WO2023078431A1

WO2023078431A1 - Synthetic t cell receptor and antigen receptor specifically binding to mesothelin and use thereof

Info

Publication number: WO2023078431A1
Application number: PCT/CN2022/130106
Authority: WO
Inventors: 林欣; 芮魏; 虞莉; 伍春燕; 赵学强
Original assignee: 清华大学; 华夏英泰（北京）生物技术有限公司
Priority date: 2021-11-05
Filing date: 2022-11-04
Publication date: 2023-05-11
Also published as: CN118139897A

Abstract

A synthetic T cell receptor and antigen receptor specifically binding to mesothelin and the use thereof. TCR and CAR are modified with an antigen-binding fragment targeting MSLN, which results in a better tumor treatment effect.

Description

A synthetic T cell receptor antigen receptor specifically binding to mesothelin and its application

technical field

The invention relates to the field of biomedicine, in particular to a synthetic T cell receptor antigen receptor (STAR) specifically binding to mesothelin, a STAR complex, an immune cell comprising STAR or a STAR complex, and its application in biomedicine field usage.

Background technique

Mesothelin, also known as MSLN, is a cell surface glycoprotein encoded by the MSLN gene. The MSLN gene encodes a proprotein, which produces two protein products, megakaryocyte-potentiating factor (MPF) and mesothelin MSLN, after proteolysis. Among them, MSLN is a protein anchored on the cell surface through glycosylphosphatidylinositol, which belongs to a differentiation antigen present on normal mesothelial cells. It is rarely expressed in normal tissues, but MSLN has been found to be overexpressed in a variety of cancers, so mesothelin MSLN may become an important target for cancer therapy. Overexpression of mesothelin has been observed in mesothelioma, ovarian cancer, lung cancer, esophageal cancer, pancreatic cancer, gastric cancer, bile duct cancer, endometrial cancer, thymus cancer, colon cancer and breast cancer. The frequency and distribution of MSLN expression varies with tumor subtype. In cancer cells, MSLN expression may be luminal/membrane or cytoplasmic. There are also certain differences in the expression position of MSLN in different types of tumors. In mesothelioma tumors, MSLN is expressed on the cell surface in a uniform distribution. In lung adenocarcinoma, MSLN is expressed in both the cytoplasm and the cell surface. In gastric cancer, cytoplasmic expression is more prevalent than membrane expression. MSLN is also expressed in solid tumors such as thyroid, kidney and synovial sarcoma tumors.

For the treatment of tumors, CAR-T and TCR-T cells are currently developing rapidly. However, STAR-T derived from natural TCR lacks co-stimulatory signals in T cell activation during activation, and its proliferation and activation capabilities are often affected. Therefore, there remains a need in the art for improved TCRs and corresponding TCR-T therapies.

Therefore, the present application uses antigen-binding fragments targeting MSLN to modify TCR and CAR, hoping to obtain better effects on tumor treatment.

Contents of the invention

In order to solve the defects of the prior art, the present invention provides a synthetic T cell receptor antigen receptor STAR and its application. The synthetic T cell receptor antigen receptor can specifically bind to mesothelin, and through antigen binding targeting MSLN Fragments are used to modify TCR and CAR to obtain better tumor treatment effect.

Specifically, the first aspect of the present invention provides a synthetic T cell receptor antigen receptor (STAR),

i) The synthetic T cell receptor antigen receptor comprises an α chain and a β chain, wherein the α chain comprises a first target binding domain and a first constant domain, and the β chain comprises a second target binding domain and a second constant domain.

or,

ii) The synthetic T cell receptor antigen receptor comprises a gamma chain and a delta chain, wherein the gamma chain comprises a first target binding domain and a first constant domain, and the delta chain comprises a second target binding domain and a second constant domain. Preferably, i) α-chain and/or β-chain have at least one functional domain connected to their C-terminus; or, ii) γ-chain and/or δ-chain have at least one functional domain connected to their C-terminus.

Further preferably, i) the at least one functional domain is directly or through a linker connected to the C-terminus of the α chain and/or the beta chain, or, ii) the at least one functional domain is directly or through a linker connected to the γ chain and/or the C-terminus of the delta chain.

Preferably, i) the intracellular region of the α chain and/or β chain in the synthetic T cell receptor antigen receptor is deleted; or, ii) the γ chain and/or the synthetic T cell receptor antigen receptor The intracellular region of the delta chain is deleted.

More preferably, i) the functional domain is connected directly or through a linker to the C-terminus of the α-chain and/or β-chain deleted in the intracellular region; or, ii) the functional domain is connected directly or through a linker To the C-terminus of the gamma chain and/or delta chain deleted in the intracellular region.

Preferably, i) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or

More functional domains, and/or, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more are connected to the C-terminus of the β chain in the synthetic T cell receptor antigen receptor multiple functional domains; or, ii) the C-terminus of the gamma chain in the synthetic T cell receptor antigen receptor is connected with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more Functional structural domains, and/or, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional structural domains are connected to the C-terminus of the δ chain in the STAR.

In a specific embodiment of the present invention, the multiple (including two or more) functional domains are directly connected or connected through a linker.

In a specific embodiment of the present invention, at least one functional domain is connected to the C-terminus of the alpha chain in the synthetic T cell receptor antigen receptor. The intracellular region of the alpha chain is deleted. The functional structural domain is connected to the C-terminus of the α-chain deleted in the intracellular region through a linker.

In a specific embodiment of the present invention, at least one functional domain is connected to the C-terminus of the β chain in the synthetic T cell receptor antigen receptor. The intracellular region of the beta chain is deleted. The functional structural domain is connected to the C-terminus of the beta chain deleted in the intracellular region through a linker.

In a specific embodiment of the present invention, at least one functional domain is connected to the C-terminus of the γ chain in the synthetic T cell receptor antigen receptor. The intracellular region of the gamma chain is deleted. The functional structural domain is connected to the C-terminus of the gamma chain deleted in the intracellular region through a linker.

In a specific embodiment of the present invention, at least one functional domain is connected to the C-terminus of the delta chain in the synthetic T cell receptor antigen receptor. The intracellular region of the delta chain is deleted. The functional structural domain is connected to the C-terminus of the delta chain deleted in the intracellular region through a linker.

Preferably, i) the functional domains linked to the C-terminus of the α chain and/or β chain in the synthetic T cell receptor antigen receptor are the same or different;

Or, ii) the functional domains connected to the C-terminals of the γ chain and/or δ chain in the synthetic T cell receptor antigen receptor are the same or different.

In a specific embodiment of the present invention, the multiple functional domains linked by the α chain in the synthetic T cell receptor antigen receptor can be the same or different.

In a specific embodiment of the present invention, multiple functional domains linked by β chains in the synthetic T cell receptor antigen receptor may be the same or different.

In a specific embodiment of the present invention, multiple functional domains linked by γ chains in the synthetic T cell receptor antigen receptor may be the same or different.

In a specific embodiment of the present invention, multiple functional domains linked by δ chains in the synthetic T cell receptor antigen receptor may be the same or different.

In a specific embodiment of the present invention, the functional domain linked by the α chain and the functional domain linked by the β chain in the synthetic T cell receptor antigen receptor may be the same or different.

In a specific embodiment of the present invention, the functional domain linked by the γ chain and the functional domain linked by the δ chain in the synthetic T cell receptor antigen receptor may be the same or different.

Preferably, the functional domain is a co-stimulatory molecule or a fragment thereof, a co-inhibitory molecule or a fragment thereof, a cytokine receptor or a fragment thereof, or an intracellular protein or a fragment thereof. Further preferably, the functional domain is an intracellular domain of a co-stimulatory molecule, an intracellular domain of a co-inhibitory molecule, an intracellular domain of a cytokine receptor, or an intracellular protein. It can also be the fusion of cytokine receptor intracellular domain directly or through a linker and human STAT5 activation module (amino acid sequence shown in SEQ ID NO: 25).

The co-stimulatory molecule is selected from CD40, OX40, ICOS, CD28, 4-1BB (CD137) or CD27.

The co-inhibitory molecule is selected from TIM3, PD1, CTLA4, LAG3.

Described cytokine receptor is selected from interleukin receptor (such as IL-2 receptor), interferon receptor, tumor necrosis factor superfamily receptor, colony-stimulating factor receptor, chemokine receptor, growth factor receptors or other membrane proteins.

The intracellular protein is a T cell regulatory factor, such as the domain of NIK.

In a specific embodiment of the present invention, the co-stimulatory molecule is CD40, and its intracellular domain includes the amino acid sequence shown in SEQ ID NO:10.

In a specific embodiment of the present invention, the co-stimulatory molecule is OX40, and its intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 11.

In a specific embodiment of the present invention, the co-stimulatory molecule is ICOS, and its intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 12.

In a specific embodiment of the present invention, the co-stimulatory molecule is CD28, and its intracellular domain includes the amino acid sequence shown in SEQ ID NO: 13.

In a specific embodiment of the present invention, the co-stimulatory molecule is 4-1BB, and its intracellular domain comprises the amino acid sequence shown in SEQ ID NO:14.

In a specific embodiment of the present invention, the co-stimulatory molecule is CD27, and its intracellular domain includes the amino acid sequence shown in SEQ ID NO: 15.

In a specific embodiment of the present invention, the cytokine receptor is IL-2β, and its intracellular domain includes the amino acid sequence shown in SEQ ID NO: 22.

In a specific embodiment of the present invention, the cytokine receptor is IL-7α, and its intracellular domain includes the amino acid sequence shown in SEQ ID NO: 23.

In a specific embodiment of the present invention, the cytokine receptor is IL-21, and its intracellular domain includes the amino acid sequence shown in SEQ ID NO: 24.

In a specific embodiment of the present invention, the functional domain is the fusion of IL-2β intracellular domain and human STAT5 activation module, which includes the amino acid sequence shown in SEQ ID NO:26.

In a specific embodiment of the present invention, the functional domain is the fusion of IL-7α intracellular domain and human STAT5 activation module, which comprises the amino acid sequence shown in SEQ ID NO:27.

Preferably, the first constant region is a TCRα chain constant region or a TCRγ chain constant region, preferably a modified TCRα chain constant region or a modified TCRγ chain constant region.

The TCR alpha chain constant region is selected from human TCR alpha chain constant region or rodent (preferably mouse, more preferably mouse) TCR alpha chain constant region. The TCRγ chain constant region is selected from human TCRγ chain constant region or rodent (preferably mouse, more preferably mouse) TCRγ chain constant region.

In a specific embodiment of the present invention, the amino acid sequence of the human TCRα chain constant region is shown in SEQ ID NO: 1, and the rodent (preferably mouse, more preferably mouse) TCRα chain constant region The amino acid sequence is shown in SEQ ID NO: 3.

In a specific embodiment of the present invention, the amino acid sequence of the human TCR gamma chain constant region is shown in SEQ ID NO: 45, the rodent (preferably mouse, more preferably mouse) TCR gamma chain constant region The amino acid sequence is shown in SEQ ID NO: 46.

The modified TCR α chain constant region is derived from the human TCR α chain constant region, which contains one or more modifications at

position

48, 116 or 119 relative to the wild-type human TCR α chain constant region, and the modification is a mutation or missing.

The modified TCR α chain constant region is derived from the human TCR α chain constant region, which, relative to the wild-type human TCR α chain constant region, contains a mutation of threonine T to cysteine C at position 48.

The modified TCR α chain constant region is derived from the human TCR α chain constant region. Compared with the wild-type human TCR α chain constant region, the 116th serine S is mutated to leucine L, and the 119th glycine G is mutated to valine Acid V.

The modified TCR α chain constant region is derived from the human TCR α chain constant region, which, relative to the wild-type human TCR α chain constant region, comprises a mutation of threonine T at position 48 to cysteine C, serine at position 116 S was mutated to leucine L, and glycine G at position 119 was mutated to valine V.

The modified TCR alpha chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCR alpha chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCR alpha chain constant region, It includes one or more modifications at

positions

6, 13, 15-18, 48, 112, 114, 115, which are mutations or deletions.

The modified TCR alpha chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCR alpha chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCR alpha chain constant region, It includes having one or more modifications at positions 13, 36, 47, 53, 58, 78, 98, 122 which are mutations or deletions.

Preferably, the modified TCRα chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCRα chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCRα chain The constant region, including the introduction of cysteine.

Further preferably, the modified TCRα chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCRα chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCRα The chain constant region, comprising an amino acid at position 48 such as threonine T was mutated to cysteine C.

In a specific embodiment of the present invention, the constant region of the TCR alpha chain of rodents (preferably mice, more preferably mice) introduced with cysteine comprises the amino acid sequence shown in SEQ ID NO:5.

Preferably, the modified TCRα chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCRα chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCRα chain The constant region contains hydrophobic amino acid mutations.

Further preferably, the modified TCRα chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCRα chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCRα Chain constant region comprising an amino acid at position 112 such as serine S changed to leucine L, an amino acid at position 114 such as methionine M changed to isoleucine I, and/or, at position 115 An amino acid such as glycine G is changed to valine V.

In a specific embodiment of the present invention, the rodent (preferably mouse, more preferably mouse) TCR alpha chain constant region comprising a hydrophobic amino acid mutation comprises the amino acid sequence shown in SEQ ID NO:7.

Preferably, the modified TCRα chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCRα chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCRα chain Constant region, including N-terminal modifications.

Further preferably, the modified TCRα chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCRα chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCRα A chain constant region, which comprises amino acids at position 6 such as E replaced by D, K at position 13 replaced by R, and amino acids at positions 15-18 deleted.

Preferably, the modified TCRα chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCRα chain constant region, which contains a mutation of lysine to arginine in the transmembrane region.

Further preferably, the modified TCRα chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCRα chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCR alpha chain constant region, which comprises amino acid K at position 122 replaced by R.

In a specific embodiment of the present invention, the rodent (preferably mouse, more preferably mouse) TCRα chain constant region comprising a transmembrane region lysine mutated to arginine comprises the amino acid sequence shown in SEQ ID NO:8.

Preferably, the modified TCRα chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCRα chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCRα chain Constant region, containing cysteine introductions and hydrophobic amino acid mutations.

Further preferably, the modified TCRα chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCRα chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCRα Chain constant region, comprising an amino acid at position 48 such as threonine T being mutated to cysteine C, an amino acid at position 112 such as serine S being changed to leucine L, an amino acid at position 114 such as methylthio The amino acid M is changed to isoleucine I, and the amino acid at position 115 such as glycine G is changed to valine V.

In a specific embodiment of the present invention, the rodent (preferably mouse, more preferably mouse) TCR alpha chain constant region comprising cysteine introduction and hydrophobic amino acid mutation comprises the amino acid sequence shown in SEQ ID NO: 31.

The modified TCR alpha chain constant region is derived from the rodent (preferably mouse, more preferably mouse) TCR alpha chain constant region, which is relative to the wild-type rodent (preferably mouse, further preferably mouse) TCR alpha chain constant region , which includes the amino acid at position 48 such as threonine T being mutated to cysteine C, and the amino acid K at position 122 being substituted by R.

The modified TCR alpha chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCR alpha chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCR alpha chain constant region, It includes substitution of amino acid at position 6 such as E with D, K at position 13 with R, deletion of amino acids 15-18, and mutation of amino acid at position 48 such as threonine T to cysteine c.

The modified TCR alpha chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCR alpha chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCR alpha chain constant region, It includes amino acid at position 48 such as threonine T being mutated to cysteine C, amino acid at position 112 such as serine S being changed to leucine L, amino acid at position 114 such as methionine M was changed to isoleucine I, amino acid at position 115 such as glycine G was changed to valine V, and amino acid K at position 122 was replaced by R.

The modified TCR alpha chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCR alpha chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCR alpha chain constant region, It contains amino acids at position 6 such as E replaced by D, K at position 13 replaced by R, and amino acids 15-18 are deleted, and amino acids at position 48 such as threonine T are mutated to cysteine C, the amino acid at position 112 such as serine S is changed to leucine L, the amino acid at position 114 such as methionine M is changed to isoleucine I, and the amino acid at position 115 such as glycine G is changed into valine V, and the amino acid K at position 122 is replaced by R.

The modified TCR alpha chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCR alpha chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCR alpha chain constant region, It includes amino acids at position 6 such as E replaced by D, K at position 13 replaced by R, and amino acids 15-18 are deleted, and amino acids at position 48 such as threonine T are mutated to cysteine C, amino acid K at position 122 was replaced by R.

The modified TCR alpha chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCR alpha chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCR alpha chain constant region, It includes amino acids at position 6 such as E being replaced by D, K at position 13 being replaced by R, and amino acids at positions 15-18 are deleted, amino acids at position 112 such as serine S are changed to leucine L, and in An amino acid at position 114 such as methionine M is changed to isoleucine I and an amino acid at position 115 such as glycine G is changed to valine V.

The modified TCR alpha chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCR alpha chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCR alpha chain constant region, It includes substitution of amino acid at position 6 such as E by D, substitution of K at position 13 by R, deletion of amino acids 15-18, and substitution of amino acid K at position 122 by R.

The modified TCR alpha chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCR alpha chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCR alpha chain constant region, It includes amino acids at position 6 such as E being replaced by D, K at position 13 being replaced by R, and amino acids at positions 15-18 are deleted, amino acids at position 112 such as serine S are changed to leucine L, and in The amino acid at position 114 such as methionine M is changed to isoleucine I, the amino acid at position 115 such as glycine G is changed to valine V, and the amino acid K at position 122 is replaced by R.

Preferably, the modified TCRα chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCRα chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCRα chain The constant region includes cysteine introduction, hydrophobic amino acid mutation and N-terminal modification.

Further preferably, the modified TCRα chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCRα chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCRα Chain constant region, which contains amino acid at position 6 such as E replaced by D, K at position 13 replaced by R, amino acids 15-18 deleted, amino acid at position 48 such as threonine T mutated to half Cystine C, an amino acid at position 112 such as Serine S is changed to Leucine L, an amino acid at position 114 such as Methionine M is changed to Isoleucine I, and an amino acid at position 115 such as Glycine G is changed to valine V.

In a specific embodiment of the present invention, the rodent (preferably mouse, more preferably mouse) TCR alpha chain constant region comprising cysteine introduction, hydrophobic amino acid mutation and intracellular region deletion comprises SEQ ID NO: 16 amino acid sequence.

Preferably, the modified TCRα chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCRα chain constant region, which includes N-terminal modification, cysteine introduction and hydrophobic amino acid mutation.

Further preferably, the modified TCRα chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCRα chain constant region, which comprises amino acids at position 6 such as E being replaced by D, and amino acids at position 13 K is replaced by R, amino acids 15-18 are deleted, and the amino acid at position 48 such as threonine T is mutated to cysteine C and the amino acid at position 112 such as serine S is changed to leucine L, the amino acid at position 114 such as methionine M is changed to isoleucine I, and the amino acid at position 115 such as glycine G is changed to valine V.

In a specific embodiment of the present invention, the rodent (preferably mouse, more preferably mouse) TCR α chain constant region comprising N-terminal modification and cysteine introduction and hydrophobic amino acid mutation comprises SEQ ID NO: 32 amino acid sequence.

Preferably, the modified TCRα chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCRα chain constant region, which includes intracellular deletion, N-terminal modification, cysteine introduction and hydrophobic amino acid mutation .

Further preferably, the modified TCRα chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCRα chain constant region, which contains an intracellular deletion, and an amino acid at position 48 such as threonine T is mutated to cysteine C, an amino acid at position 112 such as serine S is changed to leucine L, an amino acid at position 114 such as methionine M is changed to isoleucine I, and at position 115 An amino acid at position 1 such as glycine G is changed to valine V, and an amino acid at position 6 such as E is replaced by D, K at position 13 is replaced by R, and amino acids 15-18 are deleted.

In a specific embodiment of the present invention, the rodent (preferably mouse, more preferably mouse) TCRα chain constant region comprising intracellular deletion, N-terminal modification, cysteine introduction and hydrophobic amino acid mutation comprises SEQ ID NO: The amino acid sequence shown in 43.

In a specific embodiment of the present invention, the first constant region comprises the amino acid sequence shown in one of SEQ ID NO: 1, 3, 5, 7, 8, 16, 31, 32 or 43.

Preferably, the second constant region is a TCRβ chain constant region or a TCRδ chain constant region, preferably a modified TCRβ chain constant region or a modified TCRδ chain constant region.

Further preferably, the TCR β chain constant region is selected from a human TCR β chain constant region or a rodent (preferably a mouse, more preferably a mouse) TCR β chain constant region, and the TCR δ chain constant region is selected from a human TCR δ chain constant region Or rodent (preferably mouse, more preferably mouse) TCRδ chain constant region.

In a specific embodiment of the present invention, the human TCR beta chain constant region comprises the amino acid sequence shown in SEQ ID NO:2.

In a specific embodiment of the present invention, the rodent (preferably mouse, more preferably mouse) TCRβ chain constant region comprises the amino acid sequence shown in SEQ ID NO:4.

In a specific embodiment of the present invention, the human TCR δ chain constant region comprises the amino acid sequence shown in SEQ ID NO: 47.

In a specific embodiment of the present invention, the rodent (preferably mouse, more preferably mouse) TCR δ chain constant region comprises the amino acid sequence shown in SEQ ID NO: 48.

The modified TCR β chain constant region is derived from a human TCR β chain constant region, which includes one or more modifications at position 57, 173 or 175 relative to the wild-type human TCR β chain constant region, and the modification is a mutation or missing.

The modified TCR β chain constant region is derived from the human TCR β chain constant region, which includes the mutation of the 57th serine S to cysteine C relative to the wild type human TCR β chain constant region.

The modified TCR β chain constant region is derived from the human TCR β chain constant region, which includes the 173rd and 175th lysine K mutations to arginine relative to the wild type human TCR β chain constant region.

The modified TCR β chain constant region is derived from the human TCR β chain constant region, which, relative to the wild-type human TCR β chain constant region, includes the mutation of serine S at position 57 to cysteine C, and the mutation at positions 173 and 175 Lysine K was mutated to arginine.

The modified TCR beta chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCR beta chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCR beta chain constant region, It includes having one or more modifications at

positions

3, 6, 9, 11, 12, 17, 21-25, 56, 150, 168 or 170, which modifications are mutations or deletions.

The modified TCR beta chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCR beta chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCR beta chain constant region, It includes one or more modifications at

positions

9, 17, 23, 25, 49, 63, 103, 110, 150, 168, 170, which are mutations or deletions.

Preferably, the modified TCRβ chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCRβ chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCRβ chain The constant region, including the introduction of cysteine.

Further preferably, the modified TCRβ chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCRβ chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCRβ chain In the chain constant region, the amino acid at position 56 such as serine S is mutated to cysteine C.

In a specific embodiment of the present invention, the introduced rodent (preferably mouse, more preferably mouse) TCRβ chain constant region comprising cysteine comprises the amino acid sequence shown in SEQ ID NO:6.

Preferably, the modified TCRβ chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCRβ chain constant region, in which lysine in the intracellular region is replaced by arginine.

Further preferably, the modified TCR β chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCR β chain constant region, and the lysine at position 150, 168 or 170 is replaced by arginine.

In a specific embodiment of the present invention, the rodent (preferably mouse, more preferably mouse) TCR beta chain constant region comprising intracellular region lysine replaced by arginine comprises the amino acid sequence shown in SEQ ID NO: 9

Preferably, the modified TCRβ chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCRβ chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCRβ chain Constant region, including N-terminal modifications.

Further preferably, the modified TCRβ chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCRβ chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCRβ chain In the constant region of the chain, the amino acid at the 3rd position such as R is substituted by K, the amino acid at the 6th position such as T is substituted by F, the K at the 9th position is substituted by E, the S at the 11th position is substituted by A, and the L at the 12th position Substituted by V, and amino acids 17, 21-25 were deleted.

Preferably, the modified TCRβ chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCRβ chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCRβ chain The constant region, including the introduction of cysteine and the deletion of the intracellular region.

In a specific embodiment of the present invention, the rodent (preferably mouse, more preferably mouse) TCR beta chain constant region comprising the introduction of cysteine and deletion of the intracellular region comprises SEQ ID NO: 17 amino acid sequence.

The modified TCR beta chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCR beta chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCR beta chain constant region, It includes mutation of an amino acid at position 56 such as serine S to cysteine C, and substitution of lysine at position 150, 168 or 170 by arginine.

The modified TCR beta chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCR beta chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCR beta chain constant region, It contains amino acids at position 3 such as R being substituted by K, amino acids at position 6 such as T being substituted by F, K at position 9 being substituted by E, S at position 11 being substituted by A, and L at position 12 being substituted by V , and the 17th, 21-25th amino acid is deleted, the 56th amino acid such as serine S is mutated to cysteine C, and the 150th, 168th or 170th lysine is replaced by arginine.

The modified TCR beta chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCR beta chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCR beta chain constant region, It contains amino acids at position 3 such as R being substituted by K, amino acids at position 6 such as T being substituted by F, K at position 9 being substituted by E, S at position 11 being substituted by A, and L at position 12 being substituted by V , and amino acids 17, 21-25 are deleted, and lysine at position 150, 168 or 170 is replaced by arginine.

Preferably, the modified TCRβ chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCRβ chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCRβ chain Chain constant region, including N-terminal modification and cysteine introduction.

The modified TCR beta chain constant region is derived from a rodent (preferably a mouse, more preferably a mouse) TCR beta chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCR beta chain constant region, It includes amino acids at position 3 such as R being substituted by K, amino acids at position 6 such as T being substituted by F, K at position 9 being substituted by E, S at position 11 being substituted by A, and L at position 12 being substituted by V , and amino acids 17, 21-25 are deleted, and an amino acid at position 56 such as serine S is mutated to cysteine C.

In a specific embodiment of the present invention, the rodent (preferably mouse, more preferably mouse) TCR beta chain constant region comprising N-terminal modification and cysteine introduction comprises the amino acid sequence shown in SEQ ID NO: 33 .

Preferably, the modified TCRβ chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCRβ chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCRβ chain Chain constant region, including intracellular deletion, N-terminal modification and cysteine introduction.

Further preferably, the modified TCRβ chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCRβ chain constant region, which is relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCRβ chain Chain constant region, which contains an intracellular deletion, and an amino acid at position 56 such as serine S is mutated to cysteine C, and an amino acid at position 3 such as R is replaced by K, and an amino acid at position 6 such as T is replaced by F Substitution, K at position 9 is replaced by E, S at position 11 is replaced by A, L at position 12 is replaced by V, and amino acids at positions 17 and 21-25 are deleted.

In a specific embodiment of the present invention, said rodent (preferably mouse, more preferably mouse) TCR beta chain constant region comprising intracellular deletion, N-terminal modification and cysteine introduction comprises SEQ ID NO: 44 The amino acid sequence shown.

In a specific embodiment of the present invention, the second constant region comprises the amino acid sequence shown in one of SEQ ID NO: 2, 4, 6, 9, 17, 33 or 44.

The target binding region is located at the N-terminal of the constant region. The two can be connected directly or via a joint.

The first target binding region may comprise one or more same or different binding regions. The target binding region is an antigen binding region or a fragment thereof, a non-immunoglobulin antigen binding domain or a fragment thereof, an antibody binding region or a fragment thereof, a receptor or a fragment thereof, a ligand or a fragment thereof, and the receptor is preferably Natural T cell receptor.

Said antigen binding region is derived from an antibody.

The STAR comprises one or more antigen binding domains;

Preferably, the multiple antigen-binding regions are the same or different;

Further preferably, the multiple antigen-binding domains are connected directly or through a linker.

Said antibody can be monoclonal antibody or polyclonal antibody.

The antibody may also comprise fragments such as Fab _, Fab', _Fab' -SH, Fv, scFv, ( _Fab ') ₂ , _single domain antibody, diabody (dAb) or linear antibody.

The antibody can be a monospecific antibody or a multispecific antibody (eg bispecific antibody).

Preferably, the antibody may be a fully human antibody, a humanized antibody, or an animal-derived antibody. Wherein, the said animals may be rats, rabbits, cows, monkeys and so on.

Preferably, the first antigen-binding region is directly connected to the first constant region or connected through a linker, and/or the second antigen-binding region is directly connected to the second constant region or connected through a linker.

Preferably, the first antigen-binding region and the second antigen-binding region each independently or in combination specifically bind the target antigen.

Preferably, the target antigen is a disease-associated antigen, preferably a cancer-associated antigen, such as a cancer-associated antigen selected from the following: GPC3, CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123 , CD138, ErbB2 (HER2/neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40 , disialoganglioside GD2, ductal mucin, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, β-human chorionic gonadotropin, α-fetoprotein (AFP), Lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2(AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, Prostate enzyme (prostase), prostate enzyme-specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, Prostein, PSMA, survival and telomerase, prostate cancer tumor antigen-1 (PCTA-1) , MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF1)-I, IGF-II, IGFI receptor, mesothelin, major tumor-specific peptide epitope presentation Histocompatibility complex (MHC) molecules, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigen, extra domain A (EDA) and extra domain B (EDB) of fibronectin, A1 of tenascin-C domain (TnC A1), fibroblast-associated protein (fap), CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, Foxp3, B7-1(CD80), B7-2(CD86), ( UNIPROT Q9NYL4), KAMP3, ITGA8 (UNIPROT P53708), or FCRL5 (UNIPROT Q68SN8).

Preferably, the first antigen-binding domain comprises the heavy chain variable domains of one or more antibodies that specifically bind the target antigen, and the second antigen-binding domain comprises the light chain variable domains of one or more antibodies that specifically bind the target antigen. or, the first antigen-binding region comprises the light chain variable regions of one or more antibodies that specifically bind the target antigen, and the second antigen-binding region comprises the heavy chains of one or more antibodies that specifically bind the target antigen may Variable area.

Among them, the heavy chain variable regions of multiple antibodies are connected directly or through a linker. The light chain variable regions of multiple antibodies are linked directly or via a linker.

Preferably, the first antigen-binding region comprises one or more single-chain antibodies or one or more single-domain antibodies that specifically bind the target antigen; and/or the second antigen-binding region comprises one or more antibodies that specifically bind the target antigen. A single chain antibody or one or more single domain antibodies.

Among them, multiple single-chain antibodies are connected directly or through a linker. Multiple single domain antibodies are connected directly or through linkers.

Preferably, the single-chain antibody comprises a heavy chain variable region and a light chain variable region connected directly or through a linker.

Preferably, the first antigen binding domain and the second antigen binding domain bind the same or different target antigens.

Preferably, the first antigen binding region and the second antigen binding region bind different regions (eg different epitopes) of the same target antigen.

In a specific embodiment of the present invention, the target antigen is mesothelin.

In a specific embodiment of the present invention, the first antigen-binding region comprises one or more single-domain antibodies, and/or, the second antigen-binding region comprises one or more single-domain antibodies;

Preferably, the multiple single-domain antibodies contained in the first antigen-binding region are the same or different;

Preferably, the multiple single-domain antibodies contained in the second antigen-binding region are the same or different;

Further preferably, the multiple single domain antibodies are connected directly or through a linker.

Preferably, the single-domain antibody contained in the first antigen-binding region is the same as or different from the single-domain antibody contained in the second antigen-binding region.

In a specific embodiment of the present invention, the single domain antibody comprises a heavy chain variable region, and the heavy chain variable region comprises CDR1-3, wherein,

i) CDR1 includes the amino acid sequence shown in SEQ ID NO: 34, CDR2 includes the amino acid sequence shown in SEQ ID NO: 35, and the CDR3 includes the amino acid sequence shown in SEQ ID NO: 36;

or,

ii) CDR1 includes the amino acid sequence shown in SEQ ID NO: 37, CDR2 includes the amino acid sequence shown in SEQ ID NO: 38, and the CDR3 includes the amino acid sequence shown in SEQ ID NO: 39.

In a specific embodiment of the present invention, the single domain antibody comprises the amino acid sequence shown in SEQ ID NO: 28 or 29.

The second aspect of the present invention provides a STAR complex, wherein,

i) the STAR complex comprises an α chain, a β chain, CD3ε, CD3γ, CD3δ and CD3ζ, the α chain comprises a first target binding domain and a first constant domain, and the β chain comprises a second target binding domain and a second constant region;

or,

ii) the STAR complex comprises a gamma chain, a delta chain, CD3ε, CD3γ, CD3δ, and CD3ζ, the gamma chain comprising a first target binding domain and a first constant domain, and the delta chain comprising a second target binding domain and a third Two constant regions.

Preferably, i) at least one of α chain, β chain, CD3ε, CD3γ, CD3δ and CD3ζ has at least one functional domain connected to its C-terminus; or, ii) γ chain, δ chain, CD3ε, CD3γ, CD3δ and At least one of CD3ζ has at least one functional domain linked to its C-terminus.

Preferably, i) said at least one functional domain is connected directly or through a linker to the C-terminus of at least one of α-chain, β-chain, CD3ε, CD3γ, CD3δ and CD3ζ; or, ii) said at least one The functional domain is linked directly or through a linker to the C-terminus of at least one of the γ chain, δ chain, CD3ε, CD3γ, CD3δ, and CD3ζ.

Preferably, i) the intracellular region of at least one of the α chain, β chain, CD3ε, CD3γ, CD3δ and CD3ζ in the STAR complex is deleted; or, ii) the γ chain, The intracellular region of at least one of the delta chain, CD3ε, CD3γ, CD3δ, and CD3ζ is deleted.

Further preferably, i) the at least one functional domain is connected directly or through a linker to the C-terminus of at least one of the α-chain, β-chain, CD3ε, CD3γ, CD3δ and CD3ζ in which the intracellular region is deleted; or, ii ) said at least one functional domain is connected directly or through a linker to the C-terminus of at least one of the intracellular region-deleted γ chain, δ chain, CD3ε, CD3γ, CD3δ and CD3ζ.

In a specific embodiment of the present invention, i) the C-terminus of at least one of the α-chain, β-chain, CD3ε, CD3γ, CD3δ and CD3ζ in the STAR complex is linked 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10 or more functional domains; or, ii) C of at least one of γ chain, δ chain, CD3ε, CD3γ, CD3δ and CD3ζ in the

STAR complex

1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains are connected to each other.

Preferably, the multiple functional domains may be connected directly or through a linker.

Preferably, i) the C-terminal linked functional domains of at least one of the α chain, β chain, CD3ε, CD3γ, CD3δ and CD3ζ in the STAR complex are the same or different; or, ii) the STAR The C-terminal linked functional domains of at least one of the γ chain, δ chain, CD3ε, CD3γ, CD3δ and CD3ζ in the complex are the same or different.

In a specific embodiment of the present invention, the multiple functional domains linked by the α chains may be the same or different. The multiple functional domains connected by the β chains can be the same or different. The multiple functional domains connected by the γ chains can be the same or different. The multiple functional domains connected by the δ chains can be the same or different. The multiple functional domains linked by CD3ε may be the same or different. The multiple functional domains linked by CD3γ can be the same or different. The multiple functional domains linked by CD3δ can be the same or different. The multiple functional domains linked by CD3ζ can be the same or different.

In a specific embodiment of the present invention, the functional domains linked by the α chain, β chain, CD3ε, CD3γ, CD3δ and CD3ζ may be the same or different.

In a specific embodiment of the present invention, the functional domains linked by the γ chain, δ chain, CD3ε, CD3γ, CD3δ and CD3ζ can be the same or different.

Preferably, the functional domain is a co-stimulatory molecule or a fragment thereof, a co-inhibitory molecule or a fragment thereof, a cytokine receptor or a fragment thereof, or an intracellular protein or a fragment thereof. Further preferably, the functional domain is an intracellular domain of a co-stimulatory molecule, an intracellular domain of a co-inhibitory molecule, an intracellular domain of a cytokine receptor, or an intracellular protein;

Preferably, the costimulatory molecule is selected from CD40, OX40, ICOS, CD28, 4-1BB (CD137) or CD27;

Preferably, the co-inhibitory molecule is selected from TIM3, PD1, CTLA4, LAG3;

Preferably, the cytokine receptors are selected from interleukin receptors (such as IL-2 receptors), interferon receptors, tumor necrosis factor superfamily receptors, colony-stimulating factor receptors, chemokine receptors , growth factor receptors or other membrane proteins;

Preferably, the intracellular protein is a T cell regulatory factor, such as a domain of NIK.

In a specific embodiment of the present invention, the STAR complex comprises the above-mentioned STAR, as well as CD3ε, CD3γ, CD3δ and CD3ζ.

Preferably, said CD3ε, CD3γ, CD3δ and/or CD3ζ are of human origin.

In a specific embodiment of the present invention, said CD3ε comprises the amino acid sequence shown in SEQ ID NO: 20.

In a specific embodiment of the present invention, said CD3γ comprises the amino acid sequence shown in SEQ ID NO: 18.

In a specific embodiment of the present invention, said CD3δ comprises the amino acid sequence shown in SEQ ID NO: 19.

In a specific embodiment of the present invention, said CD3ζ comprises the amino acid sequence shown in SEQ ID NO: 21.

In the third aspect of the present invention, an antibody or antigen-binding fragment is provided, and the antibody or antigen-binding fragment comprises a heavy chain variable region and/or a light chain variable region.

The heavy chain variable region comprises CDR1-3, wherein,

i) CDR1 includes the amino acid sequence shown in SEQ ID NO: 34, CDR2 includes the amino acid sequence shown in SEQ ID NO: 35, and the CDR3 includes the amino acid sequence shown in SEQ ID NO: 36.

or,

In a specific embodiment of the present invention, the antibody or antigen-binding fragment is a single-chain antibody or a single-domain antibody.

In a specific embodiment of the present invention, said antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 28 or 29.

The fourth aspect of the present invention provides a single-chain antibody comprising a heavy chain variable region and/or a light chain variable region.

The heavy chain variable region comprises CDR1-3, wherein,

or,

The fifth aspect of the present invention provides a single domain antibody, the single domain antibody comprises a heavy chain variable region, and the heavy chain variable region comprises CDR1-3.

Wherein, i) CDR1 includes the amino acid sequence shown in SEQ ID NO: 34, CDR2 includes the amino acid sequence shown in SEQ ID NO: 35, and the CDR3 includes the amino acid sequence shown in SEQ ID NO: 36. Or, ii) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 37, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 38, and said CDR3 comprises the amino acid sequence shown in SEQ ID NO: 39.

The sixth aspect of the present invention provides a method for preparing the above-mentioned antibody or antigen-binding fragment or the above-mentioned single-domain antibody, the preparation method comprising: preparing a phage display library, and screening the phage display library to obtain an antibody or antigen Binding fragments or single domain antibodies.

The seventh aspect of the present invention provides an antigen receptor, which comprises a transmembrane region, an intracellular region and one or more identical or different extracellular binding domains.

The antigen receptor is TCR or CAR.

In a specific embodiment of the present invention, the antigen receptor is CAR.

The extracellular binding domains are extracellular antigen binding domains, extracellular antibody binding domains, receptors, and ligands, and the receptors are preferably natural T cell receptors.

In a specific embodiment of the present invention, the extracellular binding domain is an extracellular antigen binding domain.

Said extracellular antigen binding domain is derived from an antibody.

Preferably, the transmembrane region is directly connected to one or more extracellular antigen-binding domains or connected through a linker.

Preferably, the antigen is selected from cancer-associated antigens, for example, cancer-associated antigens selected from the following: GPC3, CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123, CD138, ErbB2 ( HER2/neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, disialyl neuro Ganglioside GD2, ductal mucin, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin response Sexual AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2(AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase , Prostate Enzyme Specific Antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, Prostein, PSMA, Survival and Telomerase, Prostate Cancer Tumor Antigen-1 (PCTA-1), MAGE, ELF2M, Neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF1)-I, IGF-II, IGFI receptor, mesothelin, major histocompatibility complex presenting tumor-specific peptide epitopes (MHC) molecule, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigen, extra domain A (EDA) and extra domain B (EDB) of fibronectin, A1 domain of tenascin-C (TnC A1 ), fibroblast-associated protein (fap), CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, Foxp3, B7-1(CD80), B7-2(CD86), GM-CSF, cell Factor receptors, endoglin, major histocompatibility complex (MHC) molecules, BCMA(CD269, TNFRSF17), TNFRSF17(UNIPROTQ02223), SLAMF7(UNIPROT Q9NQ25), GPRC5D(UNIPROT Q9NZD1), FKBP11(UNIPROT Q9NYL4), KAMP3 , ITGA8 (UNIPROT P53708) or FCRL5 (UNIPROT Q68SN8);

Preferably, the antigen is mesothelin.

Preferably, the extracellular antigen binding domain comprises CDR1-3, wherein, i) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 34, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 35, and the CDR3 Comprising the amino acid sequence shown in SEQ ID NO: 36. Or, ii) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 37, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 38, and said CDR3 comprises the amino acid sequence shown in SEQ ID NO: 39.

In a specific embodiment of the present invention, the extracellular antigen-binding domain comprises the above-mentioned antibody or antigen-binding fragment, the above-mentioned single-chain antibody, or the above-mentioned single-domain antibody.

Preferably, the transmembrane region is derived from human CD8.

Preferably, the intracellular region is derived from 4-1BB, CD28 or CD3ζ.

The eighth aspect of the present invention provides a nucleic acid encoding the above-mentioned STAR, the above-mentioned STAR complex, the above-mentioned antibody or antigen-binding fragment, the above-mentioned single-chain antibody, the above-mentioned single domain antibody, the above-mentioned antigen receptor.

The ninth aspect of the present invention provides a vector comprising the above-mentioned nucleic acid.

The vector can be expressed in vivo or in vitro or under ex vivo conditions, preferably an expression vector. Preferably, the expression vector is a prokaryotic expression vector, a virus expression vector, a plasmid, a cosmid, a phage, a virus, and the like.

Preferably, the prokaryotic expression vector is Escherichia coli series. For example pET-26b or pET28a+.

Preferably, it can be Rous sarcoma virus (RSV), lentivirus, human immunodeficiency virus (HIV), murine leukemia virus (MLV), equine infectious anemia virus (EIAV), mouse breast cancer virus (MMTV), Fujinami sarcoma virus (FuSV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine leukemia virus (Mo-MLV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A- MLV), avian myeloproliferative virus 29 (MC29) or avian erythroblastosis virus (AEV) and the like. Further preferably, the expression vector is a lentiviral expression vector. For example pHAGE-IRES-RFP.

The tenth aspect of the present invention provides a host cell comprising the above-mentioned nucleic acid or the above-mentioned vector.

Preferably, said host cell can be eukaryotic or prokaryotic. More preferably, the host cells are yeast cells, 293 cells, CHO cells, Escherichia coli and the like.

The eleventh aspect of the present invention provides an immune cell expressing the above-mentioned STAR, the above-mentioned STAR complex, the above-mentioned antibody or antigen-binding fragment, the above-mentioned single domain antibody, the above-mentioned antigen receptor .

Preferably, the immune cells contain one or more of the above-mentioned nucleic acids.

Preferably, the immune cells are selected from T cells, Treg cells, macrophages, NK cells, NKT cells, peripheral blood mononuclear cells, TIL cells or dendritic cells (DC).

Preferably, said immune cells are isolated from T cells of a subject.

In a specific embodiment of the present invention, the immune cells are T cells, NK cells, CTLs, human embryonic stem cells, lymphoid progenitor cells and/or T cell precursor cells.

The twelfth aspect of the present invention provides a CAR-T cell, the CAR-T cell comprising the above-mentioned antibody or antigen fragment, the above-mentioned single-chain antibody or the above-mentioned single-domain antibody.

The thirteenth aspect of the present invention provides a preparation method of immune cells, the preparation method comprising transfecting the above nucleic acid sequence into immune cells for expression.

The fourteenth aspect of the present invention provides a method for preparing recombinant T cells, comprising the following steps:

1) obtaining the above-mentioned nucleic acid from positive T cell clones;

2) Isolation and culture of primary T cells;

3) delivering the nucleic acid obtained in step 1) to the primary T cells described in step 2) to obtain recombinant T cells expressing the above-mentioned STAR.

In a fifteenth aspect of the present invention, a method for preparing STAR or a STAR complex is provided, comprising the following steps:

(1) obtaining the above-mentioned nucleic acid from positive T cell clones;

(2) connecting the nucleic acid obtained in step (1) to the vector backbone to obtain an expression vector;

(3) transforming the expression vector obtained in step (2) into a host cell, and then inducing its expression;

(4) Obtain STAR.

The sixteenth aspect of the present invention provides a method for preparing an antibody or an antigen-binding fragment, a single-chain antibody or a single-domain antibody, and the method includes protein immunization and/or DNA immunization.

The seventeenth aspect of the present invention provides a method for preparing an antibody or an antigen-binding fragment, a single-chain antibody or a single-domain antibody, the method comprising:

A) obtaining the encoded nucleic acid sequence;

B) Transforming the nucleic acid sequence obtained in step A) into a host cell, then inducing its expression and purifying it.

The eighteenth aspect of the present invention provides the above-mentioned STAR, the above-mentioned STAR complex, the above-mentioned antibody or antigen-binding fragment, the above-mentioned single domain antibody, the above-mentioned antigen receptor, the above-mentioned nucleic acid, and the above-mentioned immune cell. Applications in products for the diagnosis or treatment of tumors.

Preferably, the tumors include but are not limited to lymphoma, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, lung cancer, bronchial cancer Cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplasia syndrome, and sarcoma. Wherein, the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myelogenous leukemia, myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia; The lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma , T-cell lymphoma, and Waldenstrom macroglobulinemia; said sarcoma is selected from the group consisting of osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma , and chondrosarcoma.

The nineteenth aspect of the present invention provides an immunoconjugate or antibody drug conjugate, which comprises the above-mentioned antibody or antigen-binding fragment of the present invention, the above-mentioned single-chain antibody conjugated with a therapeutic agent or a diagnostic reagent Or the single domain antibodies described above.

The twentieth aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned STAR, the above-mentioned STAR complex, the above-mentioned antibody or antigen-binding fragment, the above-mentioned single domain antibody, the above-mentioned antigen Receptor, nucleic acid mentioned above, immune cell mentioned above.

Preferably, the medicament also contains pharmaceutically acceptable auxiliary materials. Further preferably, the pharmaceutically acceptable excipients include but not limited to diluents, binders, wetting agents, surfactants, lubricants or disintegrants and the like.

The twenty-first aspect of the present invention provides a kit comprising the above-mentioned STAR, the above-mentioned STAR complex, the above-mentioned antibody or antigen-binding fragment, the above-mentioned single domain antibody, the above-mentioned antigen receptor body, the aforementioned nucleic acid, and the aforementioned immune cells.

The twenty-second aspect of the present invention provides a method for treating tumors, the method comprising administering an effective amount of the STAR, STAR complex, CAR, antibody or antigen-binding fragment thereof of the present invention to a subject , single chain antibody, single domain antibody, immune cell, CAR-T cell or pharmaceutical composition.

The "linker" in the present invention includes but not limited to rigid linker, flexible linker, cleavable linker or nonsense amino acid. Preferably, the amino acid sequence of the rigid linker is selected from one or more than two of SEQ ID NO: 49-59. Preferably, the flexible linker is selected from peptides rich in glycine and/or serine; Preferably, the flexible linker is selected from one or more of SEQ ID NO: 60-112. Preferably, all The cleavable linker is selected from one or more than two of SEQ ID NO: 113-117.

The "antibody" of the present invention can be of any class (such as IgA, IgD, IgE, IgG and IgM) or subclass (such as IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2).

The "antigen-binding fragment" of the present invention includes, but is not limited to: Fab fragments, which have VL, CL, VH and CH1 domains; Fab' fragments, which have one or more cysteines at the C-terminus of the CH1 domain Fab fragment of residue; Fd fragment, it has VH and CH1 domain; Fd' fragment, it has VH and CH1 domain and one or more cysteine residues in the C-terminus of CH1 domain; Fv fragment, it has antibody The VL and VH domains of a single arm of ; a dAb fragment, which consists of a VH domain or a VL domain; an isolated CDR region; an F(ab')2 fragment, which is a fragment comprising two Fabs connected by a disulfide bridge at the hinge region Bivalent fragments of 'fragments; single-chain antibody molecules (e.g., single-chain Fv; scFv); "diabodies" with two antigen-combining sites comprising a light-chain variable domain (VL) linked in the same polypeptide chain a heavy chain variable domain (VH); a "linear antibody" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) which together with a complementary light chain polypeptide form a pair of antigen-binding regions; and A modified form of any of the foregoing, which retains antigen-binding activity.

The "CDR" in the present invention refers to the complementarity determining region in the variable sequence of an antibody. For each variable region, there are three CDRs in each variable region of the heavy and light chains, referred to as CDR1, CDR2 and CDR3. The exact boundaries of these CDRs are defined differently from system to system. The system described by Kabat et al (Kabat et al, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides unambiguous residue numbering for antibody variable regions system, but also provides residue boundaries defining three CDRs. These CDRs may be referred to as Kabat CDRs. Each complementarity determining region may contain amino acid residues from a "complementarity determining region" as defined by Kabat. Chothia et al. ( Chothia & Lesk, J.Mol.Biol, 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (-1989)) found that some sub-parts within the Kabat CDR adopt almost the same peptide backbone conformation , despite large diversity at the amino acid sequence level. These sub-segments are referred to as L1, L2, and L3 or H1, H2, and H3, respectively, where "L" and "H" denote the light and heavy chain regions, respectively. These regions may be referred to as a Chothia CDR, which has boundaries that overlap with Kabat CDRs. There are other CDR boundary definitions that may not strictly follow one of the above systems, but will still overlap with Kabat CDRs, and the methods used herein can utilize boundaries defined according to any of these systems CDR, although the preferred embodiment uses the CDR defined by Kabat or Chothia. The "antibody variable region" refers to the complementarity determining region (CDR, namely CDR1, CDR2 and CDR3) and framework in the light chain and heavy chain of the antibody molecule The portion of the amino acid sequence of the region (FR). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain.

"Diagnosis" in the present invention refers to finding out whether a patient has a disease or disorder in the past, at the time of diagnosis or in the future, or to find out the progress of a disease or possible future progress, or to evaluate a patient's response to treatment.

"Treatment" as used herein means slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, disorder, or disease, but not necessarily all disease-related signs, Complete elimination of a symptom, condition, or disorder, and refers to a therapeutic intervention that ameliorate the signs, symptoms, etc. of a disease or pathological condition after the disease has begun to develop.

The "effective amount" mentioned in the present invention refers to the STAR, STAR complex, CAR, CAR-T, STAR described in the present invention that provides the desired treatment or prevention after being administered to patients or organs in single or multiple doses. - amount or dose of T, immune cells, pharmaceutical composition, etc.

The "products" described in the present invention can be kits, chips, antibody conjugates, multifunctional antibodies, pharmaceutical compositions and the like.

The "individual" or "subject" in the present invention can be a human or a non-human animal, and the non-human animal can be a non-human mammal such as a mouse, a cow, a sheep, a rabbit, a pig, or a monkey.

The "and/or" in the present invention includes all combinations of the items connected by this term, and it should be considered that each combination has been listed separately in this question. For example, "A and/or B" includes "A", "A and B" and "B". As another example, "A, B and/or C" includes "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C ".

The "comprising" or "comprising" in the present invention is an open-ended writing method. When used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid can be composed of the sequence, or at one end of the protein or nucleic acid Or both ends may have additional amino acids or nucleotides, but still have the activity described in the present invention. In addition, those skilled in the art know that the methionine encoded by the start codon at the N-terminal of the polypeptide may be retained in some practical cases (such as when expressed in a specific expression system), but it does not substantially affect the function of the polypeptide. Therefore, when describing a specific amino acid sequence of a polypeptide in the specification and claims of this application, although it may not contain a methionine encoded by a start codon at the N-terminus, it also covers a methionine containing the methionine at this time. Sequence, correspondingly, its coding nucleotide sequence may also contain an initiation codon; and vice versa.

Description of drawings

Hereinafter, embodiments of the present invention will be described in detail in conjunction with the accompanying drawings, wherein:

Figure 1: Antibody expression and specificity identification of NM5/NM24-VHH. Among them, Figure 1A is the antibody expression of NM5-VHH, and M is Marker. Figure 1B is the antibody expression of NM24-VHH, M is Marker. Figure 1C is the specific staining result after co-incubation of the antibody with AsPC-1 cells. Figure 1D is the specific staining result after the antibody was co-incubated with 293T-MSLN cells, PC-Ab is a publicly known MSLN antibody, positive control, and NC is a negative control.

Figure 2: Schematic diagram of optimization of STAR by constant region cysteine modification, transmembrane and intracellular region modification.

Figure 3: Schematic representation of STAR optimization by adding co-stimulatory molecule receptor intracellular domains to α and/or β chains.

Figure 4: Schematic representation of optimization of STAR by addition of co-stimulatory molecule receptor intracellular domains directly or via linkers after deletion of the intracellular domains of the α and/or β chains.

Figure 5: Schematic representation of STAR optimization by adding co-stimulatory molecule receptor intracellular domains to CD3 subunits.

Figure 6: Schematic representation of STAR optimization by adding cytokine receptor intracellular signaling domains to α and/or β chains.

Figure 7: Affinity detection of MSLN single domain antibodies NM5 (A) and NM24 (B).

Figure 8: Schematic diagram of the structure of anti-MSLN-co-STAR.

Figure 9: Experimental results of infection efficiency (RFP+ subpopulation ratio) of NM5, NM13, NM24 STAR and negative control (NC).

Figure 10: Selection of target cells expressing MSLN, A is a pancreatic cancer tumor cell line, B is an antigen overexpressing cell line.

Figure 11: Comparison of killing functions of different VHHs in vitro. AsPC-1 belongs to a human metastatic pancreatic adenocarcinoma cell line and is a MSLN-positive target cell. 293T were transfected to express human hMSLN and mouse mMSLN respectively. Figure 11A shows the in vitro killing of various nanobodies on AsPC-1 and 293T cells, and Figure 11B shows that STAR T cells with four nanobody sequences of NM5, 11, 13, and 24 have a strong killing effect on Aspc-1, Figure 11B 11C shows that NM5 and NM24 STAR T cells can recognize and kill 293T-hMSLN but cannot recognize and kill 293T-mMSLN target cells.

Figure 12: Cytokine release levels of NM5/NM24-VHH based anti-MSLN STAR synthetic mutants. Among them, Figure 12A is the release level of IFN-γ, Figure 12B is the IFN-γ release level of NM5 STAR T cells, NM11 STAR T cells, NM13 STAR T cells, and NM24 STAR T cells after 24 and 48 hours, and Figure 12C is the IL -2 release level, Figure 12D is the IL-2 release level of NM5 STAR T cells, NM11 STAR T cells, NM13 STAR T cells, and NM24 STAR T cells after 24 and 48 hours.

Figure 13: The results of anti-MSLN-STAR-T cell therapy for tumors, where Figure 13A shows the in vivo anti-tumor effects of Mock T cells, NM5 STAR T cells, NM13 STAR T cells, and NM24 STAR T cells in mouse tumor models; Figure 13B is the statistical result of tumor fluorescence in mice corresponding to Figure 13A; Figure 13C is the weight change of mice after reinfusion of Mock T cells, NM5 STAR T cells, NM13 STAR T cells, and NM24 STAR T cells; Figure 13D Shows the proliferation of Mock T cells, NM5 STAR T cells, NM13 STAR T cells, and NM24 STAR T cells in mice; Figure 13E shows Mock T cells, NM5 STAR T cells, NM13 STAR T cells, NM24 STAR T cells The percentage of CD4 ⁺ T cells of T cells in the mouse tumor model; Figure 13F shows the CD8 ⁺ T cells of Mock T cells, NM5 STAR T cells, NM13 STAR T cells, and NM24 STAR T cells in the mouse tumor model percentage content.

Figure 14: The results of anti-MSLN-STAR-T cell therapy on tumors, in which Figure 14A shows the anti-MSLN-STAR-T cells constructed when (G4S)nlinker is n=0 and n=5 respectively in mouse tumors The in vivo anti-tumor effect of the model; Figure 14B is the statistical result of tumor fluorescence in mice corresponding to Figure A; Figure 14C is the anti-MSLNSTAR comprehensive variable constructed by α-del-OX40 and α-del-(G4S)5-OX40 Figure 14D is the anti-MSLN STAR comprehensive variant constructed by α-del-OX40 and α-del-(G4S)5-OX40 CD62L ⁺ CD8 ⁺ T cells in the mouse tumor model percentage content.

Figure 15: Pharmacokinetic analysis experiment of NM24 STAR-T cells in various tissues reinfused for 0-21 days, wherein Figure 15A is the copy number of NM24 STAR T cells; Figure 15B is the percentage of NM24 STAR T cells.

Figure 16: The comparative test experiment of the in vitro killing and IFN-γ secretion of NM24 STAR-T cells and the similar cell product TCR ² TC-210 (SD1-eTruC T cells), where Figure 16A shows the in vitro killing effect, and Figure 16B shows The release level of IFN-γ secretion is shown, where E/T ratio is the ratio of effector to target.

Figure 17: The in vivo drug efficacy and safety of NM24 STAR-T cells and the comparative test experiment of similar cell product TCR ² TC-210 (SD1-eTruC T cells), where Figure 17A shows Mock T cells, NM24 STAR T cells , SD1-eTruC T cells in vivo anti-tumor effect in mouse tumor models; Figure 17B is the statistical results of tumor fluorescence in mice corresponding to Figure 17A; Figure 17C is the reinfusion of Mock T cells, NM24 STAR T cells, SD1-eTruC Body weight changes of mice after T cells; FIG. 17D shows the survival rate of mice after reinfusion of Mock T cells, NM24 STAR T cells, and SD1-eTruC T cells.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Example 1: Sequence screening of single domain antibodies targeting MSLN

1. Preparation of human MSLN protein

1) Construction of expression vector

Using human MSLN highly expressed tumor cell cDNA as a template, the DNA sequence of the extracellular region of human MSLN protein is shown in SEQ ID No.120, and its amino acid sequence is shown in SEQ ID No.121. The extracellular region of MSLN was amplified by PCR. The extracellular region of the amplified MSLN was ligated into the pVRC expression vector by enzyme cutting-ligation method, and transformed into DH5α competent cells, and a single colony or plasmid was extracted for sequencing.

2) Protein expression

When the density of 293F cells grows to about 2×10 ⁶ cells/mL, transfection is carried out. 200 μg DNA is required to transfect 200 mL cells, and the mass ratio of plasmid DNA to polyethyleneimine PEI (concentration 1 mg/mL) is about 1:3, that is 200μg: 600μg, add the plasmid DNA and PEI to the anti-resistant medium respectively to a final volume of 5mL. Add the mixture of PEI and medium to the mixture of plasmid DNA and medium, and incubate for about 15-30 minutes. The volume of the incubation mixture is about 1/10 of the volume of the cells to be transfected. The incubated mixture was added to 293F cells, cultured for 72-96 hours, and the cells or medium supernatant were collected for protein extraction.

3) Protein purification

The collected medium supernatant was centrifuged to remove the precipitate, and after the supernatant was passed through a 0.22 μm filter membrane, it was concentrated and replaced with a lysate using a concentration bag. Wash 10 column volumes of the nickel column with the lysate washing solution to remove non-specifically bound impurities, and finally use the eluent to elute the target protein from the affinity chromatography column. After concentrating the replacement solution, measure the concentration, subpackage and store in freezer, and save the sample to run SDS-PAGE gel.

2. Immunization of alpaca with human MSLN protein

The human MSLN protein extracellular region (100 μg) expressed and purified by the eukaryotic system in the first step was used to immunize healthy alpacas, and the adjuvants included complete Freund's adjuvant (CFA, Sigma) and incomplete Freund's adjuvant (IFA, Sigma) ). The above-mentioned expressed and purified extracellular region of human MSLN protein was diluted with PBS, and then mixed with corresponding adjuvants 1:1. The antigen and adjuvant are completely mixed to form a stable emulsifier, and the antigen mixture is extracted with a syringe, and injected subcutaneously at multiple points under the skin of the alpaca's neck, with 100-200 μL per point. The specific procedure for immunizing animals is as follows:

1) Day 1 (first immunization): MSLN antigen (100 μg) mixed with complete Freund's adjuvant (CFA) was injected subcutaneously to immunize alpacas;

2) On the 14th day (the second immunization): the alpaca was immunized by subcutaneous injection of MSLN antigen (100 μg) mixed with incomplete Freund's adjuvant (IFA);

3) On the 28th day (the third immunization): the alpaca was immunized by subcutaneous injection of MSLN antigen (100 μg) mixed with incomplete Freund's adjuvant (IFA) again;

4) Day 42 (the first serum collection): Alpaca blood samples were taken from alpaca ear veins, serum was extracted, and antibody titer was tested. The P/N value of 200,000-fold diluted serum was greater than 2;

5) Day 42 (4th immunization): MSLN antigen (100 μg) mixed with incomplete Freund's adjuvant (IFA) was injected subcutaneously again to immunize alpacas to enhance the immune effect;

6) Day 53 (second serum collection): Alpaca blood samples were taken from alpaca ear veins, serum was extracted, and antibody titer was tested. The P/N value of 200,000-fold diluted serum was greater than 2;

7) Days 54, 57, and 60: 30-40 mL alpaca blood samples were collected from the alpaca hind leg veins for PBMC isolation.

3. PBMC isolation

1) Separate PBMCs in a biological safety cabinet, combine the blood in the anticoagulant tube into a 50mL centrifuge tube (30mL), add PBS to a total volume of 50mL, and mix gently.

2) Take a new 50mL centrifuge tube and add Ficoll separation solution, 15mL/tube. Spread blood samples on the surface of Ficol liquid, 25mL/tube. The operation process should be stable to form a layer of Ficoll and blood to prevent mixing.

3) Adjust the lifting speed of the centrifuge to 0, RT, centrifugal force 800g, and centrifuge for 30min. After centrifugation, take the sample out of the centrifuge and layer the sample as follows: upper aqueous phase-buffy coat-Ficoll layer-erythrocyte layer, in which PBMC is in the buffy coat layer, suck out the buffy coat layer and transfer to a new 50mL centrifuge tube middle.

4) Add PBS to the sample tube to 50mL, mix well, centrifuge at 2000rpm, RT for 5min, discard the supernatant, add 5mL PBS to resuspend the cell pellet.

5) Repeat step 4 while lowering the temperature to 4°C.

6) Add 5mL PBS to resuspend the cell pellet, add PBS to 40mL, and count the cells.

7) Centrifuge at 1500rpm at 4°C for 5min, discard the supernatant, add 1mL PBS, resuspend the cells, blow evenly; add 20mL Trizol, mix well, let stand at room temperature for 5min, lyse the cells, and aliquot 1mL into RNase-free 1.5mL EP Tubes, stored at -80°C for RNA extraction.

4. RNA extraction and reverse transcription

1) Take the sample out from -80°C, thaw it at room temperature, add 200 μL of chloroform, and let it stand at room temperature for 3 minutes. Centrifuge the sample at 12000g for 15min at 4°C. The sample is divided into the upper aqueous phase, the middle layer and the organic layer. Transfer the upper layer to a new RNase-free tube, add 1 μL of glycogen and 500 μL of isopropanol. Stand overnight at 4°C.

2) Centrifuge the sample at 12000g at 4°C for 20 min, remove the supernatant, add 1 mL of pre-cooled 75% alcohol to wash the precipitate, centrifuge again to remove the alcohol, and dry it in the air. Add 15 μL of RNase-free water to each tube to dissolve the RNA pellet for reverse transcription.

3) Promega reverse transcription kit was used to synthesize cDNA (20 μL system).

Step 1: Take a certain amount of template RNA and add Oligo(dT), see Table 1;

Table 1

组分components	体积(μL)Volume (μL)
RNA(<5μg/reaction)RNA(<5μg/reaction)	1111
Oligo(dT)15PrimerOligo(dT) 15 Primer	11
总体积 total capacity	1212

Step 2: Put the mixture of template RNA and Oligo(dT) at 65°C for 5 minutes for pre-denaturation, and put it back on ice after completion.

Step 3: During pre-denaturation, RT-Mix can be prepared in advance, 8 μL per tube, and the components and volumes are shown in Table 2.

Table 2

组分components	体积(μL)Volume (μL)
5×Reaction Buffer5×Reaction Buffer	4μL4μL
RiboLockRnaseInhibitor(20U/μL)RiboLockRnase Inhibitor (20U/μL)	1μL1μL
10mM dNTP Mix10mM dNTP Mix	2μL2μL
RevertAid M-MuLVRT(200U/μL)RevertAid M-MuLVRT (200U/μL)	1μL1μL
总体积total capacity	8μL8μL

Step 4: Set reverse transcription program, extension, reverse transcriptase inactivation. The cDNA was obtained after the procedure was completed.

5. Phage library construction

1) Obtain the VHH sequence by PCR

The VHH sequence was obtained by two rounds of PCR, and the homology arms of the vector were added at both ends of the sequence.

2) The first round of PCR

Step 1: See Table 3 for the preparation of the reaction system.

table 3

Step 2: See Table 4 for PCR conditions.

Table 4

The PCR product was subjected to gel electrophoresis, and the target band at 0.7kb was cut out for product recovery.

3) The second round of PCR

Step 1: See Table 5 for the preparation of the reaction system.

table 5

Step 2: See Table 6 for PCR conditions.

Table 6

The PCR product was subjected to gel electrophoresis, and the target band at 400 bp was cut out for product recovery.

4) Vector PCR

The vector region of the phagemid was obtained by PCR for expression of the VHH sequence.

Step 1: See Table 7 for the preparation of the reaction system.

Table 7

Step 2: See Table 8 for PCR conditions.

Table 8

The PCR product was subjected to gel electrophoresis, and the target band of 4000 bp was cut out for product recovery.

5) Connection, purification and concentration of connection products

The VHH fragments were ligated to the phagemid vector, and the ligation product was then concentrated.

Step 1: See Table 9 for the preparation of the reaction system.

Table 9

Step 2: Incubate the above mixture at 50° C. for 2 h, and cool on ice.

Step 3: Purify the ligation product, remove salt ions and proteins in the ligation system, and concentrate the volume to 1/10 of the original volume.

6. Electrical transfer to build library

1) Take a tube of competent E. coli and thaw it on ice.

1) Take 2 μL of the ligation product or the positive control, add it to the above-mentioned competent state, and gently blow evenly. Let stand on ice for 1-2 minutes, transfer to a pre-cooled electroporation cup, and perform electroporation.

2) Immediately after electroporation, add 1 mL of 37°C 2YT-G, rinse the electroporation cup with a pipette tip, transfer the electroporated bacterial solution to a 15mL centrifuge tube or a 2mL EP tube, and recover in a 37°C water bath until electroporation of all samples is complete. (2YT-G: 2×YT medium containing 2% Glucose) was transferred to a shaker at 37° C., 220 rpm, and recovered for 1 h.

3) Aspirate 5 μL of the above-mentioned bacterial solution from the above-mentioned bacterial solution and dilute it 10^2-10^5 times, coat a plate with 2YT-A (2YT plate containing 100 μg/mL ampicillin), and place it in a 37°C incubator overnight for colonization count.

4) Inoculate the remaining bacterial solution into 2YT-AG medium, shake to the logarithmic phase, add helper phage to infect, shake the bacteria at 30°C, 220rpm for 12-16h. (2YT-AG: 2×YT medium containing 2% Glucose and 100 μg/mL ampicillin)

5) Collect and concentrate the phage, and measure the titer.

7. Phage library antibody screening

The phage library obtained in step 7 above was subjected to three rounds of antibody screening, and each screening included a positive selection and a negative selection. Incubate the phage with the antigen peptide first, discard the phage that cannot bind to the antigen peptide, and leave the phage that binds to the antigen peptide, and then incubate with BSA for negative selection, leaving the phage that cannot bind to BSA.

1) Coat board. Dilute the antigen with PBS to a concentration of 2ng/μL, add it to a 96-well plate, 100μL/well; prepare 2% BSA with PBS, add it to the corresponding negative selection well, 100μL/well, seal with plastic wrap, and incubate at 4°C overnight.

2) Discard the coating solution, add 200 μL of washing solution (washing solution: 1% Tween 20/PBS, pH 7.4), and wash 3 times.

3) closed. Add 2% BSA blocking solution to all wells, 100 μL/well, seal with plastic wrap, and incubate at 37°C for 1 hour.

4) Discard the supernatant, add 200 μL washing solution, and wash 3 times.

5) Add phages to the positively selected wells, 1×10 ¹² phages/well, dilute the volume to 100 μL, seal with plastic wrap, and incubate at 37° C. for 1 hour.

6) Discard the supernatant, add 200 μL washing solution, and wash 10 times.

7) Elution-neutralization. Add 200 μL of eluate to positively selected wells and neutralize to pH 7-7.4.

8) Negative selection. Add the above-mentioned eluent to the negative selection well, seal it with plastic wrap, incubate at 37°C for 1 hour, absorb and save the supernatant for titer detection.

9) Dilute a small amount of phage after one round of Panning and spread on 2×YT-A plates, overnight at 37°C; count the colonies the next day, calculate the titer; and pick single clones for sequencing to analyze sequence diversity and enrichment.

10) All remaining phages were used for TG1 infection.

11) M13KO7 infection. Dilute the phage, add M13KO7 to the bacterial solution, bathe in water at 37°C for 30min; replace 2×YT-AK medium, shake the bacteria at 30°C, 220rpm for 14-16h.

12) Concentrate the phage and detect the phage titer, then the next round of screening can be carried out.

The amount of coated antigen in the second round of screening and the third round of screening is reduced, and the number of washings is increased after the phage is incubated with the positive wells, and other steps are the same as the above steps.

8. Combined detection and sequence acquisition

The phage obtained from the three rounds of screening and the M13KO7 helper phage co-infect TG1, spread on 2YT-AK plate, pick a single clone for phage expansion, collect phage for binding detection, and determine the available phage/antibody.

1) Coat board. Dilute the antigen with coating solution to 1ng/μL, 100μL/well; add 2% BSA to the control negative well; seal with plastic wrap, overnight at 4°C.

2) Discard the coating solution in the plate, add 200-250 μL of washing solution and wash 3 times.

3) Add 200 μL of 2% BSA to all wells and block for 1 h at room temperature.

4) Discard the blocking solution in the plate, add 200 μL of washing solution, and wash once.

5) Add 100 μL of phage to each of the positive and negative wells, and incubate at 37° C. for 1 h.

6) Discard the phage in the plate, add 200 μL of washing solution, and wash 3 times.

7) Dilute the anti-M13-HRP antibody, 50ng/well, and incubate at room temperature for 1h.

8) Discard the antibody in the plate, add 200 μL of washing solution, and wash 5 times.

9) Add 100 μL of TMB chromogenic solution to each well, and react at room temperature until the OD value is between 2 and 3.

10) Add stop solution to the chromogenic system, 50 μL per well.

11) Measure the absorbance at 450 nm with a spectrophotometer. The monoclonal bacterial liquid corresponding to the positive well was sent for sequencing to determine the VHH sequence.

12) The obtained antibodies were named NM1-24 respectively.

9. Functional screening at the cellular level

1) Assemble the positive antibody VHH sequence obtained from the aforementioned screening with the constant region of the STAR molecule, and use homologous recombination to insert it into a lentiviral vector to construct a complete STAR plasmid.

2) Packaging virus

Inoculate Lentix-293T cells at 5×10 ⁵ cells/mL into 10 cm culture dishes, culture them in a 37°C, 5% CO ₂ incubator, and perform transfection when the cell density reaches about 80% (observed under a microscope). dye. The four plasmids were mixed evenly with 500 μL of serum-free DMEM according to the ratio of PMD2.G:PRSV-Rev:PMDlg:transfer plamid=1:1:2:4. Mix 54 μL PEI-max with 500 μL serum-free DMEM evenly, and let stand at room temperature for 5 minutes (the volume-mass ratio of PEI-Max and plasmid is 3:1). Slowly add the PEI-max mixture to the plasmid mixture, gently pipette, mix well, and let stand at room temperature for 15 minutes. Slowly add the final mixed solution into the culture medium, mix well, put it back into the incubator to continue culturing for 12h-16h, change to 6% FBS DMEM medium to continue cultivating, and collect the virus liquid for 48h and 72h.

3) Virus titer measurement

TCR-knockout Jurkat-C4 cells were seeded in a flat-bottomed 96-well plate at 1.5×10 ⁵ cells/mL, and 100 μL of 1640 medium containing 10% FBS and 0.2 μL of 1000× polybrene was added to each well. When the virus was diluted, it was diluted 10 times with 1640 complete medium. Add the diluted cells into the virus wells, 100 μL/well, mix, 32°C, 1500rpm, centrifuge for 90min, culture in a 37°C, 5% _CO2 incubator, measure the infection efficiency with a flow cytometer after 72h, and calculate the titer , select wells with an infection rate of 2-30%, and the calculation formula is: titer (TU/mL)=1.5×10^4×positive rate÷virus volume (μL)×1000. The above viruses are used to infect T cells to express STAR.

4) Isolation, activation and infection of primary human T cells

After the primary T cells were obtained by Ficoil separation method, cultured in X-VIVO medium containing 10% FBS and 100IU/mL IL-2, the initial culture density was 1×10 ⁶ /mL, and CD3, CD28 and Fibronectin were added Activated in the orifice plate. After 24 hours of activation, virus solution was added, centrifuged at 1500 rpm for 90 minutes, and placed in a CO ₂ incubator for cultivation. After 24 hours of infection, X-VIVO medium containing 10% FBS and 100IU/mL IL-2 was supplemented, and the wells were transfected, and then subcultured at intervals of 1-2 days.

5) T cells and target cells were co-cultured in vitro to determine the killing efficiency and cytokine release

One day in advance, the target cells AsPC-1 or 293T cells overexpressing MSLN were plated in a 24-well plate at a density of 1E5/well and cultured overnight. Ratio, add the corresponding number of STAR-T cells into the target cells, and detect the killing efficiency of STAR-T cells on the target cells and the concentration of cytokines released in the supernatant after 24 hours or 48 hours of culture.

10. Antibody expression and specificity identification

The VHH region of NM5 and NM24 was fused with the Fc region of human IgG1 to construct a plasmid, which was transformed into 293F cells, and the supernatant was collected to purify the corresponding antibody. And identify the antibody size by SDS-PAGE. The purified antibody was used for staining and flow cytometry of 293T-MSLN cells to determine the specificity of the antibody. (See Figures 1A-1D)

11. Antibody Affinity Determination

The CM5 chip was used to immobilize the MSLN protein as the stationary phase, and the recombinantly expressed antibody NM5-hFc or NM24-hFc was used as the mobile phase, and the affinity KD values of the two recombinant antibodies were measured on a Biacore 8kplus instrument. The results are shown in Figure 7 and Table 10.

Table 10

immobilized ligand

Inject analyte solution

KD(M)

1:1 binding ka(1/Ms)

kd(1/s)

MSLNMSLN	NM1NM1	1.66E-091.66E-09	5.52E+055.52E+05	9.14E-049.14E-04
MSLNMSLN	NM3NM3	4.31E-114.31E-11	1.31E+061.31E+06	5.77E-055.77E-05
MSLNMSLN	NM5NM5	1.80E-101.80E-10	6.08E+056.08E+05	1.09E-041.09E-04
MSLNMSLN	NM11NM11	4.03E-104.03E-10	2.26E+062.26E+06	9.09E-049.09E-04
MSLNMSLN	NM12NM12	6.74E-106.74E-10	6.04E+076.04E+07	4.07E-024.07E-02
MSLNMSLN	NM13NM13	1.35E-101.35E-10	5.80E+075.80E+07	7.84E-037.84E-03
MSLNMSLN	NM16NM16	1.17E-091.17E-09	6.58E+076.58E+07	7.69E-047.69E-04
MSLNMSLN	NM17NM17	3.00E-093.00E-09	2.15E+052.15E+05	6.45E-046.45E-04
MSLNMSLN	NM20NM20	7.40E-107.40E-10	4.05E+054.05E+05	3.00E-043.00E-04
MSLNMSLN	NM23NM23	1.78E-091.78E-09	3.34E+053.34E+05	5.93E-045.93E-04
MSLNMSLN	NM24NM24	5.90E-105.90E-10	9.59E+069.59E+06	5.66E-035.66E-03

Example 2: Structural design of STAR targeting MSLN

The variable region (antigen-binding region) of the human TCRα and β chains was replaced with an antibody heavy chain variable region (VHH) derived from alpaca that can specifically recognize mesothelin (MSLN), and the TCRα chain The following modifications were made to the constant regions of the α and β chains: human TCR α and β chain sequences were replaced by mouse sequences; two cysteine mutations were introduced into each of the two chains; hydrophobic mutations were introduced into the transmembrane region of the α chain; Deletion of intracellular amino acids; co-stimulatory factors linked intracellularly to both chains. Thus, a mutant synthetic T-cell receptor antigen receptor (Synthetic T-Cell Receptor and AntigenReceptor, STAR) capable of specifically recognizing MSLN was constructed, and its structure is shown in Figure 2.

1. Design of wild-type T cell receptor and its constant region mutant STAR molecule

1) Prototype design of STAR

The secreted antibody (Antibody, Ab) or B cell receptor (BCR) and T cell receptor (TCR) produced by B cells have great similarities in gene structure, protein structure and spatial conformation. Antibodies and TCRs are composed of variable regions and constant regions, in which the variable region plays the role of antigen recognition and binding, while the constant region plays the role of structural interaction and signal transduction. A synthetic chimeric molecule can be constructed by replacing the variable regions of the TCR α and β chains (or TCR γ and δ chains) with the heavy chain variable region (VH) and light chain variable region (VL) of the antibody , called Synthetic T-Cell Receptor and Antigen Receptor (STAR/WT-STAR), its structure is shown in Figure 2 (left).

The STAR molecule has two chains, the first chain is obtained by fusing the antigen recognition region and the constant region of the T cell receptor, and the second chain is obtained by fusing the antigen recognition region and the constant region of the T cell receptor. In this construct, the antigen recognition domain can be derived from the antibody variable region (such as VH, VL, scFv or VHH) of human, mouse, alpaca, and rabbit, and the T cell receptor constant region can be derived from human or mouse antibody. The constant regions of TCR α and β chains or γ and δ chains, through different combinations of these antigen recognition regions TCR constant regions, can obtain a variety of constructs with different configurations but similar functions.

After the two chains of the STAR molecule are expressed in T cells, they will combine with the endogenous CD3εδ, CD3γε, and CD3ζζ chains in the endoplasmic reticulum to form a complex of 8 subunits, which will be displayed on the cell membrane in the form of complexes surface. A complete STAR complex contains 10 Immunoreceptor Tyrosine-based Activation Motifs (ITAM) (the intracellular regions of CD3ε, δ, γ, and ε chains each contain 1 ITAM sequence, and the CD3ζ chain The intracellular region contains 3 ITAM sequences) When the antigen recognition sequence of the STAR receptor binds to its specific antigen, the ITAM sequence in the cell will be phosphorylated, thereby activating downstream signaling pathways, activating NF-κΒ, NFAT and AP-1 Transcription factors such as T cell activation and effector function. The inventors' previous studies showed that compared with the conventional chimeric antigen receptor (CAR), STAR can mediate a stronger T cell activation signal, and the background activation of STAR-T cells in the absence of antigen stimulation is significantly reduced , and it is not easy to be exhausted under continuous antigen stimulation, so it has significant advantages (see Chinese invention patent application number: 201810898720.2). However, the present invention provides a further improvement on STAR.

2) Design of mutant STAR (mut-STAR) and STAR with modified transmembrane and intracellular regions (ub-STAR)

The STAR prototype design uses the constant region sequences of human TCRα and β chains (or TCRγ and δ chains) (wild-type human TCRα constant region, SEQ ID NO:1; wild-type human TCRβ constant region, SEQ ID NO:2 ). Since the constant region sequences of human, primate and murine TCRα/β chains have high functional conservation and the key amino acid sequences are the same, they can be replaced with each other.

After the STAR molecule is transfected into T cells, it will mismatch with the endogenous TCR of T cells through the constant region. On the one hand, this mismatch problem will reduce the efficiency of correct pairing of STAR molecules and weaken its function; on the other hand, it will increase the possibility of mismatching to generate unknown specific receptor molecules and increase safety risks. To solve this problem, the inventors made the following modifications to the wt-STAR molecule: a. The constant region of the STAR molecule was replaced with a mouse sequence, (wild-type mouse TCRα constant region mouse TCRaC-WT, SEQ ID NO:3 ; the wild-type mouse TCRβ constant region mouse TCRbC-WT, SEQ ID NO: 4), so that the mouse constant region cannot be mismatched with the endogenous human TCR chain and improve the distance between the two chains of the exogenously transferred STAR molecule pairing efficiency. b. Carry out a cysteine point mutation in the constant region of the STAR molecule, specifically mutating the 48th threonine T to cysteine C (mouse TCRaC-Cys, SEQ ID NO: 5) in the constant region of the TCRα chain. In the TCRβ chain constant region, the 56th serine S is mutated into cysteine C (mouse TCRbC-Cys, SEQ ID NO: 6). These two newly added cysteines will form a disulfide bond between the two chains of STAR, reduce the mismatch between the two chains of STAR and the endogenous TCR chain, help STAR molecules form a more stable complex, and obtain better functionality. In addition, in order to further increase the stability of STAR molecules and improve the function of STAR-T cells, the inventors also carried out the following modifications to STAR molecules: c. Replace the transmembrane region of STAR with hydrophobic amino acids, specifically in the constant TCRα chain In the 111th to 119th amino acid region of the transmembrane region, 3 amino acid mutations were carried out: 112-position serine S was changed to leucine L, 114-position methionine M was changed to isoleucine I, 115 Glycine G becomes valine V. The overall amino acid sequence of this region is changed from LSVMGLRIL to LLVIVLRIL. This modification is called mouse TCRaC-TM9, and the resulting constant region sequence is SEQ ID NO:7. This design increases the hydrophobicity of the transmembrane region, offsets the instability caused by the positive charge carried by the TCR transmembrane region, and enables STAR molecules to exist more stably on the cell membrane, thereby obtaining better functions. See Figure 2 (middle). d. Mutate the lysine in the intracellular region of the constant region of the alpha chain of the STAR molecule, and the transmembrane region and the intracellular region of the constant region of the beta chain to arginine to generate the constant region sequence Mouse TCRαC-Arg mut (SEQ ID NO:8) and the constant region sequence is Mouse TCRβC-Arg mut (SEQ ID NO:9), this design reduces the possibility of ubiquitination of the transmembrane region and intracellular end of the STAR molecule, thereby reducing the endocytosis of the STAR molecule phenomenon, so that STAR molecules can exist more stably on the cell membrane, and thus obtain better functions. Its structure is shown in Figure 2 (right).

2. Design of wild-type and mutant STAR molecules containing co-stimulatory molecule receptor intracellular domains

In order to improve the proliferation ability, survival time of STAR-T cells in vivo and the ability to infiltrate into the tumor microenvironment and kill target cells efficiently, the inventors designed a new structure, modified the STAR complex, and can Customize enhanced mut-STAR cells according to needs to improve the clinical response rate of STAR-T cells and achieve durable efficacy.

1) Design of a mut-STAR molecule (co-STAR) containing the intracellular domain of a co-stimulatory molecule receptor

In order to enhance STAR-T cytotoxicity and cell proliferation persistence, the present invention introduces the intracellular end sequences of human co-stimulatory molecule receptors into the C-terminus of the STAR constant region (Figure 3), and explores its effect on STAR-T cell function Influence. The STAR constant region described in the present invention includes the unmodified wt-STAR constant region, the cys-STAR constant region containing additional intermolecular disulfide bonds, the constant region of murine hm-STAR and the integration of the 1st subsection. Three modified mut-STARs. The co-stimulatory signal transduction structure includes the intracellular signal transduction domain of CD40, OX40, ICOS, CD28, 4-1BB or CD27, and the sequences are respectively SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 , SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15. The intracellular domain of co-stimulatory molecules can be connected in series at the C-terminus of the TCRα chain, or at the C-terminus of the β-chain, or in parallel at the C-terminals of the α-chain and β-chain (co-STAR), individually or simultaneously.

In addition, the costimulatory molecular domain and the C-terminal of the TCR constant region can be connected directly or through a flexible linker (SEQ ID NO:60 to NO:112) containing glycine and serine, a rigid linker containing alanine and glutamic acid ( SEQ ID NO:49 to NO:59), and the cleavable type linker (SEQ ID NO:113 to NO:117), or remove the TCR molecule intracellular end sequence and then connect (the intracellular end is removed and contains half TCRα chain constant region (mouse TCRαC-del mut, SEQ ID NO: 16) modified by cystine substitution and hydrophobic region, TCRβ chain constant region (mouse TCRβC-delmut, mouse TCRβC-delmut, SEQ ID NO:17)) (co-linker-STAR, Figure 4). Multiple co-stimulatory molecular domains can also be connected directly or through a linker to the α-chain and β-chain of the TCR constant region or the C-terminus of the α-chain or β-chain (as shown in dCo-STAR or tCo-STAR, Figure 4) γδT The TCR of cells is composed of two chains, TCRγ and TCRδ. According to the type of TCRδ chain, γδT cells can be divided into three subgroups, γδ1, γδ2 and γδ3. Different subgroups have different distributions in the human body. γδT cells primarily recognize non-peptide antigens in a non-MHC-restricted manner and play an important role in pathogen and tumor surveillance. The present inventors introduced the intracellular end sequences of human co-stimulatory molecule receptors into the C-terminals of TCRγ and TCRδ respectively, expecting to improve the performance of γδT cells.

2) Design of CD3 molecule (co-CD3-STAR) containing costimulatory receptor intracellular domain

CD3 subunits include γ chain, δ chain, ε chain and ζ chain, which form a T cell receptor complex with TCR molecules, and transmit signals from extracellular to intracellular, thereby regulating the state of cells and responding to stimuli. In order to design enhanced TCR T cells and improve the tumor killing ability, proliferation ability and retention time of T cells in vivo, the inventors modified the CD3 molecule and introduced the intracellular domain of the human co-stimulatory molecule receptor into the CD3γ chain ( C-termini of SEQ ID NO: 18), delta chain (SEQ ID NO: 19), ε chain (SEQ ID NO: 20) and zeta chain (SEQ ID NO: 21) (Figure 5). Expressing the engineered CD3 molecule in mut-STAR-T cells is expected to improve its function.

3) Design of STAR molecule (cytokine-STAR, CK-STAR) containing cytokine receptor stimulation region

Cytokines play an important role in T cell proliferation, anti-tumor, differentiation and other functions. Different cytokines combine with their receptors to transmit signals from extracellular to intracellular, thereby regulating the state of cells and responding to stimuli.

In addition, studies have shown that the downstream molecule STAT5 (SEQ ID NO: 25) is activated through a cascade reaction at the intracellular end of the IL-2 receptor, thereby enhancing the transcription of T cell proliferation-related molecules and enhancing the proliferation ability of CAR-T cells. In order to design enhanced STAR-T cells and improve the tumor-killing ability, proliferation ability and retention time of T cells in vivo, the inventors modified the STAR molecule and incorporated the intracellular signal transduction region of human cytokine receptor (such as : IL-2β receptor intracellular end IL2Rb, SEQ ID NO:22; IL-7α receptor intracellular end, SEQ ID NO:23; IL-21 receptor intracellular end, SEQ ID NO:24, etc.) in series in TCRα The C-terminal of the chain, or the C-terminal of the β-chain, or the C-terminal of the α-chain and β-chain, or the STAT5 activation module (SEQID NO:25) can be further connected to the intracellular region of IL-2β or IL-7Rα receptor through G4S end (IL-2RbQ, SEQ ID NO:26; IL-7RbQ, SEQ ID NO:27) (Figure 6).

Embodiment 3: Molecular carrier construction of anti-MSLN STAR based on NM5/24-VHH

1. Vector source

The vectors used in the present invention, including viral vectors, plasmid vectors, etc., are purchased from commercial companies or synthesized by commercial companies, and the full-length sequences of these vectors are obtained, and the definite enzyme cutting sites are known.

2. Fragment source

The STAR mentioned in the present invention includes the aforementioned wt-STAR, mut-STAR, ub-STAR, co-STAR, co-linker-STAR, CK-STAR, co-CD3-STAR, etc. The gene fragments used in the present invention, such as the constant region of TCR, the intracellular region of co-stimulatory molecule receptors, the intracellular signal transduction region of cytokine receptors, tag sequences and linkers, etc., are all synthesized from commercial companies.

The fragment of the STAR antigen-binding region used in this example is derived from the VHH of the antibody NM5 targeting MSLN (its amino acid sequence is shown in SEQ ID NO: 28, and its nucleotide sequence is shown in SEQ ID NO: 118) and/or Or the VHH of the antibody NM24 targeting MSLN (its amino acid sequence is shown in SEQ ID NO: 29, and its nucleotide sequence is shown in SEQ ID NO: 119), wherein the anti-MSLN NM5 VHH comprises SEQ ID NO Heavy chain CDR1 shown in :34, heavy chain CDR2 shown in SEQ ID NO:35 and heavy chain CDR3 shown in SEQ ID NO:36, the anti-MSLN NM24VHH includes heavy chain CDR1 shown in SEQ ID NO:37, SEQ ID Heavy chain CDR2 shown in NO:38 and heavy chain CDR3 shown in SEQ ID NO:39. VHH sequences were codon-optimized and synthesized by commercial companies.

3. Vector Construction

The lentiviral vector pCDH-IRES-RFP used in the present invention obtains a linear vector through the restriction endonuclease NotI/NheI, the gene fragments are obtained by synthesis and PCR, and the linearized vector and each gene fragment are connected by a homologous recombination method Get the full vector.

4. Construct structure

Different combinations of the constant region, antigen-binding region and intracellular region of the STAR construct can obtain constructs with different configurations but all specifically target MSLN, see Table 11 for details, and some of the receptor structures are shown in Figure 8 .

Table 11

For example, in this example, the NM5-TCRβ-co-linker-STAR structure is constructed on the basis of the pCDH-IRES-RFP vector. The antigen binding sequence used was anti-MSLN NM5 VHH (SEQ ID NO: 28). The first constant region used is mouseTCRaC-Cys-TM9 (SEQ ID NO: 31), the second constant region mouseTCRbC-Cys (SEQ ID NO: 6), and the linker connecting the co-stimulatory factor is a flexible type linker (G4S) 3 ( SEQ ID NO: 83), can also be other flexible, rigid or cleavable type linker (range includes SEQ ID NO: 49 to NO: 117). In addition, when the flexible linker is (G4S)5 (SEQ ID NO: 85), and the C-terminals of the TCRα chain and β chain constant regions are connected in parallel with two co-stimulatory factors, the anti-MSLN-STAR receptor structure also shows Excellent coating rate and killing effect.

5. Plasmid Extraction

Plasmid extraction adopts the alkaline lysis method. The general principle is that the bacterial suspension is exposed to a strong anionic detergent with high pH, which will rupture the cell wall, denature the chromosomal DNA and protein, and intertwine into a large complex, which is coated with dodecyl sulfate. Cap, when potassium ions are substituted for sodium ions, the complexes are efficiently precipitated from solution, and after removal by centrifugation, plasmid DNA can be recovered from the supernatant. Usually divided into small extraction (2-5mL), small extraction medium (10-20mL), and large extraction (100-200mL) according to the amount of bacterial liquid. After plasmid extraction, the plasmid concentration was detected by 260nm light absorption. The plasmid with the correct initial sequencing result needs to be transformed to pick a single clone, perform small extraction, and check whether the plasmid is correct by enzyme digestion.

6. Packaging virus

7. Virus titer measurement

The TCR-knockout Jurkat-C4 cells were seeded in a flat-bottomed 96-well plate at 1.5×10^5 cells/mL, and 100 μL of 1640 medium containing 10% FBS and 0.2 μL of 1000×polybrene was added to each well. When diluting the virus, use 1640 complete medium for 10-fold dilution. If it is a virus stock solution, the virus volume in the first well is 100 μL, and if it is a concentrated solution, the virus volume in the first well is 1 μL. The diluted cells were added to the virus wells, 100 μL/well, mixed, 32°C, 1500rpm, centrifuged for 90min, and cultured in a 37°C, 5% CO ₂ incubator for 72h. The cells on the 96-well flat-bottom plate were sucked into the round-bottom 96-well plate, centrifuged at 1800 rpm for 5 min at 4°C, and the supernatant was discarded. After adding 200 μL of 1×PBS, centrifuge at 1800 rpm for 5 min at 4°C, and discard the supernatant. Add 200 μL of 4% tissue fixative, store in the dark, and put on a flow cytometer. Use a flow cytometer to measure the infection efficiency. When calculating the titer, select wells with an infection rate of 2-30%. The calculation formula is: titer (TU/mL) = 1.5 × 10^4 × positive rate ÷ virus volume (μL )×1000. The above viruses are used to infect T cells to express STAR.

Example 4: Expression of STAR receptor and its mutants in T cells and detection of upper membrane

1. Isolation and activation of human primary T cells

After the primary T cells were obtained by Ficoil separation method, cultured in X-VIVO medium containing 10% FBS and 100IU/mL IL-2, the initial culture density was 1×10 ⁶ /mL, and CD3, CD28 and Fibronectin were added Activated in the orifice plate.

2. Infection of Human Primary T Cells

After the primary T cells were activated for 24 hours, the virus solution was added, centrifuged at 1500 rpm for 90 minutes, and placed in a CO ₂ incubator for culture. After 24 hours of infection, X-VIVO medium containing 10% FBS and 100IU/mL IL-2 was supplemented, and the wells were transfected, and then subcultured at intervals of 1-2 days.

3. Membrane detection on STAR

72 hours after infection, the membrane efficiency of STAR was detected by using Anti-mouse TCRβ flow antibody or MSLN protein labeled with a fluorescent label (wherein the amino acid sequence of red fluorescent protein is shown in SEQ ID NO: 30). The mismatch efficiency was evaluated by comparing the positive ratio of RFP with the positive ratio of Anti-mouse TCRβ flow antibody or MSLN protein stained with fluorescent label. The results are shown in Figure 9, based on the ratio of RFP ⁺ subpopulations, the infection efficiency of NM5, NM13, and NM24 STAR is close.

Example 5: In vitro killing ability and cytokine release level of anti-MSLN STAR comprehensive mutants based on NM5/NM24-VHH

As shown in Figures 10A and 10B, in this example, a pancreatic cancer tumor cell line (AsPC-1) and an antigen-overexpressing cell line (293T, 293T-hMSLN, and 293T-mMSLN) were selected as target cells expressing MSLN.

1. Co-culture of T cells and target cells in vitro

1) Comparison of the killing effect of MSLN-STAR based on NM5 and NM24 on AsPC-1 cells

One day in advance, the target cells were plated in a 24-well plate at a density of 1E5/well for overnight culture, and the corresponding number of STAR-T cells were collected according to the ratio of 3:1, 1:1, and 1:3 between STAR-positive T cells and target cells Add it to target cells, and detect the killing efficiency of STAR-T cells on target cells after 24 hours or 48 hours of culture.

2) Comparison of the killing efficiency of MSLN-STAR with different structures on AsPC-1 cells

MSLN-STAR-T cells with different structures shown in Table 11 were co-cultured with AsPC-1 target cells, the effect-to-target ratio (E/T ratio) was 1:1 or 1:3, and the co-culture time was 24 hours Or 48 hours, after the co-cultivation, the killing efficiency was detected.

3) Comparison of NM5/NM24-based MSLN-STAR and other antibody-based MSLN-STAR cells

The antigen-binding region of NM5/NM24-MSLN-STAR in the experimental group was anti-MSLN-NM5/24VHH (SEQ ID NO: 28; SEQ NO: 29). The antigen binding regions of the control group STAR were derived from three published antibodies against MSLN (SEQ ID NO: 40, SEQ ID NO: 41, SEQ NO: 42, and the corresponding nucleotide sequences were SEQ ID NO: 128 , SEQ ID NO: 129, SEQ NO: 130) After constructing STAR-T cells in the experimental group and the control group respectively, according to the effect-to-target ratio of 1:1, the NM5-STAR, NM24-STAR and three groups of control-STAR-T Cells were co-cultured with target cells AsPC-1 or 293-T cells overexpressing MSLN for 48 hours, and the killing efficiency was detected.

2. Detection method

1) Detection of killing efficiency: luciferase method

After co-cultivation, remove the supernatant, add 400 μL of 1× cell lysate (promega) to each well, shake at room temperature for 10 minutes, centrifuge at 12000 rpm for 10 minutes, and remove the supernatant. 20 μL of supernatant was added to a white 96-well plate, and 2 replicate wells were taken from each well, and 50 μL of luciferase substrate (promega) was added to each well. A multifunctional microplate reader was used to detect the chemiluminescence value, and the gain value was fixed at 100. Calculation of cell killing: killing efficiency=100%-(effector cells-target cell pore value)/(control cell-target cell pore value).

2) Detection of cytokine release level

After T cells are stimulated with target cells or antigens, T cells are collected, centrifuged, and the supernatant is taken. TNF-α, IFN-γ, IL-2 ELISA kits use Human IL-2 Uncoated ELISA, Human TNF-α Uncoated ELISA, Human IFN-γ Uncoated ELISA (Cat. No. 88-7025, 88-7346, 88-7316 respectively) . The specific steps are as follows: dilute 10× Coating Buffer to 1× with ddH ₂ O, add coating antibody (250×), mix well and add to 96-well plate (for ELISA), 100 μL/well. After sealing with plastic wrap, store overnight at 4°C, wash 3 times with 1×PBST (also known as Wash Buffer, 0.05% Tween 20 in 1×PBS), 260 μL/well each time, dilute 5×ELISA/ELISPOT Diluent with ddH ₂ O For 1×, add 200 μL/well to a 96-well plate, and let it stand at room temperature for 1 hour. Wash once with PBST, dilute the standard curve (range: 2-250, 4-500, 4-500), and dilute the sample 20-50 times with 1×Diluent. Add samples and standard music, 100 μL per well, duplicate wells, incubate at room temperature for 2 hours, wash with PBST three times, add 1×Diluent diluted detection antibody, incubate for 1 hour,

Wash 3 times with PBST, then add 1×Diluent diluted HRP, incubate for 30 minutes, wash 6 times, add TMB for color development, the color development time does not exceed 15 minutes, add 2N H ₂ SO ₄ to stop, and detect light absorption at 450 nm.

3. Test results

The co-culture results showed that both NM5-STAR and NM24-STAR could effectively kill MSLN-positive target cells AsPC-1 and 293T-hMSLN, but could not recognize and kill 293T-mMSLN target cells, thus showing good specificity (see Figure 11A-D). And compared with different STAR structures, the STAR-T cell killing function of only one antibody VHH is better. At the same time, compared with the STAR-T cells constructed by the reported antibodies, both NM5-STAR-T and NM24-STAR-T exhibited stronger killing ability and higher cytokine release levels (see Figure 12A-D ).

Example 6: In vivo killing activity of anti-MSLN STAR comprehensive mutants based on NM5/NM24VHH

1. Tumor model establishment

In this experiment, NSG immunodeficient mice were used. The mouse genotype is NOD-Prkdcem26 Il2rg em26/Nju, lacks T cells, B cells, NK cells, and its macrophages and dendritic cells are also defective. NSG mice are currently the most complete immunodeficiency mouse strains, because they will not produce rejection reactions to transplanted tumors and T cells, and thus are widely used in preclinical research on T cell therapy. In this experiment, female NSG mice aged 6-8 weeks were used, and the weight difference of mice in each batch of experiments was controlled within 2g. Mice were housed in individually ventilated cages within specific pathogen-free (SPF) clean-grade barriers, provided with a normal diet and drinking water with an acidic pH to prevent pathogen contamination.

In this experiment, the human pancreatic cancer cell line AsPC-1 was used for xenogeneic subcutaneous or intraperitoneal inoculation and transplantation. AsPC-1 cells are cell lines expressing the luciferase gene through lentiviral vectors, and the development and changes of tumors are monitored in real time by means of luciferin chemiluminescence and live imaging in mice. In this model, different doses of AsPC-1-luciferase cells were inoculated subcutaneously or intraperitoneally into 6-8 week old female NSG mice. The mice were intraperitoneally injected with fluorescein potassium salt solution every 3-4 days, and the fluorescent signal of the tumor cells was detected by live imaging.

All manipulations of experimental animals and mice were carried out after the approval of the Experimental Animal Research and Use Program (Animal protocol).

2. Evaluation of the xenograft tumor killing activity of STAR-T cells based on NM5/NM24

Using the aforementioned tumor model, AsPC-1 cells were inoculated subcutaneously or in the abdominal cavity of mice at a dose of 5×10 ⁵ /mouse. Three days later, live imaging was performed on the mice, and the mice were grouped according to the magnitude of the tumor fluorescence signal. After one day, , the control T cells or MSLN-STAR-T cells were reinfused intravenously, with a dose of 5×10 ⁶ RFP-positive T cells per mouse, and the total reinfusion amount of the control T cells was consistent with that of the STAR-T cell group. After reinfusion, mice were subjected to fluorescence imaging every 3-7 days to detect tumor size. At the same time, the body weight of the mice was measured to evaluate the potential toxicity of STAR-T cells to the mice, and the results are shown in Figure 13A-F.

Example 7: Effects of two or more co-stimulatory intracellular domains on the function of anti-MSLN STAR-T cells directly or in series at the C-terminus of TCR α-chain and β-chain or the C-terminus of α-chain or β-chain

1. In vitro co-culture method of T cells and target cells

For the detection of the anti-MSLN co-STAR T cell killing function, the target cells were plated in a 24-well plate at a density of 1×10 ⁵ /well one day in advance and cultured overnight, and the STAR positive T cells and target cells were 3:1, 1. :1, 1:3 ratio, take the corresponding number of STAR-T cells and add them to the target cells for co-culture, and set different co-culture time groups, respectively: 24h, 48h. For the detection of the proliferation of co-STAR T cells, target cells were co-incubated with primary T cells for 7 days to observe the changes in the number of cell proliferation and IL-2 secretion, and positive T cells were obtained by flow cytometry after 7 days. After two days of static culture without antigen stimulation, the cells were co-incubated with the target cells for 24 hours to detect the killing of T cells.

2. T cell stimulation method by target antigen

The target antigen of the present invention is generally a cell surface protein, which can be directly used in the activation of T cells to detect the function of T cells. Usually, 1×10 ⁵ /well of positive T cells is added, centrifuged, and the cell suspension is collected after 24 hours of activation or The supernatant of the culture medium was used to detect the function of T cells, or to detect the killing function of T cells after activation for 24h, 48h, 96h or 7 days.

3. Detection of T cell killing efficiency: luciferase method

The tumor killing effect of anti-MSLN-co-STAR under different effect-to-target ratios was detected by luciferase detection method. Among them, the co-stimulatory intracellular domains are connected directly or through linkers in series at the C-terminals of TCRα chains and β-chains or at the C-terminals of α-chains or β-chains, and multiple co-stimulatory intracellular domains can be connected directly or through linkers (structure shown Views are shown in Figures 4 and 8).

4. Analysis of cytokines secreted by T cells: ELISA

During the activation of T cells, a large number of cytokines are released to help T cells kill target cells or promote the expansion of T cells themselves. The common ones are TNF-α, IFN-γ, and IL-2. After T cells are stimulated with target cells or antigens, T cells are collected, centrifuged, and the supernatant is taken. TNF-α, IFN-γ, and IL-2 ELISA kits use HumanIL-2 UncoatedELISA, HumanTNF-αUncoatedELISA, and HumanIFN-γUncoatedELISA (Cat. Nos. 88-7025, 88-7346, and 88-7316, respectively). The specific steps are as follows: dilute 10×CoatingBuffer to 1× with ddH ₂ O, add coating antibody (250×), mix well and add to 96-well plate (for ELISA), 100 μL/well. After sealing with plastic wrap, store overnight at 4°C, wash 3 times with 1×PBST (also known as WashBuffer, 0.05% Tween20 in 1×PBS), 260 μL/well each time, dilute 5×ELISA/ELISPOT Diluent to 1× with ddH ₂ O , added to a 96-well plate, 200 μL/well, and allowed to stand at room temperature for 1 h. Wash once with PBST, dilute with standard song (the ranges of IL-2, TNF-α, and IFN-γ are: 2-250, 4-500, 4-500, respectively), and dilute the sample 20-50 times with 1×Diluent. Add samples and standard music, 100 μL per well, duplicate wells, incubate at room temperature for 2 hours, wash with PBST three times, add 1×Diluent diluted detection antibody, incubate for 1 hour, wash with PBST three times, then add 1×Diluent diluted HRP, incubate for 30 minutes, wash 6 times, add TMB to develop color, the color development time does not exceed 15min, add 2NH ₂ SO ₄ to stop, detect light absorption at 450nm.

5. Detection of T cell proliferation changes: counting by flow cytometry

After T cells were co-incubated with target cells for 7 days, they were centrifuged, resuspended in PBS to 200 μL, and the number of positive T cells was counted by flow cytometry. Changes in T cell proliferation: multiplication factor=number of positive T cells/initial positive T cell addition after 7 days.

The sorted anti-MSLN-co-linker-START cells and target cells were initially co-cultured at an effect-to-target ratio of 1:3, which was recorded as day 0, and then cells were collected on day 1 and day 7 for flow cytometry analysis . Among them, the medium used is 1640 complete medium without IL2, and the initial STAR-T cells are 1×10 ⁵ cells. The samples at each time point are incubated independently, and the remaining co-incubated samples are half-changed every other day. solution, add target cells. The cells used for flow cytometric analysis were first stained with anti-humanCD3 antibody, and the cells with a specified volume were collected and recorded on the machine, and the number and proportion of T cells in the system were obtained through conversion. By establishing the proliferation fold curve of absolute anti-MSLN-co-linker-STAR cell number, analyze the proliferation of T cells with co-stimulatory intracellular domains alone or two or more in series at the C-terminus of TCRα chain or β chain. Combining the results of tumor killing effect, ELISA results and T cell proliferation effect, the tumor clearance and proliferation ability of STAR-T constructed by connecting two or more co-stimulatory molecular intracellular domains in series at the C-terminus of the TCR α chain or β chain were analyzed .

Example 8. Effects of co-stimulatory intracellular domains with different lengths of G4S linkers connected in the α or β constant region of the anti-MSLN STAR receptor structure on the function of STAR-T cells

1. In vitro co-culture method of T cells and target cells

For the detection of the anti-MSLNco-START cell killing function, the target cells were plated in a 24-well plate at a density of 1×10 ⁵ /well one day in advance and cultured overnight, and the STAR positive T cells and target cells were 3:1, 1:1. , 1:3 ratio, take the corresponding number of STAR-T cells and add them to the target cells for co-culture, and set different co-culture time groups, respectively: 24h, 48h.

2. Target antigen stimulates T cells

3. Detection of T cell killing efficiency: luciferase method

After co-cultivation, remove the supernatant, add 400 μL of 1× cell lysate (Promega) to each well, shake at room temperature for 10 min, centrifuge at 12000 rpm for 10 min, and remove the supernatant. 20 μL of supernatant was added to a white 96-well plate, and 2 replicate wells were taken from each well, and 50 μL of luciferase substrate (Promega) was added to each well. The chemiluminescence value was detected with a multifunctional microplate reader, and the gain value was fixed at 100. Calculation of cell killing: killing efficiency=100%-(effector cells-target cell pore value)/(control cell-target cell pore value).

The killing function of T cells was detected by luciferase method, and the co-stimulatory intracellular domains containing different lengths of G4Slinker were connected to the intracellular end of the α or β constant region of the anti-MSLNSTAR receptor structure, and the connection mode of the α chain constant region was α -del-OX40, α-OX40, α-del-G4S-OX40, α-del-(G4S)3-OX40, α-del-(G4S)5-OX40, α-del-(G4S)7-OX40 and α-del-(G4S)10-OX40; and the linker is connected to the intracellular end of the β constant region through β-del-OX40, β-ox40, β-del-(G4S)3-OX40, β-del-( G4S)5-OX40, β-del-(G4S)7-OX40 and β-del-(G4S)10-OX40, some of which are shown in Figure 4.

4. Analysis of cytokines secreted by T cells: ELISA

During the activation of T cells, a large number of cytokines are released to help T cells kill target cells or promote the expansion of T cells themselves. The common ones are TNF-α, IFN-γ, and IL-2. After T cells are stimulated with target cells or antigens, T cells are collected, centrifuged, and the supernatant is taken. TNF-α, IFN-γ, IL-2 ELISA kits use Human IL-2 Uncoated ELISA, Human TNF-α Uncoated ELISA, Human IFN-γ Uncoated ELISA (Cat. No. 88-7025, 88-7346, 88-7316 respectively) . The specific steps are as follows: dilute 10× Coating Buffer to 1× with ddH ₂ O, add coating antibody (250×), mix well and add to 96-well plate (for ELISA), 100 μL/well. After sealing with plastic wrap, store overnight at 4°C, wash 3 times with 1×PBST (also known as Wash Buffer, 0.05% Tween 20 in 1×PBS), 260 μL/well each time, dilute 5×ELISA/ELISPOT Diluent with ddH ₂ O For 1×, add 200 μL/well to a 96-well plate, and let it stand at room temperature for 1 hour. Wash once with PBST, dilute with standard song (the ranges of IL-2, TNF-α, and IFN-γ are: 2-250, 4-500, 4-500, respectively), and dilute the sample 20-50 times with 1×Diluent. Add samples and standard music, 100 μL per well, duplicate wells, incubate at room temperature for 2 hours, wash with PBST three times, add 1×Diluent diluted detection antibody, incubate for 1 hour, wash with PBST three times, then add 1×Diluent diluted HRP, incubate for 30 minutes, wash 6 times, add TMB to develop color, the color development time does not exceed 15min, add 2NH ₂ SO ₄ to stop, detect light absorption at 450nm.

5. Detection of T cell differentiation changes: analysis by flow cytometry

During the activation of T cells, a large number of cytokines and other chemokines are released, and the signals are transduced into the nucleus through cytokine or chemokine receptors to regulate the changes of T cell differentiation. The differentiation of T cells is from naive T cells (naive) to central memory T cells (Tcm) to effector memory T cells (Tem) and finally to effector T cells (Teff). The proliferation and persistence of T cells in vivo are affected by the number of T cells that differentiate into central memory T cells (Tcm) and effector memory T cells (Tem). The classification of memory T cells is roughly divided into T cells in a stem cell state, central memory T cells, and effector memory T cells. The proportion of central memory T cell differentiation affects the persistent killing effect of T cells in vivo. The ratio of naive T cells to effector T cells affects the killing effect and persistence of T cells on tumors in vivo. The differentiation of T cells can be known by detecting the expression of CD45RA and CCR7 on the surface of T cells by flow cytometry. After co-incubating T cells with target cells for 7 days, centrifuge, stain T cells with anti-human anti-human-CD45RA-Percp-cy5.5 and anti-human anti-human-CCR7-APC flow antibodies for 30 minutes, centrifuge, After washing once with PBS, fix with 4% paraformaldehyde solution, and detect T cell differentiation by flow cytometry.

The differentiation of central memory T cells of anti-MSLNSTAR constructed by G4S linkers with different lengths was detected by flow cytometry. Combined tumor killing results, ELISA results and flow cytometry detection of cell differentiation, the tumor killing effect and IL-2 secretion of the anti-MSLNSTAR receptor structure deleted at the intracellular end of the α or β constant region by linkers of different lengths (G4S) and T cell memory cell populations were analyzed.

Example 9: In vivo killing activity of anti-MSLN STAR comprehensive mutants based on different length G4S linkers

In the same method as in Example 6, the in vivo killing activity of anti-MSLN STAR comprehensive mutants without G4S linker and with (G4S) ₅ linker were tested respectively, and the results are shown in Figure 14A-F.

Example 10 NM24 STAR-T cell pharmacokinetic analysis experiment

Tumor-bearing mice were reinfused with high-dose STAR-T cells, about 3E8/kg mouse body weight. Tissues were collected from blank control mice, mice at 4 hours, 1 day, 3 days, 7 days, 14 days and 21 days after reinfusion of T cells. The tissues selected in this embodiment include: blood, bone marrow, tumor, heart, liver, spleen, lung, kidney, brain, and ovary. The distribution and metabolism of STAR-T cells in various tissues were detected by Q-PCR.

Results As shown in Figure 15A-B, STAR copy number was increased in all tissues. Day 14 was the visit point with the highest copy number of STAR; among all tissues, the copy number of tumor, spleen, and lung proliferated the fastest.

Comparative example 1 The in vitro killing and IFN-γ secretion of NM24 STAR-T cells and the comparative test experiment of TCR ² TC-210, a cell product of the same kind

In the same method as in Example 5, the in vitro killing and IFN-γ secretion of NM24 STAR-T and the similar cell product TCR ² TC-210 were tested respectively. The results are shown in Figure 16A-B, the in vitro killing and IFN-γ secretion of NM24 STAR-T cells is superior to that of similar cell product TCR ² TC-210.

Comparative example 2 The in vivo drug efficacy and safety of NM24 STAR-T cells and the comparison test experiment of TCR ² TC-210, a cell product of the same kind

In the same way as in Example 6, the in vivo drug efficacy and safety of NM24 STAR-T and the cell product TCR ² TC-210 of the same kind were tested respectively. As shown in Figures 17A and 17B, the in vivo drug efficacy of NM24 STAR-T cells is superior to that of a similar cell product, TCR ² TC-210. As shown in Figure 17C, ^compared with the same kind of cell product TCR ² TC-210, the weight loss of mice after reinfusion of NM24 STAR-T cells was less; On day 25, a total of 3 mice died. Therefore, the safety of NM24 STAR-T cells is better than that of similar cell products TCR ² TC-210.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention. These simple modifications All belong to the protection scope of the present invention.

In addition, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way if there is no contradiction. The combination method will not be described separately.

Claims

A synthetic T cell receptor antigen receptor, characterized in that:

The synthetic T cell receptor antigen receptor comprises an alpha chain and a beta chain, wherein the alpha chain comprises a first target binding domain and a first constant domain, and the beta chain comprises a second target binding domain and a second constant domain; or,

ii) The synthetic T cell receptor antigen receptor comprises a gamma chain and a delta chain, wherein the gamma chain comprises a first target binding region and a first constant region, and the delta chain comprises a second target binding region and a second constant region;

Wherein the first target-binding region and the second target-binding region comprise one or more antigen-binding regions, the multiple antigen-binding regions are the same or different, and the multiple antigen-binding regions are connected directly or via a linker;

The antigen binding region in the first target binding region comprises the heavy chain variable region of an antibody that specifically binds mesothelin, and the antigen binding region in the second target binding region comprises the light chain variable region of an antibody that specifically binds mesothelin. chain variable region; or, the antigen-binding region in the first target-binding region comprises the light chain variable region of an antibody that specifically binds mesothelin, and the antigen-binding region in the second target-binding region comprises a light chain variable region that specifically binds mesothelin The heavy chain variable region of an antibody to a protein.
The synthetic T cell receptor antigen receptor according to claim 1, wherein the antigen-binding region in the first target-binding region comprises a single-chain antibody or a single-domain antibody specifically binding to mesothelin; and/or the second The antigen-binding region in the second target-binding region comprises a single-chain antibody or a single-domain antibody that specifically binds to mesothelin;

Preferably, the single-chain antibody comprises a heavy chain variable region and a light chain variable region connected directly or through a linker;

Preferably, the antigen binding region in the first target binding region and the antigen binding region in the second target binding region bind to different regions of mesothelin (eg different epitopes).
The synthetic T cell receptor antigen receptor according to claim 1 or 2, wherein the antigen binding region in the first target binding region comprises one or more single domain antibodies, and/or, the The antigen binding region in the second target binding region comprises one or more single domain antibodies;

Preferably, the multiple single-domain antibodies contained in the antigen-binding region in the first target-binding region are the same or different;

Preferably, the multiple single-domain antibodies contained in the antigen-binding region in the second target-binding region are the same or different;

Further preferably, the multiple single domain antibodies are connected directly or through a linker.
The synthetic T cell receptor antigen receptor according to claim 3, wherein the single domain antibody comprises a heavy chain variable region, and the heavy chain variable region comprises CDR1-3, wherein,

i) CDR1 includes the amino acid sequence shown in SEQ ID NO: 34, CDR2 includes the amino acid sequence shown in SEQ ID NO: 35, and the CDR3 includes the amino acid sequence shown in SEQ ID NO: 36;

or,

ii) CDR1 includes the amino acid sequence shown in SEQ ID NO: 37, CDR2 includes the amino acid sequence shown in SEQ ID NO: 38, and the CDR3 includes the amino acid sequence shown in SEQ ID NO: 39.
The synthetic T cell receptor antigen receptor according to claim 3, wherein the single domain antibody comprises the amino acid sequence shown in SEQ ID NO: 28 or 29.
The synthetic T cell receptor antigen receptor according to claim 1, characterized in that,

i) the α chain and/or the β chain has at least one functional domain connected to its C-terminus, and the at least one functional domain is connected to the C-terminus of the α chain and/or the β chain directly or through a linker;

or,

ii) The C-terminus of the γ-chain and/or δ-chain is connected with at least one functional domain, and the at least one functional domain is directly or through a linker connected to the C-terminus of the γ-chain and/or δ-chain.
The synthetic T cell receptor antigen receptor according to claim 1, characterized in that,

i) the intracellular region of the α chain and/or β chain in the synthetic T cell receptor antigen receptor is deleted;

or,

ii) The intracellular region of the γ chain and/or δ chain in the synthetic T cell receptor antigen receptor is deleted.
The synthetic T cell receptor antigen receptor according to claim 7, characterized in that,

i) the α chain and/or the β chain has at least one functional domain connected to its C-terminus, and the at least one functional domain is connected to the C-terminus of the α chain and/or the β chain directly or through a linker;

or,

ii) The C-terminus of the γ-chain and/or δ-chain is connected with at least one functional domain, and the at least one functional domain is directly or through a linker connected to the C-terminus of the γ-chain and/or δ-chain.
The synthetic T cell receptor antigen receptor according to any one of claims 6-8, characterized in that,

i) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains are connected to the C-terminus of the α chain in the synthetic T cell receptor antigen receptor, and/or, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains are connected to the C-terminus of the β chain in the synthetic T cell receptor antigen receptor;

or,

ii) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains are connected to the C-terminus of the gamma chain in the synthetic T cell receptor antigen receptor, and/or, The C-terminus of the delta chain in the synthetic T cell receptor antigen receptor is connected with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains;

Wherein, the functional domains are the same or different.
The synthetic T cell receptor antigen receptor according to any one of claims 6-8, wherein the functional domain is a co-stimulatory molecule or a fragment thereof, a co-inhibitory molecule or a fragment thereof, a cytokine receptor or Fragments thereof or intracellular proteins or fragments thereof;

Preferably, the co-stimulatory molecule is selected from CD40, OX40, ICOS, CD28, 4-1BB or CD27;

Preferably, the co-inhibitory molecule is selected from TIM3, PD1, CTLA4, LAG3;

Preferably, the cytokine receptors are selected from interleukin receptors, interferon receptors, tumor necrosis factor superfamily receptors, colony stimulating factor receptors, chemokine receptors, growth factor receptors or other membrane protein;

Preferably, the intracellular protein is a T cell regulatory factor, such as a domain of NIK.
The synthetic T cell receptor antigen receptor according to any one of claims 1-10, wherein the linker is selected from rigid linkers, flexible linkers, cleavable linkers or nonsense amino acids;

Preferably, the amino acid sequence of the rigid linker is selected from one or more of SEQ ID NO: 49-59;

Preferably, the flexible linker is selected from peptides rich in glycine and/or serine; preferably, the flexible linker is selected from one or more of SEQ ID NO: 60-112;

Preferably, the cleavable linker is selected from one or more than two of SEQ ID NO: 113-117.
The synthetic T cell receptor antigen receptor according to any one of claims 1-11, wherein the first constant region is a TCRα chain constant region or a TCRγ chain constant region, preferably a modified TCRα chain constant region. region or modified TCR gamma chain constant region;

Preferably, the TCRα chain constant region is selected from a human TCRα chain constant region or a mouse (preferably mouse) TCRα chain constant region;

Preferably, the TCRγ chain constant region is selected from human TCRγ chain constant region or mouse (preferably mouse) TCRγ chain constant region.
The synthetic T cell receptor antigen receptor according to claim 12, characterized in that: the modified TCRα chain constant region is derived from the mouse (preferably mouse) TCRα chain constant region, which is relative to the wild type mouse (preferably Mouse) TCRα chain constant region, which includes one or more modifications at positions 6, 13, 15-18, 48, 112, 114, 115, the modification being mutation or deletion; or

The modified TCRα chain constant region is derived from the mouse (preferably mouse) TCRα chain constant region, which is included in the 13th, 36th, 47th, 53rd, Positions 58, 78, 98, and 122 have one or more modifications, which are mutations or deletions.
The synthetic T cell receptor antigen receptor according to claim 12, characterized in that: the modified TCRα chain constant region is derived from the mouse (preferably mouse) TCRα chain constant region, which is relative to the wild type mouse (preferably Mouse) TCRα chain constant region, which includes the amino acid at position 48 such as threonine T being mutated to cysteine C;

The modified TCR α chain constant region is derived from a mouse (preferably mouse) TCR α chain constant region, which, relative to the wild-type mouse (preferably mouse) TCR α chain constant region, includes an amino acid at position 112 such as serine S replaced by to leucine L, the amino acid at position 114, such as methionine M, is changed to isoleucine I, and/or, the amino acid at position 115, such as glycine G, is changed to valine V;

The modified TCR α chain constant region is derived from the mouse (preferably mouse) TCR α chain constant region, which is relative to the wild-type mouse (preferably mouse) TCR α chain constant region, which includes amino acid at position 6 such as E replaced by D , K at position 13 is replaced by R, and amino acids at positions 15-18 are deleted;

The modified TCR alpha chain constant region is derived from the mouse (preferably mouse) TCR alpha chain constant region, which is relative to the wild-type mouse (preferably mouse) TCR alpha chain constant region, which includes amino acid K at position 122 replaced by R ;

The modified TCR α chain constant region is derived from a mouse (preferably mouse) TCR α chain constant region, which, relative to the wild-type mouse (preferably mouse) TCR α chain constant region, includes an amino acid at position 48 such as threonine T is mutated to cysteine C, an amino acid at position 112 such as serine S is changed to leucine L, an amino acid at position 114 such as methionine M is changed to isoleucine I, and at position 115 An amino acid such as glycine G is changed to valine V;

The modified TCR alpha chain constant region is derived from the mouse (preferably mouse) TCR alpha chain constant region, which is relative to the wild-type mouse (preferably mouse) TCR alpha chain constant region, which includes the 48th amino acid such as threonine Acid T is mutated to cysteine C, and amino acid K at position 122 is replaced by R;

The modified TCR α chain constant region is derived from the mouse (preferably mouse) TCR α chain constant region, which is relative to the wild-type mouse (preferably mouse) TCR α chain constant region, which includes amino acid at position 6 such as E replaced by D , the K at position 13 is replaced by R, and the amino acids at positions 15-18 are deleted, and the amino acid at position 48 such as threonine T is mutated to cysteine C;

The modified TCR α chain constant region is derived from a mouse (preferably mouse) TCR α chain constant region, which, relative to the wild-type mouse (preferably mouse) TCR α chain constant region, includes an amino acid at position 48 such as threonine T is mutated to cysteine C, an amino acid at position 112 such as serine S is changed to leucine L, an amino acid at position 114 such as methionine M is changed to isoleucine I, and at position 115 The amino acid at the position such as glycine G is changed to valine V, and the amino acid K at position 122 is replaced by R;

The modified TCR alpha chain constant region is derived from the mouse (preferably mouse) TCR alpha chain constant region, which is relative to the wild-type mouse (preferably mouse) TCR alpha chain constant region, which contains the amino acid at position 6 such as E by D Substitution, K at position 13 is replaced by R, and amino acids 15-18 are deleted, amino acid at position 48 such as threonine T is mutated to cysteine C, amino acid at position 112 such as serine S is mutated to leucine L, an amino acid at position 114 such as methionine M is changed to isoleucine I, an amino acid at position 115 such as glycine G is changed to valine V, and amino acid at position 122 is K replaced by R;

The modified TCR alpha chain constant region is derived from the mouse (preferably mouse) TCR alpha chain constant region, which is relative to the wild-type mouse (preferably mouse) TCR alpha chain constant region, which includes amino acid at position 6 such as E replaced by D , K at position 13 is replaced by R, and amino acids 15-18 are deleted, amino acid at position 48 such as threonine T is mutated to cysteine C, and amino acid at position 112 such as serine S is mutated into leucine L, the amino acid at position 114 such as methionine M is changed to isoleucine I, and the amino acid at position 115 such as glycine G is changed to valine V;

The modified TCR α chain constant region is derived from the mouse (preferably mouse) TCR α chain constant region, which is relative to the wild-type mouse (preferably mouse) TCR α chain constant region, which includes amino acid at position 6 such as E replaced by D , the K at position 13 is replaced by R, and the amino acids at positions 15-18 are deleted, the amino acid at position 48 such as threonine T is mutated to cysteine C, and the amino acid K at position 122 is replaced by R;

The modified TCR α chain constant region is derived from the mouse (preferably mouse) TCR α chain constant region, which is relative to the wild-type mouse (preferably mouse) TCR α chain constant region, which includes amino acid at position 6 such as E replaced by D , the K at position 13 is replaced by R, and the amino acids at positions 15-18 are deleted, the amino acid at position 112 such as serine S is changed to leucine L, and the amino acid at position 114 such as methionine M is changed into isoleucine I, the amino acid at position 115 such as glycine G is changed into valine V;

The modified TCR α chain constant region is derived from the mouse (preferably mouse) TCR α chain constant region, which is relative to the wild-type mouse (preferably mouse) TCR α chain constant region, which includes amino acid at position 6 such as E replaced by D , K at position 13 is substituted by R, and amino acids 15-18 are deleted, and amino acid K at position 122 is substituted by R; or

The modified TCR α chain constant region is derived from the mouse (preferably mouse) TCR α chain constant region, which is relative to the wild-type mouse (preferably mouse) TCR α chain constant region, which includes amino acid at position 6 such as E replaced by D , the K at position 13 is replaced by R, and the amino acids at positions 15-18 are deleted, the amino acid at position 112 such as serine S is changed to leucine L, and the amino acid at position 114 such as methionine M is changed into isoleucine I, the amino acid at position 115 such as glycine G is changed to valine V, and the amino acid K at position 122 is replaced by R.
The synthetic T cell receptor antigen receptor according to claim 12, wherein the first constant region comprises one of SEQ ID NO: 1, 3, 5, 7, 8, 16, 31, 32 or 43 Amino acid sequence shown.
The synthetic T cell receptor antigen receptor according to any one of claims 1-15, characterized in that: the second constant region is a TCRβ chain constant region or a TCRδ chain constant region, preferably a modified TCRβ chain constant region or a modified TCRδ chain constant region;

Preferably, the TCR beta chain constant region is selected from a human TCR beta chain constant region or a mouse (preferably mouse) TCR beta chain constant region;

Preferably, the TCRδ chain constant region is selected from human TCRδ chain constant region or mouse (preferably mouse) TCRδ chain constant region.
The synthetic T cell receptor antigen receptor according to claim 16, characterized in that: the modified TCRβ chain constant region is derived from the mouse (preferably mouse) TCRβ chain constant region, which is relative to the wild type mouse (preferably mouse) TCR beta chain constant region, which comprises one or more modifications at positions 3, 6, 9, 11, 12, 17, 21-25, 56, 150, 168 or 170, said modification being mutation or deletion ;or

The modified TCR β chain constant region is derived from the mouse (preferably mouse) TCR β chain constant region, which is included in 9, 17, 23, 25, Positions 49, 63, 103, 110, 150, 168, and 170 have one or more modifications, which are mutations or deletions.
The synthetic T cell receptor antigen receptor according to claim 16, characterized in that: the modified TCRβ chain constant region is derived from the mouse (preferably mouse) TCRβ chain constant region, which is relative to the wild type mouse (preferably mouse) TCR beta chain constant region, which includes an amino acid at position 56 such as serine S being mutated to cysteine C;

The modified TCR beta chain constant region is derived from the murine (preferably mouse) TCR beta chain constant region, which includes the 150th, 168th or 170th lysine being replaced by arginine;

The modified TCR β chain constant region is derived from the mouse (preferably mouse) TCR β chain constant region, which is relative to the wild-type mouse (preferably mouse) TCR β chain constant region, which includes the amino acid at position 3 such as R being replaced by K , the amino acid at position 6 such as T is replaced by F, K at position 9 is replaced by E, S at position 11 is replaced by A, L at position 12 is replaced by V, and amino acids at positions 17, 21-25 are deleted ;

The modified TCR beta chain constant region is derived from the murine (preferably mouse) TCR beta chain constant region, which is relative to the wild-type murine (preferably mouse) TCR beta chain constant region, which includes the amino acid at position 56 such as serine S being replaced by Mutation to cysteine C, and substitution of lysine at position 150, 168, or 170 by arginine;

The modified TCR β chain constant region is derived from the mouse (preferably mouse) TCR β chain constant region, which is relative to the wild-type mouse (preferably mouse) TCR β chain constant region, which includes the amino acid at position 3 such as R being replaced by K , the amino acid at position 6 such as T is replaced by F, K at position 9 is replaced by E, S at position 11 is replaced by A, L at position 12 is replaced by V, and amino acids at positions 17, 21-25 are deleted , and an amino acid at position 56 such as serine S is mutated to cysteine C;

The modified TCR beta chain constant region is derived from the mouse (preferably mouse) TCR beta chain constant region, which is relative to the wild-type mouse (preferably mouse) TCR beta chain constant region, which comprises the amino acid at position 3 such as R being replaced by K , the amino acid at position 6 such as T is replaced by F, K at position 9 is replaced by E, S at position 11 is replaced by A, L at position 12 is replaced by V, and amino acids at positions 17, 21-25 are deleted , the amino acid at position 56, such as serine S, is mutated to cysteine C, and the lysine at position 150, 168, or 170 is replaced by arginine; or

The modified TCR β chain constant region is derived from the mouse (preferably mouse) TCR β chain constant region, which is relative to the wild-type mouse (preferably mouse) TCR β chain constant region, which comprises the amino acid at position 3 such as R is replaced by K , the amino acid at position 6 is replaced by F, the K at position 9 is replaced by E, the S at position 11 is replaced by A, the L at position 12 is replaced by V, and the amino acids at positions 17 and 21-25 are deleted , and the lysine at position 150, 168 or 170 is replaced by arginine.
The synthetic T cell receptor antigen receptor according to claim 16, wherein the second constant region comprises an amino acid sequence shown in one of SEQ ID NO: 2, 4, 6, 9, 17, 33 or 44 .
The synthetic T cell receptor antigen receptor according to claim 1 or 2, characterized in that: the first target binding region is directly connected to the first constant region or connected through a linker, and/or, the The second target binding region is directly connected to the second constant region or connected through a linker.
The synthetic T cell receptor antigen receptor according to claim 20, characterized in that: the linker is selected from rigid linkers, flexible linkers, cleavable linkers or nonsense amino acids;

Preferably, the amino acid sequence of the rigid linker is selected from one or more of SEQ ID NO: 49-59;

Preferably, the flexible linker is selected from peptides rich in glycine and/or serine; preferably, the flexible linker is selected from one or more of SEQ ID NO: 60-112;

Preferably, the cleavable linker is selected from one or more than two of SEQ ID NO: 113-117.
A synthetic T cell receptor antigen receptor complex, characterized in that: the complex comprises the synthetic T cell receptor antigen receptor according to any one of claims 1-21, and CD3ε, CD3γ, CD3δ and CD3ζ.
The synthetic T cell receptor antigen receptor complex according to claim 22, characterized in that: said CD3ε, CD3γ, CD3δ and/or CD3ζ are of human origin;

Preferably, the CD3ε comprises the amino acid sequence shown in SEQ ID NO: 20;

Preferably, said CD3γ comprises the amino acid sequence shown in SEQ ID NO: 18;

Preferably, the CD3δ comprises the amino acid sequence shown in SEQ ID NO: 19;

Preferably, the CD3ζ comprises the amino acid sequence shown in SEQ ID NO: 21.
An antibody or antigen-binding fragment, characterized in that: the antibody or antigen-binding fragment comprises a heavy chain variable region and/or a light chain variable region, and the heavy chain variable region comprises CDR1-3, wherein,

i) CDR1 includes the amino acid sequence shown in SEQ ID NO: 34, CDR2 includes the amino acid sequence shown in SEQ ID NO: 35, and the CDR3 includes the amino acid sequence shown in SEQ ID NO: 36;

or,

ii) CDR1 includes the amino acid sequence shown in SEQ ID NO: 37, CDR2 includes the amino acid sequence shown in SEQ ID NO: 38, and the CDR3 includes the amino acid sequence shown in SEQ ID NO: 39.
The antibody or antigen-binding fragment according to claim 24, wherein the antibody or antigen-binding fragment is a single-chain antibody or a single-domain antibody.
The antibody or antigen-binding fragment according to claim 24, wherein the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 28 or 29.
An antigen receptor, characterized in that the antigen receptor comprises a transmembrane region, an intracellular region, and one or more identical or different extracellular binding domains, and the extracellular binding domains are extracellular antigen binding area;

Wherein said extracellular antigen binding domain comprises CDR1-3, wherein,

i) CDR1 includes the amino acid sequence shown in SEQ ID NO: 34, CDR2 includes the amino acid sequence shown in SEQ ID NO: 35, and the CDR3 includes the amino acid sequence shown in SEQ ID NO: 36;

or,

ii) CDR1 includes the amino acid sequence shown in SEQ ID NO: 37, CDR2 includes the amino acid sequence shown in SEQ ID NO: 38, and the CDR3 includes the amino acid sequence shown in SEQ ID NO: 39.
The antigen receptor according to claim 27, wherein the extracellular antigen-binding domain comprises the antibody or antigen-binding fragment according to any one of claims 24-26.
The antigen receptor according to claim 27, wherein the antigen receptor is STAR, TCR or CAR;

Preferably, the transmembrane region is derived from human CD8;

Preferably, the intracellular region is derived from 4-1BB, CD28 or CD3ζ.
The antigen receptor according to claim 27, wherein the transmembrane region is directly connected to one or more extracellular antigen-binding domains or connected through a linker.
The antigen receptor according to claim 30, wherein the linker is selected from rigid linkers, flexible linkers, cleavable linkers or nonsense amino acids;

Preferably, the amino acid sequence of the rigid linker is selected from one or more of SEQ ID NO: 49-59;

Preferably, the flexible linker is selected from peptides rich in glycine and/or serine; preferably, the flexible linker is selected from one or more of SEQ ID NO: 60-112;

Preferably, the cleavable linker is selected from one or more than two of SEQ ID NO: 113-117.
A nucleic acid, characterized in that: the nucleic acid encodes the synthetic T cell receptor antigen receptor described in any one of claims 1-21, and the synthetic T cell receptor antigen receptor described in any one of claims 22-23 The complex, the antibody or antigen-binding fragment according to any one of claims 24-26, the antigen receptor according to any one of claims 27-31.
A vector, characterized in that: the vector comprises the nucleic acid according to claim 32.
A host cell, characterized in that the host cell comprises the nucleic acid of claim 32 or the vector of claim 33.
An immune cell, characterized in that: the immune cell expresses the synthetic T cell receptor antigen receptor described in any one of claims 1-21, and the synthetic T cell receptor antigen described in any one of claims 22-23 The receptor complex, the antibody or antigen-binding fragment according to any one of claims 24-26, the antigen receptor according to any one of claims 27-31.
The immune cell according to claim 35, characterized in that: the immune cell comprises one or more nucleic acids according to claim 32.
The immune cell according to claim 35 or 36, characterized in that: the immune cell is selected from T cells, Treg cells, macrophages, NK cells, NKT cells, peripheral blood mononuclear cells, TIL cells or dendrites shape cells (DC).
The immune cell according to claim 35 or 36, wherein the immune cell is isolated from T cells of a subject.
A method for preparing immune cells, characterized in that the preparation method comprises transfecting the nucleic acid sequence according to claim 32 into immune cells for expression.
A method for preparing recombinant T cells, comprising the steps of:

1) obtaining the nucleic acid according to claim 32 from positive T cell clones;

2) Isolation and culture of primary T cells;

3) delivering the nucleic acid obtained in step 1) to the primary T cells described in step 2), to obtain recombinant T cells expressing the synthetic T cell receptor antigen receptor described in any one of claims 1-21.
A method for preparing a synthetic T cell receptor antigen receptor, characterized in that it comprises the steps of:

(1) obtaining the nucleic acid according to claim 32 from positive T cell clones;

(2) connecting the nucleic acid obtained in step (1) to the vector backbone to obtain an expression vector;

(3) transforming the expression vector obtained in step (2) into a host cell, and then inducing its expression;

(4) Obtaining a synthetic T cell receptor antigen receptor.
The synthetic T cell receptor antigen receptor of any one of claims 1-21, the synthetic T cell receptor antigen receptor complex of any one of claims 22-23, the synthetic T cell receptor antigen receptor complex of any one of claims 24-26 The antibody or antigen-binding fragment, the antigen receptor according to any one of claims 27-31, the nucleic acid according to claim 32, or the immune cell according to any one of claims 35-38 in the preparation of products for diagnosing or treating tumors application.
The application according to claim 42, characterized in that: said tumors include lymphoma, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer , bladder cancer, lung cancer, bronchus cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophagus cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma , melanoma, myelodysplastic syndrome, and sarcoma; wherein the leukemia is selected from acute lymphoblastic (lymphoblastic) leukemia, acute myelogenous leukemia, myelogenous leukemia, chronic lymphocytic leukemia, multiple Myeloma, plasma cell leukemia, and chronic myelogenous leukemia; said lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma tumor, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenstrom's macroglobulinemia; said sarcoma is selected from the group consisting of osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue Sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma.
A pharmaceutical composition, characterized in that: the pharmaceutical composition comprises the synthetic T cell receptor antigen receptor described in any one of claims 1-21, the synthetic T cell receptor antigen receptor described in any one of claims 22-23, Antibody antigen receptor complex, the antibody or antigen-binding fragment according to any one of claims 24-26, the antigen receptor according to any one of claims 27-31, the nucleic acid according to claim 32, the nucleic acid according to claims 35-38 Any of the immune cells described above.
A kit, characterized in that: the kit comprises the synthetic T cell receptor antigen receptor described in any one of claims 1-21, and the synthetic T cell receptor antigen described in any one of claims 22-23 Receptor complex, antibody or antigen-binding fragment according to any one of claims 24-26, antigen receptor according to any one of claims 27-31, nucleic acid according to claim 32, any one of claims 35-38 The immune cells.