Synthetic T cell receptor antigen receptor specifically combined with LILRB4 and application thereof

Abstract

The invention discloses a synthetic T cell receptor antigen receptor specifically combined with LILRB4 and application of the synthetic T cell receptor antigen receptor. According to the invention, the antigen binding fragment of the targeted LILRB4 is used for transforming the TCR and the CAR, so that a better tumor treatment effect is achieved.

Description

A synthetic T cell receptor antigen receptor specifically binding to LILRB4 and its application Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a synthetic T cell receptor antigen receptor (STAR) that specifically binds to LILRB4, as well as a STAR complex, immune cells containing STAR or the STAR complex, and uses thereof in the field of biomedicine. Background Art

[0002] LILRB4 is a member of the leukocyte immunoglobulin receptor family. It has a single transmembrane structure, two extracellular C-type Ig-like domains, and three intracellular tyrosine-based immunoreceptor inhibitory motifs (ITIMs). In the normal human body, LILRB4 is primarily expressed in myeloid immune cells, such as macrophages, DCs, and monocytes, where it exerts an immunosuppressive function. LILRB4 is highly expressed in acute myeloid-monocytic leukemia and acute monocytic leukemia, with expression levels higher than in normal monocytes. LILRB4 expression has also been detected in leukemic cancer stem cells. Furthermore, LILRB4 is highly expressed in various immunosuppressive cells within the tumor microenvironment, including TAMs, M-MDSCs, tolerogenic DCs, and Tregs. Studies have shown that LILRB4 expressed in AML promotes tumor cell migration and infiltration and inhibits T cell proliferation through Arginase-1 and uPAR. Furthermore, LILRB4 expression in other myeloid immune cells, such as macrophages, DCs, and monocytes, significantly promotes immune cell differentiation toward immune tolerance, thereby exerting an immunosuppressive effect. Based on the specific expression and immunosuppressive function of LILRB4 in leukemia cells and myeloid immune cells, LILRB4 has the potential to become an ideal therapeutic target for acute myelomonocytic and acute monocytic leukemia by eliminating tumor cells and improving the immunosuppressive environment.

[0003] Chimeric antigen receptor T cell (CAR-T) therapy is an anticancer immunotherapy that has shown promising results in recent years. Unlike natural T cells, which recognize tumor cells, CAR-T cells do so independently of MHC molecules. CAR molecules consist of three main components: an extracellular region, which is derived from the antigen recognition domain of an antibody and is responsible for recognizing the target antigen; a transmembrane region; and an intracellular region, which contains signaling and co-stimulatory molecules derived from the T cell receptor and is responsible for transmitting T cell activation signals upon stimulation. The mechanism of action is as follows: when CAR molecules bind to their corresponding antigens, they aggregate, leading to increased local phosphorylation levels and activation of downstream signaling, ultimately initiating the T cell's effector function and killing the target tumor cells.

[0004] The T cell receptor (TCR) complex molecule contains multiple chains. The TCRα chain and TCRβ chain are responsible for recognizing MHC-peptide molecules, and the other 6 CD3 subunits bind to the TCRα / β chain to perform signal transduction functions. The natural TCR complex contains a total of 10 ITAM signal sequences, which can theoretically transmit stronger signals than CAR. Previous studies have shown that although the TCR signal is slower than the CAR signal, the TCR signal is more persistent. Therefore, by utilizing the signal transduction function of the natural TCR, it will be possible to construct a new type of receptor to alleviate T cell dysfunction, enabling it to better play an anti-solid tumor role.

[0005] The extracellular region of TCR is very similar to the Fab domain of antibody. Therefore, the variable region sequence of TCR can be replaced with the variable region sequence of antibody to obtain a synthetic T cell receptor antigen receptor (Snythetic TCR and Antigen Receptor, STAR), which has both the specificity of antibody and the superior signal transduction function of natural TCR and can mediate complete T cell activation.

[0006] However, STARs derived from natural TCRs still have disadvantages such as poor membrane stability, low α / β chain pairing ability, mismatch with endogenous TCRs, and difficulty in introducing into T cells. Therefore, there is still a need in the art for improved STARs.

[0007] Summary of the Invention

[0008] To address the deficiencies of the prior art, the present invention provides a synthetic T cell receptor antigen receptor (STAR) and its application. This synthetic T cell receptor antigen receptor can specifically bind to LILRB4. By targeting the antigen binding fragment of LILRB4, TCR and CAR are modified. Based on existing results, the STAR-T cells of the present invention have greater potential in the treatment of relapsed and refractory AML FAB M4 / M5, and their therapeutic efficacy and safety are better than existing technologies such as CAR-T. Based on the high expression of LILRB4 in AML FAB M4 / M5 and its high expression in myeloid immunosuppressive cells, the development of LILRB4 dual-epitope STAR-T can provide a new immune cell therapy strategy for R / R AML FAB M4 / M5 patients by killing tumor cells and improving the tumor microenvironment.

[0009] Specifically, the first aspect of the present invention provides a synthetic T cell receptor antigen receptor (STAR),

[0010] i) the synthetic T cell receptor antigen receptor comprises an α chain and a β chain, wherein the α chain comprises a first target binding region and a first constant region, and the β chain comprises a second target binding region and a second constant region, or the α chain comprises the first target binding region and the β chain comprises the second target binding region and a second constant region; or

[0011] ii) The synthetic T cell receptor antigen receptor comprises a γ chain and a δ chain, wherein the γ chain comprises a first target binding region and a first constant region, and the δ chain comprises a second target binding region and a second constant region, or the γ chain comprises a first target binding region and a first constant region and the δ chain comprises a second target binding region and a second constant region.

[0012] Preferably, i) the α chain and / or the β chain has at least one functional domain linked to its C-terminus; or ii) the γ chain and / or the δ chain has at least one functional domain linked to its C-terminus.

[0013] Further preferably, i) the at least one functional domain is directly or via a linker connected to the C-terminus of the α chain and / or β chain, or, ii) the at least one functional domain is directly or via a linker connected to the C-terminus of the γ chain and / or δ chain.

[0014] Preferably, i) the intracellular region of the α chain and / or β chain in the synthetic T cell receptor antigen receptor is deleted; or, ii) the intracellular region of the γ chain and / or δ chain in the synthetic T cell receptor antigen receptor is deleted.

[0015] Further preferably, i) the functional domain is directly or via a linker connected to the C-terminus of the α chain and / or β chain deleted from the intracellular region; or, ii) the functional domain is directly or via a linker connected to the C-terminus of the γ chain and / or δ chain deleted from the intracellular region.

[0016] Preferably, i) the C-terminus of the α chain in the synthetic T cell receptor antigen receptor is connected 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 connected to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains; or

[0017] ii) the C-terminus of the γ chain in the synthetic T cell receptor antigen receptor is connected to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains, and / or the C-terminus of the δ chain in the STAR is connected to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains.

[0018] In a specific embodiment of the present invention, the multiple (including two or more) functional domains are directly connected or connected through a linker.

[0019] In one embodiment of the present invention, the synthetic T cell receptor antigen receptor has at least one functional domain attached to the C-terminus of the α chain. The intracellular region of the α chain is deleted. The functional domain is connected to the C-terminus of the α chain with the deleted intracellular region via a linker.

[0020] In one embodiment of the present 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 β chain is deleted. The functional domain is connected to the C-terminus of the β chain with the deleted intracellular region via a linker.

[0021] In one embodiment of the present invention, the synthetic T cell receptor antigen receptor has at least one functional domain attached to the C-terminus of the γ chain. The intracellular region of the γ chain is deleted. The functional domain is connected to the C-terminus of the γ chain with the deleted intracellular region via a linker.

[0022] In one embodiment of the present invention, the synthetic T cell receptor antigen receptor has at least one functional domain attached to the C-terminus of the delta chain. The intracellular region of the delta chain is deleted. The functional domain is connected to the C-terminus of the delta chain with the deleted intracellular region via a linker.

[0023] Preferably, i) the functional domains connected to the C-termini of the α chain and / or β chain in the synthetic T cell receptor antigen receptor are the same or different;

[0024] Alternatively, ii) the functional domains connected to the C-termini of the γ chain and / or the δ chain in the synthetic T cell receptor antigen receptor are the same or different.

[0025] In a specific embodiment of the present invention, the multiple functional domains connected to the α chain in the synthetic T cell receptor antigen receptor can be the same or different.

[0026] In a specific embodiment of the present invention, the multiple functional domains connected by the β chains in the synthetic T cell receptor antigen receptor can be the same or different.

[0027] In a specific embodiment of the present invention, the multiple functional domains connected to the γ chain in the synthetic T cell receptor antigen receptor can be the same or different.

[0028] In a specific embodiment of the present invention, the multiple functional domains connected by the δ chain in the synthetic T cell receptor antigen receptor can be the same or different.

[0029] In a specific embodiment of the present invention, the functional domain connected to the α chain and the functional domain connected to the β chain in the synthetic T cell receptor antigen receptor may be the same or different.

[0030] In a specific embodiment of the present invention, the functional domain connected to the γ chain and the functional domain connected to the δ chain in the synthetic T cell receptor antigen receptor may be the same or different.

[0031] Preferably, the functional domain is a co-stimulatory molecule or a fragment thereof, a co-inhibitory molecule or a fragment thereof, a cytokine receptor or a fragment thereof, or an intracellular protein or a fragment thereof. Further preferably, the functional domain is the intracellular domain of a co-stimulatory molecule, the intracellular domain of a co-inhibitory molecule, the intracellular domain of a cytokine receptor, or an intracellular protein. Alternatively, the functional domain may be a fusion of the intracellular domain of a cytokine receptor with the human STAT5 activation module (amino acid sequence as shown in SEQ ID NO: 25), directly or through a linker.

[0032] The co-stimulatory molecule is selected from CD40, OX40, ICOS, CD28, 4-1BB (CD137) or CD27.

[0033] The co-inhibitory molecule is selected from TIM3, PD1, CTLA4, and LAG3.

[0034] The cytokine receptor is selected from interleukin receptors (such as IL-2 receptor), interferon receptors, tumor necrosis factor superfamily receptors, colony stimulating factor receptors, chemokine receptors, growth factor receptors or other membrane proteins.

[0035] The intracellular protein is a T cell regulatory factor, such as the domain of NIK.

[0036] In a specific embodiment of the present invention, the costimulatory molecule is CD40, and its intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 10.

[0037] In a specific embodiment of the present invention, the costimulatory molecule is OX40, and its intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 11.

[0038] In a specific embodiment of the present invention, the costimulatory molecule is ICOS, and its intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 12.

[0039] In a specific embodiment of the present invention, the costimulatory molecule is CD28, and its intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 13.

[0040] In a specific embodiment of the present invention, the costimulatory molecule is 4-1BB, whose intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 14.

[0041] In a specific embodiment of the present invention, the costimulatory molecule is CD27, and its intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 15.

[0042] In a specific embodiment of the present invention, the cytokine receptor is IL-2β, and its intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 22.

[0043] In a specific embodiment of the present invention, the cytokine receptor is IL-7α, and its intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 23.

[0044] In a specific embodiment of the present invention, the cytokine receptor is IL-21, and its intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 24.

[0045] In a specific embodiment of the present invention, the functional domain is a fusion of the IL-2β intracellular domain and the human STAT5 activation module, which comprises the amino acid sequence shown in SEQ ID NO: 26.

[0046] In a specific embodiment of the present invention, the functional domain is a fusion of the IL-7α intracellular domain and the human STAT5 activation module, which comprises the amino acid sequence shown in SEQ ID NO: 27.

[0047] Preferably, the first constant region is a TCRα chain constant region or a TCRγ chain constant region, preferably a modified TCRα chain constant region or a modified TCRγ chain constant region.

[0048] The TCRα chain constant region is selected from a human TCRα chain constant region or a rodent (preferably a murine, more preferably a mouse) TCRα chain constant region. The TCRγ chain constant region is selected from a human TCRγ chain constant region or a rodent (preferably a murine, more preferably a mouse) TCRγ chain constant region.

[0049] In a specific embodiment of the present invention, the amino acid sequence of the human TCRα chain constant region is shown in SEQ ID NO: 1, and the amino acid sequence of the rodent (preferably mouse, further preferably mouse) TCRα chain constant region is shown in SEQ ID NO: 3.

[0050] In a specific embodiment of the present invention, the amino acid sequence of the human TCRγ chain constant region is shown in SEQ ID NO: 45, and the amino acid sequence of the rodent (preferably mouse, further preferably mouse) TCRγ chain constant region is shown in SEQ ID NO: 46.

[0051] The modified TCR α chain constant region is derived from a human TCR α chain constant region, and comprises one or more modifications at position 48, 116 or 119 relative to a wild-type human TCR α chain constant region, wherein the modification is a mutation or a deletion.

[0052] The modified TCR alpha chain constant region is derived from a human TCR alpha chain constant region, which comprises a mutation of Threonine T at position 48 to Cysteine ​​C relative to the wild-type human TCR alpha chain constant region.

[0053] The modified TCR α chain constant region is derived from a human TCR α chain constant region, wherein the serine S at position 116 is mutated to a leucine L, and the glycine G at position 119 is mutated to a valine V relative to the wild-type human TCR α chain constant region.

[0054] The modified TCRα chain constant region is derived from a human TCRα chain constant region, which comprises a mutation of threonine T at position 48 to cysteine ​​C, a mutation of serine S at position 116 to leucine L, and a mutation of glycine G at position 119 to valine V relative to the wild-type human TCRα chain constant region.

[0055] The modified TCRα chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCRα chain constant region, which comprises one or more modifications at positions 6, 13, 15-18, 48, 112, 114, and 115 relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCRα chain constant region, and the modifications are mutations or deletions.

[0056] The modified TCRα chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCRα chain constant region, which comprises one or more modifications at positions 13, 36, 47, 53, 58, 78, 98, and 122 relative to a wild-type rodent (preferably a mouse, further preferably a mouse) TCRα chain constant region, wherein the modification is a mutation or deletion.

[0057] Preferably, the modified TCRα chain constant region is derived from a rodent (preferably murine, further preferably mouse) TCRα chain constant region, which comprises the introduction of cysteine ​​relative to the wild-type rodent (preferably murine, further preferably mouse) TCRα chain constant region.

[0058] Further preferably, the modified TCRα chain constant region is derived from a rodent (preferably rat, further preferably mouse) TCRα chain constant region, which comprises an amino acid at position 48, such as threonine T, which is mutated to cysteine ​​C relative to the wild-type rodent (preferably rat, further preferably mouse) TCRα chain constant region.

[0059] In a specific embodiment of the present invention, the rodent (preferably murine, more preferably mouse) TCR α chain constant region containing the introduced cysteine ​​comprises the amino acid sequence shown in SEQ ID NO:5.

[0060] Preferably, the modified TCRα chain constant region is derived from a rodent (preferably murine, further preferably mouse) TCRα chain constant region, which comprises a hydrophobic amino acid mutation relative to a wild-type rodent (preferably murine, further preferably mouse) TCRα chain constant region.

[0061] Further preferably, the modified TCRα chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCRα chain constant region, which, relative to the wild-type rodent (preferably a mouse, further preferably a mouse) TCRα chain constant region, comprises that the amino acid at position 112, such as serine S, is changed to leucine L, the amino acid at position 114, such as methionine M, is changed to isoleucine I, and / or, the amino acid at position 115, such as glycine G, is changed to valine V.

[0062] In a specific embodiment of the present invention, the rodent (preferably murine, more preferably mouse) TCR α chain constant region comprising a hydrophobic amino acid mutation comprises the amino acid sequence shown in SEQ ID NO: 7.

[0063] Preferably, the modified TCRα chain constant region is derived from a rodent (preferably murine, further preferably mouse) TCRα chain constant region, which comprises an N-terminal modification relative to a wild-type rodent (preferably murine, further preferably mouse) TCRα chain constant region.

[0064] Further preferably, the modified TCRα chain constant region is derived from a rodent (preferably rat, further preferably mouse) TCRα chain constant region, which comprises that the amino acid at position 6, such as E, is replaced by D, K at position 13 is replaced by R, and amino acids 15-18 are deleted relative to the wild-type rodent (preferably rat, further preferably mouse) TCRα chain constant region.

[0065] Preferably, the modified TCR α chain constant region is derived from a rodent (preferably murine, further preferably mouse) TCR α chain constant region, which comprises a lysine mutation in the transmembrane region to an arginine.

[0066] Further preferably, the modified TCRα chain constant region is derived from a rodent (preferably rat, further preferably mouse) TCRα chain constant region, which comprises the amino acid K at position 122 replaced by R relative to the wild-type rodent (preferably rat, further preferably mouse) TCRα chain constant region.

[0067] In a specific embodiment of the present invention, the rodent (preferably murine, more preferably mouse) TCR α chain constant region comprising a transmembrane region lysine mutation to arginine comprises the amino acid sequence shown in SEQ ID NO: 8.

[0068] Preferably, the modified TCRα chain constant region is derived from a rodent (preferably rat, further preferably mouse) TCRα chain constant region, which comprises cysteine ​​introduction and hydrophobic amino acid mutation relative to the wild-type rodent (preferably rat, further preferably mouse) TCRα chain constant region.

[0069] Further preferably, the modified TCRα chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCRα chain constant region, which, relative to the wild-type rodent (preferably a mouse, further preferably a mouse) TCRα chain constant region, comprises that the amino acid at position 48, such as threonine T, is mutated to cysteine ​​C, the amino acid at position 112, such as serine S, is changed to leucine L, the amino acid at position 114, such as methionine M, is changed to isoleucine I, and the amino acid at position 115, such as glycine G, is changed to valine V.

[0070] In a specific embodiment of the present invention, the rodent (preferably murine, more preferably mouse) TCR α chain constant region comprising cysteine ​​introduction and hydrophobic amino acid mutation comprises the amino acid sequence shown in SEQ ID NO: 30.

[0071] The modified TCRα chain constant region is derived from a rodent (preferably a mouse, further preferably a rat) TCRα chain constant region, which includes a mutation of the amino acid at position 48, such as threonine T, to cysteine ​​C, and a substitution of the amino acid K at position 122 by R, relative to the wild-type rodent (preferably a mouse, further preferably a rat) TCRα chain constant region.

[0072] The modified TCRα chain constant region is derived from a rodent (preferably murine, further preferably mouse) TCRα chain constant region, which comprises, relative to the wild-type rodent (preferably murine, further preferably mouse) TCRα chain constant region, an amino acid at position 6, such as E, being replaced by D, K at position 13 being replaced by R, amino acids at positions 15-18 being deleted, and an amino acid at position 48, such as threonine T, being mutated to cysteine ​​C.

[0073] The modified TCRα chain constant region is derived from a rodent (preferably murine, further preferably mouse) TCRα chain constant region, which, relative to the wild-type rodent (preferably murine, further preferably mouse) TCRα chain constant region, includes that the amino acid at position 48, such as threonine T, is mutated to cysteine ​​C, the amino acid at position 112, such as serine S, is changed to leucine L, the amino acid at position 114, such as methionine M, is changed to isoleucine I, the amino acid at position 115, such as glycine G, is changed to valine V, and the amino acid K at position 122 is replaced by R.

[0074] The modified TCRα chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCRα chain constant region, which comprises, relative to the wild-type rodent (preferably a mouse, further preferably a mouse) TCRα chain constant region, an amino acid at position 6, such as E, being replaced by D, K at position 13 being replaced by R, and amino acids 15-18 being deleted, an amino acid at position 48, such as threonine T, being mutated to cysteine ​​C, an amino acid at position 112, such as serine S, being changed to leucine L, an amino acid at position 114, such as methionine M, being changed to isoleucine I, an amino acid at position 115, such as glycine G, being changed to valine V, and an amino acid K at position 122 being replaced by R.

[0075] The modified TCRα chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCRα chain constant region, which comprises, relative to the wild-type rodent (preferably a mouse, further preferably a mouse) TCRα chain constant region, an amino acid at position 6, such as E, being replaced by D, K at position 13 being replaced by R, and amino acids 15-18 being deleted, an amino acid at position 48, such as threonine T, being mutated to cysteine ​​C, and amino acid K at position 122 being replaced by R.

[0076] The modified TCRα chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCRα chain constant region, which comprises, relative to the wild-type rodent (preferably a mouse, further preferably a mouse) TCRα chain constant region, an amino acid at position 6, such as E, being replaced by D, K at position 13 being replaced by R, and amino acids at positions 15-18 being deleted, an amino acid at position 112, such as serine S, being changed to leucine L, an amino acid at position 114, such as methionine M, being changed to isoleucine I, and an amino acid at position 115, such as glycine G, being changed to valine V.

[0077] The modified TCRα chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCRα chain constant region, which comprises that the amino acid at position 6, such as E, is replaced by D, the K at position 13 is replaced by R, the amino acids at positions 15-18 are deleted, and the amino acid K at position 122 is replaced by R relative to the wild-type rodent (preferably a mouse, further preferably a mouse) TCRα chain constant region.

[0078] The modified TCRα chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCRα chain constant region, which comprises, relative to the wild-type rodent (preferably a mouse, further preferably a mouse) TCRα chain constant region, an amino acid at position 6, such as E, being replaced by D, K at position 13 being replaced by R, and amino acids 15-18 being deleted, an amino acid at position 112, such as serine S, being changed to leucine L, an amino acid at position 114, such as methionine M, being changed to isoleucine I, an amino acid at position 115, such as glycine G, being changed to valine V, and an amino acid K at position 122 being replaced by R.

[0079] Preferably, the modified TCRα chain constant region is derived from a rodent (preferably rat, further preferably mouse) TCRα chain constant region, which comprises cysteine ​​introduction, hydrophobic amino acid mutation and N-terminal modification relative to the wild-type rodent (preferably rat, further preferably mouse) TCRα chain constant region.

[0080] Further preferably, the modified TCRα chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCRα chain constant region, which comprises, relative to the wild-type rodent (preferably a mouse, further preferably a mouse) TCRα chain constant region, that the amino acid at position 6, such as E, is replaced by D, K at position 13 is replaced by R, amino acids 15-18 are deleted, the amino acid at position 48, such as threonine T, is mutated to cysteine ​​C, the amino acid at position 112, such as serine S, is changed to leucine L, the amino acid at position 114, such as methionine M, is changed to isoleucine I, and the amino acid at position 115, such as glycine G, is changed to valine V.

[0081] In a specific embodiment of the present invention, the rodent (preferably murine, more preferably mouse) TCR α chain constant region comprising cysteine ​​introduction, hydrophobic amino acid mutation and intracellular region deletion comprises the amino acid sequence shown in SEQ ID NO: 16.

[0082] Preferably, the modified TCR α chain constant region is derived from a rodent (preferably murine, further preferably mouse) TCR α chain constant region, which comprises an N-terminal modification, the introduction of cysteine, and a hydrophobic amino acid mutation.

[0083] Further preferably, the modified TCRα chain constant region is derived from a rodent (preferably mouse, further preferably mouse) TCRα chain constant region, which comprises that the amino acid at position 6, such as E, is replaced by D, K at position 13 is replaced by R, amino acids 15-18 are deleted, and the amino acid at position 48, such as threonine T, is mutated to cysteine ​​C, the amino acid at position 112, such as serine S, is changed to leucine L, the amino acid at position 114, such as methionine M, is changed to isoleucine I, and the amino acid at position 115, such as glycine G, is changed to valine V.

[0084] In a specific embodiment of the present invention, the rodent (preferably murine, more preferably mouse) TCR α chain constant region comprising N-terminal modification, cysteine ​​introduction, and hydrophobic amino acid mutation comprises the amino acid sequence shown in SEQ ID NO: 31.

[0085] Preferably, the modified TCR α chain constant region is derived from a rodent (preferably murine, further preferably mouse) TCR α chain constant region, which comprises an intracellular deletion, an N-terminal modification, and the introduction of cysteine ​​and hydrophobic amino acid mutations.

[0086] Further preferably, the modified TCRα chain constant region is derived from a rodent (preferably mouse, further preferably mouse) TCRα chain constant region, which comprises an intracellular deletion, and the amino acid at position 48, such as threonine T, is mutated to cysteine ​​C, the amino acid at position 112, such as serine S, is changed to leucine L, the amino acid at position 114, such as methionine M, is changed to isoleucine I, the amino acid at position 115, such as glycine G, is changed to valine V, and the amino acid at position 6, such as E, is substituted by D, K at position 13 is substituted by R, and amino acids 15-18 are deleted.

[0087] In a specific embodiment of the present invention, the rodent (preferably murine, further preferably mouse) TCR α chain constant region comprising intracellular deletion, N-terminal modification, cysteine ​​introduction and hydrophobic amino acid mutation comprises the amino acid sequence shown in SEQ ID NO: 43.

[0088] In a specific embodiment of the present invention, the first constant region comprises the amino acid sequence shown in one of SEQ ID NOs: 1, 3, 5, 7, 8, 16, 30, 31 or 43.

[0089] 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.

[0090] Further preferably, the TCRβ chain constant region is selected from the human TCRβ chain constant region or the rodent (preferably rat, further preferably mouse) TCRβ chain constant region, and the TCRδ chain constant region is selected from the human TCRδ chain constant region or the rodent (preferably rat, further preferably mouse) TCRδ chain constant region.

[0091] In a specific embodiment of the present invention, the human TCR β chain constant region comprises the amino acid sequence shown in SEQ ID NO:2.

[0092] In a specific embodiment of the present invention, the rodent (preferably murine, more preferably mouse) TCR β chain constant region comprises the amino acid sequence shown in SEQ ID NO:4.

[0093] In a specific embodiment of the present invention, the human TCR δ chain constant region comprises the amino acid sequence shown in SEQ ID NO: 47.

[0094] In a specific embodiment of the present invention, the rodent (preferably murine, more preferably mouse) TCR δ chain constant region comprises the amino acid sequence shown in SEQ ID NO: 48.

[0095] The modified TCRβ chain constant region is derived from a human TCRβ chain constant region, and comprises one or more modifications at positions 57, 173 or 175 relative to a wild-type human TCRβ chain constant region, wherein the modification is a mutation or a deletion.

[0096] The modified TCR β chain constant region is derived from a human TCR β chain constant region, and comprises a mutation of serine S at position 57 to cysteine ​​C relative to a wild-type human TCR β chain constant region.

[0097] The modified TCR β chain constant region is derived from a human TCR β chain constant region, and comprises a mutation of lysine K at positions 173 and 175 to arginine relative to the wild-type human TCR β chain constant region.

[0098] The modified TCRβ chain constant region is derived from the human TCRβ chain constant region, which comprises a mutation of serine S at position 57 to cysteine ​​C, and a mutation of lysine K at positions 173 and 175 to arginine relative to the wild-type human TCRβ chain constant region.

[0099] The modified TCRβ chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCRβ chain constant region, which comprises one or more modifications at positions 3, 6, 9, 11, 12, 17, 21-25, 56, 150, 168 or 170 relative to the wild-type rodent (preferably a mouse, further preferably a mouse) TCRβ chain constant region, and the modification is a mutation or deletion.

[0100] The modified TCRβ chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCRβ chain constant region, which comprises one or more modifications at positions 9, 17, 23, 25, 49, 63, 103, 110, 150, 168, and 170 relative to the wild-type rodent (preferably a mouse, further preferably a mouse) TCRβ chain constant region, and the modifications are mutations or deletions.

[0101] Preferably, the modified TCRβ chain constant region is derived from a rodent (preferably rat, further preferably mouse) TCRβ chain constant region, which comprises the introduction of cysteine ​​relative to the wild-type rodent (preferably rat, further preferably mouse) TCRβ chain constant region.

[0102] Further preferably, the modified TCRβ chain constant region is derived from a rodent (preferably rat, further preferably mouse) TCRβ chain constant region, and relative to the wild-type rodent (preferably rat, further preferably mouse) TCRβ chain constant region, the amino acid at position 56, e.g., serine S, is mutated to cysteine ​​C.

[0103] In a specific embodiment of the present invention, the rodent (preferably mouse, more preferably mouse) TCRβ chain constant region containing the introduction of cysteine ​​comprises the amino acid sequence shown in SEQ ID NO:6.

[0104] Preferably, the modified TCRβ chain constant region is derived from a rodent (preferably murine, further preferably mouse) TCRβ chain constant region, wherein lysine in the intracellular region is substituted by arginine.

[0105] Further preferably, the modified TCRβ chain constant region is derived from a rodent (preferably murine, further preferably mouse) TCRβ chain constant region, wherein the lysine at position 150, 168 or 170 is substituted by arginine.

[0106] In one embodiment of the present invention, the rodent (preferably rat, more preferably mouse) TCRβ chain constant region comprising an intracellular region lysine substituted with arginine comprises the amino acid sequence shown in SEQ ID NO: 9

[0107] Preferably, the modified TCRβ chain constant region is derived from a rodent (preferably rat, further preferably mouse) TCRβ chain constant region, which comprises an N-terminal modification relative to a wild-type rodent (preferably rat, further preferably mouse) TCRβ chain constant region.

[0108] Further preferably, the modified TCRβ chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCRβ chain constant region, and relative to the wild-type rodent (preferably a mouse, further preferably a mouse) TCRβ chain constant region, the amino acid at position 3, such as R, is replaced by K, the amino acid at position 6, such as T, is replaced by F, K at position 9 is replaced by E, S at position 11 is replaced by A, L at position 12 is replaced by V, and amino acids at positions 17 and 21-25 are deleted.

[0109] Preferably, the modified TCRβ chain constant region is derived from a rodent (preferably a rat, further preferably a mouse) TCRβ chain constant region, which comprises the introduction of cysteine ​​and the deletion of the intracellular region relative to the wild-type rodent (preferably a rat, further preferably a mouse) TCRβ chain constant region.

[0110] In a specific embodiment of the present invention, the rodent (preferably mouse, more preferably mouse) TCR β chain constant region comprising the introduction of cysteine ​​and the deletion of the intracellular region comprises the amino acid sequence shown in SEQ ID NO: 17.

[0111] The modified TCRβ chain constant region is derived from a rodent (preferably murine, further preferably mouse) TCRβ chain constant region, which includes a mutation of the amino acid at position 56, such as serine S, to cysteine ​​C, and a substitution of lysine at position 150, 168 or 170 by arginine relative to the wild-type rodent (preferably murine, further preferably mouse) TCRβ chain constant region.

[0112] The modified TCRβ chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCRβ chain constant region, which comprises, relative to the wild-type rodent (preferably a mouse, further preferably a mouse) TCRβ chain constant region, an amino acid at position 3, such as R, replaced by K, an amino acid at position 6, such as T, replaced by F, K at position 9 replaced by E, S at position 11 replaced by A, L at position 12 replaced by V, and amino acids at positions 17, 21-25 deleted, an amino acid at position 56, such as serine S, mutated to cysteine ​​C, and lysine at position 150, 168 or 170 replaced by arginine.

[0113] The modified TCRβ chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCRβ chain constant region, which comprises, relative to the wild-type rodent (preferably a mouse, further preferably a mouse) TCRβ chain constant region, an amino acid at position 3, such as R, replaced by K, an amino acid at position 6, such as T, replaced by F, K at position 9 replaced by E, S at position 11 replaced by A, L at position 12 replaced by V, and amino acids at positions 17, 21-25 are deleted, and lysine at position 150, 168 or 170 is replaced by arginine.

[0114] Preferably, the modified TCRβ chain constant region is derived from a rodent (preferably a rat, further preferably a mouse) TCRβ chain constant region, which comprises an N-terminal modification and cysteine ​​introduction relative to a wild-type rodent (preferably a rat, further preferably a mouse) TCRβ chain constant region.

[0115] The modified TCRβ chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCRβ chain constant region, which comprises, relative to the wild-type rodent (preferably a mouse, further preferably a mouse) TCRβ chain constant region, an amino acid at position 3, such as R, being replaced by K, an amino acid at position 6, such as T, being replaced by F, K at position 9 being replaced by E, S at position 11 being replaced by A, L at position 12 being replaced by V, and amino acids at positions 17, 21-25 being deleted, and an amino acid at position 56, such as serine S, being mutated to cysteine ​​C.

[0116] In a specific embodiment of the present invention, the rodent (preferably mouse, further preferably mouse) TCR β chain constant region comprising N-terminal modification and cysteine ​​introduction comprises the amino acid sequence shown in SEQ ID NO: 32.

[0117] Preferably, the modified TCRβ chain constant region is derived from a rodent (preferably a rat, further preferably a mouse) TCRβ chain constant region, which comprises an intracellular deletion, an N-terminal modification and a cysteine ​​introduction relative to a wild-type rodent (preferably a rat, further preferably a mouse) TCRβ chain constant region.

[0118] Further preferably, the modified TCRβ chain constant region is derived from a rodent (preferably a mouse, further preferably a mouse) TCRβ chain constant region, which comprises an intracellular deletion relative to the wild-type rodent (preferably a mouse, further preferably a mouse) TCRβ chain constant region, and the amino acid at position 56, such as serine S, is mutated to cysteine ​​C, and the amino acid at position 3, such as R, is replaced by K, the amino acid at position 6, such as T, is replaced by F, K at position 9 is replaced by E, S at position 11 is replaced by A, L at position 12 is replaced by V, and amino acids at positions 17, 21-25 are deleted.

[0119] In a specific embodiment of the present invention, the rodent (preferably mouse, further preferably mouse) TCRβ chain constant region comprising intracellular deletion, N-terminal modification and cysteine ​​introduction comprises the amino acid sequence shown in SEQ ID NO:44.

[0120] In a specific embodiment of the present invention, the second constant region comprises the amino acid sequence shown in one of SEQ ID NOs: 2, 4, 6, 9, 17, 32 or 44.

[0121] The target binding region is located at the N-terminus of the constant region. The two can be directly connected or connected through a linker.

[0122] The first target binding region may comprise one or more identical or different binding regions, wherein the binding region is an antigen binding region or a fragment thereof, a non-immunoglobulin antigen binding domain or a fragment thereof, an antibody binding region or a fragment thereof, a receptor or a fragment thereof, or a ligand or a fragment thereof, wherein the receptor is preferably a natural T cell receptor.

[0123] The antigen binding region is derived from an antibody.

[0124] The STAR comprises one or more antigen binding regions;

[0125] Preferably, the multiple antigen binding regions are the same or different;

[0126] Further preferably, the multiple antigen binding regions are directly connected or connected through a linker.

[0127] The antibody can be a monoclonal antibody or a polyclonal antibody.

[0128] The antibody may also include F ab 、F ab '、F ab '-SH, Fv, scFv, (F ab ')2. Fragments of single domain antibodies, diabodies (dAbs) or linear antibodies.

[0129] The antibody can be a monospecific antibody or a multispecific antibody (eg, a bispecific antibody).

[0130] Preferably, the antibody can be a fully human antibody, a humanized antibody, or an antibody of animal origin, wherein the animal can be a mouse, rabbit, cow, monkey, etc.

[0131] Preferably, the first target binding region is directly linked to the first constant region or linked via a linker, and / or the second target binding region is directly linked to the second constant region or linked via a linker.

[0132] Preferably, the first target binding region and the second target binding region each independently or in combination specifically bind to the target antigen.

[0133] Preferably, the target antigen is a disease-associated antigen, preferably a cancer-associated antigen, for example, a cancer-associated antigen selected from the following: LILRB4, GPC3, CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123, CD138, ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, β-human chorionic gonadotropin, alpha fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate enzyme-specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, Prostein, PSMA, survival and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF1)-I, IGF-II, IGF1 receptor, mesothelin, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigen, extra domain A (EDA) and extra domain B (EDB) of fibronectin, A1 domain of tenascin-C (TnC A1), fibroblast-associated protein (fap), CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, Foxp3, B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, endoglin, major histocompatibility complex (MHC) molecules, BCMA (CD269, TNFRSF17), TNFRSF17 (UNIPROT Q02223), SLAMF7 (UNIPROT Q9NQ25), GPRC5D (UNIPROT Q9NZD1), FKBP11 (UNIPROT Q9NYL4), KAMP3, ITGA8 (UNIPROT P53708), or FCRL5 (UNIPROT Q68SN8).

[0134] Preferably, the first target binding region comprises one or more antibodies or antibody fragments that specifically bind to the target antigen, and the second target binding region comprises one or more antibodies or antibody fragments that specifically bind to the target antigen.

[0135] Wherein, multiple antibodies or antibody fragments are directly connected or connected through a linker.

[0136] Preferably, the first target 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 target binding region comprises one or more single chain antibodies or one or more single domain antibodies that specifically bind to the target antigen.

[0137] Multiple single-chain antibodies are directly connected or connected via a linker. Multiple single-domain antibodies are directly connected or connected via a linker.

[0138] Preferably, the single-chain antibody comprises a heavy chain variable region and a light chain variable region directly connected or connected through a linker.

[0139] Preferably, the multiple antigen binding regions in the first target binding region and / or the second target binding region bind to the same or different target antigens.

[0140] Preferably, the multiple antigen binding regions in the first target binding region and / or the second target binding region bind to different regions, such as different epitopes, of the same target antigen.

[0141] In a specific embodiment of the present invention, the target antigen is LILRB4.

[0142] In one embodiment of the present invention, the antigen binding region in the first target binding region comprises one or more single domain antibodies, and / or the antigen binding region in the second target binding region comprises one or more single domain antibodies;

[0143] Preferably, the antigen binding regions in the first target binding region comprise multiple single domain antibodies that are the same or different;

[0144] Preferably, the antigen binding regions in the second target binding region comprise multiple single domain antibodies that are the same or different;

[0145] Further preferably, the multiple single-domain antibodies are directly connected or connected through a linker.

[0146] Preferably, the single domain antibody contained in the antigen binding region of the first target binding region is the same as or different from the single domain antibody contained in the antigen binding region of the second target binding region.

[0147] In one embodiment of the present invention, the single-domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises CDR1-3, wherein

[0148] i) CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 33, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 34, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 35;

[0149] or,

[0150] ii) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 36, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 37, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 38.

[0151] In a specific embodiment of the present invention, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO: 28 or 29.

[0152] In a second aspect of the present invention, a STAR complex is provided, wherein:

[0153] i) the STAR complex comprises an α chain, a β chain, CD3ε, CD3γ, CD3δ and CD3ζ, wherein the α chain comprises a first target binding region and a first constant region, the β chain comprises a second target binding region and a second constant region, or the α chain comprises a first target binding region and the β chain comprises a second target binding region and a second constant region; or

[0154] ii) the STAR complex comprises a γ chain, a δ chain, CD3ε, CD3γ, CD3δ and CD3ζ, wherein the γ chain comprises a first target binding region and a first constant region, the δ chain comprises a second target binding region and a second constant region, or the γ chain comprises a first target binding region and a first constant region and the δ chain comprises a second target binding region and a second constant region.

[0155] Preferably, i) at least one of the α chain, β chain, CD3ε, CD3γ, CD3δ and CD3ζ has at least one functional domain connected to its C-terminus; or, ii) at least one of the γ chain, δ chain, CD3ε, CD3γ, CD3δ and CD3ζ has at least one functional domain connected to its C-terminus.

[0156] Preferably, i) the at least one functional domain is directly or via a linker connected to the C-terminus of at least one of the α chain, β chain, CD3ε, CD3γ, CD3δ and CD3ζ; or, ii) the at least one functional domain is directly or via a linker connected to the C-terminus of at least one of the γ chain, δ chain, CD3ε, CD3γ, CD3δ and CD3ζ.

[0157] Preferably, i) the intracellular region of at least one of the α chain, β chain, CD3ε, CD3γ, CD3δ and CD3ζ in the STAR complex is deleted; or, ii) the intracellular region of at least one of the γ chain, δ chain, CD3ε, CD3γ, CD3δ and CD3ζ in the STAR complex is deleted.

[0158] Further preferably, i) the at least one functional domain is directly or via a linker connected to the C-terminus of at least one of the α chain, β chain, CD3ε, CD3γ, CD3δ and CD3ζ whose intracellular region is deleted; or, ii) the at least one functional domain is directly or via a linker connected to the C-terminus of at least one of the γ chain, δ chain, CD3ε, CD3γ, CD3δ and CD3ζ whose intracellular region is deleted.

[0159] In a specific embodiment of the present invention, i) the C-terminus of at least one of the α chain, β chain, CD3ε, CD3γ, CD3δ and CD3ζ in the STAR complex is connected to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains; or, ii) the C-terminus of at least one of the γ chain, δ chain, CD3ε, CD3γ, CD3δ and CD3ζ in the STAR complex is connected to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains.

[0160] Preferably, the multiple functional domains can be connected directly or through a linker.

[0161] Preferably, i) the functional domains connected to the C-terminus of at least one of the α chain, β chain, CD3ε, CD3γ, CD3δ and CD3ζ in the STAR complex are the same or different; or, ii) the functional domains connected to the C-terminus of at least one of the γ chain, δ chain, CD3ε, CD3γ, CD3δ and CD3ζ in the STAR complex are the same or different.

[0162] In a specific embodiment of the present invention, the multiple functional domains connected by the α chain may be the same or different. The multiple functional domains connected by the β chain may be the same or different. The multiple functional domains connected by the γ chain may be the same or different. The multiple functional domains connected by the δ chain may be the same or different. The multiple functional domains connected by the CD3ε may be the same or different. The multiple functional domains connected by the CD3γ may be the same or different. The multiple functional domains connected by the CD3δ may be the same or different. The multiple functional domains connected by the CD3ζ may be the same or different.

[0163] In a specific embodiment of the present invention, the functional domains connected to the α chain, β chain, CD3ε, CD3γ, CD3δ and CD3ζ respectively may be the same or different.

[0164] In a specific embodiment of the present invention, the functional domains to which the γ chain, δ chain, CD3ε, CD3γ, CD3δ and CD3ζ are respectively connected may be the same or different.

[0165] Preferably, the functional domain is a co-stimulatory molecule or a fragment thereof, a co-inhibitory molecule or a fragment thereof, a cytokine receptor or a fragment thereof, or an intracellular protein or a fragment thereof. Further preferably, the functional domain is an intracellular domain of a co-stimulatory molecule, an intracellular domain of a co-inhibitory molecule, an intracellular domain of a cytokine receptor, or an intracellular protein;

[0166] Preferably, the costimulatory molecule is selected from CD40, OX40, ICOS, CD28, 4-1BB (CD137) or CD27;

[0167] Preferably, the co-inhibitory molecule is selected from TIM3, PD1, CTLA4, and LAG3;

[0168] Preferably, the cytokine receptor is selected from interleukin receptors (such as IL-2 receptor), interferon receptors, tumor necrosis factor superfamily receptors, colony stimulating factor receptors, chemokine receptors, growth factor receptors or other membrane proteins;

[0169] Preferably, the intracellular protein is a T cell regulatory factor, such as the domain of NIK.

[0170] In a specific embodiment of the present invention, the costimulatory molecule is CD40, and its intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 10.

[0171] In a specific embodiment of the present invention, the costimulatory molecule is OX40, and its intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 11.

[0172] In a specific embodiment of the present invention, the costimulatory molecule is ICOS, and its intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 12.

[0173] In a specific embodiment of the present invention, the costimulatory molecule is CD28, and its intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 13.

[0174] In a specific embodiment of the present invention, the costimulatory molecule is 4-1BB, whose intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 14.

[0175] In a specific embodiment of the present invention, the costimulatory molecule is CD27, and its intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 15.

[0176] In a specific embodiment of the present invention, the STAR complex comprises the above-mentioned STAR, as well as CD3ε, CD3γ, CD3δ and CD3ζ.

[0177] Preferably, the CD3ε, CD3γ, CD3δ and / or CD3ζ are of human origin.

[0178] In a specific embodiment of the present invention, the CD3ε comprises the amino acid sequence shown in SEQ ID NO: 20.

[0179] In a specific embodiment of the present invention, the CD3γ comprises the amino acid sequence shown in SEQ ID NO:18.

[0180] In a specific embodiment of the present invention, the CD3δ comprises the amino acid sequence shown in SEQ ID NO:19.

[0181] In a specific embodiment of the present invention, the CD3ζ comprises the amino acid sequence shown in SEQ ID NO: 21.

[0182] In a third aspect, the present invention provides an antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and / or a light chain variable region.

[0183] The heavy chain variable region comprises CDR1-3, wherein,

[0184] i) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 33, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 34, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 35.

[0185] or,

[0186] ii) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 36, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 37, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 38.

[0187] The antibody may also include F ab 、Fab '、F ab '-SH, Fv, scFv, (F ab ')2. Fragments of single domain antibodies, diabodies (dAbs) or linear antibodies.

[0188] The antibody can be a monospecific antibody or a multispecific antibody (eg, a bispecific antibody).

[0189] Preferably, the antibody can be a fully human antibody, a humanized antibody, or an antibody of animal origin, wherein the animal can be a mouse, rabbit, cow, monkey, etc.

[0190] In a specific embodiment of the present invention, the antibody or antigen-binding fragment is a single-chain antibody or a single-domain antibody.

[0191] In a specific embodiment of the present invention, the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 28 or 29.

[0192] In a fourth aspect, the present invention provides a single-chain antibody comprising a heavy chain variable region and / or a light chain variable region.

[0193] The heavy chain variable region comprises CDR1-3, wherein,

[0194] i) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 33, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 34, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 35.

[0195] or,

[0196] ii) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 36, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 37, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 38.

[0197] In a specific embodiment of the present invention, the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 28 or 29.

[0198] In a fifth aspect, the present invention provides a single-domain antibody, wherein the single-domain antibody comprises a heavy chain variable region, and the heavy chain variable region comprises CDR1-3.

[0199] wherein i) CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 33, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 34, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 35. Alternatively, ii) CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 36, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 37, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 38.

[0200] In a specific embodiment of the present invention, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO: 28 or 29.

[0201] In a sixth aspect of the present invention, a method for preparing the above-mentioned antibody or antigen-binding fragment or the above-mentioned single-domain antibody is provided, wherein the preparation method comprises: preparing a phage display library, and screening the antibody or antigen-binding fragment or single-domain antibody from the phage display library.

[0202] In a seventh aspect, the present invention provides an antigen receptor comprising a transmembrane region, an intracellular region, and one or more identical or different extracellular binding domains.

[0203] The antigen receptor is TCR or CAR.

[0204] In a specific embodiment of the present invention, the antigen receptor is CAR.

[0205] The extracellular binding domain is an extracellular antigen binding domain, an extracellular antibody binding domain, a receptor, or a ligand, and the receptor is preferably a natural T cell receptor.

[0206] In a specific embodiment of the present invention, the extracellular binding domain is an extracellular antigen binding domain.

[0207] The extracellular antigen binding domain is derived from an antibody.

[0208] Preferably, the transmembrane region is directly connected to one or more extracellular antigen binding domains or is connected via a linker.

[0209] Preferably, the antigen is selected from cancer-associated antigens, for example, selected from the following cancer-associated antigens: LILRB4, GPC3, CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123, CD138, ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glycosphingolipids, glioma-associated antigens, β-human chorionic gonadotropin, alpha fetoprotein (AFP), exogenous lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate enzyme-specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, Prostein, PSMA, survival and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF1)-I, IGF-II, IGF1 receptor, mesothelin, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigen, extra domain A (EDA) and extra domain B (EDB) of fibronectin, A1 domain of tenascin-C (TnC A1), fibroblast-associated protein (fap), CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, Foxp3, B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, endoglin, major histocompatibility complex (MHC) molecules, BCMA (CD269, TNFRSF17), TNFRSF17 (UNIPROT Q02223), SLAMF7 (UNIPROT Q9NQ25), GPRC5D (UNIPROT Q9NZD1), FKBP11 (UNIPROT Q9NYL4), KAMP3, ITGA8 (UNIPROT P53708), or FCRL5 (UNIPROT Q68SN8);

[0210] Preferably, the antigen is LILRB4.

[0211] Preferably, the extracellular antigen-binding domain comprises CDR1-3, wherein i) CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 33, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 34, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 35. Alternatively, ii) CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 36, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 37, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 38.

[0212] In a specific embodiment of the present invention, the extracellular antigen-binding domain comprises the above-mentioned antibody or antigen-binding fragment, the above-mentioned single-chain antibody, or the above-mentioned single-domain antibody.

[0213] Preferably, the transmembrane region is derived from human CD8.

[0214] Preferably, the intracellular region is derived from 4-1BB, CD28 or CD3ζ.

[0215] In an eighth aspect, the present invention provides a nucleic acid encoding the aforementioned STAR, the aforementioned STAR complex, the aforementioned antibody or antigen-binding fragment, the aforementioned single-chain antibody, the aforementioned single-domain antibody, or the aforementioned antigen receptor.

[0216] The ninth aspect of the present invention provides a vector comprising the above-mentioned nucleic acid.

[0217] The vector can be expressed in vivo, in vitro or in vitro, and is 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.

[0218] Preferably, the prokaryotic expression vector is an Escherichia coli series, such as pET-26b or pET28a+.

[0219] Preferably, the expression vector may be Rous sarcoma virus (RSV), lentivirus, human immunodeficiency virus (HIV), murine leukemia virus (MLV), equine infectious anemia virus (EIAV), mouse mammary cancer virus (MMTV), Fujinami sarcoma virus (FuSV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine leukemia virus (Mo-MLV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myeloproliferative virus 29 (MC29), or avian erythroblastosis virus (AEV). More preferably, the expression vector is a lentiviral expression vector, such as pHAGE-IRES-RFP.

[0220] The tenth aspect of the present invention provides a host cell, wherein the host cell comprises the above-mentioned nucleic acid or the above-mentioned vector.

[0221] Preferably, the host cell can be eukaryotic or prokaryotic. More preferably, the host cell is yeast cell, 293 cell, CHO cell, Escherichia coli, etc.

[0222] In an eleventh aspect of the present invention, an immune cell is provided, wherein the immune cell expresses the aforementioned STAR, the aforementioned STAR complex, the aforementioned antibody or antigen-binding fragment, the aforementioned single domain antibody, and the aforementioned antigen receptor.

[0223] Preferably, the immune cells contain one or more of the above-mentioned nucleic acids.

[0224] Preferably, the immune cells are selected from T cells, Treg cells, macrophages, NK cells, NKT cells, peripheral blood monocytes, TIL cells or dendritic cells (DC).

[0225] Preferably, the immune cells are isolated and derived from T cells of the subject.

[0226] In a specific embodiment of the present invention, the immune cells are selected from T cells, NK cells, CTLs, human embryonic stem cells, lymphocyte progenitor cells and / or T cell precursor cells.

[0227] The twelfth aspect of the present invention provides a CAR-T cell, wherein the CAR-T cell comprises the above-mentioned antibody or antigen fragment, the above-mentioned single-chain antibody or the above-mentioned single-domain antibody.

[0228] The thirteenth aspect of the present invention provides a method for preparing immune cells, which comprises transfecting the above-mentioned nucleic acid sequence into immune cells for expression.

[0229] A fourteenth aspect of the present invention provides a method for preparing recombinant T cells, comprising the following steps:

[0230] 1) Obtaining the aforementioned nucleic acid from a positive T cell clone;

[0231] 2) Isolation and culture of primary T cells;

[0232] 3) delivering the nucleic acid obtained in step 1) into the primary T cells described in step 2) to obtain recombinant T cells expressing the STAR.

[0233] A fifteenth aspect of the present invention provides a method for preparing a STAR or a STAR complex, comprising the following steps:

[0234] (1) obtaining the aforementioned nucleic acid from a positive T cell clone;

[0235] (2) connecting the nucleic acid obtained in step (1) to a vector backbone to obtain an expression vector;

[0236] (3) transforming the expression vector obtained in step (2) into a host cell and then inducing its expression;

[0237] (4) Obtain STAR.

[0238] In a sixteenth aspect, the present invention provides a method for preparing an antibody or antigen-binding fragment, a single-chain antibody or a single-domain antibody, wherein the method comprises protein immunization and / or DNA immunization.

[0239] A seventeenth aspect of the present invention provides a method for preparing an antibody or antigen-binding fragment, a single-chain antibody or a single-domain antibody, the method comprising:

[0240] A) obtaining the encoding nucleic acid sequence;

[0241] B) Transforming the nucleic acid sequence obtained in step A) into host cells, then inducing its expression and purifying it.

[0242] The eighteenth aspect of the present invention provides the use of the above-mentioned STAR, the above-mentioned STAR complex, the above-mentioned antibody or antigen-binding fragment, the above-mentioned single-domain antibody, the above-mentioned antigen receptor, the above-mentioned nucleic acid, and the above-mentioned immune cell in the preparation of products for diagnosing or treating tumors.

[0243] Preferably, the tumor 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, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma.

[0244] Preferably, the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myeloid leukemia;

[0245] Preferably, 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;

[0246] Preferably, the sarcoma is selected from osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma.

[0247] Preferably, the acute myeloid leukemia is M4 or M5 acute myeloid leukemia;

[0248] Preferably, the chronic myeloid leukemia is chronic myelomonocytic leukemia.

[0249] In a nineteenth aspect, the present invention provides an immunoconjugate or antibody-drug conjugate comprising the above-mentioned antibody or antigen-binding fragment, the above-mentioned single-chain antibody or the above-mentioned single-domain antibody of the present invention conjugated to a therapeutic agent or a diagnostic agent.

[0250] The twentieth aspect of the present invention provides a pharmaceutical composition, which comprises the above-mentioned STAR, the above-mentioned STAR complex, the above-mentioned antibody or antigen-binding fragment, the above-mentioned single-domain antibody, the above-mentioned antigen receptor, the above-mentioned nucleic acid, and the above-mentioned immune cell.

[0251] Preferably, the drug further comprises a pharmaceutically acceptable excipient. Further preferably, the pharmaceutically acceptable excipient includes but is not limited to a diluent, a binder, a wetting agent, a surfactant, a lubricant or a disintegrant, etc.

[0252] In the twenty-first aspect of the present invention, a kit is provided, which comprises the above-mentioned STAR, the above-mentioned STAR complex, the above-mentioned antibody or antigen-binding fragment, the above-mentioned single-domain antibody, the above-mentioned antigen receptor, the above-mentioned nucleic acid, and the above-mentioned immune cell.

[0253] The twenty-second aspect of the present invention provides a method for treating tumors, which comprises applying to a subject an effective amount of the STAR, STAR complex, CAR, antibody or antigen-binding fragment thereof, single-chain antibody, single-domain antibody, immune cell, CAR-T cell or pharmaceutical composition described in the present invention.

[0254] The "linker" of the present invention includes, but is not limited to, a rigid linker, a flexible linker, a cleavable linker, or a nonsense amino acid. Preferably, the amino acid sequence of the rigid linker is selected from one or more of SEQ ID NOs: 49-59. Preferably, the flexible linker is selected from a peptide rich in glycine and / or serine; preferably, the flexible linker is selected from one or more of SEQ ID NOs: 60-112. Preferably, the cleavable linker is selected from one or more of SEQ ID NOs: 113-117.

[0255] The "antibody" of the present invention may be of any class (such as IgA, IgD, IgE, IgG and IgM) or subclass (such as IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2).

[0256] 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 having one or more cysteine ​​residues at the C-terminus of the CH1 domain; an Fd fragment having VH and CH1 domains; an Fd' fragment having VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain; an Fv fragment having the VL and VH domains of a single arm of an antibody; a dAb fragment consisting of either a VH domain or a VL domain; isolated CDR regions; an F(ab ')2 fragment, which is a bivalent fragment comprising two Fab' fragments connected by a disulfide bridge at the hinge region; a single-chain antibody molecule (e.g., single-chain Fv; scFv); a "diabody" with two antigen-binding sites, which comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain; a "linear antibody" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) that, together with complementary light chain polypeptides, form a pair of antigen-binding regions; and modified forms of any of the foregoing that retain antigen-binding activity.

[0257] The "CDR" of the present invention refers to the complementary determining region within the variable sequence of an antibody. For each variable region, there are three CDRs in each variable region of the heavy chain and light chain, which are called CDR1, CDR2 and CDR3. The exact boundaries of these CDRs are defined differently according to 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)) not only provides a clear residue numbering system applicable to antibody variable regions, but also provides residue boundaries that define the three CDRs. These CDRs can be referred to as Kabat CDRs. Each complementary determining region can include 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 Kabat In some sub-portions of the CDR, almost identical peptide backbone conformations are used, although there is great diversity at the amino acid sequence level. These sub-portions are respectively referred to as L1, L2 and L3 or H1, H2 and H3, wherein "L" and "H" represent the light chain and heavy chain regions, respectively. These regions can be referred to as Chothia CDRs, which have borders that overlap with Kabat CDRs. Other CDR boundary definitions may not strictly follow one of the above-mentioned systems, but will still overlap with Kabat CDRs, and the methods used herein can utilize CDRs defined according to any of these systems, although preferred embodiments use CDRs defined by Kabat or Chothia. The "antibody variable region" refers to the portion of the amino acid sequence that includes the complementary determining regions (CDRs, i.e., CDR1, CDR2 and CDR3) and framework regions (FRs) in the light and heavy chains of an antibody molecule. VH refers to the variable domains of the heavy chain. VL refers to the variable domains of the light chain.

[0258] The term "diagnosis" as used herein refers to determining whether a patient has had a disease or condition in the past, at the time of diagnosis, or in the future, or to determining the progression or possible future progression of a disease, or to evaluating a patient's response to treatment.

[0259] "Treatment" as used herein means to slow down, interrupt, prevent, control, stop, alleviate, or reverse the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders, and refers to therapeutic intervention that improves the signs, symptoms, etc. of a disease or pathological state after the disease has begun to develop.

[0260] The "effective amount" described in the present invention refers to the amount or dose of the STAR, STAR complex, CAR, CAR-T, STAR-T, immune cell, pharmaceutical composition, etc. described in the present invention that provides the desired treatment or prevention after administration to a patient or organ in a single or multiple doses.

[0261] The "product" described in the present invention may be a kit, a chip, an antibody conjugate, a multifunctional antibody, a pharmaceutical composition, and the like.

[0262] The "individual" or "subject" described in the present invention can be a human or a non-human animal. The non-human animal can be a non-human mammal such as a mouse, cow, sheep, rabbit, pig, monkey, etc.

[0263] The term "and / or" as used herein includes all combinations of the items connected by the term, and each combination should be deemed to have been listed separately herein. For example, "A and / or B" includes "A," "A and B," and "B." For another example, "A, B, and / or C" includes "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0264] The terms “comprising” or “including” described in the present invention are open-ended. When used to describe a protein or nucleic acid sequence, the protein or nucleic acid may be composed 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 in the present invention. In addition, it is clear to those skilled in the art that the methionine encoded by the start codon at the N-terminus of the polypeptide may be retained in certain practical situations (for example, when expressed in a specific expression system), but it does not substantially affect the function of the polypeptide. Therefore, when describing a specific polypeptide amino acid sequence in the specification and claims of this application, although it may not contain a methionine encoded by a start codon at the N-terminus, a sequence containing the methionine is also covered, and accordingly, its encoding nucleotide sequence may also contain a start codon; and vice versa. BRIEF DESCRIPTION OF THE DRAWINGS

[0265] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0266] Figure 1A-1B: Detection results of LILRB4 expression levels in different AML cell lines;

[0267] Figure 2: Schematic diagram of the structure of STAR targeting LILRB4;

[0268] Figure 3: Killing effects of different antibodies on target cells;

[0269] Figure 4A-4B: Affinity results of NLB4 / NLB14 nanobody detected by SPR;

[0270] Figure 5A-Figure 5B: Competition binding results of NLB4 / NLB14 nanobody detected by SPR;

[0271] Figure 6: Schematic diagram of the structure of LILRB4 protein;

[0272] Figure 7: Epitope recognition of LILRB4 by NLB4 / NLB14 nanobody;

[0273] Figure 8: Flow cytometry analysis of the non-specific binding of NLB4 / NLB14 nanobodies to various LILRB4 family proteins.

[0274] Figures 9A-9B: Flow cytometry analysis of the non-specific binding of NLB4 / NLB14 nanobodies to different human tissues;

[0275] Figure 10: Flow cytometry analysis of the binding of NLB4 / NLB14 nanobodies to human LILRB4 and mouse LILRB4;

[0276] Figure 11: Schematic diagram of the structures of single-epitope and dual-epitope STARs targeting LILRB4;

[0277] FIG12 is a flow cytometry analysis showing the expression of single-epitope and dual-epitope LILRB4 STAR on the upper membrane;

[0278] Figure 13: Recognition and killing effect of LILRB4 STAR-T cells on target cell line THP1;

[0279] Figure 14A-14B: Killing effect of LILRB4 STAR-T cells on target cells;

[0280] Figure 15: Cytokine secretion levels of LILRB4 STAR-T;

[0281] Figure 16A-Figure 16D: Killing effect of LILRB4 STAR-T on mouse tumor model;

[0282] Figure 17: HE staining results of different tissues of mice after LILRB4 STAR-T transfusion;

[0283] Figure 18: In vitro tumorigenicity assay results of LILRB4 STAR-T cells;

[0284] 19A-19C : Relationship between NLB4 / NLB14 and the LILRB4 ligand ApoE. DETAILED DESCRIPTION

[0285] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0286] Experimental Materials and Methods

[0287] Vector construction

[0288] The lentiviral vectors and lentiviral packaging plasmids used in the examples of the present application were purchased from commercial companies or synthesized by commercial companies. The gene fragments used in the examples of the present application, including signal peptides, antibody binding regions, hinge regions, TCR constant regions, tag proteins, etc., were synthesized by commercial companies. By means of synthetic primer PCR, one or more target fragments were connected to obtain the corresponding functional sequence. The lentiviral vector used in the present invention is pHAGE-hEF1α-RFP, and the pHAGE-hEF1A-WPRE-AMP vector was obtained by restriction endonuclease SpeI / SalI. The fragment gene was obtained by synthesis and PCR, and the complete vector was obtained under the action of the recombinase by homologous recombination.

[0289] The α chain constant region is of mouse origin, and the wild-type is named TRAC. The β chain constant region is of mouse origin, and the wild-type is named TRBC. The costimulatory molecule is OX40.

[0290] Lentiviral packaging

[0291] LentiX-293T cells were cultured at a rate of 5 × 10 5 Inoculate cells / mL into a 10cm culture dish and culture in a 37°C, 5% CO2 incubator. Transfection is performed when the cell density reaches approximately 80% (observed under a microscope). Mix the four plasmids with 500uL of serum-free DMEM in a ratio of PMD2.G:REV:PMDLG:transfer plamid = 1:1:2:4. Mix 54uL of PEI-max with 500uL of serum-free DMEM and let it stand at room temperature for 5 minutes (the volume-to-mass ratio of PEI-Max to plasmid is 3:1). Slowly add the PEI-max mixture to the plasmid mixture, gently pipette to mix, and let it stand at room temperature for 15 minutes. Slowly add the final mixture to the culture medium, mix thoroughly, return to the incubator, and continue culturing for 12-16 hours. Then switch to 6% FBS DMEM culture medium for further culturing, and collect the virus liquid after 48 hours and 72 hours.

[0292] T cell culture and infection

[0293] 1) Jurkat T cell line culture method

[0294] Jurkat T cell lines were cultured in RPMI 1640 medium containing 10% FBS. The culture density was 3*10 5 / ml, the maximum is no more than 3*10 6 / ml, and subculture every 1-2 days. After cell counting, take the required amount of cells, add culture medium to adjust to the above density, and culture in a CO2 incubator.

[0295] 2) Jurkat T cell line infection method

[0296] Cell counting, take 1*10 6 1 ml of cells were centrifuged and resuspended in 1 ml of RPMI 1640 medium containing 10% FBS. The cells were plated in a 24-well plate and the virus solution (MOI = 1) was added. The cells were centrifuged at 1500 rpm for 90 minutes and then incubated in a CO2 incubator. 12 hours after infection, the medium was completely replaced with fresh RPMI 1640 medium containing 10% FBS. The positive rate was tested after 72 hours.

[0297] 3) Culture methods of primary human T cells

[0298] After obtaining primary T cells by Ficoll separation, they were cultured in X-VIVO medium containing 10% FBS and 100 IU / ml IL-2, with an initial culture density of 1*10 6 / ml, and added to the CD3 and RetroNectin (final concentration of 5μg / ml) pre-coated well plates. The later culture density was 5*10 5 / ml, the maximum is no more than 3*10 6 / ml, and passage was performed every 1-2 days.

[0299] 4) Infection method of primary human T cells

[0300] After 48 hours of culture, primary T cells were incubated with virus at an MOI of 20, centrifuged at 1500 rpm for 90 minutes, and incubated in a CO2 incubator. 24 hours after infection, the cells were supplemented with X-VIVO medium containing 10% FBS and 100 IU / ml IL-2 and transfected. After 72 hours, infection efficiency was detected using labeled proteins or antibodies.

[0301] 5) Infection efficiency detection method

[0302] After 72 hours of infection, the cells were evenly blown and counted, and 5*10 5 / ml centrifugation, discard the supernatant, staining solution is PBS + 2% FBS + 2mM EDTA, add the corresponding antibody for incubation, incubate for 30 minutes, then add PBS to wash twice, and detect on the machine.

[0303] In vitro functional assay of STAR-T cells

[0304] Positive T cells were co-cultured with target cells expressing Luciferase (Raji cells, CD19-KO Raji cells or CD22-KO Raji cells) at the specified effector-target ratio.

[0305] After 24 hours of co-culture, gently pipette the cell suspension to homogenize. Transfer 150 μL of the cell suspension to each well of a white 96-well plate. Samples are plated in duplicate and luciferase substrate is added. Shake (low speed) and incubate for 10 minutes. Chemiluminescence is then measured using a multi-function microplate reader. Calculate cell killing efficiency as follows: killing efficiency = 100% - (value of effector cells minus value of target cells) / (value of control cells minus value of target cells). Alternatively, after 24 hours of co-culture, collect the cell culture supernatant and assay for cytokine levels using commercially available kits.

[0306] In vivo functional assay of STAR-T cells

[0307] The model was constructed using NSG immunodeficient mice. These mice, with the genotype NOD-Prkdcem26Il2rgem26 / Nju, are deficient in T cells, B cells, and NK cells, and also have defects in macrophages and dendritic cells. Female NSG mice aged 6 to 8 weeks were used in this experiment, with the weight variation within each batch controlled to within 2 g. Mice were housed in individually ventilated cages in a specific pathogen-free (SPF) environment and provided with a normal diet and drinking water with a slightly acidic pH to prevent pathogen contamination.

[0308] A tumor model was constructed by xenografting Raji cells, a human Burkitt's lymphoma cell line. Raji cells are a cell line expressing the luciferase gene via a lentiviral vector. The development and changes of Raji tumors were monitored in real time in mice using luciferin chemiluminescence and in vivo imaging. In this model, 1 to 3 x 10 6 Raji-luciferase cells were inoculated into 6-8 week old female NSG mice via tail vein infusion. Luciferin potassium salt solution was injected into the mice intraperitoneally, and the fluorescence signal of the tumor cells in vivo was detected by in vivo imaging.

[0309] Example 1 Construction of target cells

[0310] To screen nanobodies targeting LILRB4 and test the killing effect of STAR-T on AML tumor cells, the inventors detected LILRB4 antigen expression in various AML cells by flow cytometry. The results, as shown in Figures 1A and 1B, showed high LILRB4 antigen expression in THP1, MV4-11, and OCI-AML3, while low LILRB4 antigen expression in mitochondrial KASUMI-1 cells.

[0311] Example 2 Screening of Nanobodies Targeting LILRB4

[0312] 1. Immunization of alpacas with human LILRB4 protein

[0313] Healthy alpacas were immunized with the commercially available extracellular domain of human LILRB4 protein (100 μg) using complete Freund's adjuvant (CFA, Sigma) and incomplete Freund's adjuvant (IFA, Sigma). The expressed and purified extracellular domain of human LILRB4 protein was diluted with PBS and then mixed with the corresponding adjuvant in a 1:1 ratio. The antigen and adjuvant were thoroughly mixed to form a stable emulsion. The antigen mixture was then drawn with a syringe and injected subcutaneously at multiple points under the skin of the alpaca's neck, with 100-200 μL injected at each site. The specific animal immunization process is as follows:

[0314] 1) Day 1 (first immunization): Alpacas were immunized subcutaneously with LILRB4 antigen (100 μg) mixed with complete Freund's adjuvant (CFA);

[0315] 2) Day 14 (second immunization): Alpacas were immunized subcutaneously with LILRB4 antigen (100 μg) mixed with incomplete Freund's adjuvant (IFA);

[0316] 3) Day 28 (third immunization): LILRB4 antigen (100 μg) mixed with incomplete Freund's adjuvant (IFA) was again injected subcutaneously to immunize the alpaca;

[0317] 4) Day 42 (first serum collection): Blood samples were collected from the alpaca's ear vein, serum was extracted, and antibody titer was tested. The P / N value of the 200,000-fold diluted serum was greater than 2;

[0318] 5) Day 42 (4th immunization): LILRB4 antigen (100 μg) mixed with incomplete Freund's adjuvant (IFA) was again injected subcutaneously to immunize the alpaca to enhance the immune effect;

[0319] 6) Day 53 (Second serum collection): Blood samples were collected from the alpaca's ear vein, serum was extracted, and antibody titer was tested. The P / N value of the 200,000-fold diluted serum was greater than 2;

[0320] 7) Days 54, 57, and 60: 30-40 mL of alpaca blood samples were collected from the alpaca's hind leg vein for PBMC isolation.

[0321] 2. PBMC Isolation

[0322] 1) Isolate PBMCs in a biosafety cabinet. Combine the blood from the anticoagulant tube into a 50 mL centrifuge tube (30 mL). Add PBS to a total volume of 50 mL and mix gently.

[0323] 2) In a new 50mL centrifuge tube, add Ficoll separation solution at 15mL / tube. Layer the blood sample on top of the Ficoll solution at 25mL / tube. Operate gently to separate the Ficoll and blood into separate layers to prevent mixing.

[0324] 3) Set the centrifuge speed to zero, RT, and centrifuge at 800 g for 30 minutes. After centrifugation, remove the sample from the centrifuge and separate into the following layers: upper aqueous phase - buffy coat - Ficoll layer - red blood cell layer. PBMCs are in the buffy coat layer. Aspirate the buffy coat layer and transfer it to a new 50 mL centrifuge tube.

[0325] 4) Add PBS to 50 mL into the sample tube, mix well, centrifuge at 2000 rpm, RT for 5 min, discard the supernatant, and resuspend the cell pellet in 5 mL of PBS.

[0326] 5) Repeat step 4 and cool down to 4°C.

[0327] 6) Resuspend the cell pellet in 5 mL of PBS, add more PBS to 40 mL, and count the cells.

[0328] 7) Centrifuge at 1500 rpm, 4°C for 5 min, discard the supernatant, add 1 mL of PBS, resuspend the cells, and blow evenly; add 20 mL of Trizol, mix well, let stand at room temperature for 5 min, lyse the cells, and aliquot 1 mL into RNase-free 1.5 mL EP tubes. Freeze at -80°C for RNA extraction.

[0329] 3. RNA Extraction and Reverse Transcription

[0330] 1) Remove the sample from -80°C, thaw at room temperature, add 200 μL of chloroform, and let it stand at room temperature for 3 minutes. Centrifuge the sample at 12,000 g for 15 minutes at 4°C to separate the sample into an upper aqueous phase, an intermediate phase, and an organic phase. Transfer the upper phase to a new RNase-free tube, add 1 μL of glycogen and 500 μL of isopropanol, and let it stand at 4°C overnight.

[0331] 2) Centrifuge the sample at 12,000 g for 20 minutes at 4°C. Aspirate the supernatant and wash the precipitate with 1 mL of pre-chilled 75% ethanol. Centrifuge again to remove the alcohol and air dry. Add 15 μL of RNase-free water to each tube to dissolve the RNA precipitate and proceed with reverse transcription.

[0332] 3) cDNA was synthesized using the Promega reverse transcription kit (20 μL system).

[0333] Step 1: Take a certain amount of template RNA and add Oligo(dT), see Table 1;

[0334] Table 1

[0335] Step 2: Denature the mixture of template RNA and Oligo (dT) at 65°C for 5 minutes and place it back on ice.

[0336] Step 3: During pre-denaturation, RT-Mix can be prepared in advance, 8 μL per tube. The components and volumes are shown in Table 2.

[0337] Table 2

[0338] Step 4: Set up the reverse transcription program, extension, and reverse transcriptase inactivation. Once the program is completed, cDNA is obtained.

[0339] 4. Phage Library Construction

[0340] 1) PCR to obtain VHH sequences

[0341] The VHH sequence was obtained by two rounds of PCR, and homology arms of the vector were added to both ends of the sequence.

[0342] 2) First round of PCR

[0343] Step 1: Prepare the reaction system as shown in Table 3.

[0344] Table 3

[0345] Step 2: PCR conditions are shown in Table 4.

[0346] Table 4

[0347] The PCR products were subjected to gel electrophoresis, and the target band at 0.7 kb was cut out for product recovery.

[0348] 3) Second round of PCR

[0349] Step 1: Prepare the reaction system as shown in Table 5.

[0350] Table 5

[0351] Step 2: PCR conditions are shown in Table 6.

[0352] Table 6

[0353] The PCR products were subjected to gel electrophoresis, and the target band at 400 bp was cut out for product recovery.

[0354] 4) Vector PCR

[0355] The vector region of phagemid was obtained by PCR and used to express the VHH sequence.

[0356] Step 1: Prepare the reaction system as shown in Table 7.

[0357] Table 7

[0358] Step 2: PCR conditions are shown in Table 8.

[0359] Table 8

[0360] The PCR products were subjected to gel electrophoresis, and the 4000 bp target band was cut out for product recovery.

[0361] 5) Ligation, purification and concentration of ligation products

[0362] The VHH fragment was ligated to the phagemid vector and the ligation product was subsequently concentrated.

[0363] Step 1: Prepare the reaction system as shown in Table 9.

[0364] Table 9

[0365] Step 2: Incubate the above mixture at 50°C for 2 h and cool on ice.

[0366] Step 3: Purify the ligation product, remove salt ions, proteins and other components in the ligation system, and concentrate the volume to 1 / 10 of the original volume.

[0367] 5. Electroporation and library construction

[0368] 1) Take a tube of competent E. coli and thaw it on ice.

[0369] 2) Take 2 μL of the ligation product or positive control and add it to the competent medium. Gently pipette to mix thoroughly. Let it stand on ice for 1-2 minutes. Transfer it to a pre-chilled electroporation cuvette and electroporate.

[0370] 3) Immediately after electroporation, add 1 mL of 37°C 2YT-G medium. Rinse the electroporation cuvette with a pipette tip. Transfer the electroporated culture to a 15 mL centrifuge tube or 2 mL EP tube. Resuscitate in a 37°C water bath until all cells are electroporated. (2YT-G: 2xYT medium containing 2% glucose.) Transfer to a 37°C shaker at 220 rpm and resuscitate for 1 hour.

[0371] 4) Aspirate 5 μL of the above bacterial solution and dilute it 10^2-10^5 times. Apply it to 2YT-A plates (2YT plates containing 100 μg / mL ampicillin) and incubate at 37°C overnight for colony counting.

[0372] 5) Inoculate the remaining bacterial suspension into 2YT-AG medium, shake until the culture reaches logarithmic phase, add helper phage for infection, and shake at 30°C, 220 rpm for 12-16 hours. (2YT-AG: 2xYT medium containing 2% glucose and 100 μg / mL ampicillin)

[0373] 6) Collect and concentrate the phage and determine the titer.

[0374] 6. Phage library antibody screening

[0375] The phage library obtained in step 7 above was subjected to three rounds of antibody screening, each including a positive selection and a negative selection. The phage were first incubated with the antigenic peptide, and those that did not bind were discarded, retaining those that bound to the antigenic peptide. The phage were then incubated with BSA for negative selection, retaining those that did not bind to BSA.

[0376] 1) Coat the plate. Dilute the antigen to a concentration of 2 ng / μL in PBS and add 100 μL / well to a 96-well plate. Prepare 2% BSA in PBS and add 100 μL / well to the corresponding negative selection wells. Seal with plastic wrap and incubate overnight at 4°C.

[0377] 2) Discard the coating solution, add 200 μL of washing solution (washing solution: 1% Tween 20 / PBS, pH 7.4), and wash three times.

[0378] 3) Blocking: Add 2% BSA blocking solution to all wells at 100 μL / well, seal with plastic wrap, and incubate at 37°C for 1 hour.

[0379] 4) Discard the supernatant, add 200 μL of washing solution, and wash three times.

[0380] 5) Add phage to the positive selection wells, 1×10 12 phages / well, dilute to 100 μL, seal with plastic wrap, and incubate at 37°C for 1 h.

[0381] 6) Discard the supernatant, add 200 μL of washing solution, and wash 10 times.

[0382] 7) Elution-Neutralization: Add 200 μL of elution buffer to the positively selected wells and neutralize to pH 7-7.4.

[0383] 8) Negative selection: Add the above eluate to the negative selection wells, seal with plastic wrap, incubate at 37°C for 1 hour, aspirate and retain the supernatant, and perform titer detection.

[0384] 9) Take a small amount of phage after one round of panning, dilute it, and spread it on 2×YT-A plates. Incubate at 37°C overnight. Count the colonies the next day and calculate the titer. Select single clones for sequencing and analyze sequence diversity and enrichment.

[0385] 10) All remaining phages are used for TG1 infection.

[0386] 11) Infection with M13KO7: Dilute the phage and add M13KO7 to the bacterial suspension. Incubate at 37°C in a water bath for 30 minutes. Replace with 2×YT-AK medium and shake at 30°C, 220 rpm, for 14-16 hours.

[0387] 12) Concentrate the phage and test the phage titer before proceeding to the next round of screening.

[0388] The amount of coated antigen in the second and third rounds of screening was reduced, and the number of washes was increased after incubation of phage with positive wells. The other steps were the same as above.

[0389] 7. Combined detection and sequence acquisition

[0390] The phages obtained from the three rounds of screening were co-infected with M13KO7 helper phage on TG1, coated on 2YT-AK plates, single clones were picked for phage expansion, and phages were collected for binding detection to determine the usable phage / antibody.

[0391] 1) Coat the plate. Dilute the antigen to 1 ng / μL with coating solution, 100 μL / well. Add 2% BSA to the negative control wells. Seal with plastic wrap and incubate at 4°C overnight.

[0392] 2) Discard the coating solution in the plate and add 200-250 μL of washing solution and wash three times.

[0393] 3) Add 200 μL of 2% BSA to all wells and block at room temperature for 1 hour.

[0394] 4) Discard the blocking solution in the plate, add 200 μL of washing solution, and wash once.

[0395] 5) Add 100 μL of phage to each of the positive and negative wells and incubate at 37°C for 1 h.

[0396] 6) Discard the phages in the plate, add 200 μL of washing solution, and wash three times.

[0397] 7) Dilute anti-M13-HRP antibody to 50 ng / well and incubate at room temperature for 1 hour.

[0398] 8) Discard the antibody in the plate, add 200 μL of washing solution, and wash 5 times.

[0399] 9) Add 100 μL of TMB colorimetric solution to each well and react at room temperature until the OD value reaches between 2 and 3.

[0400] 10) Add stop solution to the color development system, 50 μL per well.

[0401] 11) Measure the absorbance at 450 nm using a spectrophotometer. Send the monoclonal bacterial solution corresponding to the positive wells for sequencing to determine the VHH sequence.

[0402] 12) The obtained antibodies were named NLB1-NLB23

[0403] 8. Cell-level functional screening

[0404] 1) A STAR structure targeting LILRB4 was constructed according to the structure shown in Figure 2. The positive antibody heavy chain (VHH) sequence obtained from the previous screening was assembled with the STAR molecule constant region and inserted into a lentiviral vector using homologous recombination to construct a complete STAR plasmid.

[0405] 2) Packaging virus

[0406] LentiX-293T cells were cultured at a rate of 5 × 10 5 Cells were inoculated into 10 cm culture dishes at 500 μL of culture medium at a ratio of PMD2.G:PRSV-Rev:PMD1g:transfer plamid = 1:1:2:4. The four plasmids were mixed evenly with 500 μL of serum-free DMEM. 54 μL of PEI-max was mixed evenly with 500 μL of serum-free DMEM and allowed to stand at room temperature for 5 minutes (the volume-to-mass ratio of PEI-Max to plasmid was 3:1). The PEI-max mixture was slowly added to the plasmid mixture, gently pipetted, mixed, and allowed to stand at room temperature for 15 minutes. The final mixture was slowly added to the culture medium, mixed thoroughly, and returned to the incubator for 12-16 hours. The culture medium was then switched to 6% FBS DMEM for further culture, and the virus solution was collected at 48 and 72 hours.

[0407] 3) Viral titer measurement

[0408] TCR knockout Jurkat-C4 cells were cultured at 1.5×10 5 Cells were seeded at 100 μL / mL in a flat-bottom 96-well plate. 100 μL of 1640 medium containing 10% FBS and 0.2 μL of 1000× polybrene was added to each well. Virus was diluted 10-fold using complete 1640 medium. The diluted cells were added to the virus-containing wells at 100 μL / well, mixed, and centrifuged at 32°C, 1500 rpm, for 90 minutes. The cells were incubated in a 37°C, 5% CO2 incubator. After 72 hours, infection efficiency was measured using flow cytometry. Wells with an infection rate of 2-30% were selected for titer calculation using the following formula: Titer (TU / mL) = 1.5 × 10^4 × positive rate ÷ virus volume (μL) × 1000. The above virus was used to infect T cells expressing STAR.

[0409] 4) Isolation, activation and infection of primary human T cells

[0410] After obtaining primary T cells using the Ficoil separation method, they were cultured in X-VIVO medium containing 10% FBS and 100 IU / mL IL-2 at an initial culture density of 1 × 10 6 / mL, and activated in CD3, CD28, and Fibronectin-precoated plates. 24 hours after activation, add the viral solution, centrifuge at 1500 rpm for 90 minutes, and incubate in a CO2 incubator. 24 hours after infection, supplement with X-VIVO medium supplemented with 10% FBS and 100 IU / mL IL-2, and transfer the cells to the wells. Subculture every 1-2 days.

[0411] 5) Determination of killing efficiency by co-culture of T cells and target cells in vitro

[0412] One day in advance, target THP1 cells or LILRB4-overexpressing 293T cells were plated in a 24-well plate at a density of 1E5 / well and cultured overnight. According to the ratio of STAR-positive T cells to target cells of 0.5:1, the corresponding number of STAR-T cells were added to the target cells, and the killing effect of STAR-T cells on target cells was detected after 24 hours or 48 hours of culture.

[0413] The results are shown in Figure 3. NLB3, 4, 5, 6, 7, 8, 9, 10, 11, 14, and PC specifically recognized LILRB4 target cells; NLB1, 2, 12, 13, 21, 22, and 23 did not recognize LILRB4; and NLB15, 16, 17, 18, 19, and 20 nonspecifically recognized and killed LILRB4-negative cells.

[0414] Example 3 Antibody Affinity Determination (SPR)

[0415] Antibody affinity was determined using surface plasmon resonance (SPR). Mouse anti-human IgG (Fc) antibodies were immobilized on the CM5 chip surface via amino conjugation. VHH antibodies fused to human IgG1 (Fc) were then injected into the capture experimental channel (Fc2). The reference channel (Fc1) remained untouched. LILRB4 protein was diluted two-fold to a concentration gradient of 62.5 nM, 31.25 nM, 15.625 nM, 7.813 nM, 3.906 nM, 1.953 nM, and 0.977 nM. The diluted protein was sequentially injected into the experimental and reference channels, and the binding and dissociation times were measured. Sample KD values ​​were calculated using Biacore 8K analysis software, with the reference channel (Fc1) used for background subtraction. The results are shown in Table 1 and Figure 4 . The affinities of NLB4 and NLB14 for LILRB4 protein were 1.32 nM and 0.493 nM, respectively.

[0416] Table 1 Affinity determination data of NLB4 and NLB14

[0417] Example 4 Nanobody Competition Binding Assay (SPR)

[0418] Competition for antibody binding was detected using surface plasmon resonance (SPR). The stationary phase was LILRB4 protein, and the mobile phase was NLB(G4S)IGG1FC. As shown in Figures 5A and 5B, NLB4 and NLB14 did not compete for LILRB4 binding.

[0419] Example 5 Recognition epitope of LILRB4 by NLB4 / NLB14 nanobody

[0420] The structure of LILRB4 is shown in Figure 6. Flow cytometry was used to examine the epitope recognition of LILRB4 by the NLB4 / NLB14 nanobodies. As shown in Figure 7, NLB4 or NLB14 recognized the full-length extracellular region of LILRB4 but could not bind to the individual Ig domains or the stock region.

[0421] Example 6 Antibody Binding Specificity Assay (SPR)

[0422] To detect whether the antibodies bind nonspecifically to other LILRB4 family proteins (LILRA1, LILRA2, LILRA3, LILRA4, LILRA5, LILRA6, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5), this experiment first constructed a 293T cell line expressing the same family proteins by lentiviral infection. RFP was co-expressed behind these proteins. The RFP positivity rate was detected by flow cytometry to determine the infection efficiency. LILRB4 antibody staining was used to detect nonspecific binding to the same family proteins. The results are shown in Figure 8, indicating that the two LILRB4 nanoantibodies, NLB4 and NLB14, significantly bound to LILRB4 but did not bind to other proteins in the same family.

[0423] Example 7 LILRB4 Safety Evaluation Cell Line Binding Assay

[0424] To test the accuracy of target recognition by the NLB4 and NLB14 antibodies and whether they bind nonspecifically to other components of human tissues, we established a safety evaluation cell library consisting of nine cell lines derived from different human tissues (lung, muscle, blood vessels, prostate, liver, kidney, brain, intestine, and skin). Flow cytometry was used to preliminarily evaluate the safety of the antibodies. We incubated NLB4 and NLB14 antibodies fused with the FC (Human IGG1FC) tag with the safety evaluation cell lines, then labeled them with an anti-FC fluorescent secondary antibody and analyzed them by flow cytometry. The results, as shown in Figures 9A and 9B, show that NLB4 and NLB14 showed no nonspecific binding to any of the nine tissue cell lines.

[0425] Example 8 Species Specificity Evaluation of NLB4 and NLB14 Nanobodies - Mice

[0426] In this example, 293T-human LILRB4 and 293T-mouse LILRB4 were established, and flow cytometry was used to test the species specificity of the NLB4 and NLB14 antibodies. As shown in Figure 10, the NLB4 and NLB14 antibodies only recognized human LILRB4 and did not recognize mouse LILRB4.

[0427] Example 9: Single-epitope and dual-epitope LILRB4 STAR structures and membrane expression

[0428] In order to detect the membrane expression, in vitro killing effect, and in vivo killing effect of single-epitope and dual-epitope LILRB4 STAR, we constructed the STAR structure targeting LILRB4 as shown in Figure 11, wherein the NLB4 STAR amino acid sequence is shown in SEQ ID NO:39, the NLB14 STAR amino acid sequence is shown in SEQ ID NO:40, the NLB4 / NLB 14 STAR amino acid sequence is shown in SEQ ID NO:41, and the NLB4 / (myc)NLB14 STAR amino acid sequence is shown in SEQ ID NO:42.

[0429] For STAR-T cells, we infected primary T cells with lentivirus and examined membrane expression using flow cytometry. We co-expressed the RFP fluorescent protein via an IRES at the end of STAR. We confirmed membrane expression of the bi-epitope STAR using an anti-mouse TCRβ chain antibody and RFP expression efficiency. The results, as shown in Figure 12, show high STAR infection efficiency, with positive results for both the mouse TCRβ chain and RFP, demonstrating membrane expression. After verifying membrane expression, the IRES-RFP sequence was removed from the vector.

[0430] Example 10 Cytotoxicity and specificity of LILRB4 STAR-T cells against target cell line THP1

[0431] To verify the specific killing of LILRB4 target cells by STAR-T, we stably overexpressed firefly luciferase in the AML cell line THP1. Based on this, we used CRISPR to knock out the LILRB4 gene in THP1 cells (Figure 13A). We screened for LILRB4-recognizing nanobodies, NLB4 and NLB14, using alpaca. NLB4, NLB14, and NLB4 / NLB14 were simultaneously constructed into STAR vectors, resulting in LILRB4-1 STAR, LILRB4-2 STAR, and biparatopic STAR. These STARs were then introduced into T cells using the aforementioned lentiviral vectors to generate LILRB4-1 STAR-T, LILRB4-2 STAR-T, and LILRB4 biparatopic STAR-T. Functional evaluation of these STAR-Ts was performed. After co-culturing STAR-T with target cells for 24 hours, luciferase substrate was added, and the killing effect of STAR-T on target cells was detected by chemiluminescence. The experimental results are shown in Figure 13. LILRB4-1 STAR-T, LILRB4-2 STAR-T and dual-epitope STAR-T all have a strong killing effect only on THP1, but have no killing effect on THP1-LILRB4KO cells, indicating that LILRB4-1 STAR-T, LILRB4-2 STAR-T and dual-epitope STAR-T can specifically recognize and kill LILRB4 target cells.

[0432] Example 11 Cytotoxicity of LILRB4 STAR-T Cells against Different AML Cell Lines

[0433] We used two single-epitope STAR-Ts and one dual-epitope STAR-T to test the cytotoxicity of these four target cells. As shown in Figure 14A, after 24 hours of co-culture, LILRB4-1 STAR-T, LILRB4-2 STAR-T, and dual-epitope STAR-T cells all showed strong cytotoxicity against the high-antigen-density AML target cell lines THP1, MV-11, and OCI-AML3. However, LILRB4 dual-epitope STAR-T showed no significant advantage over single-epitope STAR-T. As shown in Figure 14B, after 6, 15, and 24 hours of co-culture, LILRB4-1 STAR-T, LILRB4-2 STAR-T, and dual-epitope STAR-T cells also showed strong cytotoxicity against KASUMI-1, which lowly expresses LILRB4. Furthermore, LILRB4 bi-epitope STAR-T was significantly more effective at killing target cells with low antigen density than single-epitope STAR-T, suggesting that bi-epitope STAR-T is more effective in eliminating target cells with low LILRB4 expression. In vitro killing assays demonstrated that bi-epitope STAR-T can effectively and specifically kill LILRB4 target cells at the cellular level in vitro.

[0434] Example 12 Cytokine secretion levels of LILRB4 STAR-T cells

[0435] After 24 hours of co-culture of LILRB4 STAR-T with MV4-11 target cells, we collected culture supernatants and assayed the cytokines IL-2, IFN-γ, and TNF-α. As shown in Figure 15, both single-epitope and dual-epitope STAR-T cells significantly elevated IL-2 and IFN-γ levels after co-culture with MV4-11 target cells, while TNF-α levels were relatively low. This is because TNF-α can directly stimulate macrophages, neutrophils, and other cells to secrete IL-6, a major cytokine involved in cytokine storms in cell therapy. This suggests that dual-epitope STAR-T may exhibit a better safety profile in clinical applications. Furthermore, compared with LILRB4-1 and LILRB4-2 STAR-T cells, dual-epitope STAR-T cells exhibited significantly higher levels of IL-2 and IFN-γ, suggesting that dual-epitope STAR-T cells may have a more effective tumorigenic effect.

[0436] Example 13 Evaluation of the killing effect of LILRB4 STAR-T on mouse tumor models

[0437] To validate the in vivo killing effect of STAR-T cells and address potential safety concerns, we established an MV4-11 cell-based NCG mouse tumor model by intravenous infusion (tail vein infusion, 1E6 / mouse) of luciferase-expressing MV4-11 cells into immunodeficient mice. Furthermore, to validate the tumorigenicity of bi-epitope STAR-T, we constructed a STAR-T model using a previously published TEXAS antibody scFv as a positive control (PC STAR-T). Seven days after MV4-11 tumor cell infusion, LILRB4-1 STAR-T cells, LILRB4-2 STAR-T cells, bi-epitope STAR-T cells, control T cells (PC STAR-T), and mock-T cells (uninfected STAR T cells) were infused into the tail vein at a rate of 4E6 / mouse. In vivo imaging was performed beginning on day 7 to monitor tumor growth, with imaging performed twice a week in the early stages and once a week in the later stages. The results are shown in Figures 16A and 16B . The dual-epitope STAR-T exhibited significant tumor growth inhibition in the mouse tumor model and was significantly superior to the PC positive control group, LILRB4-1 STAR-T, and LILRB4-2 STAR-T groups.

[0438] Example 14 LILRB4 STAR-T Cell Safety Experimental Data

[0439] In terms of animal safety, the performance of mice (such as appearance, behavior, response to stimulation, excretion, etc.), mortality and weight changes were observed before and after cell infusion. In the STAR-T cell group, there was no change in the appearance of mice (such as hair, color) before and after STAR-T cell infusion, the mice behaved normally, there was no increased sensitivity to sudden stimulation, and there was no significant change in excretion (observed, not quantified). As shown in Figure 16A, the death data results showed that the animals in the Mock-T group died on the 28th day due to the increasing tumor load, while STAR-T did not die until the 34th day. At the same time, as shown in Figure 16C, STAR-T re-infusion had no significant effect on the weight of mice, suggesting that STAR-T has good safety.

[0440] Example 15 In vivo pharmacokinetic experiments of LILRB4 STAR-T cells

[0441] Regarding STAR-T cell metabolism in vivo, we first collected blood from the mouse orbits to examine their in vivo expansion. As shown in Figure 16D, bi-epitope STAR-T cells rapidly expanded after infusion, with the number significantly higher than in the PC control group. However, as tumor cells were eliminated, the number of STAR-T cells subsequently declined and remained at a low level. Furthermore, the number of bi-epitope STAR-T cells in the mouse peripheral blood, particularly on days 10 and 14 after T cell infusion, was significantly higher than in the other groups, indicating that STAR-T cells expanded more effectively in the mice.

[0442] Example 16 In vivo systemic toxicity experiment of LILRB4 STAR-T cells

[0443] To further test the safety of LILRB4 STAR-T in mice, we infused STAR-T cells and mock-T cells into the tail vein on day 7 after transfusion of luciferase-expressing MV4-11 cells, with an effective dose of 4E6 cells per mouse. On days 14 and 28 after T cell transfusion, the heart, liver, spleen, lung, kidney, small intestine, pancreas, and brain of the mice were fixed and stained with HE to observe pathological changes in the mouse tissues. The results showed that no obvious lesions were observed in the mouse tissues on days 14 and 28 after STAR-T transfusion (Figure 17).

[0444] Example 17 In vitro tumorigenicity assay of LILRB4 STAR-T cells

[0445] To verify the tumorigenicity of LILRB4 STAR-T cells, we performed a soft agar cloning assay. LILRB4 STAR-T cells were divided into three dose groups: low, medium, and high doses of 0.5 x 103, 1.0 x 103, and 2.0 x 103. The AML tumor cell line OCI-AML3 was used at a dose of 1.0 x 103 as a positive control, and mock-T cells (uninfected STAR T cells, at a dose of 2.0 x 103) were used as a negative control. The cells from the above groups were subjected to clonal growth experiments on the lower 0.6% and upper 0.35% soft agar layers, respectively. Observation times were 8, 15, and 21 days after plating. As shown in Figure 11, OCI-AML3 tumor cells (positive control group) formed small colonies on day 8 after plating and larger colonies on day 21. However, LILRB4 STAR-T cells and mock-T cells at all three doses failed to form colonies by day 21. These results indicate that LILRB4 STAR-T cells are unable to form colonies in the soft agar cloning assay, suggesting that they lack tumorigenicity in vitro.

[0446] Example 18 Relationship between NLB4 / NLB14 and LILRB4 ligand ApoE

[0447] LILRB4 binds to ApoE and, through the ApoE-LILRB4-SHP-2-uPAR-Arginase-1 signaling pathway, supports tumor cell infiltration into tissues and suppresses T cell activity in acute myeloid leukemia (AML) cells. Competitive binding of NLB4 / NLB14 with ApoE was tested using LILRB4 protein as the stationary phase, ApoE as mobile phase 1, and NLB(G4S)IGG1FC as mobile phase 2. As shown in Figure 19, NLB4 / NLB14 and ApoE did not compete for LILRB4 binding, and ApoE did not affect NLB4 / 14 binding to LILRB4. Flow cytometry analysis of the target cell line, KASUMI-1, revealed that the AML cell line, myeloid KASUMI-1, does not express ApoE. ApoE concentrations in normal peripheral blood range from 29 to 70 ng / μL. Therefore, the inventors selected ApoE concentrations of 0, 40 ng / μL, and 80 ng / μL to test the killing effects of single-epitope STAR-T and dual-epitope STAR-T on target cells. As shown in Figure 19C, ApoE did not inhibit the killing of target cells by LILRB4 STAR-T.

[0448] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0449] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0450] The sequences involved in this application are as follows:

[0451] SEQ ID NO: 1 Wild-type human TCRα constant region amino acid sequence

[0452] SEQ ID NO: 2 Wild-type human TCRβ constant region amino acid sequence

[0453] SEQ ID NO:3 Wild-type mouse TCRα constant region amino acid sequence

[0454] SEQ ID NO:4 Wild-type mouse TCRβ constant region amino acid sequence

[0455] SEQ ID NO: 5 Mouse T cell receptor alpha chain constant region containing a cysteine ​​substitution (MouseTCRaC-Cys)

[0456] SEQ ID NO:6 Mouse T cell receptor β chain constant region containing cysteine ​​substitution (MouseTCRβC-Cys)

[0457] SEQ ID NO: 7 Mouse T cell receptor alpha chain constant region containing hydrophobic amino acid substitutions (mouseTCRaC-TM9)

[0458] SEQ ID NO: 8 Mouse T cell receptor alpha chain constant region comprising a transmembrane lysine substitution (mouseTCRaC-Argmut)

[0459] SEQ ID NO: 9 comprises a mouse T cell receptor β chain constant region with lysine substitutions in the transmembrane region and the intracellular region (mouseTCRβC-Argmut),

[0460] SEQ ID NO: 10CD40 intracellular domain amino acid sequence

[0461] SEQ ID NO:110X40 intracellular domain amino acid sequence

[0462] SEQ ID NO: 12 ICOS intracellular domain amino acid sequence

[0463] SEQ ID NO: 13CD28 intracellular domain amino acid sequence

[0464] SEQ ID NO:144-1BB intracellular domain amino acid sequence

[0465] SEQ ID NO: 15CD27 intracellular domain amino acid sequence

[0466] SEQ ID NO: 16 Mouse T cell receptor α chain constant region lacking the intracellular region and comprising cysteine ​​substitutions and hydrophobic region modifications (mouseTCRαC-delmut)

[0467] SEQ ID NO: 17 Mouse T cell receptor β chain constant region deleted, containing cysteine ​​substitution (mouseTCRβC-delmut)

[0468] SEQ ID NO: 18 human CD3γ amino acid sequence

[0469] SEQ ID NO: 19 human CD3δ amino acid sequence

[0470] SEQ ID NO: 20 Human CD3ε amino acid sequence

[0471] SEQ ID NO: 21 human CD3ζ amino acid sequence

[0472] SEQ ID NO: 22 Human IL-2β receptor intracellular terminal amino acid sequence

[0473] SEQ ID NO: 23 Human IL-7α receptor intracellular terminal amino acid sequence

[0474] SEQ ID NO: 24 Human IL-21 receptor intracellular terminal amino acid sequence

[0475] SEQ ID NO: 25 amino acid sequence of human STAT5 activation module

[0476] SEQ ID NO:26 Amino acid sequence of the intracellular end of human IL-2β receptor and human STAT5 activation module IL-2RbQ

[0477] SEQ ID NO:27 amino acid sequence of the intracellular end of human IL-7α receptor and human STAT5 activation module IL-7RbQ

[0478] SEQ ID NO:28 VHH amino acid sequence of LILRB4 antibody NLB4

[0479] SEQ ID NO:29 VHH amino acid sequence of LILRB4 antibody NLB14

[0480] SEQ ID NO:30 mouseTCRaC-Cys-TM9 (hmctSTARTCRaC) amino acid sequence

[0481] SEQ ID NO:31mouseTCRaC-Cys-TM9-N.Rec (NrecSTARTCRaC) amino acid sequence

[0482] SEQ ID NO: 32 Amino acid sequence of mouse T cell receptor β chain constant region containing N-terminal modification and cysteine ​​substitution (MouseTCRbC-Cys-N.Rec, NrecSTARTCRbC)

[0483] SEQ ID NO:33 anti-LILRB4NLB4CDR1 amino acid sequence

[0484] SEQ ID NO:34 anti-LILRB4NLB4CDR2 amino acid sequence

[0485] SEQ ID NO:35 anti-LILRB4NLB4CDR3 amino acid sequence

[0486] SEQ ID NO:36 anti-LILRB4NLB14CDR1 amino acid sequence

[0487] SEQ ID NO:37 anti-LILRB4NLB14CDR2 amino acid sequence

[0488] SEQ ID NO:38 anti-LILRB4NLB14CDR3 amino acid sequence

[0489] SEQ ID NO:39 NLB4 STAR amino acid sequence

[0490] SEQ ID NO:40 NLB14 STAR amino acid sequence

[0491] SEQ ID NO:41 NLB4 / NLB14 STAR

[0492] SEQ ID NO:42 NLB4 / (myc)NLB14 STAR

[0493] SEQ ID NO: 43 Mouse T cell receptor alpha chain constant region lacking the intracellular region, comprising N-terminal modification, cysteine ​​substitution, and hydrophobic modification of the transmembrane region

[0494] SEQ ID NO: 44 Mouse T cell receptor β chain constant region lacking the intracellular region, comprising an N-terminal modification and a cysteine ​​substitution

[0495] SEQ ID NO:45 Wild-type human TCRγ chain constant region amino acid sequence:

[0496] SEQ ID NO:46 Wild-type mouse TCRγ chain constant region amino acid sequence:

[0497] SEQ ID NO: 47 Wild-type human TCR δ chain constant region amino acid sequence:

[0498] SEQ ID NO: 48 Wild-type murine TCR δ chain constant region amino acid sequence:

[0499] SEQ ID NO:49EAAAK linker amino acid sequence

[0500] SEQ ID NO:50 (EAAAK) 2 linker amino acid sequence

[0501] SEQ ID NO:51(EAAAK)3 linker amino acid sequence

[0502] SEQ ID NO:52 EAAK linker amino acid sequence

[0503] SEQ ID NO:53 (EAAK) 2 linker amino acid sequence

[0504] SEQ ID NO:54 (EAAK) 3 linker amino acid sequence

[0505] SEQ ID NO:55A2(EAAAK)2A linker amino acid sequence

[0506] SEQ ID NO:56A3(EAAAK)3A linker amino acid sequence

[0507] SEQ ID NO:57A4(EAAAK)4A linker amino acid sequence

[0508] SEQ ID NO:58A5(EAAAK)5A linker amino acid sequence

[0509] AAAAAEAAAKEAAAKEAAAKEAAAKEAAAKEAAAKA linker amino acid sequence

[0510] SEQ ID NO:59A(EAAAK)4ALEA(EAAAK)4A linker amino acid sequence

[0511] SEQ ID NO:60G4S2 linker amino acid sequence

[0512] SEQ ID NO:61(G4S2)2 linker amino acid sequence

[0513] SEQ ID NO:62(G4S2)3 linker amino acid sequence

[0514] SEQ ID NO:63(G4S2)4 linker amino acid sequence

[0515] SEQ ID NO:64(G4S2)5 linker amino acid sequence

[0516] SEQ ID NO:65(G4S2)6 linker amino acid sequence

[0517] SEQ ID NO:66(G4S2)7 linker amino acid sequence

[0518] SEQ ID NO:67(G4S2)8 linker amino acid sequence

[0519] SEQ ID NO:68(G4S2)9 linker amino acid sequence

[0520] SEQ ID NO:69(G4S2)10 linker amino acid sequence

[0521] SEQ ID NO:70S7 linker amino acid sequence

[0522] SEQ ID NO:71G3S linker amino acid sequence

[0523] SEQ ID NO:72(G3S)2 linker amino acid sequence

[0524] SEQ ID NO:73(G3S)3 linker amino acid sequence

[0525] SEQ ID NO:74(G3S)4 linker amino acid sequence

[0526] SEQ ID NO:75(G3S)5 linker amino acid sequence

[0527] SEQ ID NO:76(G3S)6 linker amino acid sequence

[0528] SEQ ID NO:77(G3S)7 linker amino acid sequence

[0529] SEQ ID NO:78(G3S)8 linker amino acid sequence

[0530] SEQ ID NO:79(G3S)9 linker amino acid sequence

[0531] SEQ ID NO:80(G3S)10 linker amino acid sequence

[0532] SEQ ID NO:81G4S linker amino acid sequence

[0533] SEQ ID NO:82(G4S)2 linker amino acid sequence

[0534] SEQ ID NO:83(G4S)3 linker amino acid sequence

[0535] SEQ ID NO:84(G4S)4 linker amino acid sequence

[0536] SEQ ID NO:85(G4S)5 linker amino acid sequence

[0537] SEQ ID NO:86(G4S)6 linker amino acid sequence

[0538] SEQ ID NO:87(G4S)7 linker amino acid sequence

[0539] SEQ ID NO:88(G4S)8 linker amino acid sequence

[0540] SEQ ID NO:89(G4S)9 linker amino acid sequence

[0541] SEQ ID NO:90(G4S)10 linker amino acid sequence

[0542] SEQ ID NO:91G2S linker amino acid sequence

[0543] SEQ ID NO:92(G2S)2 linker amino acid sequence

[0544] SEQ ID NO:93(G2S)3 linker amino acid sequence

[0545] SEQ ID NO:94(G2S)4 linker amino acid sequence

[0546] SEQ ID NO:95(G2S)5 linker amino acid sequence

[0547] SEQ ID NO:96(G2S)6 linker amino acid sequence

[0548] SEQ ID NO:97(G2S)7 linker amino acid sequence

[0549] SEQ ID NO:98(G2S)8 linker amino acid sequence

[0550] SEQ ID NO:99(G2S)9 linker amino acid sequence

[0551] SEQ ID NO:100(G2S)10 linker amino acid sequence

[0552] SEQ ID NO: 101 GS linker amino acid sequence

[0553] SEQ ID NO:102 (GS) 2 linker amino acid sequence

[0554] SEQ ID NO: 103 (GS) 3 linker amino acid sequence

[0555] SEQ ID NO: 104 (GS) 4 linker amino acid sequence

[0556] SEQ ID NO: 105 (GS) 5 linker amino acid sequence

[0557] SEQ ID NO: 106 (GS) 6 linker amino acid sequence

[0558] SEQ ID NO: 107 (GS) 7 linker amino acid sequence

[0559] SEQ ID NO: 108 (GS) 8 linker amino acid sequence

[0560] SEQ ID NO: 109 (GS) 9 linker amino acid sequence

[0561] SEQ ID NO:110(GS)10 linker amino acid sequence

[0562] SEQ ID NO:111(G)5 linker amino acid sequence

[0563] SEQ ID NO:112(A)11 linker amino acid sequence

[0564] SEQ ID NO:113F2A cleavable linker amino acid sequence

[0565] SEQ ID NO:114 P2A cleavable linker amino acid sequence

[0566] SEQ ID NO:115 T2A cleavable linker amino acid sequence

[0567] SEQ ID NO: 116E2A cleavable linker amino acid sequence

[0568] SEQ ID NO: 117 Disulfide bond type cleavable linker amino acid sequence

[0569] SEQ ID NO: 118 NLB4 VHH nucleotide sequence

[0570] SEQ ID NO: 119 NLB14 VHH nucleotide sequence

[0571] SEQ ID NO: 120 NLB4 STAR nucleotide sequence

[0572] SEQ ID NO: 121 NLB14 STAR nucleotide sequence

[0573] SEQ ID NO:122 NLB4 / NLB14 STAR nucleotide sequence

[0574] SEQ ID NO:123 NLB4 / (myc)NLB14 STAR nucleotide sequence

Claims

1. A synthetic T cell receptor antigen receptor, characterized in that: The synthetic T cell receptor antigen receptor comprises an α chain and a β chain, wherein the α chain comprises a first target binding region and a first constant region, and the β chain comprises a second target binding region and a second constant region, or the α chain comprises the first target binding region and the β chain comprises the second target binding region and a second constant region; or, ii) the synthetic T cell receptor antigen receptor comprises a γ chain and a δ chain, wherein the γ chain comprises a first target binding region and a first constant region, and the δ chain comprises a second target binding region and a second constant region, or the γ chain comprises a first target binding region and a first constant region and the δ chain comprises a second target binding region and a second constant region; wherein the first target binding region and / or the second target binding region comprises one or more antigen binding regions, the multiple antigen binding regions are the same or different, and the multiple antigen binding regions are directly connected or connected through a linker; The antigen binding region in the first target binding region comprises an antibody or antibody fragment that specifically binds to LILRB4, and the antigen binding region in the second target binding region comprises an antibody or antibody fragment that specifically binds to LILRB4.

2. The synthetic T cell receptor antigen receptor according to claim 1, characterized in that The antigen binding region in the first target binding region comprises a single chain antibody or a single domain antibody that specifically binds to LILRB4; 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 to LILRB4; Preferably, the single-chain antibody comprises a heavy chain variable region and a light chain variable region connected directly or through a linker; Preferably, the multiple antigen binding regions in the first target binding region and / or the second target binding region bind to different regions of LILRB4, such as different epitopes.

3. The synthetic T cell receptor antigen receptor according to claim 1 or 2, characterized in that The antigen binding region in the first target binding region comprises one or more single domain antibodies, and / or the antigen binding region in the second target binding region comprises one or more single domain antibodies; Preferably, the multiple single domain antibodies comprised in the antigen binding region in the first target binding region are the same or different; Preferably, the multiple single domain antibodies comprised in the antigen binding region in the second target binding region are the same or different; Further preferably, the multiple single-domain antibodies are directly connected or connected via a linker.

4. The synthetic T cell receptor antigen receptor according to claim 3, characterized in that The single-domain antibody comprises a heavy chain variable region, and the heavy chain variable region comprises CDR1-3, wherein: i) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 33, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 34, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 35; or, ii) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 36, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 37, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO:

38.

5. The synthetic T cell receptor antigen receptor according to claim 3, characterized in that: The single-domain antibody comprises the amino acid sequence shown in SEQ ID NO: 28 or 29.

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

7. The synthetic T cell receptor antigen receptor according to claim 1, characterized in that i) the intracellular region of the α chain and / or β chain in the synthetic T cell receptor antigen receptor is deleted; or, ii) The intracellular region of the γ chain and / or δ chain in the synthetic T cell receptor antigen receptor is deleted.

8. The synthetic T cell receptor antigen receptor according to claim 7, characterized in that i) at least one functional domain is connected to the C-terminus of the α chain and / or the β chain, and the at least one functional domain is connected to the C-terminus of the α chain and / or the β chain directly or through a linker; or, ii) at least one functional domain is linked to the C-terminus of the γ chain and / or the δ chain, and the at least one functional domain is linked to the C-terminus of the γ chain and / or the δ chain directly or via a linker.

9. The synthetic T cell receptor antigen receptor according to any one of claims 6 to 8, characterized in that i) the C-terminus of the α chain in the synthetic T cell receptor antigen receptor is connected 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 connected to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains; or, ii) the C-terminus of the γ chain in the synthetic T cell receptor antigen receptor is connected to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains, and / or the C-terminus of the δ chain in the synthetic T cell receptor antigen receptor is connected to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more functional domains; Wherein, the functional domains are the same or different.

10. The synthetic T cell receptor antigen receptor according to any one of claims 6 to 8, characterized in that 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; Preferably, the co-stimulatory molecule is selected from CD40, OX40, ICOS, CD28, 4-1BB or CD27; Preferably, the co-inhibitory molecule is selected from TIM3, PD1, CTLA4, and LAG3; Preferably, the cytokine receptor is selected from interleukin receptors, interferon receptors, tumor necrosis factor superfamily receptors, colony stimulating factor receptors, chemokine receptors, growth factor receptors or other membrane proteins; Preferably, the intracellular protein is a T cell regulatory factor, such as the domain of NIK.

11. The synthetic T cell receptor antigen receptor according to any one of claims 1 to 10, characterized in that The linker is selected from a rigid linker, a flexible linker, a cleavable linker or a meaningless amino acid; Preferably, the amino acid sequence of the rigid linker is selected from one or more of SEQ ID NOs: 49-59; Preferably, the flexible linker is selected from a peptide segment rich in glycine and / or serine; preferably, the flexible linker is selected from one or more of SEQ ID NOs: 60-112; Preferably, the cleavable linker is selected from one or more of SEQ ID NOs: 113-117.

12. The synthetic T cell receptor antigen receptor according to any one of claims 1 to 11, characterized in that The first constant region is a TCRα chain constant region or a TCRγ chain constant region, preferably a modified TCRα chain constant region or a modified TCRγ chain constant region; 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 TCRγ chain constant region is selected from the human TCRγ chain constant region or the murine (preferably mouse) TCRγ chain constant region.

13. The synthetic T cell receptor antigen receptor according to claim 12, characterized in that: The modified TCR alpha chain constant region is derived from a murine (preferably mouse) TCR alpha chain constant region, which comprises one or more modifications at positions 6, 13, 15-18, 48, 112, 114, 115 relative to a wild-type murine (preferably mouse) TCR alpha chain constant region, wherein the modification is a mutation or a deletion; or The modified TCRα chain constant region is derived from a murine (preferably mouse) TCRα chain constant region, which comprises one or more modifications at positions 13, 36, 47, 53, 58, 78, 98, and 122 relative to a wild-type murine (preferably mouse) TCRα chain constant region, wherein the modifications are mutations or deletions.

14. The synthetic T cell receptor antigen receptor according to claim 12, characterized in that: The modified TCR alpha chain constant region is derived from a murine (preferably mouse) TCR alpha chain constant region, which comprises a mutation at position 48, such as threonine T, to cysteine ​​C relative to a wild-type murine (preferably mouse) TCR alpha chain constant region; The modified TCR α chain constant region is derived from a murine (preferably mouse) TCR α chain constant region, which comprises, relative to a wild-type murine (preferably mouse) TCR α chain constant region, that the amino acid at position 112, e.g., serine S, is changed to leucine L, the amino acid at position 114, e.g., methionine M, is changed to isoleucine I, and / or, the amino acid at position 115, e.g., glycine G, is changed to valine V; The modified TCR α chain constant region is derived from a murine (preferably mouse) TCR α chain constant region, which comprises a substitution of amino acid E at position 6 with D, K at position 13 with R, and amino acids 15-18 are deleted 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 a substitution of amino acid K at position 122 by 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, which comprises, relative to a wild-type murine (preferably mouse) TCR α chain constant region, a mutation at position 48, such as threonine T, to cysteine ​​C, a mutation at position 112, such as serine S, to leucine L, a mutation at position 114, such as methionine M, to isoleucine I, and a mutation at position 115, such as glycine G, to valine V; The modified TCR α chain constant region is derived from a murine (preferably mouse) TCR α chain constant region, which comprises a mutation of the amino acid at position 48, such as threonine T, to cysteine ​​C, and a substitution of the amino acid K at position 122 by R relative to the 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, relative to a wild-type murine (preferably mouse) TCR α chain constant region, a substitution of amino acid at position 6, such as E, with D, a substitution of K at position 13 with R, and a deletion of amino acids at positions 15-18, and a mutation of amino acid at position 48, such as threonine T, to cysteine ​​C; The modified TCR α chain constant region is derived from a murine (preferably mouse) TCR α chain constant region, which comprises, relative to a wild-type murine (preferably mouse) TCR α chain constant region, a mutation at position 48, such as threonine T, to cysteine ​​C, a mutation at position 112, such as serine S, to leucine L, a mutation at position 114, such as methionine M, to isoleucine I, a mutation at position 115, such as glycine G, to valine V, and a substitution at position 122, such as amino acid K, to R; The modified TCR alpha chain constant region is derived from a murine (preferably mouse) TCR alpha chain constant region, which comprises, relative to a wild-type murine (preferably mouse) TCR alpha chain constant region, a substitution of D for an amino acid such as E at position 6, a substitution of R for K at position 13, and a deletion of amino acids 15-18, a mutation of threonine T at position 48 to cysteine ​​C, a mutation of leucine S at position 112 to leucine L, a mutation of methionine M at position 114 to isoleucine I, a substitution of valine V for an amino acid such as glycine G at position 115, and a substitution of R for amino acid K at position 122; The modified TCR α chain constant region is derived from a murine (preferably mouse) TCR α chain constant region, which, relative to a wild-type murine (preferably mouse) TCR α chain constant region, includes a substitution of an amino acid at position 6, such as E, with a substitution of D, a substitution of K at position 13 with R, and a deletion of amino acids 15-18, a mutation of an amino acid at position 48, such as threonine T, with a mutation of cysteine ​​C, a mutation of an amino acid at position 112, such as serine S, with a mutation of leucine L, a mutation of an amino acid at position 114, such as methionine M, with a mutation of isoleucine I, and a mutation of an amino acid at position 115, such as glycine G, with a mutation of valine V; The modified TCR α chain constant region is derived from a murine (preferably mouse) TCR α chain constant region, which comprises, relative to a wild-type murine (preferably mouse) TCR α chain constant region, a substitution of the amino acid at position 6, such as E, with D, a substitution of the K at position 13 with R, and a deletion of the amino acids at positions 15-18, a mutation of the amino acid at position 48, such as threonine T, to cysteine ​​C, and a substitution of the amino acid K at position 122 with R; The modified TCR α chain constant region is derived from a murine (preferably mouse) TCR α chain constant region, which comprises, relative to a wild-type murine (preferably mouse) TCR α chain constant region, a substitution of an amino acid at position 6, such as E, with a substitution of an amino acid at position 13 with an amino acid at position 15 to 18 with an amino acid at position 112, such as serine S, with a substitution of an amino acid at position 114, such as methionine M, with a substitution of an amino acid at position 115, such as glycine G, with a substitution of an amino acid at position 115 with a valine V; The modified TCR alpha chain constant region is derived from a murine (preferably mouse) TCR alpha chain constant region, which comprises a substitution of amino acid E at position 6 with D, K at position 13 with R, and amino acids 15-18 are deleted, and amino acid K at position 122 is substituted with R relative to a wild-type murine (preferably mouse) TCR alpha chain constant region; or The modified TCR α chain constant region is derived from a murine (preferably mouse) TCR α chain constant region, which, relative to the wild-type murine (preferably mouse) TCR α chain constant region, includes the amino acid at position 6, such as E, being replaced by D, the K at position 13 being replaced by R, and the amino acids at positions 15-18 being deleted, the amino acid at position 112, such as serine S, being changed to leucine L, the amino acid at position 114, such as methionine M, being changed to isoleucine I, the amino acid at position 115, such as glycine G, being changed to valine V, and the amino acid K at position 122 being replaced by R.

15. The synthetic T cell receptor antigen receptor according to claim 12, characterized in that: The first constant region comprises the amino acid sequence shown in one of SEQ ID NOs: 1, 3, 5, 7, 8, 16, 30, 31 or 43.

16. The synthetic T cell receptor antigen receptor according to any one of claims 1 to 15, characterized in that: 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 TCRβ chain constant region is selected from a human TCRβ chain constant region or a murine (preferably mouse) TCRβ chain constant region; Preferably, the TCRδ chain constant region is selected from the human TCRδ chain constant region or the murine (preferably mouse) TCRδ chain constant region.

17. The synthetic T cell receptor antigen receptor according to claim 16, characterized in that: 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 at positions 3, 6, 9, 11, 12, 17, 21-25, 56, 150, 168 or 170 relative to a wild-type murine (preferably mouse) TCR β chain constant region, wherein the modification is a mutation or a deletion; or The modified TCRβ chain constant region is derived from a murine (preferably mouse) TCRβ chain constant region, which comprises one or more modifications at positions 9, 17, 23, 25, 49, 63, 103, 110, 150, 168, and 170 relative to a wild-type murine (preferably mouse) TCRβ chain constant region, wherein the modifications are mutations or deletions.

18. The synthetic T cell receptor antigen receptor according to claim 16, characterized in that: The modified TCR β chain constant region is derived from a murine (preferably mouse) TCR β chain constant region, which comprises a mutation at position 56, such as serine S, 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, which includes a substitution of lysine at position 150, 168 or 170 by arginine; The modified TCR β chain constant region is derived from a murine (preferably mouse) TCR β chain constant region, which comprises, relative to a wild-type murine (preferably mouse) TCR β chain constant region, a substitution of the amino acid at position 3, such as R, with K, a substitution of the amino acid at position 6, such as T, with F, a substitution of K at position 9 with E, a substitution of S at position 11 with A, a substitution of L at position 12 with V, and deletions of amino acids 17, 21-25; The modified TCR β chain constant region is derived from a murine (preferably mouse) TCR β chain constant region, which comprises a mutation of the amino acid at position 56, such as serine S, to cysteine ​​C, and a substitution of lysine at position 150, 168 or 170 by arginine relative to the 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, which, relative to a wild-type murine (preferably mouse) TCR β chain constant region, includes a substitution of the amino acid at position 3, such as R, by K, a substitution of the amino acid at position 6, such as T, by F, a substitution of K at position 9 by E, a substitution of S at position 11 by A, a substitution of L at position 12 by V, and deletions of amino acids at positions 17, 21-25, and a mutation of the amino acid at position 56, such as serine S, to cysteine ​​C; The modified TCR β chain constant region is derived from a murine (preferably mouse) TCR β chain constant region, which comprises, relative to a wild-type murine (preferably mouse) TCR β chain constant region, an amino acid at position 3, such as R, replaced by K, an amino acid at position 6, such as T, replaced by F, K at position 9 replaced by E, S at position 11 replaced by A, L at position 12 replaced by V, and amino acids at positions 17, 21-25 are deleted, an amino acid at position 56, such as serine S, is mutated to cysteine ​​C, and lysine at positions 150, 168 or 170 is replaced by arginine; The modified TCRβ chain constant region is derived from a murine (preferably mouse) TCRβ chain constant region, which comprises, relative to the wild-type murine (preferably mouse) TCRβ chain constant region, an amino acid at position 3 such as R being replaced by K, an amino acid at position 6 such as T being replaced by F, K at position 9 being replaced by E, S at position 11 being replaced by A, L at position 12 being replaced by V, and amino acids at positions 17, 21-25 being deleted, and lysine at positions 150, 168 or 170 being replaced by arginine.

19. The synthetic T cell receptor antigen receptor according to claim 16, characterized in that: The second constant region comprises the amino acid sequence shown in one of SEQ ID NOs: 2, 4, 6, 9, 17, 32 or 44.

20. The synthetic T cell receptor antigen receptor according to claim 1 or 2, characterized in that: The first target binding region is directly connected to the first constant region or connected via a linker, and / or the second target binding region is directly connected to the second constant region or connected via a linker.

21. The synthetic T cell receptor antigen receptor according to claim 20, characterized in that: The linker is selected from a rigid linker, a flexible linker, a cleavable linker or a meaningless amino acid; Preferably, the amino acid sequence of the rigid linker is selected from one or more of SEQ ID NOs: 49-59; Preferably, the flexible linker is selected from a peptide segment rich in glycine and / or serine; preferably, the flexible linker is selected from one or more of SEQ ID NOs: 60-112; Preferably, the cleavable linker is selected from one or more of SEQ ID NOs: 113-117.

22. A synthetic T cell receptor antigen receptor complex, characterized in that: The complex comprises the synthetic T cell receptor antigen receptor according to any one of claims 1-21, and CD3ε, CD3γ, CD3δ and CD3ζ.

23. The synthetic T cell receptor antigen receptor complex according to claim 22, characterized in that: The CD3ε, CD3γ, CD3δ and / or CD3ζ are of human origin; Preferably, the CD3ε comprises the amino acid sequence shown in SEQ ID NO: 20; Preferably, the CD3γ comprises the amino acid sequence shown in SEQ ID NO: 18; Preferably, the CD3δ comprises the amino acid sequence shown in SEQ ID NO: 19; Preferably, the CD3ζ comprises the amino acid sequence shown in SEQ ID NO:

21.

24. An antibody or antigen-binding fragment, characterized in that: The antibody or antigen-binding fragment comprises a heavy chain variable region, wherein the heavy chain variable region comprises CDR1-3, wherein i) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 33, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 34, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 35; or, ii) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 36, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 37, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO:

38.

25. The antibody or antigen-binding fragment according to claim 24, characterized in that: The antibody or antigen-binding fragment is a single-chain antibody or a single-domain antibody.

26. The antibody or antigen-binding fragment according to claim 24, characterized in that: The antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 28 or 29.

27. An antigen receptor, characterized in that The antigen receptor comprises a transmembrane region, an intracellular region and one or more identical or different extracellular binding domains, wherein the extracellular binding domain is an extracellular antigen binding domain; The extracellular antigen binding domain comprises CDR1-3, wherein: i) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 33, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 34, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 35; or, ii) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 36, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 37, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO:

38.

28. The antigen receptor according to claim 27, characterized in that The extracellular antigen binding domain comprises the antibody or antigen binding fragment described in any one of claims 24-26.

29. The antigen receptor according to claim 27, characterized in that The antigen receptor is STAR, TCR or CAR; Preferably, the transmembrane region is derived from human CD8; Preferably, the intracellular region is derived from 4-1BB, CD28 or CD3ζ.

30. The antigen receptor according to claim 27, characterized in that The transmembrane region is directly connected to one or more extracellular antigen binding domains or connected via a linker.

31. The antigen receptor according to claim 30, characterized in that The linker is selected from a rigid linker, a flexible linker, a cleavable linker or a meaningless amino acid; Preferably, the amino acid sequence of the rigid linker is selected from one or more of SEQ ID NOs: 49-59; Preferably, the flexible linker is selected from a peptide segment rich in glycine and / or serine; preferably, the flexible linker is selected from one or more of SEQ ID NOs: 60-112; Preferably, the cleavable linker is selected from one or more of SEQ ID NOs: 113-117.

32. A nucleic acid, characterized in that: The nucleic acid encodes the synthetic T cell receptor antigen receptor described in any one of claims 1-21, the synthetic T cell receptor antigen receptor complex described in any one of claims 22-23, the antibody or antigen binding fragment described in any one of claims 24-26, and the antigen receptor described in any one of claims 27-31.

33. A carrier, characterized in that: The vector comprises the nucleic acid of claim 32.

34. A host cell, characterized in that: The host cell comprises the nucleic acid of claim 32 or the vector of claim 33.

35. An immune cell, characterized in that: The immune cells express the synthetic T cell receptor antigen receptor described in any one of claims 1-21, the synthetic T cell receptor antigen receptor complex described in any one of claims 22-23, the antibody or antigen binding fragment described in any one of claims 24-26, and the antigen receptor described in any one of claims 27-31.

36. The immune cell according to claim 35, characterized in that: The immune cell comprises one or more nucleic acids according to claim 32.

37. The immune cell according to claim 35 or 36, characterized in that: The immune cells are selected from T cells, Treg cells, macrophages, NK cells, NKT cells, peripheral blood monocytes, TIL cells or dendritic cells (DC).

38. The immune cell according to claim 35 or 36, characterized in that The immune cells are isolated and derived from T cells of the subject.

39. A method for preparing immune cells, characterized in that: The preparation method comprises transfecting the nucleic acid sequence of claim 32 into immune cells for expression.

40. A method for preparing a recombinant T cell, characterized in that: The steps include: 1) Obtaining the nucleic acid of claim 32 from a positive T cell clone; 2) Isolation and culture of 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 the synthetic T cell receptor antigen receptor described in any one of claims 1-21.

41. A method for preparing a synthetic T cell receptor antigen receptor, characterized in that: The steps include: (1) obtaining the nucleic acid of claim 32 from a positive T cell clone; (2) connecting the nucleic acid obtained in step (1) to a vector backbone to obtain an expression vector; (3) transforming the expression vector obtained in step (2) into a host cell and then inducing its expression; (4) Obtaining synthetic T cell receptor antigen receptor.

42. Use of the synthetic T cell receptor antigen receptor described in any one of claims 1-21, the synthetic T cell receptor antigen receptor complex described in any one of claims 22-23, the antibody or antigen binding fragment described in any one of claims 24-26, the antigen receptor described in any one of claims 27-31, the nucleic acid described in claim 32, and the immune cell described in any one of claims 35-38 in the preparation of products for diagnosing or treating tumors.

43. The use according to claim 42, characterized in that: 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, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma; Preferably, the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myeloid leukemia; Preferably, 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; Preferably, the sarcoma is selected from osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma; Preferably, the acute myeloid leukemia is M4 or M5 acute myeloid leukemia; Preferably, the chronic myeloid leukemia is chronic myelomonocytic leukemia.

44. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the synthetic T cell receptor antigen receptor described in any one of claims 1-21, the synthetic T cell receptor antigen receptor complex described in any one of claims 22-23, the antibody or antigen binding fragment described in any one of claims 24-26, the antigen receptor described in any one of claims 27-31, the nucleic acid described in claim 32, and the immune cell described in any one of claims 35-38.

45. A kit, characterized in that: The kit comprises the synthetic T cell receptor antigen receptor according to any one of claims 1-21, the synthetic T cell receptor antigen receptor complex according to any one of claims 22-23, the antibody or antigen binding fragment according to any one of claims 24-26, the antigen receptor according to any one of claims 27-31, the nucleic acid according to claim 32, and the immune cell according to any one of claims 35-38.

46. ​​A method for treating a tumor, comprising administering to a subject an effective amount of the antibody or antigen-binding fragment of any one of claims 24-26, the immune cell of any one of claims 35-38, or the pharmaceutical composition of claim 44.

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