding 96-well plate and incubated in the cell incubator for 18h. Centrifuging at 500g for 10min, discarding 100 μL supernatant, adding 100 μL substrate, mixing, incubating at room temperature for 15min, collecting 100 μL to 96-well ELISA plate, reading fluorescence value with ELISA reader, and analyzing experimental result

(4) Experimental results

Experimental results as shown in fig. 14, each set of CAR-T cells treated with tgfβ -1 cytokines were co-incubated with target cells to assess the effect of tgfβ -1 cytokines on the ability of the CAR-T cells to kill tumor cells,

MSLN-CAR-T cells (P01), P01, P43 double virus infected CD19-MSLN-CAR-T cells (P01+P43) group after TGF beta-1 cytokine treatment has obviously reduced killing ability to target cells. Whereas CAR-T cell group with dntgfbetarii receptor: dnTGF beta RII-MSLN-CAR-T cells (P17), P17, P43 double virus infected dnTGF beta RII-CD19-MSLN-CAR-T cells (P17+P43), after TGF beta-1 cytokine treatment has little effect on target cell killing ability.

Example 12: in vivo efficacy evaluation of CAR-T cell mouse ovarian cancer subcutaneous transplantation tumor model

In order to evaluate the therapeutic effect of enhanced CAR-T cells on a mouse engraftment model, a constructed ovarian cancer cell line subcutaneous engraftment model was selected from severe immunodeficiency NCG mice (purchased by the collectable drug rehabilitation company), females, 6-8 weeks old, feeding environment: SPF stage. After one week of adaptive feeding, the mice were randomly divided into 4 groups of 6 mice each. Each mouse was used 5X 10 ⁶ The right shoulder blade part of each SK-OV3 tumor cell is inoculated subcutaneously for molding,

to average tumor volume to 150cm ³ Randomly grouping, 2X 10 mice were returned each ⁷ The body weight and the tumor size of the mice are recorded 2 to 3 times per week by CART cells, and the killing condition of different CART on SK-OV3 tumor cells in vivo is compared.

The results of tumor size and mouse weight change after mouse modeling and CAR-T cell reinfusion are shown in fig. 15 and 16. As can be seen from fig. 15, the tumor volume of the UTD-injected control mice continued to increase, the MSLN-CAR-T (P01) -injected dntgfbetarii-MSLN-CAR-T (P17) -dntgfbetarii-CD 19-MSLN-CAR-T (p17+p43) -group mice tumor volume began to decrease after one week of CAR-T cell injection, three weeks later tumor disappeared, indicating that MSLN-targeted CAR-T cells could effectively kill SK-OV3 tumor cells in the mouse engraftment tumor model. Figure 16 shows that the mice of each group injected with CAR-T cells did not lose weight, the mice were in a normal state of mind, and the hair was in a normal state, indicating that the injected MSLN-CAR-T had no significant toxic side effects on the mice.

Example 13: pharmacokinetic detection of CAR-T cells in vivo in transplanted tumor mice

Proliferation assay of CAR-T cells in transplanted tumor mice, NCG mice (purchased from collectable drug rehabilitation), females, 6-8 weeks, feeding environment: SPF stage. After one week of adaptive feeding, the mice were randomly divided into 4 groups of 6 mice each. Each mouse was used 5X 10 ⁶ Subcutaneous inoculation and molding of right shoulder blade part of SK-OV3 tumor cells until the average tumor volume is 150cm ³ Randomly grouping, 2X 10 mice were returned each ⁷ After the CART cells are infused back, the blood of the mice is collected by the orbital veins on days 1, 7, 14 and 21, and the quantitative PCR method is used for detecting the MSLN-CAR molecular copy number of the CART cells in the peripheral blood of the mice so as to reflect the expansion number of the CAR-T cells in the mice after the specific stimulation of SK-OV3 tumor cells in the mice. As shown in fig. 17, there was no significant expansion after UTD cell reinfusion in the control group, MSLN-CAR-T (P01) group, dntgfbetarii-MSLN-CAR-T (P17) group, dntgfbetarii-CD 19-MSLN-CAR-T (p17+p43) group, CAR-T cells were significantly expanded on day 7 after reinfusion, and CAR-T cells returned to basal levels on day 14.

Example 14: establishment of human SK-OV-3 cell line NCG mouse subcutaneous transplantation tumor model and pharmacodynamics of CAR T cells on model

1. Purpose of experiment

Pharmacodynamic evaluation of CAR-T cell drug antitumor effect was performed on this tumor model by subcutaneously seeding NCG mice with human ovarian adenocarcinoma cells (SK-OV-3 cells).

2. Experimental materials

2.1 cell lines

SK-OV-3 cells from Shanghai Reindeer Biotechnology Co., ltd under conditions of Mc 'oy's 5a medium with 10% heat-inactivated Fetal Bovine Serum (FBS), and placing at 37deg.C and 5% CO ₂ Culturing in an incubator. Cells in log phase will be used for subcutaneous tumor inoculation in NCG mice.

2.2 laboratory animals

2.2.1 laboratory animal sources

162 NCG mice, females, weighing approximately 18-24g, were purchased from Jiangsu Jiugang Biotech Inc. Feeding environment: SPF stage. The animals were acclimatized for at least 3 days prior to the official experiments.

2.2.2 laboratory animal feeding

All experimental mice are fed into an IVC constant temperature and constant pressure system of an SPF-class animal house, wherein the temperature is 20-26 ℃, the humidity is 40-70%, and the illumination period is 12 hours and is dark. And raising no more than 6 mice in each cage box, wherein the size of the cage box is 325mm multiplied by 210mm multiplied by 180mm, and the cage box is filled with the padding material which is autoclaved corncob, and the replacement is carried out twice a week. During the whole experimental process, all experimental mice can eat and drink freely, the feed is sterilized by Co60 irradiation, and the drinking water is sterilized under high pressure, so that the feed and the drinking water are kept in sufficient supply. All people who come in and go out of the animal feeding room or experiment operators wear sterilizing experiment clothes, disposable medical masks and gloves. Each feeder cage has a corresponding well-defined detail tag, the tag content comprising: IACUC approval number ldiacauc 006, number of animals, sex, strain, date received, project number, group, current experimental stage and person in charge of the experiment, etc. All experimental animals will be acclimatized for at least 3 days prior to experimental use.

2.3 test drug

The test agents were performed on 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, and P17+P43 cells are the same as described in example 4, and C1+P43 cells are cells dnTGFβRII (C1) +pCDH-CD19-CAR-tEGFR (P43) double-virus-infected dnTGFβRII-CD19-MSLN-CAR-T cells (C1+P43), and C2+P43 cells are cells dnTGFβRII-CD19-MSLN-CAR-T cells (C2+P43) double-virus-infected with pCDH-MSLN-CAR-dnTGFβRII (C2) +pCDH-CD19-CAR-tEGFR (P43) double-virus-infected. The pCDH-CD 19-CAR-tgfr (P43) virus used for infection of c1+p43 cells and c2+p43 cells was identical to that described in example 2 and example 4, the basic structure of C1 and C2 was identical to that of lentivirus P17 described in example 2 and example 4, except that the single chain antibody scFv sequence targeting MSLN in the C1 structure was as set forth in SEQ ID NO:101, the scFv sequence of the single-chain antibody targeting MSLN in the C2 structure is shown in SEQ ID NO: shown at 90.

3. Experimental procedure and method

3.1 Culture of SK-OV-3 tumor cells

SK-OV-3 cells were cultured in Mc 'oy'5A medium supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS), and placed at 37deg.C in 5% CO ₂ Culturing in an incubator. Cells in logarithmic growth phase to be used in NCG mouse skinLower tumor inoculation.

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

SK-OV-3 cells in the logarithmic growth phase were harvested at an inoculum size of 8X 106 cells/mouse, respectively, and inoculated at a cell volume of 0.2 ml/mouse (50% matrigel) and inoculated subcutaneously on the right side of NCG mice. When the tumor volume of the mice grows to 150-200mm ³ Groups were performed according to mouse tumor volume, 108 were selected, randomly divided into 18 groups of 6, and day 0 (D0) of group administration. D0, in the low dose group (G11-G18), before CAR-T injection, tail vein injection was 1X 10 ⁶ NALM6 cells (3 h after MMC treatment) and NALM6 cells treated similarly were injected at D7, D14, and D21. Detailed methods of administration, doses and routes of administration are shown in Table 2.

TABLE 2 grouping and administration

High dose dosing group:

note that: n: the number of animals used; v. the following: administering by tail vein injection; and (i) p: intraperitoneal injection administration

Dosing volume: the dosing volume was adjusted to 200 μl based on the tumor-bearing mice body weight.

Low dose dosing group:

note that: n: the number of animals used; v. the following: tail intravenous injection administration

Day0: administration on day0

3.3 experimental observations

Throughout the experiment, the use and observation of the experimental animals were performed in accordance with the relevant regulations for AAALAC animal use and administration. After the experimental animals are inoculated with tumor tissues, the experimental animals are observed every day, and the morbidity and the mortality of the experimental animals are recorded. All experimental animals were monitored and recorded for behavior, feeding, water intake, weight changes, hair shine and some other abnormalities during the course of the routine experiment.

3.4 evaluation index

The method is mainly used for detecting the growth inhibition effect or the complete cure capacity of the CAR-T on the SK-OV-3 cell line tumor model by the tumor growth curve of the SK-OV-3 cells on NCG mice.

3.4.1 tumor volumes and tumor-bearing mouse body weight measurements: using vernier calipers for twice weekly measurement, the calculation formula of tumor volume is v=0.5a×b2, a, b represent the long diameter and the wide diameter of the tumor respectively;

3.4.2 tumor growth inhibition ratio TGI (%) =100- (delta T/delta C) ×100=100- (Ti-T0)/(Vi-V0) ×100%, ti is the mean tumor volume after the CAR-T cell group starts administration, T0 is the mean tumor volume at the first administration of the CAR-T cell group, vi is the mean tumor volume after the vehicle control group starts administration, and V0 is the mean tumor volume at the first administration of the vehicle control group; T/C% = TRTV/CRTV x 100%, RTV = Vt/V0, T/C% is the tumor relative proliferation rate, and T and C are the Tumor Volumes (TV) of the dosing group and the control group, respectively, at a certain time point.

3.4.3 body weights of all tumor-bearing mice were measured twice a week. Weight change formula RCBW% = (BWi-BW 0)/bw0×100%, BWi being the current weight of the mice, BW0 being the weight of the mice on the day of the group;

3.5 sample collection

Pharmacodynamic groups 100 μl of blood was collected orbit using EDTA anticoagulant tubes D1, D7, D14, D21, D28 days after CAR-T injection.

3.6 data analysis

All data are expressed as mean±sem and One-Way ANOVA was used to compare the presence or absence of significant differences in tumor volume between the dosed and control groups. All data were analyzed with Graphpad and P < 0.05 was considered significant differences.

4. Experimental results

At 22 days of administration, some mice had tumor volumes exceeding 2000mm ³ It was euthanized and all statistics were taken at day 22.

On day 22 post-dose, the average tumor volume of the PBS control group was 1861.70 + -178.19 mm ³ HD UTD,2×107/mouse-administered group, HD P43,2×107/mouse-administered group, HD P01,2×107/mouse-administered group, HD P17,2×107/mouse-administered group, HD P01+P43,2×107/mouse-administered group, HD P17+P43,2×107/mouse-administered group, i.v. administered group, HD C1+P43,2×107/mouse-administered group, HD C2+P43,2×107/mouse-administered group, HD P17+P43,2×107/mouse, i.p. administered 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 Group of mouse administration, LD P01+P43,5×10 ⁶ Mouse administration group, LD C1+P43,5×10 ⁶ Mouse administration group and LD C2+P43,5×10 ⁶ Tumor mean volumes of the/mouse-administered groups were 1761.94.+ -. 157.75mm, respectively 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 ³ 、42.39±1.24mm ³ 、2115.27±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 220.16 + -111.43 mm ³ . Tumor Growth Inhibition (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%, respectively, compared to its control group.

HD P01,2×10 compared to PBS control group (day 22) ⁷ Mouse administration group, HD P17, 2X 10 ⁷ Mouse administration group, HD P01+P43,2×10 ⁷ Group/mouse administration, HD P17+P43,2×10 ⁷ Mouse, i.v. dosing group, HD C1+P43,2×10 ⁷ Mouse administration group, HD C2+P43,2×10 ⁷ Group/mouse administration, HD P17+P43,2×10 ⁷ Mouse, i.p. administration group, LD P17, 5X 10 ⁶ Group of mouse administration, LD P01+P43,5×10 ⁶ Mouse administration group, and LD C2+P43,5×10 ⁶ The tumor volume of the mouse administration group is obviously reduced (P is less than 0.001), which shows that the CAR-T has very obvious tumor inhibiting effect in mice and longer tumor inhibiting effect.

And on day 36 after administration, the experiment was ended, HD P17,2×10 ⁷ Mouse administration group, HD P01+P43,2×10 ⁷ Group/mouse administration, HD P17+P43,2×10 ⁷ The tumor of the i.p. administration group completely regressed, and showed very remarkable tumor-inhibiting effect.

In the course of this experiment, LD P43,5×10 ⁶ Mouse-administered group 70# mice and LD C1+P43,5×10 ⁶ Abnormal death of mice # 102 in the mouse-administered group was not significantly abnormal due to individual differences in mice, and the body weight and preclinical behaviours of the remaining mice in each group were not significantly changed, indicating that tumor-bearing mice had good tolerance to each of the test drugs at the test dose (see fig. 18, 19).

5. Summary and discussion of the experiments

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

The pharmacodynamics experiment result shows that compared with the control group, the HD P01,2 multiplied by 10 ⁷ Mouse administration group, HD P17, 2X 10 ⁷ Mouse administration group, HD P01+P43,2×10 ⁷ Group/mouse administration, HD P17+P43,2×10 ⁷ Mouse, i.v. dosing group, HD C1+P43,2×10 ⁷ Mouse administration group, HD C2+P43,2×10 ⁷ Group/mouse administration, HD P17+P43,2×10 ⁷ Mouse, i.p. administration group, LD P17, 5X 10 ⁶ Group of mouse administration, LD P01+P43,5×10 ⁶ Mouse administration group, LD P17+P43,5×10 ⁶ Mouse administration group and LD C2+P43,5×10 ⁶ The tumor volume of the mouse administration group is obviously reduced (P is less than 0.001), which shows that the CAR-T has very obvious tumor inhibiting effect in mice and longer tumor inhibiting effect.

Meanwhile, the weight and pre-clinical behaviours of all mice in each group have no obvious abnormal change, which indicates that the tumor-bearing mice have good tolerance to various tested drugs under the test dose.

EXAMPLE 15 enrichment and screening of MSLN antibodies Using phage antibody libraries

Specific antibody clones targeting MSLN protein were enriched from phage antibody libraries using appropriate negative and positive panning strategies.

Phage antibody libraries used were self-constructed by the applicant, including natural libraries, semisynthetic libraries and single domain libraries. The semisynthetic phage antibody library is used together with the natural library to solve the problem that the natural library may lack MSLN high affinity antibody clones. The single domain phage antibody library is an antibody library consisting of only the variable region amino acids of heavy chain antibodies, which has a molecular weight of only 12-15kDa, but has similar or higher specificity and affinity than conventional antibodies.

A significant increase in recovery was observed for each panning using a different antibody pool, demonstrating an effective enrichment of antibody clones, followed by screening of specific clones from the enriched phage pool using enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS).

Purpose and principle of screening: phage pools enriched by the affinity panning step contain phage antibodies of various properties: specific clones, non-specific clones and negative clones. To obtain specific clones, we need to isolate the monoclonal from them, package the monoclonal phage, and perform a preliminary screening of a large number of monoclonal by enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS), from which to select the monoclonal that specifically binds both MSLN protein and MSLN positive cell line CHO-K1-MSLN. The specific monoclonal antibody sequence contained therein was further determined by DNA sequencing.

In ELISA primary screening, the biotinylated target protein KACTUS-MSLN-Bio (Glu 296-Gly 588) was brought closer to the native antigen conformation in the reaction solution by binding of Streptavidin to Biotin Biotin. Only KACTUS-MSLN-Bio (Glu 296-Gly 588) and not Streptavidin was identified as specific clones, and some of these specific clones bound to KACTUS-MSLN-Bio (Glu 296-Asn 494). FACS preliminary screening was performed using the positive cell line CHO-K1-MSLN with high expression of MSLN and the cell line CHO-K1 negative for MSLN, which was identified as a specific clone binding only CHO-K1-MSLN cells and not CHO-K1 cells. By both ELISA and FACS primary screening we could obtain candidate antibodies that bind both to the recombinantly expressed KACTUS-MSLN-Bio (Glu 296-Gly 588) protein and recognize the MSLN molecule in its native state on the cell surface for subsequent further screening.

Brief steps of ELISA experiments:

1) Culturing and packaging monoclonal phage with deep hole 96-well plate;

2) Diluting strepitavidin to 2 mug/mL with PBS, adding 100 mug/hole into a high-binding ELISA plate, and binding for 2h at room temperature;

3) Discarding the coating liquid, adding 250 mu L of sealing liquid into each hole, and sealing at 4 ℃ overnight;

4) Washing the plate 2 times with 250 mu L rinsing liquid;

5) Diluting the target protein with biotin label to 2 mug/mL by PBS, adding 100 mug/hole into the pre-coated strepitavidin ELISA plate, and combining for 0.5h at room temperature;

6) Washing the plate 2 times with 250 mu L rinsing liquid;

7) Adding 100 mu L of the cultured phage supernatant obtained in the step 1) to the well coated with the target antigen, and combining for 2 hours at 4 ℃;

8) Washing the plate 4 times with 250 μl of rinse solution;

9) Adding 1:2000 dilution of mouse anti-M13 primary antibody, 100 mu L/well, and incubating for 45min at room temperature;

10 250. Mu.L rinse wash plate 4 times;

11 Adding HRP Donkey anti-mouse IgG secondary antibody with 1:2000 dilution, 100 μl/well, and incubating at room temperature for 45min;

12 250. Mu.L rinse wash plate 6 times;

13 Adding 100 mu L of TMB chromogenic substrate, and developing for 5 to 10min;

14 100. Mu.L of 2M H) ₂ SO ₄ The reaction was terminated and the results were read on a microplate reader.

Brief step of FACS preliminary screening experiment:

1) Culturing and packaging monoclonal phage with deep hole 96-well plate;

2) CHO-K1-MSLN and CHO-K1 cells were washed 2 times with PBS and resuspended to 1X10 with PBS ⁷ The concentration of the solution is/mL, and 50 mu L of the solution is split into 96-well U-bottom hole plates;

3) Adding 50 mu L of packaged monoclonal phage into each hole, uniformly mixing, and combining at 4 ℃ for 1h;

4) Washing 2 times with 180. Mu.L PBS;

5) Adding 1:2000 diluted mouse anti M13 primary antibody, 100 mu L/hole, blowing and mixing uniformly, and incubating at 4 ℃ for 30min;

6) Washing 1 time with 180. Mu.L PBS;

7) Adding FITC horse anti mouse-IgG (H+L) secondary antibody with the dilution of 1:300, blowing and mixing at 100 mu L/hole, and incubating at 4 ℃ for 30min;

8) Washing 2 times with 180. Mu.L PBS; finally, the cells were resuspended with 100. Mu.L PBS;

9) And detecting the fluorescence intensity of the FITC channel of the sample on a flow cytometer, and analyzing the result.

Main materials and reagents:

helper phage KO7, thermo/Invitrogen,18311019

Streptavidin(SA)，Pierce，21125

KACTUS-MSLN-Bio(Glu296-Gly588)，Biotinylated Human Mesothelin，Avitag ^TM ，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

Sealing liquid: PBS+3% BSA

Rinsing liquid: 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:

after randomly selecting monoclonal from the enriched phage antibody pool and packaging into phage, detecting the binding of the monoclonal phage to KACTUS-MSLN-Bio protein, KACTUS-MSLN-Bio (Glu 296-Asn 494) protein and SA protein by phage ELISA, and finding out specific phage antibody clones which are only bound to KACTUS-MSLN-Bio (Glu 296-Gly 588), are not bound to KACTUS-MSLN-Bio (Glu 296-Asn 494) or are bound to KACTUS-MSLN-Bio (Glu 296-Gly 588) and KACTUS-MSLN-Bio (Glu 296-Asn 494) simultaneously. The ELISA results of the partial clones are shown in FIG. 20. Wherein the negative control is the negative control of phage, the positive control 1 is the positive control added with MSLN antibody MSLN Ab (RD), and the positive control 2 is the positive control added with MSLNAb (Biolegend); as can be seen from the figure, clones G1 to G9 bind well to the target antigen KACTUS-MSLN-Bio (Glu 296-Gly 588) and not to the control antigen streptavidin Streptavidin (SA), and have good specificity, and among them, clones G2, G4 to G7 bind also to KACTUS-MSLN-Bio (Glu 296-Asn 494), indicating that these clones can bind to different regions of the MSLN antigen and have good specificity. G10 clone was negative clone, which did not bind to the target antigen KACTUS-MSLN-Bio (Glu 296-Gly 588), KACTUS-MSLN-Bio (Glu 296-Asn 494) and streptavidin Streptavidin (SA).

The results of FACS preliminary screening of partial clones are shown in fig. 21. Wherein the negative control is a negative control of phage; as can be seen from the figures, clones G1 to G9 bind to the MSLN positive cell line CHO-K1-MSLN, do not bind to the MSLN negative cell line CHO-K1, are specific clones, and clone G10 is a negative clone (does not bind to 2 cells).

By ELISA detection and FACS preliminary screening, we obtained 359 specific clones in total.

EXAMPLE 16 identification of monoclonal specificity by FACS Using multiple cell lines

Experimental purposes and principles: antibodies for therapeutic use must have very good target specificity, bind only to the target antigen, and not to any unrelated antigen; on the other hand, the amino acid sequence of the same antigen on different cell lines will differ (isomer or mutant) or the ligand bound will be different, and it will be necessary to examine whether our antibodies bind to cells positive for various target proteins. To further analyze the specificity and universality of these monoclonal clones, we further assessed the specificity of the primary clones by flow cytometry, looking for the best candidate clones. In this experiment we used a variety of MSLN positive and a variety of MSLN negative cell lines to react with these monoclonal phage antibodies to analyze whether these clones could bind to the MSLN antigen on the cell line and any non-specific binding to other cell lines that do not express MSLN. Through this experiment we obtained several clones with good specificity.

The experimental method comprises the following steps: the same as FACS prescreening;

major samples and reagents:

CHO-K1-MSLN, OVCAR-3, aspc-1 cell line, MSLN positive cell line (+);

CHO-K1, nalm6 cell line, MSLN negative cell line (-);

the remaining reagents were identical to FACS prescreening.

Experimental results:

antibodies for therapeutic use must have very good target specificity. To further analyze the specificity of these monoclonal antibodies, we identified the unique clone obtained in example 2 on more antigens and cell lines using enzyme-linked immunosorbent and flow cytometry. The results are shown in FIGS. 22A, 22B, 22C, 22D, in which negative controls were negative phage antibody clones, and it was found that clones #2, #3, #5, #7, #8, #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, #90, #91, #93, #96, #97, #107, #110 (FIG. 22C), #116, #118, #119, #120, #121, #122 (FIG. 22D) were all bound to the MSLN positive cell line CHO-K1-MSLN, and none were bound to the MSLN negative cell line, clones #24, #26, #28, #29 (FIG. 22A), #30, #43 (FIG. 22B), #86, #96 (FIG. 22C), #118-122 (FIG. 22D) were also combined with the MSLN positive cell line OVCAR-3, clones #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) were also combined with the MSLN positive cell line ASPC-1, and the specificity was good.

EXAMPLE 17 identification of monoclonal specificity by ELISA Using different antigens

Experimental purposes and principles: antibodies for use in therapy must have very good target specificity, bind only to the target antigen, and not to any unrelated antigen. To further analyze the specificity and universality of these monoclonal clones, we further assessed the specificity of the primary clones by enzyme-linked immunosorbent assay (ELISA) to find the best candidate clone. In this experiment we used MSLN antigens purchased from different companies and a number of MSLN-unrelated antigens to react with these monoclonal phage antibodies to analyze whether these clones could bind to different MSLN antigens and whether there was any non-specific binding to other MSLN-unrelated antigens. Through this experiment we obtained several clones with good specificity.

The experimental method comprises the following steps: the same as ELISA primary screening;

the main samples and reagents are shown in table 3:

TABLE 3 Table 3

The remaining reagents were identical to ELISA primary screen.

Experimental results:

antibodies for therapeutic use must have very good target specificity. To further analyze the specificity of these monoclonal antibodies, we identified the clones obtained in example 2 on a variety of antigens using enzyme-linked immunosorbent assay (ELISA). The results are shown in fig. 23A, 23B, 23C, 23D, the negative control was that of phage, positive control 1 was that of MSLN antibody MSLN Ab (RD) added, and positive control 2 was that of MSLN Ab (Biolegend) added; as can be seen from the figure, clone #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 (FIG. 23C), #116, #118, #119, #120, #121, #122 (FIG. 23D) strongly bound to KACTUS-MSLN-Bio (Glu 296-Gly 588), and not associated antigens KACTUS BAFFR-Bio, KACTUS CD5-Bio and SA, wherein #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 (fig. 23C) clone and KACTUS-MSLN-Bio (Glu 296-Asn 494) protein, indicating that these clones can bind to different regions of MSLN antigen and are well specific; in addition, clone #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 (FIG. 23C), #116, #119, #122 (FIG. 23D) were combined with ACRO-MSLN-cyno, there were monkey crossings, #28, #29 (FIG. 23A), #30, #39, #46, #75, #76, #77 (FIG. 23B), #84, #85, #86, #91, #93, #97, #107, #110 (FIG. 23C) were combined with ACRO-MSLN-mo, and there were mouse crossings.

EXAMPLE 18 characterization of antibody specificity by FACS using multiple cell lines after recombinant expression

Experimental purposes and principles: antibodies for therapeutic use must have very good target specificity, bind only to the target antigen, and not to any unrelated antigen; on the other hand, the amino acid sequence of the same antigen on different cell lines will differ (isomer or mutant) or the ligand bound will be different, and it will be necessary to examine whether our antibodies bind to cells positive for various target proteins. To further analyze the specificity and universality of these monoclonal clones, we found the best candidate clones by recombinant expression, i.e. constructing the specific sequences identified on phage level on expression vectors, electrotransferring into cells for culture, collecting the supernatant of cell expression, and further evaluating the specificity of the clones on protein level by flow cytometry. In this experiment we used a variety of MSLN positive and a variety of MSLN negative cell lines to react with these recombinantly expressed antibodies to analyze whether these clones can bind to the MSLN antigen on the cell line and whether there is any non-specific binding to other cell lines that do not express MSLN. Through this experiment we obtained several clones with good specificity.

Brief steps of FACS experiment:

1) Preparing a recombinant expressed protein supernatant to be identified;

2) Cells to be used were washed 2 times with PBS and resuspended to 5X10 with PBS containing 5% FBS ⁶ The concentration of the solution is/mL, and 100 mu L of the solution is split into 96-well U-bottom hole plates;

3) Placing at 4deg.C, and sealing for 30min;

4) Adding protein supernatant, and incubating for 1h at 4 ℃;

5) Washing 2 times with 180. Mu.L PBS;

6) Adding Anti-human IgG (647-conjugated) secondary antibody at a ratio of 1:300, blowing and mixing at 100 μl/well, and incubating at 4deg.C for 30min;

7) Washing 2 times with 180. Mu.L PBS; finally, the cells were resuspended with 100. Mu.L PBS;

8) The fluorescence intensity of the sample APC channel was measured on a flow cytometer, and the results were analyzed.

Major samples and reagents:

CHO-K1-MSLN, nalm6-977, ovcar-3, aspc-1 cell line, MSLN positive cell line (+);

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

anti-human IgG (647-conjugated) secondary antibody, jackson, cat: 146154

Experimental results:

antibodies for therapeutic use must have very good target specificity. To further analyze the specificity of these monoclonal antibodies, we expressed the specific clone recombinations obtained in examples 4 and 5 as protein supernatants and then identified on more antigens and cell lines using enzyme-linked immunosorbent assay and flow cytometry. The results are shown in FIGS. 24A, 24B, 24C, in which negative controls are those without protein supernatant plus only secondary antibodies, and it can be seen that #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) were all bound to 2 MSLN positive cell lines CHO-K1-MSLN, nalm6-977, and that Median Fluorescence Intensity (MFI) was relatively strong, and that MSLN negative cell lines were all not bound, with low MFI, and that specific clones, among which #5, #8, #14, #24, #26, #28, #29, #30, #33 (FIG. 24A) #55, #76, #87, #90, # 21, #96, # 21 (FIG. 24B, #96, # and #96, #93, #3, # and, # 93) were specifically; clones #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) were also combined with the MSLN positive cell line ASPC-1; in addition, clones #34, #39, #40, #43, #46 (FIG. 24B), #97, #107, #110, #118, #121, #120, #122 (FIG. 24C) had weak binding to MSLN negative cell lines, and were non-specific antibodies, which did not meet the experimental requirements.

EXAMPLE 19 identification of antibody specificity by ELISA Using different antigens after recombinant expression

Experimental purposes and principles: antibodies for use in therapy must have very good target specificity, bind only to the target antigen, and not to any unrelated antigen. To further analyze the specificity and universality of these monoclonal clones, we found the best candidate clones by recombinant expression, i.e. constructing the specific sequences identified on phage level on expression vectors, electrotransferring into cells for culture, collecting the supernatant of cell expression, and further evaluating the specificity of the clones on protein level by enzyme-linked immunosorbent assay (ELISA). In this experiment we used MSLN antigens purchased from different companies and a number of MSLN-unrelated antigens to react with recombinantly expressed antibodies to analyze whether these clones could bind to different MSLN antigens and whether there was any non-specific binding to other MSLN-unrelated antigens. Through this experiment we obtained several clones with good specificity.

Brief steps of ELISA experiments:

1) Preparing a recombinant expressed protein supernatant to be identified;

4) Washing the plate 2 times with 250 mu L rinsing liquid;

6) Washing the plate 2 times with 250 mu L rinsing liquid;

7) Adding 100 mu L of the protein supernatant prepared in the step 1) to the well coated with the target antigen, and combining for 45min at 4 ℃;

8) Washing the plate 4 times with 250 μl of rinse solution;

9) Adding 1:2000 diluted anti-human-HRP secondary antibody, 100 mu L/hole, and incubating for 45min at room temperature;

10 250. Mu.L rinse wash plate 6 times;

11 Adding 100 mu L of TMB chromogenic substrate, and developing for 5 to 10min;

12 100. Mu.L of 2M H) ₂ SO ₄ The reaction was terminated and the results were read on a microplate reader.

The main samples and reagents are shown in table 4:

TABLE 4 Table 4

The remaining reagents were identical to ELISA primary screen.

Experimental results:

antibodies for therapeutic use must have very good target specificity. To further analyze the specificity of these monoclonal antibodies, we expressed the specific clone recombinations obtained in examples 4 and 5 as protein supernatants and then identified using enzyme-linked immunosorbent assay (ELISA). The results are shown in fig. 25A, 25B, 25C, where the secondary antibody control is a control without protein supernatant plus only secondary antibody, positive control 1 is a positive control plus MSLN antibody MSLN Ab (RD), positive control 3 is a positive control plus HUYP218 (MSLN clinically positive antibody); as can be seen from the figure, clone numbers #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 (FIG. 25C) bind to the KACTUS-MSLN-Bio (Glu 296-Gly 588) protein to a different extent and to the non-associated antigen KACTUS BAFFR-Bio, KACTUS IL10-Bio and SA did not bind, with #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 bound to different degrees to KACTUS-MSLN-Bio (Glu 296-Asn 494) proteins, indicating that these clones can bind to different regions of the MSLN antigen and are well specific; in addition, clones #2, #12, #14, #24, #26, #28, #29, #30, #33 (FIG. 25A), #34, #39, #40, #46, #75, #76, #77, #84, #85, #86, #87 (FIG. 25B), #91, #93, #107, #110, #116, #119, #122 (FIG. 25C) were combined with ACRO-MSLN-cyno, there were monkey crossings, #28, #29, #30, #33 (FIG. 25A), #34, #39, #43, #46, #76, #77, #84, #85, #86 (FIG. 25B), #91, #93, #107 (FIG. 25C) were combined with ACRO-MSLN-mosuse, and there were mouse crossings. At the same time clone #97 (FIG. 25C) did not meet the experimental requirements as it bound weakly non-specifically to the MSLN-unrelated antigen.

The invention uses the fully human phage to screen the antibody, directly obtains the fully human monoclonal antibody. Compared with the traditional hybridoma technology, the method omits the difficult step of humanized murine antibody, and the fully humanized antibody has lower immunogenicity than the humanized murine antibody, and has better potential in the application of antibody drugs, detection reagents and the like.

Example 20 affinity assay of antibodies

Experimental purposes and principles:

the magnitude of the affinity between the antibody and antigen may have an important effect on the killing and duration of the CAR-T or antibody drug in the patient, and to determine this important property we used the Sartorius molecular interaction technique (BLI) to screen examples 16-19 for clones #2, #5, #118 and #119, and the reference antibody huYP218 for affinity measurements.

The biomembrane interference technology applied by the system is a label-free technology and provides high-flux biomolecular interaction information in real time. The instrument emits white light onto the sensor surface and collects reflected light, the reflection spectrum of different frequencies being affected by the thickness of the optical film of the biosensor, some of the reflected light at frequencies forming constructive interference (blue) and others being subject to destructive interference (red). These interferences are detected by a spectrometer and form an interference spectrum, which is displayed as phase shift intensities (nm) of the interference spectrum. Therefore, once the number of molecules bound to the sensor surface increases or decreases, the spectrometer detects the shift of the interference spectrum in real time, and the shift directly reflects the thickness of the biological film on the sensor surface, so that high-quality data of the biological molecular interaction can be obtained, and the biological molecular interaction kinetic parameter measurement (Kon, kdis and KD) can be carried out, thereby providing important information for the research and development process.

Brief experimental procedure:

1) anti-MSLN IgG (composed of MSLN scFv sequence fused to human IgG4 Fc) was diluted to 10. Mu.g/mL with loading buffer (1 XPBS, pH 7.2,0.1% BSA and 0.02% Tween 20) and loaded onto the biosensor.

2) After the 300s equilibration period, the binding kinetics of the MSLN antibodies and MSLN antigen multi-concentrations (see table 5) were monitored. Binding and dissociation 60s were performed in parallel at each concentration, respectively.

TABLE 5 detection conditions

Remarks: mAb 02 was obtained by fusing the #2 cloned scFv sequence with human IgG4Fc, similarly to mAb 05, mAb 118, mAb 119.

3) The chip was regenerated by washing 3 times with 10mM Glycine-HCl, pH 1.5.

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 binding of a single molecule to its ligand, typically measured and reported by the equilibrium dissociation constant (KD), which can be used to assess the strength of interaction between two molecules and order this. 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: clone #2, #5, #118 and #119 all bind to the MSLN antigen, and clone #119 has slightly higher affinity than the other clones.

TABLE 6 affinity assay results

Ligand	KD(M)	kon(1/Ms)	kdis(1/s)	R^2
huYP218	2.36E-09	2.97E+05	7.00E-04	0.9983
mAb 02	3.19E-07	2.69E+05	8.90E-04	0.9941
mAb 05	2.51E-07	4.52E+05	1.13E-01	0.9971
mAb 118	1.15E-06	2.83E+05	3.27E-01	0.9933
mAb 119	4.32E-09	2.26E+05	9.76E-04	0.9991

Reference to the literature

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3.Fry，T.J.，et al.(2017“."CD22-targeted CAR T cells induce remission in B-ALL that Iive or resistant to CD19-targeted CAR immuno”herapy." Nat Med.

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Claims

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

(a) A first class of Chimeric Antigen Receptors (CARs);

(b) A second class of Chimeric Antigen Receptors (CARs);

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

(d) Truncated forms of EGFR molecules (tgfr).
The immune cell of claim 1, wherein the first type of CAR is a fully human CAR.
The immune cell of claim 1 or 2, wherein the first class of CAR is selected from the group consisting of CD19, CD20, CD22, CD5, CD7, and BCMA CAR.
The immune cell of any one of claims 1-3, wherein the second type of CAR is different from the first type of CAR and is selected from CARs of any one or more of the following targets MSLN, HER2, GPC3, egfrvlll, claudin18.2, CD70, GD2, CEA, CS1, DLL3, EGFR, erbB1, FAP, fonate Receptor, GPC1, gp100, MUC16, MUC1, NKG2D, PSCA, PSMA, ROR1, or VEGFR 2.
The immune cell of any one of claims 1-4, wherein the loss-of-function immunosuppressive receptor comprises dntgfbetarii, dnPD1.
The immune cell of any one of claims 1-5, wherein the first class of CARs comprises a first binding domain comprising one or more first antibodies or fragments thereof that specifically bind CD19, wherein the first antibodies or fragments thereof comprise a first heavy chain complementarity determining region 1 (HCDR 1), a first heavy chain complementarity determining region 2 (HCDR 2), and a first heavy chain complementarity determining region 3 (HCDR 3), the amino acid sequences of HCDR1, HCDR2, and HCDR3 being as set forth in SEQ ID NOs: 47. SEQ ID NO:48 and SEQ ID NO: shown at 49.
The immune cell of claims 1-6, wherein the first antibody or fragment thereof further comprises a first light chain complementarity determining region 1 (LCDR 1), a first light chain complementarity determining region 2 (LCDR 2), and a first light chain complementarity determining region 3 (LCDR 3), the amino acid sequences of LCDR1, LCDR2, and LCDR3 being as set forth in SEQ ID NOs: 50. SEQ ID NO:51 and SEQ ID NO: 52.
The immune cell of any one of claims 1-7, wherein the second class of CARs comprises a second binding domain comprising 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 (HCDR 1), a second heavy chain complementarity determining region 2 (HCDR 2), and a second heavy chain complementarity determining region 3 (HCDR 3), the amino acid sequences of the HCDR1, HCDR2, and HCDR3 being independently selected from the group consisting of:

(1) As set forth in SEQ ID NO:37, and HCDR1 of the amino acid sequence of SEQ ID NO:38, and HCDR2 of the amino acid sequence of SEQ ID NO:39, HCDR3 of the amino acid sequence;

(2) As set forth in SEQ ID NO:58, and HCDR1 of the amino acid sequence of SEQ ID NO:59, and HCDR2 of the amino acid sequence of SEQ ID NO:60, HCDR3 of the amino acid sequence of seq id no;

(3) As set forth in SEQ ID NO:69, and HCDR1 of the amino acid sequence of SEQ ID NO:70, and HCDR2 of the amino acid sequence of SEQ ID NO:71, HCDR3 of the amino acid sequence;

(4) As set forth in SEQ ID NO:80, and HCDR1 of the amino acid sequence of SEQ ID NO:81, and HCDR2 of the amino acid sequence of SEQ ID NO:82, HCDR3 of the amino acid sequence; and

(5) As set forth in SEQ ID NO:91, HCDR1 of the amino acid sequence of SEQ ID NO:92, and HCDR2 of the amino acid sequence of SEQ ID NO:93, and HCDR3 of the amino acid sequence of seq id no.
The immune cell of any one of claims 1-8, wherein the second antibody or fragment thereof that specifically binds MSLN (mesothelin) further comprises a second light chain complementarity determining region 1 (LCDR 1), a second light chain complementarity determining region 2 (LCDR 2), and a second light chain complementarity determining region 3 (LCDR 3), the amino acid sequences of LCDR1, LCDR2, and LCDR3 being independently selected from the group consisting of:

(1) As set forth in SEQ ID NO:40, as set forth in SEQ ID NO:41, and LCDR2 as set forth in SEQ ID NO:42, LCDR3 of the amino acid sequence;

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

(3) As set forth in SEQ ID NO:72, as set forth in SEQ ID NO:73, and LCDR2 as set forth in SEQ ID NO:74, LCDR3 of the amino acid sequence;

(4) As set forth in SEQ ID NO:83, as set forth in SEQ ID NO:84, and LCDR2 as set forth in SEQ ID NO:85, LCDR3 of the amino acid sequence; and

(5) As set forth in SEQ ID NO:94, and LCDR1 of the amino acid sequence of SEQ ID NO:95, and LCDR2 as set forth in SEQ ID NO:96, and LCDR3 of the amino acid sequence.
The immune cell of any one of claims 1-9, wherein the first antigen binding domain comprises a first heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:44, and a polypeptide having the amino acid sequence shown in seq id no.
The immune cell of any one of claims 1-10, wherein the second antigen binding domain comprises a second heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 34. 54, 65, 76 and 87.
The immune cell of any one of claims 1-11, wherein the first antigen binding domain comprises a first light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 46.
The immune cell of any one of claims 1-12, wherein the second antigen binding domain comprises a second light chain variable region (VL) comprising an amino acid sequence as set forth in SEQ ID NO: 36. 56, 67, 78 and 89.
The immune cell of any one of claims 1-13, wherein the first light chain variable region and the first heavy chain variable region are joined by a first linker and/or the second light chain variable region and the second heavy chain variable region are joined 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 sequence set forth in SEQ ID NO: 9.
The immune cell of 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 of any one of claims 1-16, wherein the CD19 antigen binding domain comprises an scFv comprising an amino acid sequence of SEQ ID No:2.
the immune cell of any one of claims 1-17, wherein the MSLN binding domain comprises an scFv comprising an amino acid sequence selected from any one of the following: SEQ ID No: 5. 57, 68, 101, 79 and 90.
An immune cell according to any one of claims 1-18, wherein the dntgfbetarii receptor comprises the amino acid sequence of SEQ ID No:28 or a functional variant thereof.
The immune cell of any one of claims 1-19, wherein the truncated form of the EGFR molecule comprises the amino acid sequence of SEQ ID No:27 or a functional variant thereof.
The immune cell of any one of claims 1-20, wherein the first type of CAR is co-expressed in the immune cell with the immunosuppressive receptor by 2A peptide action and the second type of CAR is co-expressed in the immune cell with the tgfr by 2A peptide action.
The immune cell of any one of claims 1-21, wherein the 2A peptide is selected from P2A, T a or F2A, and wherein the P2A comprises the amino acid sequence of SEQ ID NO:29 or a functional variant thereof, said T2A comprising the amino acid sequence set forth in SEQ ID NO:30 or a functional variant thereof.
The immune cell of any one of claims 1-22, wherein the first and/or second transmembrane domain comprises a polypeptide selected from the group consisting of: the 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 immune cell of any one of claims 1-23, wherein the first and/or second transmembrane domain comprises SEQ ID No:13 or a functional variant thereof.
The immune cell of 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 of any one of claims 1-25, wherein the first and/or second co-stimulatory domain comprises SEQ ID No:15 or a functional variant thereof.
The immune cell of any one of claims 1-26, wherein the first and/or second intracellular signaling domain comprises a signaling domain from CD3 z.
The immune cell of any one of claims 1-27, wherein the first and/or second intracellular signaling domain comprises SEQ ID No:17 or a functional variant thereof.
The immune cell of any one of claims 1-28, wherein the first and/or second CAR further comprises a hinge region that connects the first antigen binding domain and the first transmembrane domain or the second antigen binding domain and the second transmembrane domain.
The immune cell of any one of claims 1-29, wherein the hinge region comprises SEQ ID No:11 or a functional variant thereof.
The immune cell of any one of claims 1-30, wherein the first and/or second CAR is further linked to a signal peptide.
The immune cell of any one of claims 1-31, wherein the signal peptide comprises the amino acid sequence of SEQ ID No:8 or a functional variant thereof.
The immune cell of any one of claims 1-32, wherein the first class CAR thereof comprises the amino acid sequence of SEQ ID No:19 or a functional variant thereof, and/or the second CAR comprises the amino acid sequence set forth in SEQ ID No: 32. 97, 98, 99 and 100 or a functional variant thereof.
An isolated nucleic acid molecule encoding any one of the components expressed on the immune cells of any one of claims 1-33, or a combination thereof.
The isolated nucleic acid molecule of claim 34, 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, or a functional variant thereof.
A vector comprising the nucleic acid molecule of claim 34 or 35.
The vector of claim 36, wherein the vector is selected from the group consisting of a plasmid, a retroviral vector, and a lentiviral vector.
The immune cell of claims 1-33, wherein the immune cell is selected from the group consisting of a T lymphocyte and a Natural Killer (NK) cell.
A method of making an immune effector cell comprising introducing into an immune cell the vector of claim 36.
A pharmaceutical composition comprising the immune cell of any one of claims 1-33.
Use of the immune cell of any one of claims 1-33, or the nucleic acid molecule of claim 34 or 35, or the vector of claim 36 or 37, for the manufacture of a medicament for treating a disease or disorder associated with expression of a second class of CAR-targeted target antigens.
The use according to claim 41, wherein the disease or disorder associated with expression of a second class of CAR-targeted target antigen is cancer or malignancy.
The use according to claim 42, wherein the tumour is a solid tumour.
A MSLN (mesothelin) -targeted CAR comprising an antigen binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain, the antigen binding domain comprising a combination of one or more antibodies or fragments thereof that specifically bind MSLN (mesothelin), wherein each of the antibodies comprises a heavy chain complementarity determining region 1 (HCDR 1), a heavy chain complementarity determining region 2 (HCDR 2), and a heavy chain complementarity determining region 3 (HCDR 3), the amino acid sequences of HCDR1, HCDR2, and HCDR3 being independently selected from the group consisting of:

(1) As set forth in SEQ ID NO:58, and HCDR1 of the amino acid sequence of SEQ ID NO:59, and HCDR2 of the amino acid sequence of SEQ ID NO:60, HCDR3 of the amino acid sequence of seq id no;

(2) As set forth in SEQ ID NO:69, and HCDR1 of the amino acid sequence of SEQ ID NO:70, and HCDR2 of the amino acid sequence of SEQ ID NO:71, HCDR3 of the amino acid sequence;

(3) As set forth in SEQ ID NO:80, and HCDR1 of the amino acid sequence of SEQ ID NO:81, and HCDR2 of the amino acid sequence of SEQ ID NO:82, HCDR3 of the amino acid sequence; and

(4) As set forth in SEQ ID NO:91, HCDR1 of the amino acid sequence of SEQ ID NO:92, and HCDR2 of the amino acid sequence of SEQ ID NO:93, and HCDR3 of the amino acid sequence of seq id no.
The CAR of claim 44, wherein the combination of antibodies or fragments thereof further comprises light chain complementarity determining region 1 (LCDR 1), light chain complementarity determining region 2 (LCDR 2), and light chain complementarity determining region 3 (LCDR 3), the amino acid sequences of LCDR1, LCDR2, and LCDR3 being independently selected from the group consisting of:

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

(2) As set forth in SEQ ID NO:72, as set forth in SEQ ID NO:73, and LCDR2 as set forth in SEQ ID NO:74, LCDR3 of the amino acid sequence;

(3) As set forth in SEQ ID NO:83, as set forth in SEQ ID NO:84, and LCDR2 as set forth in SEQ ID NO:85, LCDR3 of the amino acid sequence; and

(4) As set forth in SEQ ID NO:94, and LCDR1 of the amino acid sequence of SEQ ID NO:95, and LCDR2 as set forth in SEQ ID NO:96, and LCDR3 of the amino acid sequence.
The CAR of any one of claims 44-45, wherein the combination of antibodies or fragments thereof comprises a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 34. 54, 65, 76 and 87.
The CAR of any one of claims 44-46, wherein the combination of antibodies or fragments thereof further comprises a light chain variable region having an amino acid sequence set forth in SEQ ID NO: 36. 56, 67, 78 and 89.
The CAR of any one of claims 44-47, wherein the combination of antibodies or fragments thereof comprises a light chain variable region and a heavy chain variable region linked by a linker.
The CAR of any one of claims 44-48, wherein the linker comprises the amino acid sequence of SEQ ID NO: shown at 9.
The CAR of 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 of any one of claims 44-50, wherein the MSLN binding domain (scFv) comprises an amino acid sequence selected from any one of the following: SEQ ID No: 5. 57, 68, 101, 79 and 90.
The CAR of any one of claims 44-51, wherein the CAR is further linked to a truncated form of EGFR molecule (tgfr) by a self-cleaving peptide.
The CAR of claim 52, wherein the self-cleaving peptide comprises P2A, T a or F2A.
The CAR of claim 52 or 53, wherein the truncated form of the EGFR molecule comprises the amino acid sequence of SEQ ID No:27 or a functional variant thereof; the P2A comprises SEQ ID NO:29 or a functional variant thereof; the T2A comprises SEQ ID NO:30 or a functional variant thereof.
The CAR of any one of claims 44-54, wherein the transmembrane domain comprises a polypeptide selected from the group consisting of: the 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 of any one of claims 44-55, wherein the transmembrane domain comprises SEQ ID No:13 or a functional variant thereof.
The CAR of any one of claims 44-56, wherein the co-stimulatory domain comprises a polypeptide selected from the group consisting of: CD28, 4-1BB, OX-40 and ICOS.
The CAR of any one of claims 44-57, wherein the co-stimulatory domain comprises SEQ ID No:15 or a functional variant thereof.
The CAR of any one of claims 44-58, wherein the intracellular signaling domain comprises a signaling domain from CD3 z.
The CAR of any one of claims 44-59, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID No:17 or a functional variant thereof.
The CAR of any one of claims 44-60, wherein the CAR further comprises a hinge region that connects the antigen binding domain and the transmembrane domain.
The CAR of any one of claims 44-61, wherein the hinge region comprises SEQ ID No:11 or a functional variant thereof.
The CAR of any one of claims 44-62, wherein the CAR is further linked to a signal peptide.
The CAR of any one of claims 44-63, wherein the signal peptide comprises SEQ ID No:8 or a functional variant thereof.
The CAR of any one of claims 44-64, comprising 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, 66, 75, 77, 86 and 88, or a functional variant thereof.
A vector comprising the nucleic acid molecule of claim 66 or 67.
The vector of claim 68, wherein the vector is selected from the group consisting of a plasmid, a retroviral vector, and a lentiviral vector.
An immune cell comprising the CAR of claims 44-65, the nucleic acid molecule of claim 66 or 67, or the vector of claim 68 or 69.
The immune cell of claim 70, wherein the immune cell is selected from the group consisting of a T lymphocyte and a Natural Killer (NK) cell.
A method of making an immune cell comprising introducing into an immune cell the vector of claim 68 or 69.
A pharmaceutical composition comprising the immune cell of claim 70 or 71, and a pharmaceutically acceptable adjuvant.
Use of the CAR of claims 44-65, the nucleic acid molecule of claim 66 or 67, the vector of claim 68 or 69, or the immune cell of claim 70 or 71 in the manufacture of a medicament for treating a disease or disorder associated with MSLN expression.
The use of claim 74, wherein the disease or disorder associated with MSLN expression is cancer or malignancy.
The use of claim 75, wherein the tumor is a solid tumor.