CN118139897A

CN118139897A - Synthetic T cell receptor antigen receptor specifically combined with mesothelin and application thereof

Info

Publication number: CN118139897A
Application number: CN202280071907.XA
Authority: CN
Inventors: 林欣; 芮魏; 虞莉; 伍春燕; 赵学强
Original assignee: Tsinghua University; Bristar Immunotech Ltd
Current assignee: Tsinghua University; Bristar Immunotech Ltd
Priority date: 2021-11-05
Filing date: 2022-11-04
Publication date: 2024-06-04
Also published as: WO2023078431A1

Abstract

Synthetic T cell receptor antigen receptor specifically binding mesothelin and its application are provided. The antigen binding fragments of the targeting MSLN are used for modifying TCR and CAR, so that better effect on tumor treatment is obtained.

Description

Synthetic T cell receptor antigen receptor specifically combined with mesothelin and application thereof

Technical Field

The invention relates to the field of biological medicine, in particular to a synthetic T cell receptor antigen receptor (STAR) specifically binding to mesothelin, a STAR complex, immune cells containing the STAR or STAR complex, and application thereof in the field of biological medicine.

Background

Mesothelin (Mesothelin), also known as MSLN, is a cell surface glycoprotein encoded by the MSLN gene. The MSLN gene encodes a proprotein that is proteolytically produced to produce both megakaryocyte potentiators (megakaryocyte-potentiating factor, MPF) and mesothelin MSLN protein products. Wherein MSLN is a protein anchored to the cell surface by glycosyl phosphatidylinositol, a differentiation antigen present on normal mesothelial cells. MSLN is rarely expressed in normal tissues, but MSLN is found to be overexpressed in a variety of cancers, and thus mesothelin MSLN is likely to be an important target for cancer treatment. The overexpression of mesothelin is currently observed in mesothelioma, ovarian cancer, lung cancer, esophageal cancer, pancreatic cancer, gastric cancer, cholangiocarcinoma, 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 in the lumen/membrane or cytoplasm. There is also a certain difference in the expression position of different types of tumor MSLNs. In mesothelioma tumors, MSLN is expressed on the cell surface in a uniform distribution. In lung adenocarcinoma, MSLN is expressed both on the cytoplasm and on the cell surface. In gastric cancer, cytoplasmic expression is more prevalent than membrane expression. MSLN is also expressed in solid tumors such as thyroid, renal and synovial sarcoma tumors.

For tumor treatment, CAR-T and TCR-T cells currently develop rapidly, but STAR-T derived from the native TCR lacks co-stimulatory signals in T cell activation during activation, and its proliferation and activation capacity is often affected. Thus, there remains a need in the art for improved TCRs and corresponding TCR-T therapies.

Therefore, the application modifies TCR and CAR with antigen binding fragments targeting MSLN, and is expected to obtain better effect on tumor treatment.

Disclosure of Invention

In order to solve the defects in the prior art, the invention provides a synthetic T cell receptor antigen receptor STAR and application thereof, wherein the synthetic T cell receptor antigen receptor can specifically bind mesothelin, and the TCR and the CAR are modified through an antigen binding fragment of targeting MSLN, so that a better tumor treatment effect is obtained.

In particular, in a first aspect of the invention, there is provided a synthetic T cell receptor antigen receptor (STAR),

I) The synthetic T cell receptor antigen receptor comprises an alpha chain comprising a first target binding region and a first constant region, and a beta chain comprising a second target binding region and a second constant region.

Or alternatively

Ii) the synthetic T cell receptor antigen receptor comprises a gamma chain, 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. Preferably, i) the alpha chain and/or the beta chain has attached at its C-terminal end at least one functional domain; or ii) the gamma and/or delta chain has at least one functional domain attached to its C-terminus.

It is further preferred that i) said at least one functional domain is linked directly or via a linker to the C-terminus of the alpha and/or beta chain, or ii) said at least one functional domain is linked directly or via a linker to the C-terminus of the gamma and/or delta chain.

Preferably, i) the intracellular regions of the alpha and/or beta chain in the synthetic T cell receptor antigen receptor are deleted; or ii) the intracellular region of the gamma and/or delta chain in the synthetic T cell receptor antigen receptor is deleted.

Still further preferred, the functional domain of i) is linked directly or via a linker to the C-terminus of the alpha and/or beta chain deleted from the intracellular region; or ii) the functional domain is linked directly or via a linker to the C-terminus of the gamma and/or delta chain deleted from the intracellular region.

Preferably, i) the C-terminal linkage of the alpha chain in the synthetic T cell receptor antigen receptor is 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 functional domains linked at the C-terminus of the β -chain in said synthetic T-cell receptor antigen receptor; or ii) 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains are C-terminally linked to the gamma 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 C-terminally linked to the delta chain in the STAR.

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

In one embodiment of the invention, the synthetic T cell receptor antigen receptor has at least one functional domain attached to the C-terminus of the alpha chain. The intracellular region of the alpha chain is deleted. The functional domain is linked to the C-terminus of the alpha chain deleted in the intracellular region by a linker.

In one embodiment of the invention, the synthetic T cell receptor antigen receptor has at least one functional domain attached to the C-terminus of the β -chain. The intracellular region of the beta chain is deleted. The functional domain is linked to the C-terminus of the beta chain deleted in the intracellular region by a linker.

In one embodiment of the invention, the synthetic T cell receptor antigen receptor has at least one functional domain attached to the C-terminus of the gamma chain. The intracellular region of the gamma chain is deleted. The functional domain is linked to the C-terminus of the gamma chain deleted in the intracellular region by a linker.

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

Preferably, i) the functional domains of the C-terminal linkage of the alpha and/or beta chains in the synthetic T cell receptor antigen receptor are the same or different;

Or ii) the functional domains of the gamma chain and/or delta chain C-terminal linkage in the synthetic T cell receptor antigen receptor are the same or different.

In one embodiment of the invention, the multiple functional domains of the synthetic T cell receptor antigen receptor to which the alpha chain is linked may be the same or different.

In one embodiment of the invention, the plurality of functional domains of the β -chain linkage in the synthetic T-cell receptor antigen receptor may be the same or different.

In one embodiment of the invention, the plurality of functional domains linked by the gamma chain in the synthetic T cell receptor antigen receptor may be the same or different.

In one embodiment of the invention, the multiple functional domains linked by the delta chain in the synthetic T cell receptor antigen receptor may be the same or different.

In one embodiment of the invention, the functional domain of the alpha chain linkage and the functional domain of the beta chain linkage in the synthetic T cell receptor antigen receptor may be the same or different.

In one embodiment of the invention, the functional domain of the gamma chain linkage and the functional domain of the delta chain linkage 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 preferred, 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 may also be a fusion of the intracellular domain of the cytokine receptor with a human STAT5 activation module (amino acid sequence shown in SEQ ID NO: 25), either directly or via a linker.

The co-stimulatory molecule is selected from the group consisting of CD40, OX40, ICOS, CD28, 4-1BB (CD 137) or CD27.

The co-inhibitory molecules are selected from TIM3, PD1, CTLA4 and LAG3.

The 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 receptor or other membrane proteins.

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

In one embodiment of the invention, the co-stimulatory molecule is CD40, the intracellular domain of which comprises the amino acid sequence of SEQ ID NO:10, and a polypeptide having the amino acid sequence shown in FIG. 10.

In one embodiment of the invention, the co-stimulatory molecule is OX40, the intracellular domain of which comprises the amino acid sequence of SEQ ID NO:11, and a polypeptide comprising the amino acid sequence shown in seq id no.

In one embodiment of the invention, the co-stimulatory molecule is ICOS, the intracellular domain of which comprises the amino acid sequence of SEQ ID NO:12, and a polypeptide having the amino acid sequence shown in FIG. 12.

In one embodiment of the invention, the co-stimulatory molecule is CD28, the intracellular domain of which comprises the amino acid sequence of SEQ ID NO:13, and a nucleotide sequence shown in seq id no.

In one embodiment of the invention, the co-stimulatory molecule is 4-1BB, the intracellular domain of which comprises the amino acid sequence of SEQ ID NO:14, and a polypeptide having the amino acid sequence shown in seq id no.

In one embodiment of the invention, the co-stimulatory molecule is CD27, the intracellular domain of which comprises the amino acid sequence of SEQ ID NO:15, and a polypeptide having the amino acid sequence shown in seq id no.

In one embodiment of the invention, the cytokine receptor is IL-2β, the intracellular domain of which comprises the sequence set forth in SEQ ID NO:22, and a polypeptide comprising the amino acid sequence shown in seq id no.

In one embodiment of the invention, the cytokine receptor is IL-7α, the intracellular domain of which comprises the sequence set forth in SEQ ID NO: 23.

In one embodiment of the invention, the cytokine receptor is IL-21, the intracellular domain of which comprises the amino acid sequence of SEQ ID NO:24, and a nucleotide sequence shown in seq id no.

In a specific embodiment of the invention, the functional domain is a fusion of the IL-2β intracellular domain with a human STAT5 activating module comprising the amino acid sequence of SEQ ID NO:26, and a polypeptide comprising the amino acid sequence shown in seq id no.

In a specific embodiment of the invention, the functional domain is a fusion of the IL-7α intracellular domain with a human STAT5 activation module comprising the sequence of SEQ ID NO: 27.

Preferably, the first constant region is a TCR α chain constant region or a TCR γ chain constant region, preferably a modified TCR α chain constant region or a modified TCR γ chain constant region.

The TCR a chain constant region is selected from a human TCR a chain constant region or a rodent (preferably murine, further preferably mouse) TCR a chain constant region. The TCR gamma chain constant region is selected from a human TCR gamma chain constant region or a rodent (preferably murine, further preferably mouse) TCR gamma chain constant region.

In one embodiment of the invention, the amino acid sequence of the human TCR α chain constant region is as set forth in SEQ ID NO:1, the amino acid sequence of the constant region of the TCR alpha chain of said rodent (preferably murine, more preferably mouse) is as set forth in SEQ ID NO: 3.

In one embodiment of the present invention, the amino acid sequence of the human TCR gamma chain constant region is as set forth in SEQ ID NO:45, the amino acid sequence of the constant region of the murine (preferably murine, more preferably mouse) TCR gamma chain is as set forth in SEQ ID NO: 46.

The modified TCR a chain constant region is derived from a human TCR a chain constant region comprising one or more modifications at positions 48, 116 or 119 relative to a wild-type human TCR a chain constant region, the modifications being mutations or deletions.

The modified TCR a chain constant region is derived from a human TCR a chain constant region comprising a threonine T mutation at position 48 to cysteine C relative to a wild-type human TCR a chain constant region.

The modified TCR alpha chain constant region is derived from a human TCR alpha chain constant region which is mutated to leucine L at serine S116 and valine V at glycine G119 relative to a wild-type human TCR alpha chain constant region.

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

The modified TCR a chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCR a chain constant region comprising one or more modifications at positions 6, 13, 15-18, 48, 112, 114, 115, relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCR a chain constant region, the modifications being mutations or deletions.

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

Preferably, the modified TCR a chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCR a chain constant region comprising the introduction of cysteine relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCR a chain constant region.

Further preferred, the modified TCR a chain constant region is derived from a rodent (preferably murine, further preferred mouse) TCR a chain constant region comprising an amino acid, e.g. threonine T, at position 48 mutated to cysteine C relative to a wild-type rodent (preferably murine, further preferred mouse) TCR a chain constant region.

In a specific embodiment of the invention, a rodent (preferably a mouse, more preferably a mouse) TCR a chain constant region comprising a cysteine introduction comprises the amino acid sequence of SEQ ID NO: 5.

Preferably, the modified TCR a chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCR a chain constant region comprising a hydrophobic amino acid mutation relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCR a chain constant region.

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

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

Preferably, the modified TCR a chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCR a chain constant region comprising an N-terminal modification relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCR a chain constant region.

Further preferred, the modified TCR α chain constant region is derived from a rodent (preferably murine, further preferred mouse) TCR α chain constant region comprising an amino acid at position 6, e.g. E, substituted with D, K at position 13, substituted with R, and amino acids 15-18 deleted relative to a wild-type rodent (preferably murine, further preferred mouse) TCR α chain constant region.

Preferably, the modified TCR a chain constant region is derived from a rodent (preferably murine, further preferably mouse) TCR a chain constant region comprising a lysine to arginine mutation of the transmembrane region.

Further preferred, the modified TCR α chain constant region is derived from a rodent (preferably murine, further preferred mouse) TCR α chain constant region comprising the amino acid K at position 122 substituted with R relative to a wild-type rodent (preferably murine, further preferred mouse) TCR α chain constant region.

In a specific embodiment of the invention, a rodent (preferably murine, further preferably mouse) TCR a chain constant region comprising a transmembrane region lysine mutated to arginine comprises SEQ ID NO: 8.

Preferably, the modified TCR a chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCR a chain constant region comprising a cysteine introduction and a hydrophobic amino acid mutation relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCR a chain constant region.

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

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

The modified TCR a chain constant region is derived from a rodent (preferably murine, further preferably mouse) TCR a chain constant region comprising an amino acid at position 48, e.g. threonine T, mutated to cysteine C, with amino acid K at position 122 being substituted with R, relative to a wild-type rodent (preferably murine, further preferably mouse) TCR a chain constant region.

The modified TCR a chain constant region is derived from a rodent (preferably murine, further preferably mouse) TCR a chain constant region comprising an amino acid such as E at position 6 substituted with D, K at position 13 substituted with R, and amino acids 15-18 deleted, and an amino acid such as threonine T at position 48 mutated to cysteine C, relative to a wild-type rodent (preferably murine, further preferably mouse) TCR a chain constant region.

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

The modified TCR α chain constant region is derived from a rodent (preferably murine, further preferably mouse) TCR α chain constant region comprising an amino acid such as E substituted at position 6 with D, K substituted at position 13 with R, and amino acids 15-18 deleted, an amino acid such as threonine T mutated to cysteine C, an amino acid such as serine S at position 112 with leucine L, an amino acid such as methionine M at position 114 with isoleucine I, an amino acid such as glycine G at position 115 with valine V, an amino acid K substituted at position 122 with R, relative to a wild-type rodent (preferably murine, further preferably mouse) TCR α chain constant region.

The modified TCR α chain constant region is derived from a rodent (preferably murine, further preferably mouse) TCR α chain constant region comprising an amino acid such as E at position 6 substituted with D, K at position 13 substituted with R, and amino acids 15-18 deleted, an amino acid such as threonine T at position 48 mutated to cysteine C, amino acid K at position 122 substituted with R, relative to a wild-type rodent (preferably murine, further preferably mouse) TCR α chain constant region.

The modified TCR α chain constant region is derived from a rodent (preferably murine, further preferably mouse) TCR α chain constant region comprising an amino acid such as E at position 6 substituted with D, K at position 13 substituted with R, and amino acids 15-18 deleted, an amino acid such as serine S at position 112 changed to leucine L, an amino acid such as methionine M at position 114 changed to isoleucine I, an amino acid such as glycine G at position 115 changed to valine V, relative to a wild-type rodent (preferably murine, further preferably mouse) TCR α chain constant region.

The modified TCR α chain constant region is derived from a rodent (preferably murine, further preferably mouse) TCR α chain constant region comprising an amino acid such as E at position 6 substituted with D, K at position 13 substituted with R, and amino acids 15-18 deleted, and amino acid K at position 122 substituted with R, relative to a wild-type rodent (preferably murine, further preferably mouse) TCR α chain constant region.

The modified TCR α chain constant region is derived from a rodent (preferably murine, further preferably mouse) TCR α chain constant region comprising an amino acid such as E at position 6 substituted with D, K at position 13 substituted with R, and amino acids 15-18 deleted, an amino acid such as serine S at position 112 changed to leucine L, an amino acid such as methionine M at position 114 changed to isoleucine I, an amino acid such as glycine G at position 115 changed to valine V, and an amino acid K at position 122 replaced with R, relative to a wild-type rodent (preferably murine, further preferably mouse) TCR α chain constant region.

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

Further preferred, the modified TCR α chain constant region is derived from a rodent (preferably murine, further preferred mouse) TCR α chain constant region comprising an amino acid such as E at position 6 substituted with D, K at position 13 substituted with R, amino acids 15-18 deleted, amino acid such as threonine T mutated to cysteine C at position 48, amino acid such as serine S to leucine L, amino acid such as methionine M at position 114 to isoleucine I, amino acid such as glycine G at position 115 to valine V, relative to a wild-type rodent (preferably murine, further preferred mouse) TCR α chain constant region.

In a specific embodiment of the invention, a rodent (preferably murine, further preferably mouse) TCR a chain constant region comprising a cysteine introduction, a hydrophobic amino acid mutation, and a deletion of an intracellular region comprises the amino acid sequence of SEQ ID NO:16, and a polypeptide having the amino acid sequence shown in 16.

Preferably, the modified TCR a chain constant region is derived from a rodent (preferably murine, further preferably mouse) TCR a chain constant region comprising an N-terminal modification, a cysteine introduction and a hydrophobic amino acid mutation.

Further preferred, the modified TCR α chain constant region is derived from a rodent (preferably murine, further preferably mouse) TCR α chain constant region comprising an amino acid such as E substituted with D, K substituted with R at position 13, amino acids 15-18 deleted, and amino acid such as threonine T mutated to cysteine C at position 48, amino acid such as serine S changed to leucine L at position 112, amino acid such as methionine M changed to isoleucine I at position 114, amino acid such as glycine G changed to valine V at position 115.

In a specific embodiment of the invention, a rodent (preferably murine, further preferably mouse) TCR a chain constant region comprising an N-terminal modification, a cysteine introduction and a hydrophobic amino acid mutation comprises SEQ ID NO:32, and a polypeptide having the amino acid sequence shown in seq id no.

Preferably, the modified TCR a chain constant region is derived from a rodent (preferably murine, further preferably mouse) TCR a chain constant region comprising an intracellular deletion, an N-terminal modification, a cysteine introduction and a hydrophobic amino acid mutation.

Further preferred, the modified TCR α chain constant region is derived from a rodent (preferably murine, further preferably mouse) TCR α chain constant region comprising an intracellular deletion, and the amino acid, e.g. threonine T, at position 48 is mutated to cysteine C, the amino acid, e.g. serine S, at position 112 is changed to leucine L, the amino acid, e.g. methionine M, at position 114 is changed to isoleucine I, the amino acid, e.g. glycine G, at position 115 is changed to valine V, and the amino acid, e.g. E, at position 6 is substituted with D, K at position 13 is substituted with R, and the amino acids at positions 15-18 are deleted.

In a specific embodiment of the invention, a rodent (preferably murine, further preferably mouse) TCR a chain constant region comprising an intracellular deletion, an N-terminal modification, a cysteine introduction and a hydrophobic amino acid mutation comprises the amino acid sequence of SEQ ID NO: 43.

In a specific embodiment of the invention, the first constant region comprises SEQ ID NO: 1.3, 5, 7, 8, 16, 31, 32 or 43.

Preferably, the second constant region is a TCR β chain constant region or a TCR δ chain constant region, preferably a modified TCR β chain constant region or a modified TCR δ chain constant region.

Further preferred, the TCR β chain constant region is selected from a human TCR β chain constant region or a rodent (preferably murine, further preferred mouse) TCR β chain constant region, and the TCR δ chain constant region is selected from a human TCR δ chain constant region or a rodent (preferably murine, further preferred mouse) TCR δ chain constant region.

In one embodiment of the invention, the human TCR β chain constant region comprises SEQ ID NO:2, and a polypeptide having the amino acid sequence shown in 2.

In a specific embodiment of the invention, the rodent (preferably murine, further preferably mouse) TCR β chain constant region comprises the amino acid sequence of SEQ ID NO:4, and a polypeptide having the amino acid sequence shown in (a) and (b).

In one embodiment of the invention, the human TCR delta chain constant region comprises SEQ ID NO: 47.

In a specific embodiment of the invention, the rodent (preferably murine, further preferably mouse) TCR delta chain constant region comprises the amino acid sequence of SEQ ID NO: 48.

The modified TCR β chain constant region is derived from a human TCR β chain constant region comprising one or more modifications at position 57, 173, or 175 relative to a wild-type human TCR β chain constant region, the modifications being mutations or deletions.

The modified TCR β chain constant region is derived from a human TCR β chain constant region comprising a serine S mutation at position 57 to cysteine C relative to a wild-type human TCR β chain constant region.

The modified TCR β chain constant region is derived from a human TCR β chain constant region comprising lysine K mutations to arginine at positions 173 and 175 relative to a wild-type human TCR β chain constant region.

The modified TCR β chain constant region is derived from a human TCR β chain constant region comprising a serine S mutation at position 57 to cysteine C, and lysine K mutations at positions 173 and 175 to arginine, relative to a wild-type human TCR β chain constant region.

The modified TCR β chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCR β chain constant region comprising one or more modifications at positions 3, 6, 9, 11, 12, 17, 21-25, 56, 150, 168 or 170 relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCR β chain constant region, the modifications being mutations or deletions.

The modified TCR β chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCR β chain constant region comprising one or more modifications at positions 9, 17, 23, 25, 49, 63, 103, 110, 150, 168, 170 relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCR β chain constant region, the modifications being mutations or deletions.

Preferably, the modified TCR β chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCR β chain constant region comprising the introduction of cysteine relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCR β chain constant region.

Further preferred, the modified TCR β chain constant region is derived from a rodent (preferably murine, further preferred mouse) TCR β chain constant region, which is mutated to cysteine C at amino acid 56, e.g. serine S, relative to a wild-type rodent (preferably murine, further preferred mouse) TCR β chain constant region.

In a specific embodiment of the invention, the cysteine-containing introduced rodent (preferably murine, further preferably mouse) TCR β chain constant region comprises the amino acid sequence of SEQ ID NO:6, and a polypeptide having the amino acid sequence shown in FIG. 6.

Preferably, the modified TCR β chain constant region is derived from a rodent (preferably murine, further preferably mouse) TCR β chain constant region, the intracellular region lysine of which is replaced by arginine.

Further preferred, the modified TCR β chain constant region is derived from a rodent (preferably murine, further preferred mouse) TCR β chain constant region, the lysine at position 150, 168 or 170 of which is substituted with arginine.

In a specific embodiment of the invention, a rodent (preferably murine, further preferably mouse) TCR β chain constant region comprising an intracellular region lysine replaced with arginine comprises SEQ ID NO:9, and a nucleotide sequence shown in FIG. 9

Preferably, the modified TCR β chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCR β chain constant region comprising an N-terminal modification relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCR β chain constant region.

Further preferred, the modified TCR β chain constant region is derived from a rodent (preferably murine, further preferred mouse) TCR β chain constant region which is substituted with K for amino acids such as R at position 3, F for amino acids such as T at position 6, E for K at position 9, a for S at position 11, V for L at position 12, and deleted at amino acids 17, 21-25 relative to a wild-type rodent (preferably murine, further preferred mouse) TCR β chain constant region.

Preferably, the modified TCR β chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCR β chain constant region comprising the introduction of cysteines and the deletion of an intracellular region relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCR β chain constant region.

In a specific embodiment of the invention, the rodent (preferably murine, further preferably mouse) TCR β chain constant region comprising the introduction of a cysteine and the deletion of an intracellular region comprises the sequence set forth in SEQ ID NO:17, and a sequence of amino acids shown in seq id no.

The modified TCR β chain constant region is derived from a rodent (preferably murine, further preferably mouse) TCR β chain constant region which comprises an amino acid at position 56, such as serine S, mutated to cysteine C, and a lysine at position 150, 168 or 170 substituted with arginine relative to a wild-type rodent (preferably murine, further preferably mouse) TCR β chain constant region.

The modified TCR β chain constant region is derived from a rodent (preferably murine, further preferably mouse) TCR β chain constant region comprising an amino acid such as R substituted with K, an amino acid such as T substituted with F, K substituted with E, S substituted with a, L substituted with V, and amino acids 17, 21-25 deleted, an amino acid such as serine S mutated to cysteine C, at position 56, and lysine substituted with arginine at position 150, 168 or 170, at position 6, such as T substituted with F, K substituted with E, S substituted with S at position 11, V substituted with L at position 12.

The modified TCR β chain constant region is derived from a rodent (preferably murine, further preferably mouse) TCR β chain constant region comprising an amino acid such as R substituted with K, an amino acid such as T substituted with F, K substituted with E, S substituted with a, L substituted with V, and amino acids 17, 21-25 deleted, and lysine substituted with arginine at positions 150, 168 or 170 at position 6, such as T, at position 9, at position 11, relative to a wild-type rodent (preferably murine, further preferably mouse).

Preferably, the modified TCR β chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCR β chain constant region comprising an N-terminal modification and a cysteine introduction relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCR β chain constant region.

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

In a specific embodiment of the invention, the rodent (preferably murine, further preferably mouse) TCR β chain constant region comprising an N-terminal modification and a cysteine introduction comprises SEQ ID NO:33, and a nucleotide sequence shown in seq id no.

Preferably, the modified TCR β chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCR β chain constant region comprising an intracellular deletion, an N-terminal modification and a cysteine introduction relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCR β chain constant region.

Further preferred, the modified TCR β chain constant region is derived from a rodent (preferably murine, further preferred mouse) TCR β chain constant region comprising an intracellular deletion relative to a wild-type rodent (preferably murine, further preferred mouse) TCR β chain constant region, and the amino acid at position 56, e.g. serine S, is mutated to cysteine C, and the amino acid at position 3, e.g. R, is substituted with K, the amino acid at position 6, e.g. T, is substituted with F, the K at position 9 is substituted with E, the S at position 11 is substituted with a, the L at position 12 is substituted with V, and the amino acids at positions 17, 21-25 are deleted.

In a specific embodiment of the invention, the rodent (preferably murine, further preferably mouse) TCR β chain constant region comprising an intracellular deletion, an N-terminal modification and a cysteine introduction comprises SEQ ID NO:44, and a polypeptide having the amino acid sequence shown in seq id no.

In a specific embodiment of the invention, the second constant region comprises SEQ ID NO: 2.4, 6, 9, 17, 33 or 44.

The target binding region is located at the N-terminus of the constant region. The two may be connected directly or through a joint.

The first target binding zone may comprise one or more identical or different binding zones. The target binding region is an antigen binding region or fragment thereof, a non-immunoglobulin antigen binding domain or fragment thereof, an antibody binding region or fragment thereof, a receptor or fragment thereof, a ligand or fragment thereof, preferably a natural T cell receptor.

The antigen binding region is derived from an antibody.

The STAR comprises one or more antigen binding regions;

preferably, the plurality of antigen binding regions are the same or different;

Further preferred, the plurality of antigen binding regions are linked directly or through a linker.

The antibody may be a monoclonal antibody or a polyclonal antibody.

The antibody may also comprise a fragment such as F _ab、F _ab'、F _ab'-SH、Fv、scFv、(F _ab') ₂, a single domain antibody, a diabody (dAb) or a linear antibody.

The antibody may be a monospecific antibody or a multispecific antibody (e.g., bispecific antibody).

Preferably, the antibody may be a fully human antibody, a humanized antibody, an animal-derived antibody. Wherein the animal can be mouse, rabbit, cow, monkey, etc.

Preferably, the first antigen binding region is linked directly to the first constant region or via a linker and/or the second antigen binding region is linked directly to the second constant region or via a linker.

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

Preferably, the target antigen is a disease-associated antigen, preferably a cancer-associated antigen, for example a cancer-associated antigen selected from the group consisting of: GPC3, CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123, CD138, erbB2 (HER 2/neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal Growth Factor Receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, bissialoglycganglioside GD2, ductal mucin, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, beta-human chorionic gonadotrophin, alpha Fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), enterocarboxylesterase, mut hsp70-2, M-CSF prostase (prostase), prostase 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 (IGF 1) -I, IGF-II, IGFI receptor, mesothelin, major Histocompatibility Complex (MHC) molecules presenting tumor specific peptide epitopes, 5T4, ROR1, nkp, NKG2D, tumor matrix antigen, additional domain a (EDA) and additional domain B (EDB) of fibronectin, A1 domain of tenascin-C (TnC A1), fibroblast-related protein (fap), CD3, CD4, CD8, CD24, fibronectin, CD25, CD33, CD34, CD133, CD138, foxp3, B7-1 (CD 80), B7-2 (CD 86), GM-CSF, cytokine receptor, endoglin, major Histocompatibility Complex (MHC) molecule 、BCMA(CD269、TNFRSF17)、TNFRSF17(UNIPROTQ02223)、SLAMF7(UNIPROT Q9NQ25)、GPRC5D(UNIPROT Q9NZD1)、FKBP11(UNIPROT Q9NYL4)、KAMP3、ITGA8(UNIPROT P53708), or FCRL5 (UNPROT Q68SN 8).

Preferably, the first antigen binding region comprises the heavy chain variable region of one or more antibodies that specifically bind the target antigen, and the second antigen binding region comprises the light chain variable region of one or more antibodies that specifically bind the target antigen; or the first antigen binding region comprises the light chain variable region of one or more antibodies that specifically bind to the target antigen and the second antigen binding region comprises the heavy chain variable region of one or more antibodies that specifically bind to the target antigen.

Wherein the heavy chain variable regions of the plurality of antibodies are linked directly or through a linker. The light chain variable regions of the plurality of antibodies are linked directly or through 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 to the target antigen; and/or the second antigen binding region comprises one or more single chain antibodies or one or more single domain antibodies that specifically bind to the target antigen.

Wherein the plurality of single chain antibodies are directly linked or linked by a linker. The plurality of single domain antibodies are linked directly or through a linker.

Preferably, the single chain antibody comprises a heavy chain variable region and a light chain variable region linked directly or via a linker.

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

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

In one embodiment of the invention, the target antigen is mesothelin.

In one embodiment of the 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 plurality of single domain antibodies comprised in the first antigen binding region are the same or different;

Preferably, the second antigen binding region comprises a plurality of single domain antibodies that are the same or different;

Further preferred, the plurality of single domain antibodies are linked directly or through a linker.

Preferably, the first antigen binding region comprises a single domain antibody that is the same or different from the single domain antibody comprising the second antigen binding region.

In one embodiment of the invention, the single domain antibody comprises a heavy chain variable region comprising CDR1-3, wherein,

I) CDR1 comprises SEQ ID NO:34, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:35, said CDR3 comprises the amino acid sequence set forth in SEQ ID NO:36, and a nucleotide sequence shown in seq id no;

Or alternatively

Ii) CDR1 comprises SEQ ID NO:37, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:38, said CDR3 comprises the amino acid sequence set forth in SEQ ID NO:39, and a polypeptide having the amino acid sequence shown in seq id no.

In one embodiment of the invention, the single domain antibody comprises SEQ ID NO:28 or 29.

In a second aspect of the present invention, there is provided a STAR complex, wherein,

I) The STAR complex comprises an alpha chain comprising a first target binding region and a first constant region, a beta chain comprising a second target binding region and a second constant region, CD3 epsilon, CD3 gamma, CD3 delta, and CD3 zeta;

Or alternatively

Ii) the STAR complex comprises a gamma chain comprising a first target binding region and a first constant region, a delta chain comprising a second target binding region and a second constant region, CD3 epsilon, CD3 gamma, CD3 delta, and CD3 zeta.

Preferably, i) at least one of the α chain, β chain, cd3ε, cd3γ, cd3δ and cd3ζ has at least one functional domain attached at its C-terminus; or ii) at least one of the gamma chain, delta chain, CD3 epsilon, CD3 gamma, CD3 delta and CD3 zeta has at least one functional domain attached to its C-terminus.

Preferably, i) said at least one functional domain is linked directly or through a linker to the C-terminus of at least one of the α chain, β chain, CD3 epsilon, CD3 gamma, CD3 delta and CD3 zeta; or ii) said at least one functional domain is linked directly or through a linker to the C-terminus of at least one of the gamma chain, delta chain, CD3 epsilon, CD3 gamma, CD3 delta and CD3 zeta.

Preferably, the intracellular region of at least one of the α chain, β chain, cd3ε, cd3γ, cd3δ, and cd3ζ in said STAR complex is deleted; or ii) the intracellular region of at least one of the gamma chain, delta chain, CD3 epsilon, CD3 gamma, CD3 delta, and CD3 zeta in said STAR complex.

Further preferred, i) said at least one functional domain is linked directly or via a linker to the C-terminus of at least one of the alpha chain, beta chain, CD3 epsilon, CD3 gamma, CD3 delta and CD3 zeta deleted of the intracellular region; or ii) said at least one functional domain is linked directly or through a linker to the C-terminus of at least one of the gamma chain, delta chain, CD3 epsilon, CD3 gamma, CD3 delta, and CD3 zeta deleted of the intracellular region.

In a specific embodiment of the invention, i) the C-terminal of at least one of the α chain, β chain, CD3 epsilon, CD3 gamma, CD3 delta, and CD3 zeta in the STAR complex is linked to 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains; or ii) at least one of the C-terminal linkages 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains of at least one of the gamma chain, delta chain, CD3 epsilon, CD3 gamma, CD3 delta, and CD3 zeta in the STAR complex.

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

Preferably, the C-terminal linked functional domains of at least one of the α chain, β chain, cd3ε, cd3γ, cd3δ, and cd3ζ in said STAR complex are the same or different; or ii) the C-terminal linked functional domains of at least one of the gamma chain, delta chain, CD3 epsilon, CD3 gamma, CD3 delta, and CD3 zeta in said STAR complex are the same or different.

In one embodiment of the invention, the multiple functional domains of the alpha chain linkage may be the same or different. The multiple functional domains of the beta chain linkage may be the same or different. The multiple functional domains of the gamma chain linkage may be the same or different. The multiple functional domains of the delta chain linkage may be the same or different. The multiple functional domains of the CD3 epsilon linkage may be the same or different. The multiple functional domains of the cd3γ linkage may be the same or different. The multiple functional domains of the CD3 delta linkage may be the same or different. The multiple functional domains of the cd3ζ connection may be the same or different.

In one embodiment of the invention, the functional domains to which the α chain, β chain, cd3ε, cd3γ, cd3δ and cd3ζ are linked, respectively, may be the same or different.

In one embodiment of the invention, the functional domains to which the gamma chain, delta chain, CD3 epsilon, CD3 gamma, CD3 delta and CD3 zeta chains are linked, respectively, 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 preferred, 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 co-stimulatory molecule is selected from the group consisting of CD40, OX40, ICOS, CD28, 4-1BB (CD 137) or CD27;

preferably, the co-inhibitory molecule is selected from the group consisting of TIM3, PD1, CTLA4, LAG3;

Preferably, the cytokine receptor is selected from the group consisting of interleukin receptor (e.g., IL-2 receptor), interferon receptor, tumor necrosis factor superfamily receptor, colony stimulating factor receptor, chemokine receptor, growth factor receptor, and other membrane proteins;

preferably, the intracellular protein is a domain of a T cell regulator, such as NIK.

In one embodiment of the present invention, the STAR complex comprises the STAR described above, as well as CD3 ε, CD3 γ, CD3 δ, and CD3 ζ.

Preferably, said CD3 epsilon, CD3 gamma, CD3 delta and/or CD3 zeta are of human origin.

In a specific embodiment of the present invention, said CD3 epsilon comprises the amino acid sequence of SEQ ID NO:20, and a polypeptide having the amino acid sequence shown in seq id no.

In a specific embodiment of the invention, the cd3γ comprises the amino acid sequence of SEQ ID NO:18, and a polypeptide having the amino acid sequence shown in seq id no.

In a specific embodiment of the invention, the cd3δ comprises the amino acid sequence of SEQ ID NO:19, and a polypeptide comprising the amino acid sequence shown in seq id no.

In a specific embodiment of the invention, said cd3ζ comprises the amino acid sequence of SEQ ID NO:21, and a polypeptide comprising the amino acid sequence shown in seq id no.

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

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

I) CDR1 comprises SEQ ID NO:34, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:35, said CDR3 comprises the amino acid sequence set forth in SEQ ID NO:36, and a nucleotide sequence shown in seq id no.

Or alternatively

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

In one embodiment of the invention, the antibody or antigen binding fragment comprises SEQ ID NO:28 or 29.

In a fourth aspect of the invention, there is provided 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 alternatively

In a fifth aspect of the invention, there is provided a single domain antibody comprising a heavy chain variable region comprising CDRs 1-3.

Wherein i) CDR1 comprises SEQ ID NO:34, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:35, said CDR3 comprises the amino acid sequence set forth in SEQ ID NO:36, and a nucleotide sequence shown in seq id no. Or ii) CDR1 comprises SEQ ID NO:37, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:38, said CDR3 comprises the amino acid sequence set forth in SEQ ID NO:39, and a polypeptide having the amino acid sequence shown in seq id no.

In a sixth aspect of the present invention, there is provided a method for producing the above antibody or antigen-binding fragment or the above single domain antibody, the method comprising: preparing a phage display library, and screening the phage display library to obtain antibodies or antigen binding fragments or single domain antibodies.

In a seventh aspect of the invention, there is provided an antigen receptor comprising a transmembrane region, an intracellular region and one or more identical or different extracellular binding domains.

The antigen receptor is a TCR or CAR.

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

The extracellular binding domain is an extracellular antigen binding domain, an extracellular antibody binding domain, a receptor and a ligand, wherein the receptor is preferably a natural T cell receptor.

In one embodiment of the invention, the extracellular binding domain is an extracellular antigen binding domain.

The extracellular antigen-binding domain is derived from an antibody.

Preferably, the transmembrane region is linked directly or via a linker to one or more extracellular antigen binding domains.

Preferably, the antigen is selected from cancer-related antigens, for example from the group consisting of: GPC3, CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123, CD138, erbB2 (HER 2/neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal Growth Factor Receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, bissialoglycganglioside GD2, ductal mucin, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, beta-human chorionic gonadotrophin, alpha Fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), enterocarboxylesterase, mut hsp70-2, M-CSF prostase (prostase), prostase 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 (IGF 1) -I, IGF-II, IGFI receptor, mesothelin, major Histocompatibility Complex (MHC) molecules presenting tumor specific peptide epitopes, 5T4, ROR1, nkp, NKG2D, tumor matrix antigen, additional domain a (EDA) and additional domain B (EDB) of fibronectin, A1 domain of tenascin-C (TnC A1), fibroblast-related protein (fap), CD3, CD4, CD8, CD24, fibronectin, CD25, CD33, CD34, CD133, CD138, foxp3, B7-1 (CD 80), B7-2 (CD 86), GM-CSF, cytokine receptor, endothelial factor, major Histocompatibility Complex (MHC) molecule 、BCMA(CD269、TNFRSF17)、TNFRSF17(UNIPROTQ02223)、SLAMF7(UNIPROT Q9NQ25)、GPRC5D(UNIPROT Q9NZD1)、FKBP11(UNIPROT Q9NYL4)、KAMP3、ITGA8(UNIPROT P53708), or FCRL5 (UNPROT Q68SN 8);

Preferably, the antigen is mesothelin.

Preferably, the extracellular antigen-binding domain comprises CDR1-3, wherein i) CDR1 comprises SEQ ID NO:34, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:35, said CDR3 comprises the amino acid sequence set forth in SEQ ID NO:36, and a nucleotide sequence shown in seq id no. Or ii) CDR1 comprises SEQ ID NO:37, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:38, said CDR3 comprises the amino acid sequence set forth in SEQ ID NO:39, and a polypeptide having the amino acid sequence shown in seq id no.

In one embodiment of the invention, the extracellular antigen-binding domain comprises an antibody or antigen-binding fragment as described above, a single chain antibody as described above, or a single domain antibody as described above.

Preferably, the transmembrane region is derived from human CD8.

Preferably, the intracellular domain is derived from 4-1BB, CD28 or CD3 zeta.

In an eighth aspect of the invention, there is provided a nucleic acid encoding the STAR, the STAR complex, the antibody or antigen binding fragment, the single chain antibody, the single domain antibody, the antigen receptor.

In a ninth aspect of the invention, there is provided a vector comprising a nucleic acid as described above.

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

Preferably, the prokaryotic expression vector is escherichia coli series. Such as pET-26b or pET28a+.

Preferably, it may 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 (MC 29) or avian myeloblastosis virus (AEV), etc. Still more preferably, the expression vector is a lentiviral expression vector. Such as pHAGE-IRES-RFP.

In a tenth aspect of the invention, there is provided a host cell comprising a nucleic acid as described above or a vector as described above.

Preferably, the host cell may be eukaryotic or prokaryotic. More preferably, the host cell is a yeast cell, 293 cell, CHO cell, escherichia coli, or the like.

In an eleventh aspect of the invention, an immune cell is provided, said immune cell expressing the STAR, the STAR complex, the antibody or antigen binding fragment, the single domain antibody, the antigen receptor.

Preferably, the immune cell comprises one or more of the above-described 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 (DCs).

Preferably, the immune cells are derived from T cells of the subject.

In one embodiment of the invention, the immune cells are selected from T cells, NK cells, CTLs, human embryonic stem cells, lymphoid progenitor cells and/or T cell precursor cells.

In a twelfth aspect of the invention, there is provided a CAR-T cell comprising an antibody or antigen fragment as described above, a single chain antibody as described above or a single domain antibody as described above.

According to a thirteenth aspect of the present invention, there is provided a method for producing an immune cell, which comprises transfecting the above-described nucleic acid sequence into an immune cell to express the nucleic acid sequence.

In a fourteenth aspect of the present invention, there is provided a method for preparing a recombinant T cell, comprising the steps of:

1) Cloning from positive T cells to obtain the nucleic acid described above;

2) Isolating and culturing primary T cells;

3) Delivering the nucleic acid obtained in step 1) into the primary T cells described in step 2) to obtain recombinant T cells expressing STAR as described above.

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

(1) Cloning from positive T cells to obtain the nucleic acid described above;

(2) Connecting the nucleic acid obtained in the step (1) to a vector skeleton to obtain an expression vector;

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

(4) STAR is obtained.

In a sixteenth aspect of the invention, there is provided a method of producing an antibody or antigen-binding fragment, single chain antibody or single domain antibody, said method comprising protein and/or DNA immunization.

In a seventeenth aspect of the present invention, there is provided a method for producing an antibody or antigen-binding fragment, single chain antibody or single domain antibody, said method comprising:

A) Obtaining a coded nucleic acid sequence;

b) Transforming the nucleic acid sequence obtained in the step A) into a host cell, and then inducing the expression and purification of the host cell.

In an eighteenth aspect of the invention, there is provided the use of a STAR as defined above, a STAR complex as defined above, an antibody or antigen binding fragment as defined above, a single domain antibody as defined above, an antigen receptor as defined above, a nucleic acid as defined above, or an immune cell as defined above for the preparation of a product for the diagnosis or treatment of a tumor.

Preferably, the neoplasm includes, but is 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, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, renal 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, 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's macroglobulinemia; the sarcoma is selected from osteosarcoma, ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma.

In a nineteenth aspect of the invention, there is provided an immunoconjugate or antibody drug conjugate comprising an antibody or antigen-binding fragment of the invention as described above, a single chain antibody as described above, or a single domain antibody as described above conjugated to a therapeutic or diagnostic agent.

In a twentieth aspect of the present invention, a pharmaceutical composition comprising the STAR, the STAR complex, the antibody or antigen binding fragment, the single domain antibody, the antigen receptor, the nucleic acid, and the immune cell is provided.

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

In a twenty-first aspect of the present invention, a kit is provided, said kit comprising the STAR, the STAR complex, the antibody or antigen binding fragment, the single domain antibody, the antigen receptor, the nucleic acid, and the immune cell.

In a twenty-second aspect of the invention, there is provided a method of treating a tumor, the method comprising administering to a subject an effective amount of a STAR, STAR complex, CAR, antibody or antigen binding fragment thereof, single chain antibody, single domain antibody, immune cell, CAR-T cell or pharmaceutical composition of the invention.

"Linker" as used herein includes, but is not limited to, rigid linkers, flexible linkers, cleavable linkers, or nonsensical amino acids. Preferably, the amino acid sequence of the rigid linker is selected from the group consisting of SEQ ID NO:49-59, and one or more than two thereof. Preferably, the flexible linker is selected from glycine and/or serine rich peptide fragments; preferably, the flexible linker is selected from the group consisting of SEQ ID NO:60-112, wherein said cleavable linker is selected from the group consisting of SEQ ID NO: 113-117.

The "antibodies" described herein may be of any class (e.g., igA, igD, igE, igG and IgM) or subclass (e.g., igG1, igG2, igG3, igG4, igA1, or IgA 2).

The "antigen binding fragments" of the present invention include, but are not limited to: a Fab fragment having VL, CL, VH and CH1 domains; a Fab' fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CH1 domain; fd fragment with VH and CH1 domains; fd' fragments having VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain; fv fragments having the VL and VH domains of a single arm of an antibody; a dAb fragment consisting of a VH domain or a VL domain; an isolated CDR region; a F (ab ') 2 fragment, which is a bivalent fragment comprising two Fab' fragments linked by a disulfide bridge at the hinge region; single chain antibody molecules (e.g., single chain Fv; scFv); a "diabody" having two antigen binding sites comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain; a "linear antibody" comprising a pair of tandem Fd segments (VH-CH 1-VH-CH 1) that form a pair of antigen-binding regions with complementary light chain polypeptides; and modified versions of any of the foregoing that retain antigen binding activity.

"CDR" as used herein refers to complementarity determining regions within the variable sequences of an antibody. For each variable region, there are three CDRs in each of the heavy and light chains, which are referred to as CDR1, CDR2, and CDR3. The exact boundaries of these CDRs are defined differently for different systems. 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)) provides not only a well-defined residue numbering system for the variable regions of antibodies, but also residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Each complementarity determining region may comprise 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 of the sub-portions within the Kabat CDRs employed nearly identical peptide backbone conformations, albeit with great diversity at the amino acid sequence level. These subfractions are referred to as L1, L2 and L3 or H1, H2 and H3, respectively, wherein "L" and "H" represent the light and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Still other CDR boundary definitions may not strictly follow one of the above systems, but will still overlap with Kabat CDRs, and the methods used herein may utilize CDRs defined according to any of these systems, although preferred embodiments use Kabat or Chothia defined CDRs. The "antibody variable region" refers to a portion of the light and heavy chains of an antibody molecule that includes the amino acid sequences of complementarity determining regions (CDRs, i.e., CDR1, CDR2, and CDR 3) and Framework Regions (FR). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain.

The term "diagnosis" as used herein refers to ascertaining whether a patient has a disease or condition in the past, at the time of diagnosis, or in the future, or ascertaining the progression or likely progression of the disease in the future, or assessing the 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, condition, or disease, but does not necessarily refer to the complete elimination of all disease-related signs, symptoms, conditions, or disorders, and refers to therapeutic intervention to ameliorate the signs, symptoms, etc. of a disease or pathological condition after the disease has begun to develop.

An "effective amount" as used herein refers to an amount or dose of STAR, STAR complex, CAR-T, STAR-T, immune cells, pharmaceutical compositions, etc. as described herein that provides the desired treatment or prevention after administration to a patient or organ in single or multiple doses.

The "product" of the present invention may be a kit, a chip, an antibody conjugate, a multifunctional antibody, a pharmaceutical composition, or the like.

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

All combinations of items to which the term "and/or" is attached "in this description shall be considered as being individually listed in this document. For example, "a and/or B" includes "a", "a and B", and "B". Also for example, "A, B and/or C" include "a", "B", "C", "a and B", "a and C", "B and C" and "a and B and C".

The term "comprising" or "comprising" as used herein is an open reading frame, and when used to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described herein. Furthermore, it will be clear to those skilled in the art that the methionine encoded by the start codon at the N-terminus of a polypeptide may be retained in some practical situations (e.g., when expressed in a particular expression system) without substantially affecting the function of the polypeptide. Thus, in describing a particular polypeptide amino acid sequence in the present specification and claims, although it may not comprise a methionine encoded at the N-terminus by the initiation codon, a sequence comprising such methionine is also contemplated at this time, and accordingly, the encoding nucleotide sequence may also comprise the initiation codon; and vice versa.

Drawings

Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:

Fig. 1: antibody expression and specificity identification of NM5/NM 24-VHH. Wherein, FIG. 1A shows the antibody expression of NM5-VHH, and M is Marker. FIG. 1B shows the antibody expression of NM24-VHH, M is Marker. FIG. 1C shows the results of specific staining after incubation of antibodies with AsPC-1 cells. FIG. 1D shows the results of specific staining of antibodies after co-incubation with 293T-MSLN cells, PC-Ab for publicly known MSLN antibodies, positive control, NC for negative control.

Fig. 2: schematic representation of optimization of STAR by constant region cysteine modification, transmembrane and intracellular region modification.

Fig. 3: schematic representation of optimization of STAR by addition of co-stimulatory molecule receptor intracellular domains to the alpha and/or beta chain.

Fig. 4: schematic representation of optimization of STAR by deletion of the intracellular regions of the alpha and/or beta chains followed by addition of co-stimulatory molecule receptor intracellular domains either directly or through linkers.

Fig. 5: schematic representation of STAR optimization by addition of co-stimulatory molecule receptor intracellular domains to the CD3 subunit.

Fig. 6: schematic representation of optimization of STAR by addition of cytokine receptor intracellular signaling regions to the alpha and/or beta chain.

Fig. 7: affinity detection of MSLN single domain antibodies NM5 (a) and NM24 (B).

Fig. 8: the anti-MSLN-co-STAR structure is schematically shown.

Fig. 9: experimental results of infection efficiency (rfp+ subgroup ratio) of NM5, NM13, NM24 STAR and Negative Control (NC).

Fig. 10: target cells expressing MSLN were selected, a being pancreatic cancer tumor cell line, B being antigen overexpressing cell line.

Fig. 11: comparison of killing function in vitro for different VHHs. AsPC-1 belongs to a human metastatic pancreatic adenocarcinoma cell line and is a MSLN positive target cell. 293T was transfected to express human hMSLN and murine mMSLN, respectively. FIG. 11A shows in vitro killing of various nanobodies on AsPC-1 and 293T cells, FIG. 11B shows that STAR T cells of four nanobody sequences NM5, 11, 13, 24 have a strong killing effect on Aspc-1, and FIG. 11C shows that NM5 and NM24 STAR T cells are capable of recognizing and killing 293T-hMSLN but not 293T-mMSLN target cells.

Fig. 12: cytokine release levels of anti-MSLN STAR comprehensive mutants based on NM5/NM 24-VHH. Of these, FIG. 12A shows IFN- γ release levels, FIG. 12B shows IFN- γ release levels of NM5 STAR T cells, NM11 STAR T cells, NM13 STAR T cells, NM24STAR T cells after 24 and 48 hours, FIG. 12C shows IL-2 release levels, and FIG. 12D shows IL-2 release levels of NM5 STAR T cells, NM11 STAR T cells, NM13 STAR T cells, NM24STAR T cells after 24 and 48 hours.

Fig. 13: an anti-MSLN-STAR-T cell tumor treatment result graph, wherein FIG. 13A is an in vivo anti-tumor effect of Mock T cells, NM5 STAR T cells, NM13 STAR T cells and NM24 STAR T cells in a mouse tumor model; FIG. 13B is a graph showing tumor fluorescence statistics in mice corresponding to FIG. 13A; FIG. 13C is the change in body weight of mice after back transfusion of Mock T cells, NM5 STAR T cells, NM13 STAR T cells, NM24 STAR T cells; FIG. 13D shows proliferation of Mock T cells, NM5 STAR T cells, NM13 STAR T cells, NM24 STAR T cells in mice; FIG. 13E shows the percentage of CD4 ⁺ T cells in a mouse tumor model for Mock T cells, NM5 STAR T cells, NM13 STAR T cells, NM24 STAR T cells; FIG. 13F shows the percentage of CD8 ⁺ T cells in a mouse tumor model for Mock T cells, NM5 STAR T cells, NM13 STAR T cells, NM24 STAR T cells.

Fig. 14: an anti-MSLN-STAR-T cell tumor therapeutic outcome graph, wherein fig. 14A is an in vivo anti-tumor effect of the constructed anti-MSLN-STAR-T cells in a mouse tumor model when (G4S) nlinker is n=0 and n=5, respectively; FIG. 14B is a graph of in vivo tumor fluorescence statistics of mice corresponding to graph A; FIG. 14C shows proliferation of integrated variants of anti-MSLNSTAR constructed from α -del-OX40 and α -del- (G4S) 5-OX40 in mice; FIG. 14D is the percentage of CD62L ⁺CD8 ⁺ T cells in a mouse tumor model for the anti-MSLN STAR integrated variants constructed with alpha-del-OX 40 and alpha-del- (G4S) 5-OX 40.

Fig. 15: pharmacokinetic analysis experiments of NM24 STAR-T cells in tissues returned for 0-21 days, wherein FIG. 15A is the copy number of NM24 STAR T cells; FIG. 15B is the percentage of NM24 STAR T cells.

Fig. 16: in vitro killing and IFN-gamma secretion of NM24 STAR-T cells and similar cell products TCR ² TC-210 (SD 1-eTruC T cells) are compared, wherein FIG. 16A shows the in vitro killing effect, and FIG. 16B shows the release level of IFN-gamma secretion, wherein E/T ratio is the effective target ratio.

Fig. 17: the in vivo efficacy and safety of NM24 STAR-T cells were compared with those of similar cell products TCR ² TC-210 (SD 1-eTruC T cells), wherein FIG. 17A shows the in vivo anti-tumor effects of Mock T cells, NM24 STAR T cells, SD1-eTruC T cells in a mouse tumor model; FIG. 17B is a graph showing tumor fluorescence statistics in mice corresponding to FIG. 17A; FIG. 17C is the change in body weight of mice after back transfusion of Mock T cells, NM24 STAR T cells, SD1-eTruC T cells; FIG. 17D shows the survival rate of mice after back transfusion of Mock T cells, NM24 STAR T cells, SD1-eTruC T cells.

Detailed Description

The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1: single domain antibody sequence screening targeting MSLN

1. Preparation of human MSLN proteins

1) Construction of expression vectors

The cDNA of human MSLN high expression tumor cell is used as template, the DNA sequence of human MSLN protein extracellular region is shown as SEQ ID No.120, and the amino acid sequence is shown as SEQ ID No. 121. The extracellular domain of MSLN was obtained by PCR amplification. The amplified MSLN extracellular region is connected to pVRC expression vector by enzyme cutting-connecting method, and is transformed into DH5 alpha competent cell, single colony is extracted or plasmid is extracted, and sequencing is carried out.

2) Protein expression

Transfection was performed when 293F cell densities were as high as about 2X 10 ⁶ cells/mL, 200. Mu.g of DNA was required to transfect 200mL cells, and the mass ratio of plasmid DNA to polyethyleneimine PEI (concentration 1 mg/mL) was about 1:3, 200 μg:600 μg, plasmid DNA and PEI were each added to a final volume of 5mL of non-resistant medium. The PEI and medium mixture was added to the plasmid DNA and medium mixture and incubated for about 15-30min, the volume of the incubation mixture being about 1/10 of the volume of the cells to be transfected. And adding the incubated mixed solution into 293F cells, culturing for 72-96h, and collecting cells or culture medium supernatant for protein extraction.

3) Protein purification

The collected culture supernatant was centrifuged to remove the precipitate, and the supernatant was filtered through a 0.22 μm filter membrane and concentrated and replaced with a lysate using a concentrate bag. Washing 10 column volumes of nickel columns with lysate washing liquid respectively to remove nonspecifically bound impurity proteins, and eluting target proteins from the affinity chromatography column with eluent. After concentration of the displacement solution, the concentration was determined, the samples were frozen and left for SDS-PAGE.

2. Human MSLN protein immune alpaca

Human MSLN protein extracellular region (100 μg) expressed and purified using the eukaryotic system at step 1 above was used to immunize healthy alpaca, and adjuvants include complete Freund's adjuvant (CFA, sigma) and incomplete Freund's adjuvant (IFA, sigma). The extracellular domain of the expressed and purified human MSLN protein was diluted with PBS and then mixed with the corresponding adjuvant 1:1. The antigen and adjuvant were thoroughly mixed to form a stable emulsion, the antigen mixture was withdrawn with a syringe and injected subcutaneously under the skin of the alpaca neck in multiple spots of 100-200 μl each. The specific immunization animal procedure is as follows:

1) Day 1 (immunization 1): immunization of alpaca using Complete Freund's Adjuvant (CFA) mixed MSLN antigen (100 μg) subcutaneously;

2) Day 14 (immunization 2): immunization of alpaca using Incomplete Freund's Adjuvant (IFA) mixed MSLN antigen (100 μg) subcutaneously;

3) Day 28 (immunization 3): the alpaca was immunized again by subcutaneous injection with Incomplete Freund's Adjuvant (IFA) mixed MSLN antigen (100 μg);

4) Day 42 (serum 1): taking alpaca blood samples from alpaca veins, extracting serum, and detecting antibody titer, wherein the P/N value of the serum diluted by 20 ten thousand times is more than 2;

5) Day 42 (immunization 4): subcutaneously injecting MSLN antigen (100 mug) mixed with Incomplete Freund's Adjuvant (IFA) again to immunize alpaca and enhance the immune effect;

6) Day 53 (serum 2): taking alpaca blood samples from alpaca veins, extracting serum, and detecting antibody titer, wherein the P/N value of the serum diluted by 20 ten thousand times is more than 2;

7) Day 54, 57, 60: 30-40mL alpaca blood samples were taken from the alpaca hindleg vein and PBMC isolation was performed.

PBMC isolation

1) PBMCs were isolated in a biosafety cabinet, and the blood in the anticoagulation tube was combined into one tube into 50mL centrifuge tube (30 mL), PBS was added to a total volume of 50mL, and gently mixed.

2) A new 50mL centrifuge tube was added with Ficoll separation solution, 15mL/tube. A blood sample, 25mL/tube, was placed on the Ficol level. The operation process is stable, and the layering of Ficoll and blood is formed to prevent mixing.

3) The lifting speed of the centrifuge is adjusted to 0, RT, centrifugal force is 800g, and the centrifuge is centrifuged for 30min. After centrifugation, the sample was removed from the centrifuge and the samples were stratified as follows: upper aqueous phase-buffy coat-Ficoll layer-erythrocyte layer, wherein PBMC are in the buffy coat, and the buffy coat is aspirated and transferred to a new 50mL centrifuge tube.

4) PBS was added to the sample tube to 50mL, mixed well, centrifuged at 2000rpm, at RT for 5min, the supernatant was discarded, and 5mL PBS was added to resuspend the cell pellet.

5) And (4) repeating the step (4) and simultaneously cooling to 4 ℃.

6) Cell pellet was resuspended in 5mL PBS, added PBS to 40mL, and cell counted.

7) Centrifuging at 1500rpm at 4deg.C for 5min, discarding supernatant, adding 1mL PBS, resuspending cells, and blowing well; adding 20mL Trizol, mixing, standing at room temperature for 5min, lysing cells, and sub-packaging into 1.5mL EP tube of RNase-free, and freezing at-80deg.C for extracting RNA.

4. RNA extraction and reverse transcription

1) The sample was taken out from-80 ℃, thawed at room temperature, 200. Mu.L of chloroform was added, and left standing at room temperature for 3min. The sample was centrifuged at 12000g for 15min at 4℃and separated into an upper aqueous phase, an intermediate layer and an organic layer, the upper layer was transferred to a new RNase-free tube, 1. Mu.L glycogen was added, and isopropanol was added in a proportion of 500. Mu.L isopropanol, and the mixture was allowed to stand at 4℃overnight.

2) The sample was centrifuged at 12000g at 4℃for 20min, the supernatant was aspirated, 1mL of pre-chilled 75% alcohol was added to wash the pellet, the supernatant was again centrifuged to aspirate alcohol, and the pellet was dried. RNA pellet was dissolved in 15. Mu.L of RNase-free water per tube, and reverse transcription was performed.

3) CDNA synthesis (20. Mu.L system) was performed using Promega reverse transcription kit.

The first step: taking a certain template RNA, and adding Oligo (dT), wherein the table 1 is shown;

TABLE 1

Component (A)	Volume (mu L)
RNA(<5μg/reaction)	11
Oligo(dT)15Primer	1
Total volume of	12

And a second step of: the mixture of template RNA and Oligo (dT) was pre-denatured at 65℃for 5min, and placed back on ice after completion.

And a third step of: in the case of pre-denaturation, RT-Mix was prepared in advance, 8. Mu.L per tube, and the composition and volume are shown in Table 2.

TABLE 2

Component (A)	Volume (mu L)
5×Reaction Buffer	4μL
RiboLockRnaseInhibitor(20U/μL)	1μL
10mM dNTP Mix	2μL
RevertAid M-MuLVRT(200U/μL)	1μL
Total volume of	8μL

Fourth step: reverse transcription program is set, and extension and reverse transcriptase are inactivated. After the procedure was completed, cDNA was obtained.

5. Phage library construction

1) PCR acquisition of VHH sequences

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

2) First round PCR

Step 1: the preparation reaction system is shown in Table 3.

TABLE 3 Table 3

Step 2: the PCR conditions are shown in Table 4.

TABLE 4 Table 4

The PCR product was subjected to gel electrophoresis, and the target band at 0.7kb was excised and the product was recovered.

3) Second round PCR

Step 1: the preparation reaction system is shown in Table 5.

TABLE 5

Step 2: the PCR conditions are shown in Table 6.

TABLE 6

And (3) carrying out gel electrophoresis on the PCR product, cutting a 400bp target band, and recovering the product.

4) Vector PCR

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

Step 1: the preparation reaction system is shown in Table 7.

TABLE 7

Step 2: the PCR conditions are shown in Table 8.

TABLE 8

The PCR product is subjected to gel electrophoresis, a 4000bp target band is cut, and the product is recovered.

5) Ligation, ligation product purification and concentration

The VHH fragment was ligated to phagemid vector and the ligation product was subsequently concentrated.

Step 1: the preparation reaction system is shown in Table 9.

TABLE 9

Step 2: the mixture was incubated at 50℃for 2h and cooled on ice.

Step 3: purifying the connection product, removing salt ions, proteins and other components in the connection system, and concentrating the volume to 1/10 of the original volume.

6. Electric transfer warehouse

1) One tube of E.coli was competent and thawed on ice.

1) Mu.L of the ligation product or positive control was added to the competence and gently blown. Standing on ice for 1-2min, transferring to a precooled electric rotating cup, and performing electric rotation.

2) After electrotransformation, 1mL of 2YT-G at 37 ℃ is immediately added, a gun head is used for flushing an electrotransformation cup, bacterial liquid after electrotransformation is transferred into a 15mL centrifuge tube or a 2mL EP tube, and water bath at 37 ℃ is recovered until all samples are electrotransformed. (2 YT-G: 2 XYT medium containing 2% glucose) was transferred to a shaking table at 37℃and recovered at 220rpm for 1 hour.

3) Mu.L of the bacterial liquid was aspirated from the bacterial liquid, diluted 10-5 times, plated with 2YT-A (2 YT plate containing 100. Mu.g/mL ampicillin) and incubated overnight at 37℃in an incubator for colony counting.

4) The rest bacterial liquid is inoculated into a 2YT-AG culture medium, shaken to a logarithmic phase, added with auxiliary phage for infection, and shaken at 30 ℃ and 220rpm for 12-16 hours. ( 2YT-AG: 2 XYT medium containing 2% Glucose and 100. Mu.g/mL ampicillin )

5) Phage were harvested and concentrated and titers were determined.

7. Phage library antibody screening

Three rounds of antibody screening are carried out on the phage library obtained in the step 7, and each screening comprises one positive selection and one negative selection. The phage were incubated with the antigen peptide and the unbound phage was discarded, leaving phage bound to the antigen peptide, and then incubated with BSA for negative selection, leaving phage unbound to BSA.

1) Coat plate. The antigen was diluted to a concentration of 2 ng/. Mu.L with PBS and added to a 96-well plate at 100. Mu.L/well; 2% BSA was prepared with PBS and added to the corresponding wells, 100. Mu.L/well, sealed with preservative film and incubated overnight at 4 ℃.

2) The coating solution was discarded, and 200. Mu.L of a washing solution (washing solution: 1% Tween 20/PBS, pH 7.4) and washed 3 times.

3) And (5) sealing. All wells were added with 2% BSA blocking solution, 100. Mu.L/well, sealed with preservative film and incubated at 37℃for 1h.

4) The supernatant was discarded, and 200. Mu.L of the washing solution was added thereto, followed by washing 3 times.

5) Phage were added to the wells of the positive selection, diluted to 100. Mu.L in volume at 1X 10 ¹² pages/well, sealed with preservative film and incubated for 1h at 37 ℃.

6) The supernatant was discarded, 200. Mu.L of wash solution was added, and the mixture was washed 10 times.

7) Elution-neutralization. 200. Mu.L of eluent was added to the wells of the cation selection and neutralized to pH 7-7.4.

8) Selecting yin. And adding the eluent into the anion-selective holes, sealing by using a preservative film, incubating for 1h at 37 ℃, sucking and retaining the supernatant, and detecting the titer.

9) A small amount of phage after Panning rounds of dilution is coated on a2 XYT-A plate at 37 ℃ overnight; counting the colonies the next day, and calculating the titer; and monoclonal sequencing is selected, and sequence diversity and enrichment conditions are analyzed.

10 All remaining phages were used for TG1 infection.

11 M13KO7 infection. Diluting phage, adding M13KO7 into the bacterial liquid, and carrying out water bath at 37 ℃ for 30min; 2 XYT-AK medium was changed, and shaking was performed at 30℃and 220rpm for 14-16 hours.

12 Concentrating phage and detecting phage titer, then the next round of screening can be performed.

The amount of coated antigen of two-round screening and three-round screening is reduced, the washing times are increased after the phage and the positive hole are incubated, and other steps are the same as the above steps.

8. Binding detection and sequence acquisition

The phage obtained by three rounds of screening and M13KO7 auxiliary phage are jointly infected with TG1, a 2YT-AK plate is coated, monoclonal is selected for phage expansion, phage is collected for combination detection, and available phage/antibodies are determined.

1) Coat plate. Diluting the antigen with the coating solution to 1 ng/. Mu.L, 100. Mu.L/well; control negative wells were added with 2% bsa; sealing with fresh-keeping film, and standing at 4deg.C overnight.

2) The coating solution in the plate was discarded, and 200-250. Mu.L of wash solution was added to wash 3 times.

3) 200. Mu.L of 2% BSA was added to all wells and blocked at room temperature for 1h.

4) The blocking solution in the plate was discarded, 200. Mu.L of wash solution was added, and the solution was washed 1 time.

5) 100. Mu.L of phage was added to each of the positive and negative wells and incubated at 37℃for 1h.

6) Phage in the plates were discarded, 200. Mu.L of wash was added and washed 3 times.

7) The anti-M13-HRP antibody was diluted, 50ng/well and incubated for 1h at room temperature.

8) The antibody in the plate was discarded, 200. Mu.L of wash solution was added, and the solution was washed 5 times.

9) 100 Mu L of TMB color development liquid is added into each hole, and the reaction is carried out at room temperature until the OD value is between 2 and 3.

10 Stop solution was added to the color development system at 50. Mu.L per well.

11A spectrophotometric measurement of the absorbance at 450 nm. And (5) sequencing the monoclonal bacterial liquid corresponding to the positive hole, and determining the VHH sequence.

12 The antibodies obtained were designated NM1-24, respectively.

9. Cell level functional screening

1) Assembling the positive antibody VHH sequence obtained by screening with a STAR molecule constant region, and inserting a lentiviral vector by using a homologous recombination method to construct a complete STAR plasmid.

2) Packaging virus

Lentix-293T cells were inoculated into 10cm dishes at 5X 10 ⁵/mL, incubated in a 5% CO ₂ incubator at 37℃and transfected when cell density reached about 80% (observed under a microscope). The four plasmids were homogeneously mixed with 500. Mu.L of serum-free DMEM in a PMD2.G: PRSV-Rev: PMDlg: TRANSFER PLAMID =1:1:2:4 ratio. mu.L of PEI-Max was mixed with 500. Mu.L of serum-free DMEM and allowed to stand at room temperature for 5min (PEI-Max to plasmid volume/mass ratio 3:1). The PEI-max mixture was slowly added to the plasmid mixture, gently swirled, mixed well and allowed to stand at room temperature for 15min. And slowly adding the final mixed solution into a culture medium, fully and uniformly mixing, putting the mixture back into an incubator for continuous culture for 12-16 h, changing the culture medium into a 6% FBS DMEM culture medium for continuous culture, and collecting the virus liquid for 48h and 72 h.

3) Virus titer measurement

The TCR knocked-out Jurkat-C4 cells were seeded at 1.5X10 ⁵/mL in flat bottom 96-well plates, and 100. Mu.L of 1640 medium containing 10% FBS, 0.2. Mu.L of 1000 Xpolybrene was added to each well. The virus was diluted 10-fold with 1640 complete medium. Adding diluted cells into a virus hole, mixing, centrifuging for 90min at 32 ℃ and 1500rpm, culturing in a 5% CO ₂ incubator at 37 ℃ for 72 hours, measuring infection efficiency by a flow cytometer, and selecting a hole with the infection rate of 2-30% when the titer is calculated, wherein the calculation formula is as follows: titer (TU/mL) =1.5X10≡4×positive rate ≡viral volume (. Mu.L) ×1000. The above viruses were infected with T cells and STAR was expressed.

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

After primary T cells were obtained by Ficoil isolation, they were cultured in X-VIVO medium containing 10% FBS and 100IU/mL IL-2 at an initial culture density of 1X 10 ⁶/mL and activated by addition to CD3, CD28 and fibractin pre-coated well plates. After 24h activation, adding the virus liquid, centrifuging at 1500rpm for 90min, and culturing in a CO ₂ incubator. X-VIVO medium containing 10% FBS and 100IU/mL IL-2 was supplemented 24h after infection and plated out, and subcultured at 1-2 days later.

5) Determination of killing efficiency and cytokine Release by in vitro Co-culture of T cells and target cells

Target cells AsPC-1 or 293T cells overexpressing MSLN are plated in 24-well plates overnight at a density of 1E 5/well and cultured, and corresponding numbers of STAR-T cells are added to the target cells according to the ratio of STAR-positive T cells to target cells of 3:1, 1:1 and 1:3, and the killing efficiency of the STAR-T cells on the target cells and the concentration of cytokines released from the supernatant after 24 hours or 48 hours of culture are detected.

10. Antibody expression and specificity identification

The VHH regions of NM5 and NM24 are fused with the Fc region of human IgG1 to construct plasmids, the plasmids are transferred into 293F cells, and the supernatants are collected and purified to obtain the corresponding antibodies. And the antibody size was identified by SDS-PAGE. And (3) staining and flow-type determination are carried out on 293T-MSLN cells by using the purified antibody, so as to determine the specificity of the antibody. (see FIGS. 1A-1D)

11. Antibody affinity assay

The CM5 chip is used for fixing MSLN protein as a stationary phase, the recombinant expressed antibody NM5-hFc or NM24-hFc is used as a mobile phase, and the affinity KD value of the two recombinant antibodies is measured on a Biacore 8kplus instrument. The results are shown in FIG. 7 and Table 10.

Table 10

Immobilized ligands

Sample injection analyte solution

KD(M)

1:1 Binding ka (1/Ms)

kd(1/s)

MSLN	NM1	1.66E-09	5.52E+05	9.14E-04
MSLN	NM3	4.31E-11	1.31E+06	5.77E-05
MSLN	NM5	1.80E-10	6.08E+05	1.09E-04
MSLN	NM11	4.03E-10	2.26E+06	9.09E-04
MSLN	NM12	6.74E-10	6.04E+07	4.07E-02
MSLN	NM13	1.35E-10	5.80E+07	7.84E-03
MSLN	NM16	1.17E-09	6.58E+07	7.69E-04
MSLN	NM17	3.00E-09	2.15E+05	6.45E-04
MSLN	NM20	7.40E-10	4.05E+05	3.00E-04
MSLN	NM23	1.78E-09	3.34E+05	5.93E-04
MSLN	NM24	5.90E-10	9.59E+06	5.66E-03

Example 2: structural design of MSLN-targeted STAR

The variable regions (antigen binding regions) of the human TCR alpha and beta chains were replaced with alpaca-derived antibody heavy chain variable regions (VHH) capable of specifically recognizing Mesothelin (MSLN) and the following modifications were made in the constant regions of TCR alpha and beta chains: the human TCR alpha and beta chain sequences were replaced with murine sequences; two chains each introduced a cysteine mutation; introducing hydrophobic mutation into the transmembrane region of the alpha chain; deletion of two chains of intracellular amino acids; the intracellular connection of the two chains co-stimulatory factors. Thus, a mutant synthetic T cell receptor antigen receptor (SYNTHETIC T-Cell Receptor and AntigenReceptor, STAR) capable of specifically recognizing MSLN was constructed, the structure of which is shown in FIG. 2.

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

1) Prototype design of STAR

The secreted antibodies (abs) or B Cell Receptors (BCR) produced by B cells have a great similarity to the T Cell Receptor (TCR) in terms of genetic structure, protein structure and spatial conformation. Both antibodies and TCRs are composed of variable and constant regions, with the variable region serving for antigen recognition and binding and the constant region serving for structural interactions and signal transduction. By replacing the variable regions of the TCR alpha and beta chains (or TCR gamma and delta chains) with the heavy chain variable region (VH) and the light chain variable region (VL) of the antibody, an artificially synthesized chimeric molecule, termed a synthetic T Cell Receptor antigen Receptor (SYNTHETIC T-Cell Receptor AND ANTIGEN Receptor, STAR/WT-STAR), can be constructed, the structure of which is shown in figure 2 (left).

The STAR molecule has two chains, the first chain being obtained by fusing the antigen recognition region to the constant region of the T cell receptor and the second chain being obtained by fusing the antigen recognition region to the constant region of the T cell receptor. The antigen recognition domain in the construct can be derived from antibody variable regions (such as VH, VL, scFv or VHH) of human, mouse, alpaca and rabbit, the T cell receptor constant region can be derived from constant regions of TCR alpha and beta chains or gamma and delta chains of human or mouse, and a plurality of constructs with different configurations and similar functions can be obtained through different combinations of the TCR constant regions of the antigen recognition regions.

After expression of both chains of STAR molecules in T cells, they bind to the CD3 epsilon delta, CD3 gamma epsilon, CD3 zeta chains endogenous to the cells in the endoplasmic reticulum to form complexes of 8 subunits and are displayed on the cell membrane surface as complexes. An intact STAR complex contains 10 immune receptor tyrosine activation motifs (Immunoreceptor Tyrosine-based Activation Motif, ITAM) (1 ITAM sequence in each of the intracellular domains of CD3 epsilon, delta, gamma, epsilon chains, 3 ITAM sequences in each of the intracellular domains of CD3 zeta chains) when the antigen recognition sequence of the STAR receptor binds to its specific antigen, the intracellular ITAM sequences are phosphorylated, thereby activating downstream signaling pathways, activating transcription factors such as NF-. Kappa.beta., NFAT, and AP-1, triggering T cell activation, and producing effector functions. Previous studies by the present inventors have shown that STAR is capable of mediating a stronger T cell activation signal than conventional chimeric antigen receptors (Chimericantigenreceptor, CAR), that STAR-T cells have significantly reduced background activation in the absence of antigen stimulation and are less prone to exhaustion with continued antigen stimulation, thus having significant advantages (see chinese patent application No. 201810898720.2). However, the present invention provides further improvements to STAR.

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

STAR prototypes used constant region sequences for human TCR alpha and beta chains (or TCR gamma and delta chains) (wild type human TCR alpha constant region, SEQ ID NO:1; wild type human TCR beta constant region, SEQ ID NO: 2). The constant region sequences of human, primate and murine TCR alpha/beta chains can be interchanged due to their high functional conservation and identical key amino acid sequences.

After the STAR molecule is transduced into a T cell, a mismatch will occur with the TCR endogenous to the T cell via the constant region. This mismatch problem reduces the efficiency of correct pairing of STAR molecules, impairing their function, on the one hand, and increases the likelihood of mismatches generating unknown specific receptor molecules, increasing the safety risk, on the other hand. To solve this problem, the inventors have made the following modifications to the wt-STAR molecule: a. the constant region of the STAR molecule is replaced by a murine sequence, (wild-type murine TCR alpha constant region mouse TCRaC-WT, SEQ ID NO:3; wild-type murine TCR beta constant region mouse TCRbC-WT, SEQ ID NO: 4) so that the murine constant region cannot be mismatched with an endogenous human TCR chain to increase the efficiency of pairing between the two chains of the exogenous transferred STAR molecule. b. The STAR molecule constant region is subjected to cysteine point mutation, specifically, threonine T at position 48 is mutated into cysteine C (mouse TCRaC-Cys, SEQ ID NO: 5) in the TCR alpha chain constant region, and serine S at position 56 is mutated into cysteine C (mouse TCRbC-Cys, SEQ ID NO: 6) in the TCR beta chain constant region. These two newly added cysteines form disulfide bonds between the two chains of STAR, reduce mismatches between the two chains of STAR and the endogenous TCR chain, and help the STAR molecule form a more stable complex, thereby achieving better functions. In addition, to further increase the stability of STAR molecules and to increase the function of STAR-T cells, the inventors have also performed the following modifications to STAR molecules: c. hydrophobic amino acid substitutions are made in the transmembrane region of STAR, specifically 3 amino acid mutations in the 111 to 119 amino acid regions of the transmembrane region of the constant region of TCR alpha chain: serine S at position 112 is changed to leucine L, methionine M at position 114 is changed to isoleucine I, glycine G at position 115 is changed to valine V. The overall amino acid sequence of this region was changed from LSVMGLRIL to LLVIVLRIL, and this modification was designated mouse TCRaC-TM9, resulting in a constant region sequence of SEQ ID NO. 7. This design increases the hydrophobicity of the transmembrane region, counteracts the instability caused by the positive charge carried by the TCR transmembrane region, and allows the STAR molecule to exist more stably on the cell membrane, thus achieving better function, the structure of which is shown in figure 2 (middle). d. Mutation of lysine in the constant region of the alpha chain and the constant region of the beta chain of the STAR molecule into arginine produces Mouse TCR alpha C-Arg mut (SEQ ID NO: 8) and Mouse TCR beta C-Arg mut (SEQ ID NO: 9) as constant region sequences, respectively, which reduces the possibility of ubiquitination of the STAR molecule transmembrane region and intracellular end, thereby reducing endocytosis of the STAR molecule, enabling the STAR molecule to exist more stably on the cell membrane, and further obtaining better functions, the structure is shown in figure 2 (right).

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

In order to improve the proliferation capacity, survival time and the capability of infiltrating into tumor microenvironment of STAR-T cells in vivo and killing target cells with high efficiency, the inventor designs a novel structure, modifies STAR compound, and can customize enhanced mut-STAR cells according to requirements so as to improve the clinical response rate of STAR-T cells and realize durable curative effect.

1) Design of mut-STAR molecule (co-STAR) comprising co-stimulatory molecule receptor intracellular domains

In order to enhance STAR-T cytotoxicity and cell proliferation persistence, the present invention introduces human co-stimulatory molecule receptor intracellular end sequences into the C-terminus of the STAR constant region (FIG. 3), respectively, exploring their effect on STAR-T cell function. The STAR constant regions described herein include unmodified wt-STAR constant regions, cys-STAR constant regions containing additional intermolecular disulfide bonds, murine hm-STAR constant regions, and mut-STAR incorporating the three modifications described in section 1. The co-stimulatory signaling structure comprises an intracellular signaling domain of CD40, OX40, ICOS, CD28, 4-1BB or CD27, having the sequence 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 co-stimulatory molecule intracellular domains may be either alone or simultaneously in tandem at the C-terminus of the alpha chain of the TCR, or the C-terminus of the beta chain, or in parallel at the C-terminus of both the alpha and beta chains (co-STAR).

In addition, the co-stimulatory molecule domains may be linked directly to the C-terminus of the TCR constant region or through flexible linkers comprising glycine and serine (SEQ ID NO:60 to NO: 112), rigid linkers comprising alanine and glutamic acid (SEQ ID NO:49 to NO: 59), and cleavable-type linkers (SEQ ID NO:113 to NO: 117), or linked after removal of the intracellular end sequence of the TCR molecule (intracellular end removed and comprising cysteine-substituted and hydrophobic region engineered TCR alpha chain constant region (mouse TCRaC-del mut, SEQ ID NO: 16), intracellular end removed TCR beta chain constant region comprising cysteine-substituted (mouse TCR beta C-del mut, SEQ ID NO: 17)) (co-linker-STAR, FIG. 4). The multiple co-stimulatory molecule domains may also be linked directly or via a linker to the α and β chains or the C-terminus of the α or β chain of the TCR constant region (as shown by dCo-STAR or tCo-STAR, fig. 4) the TCR of γδ T cells consists of two chains, TCR γ and TCR δ, and γδ T cells may be divided into three subsets depending on the type of TCR δ chain, γδ1, γδ2 and γδ3, the different subsets being distributed differently in humans. γδ T cells recognize non-peptide antigens primarily in a non-MHC restricted manner, playing an important role in pathogen and tumor monitoring. The inventors introduced human co-stimulatory molecule receptor intracellular end sequences into the C-terminal ends of tcrγ and tcrδ, respectively, and expected to improve performance of γδ T cells.

2) Design of CD3 molecule (co-CD 3-STAR) comprising co-stimulatory molecule receptor intracellular domains

The CD3 subunits, including the gamma, delta, epsilon and zeta chains, form T cell receptor complexes with TCR molecules, transmitting signals from extracellular to intracellular, thereby regulating the state of the cell in response to stimuli. To design enhanced TCR T cells, the present inventors engineered the CD3 molecule to increase the tumor killing, proliferation and residence time of the T cells in vivo, the human co-stimulatory receptor intracellular domain was introduced into the C-terminus of the CD3 gamma chain (SEQ ID NO: 18), delta chain (SEQ ID NO: 19), epsilon chain (SEQ ID NO: 20) and zeta chain (SEQ ID NO: 21) (FIG. 5). Expression of the engineered CD3 molecule in mut-STAR-T cells is desirable to increase its function.

3) Design of STAR molecules (cytokine-STAR, CK-STAR) comprising cytokine receptor stimulation regions

Cytokines play an important role in the proliferation, anti-tumor, differentiation and other functions of T cells. Binding of different cytokines to their receptors transmits signals from the extracellular to the intracellular end, thereby regulating the state of the cell in response to stimulation.

In addition, studies have shown that downstream molecule STAT5 (SEQ ID NO: 25) is activated at the intracellular end of the IL-2 receptor by a cascade, thereby enhancing transcription of T cell proliferation-related molecules, enhancing the proliferative capacity of CAR-T cells. To design enhanced STAR-T cells, to improve tumor killing, proliferation and residence time of T cells in vivo, the present inventors engineered STAR molecules to connect human cytokine receptor intracellular signaling regions (e.g., IL-2. Beta. Receptor intracellular IL2Rb, SEQ ID NO:22; IL-7. Alpha. Receptor intracellular end, SEQ ID NO:23; IL-21 receptor intracellular end, SEQ ID NO:24, etc.) in tandem with the C-terminus of TCR. Alpha. Chain, or the C-terminus of the beta. Chain, or the C-terminus of the alpha and beta. Chain, or alternatively, to connect STAT5 activating module (SEQ ID NO: 25) to IL-2. Beta. Or IL-7. Alpha. Receptor intracellular region ends (IL-2RbQ,SEQID NO:26;IL-7RbQ,SEQ ID NO:27) via G4S (FIG. 6).

Example 3: NM 5/24-VHH-based anti-MSLN STAR molecular vector construction

1. Carrier source

Vectors used in the present invention, including viral vectors, plasmid vectors, etc., are purchased from or synthesized by commercial companies, and the full-length sequences of these vectors are obtained, with well-defined cleavage sites known.

2. Fragment Source

The STAR mentioned in the present invention includes the above-mentioned wt-STAR, mut-STAR, ub-STAR, co-linker-STAR, CK-STAR, co-CD3-STAR, etc. Gene fragments such as the constant region of TCR, the intracellular region of the co-stimulatory molecule receptor, the intracellular signal transduction region of the cytokine receptor, the tag sequence, linker (linker) and the like are all derived from commercial sources for synthesis in the present invention.

The STAR antigen binding region fragment used in this example was derived from VHH of antibody NM5 targeting MSLN (its amino acid sequence is shown as SEQ ID NO:28, its nucleotide sequence is shown as SEQ ID NO: 118) and/or VHH of antibody NM24 targeting MSLN (its amino acid sequence is shown as SEQ ID NO:29, its nucleotide sequence is shown as SEQ ID NO: 119), wherein the anti-MSLN NM5 VHH comprises heavy chain CDR1 shown as SEQ ID NO:34, heavy chain CDR2 shown as SEQ ID NO:35 and heavy chain CDR3 shown as SEQ ID NO:36, and the anti-MSLN 24VHH comprises heavy chain CDR1 shown as SEQ ID NO:37, heavy chain CDR2 shown as SEQ ID NO:38 and heavy chain CDR3 shown as 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 invention is obtained by restriction endonuclease NotI/NheI to obtain a linear vector, the gene fragments are obtained by synthesis and PCR methods, and the linear vector and each gene fragment are connected by a homologous recombination method to obtain a complete vector.

4. Construct structure

Different combinations of constant, antigen binding and intracellular regions of STAR constructs can result in constructs of different configurations but all specifically targeting MSLN, see in particular table 11, where part of the receptor structure is shown in figure 8.

TABLE 11

For example, this example constructs NM5-TCR β -co-linker-STAR structure on the basis of pCDH-IRES-RFP vector. The antigen binding sequence used was anti-MSLN NM5 VHH (SEQ ID NO: 28). The first constant region mouse TCRaC-Cys-TM9 (SEQ ID NO: 31) and the second constant region mouseTCRbC-Cys (SEQ ID NO: 6) are used, the linker to which the co-stimulatory factor is attached is a flexible type linker (G4S) 3 (SEQ ID NO: 83), but may also be other flexible, rigid or cleavable type linkers (ranging from SEQ ID NO:49 to NO: 117). In addition, when the flexible linker is (G4S) 5 (SEQ ID NO: 85) and the C-terminal ends of the TCR alpha and beta chain constant regions are connected in parallel to two co-stimulatory factors, the anti-MSLN-STAR receptor structure also exhibits excellent membranous rate and killing effect.

5. Plasmid extraction

The basic lysis method is adopted for plasmid extraction, and the general principle is that bacterial suspension is exposed to strong anionic detergent with high pH, cell walls are broken, chromosome DNA and protein are denatured, the bacterial suspension is mutually entangled into large-scale complexes, the complexes are covered by dodecyl sulfate, when potassium ions are used for replacing sodium ions, the complexes effectively precipitate from the solution, and plasmid DNA can be recovered from supernatant after centrifugal removal. The plasmid concentration is usually detected by 260nm light absorption after plasmid extraction according to the bacterial liquid which is divided into small extract (2-5 mL), small extract medium amount (10-20 mL) and large extract (100-200 mL). The plasmid with correct primary sequencing result needs to be transformed to pick up a monoclonal, carry out small extraction and enzyme digestion to identify whether the plasmid is correct or not.

6. Packaging virus

Lentix-293T cells were inoculated into 10cm dishes at 5X 10 ⁵/mL, incubated in a 5% CO ₂ incubator at 37℃and transfected when cell density reached about 80% (observed under a microscope). Four plasmids were prepared according to PMD2.G: PRSV-Rev: PMDlg: TRANSFER PLAMID = 1:1:2:4 ratio, mixed homogeneously with 500 μl serum-free DMEM. mu.L of PEI-Max was mixed with 500. Mu.L of serum-free DMEM and allowed to stand at room temperature for 5min (PEI-Max to plasmid volume/mass ratio 3:1). The PEI-max mixture was slowly added to the plasmid mixture, gently swirled, mixed well and allowed to stand at room temperature for 15min. And slowly adding the final mixed solution into a culture medium, fully and uniformly mixing, putting the mixture back into an incubator for continuous culture for 12-16 h, changing the culture medium into a 6% FBS DMEM culture medium for continuous culture, and collecting the virus liquid for 48h and 72 h.

7. Virus titer measurement

The TCR knocked-out Jurkat-C4 cells were seeded at 1.5X10-5/mL in flat bottom 96-well plates, and 100. Mu.L of 1640 medium containing 10% FBS, 0.2. Mu.L of 1000 Xpolybrene was added to each well. The virus was diluted 10-fold in 1640 complete medium, and the amount of the first pore virus was 100. Mu.L in the case of the virus stock solution and 1. Mu.L in the case of the concentrate. The diluted cells were added to the virus wells, mixed at 32℃and 1500rpm, centrifuged for 90min, and cultured in a 5% CO ₂ incubator at 37℃for 72 hours. Cells on a 96-well flat bottom plate were aspirated into a round bottom 96-well plate, centrifuged at 1800rpm at 4℃for 5min, and the supernatant was discarded. After 200. Mu.L of 1 XPBS was added, the mixture was centrifuged at 1800rpm at 4℃for 5min, and the supernatant was discarded. 200. Mu.L of 4% tissue fixative was added and stored in the dark and up-flow cytometer. Measuring infection efficiency by using a flow cytometer, selecting a hole with the infection rate of 2-30% when calculating titer, wherein the calculation formula is as follows: titer (TU/mL) =1.5X10≡4×positive rate ≡viral volume (. Mu.L) ×1000. The above viruses were infected with T cells and STAR was expressed.

Example 4: expression of STAR receptor and mutant thereof in T cells and detection of upper membrane

1. Isolation and activation of human primary T cells

After primary T cells were obtained by Ficoil isolation, they were cultured in X-VIVO medium containing 10% FBS and 100IU/mL IL-2 at an initial culture density of 1X 10 ⁶/mL and activated by addition to CD3, CD28 and fibractin pre-coated well plates.

2. Infection of human primary T cells

After the primary T cells are activated for 24 hours, adding a virus liquid, centrifuging at 1500rpm for 90 minutes, and placing the mixture in a CO ₂ incubator for culture. X-VIVO medium containing 10% FBS and 100IU/mL IL-2 was supplemented 24h after infection and plated out, and subcultured at 1-2 days later.

STAR upper Membrane detection

After 72 hours post infection, STAR upper membrane efficiency was measured by using Anti-mouse TCR β flow antibodies or MSLN protein labeled with a fluorescent tag (wherein the amino acid sequence of the red fluorescent protein is shown in SEQ ID NO: 30). Mismatch efficiency was assessed by comparing the RFP positive ratio to the positive ratio of Anti-mouse tcrp β flow antibody or fluorescent tagged MSLN protein staining. The results are shown in FIG. 9, where NM5, NM13, NM24STAR infection efficiencies are close based on RFP ⁺ subgroup ratio.

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

As shown in FIGS. 10A and 10B, the present example selects pancreatic cancer tumor cell line (AsPC-1) and antigen-overexpressing cell lines (293T, 293T-hMSLN, and 293T-mMSLN) as target cells expressing MSLN.

In vitro co-culture of T cells and target cells

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

Target cells were plated in 24 well plates overnight at a 1E 5/well density one day in advance for culture, and corresponding numbers of STAR-T cells were added to the target cells at a ratio of STAR positive T cells to target cells of 3:1, 1:1, 1:3, and the killing efficiency of the STAR-T cells on the target cells after 24 hours or 48 hours of culture was examined.

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

MSLN-STAR-T cells of different structures shown in Table 11 were co-cultured with AsPC-1 target cells at an effective target ratio (E/T ratio) of 1:1 or 1:3, the co-cultivation time was 24 hours or 48 hours, and the killing efficiency was measured after co-cultivation.

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

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

2. Detection method

1) And (3) killing efficiency detection: luciferase method

After co-cultivation, the supernatant was removed, 400. Mu.L of 1 Xcell lysate (promega) was added to each well, and the mixture was centrifuged at 12000rpm for 10min with shaking at room temperature, and the supernatant was removed. mu.L of the supernatant was added to a white 96-well plate, 2 multiplex wells were used per well, and 50. Mu.L of luciferase substrate (promega) was added per well. The chemiluminescent value was detected using a multifunctional microplate reader with a gain value fixed at 100. Cell killing calculation: killing efficiency = 100% - (effector cell-target cell pore value)/(control cell-target cell pore value).

2) Cytokine release level detection

After T cells are stimulated with target cells or antigens, T cells are collected, centrifuged, and the supernatant is taken. TNF-alpha, IFN-gamma, IL-2ELISA kits were used as Human IL-2 Uncoated ELISA, human TNF-alpha Uncoated ELISA, human IFN-gamma Uncoated ELISA (accession numbers 88-7025, 88-7346, 88-7316, respectively). The method comprises the following specific steps: 10 Xcoating Buffer was diluted to 1X with ddH ₂ O, coated antibody (250X) was added, mixed well and then added to a 96-well plate (ELISA-specific) at 100. Mu.L/well. After sealing with the preservative film, the film was washed 3 times with 1 XPBST (also known as Wash Buffer,1 XPBS plus 0.05% Tween 20) at 260. Mu.L/well each time, diluted 1X with ddH ₂ O5 XPELISA/ELISPOT reagent, added to 96-well plates at 200. Mu.L/well and allowed to stand at room temperature for 1h overnight. PBST was washed 1 time, standard curves were diluted (ranges: 2-250, 4-500, respectively), and samples were diluted 20-50 times using 1 XDiluent. Adding sample and standard curve, incubating for 2 hr at normal temperature in each well with 100 μl, washing with PBST three times, adding 1×Diluent diluted detection antibody, incubating for 1 hr,

PBST was washed 3 times, then 1 XDluent diluted HRP was added, incubated for 30 minutes, washed 6 times, TMB was added for development, development time was not more than 15 minutes, addition was stopped by 2N H ₂SO ₄, and light absorption was measured at 450 nm.

3. Test results

Co-culture results show that both NM5-STAR and NM24-STAR can effectively kill MSLN positive target cells AsPC-1 and 293T-hMSLN, but cannot recognize and kill 293T-mMSLN target cells, thus showing good specificity (see FIGS. 11A-D). And compared with different STAR structures, the STAR-T cell killing function of only one antibody VHH is better. Meanwhile, both NM5-STAR-T and NM24-STAR-T exhibited stronger killing ability and higher cytokine release level than STAR-T cells constructed with the reported antibodies (see FIGS. 12A-D).

Example 6: in vivo killing Activity of anti-MSLN STAR comprehensive mutant based on NM5/NM24VHH

1. Tumor model establishment

NSG immunodeficient mice were used in this experiment. The mouse genotype is NOD-Prkdcem IL2rg em26/Nju, lacks T cells, B cells and NK cells, and has defects in macrophages and dendritic cells. NSG mice are the most complete mouse strain with immunodeficiency at present, and can not generate rejection reaction to transplanted tumor and T cells, so that the NSG mice are widely applied to preclinical research of T cell treatment. Female NSG mice of 6-8 weeks of age were used in this experiment, and the weight difference of the mice in each experiment was controlled within 2 g. Mice were housed in separate ventilated cages within clean grade barriers free of Specific Pathogens (SPF), providing normal diet and pH biased drinking water to prevent pathogen contamination.

The experiment adopts human pancreatic cancer cell line AsPC-1 cells for xenogeneic subcutaneous or intraperitoneal inoculation transplantation. The AsPC-1 cells are cell lines expressing luciferase genes through lentiviral vectors, and the development and change of tumors are monitored in real time in mice in a manner of chemiluminescence through luciferin and in vivo imaging. In this model, different doses of AsPC-1-luciferase cells were inoculated into 6-8 week old female NSG mice by subcutaneous or intraperitoneal inoculation. Mice were intraperitoneally injected with a potassium salt solution of fluorescein every 3-4 days, and tumor cells were examined for fluorescent signals by in vivo imaging.

All manipulations of experimental mice were performed after passing the trial of experimental Animal study and use plan (Animal protocol).

2. Evaluation of xenograft tumor killing Activity by NM5/NM24 based STAR-T cells

Using the tumor model described above, asPC-1 cells were inoculated subcutaneously or intraperitoneally into mice at a dose of 5X 10 ⁵/mouse, the mice were imaged in vivo after 3 days, the mice were grouped according to the size of the tumor fluorescent signal values, and after 1 day, control T cells or MSLN-STAR-T cells were reinfused by intravenous inoculation at a dose of 5X 10 ⁶ RFP positive T cells/mouse, and the total reinfusion number of control T cells was kept consistent with that of STAR-T cell groups. After reinfusion, mice were subjected to fluorescent imaging every 3-7 days to detect tumor size. At the same time, the mouse body weight was measured to evaluate the potential toxicity of STAR-T cells to mice, and the results are shown in FIGS. 13A-F.

Example 7: effect of two or more co-stimulatory intracellular domains connected directly or via a linker in series to the C-terminus of the alpha and beta chains of a TCR or to the C-terminus of the alpha or beta chains on anti-MSLN STAR-T cell function

T cell and target cell in vitro co-culture method

For detection of anti-MSLN co-STAR T cell killing function, target cells are spread in 24-well plates for overnight culture according to the density of 1X 10 ⁵/well one day in advance, and STAR positive T cells and target cells are added into the target cells for co-culture according to the ratio of STAR positive T cells to target cells of 3:1, 1:1 and 1:3, and different co-culture time groups are set, wherein the co-culture time groups are as follows: 24h,48h. For detection of proliferation of co-STAR T cells, target cells were incubated with primary T cells for 7 days to observe changes in cell proliferation number and IL-2 secretion, and positive T cells were obtained by flow cytometry after 7 days, were resting for two days in the absence of antigen stimulation, and were again incubated with target cells for 24h to detect T cell killing.

T cell stimulation by target antigen

The target antigen of the invention is generally cell surface protein, can be directly used for activating T cells to detect the functions of the T cells, is usually added with positive T cells of 1X 10 ⁵/hole, is centrifuged, collects cell suspension or culture supernatant after 24h activation to detect the functions of the T cells, or is activated for 24h,48h,96h or 7 days to detect the killing functions of the T cells.

T cell killing efficiency detection: luciferase method

And detecting the tumor killing effect of the anti-MSLN-co-STAR under the condition of different effective target ratios by using a luciferase detection method. Wherein the co-stimulatory intracellular domains are connected directly or via a linker in series to the C-terminus of the alpha and beta chains of the TCR or to the C-terminus of the alpha or beta chain, and wherein a plurality of co-stimulatory intracellular domains may be connected directly or via a linker (structure shown in FIGS. 4 and 8).

4.T cell secretion cytokine assay: ELISA (enzyme-Linked immuno sorbent assay)

A large amount of cytokines are released during T cell activation to help T cells kill target cells or promote expansion of T cells themselves, commonly referred to as TNF-alpha, IFN-gamma, IL-2. After T cells are stimulated with target cells or antigens, the T cells are collected and centrifuged, and the supernatant is taken. TNF- α, IFN- γ, IL-2ELISA kits were used at HumanIL-2UncoatedELISA, humanTNF- α UncoatedELISA, humanIFN- γ UncoatedELISA (88-7025, 88-7346, 88-7316, respectively). The method comprises the following specific steps: 10X CoatingBuffer was diluted to 1X with ddH ₂ O, coated antibody (250X) was added, mixed well and then added to a 96-well plate (ELISA-specific), 100. Mu.L/well. After sealing with the preservative film, 1 XPBST (also known as WashBuffer,1 XPBS plus 0.05% Tween 20) was washed 3 times at 260. Mu.L/well, diluted 1 XELISA/ELISPOTDiluent with ddH ₂ O, added to 96-well plates, 200. Mu.L/well and allowed to stand at room temperature for 1h overnight. PBST was washed 1 time, standard yeast dilutions (IL-2, TNF-alpha, IFN-gamma ranges 2-250, 4-500, respectively) were used, and samples were diluted 20-50 fold using 1 XDiluent. Adding a sample and standard yeast, incubating for 2h at normal temperature in 100 mu L of each well, washing with PBST three times, adding a1 xDiluent diluted detection antibody, incubating for 1h, washing with PBST 3 times, adding a1 xDiluent diluted HRP, incubating for 30 min, washing for 6 times, adding TMB for color development, keeping the color development time at no more than 15min, adding 2NH ₂SO ₄, and detecting light absorption at 450 nm.

5.T cell proliferation change detection: flow cytometer count

After 7 days of T cell co-incubation with target cells, centrifugation, PBS was resuspended to 200 μl and the change in positive T cell numbers was counted by flow cytometry. Change in T cell proliferation positive T cell number/initial positive T cell addition after fold proliferation = 7 days.

The separated anti-MSLN-co-linker-START cells and target cells were initially co-cultured according to an effective target ratio of 1:3, recorded as 0 days, and then the cells were collected for flow analysis at 1 day and 7 days, respectively. Wherein the culture medium is 1640 complete culture medium without IL2, the initial STAR-T cells are 1×10 ⁵ cells, the samples at each time point are independently incubated, and the rest of the co-incubated samples are subjected to half-liquid exchange every other day, and the target cells are supplemented. Cells used for flow analysis are firstly stained with anti-humanCD antibody, and cells with specified volumes are collected and recorded when the system is on machine, and the number and the proportion of T cells in the system are obtained through conversion. The proliferation of T cells with co-stimulated intracellular domains connected in series at the C-terminal of the alpha chain or beta chain of TCR is analyzed by establishing a proliferation fold curve of the absolute anti-MSLN-co-linker-STAR cell number. And (3) analyzing the tumor clearance and proliferation capacities of STAR-T constructed by connecting two or more co-stimulatory molecule intracellular domains in series at the C end of the TCR alpha chain or the beta chain by combining a tumor killing effect result, an ELISA result and a T cell proliferation effect.

Example 8 Effect of Co-stimulatory intracellular domains containing different lengths of the G4S linker (linker) on STAR-T cell function by linking the alpha or beta constant region of the anti-MSLNSTAR receptor structure

T cell and target cell in vitro co-culture method

For detection of anti-MSLNco-START cell killing function, target cells are spread in 24 well plates for overnight culture according to the density of 1X 10 ⁵/well one day in advance, and STAR-T cells with corresponding numbers are added into the target cells for co-culture according to the ratio of STAR positive T cells to target cells of 3:1, 1:1 and 1:3, and different co-culture time groups are set, wherein the co-culture time groups are as follows: 24h,48h.

2. Target antigen stimulates T cells

T cell killing efficiency detection: luciferase method

Detecting T cell killing function by luciferase method, wherein co-stimulated intracellular domain containing G4Slinker with different length is connected at the intracellular end of alpha or beta constant region of anti-MSLNSTAR acceptor structure, and the connection mode of alpha chain constant region is alpha-del-OX 40, alpha-del-G4S-OX 40, alpha-del- (G4S) 3-OX40, alpha-del- (G4S) 5-OX40, alpha-del- (G4S) 7-OX40 and alpha-del- (G4S) 10-OX40; the linker is connected to the intracellular end of the beta constant region in the manner of beta-del-OX 40, beta-OX 40, beta-del- (G4S) 3-OX40, beta-del- (G4S) 5-OX40, beta-del- (G4S) 7-OX40 and beta-del- (G4S) 10-OX40, wherein partial structures are shown in the figure 4.

4.T cell secretion cytokine assay: ELISA (enzyme-Linked immuno sorbent assay)

A large amount of cytokines are released during T cell activation to help T cells kill target cells or promote expansion of T cells themselves, commonly referred to as TNF-alpha, IFN-gamma, IL-2. After T cells are stimulated with target cells or antigens, T cells are collected, centrifuged, and the supernatant is taken. TNF-alpha, IFN-gamma, IL-2ELISA kits were used as Human IL-2Uncoated ELISA, human TNF-alpha Uncoated ELISA, human IFN-gamma Uncoated ELISA (accession numbers 88-7025, 88-7346, 88-7316, respectively). The method comprises the following specific steps: 10 Xcoating Buffer was diluted to 1X with ddH ₂ O, coated antibody (250X) was added, mixed well and then added to a 96-well plate (ELISA-specific) at 100. Mu.L/well. After sealing with the preservative film, the film was washed 3 times with 1 XPBST (also known as Wash Buffer,1 XPBS plus 0.05% Tween 20) at 260. Mu.L/well each time, diluted 1X with ddH ₂ O5 XPELISA/ELISPOT reagent, added to 96-well plates at 200. Mu.L/well and allowed to stand at room temperature for 1h overnight. PBST was washed 1 time, standard yeast dilutions (IL-2, TNF-alpha, IFN-gamma ranges 2-250, 4-500, respectively) were used, and samples were diluted 20-50 fold using 1 XDiluent. Adding a sample and standard yeast, incubating for 2h at normal temperature in 100 mu L of each well, washing with PBST three times, adding a1 xDiluent diluted detection antibody, incubating for 1h, washing with PBST 3 times, adding a1 xDiluent diluted HRP, incubating for 30 min, washing for 6 times, adding TMB for color development, keeping the color development time at no more than 15min, adding 2NH ₂SO ₄, and detecting light absorption at 450 nm.

5.T cell differentiation change detection: flow cytometer analysis

During T cell activation, a large amount of cytokines and other chemokines are released, and signals are transduced into the nucleus through cytokines or chemokine receptors, regulating the change in T cell differentiation. T cell differentiation is from primitive T cells (naive) to central memory T cells (Tcm) to effector memory T cells (Tem) and finally to effector T cells (Teff). While the proliferation and persistence of T cells in the body is affected by the number of T cells that differentiate into central memory T cells (Tcm) to effector memory T cell (Tem) cell types. Memory T cells are classified into stem cell-state T cells, central memory T cells, and effector memory T cells. The proportion of central memory T cell differentiation affects the sustained killing of T cells in vivo. The ratio of primitive T cells to effector T cells affects the killing effect and persistence of T cells on tumors in vivo. The differentiation of T cells was known by measuring the expression of CD45RA and CCR7 on the surface of T cells using a flow cytometer. After 7 days of co-incubation of T cells with target cells, the T cells were stained with anti-human-CD45RA-Percp-cy5.5 and anti-human-CCR7-APC flow antibody for 30 minutes, centrifuged, washed once with PBS, fixed with 4% paraformaldehyde solution, and examined for T cell differentiation by flow cytometry.

Differentiation of central memory T cells of anti-MSLNSTAR constructed by different lengths of G4S linker was examined by flow cytometry. And (3) combining tumor killing results, ELISA results and flow-type detection of cell differentiation, and analyzing tumor killing effects of anti-MSLNSTAR receptor structures deleted at intracellular ends of alpha or beta constant regions by different lengths (G4S) linker connection and differentiation conditions of memory cell groups of IL-2 secretion and T cells.

Example 9: in vivo killing Activity of anti-MSLN STAR comprehensive mutants based on G4S linkers of different lengths

In vivo killing activity of anti-MSLN STAR complex mutants without the G4S linker and with the (G4S) ₅ linker were tested separately in the same manner as in example 6, and the results are shown in FIGS. 14A-F.

EXAMPLE 10 NM24 STAR-T cell pharmacokinetic analysis experiments

High doses of STAR-T cells were returned in tumor-bearing mice, approximately 3E8/kg of mouse body weight. The tissues of the mice were collected from the placebo mice, 4 hours, 1 day, 3 days, 7 days, 14 days and 21 days after T cell reinfusion, respectively. The tissue selected in this embodiment includes: blood, bone marrow, tumor, heart, liver, spleen, lung, kidney, brain, ovary. The distribution and metabolism of STAR-T cells in various tissues was examined using Q-PCR.

The results are shown in FIGS. 15A-B, where STAR copy number is increased in all tissues. Day 14 this visit point has the highest STAR copy number; tumor, spleen, lung copy number proliferate most rapidly in all tissues.

Comparative example 1 in vitro injury killing and IFN-gamma secretion of NM24 STAR-T cells comparative test experiment of TCR ² TC-210, a similar cell product

In the same manner as in example 5, cells of NM24 STAR-T and the like cell product TCR ² TC-210 were tested for in vitro killing and IFN-gamma secretion, respectively. As shown in FIGS. 16A-B, the in vitro killing of NM24 STAR-T cells and IFN-gamma secretion was superior to that of the same cell product TCR ² TC-210.

Comparative example 2 in vivo efficacy and safety of NM24 STAR-T cells test experiment with similar cell products TCR ² TC-210

In vivo efficacy and safety of NM24 STAR-T and cells of the same cell product TCR ² TC-210, respectively, were tested in the same manner as in example 6. As shown in FIGS. 17A and 17B, NM24 STAR-T cells have better in vivo potency than the like cell product TCR ² TC-210. As shown in fig. 17C, mice lost less weight after reinfusion of NM24 STAR-T cells compared to the like cell product TCR ² TC-210; as shown in FIG. 17D, a total of 3 mice died on day 25 of the same cell product TCR ² TC-210 after reinfusion. Therefore, NM24 STAR-T cells are safer than the same cell product 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 of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Claims

A synthetic T cell receptor antigen receptor, characterized by:

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

Ii) the synthetic T cell receptor antigen receptor comprises a gamma chain, 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 plurality of antigen binding regions being the same or different, the plurality of antigen binding regions being linked directly or through a linker;

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

Preferably, the single chain antibody comprises a heavy chain variable region and a light chain variable region linked 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 (e.g., different epitopes) of mesothelin.
The synthetic T cell receptor antigen receptor of claim 1 or 2, wherein the antigen binding region in the first target binding region comprises one or more single domain antibodies and/or wherein the antigen binding region in the second target binding region comprises one or more single domain antibodies;

preferably, the antigen binding region of the first target binding region comprises a plurality of single domain antibodies that are the same or different;

Preferably, the antigen binding region of the second target binding region comprises a plurality of single domain antibodies that are the same or different;

Further preferred, the plurality of single domain antibodies are linked 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 comprising CDR1-3, wherein,

I) CDR1 comprises SEQ ID NO:34, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:35, said CDR3 comprises the amino acid sequence set forth in SEQ ID NO:36, and a nucleotide sequence shown in seq id no;

Or alternatively

Ii) CDR1 comprises SEQ ID NO:37, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:38, said CDR3 comprises the amino acid sequence set forth in SEQ ID NO:39, and a polypeptide having the amino acid sequence shown in seq id no.
The synthetic T cell receptor antigen receptor of claim 3, wherein: the single domain antibody comprises SEQ ID NO:28 or 29.
The synthetic T cell receptor antigen receptor of claim 1 wherein the antigen receptor is a cell receptor,

I) The alpha chain and/or the beta chain has attached at its C-terminal end at least one functional domain attached directly or through a linker to the C-terminal end of the alpha chain and/or the beta chain;

Or alternatively

Ii) the gamma and/or delta chain has attached at its C-terminal end at least one functional domain attached directly or via a linker to the C-terminal end of the gamma and/or delta chain.
The synthetic T cell receptor antigen receptor of claim 1 wherein the antigen receptor is a cell receptor,

I) The intracellular regions of the alpha and/or beta chains in the synthetic T cell receptor antigen receptor are deleted;

Or alternatively

Ii) the intracellular region of the gamma and/or delta chain in the synthetic T cell receptor antigen receptor is deleted.
The synthetic T cell receptor antigen receptor of claim 7 wherein the antigen receptor is a cell receptor,

I) The alpha chain and/or the beta chain has attached at its C-terminal end at least one functional domain attached directly or through a linker to the C-terminal end of the alpha chain and/or the beta chain;

Or alternatively

Ii) the gamma and/or delta chain has attached at its C-terminal end at least one functional domain attached directly or via a linker to the C-terminal end of the gamma and/or delta chain.
The synthetic T cell receptor antigen receptor according to any one of claims 6 to 8, wherein,

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

Or alternatively

Ii) 1,2,3,4,5, 6, 7, 8, 9, 10 or more functional domains are C-terminally linked to the gamma 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 C-terminally linked to the delta chain in the synthetic T cell receptor antigen receptor;

wherein the functional domains are the same or different.
The synthetic T cell receptor antigen receptor of any one of claims 6-8, wherein the functional domain is a costimulatory molecule or fragment thereof, a co-inhibitory molecule or fragment thereof, a cytokine receptor or fragment thereof, or an intracellular protein or fragment thereof;

Preferably, the costimulatory molecule is selected from the group consisting of CD40, OX40, ICOS, CD28, 4-1BB or CD27;

preferably, the co-inhibitory molecule is selected from the group consisting of TIM3, PD1, CTLA4, LAG3;

Preferably, the cytokine receptor is selected from the group consisting of interleukin receptor, interferon receptor, tumor necrosis factor superfamily receptor, colony stimulating factor receptor, chemokine receptor, growth factor receptor, and other membrane proteins;

preferably, the intracellular protein is a domain of a T cell regulator, such as NIK.
The synthetic T cell receptor antigen receptor of any one of claims 1-10, wherein the linker is selected from the group consisting of a rigid linker, a flexible linker, a cleavable linker, and a nonsensical amino acid;

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

Preferably, the flexible linker is selected from glycine and/or serine rich peptide fragments; preferably, the flexible linker is selected from the group consisting of SEQ ID NO:60-112, one or more than two;

Preferably, the cleavable linker is selected from the group consisting of SEQ ID NO: 113-117.
The synthetic T cell receptor antigen receptor of any one of claims 1 to 11, wherein the first constant region is a TCR α chain constant region or a TCR γ chain constant region, preferably a modified TCR α chain constant region or a modified TCR γ chain constant region;

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

preferably, the TCR γ chain constant region is selected from a human TCR γ chain constant region or a murine (preferably mouse) TCR γ chain constant region.
The synthetic T cell receptor antigen receptor of claim 12, wherein: the modified TCR a chain constant region is derived from a murine (preferably mouse) TCR a chain constant region, which comprises one or more modifications, relative to a wild-type murine (preferably mouse) TCR a chain constant region, at positions 6, 13, 15-18, 48, 112, 114, 115, which modifications are mutations or deletions; or alternatively

The modified TCR a chain constant region is derived from a murine (preferably mouse) TCR a chain constant region, which comprises one or more modifications, relative to a wild-type murine (preferably mouse) TCR a chain constant region, at positions 13, 36, 47, 53, 58, 78, 98, 122, which modifications are mutations or deletions.
The synthetic T cell receptor antigen receptor of claim 12, wherein: the modified TCR a chain constant region is derived from a murine (preferably mouse) TCR a chain constant region that comprises an amino acid at position 48, e.g., threonine T, mutated to cysteine C, relative to a wild-type murine (preferably mouse) TCR a chain constant region;

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

The modified TCR a chain constant region is derived from a murine (preferably mouse) TCR a chain constant region comprising an amino acid such as E at position 6 substituted with D, K at position 13 substituted with R, and amino acids 15-18 deleted relative to a wild-type murine (preferably mouse) TCR a chain constant region;

the modified TCR a chain constant region is derived from a murine (preferably mouse) TCR a chain constant region comprising the amino acid K at position 122 substituted with R relative to a wild-type murine (preferably mouse) TCR a chain constant region;

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

The modified TCR α chain constant region is derived from a murine (preferably mouse) TCR α chain constant region which comprises an amino acid such as threonine T at position 48 mutated to cysteine C, with amino acid K at position 122 substituted with R, relative to a wild-type murine (preferably mouse) TCR α chain constant region;

The modified TCR a chain constant region is derived from a murine (preferably mouse) TCR a chain constant region comprising an amino acid such as E at position 6 substituted with D, K at position 13 substituted with R, and amino acids 15-18 deleted, and an amino acid such as threonine T at position 48 mutated to cysteine C, relative to a wild-type murine (preferably mouse) TCR a chain constant region;

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

The modified TCR α chain constant region is derived from a murine (preferably mouse) TCR α chain constant region comprising an amino acid such as E at position 6 substituted with D, K at position 13 substituted with R, and amino acids 15-18 deleted, an amino acid such as threonine T at position 48 mutated to cysteine C, an amino acid such as serine S at position 112 changed to leucine L, an amino acid such as methionine M at position 114 changed to isoleucine I, an amino acid such as glycine G at position 115 changed to valine V, an amino acid K at position 122 substituted with R;

The modified TCR α chain constant region is derived from a murine (preferably mouse) TCR α chain constant region comprising an amino acid such as E at position 6 substituted with D, K at position 13 substituted with R, and amino acids 15-18 deleted, an amino acid such as threonine T at position 48 mutated to cysteine C, an amino acid such as serine S at position 112 changed to leucine L, an amino acid such as methionine M at position 114 changed to isoleucine I, an amino acid such as glycine G at position 115 changed to valine V;

The modified TCR α chain constant region is derived from a murine (preferably mouse) TCR α chain constant region comprising an amino acid such as E at position 6 substituted with D, K at position 13 substituted with R, and amino acids 15-18 deleted, an amino acid such as threonine T at position 48 mutated to cysteine C, amino acid K at position 122 substituted with R, relative to a wild-type murine (preferably mouse) TCR α chain constant region;

The modified TCR α chain constant region is derived from a murine (preferably mouse) TCR α chain constant region comprising an amino acid such as E at position 6 substituted with D, K at position 13 substituted with R, and amino acids 15-18 deleted, an amino acid such as serine S at position 112 changed to leucine L, an amino acid such as methionine M at position 114 changed to isoleucine I, an amino acid such as glycine G at position 115 changed to valine V, relative to a wild-type murine (preferably mouse) TCR α chain constant region;

The modified TCR α chain constant region is derived from a murine (preferably mouse) TCR α chain constant region comprising an amino acid such as E at position 6 substituted with D, K at position 13 substituted with R, and amino acids 15-18 deleted, and amino acid K at position 122 substituted with R, relative to a wild-type murine (preferably mouse) TCR α chain constant region; or alternatively

The modified TCR α chain constant region is derived from a murine (preferably mouse) TCR α chain constant region comprising an amino acid such as E at position 6 substituted with D, K at position 13 substituted with R, and amino acids 15-18 deleted, an amino acid such as serine S at position 112 changed to leucine L, an amino acid such as methionine M at position 114 changed to isoleucine I, an amino acid such as glycine G at position 115 changed to valine V, and an amino acid K at position 122 changed to R, relative to a wild-type murine (preferably mouse) TCR α chain constant region.
The synthetic T cell receptor antigen receptor of claim 12, wherein: the first constant region comprises SEQ ID NO: 1.3, 5, 7, 8, 16, 31, 32 or 43.
The synthetic T cell receptor antigen receptor of any one of claims 1-15, wherein: the second constant region is a TCR β chain constant region or a TCR δ chain constant region, preferably a modified TCR β chain constant region or a modified TCR δ chain constant region;

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

Preferably, the TCR delta chain constant region is selected from a human TCR delta chain constant region or a murine (preferably mouse) TCR delta chain constant region.
The synthetic T cell receptor antigen receptor of claim 16, wherein: the modified TCR β chain constant region is derived from a murine (preferably mouse) TCR β chain constant region comprising one or more modifications, relative to a wild-type murine (preferably mouse) TCR β chain constant region, at positions 3, 6, 9, 11, 12, 17, 21-25, 56, 150, 168 or 170, which modifications are mutations or deletions; or alternatively

The modified TCR β chain constant region is derived from a murine (preferably mouse) TCR β chain constant region, which comprises one or more modifications, relative to a wild-type murine (preferably mouse) TCR β chain constant region, at positions 9, 17, 23, 25, 49, 63, 103, 110, 150, 168, 170, which modifications are mutations or deletions.
The synthetic T cell receptor antigen receptor of claim 16, wherein: the modified TCR β chain constant region is derived from a murine (preferably mouse) TCR β chain constant region comprising an amino acid at position 56, e.g., serine S, mutated to cysteine C, relative to a wild-type murine (preferably mouse) TCR β chain constant region;

The modified TCR β chain constant region is derived from a murine (preferably mouse) TCR β chain constant region comprising a substitution of lysine at position 150, 168 or 170 with arginine;

The modified TCR β chain constant region is derived from a murine (preferably mouse) TCR β chain constant region comprising an amino acid such as R at position 3 substituted with K, an amino acid such as T at position 6 substituted with F, K at position 9 substituted with E, S at position 11 substituted with a, L at position 12 substituted with V, and amino acids at positions 17, 21-25 deleted, relative to a wild-type murine (preferably mouse) TCR β chain constant region;

The modified TCR β chain constant region is derived from a murine (preferably mouse) TCR β chain constant region comprising an amino acid at position 56, e.g. serine S, mutated to cysteine C, and lysine at position 150, 168 or 170 substituted with arginine, relative to a wild-type murine (preferably mouse) TCR β chain constant region;

The modified TCR β chain constant region is derived from a murine (preferably mouse) TCR β chain constant region comprising an amino acid such as R at position 3 substituted with K, an amino acid such as T at position 6 substituted with F, K at position 9 substituted with E, S at position 11 substituted with a, L at position 12 substituted with V, and amino acids 17, 21-25 deleted, and an amino acid such as serine S at position 56 mutated to cysteine C, relative to a wild-type murine (preferably mouse) TCR β chain constant region;

The modified TCR β chain constant region is derived from a murine (preferably mouse) TCR β chain constant region comprising an amino acid such as R at position 3 substituted with K, an amino acid such as T at position 6 substituted with F, K at position 9 substituted with E, S at position 11 substituted with a, L at position 12 substituted with V, and amino acids 17, 21-25 deleted, an amino acid such as serine S at position 56 mutated to cysteine C, and lysine at position 150, 168 or 170 substituted with arginine, relative to a wild-type murine (preferably mouse) TCR β chain constant region; or alternatively

The modified TCR β chain constant region is derived from a murine (preferably mouse) TCR β chain constant region comprising an amino acid such as R at position 3 substituted with K, an amino acid such as T at position 6 substituted with F, K at position 9 substituted with E, S at position 11 substituted with a, L at position 12 substituted with V, and amino acids 17, 21-25 deleted, and lysine at position 150, 168 or 170 substituted with arginine, relative to a wild-type murine (preferably mouse) TCR β chain constant region.
The synthetic T cell receptor antigen receptor of claim 16, wherein: the second constant region comprises SEQ ID NO: 2. 4, 6, 9, 17, 33 or 44.
The synthetic T cell receptor antigen receptor of claim 1 or 2, wherein: the first target binding region is linked directly to the first constant region or via a linker, and/or the second target binding region is linked directly to the second constant region or via a linker.
The synthetic T cell receptor antigen receptor of claim 20, wherein: the linker is selected from a rigid linker, a flexible linker, a cleavable linker or a nonsensical amino acid;

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

Preferably, the flexible linker is selected from glycine and/or serine rich peptide fragments; preferably, the flexible linker is selected from the group consisting of SEQ ID NO:60-112, one or more than two;

Preferably, the cleavable linker is selected from the group consisting of SEQ ID NO: 113-117.
A synthetic T cell receptor antigen receptor complex, characterized by: the complex comprising the synthetic T cell receptor antigen receptor of any one of claims 1-21, and CD3 epsilon, CD3 gamma, CD3 delta, and CD3 zeta.
The synthetic T cell receptor antigen receptor complex of claim 22, wherein: the CD3 epsilon, CD3 gamma, CD3 delta and/or CD3 zeta are of human origin;

preferably, the CD3 epsilon comprises SEQ ID NO:20, and a polypeptide comprising the amino acid sequence shown in seq id no;

preferably, the CD3 γ comprises SEQ ID NO:18, an amino acid sequence shown in seq id no;

Preferably, the CD3 delta comprises SEQ ID NO:19, an amino acid sequence shown in seq id no;

preferably, said CD3 ζ comprises SEQ ID NO:21, and a polypeptide comprising the amino acid sequence shown in seq id no.
An antibody or antigen-binding fragment, characterized in that: the antibody or antigen binding fragment comprises a heavy chain variable region comprising CDR1-3 and/or a light chain variable region wherein,

I) CDR1 comprises SEQ ID NO:34, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:35, said CDR3 comprises the amino acid sequence set forth in SEQ ID NO:36, and a nucleotide sequence shown in seq id no;

Or alternatively

Ii) CDR1 comprises SEQ ID NO:37, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:38, said CDR3 comprises the amino acid sequence set forth in SEQ ID NO:39, and a polypeptide having the amino acid sequence shown in seq id no.
The antibody or antigen-binding fragment of 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 of claim 24, wherein: the antibody or antigen binding fragment comprises SEQ ID NO:28 or 29.
An antigen receptor comprising a transmembrane region, an intracellular region and one or more identical or different extracellular binding domains, wherein the extracellular binding domain is an extracellular antigen binding domain;

wherein the extracellular antigen-binding domain comprises CDR1-3, wherein,

I) CDR1 comprises SEQ ID NO:34, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:35, said CDR3 comprises the amino acid sequence set forth in SEQ ID NO:36, and a nucleotide sequence shown in seq id no;

Or alternatively

Ii) CDR1 comprises SEQ ID NO:37, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:38, said CDR3 comprises the amino acid sequence set forth in SEQ ID NO:39, and a polypeptide having the amino acid sequence shown in seq id no.
The antigen receptor of claim 27, wherein the extracellular antigen-binding domain comprises an antibody or antigen-binding fragment of any one of claims 24-26.
The antigen receptor of claim 27, wherein the antigen receptor is a STAR, TCR, or CAR;

Preferably, the transmembrane region is derived from human CD8;

preferably, the intracellular domain is derived from 4-1BB, CD28 or CD3 zeta.
The antigen receptor of claim 27, wherein the transmembrane region is linked directly or via a linker to one or more extracellular antigen binding domains.
The antigen receptor of claim 30, wherein the linker is selected from the group consisting of a rigid linker, a flexible linker, a cleavable linker, and a nonsensical amino acid;

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

Preferably, the flexible linker is selected from glycine and/or serine rich peptide fragments; preferably, the flexible linker is selected from the group consisting of SEQ ID NO:60-112, one or more than two;

Preferably, the cleavable linker is selected from the group consisting of SEQ ID NO: 113-117.
A nucleic acid, characterized in that: the nucleic acid encodes 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 antibody or antigen binding fragment of any one of claims 24-26, the antigen receptor of any one of claims 27-31.
A carrier, characterized in that: the vector comprising the nucleic acid of 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 of any one of claims 1-21, the synthetic T cell receptor antigen receptor complex of any one of claims 22-23, the antibody or antigen binding fragment of any one of claims 24-26, the antigen receptor of any one of claims 27-31.
The immune cell of claim 35, wherein: the immune cell contains one or more of the nucleic acid of claim 32.
The immune cell of claim 35 or 36, wherein: 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).
The immune cell of claim 35 or 36, wherein the immune cell is derived from a T cell of the subject.
A method for preparing an immune cell, comprising transfecting the nucleic acid sequence of claim 32 into an immune cell for expression.
A method for preparing a recombinant T cell, comprising the steps of:

1) Obtaining the nucleic acid of claim 32 from a positive T cell clone;

2) Isolating and culturing primary T cells;

3) Delivering the nucleic acid obtained in step 1) to the primary T cell of step 2) to obtain a recombinant T cell expressing the synthetic T cell receptor antigen receptor of any one of claims 1-21.
A method for preparing a synthetic T cell receptor antigen receptor, comprising the steps of:

(1) Obtaining the nucleic acid of claim 32 from a positive T cell clone;

(2) Connecting the nucleic acid obtained in the step (1) to a vector skeleton to obtain an expression vector;

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

(4) Obtaining the synthetic T cell receptor antigen receptor.
Use of a synthetic T cell receptor antigen receptor according to any one of claims 1 to 21, a synthetic T cell receptor antigen receptor complex according to any one of claims 22 to 23, an antibody or antigen binding fragment according to any one of claims 24 to 26, an antigen receptor according to any one of claims 27 to 31, a nucleic acid according to claim 32, an immune cell according to any one of claims 35 to 38 for the preparation of a product for the diagnosis or treatment of a tumor.
The use according to claim 42, wherein: the 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, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, renal 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, 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's macroglobulinemia; the sarcoma is selected from osteosarcoma, ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma.
A pharmaceutical composition characterized by: the pharmaceutical composition comprises 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 antibody or antigen binding fragment of any one of claims 24-26, the antigen receptor of any one of claims 27-31, the nucleic acid of claim 32, the immune cell of any one of claims 35-38.
A kit, characterized in that: the kit comprises 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 antibody or antigen binding fragment of any one of claims 24-26, the antigen receptor of any one of claims 27-31, the nucleic acid of claim 32, and the immune cell of any one of claims 35-38.