Polypeptides comprising membrane surface domains and uses thereof

Abstract

The present invention relates to polypeptides comprising a membrane surface domain and uses thereof. In particular, the present invention provides an engineered polypeptide comprising a membrane surface domain comprising an extracellular domain of a B cell surface antigen or a variant thereof.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a polypeptide construct and its application. BACKGROUND

[0002] Tumor immune cell therapy has now occupied an increasingly important position in tumor treatment. At present, a variety of effector immune cells, i.e. killer immune cells, are used for clinical treatment, mainly including CAR-T cells, TCR-T cells, nature killer (NK) cells, CAR-NK cells, cytokine-activated killer cells (CIK), dendritic cell-stimulated cytokine-activated killer cells (DC-CIK), cytotoxic T lymphocytes (CTL), γδ T cells, tumor infiltrating lymphocytes (TIL), etc. Modification of natural effector immune cells to introduce overexpression of exogenous genes such as CAR, TCR or other transmembrane molecule encoding genes or secretory molecules (such as cytokines, antibodies, etc.) encoding genes is an important means to enhance the anti-tumor effect of immune effector cells and improve the effect of immune cell therapy. The adverse reaction of cytokine release syndrome (CRS) caused by the on target / off tumor effect triggered thereby is still a potential important risk of immune cell therapy. To solve this problem, the commonly used means is to introduce a molecular brake element into the immune effector cells, so as to timely eliminate the immune effector cells expressing the molecular brake element when the adverse reaction occurs, thereby improving the safety. The molecular brake elements reported at present mainly include metabolic suicide gene HSV-TK system, apoptosis gene iCasp9 system. CN109750067A and CN109750066A also disclose a fusion protein comprising an immunosuppressive antibody and tEGFR, which can be eliminated by immune effector cells through ADCC, ADCP and CDC by administering an antibody drug targeting EGFR such as cetuximab in use.

[0003] In addition, for immune effector cells overexpressing exogenous genes, there is still a lack of effective means for detecting cells positive for exogenous gene expression and sorting cells expressing exogenous genes. For exogenous genes whose expression products are membrane surface proteins or transmembrane proteins, different detection systems and sorting methods need to be designed for different extracellular domains of the expression products, increasing the complexity of detection and sorting operations and the difficulty of comparing the consistency of detection results. For exogenous genes whose expression products are secreted proteins or polypeptides, such as cytokines and antibodies, there is still no particularly good method for detecting and / or sorting cells positive for exogenous gene expression.

[0004] There is still a need for a polypeptide molecular tag that can be universal, which can function as a molecular brake element while also serving as a detection and sorting tag for exogenous gene expression and as a linker between different domains. This will be of great significance to the production quality control and potential clinical application of cell therapy products. SUMMARY

[0005] The present application provides an engineered polypeptide comprising a membrane surface domain, wherein the membrane surface domain comprises an extracellular domain of a B cell surface antigen or a variant thereof.

[0006] In one or more embodiments, the B cell surface antigen is BCMA, and the extracellular domain of the B cell surface antigen or the variant thereof is a BCMA extracellular domain or a variant thereof.

[0007] In one or more embodiments, the polypeptide further comprises a transmembrane region. The membrane surface domain is located in the N-terminal direction of the transmembrane region.

[0008] In one or more embodiments, the membrane surface domain is connected to the transmembrane region via a hinge region or a linker. The linker is, for example, a soft linker or a rigid linker; preferably, the amino acid sequence of the rigid linker is as set forth in any one of SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, and SEQ ID NO: 53.

[0009] In one or more embodiments, the polypeptide further comprises an extracellular recognition region located at the N-terminal or C-terminal of the membrane surface domain; preferably, the membrane surface domain and the extracellular recognition region are connected via a hinge region or a linker. The extracellular recognition region is selected from one or more of the following: an antigen binding region of a chimeric antigen receptor, a cytokine region of a membrane surface cytokine, or a signal extracellular region of a signal conversion receptor.

[0010] In one or more embodiments, the linker is a soft linker or a rigid linker or a multiple expression linker.

[0011] In one or more embodiments, the linker is a rigid linker.

[0012] In one or more embodiments, the amino acid sequence of the rigid linker is set forth in any one of SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 and SEQ ID NO: 53.

[0013] In one or more embodiments, the multiple expression linker is a polypeptide linker sequence that can be cleaved in vivo, such as F2A, P2A, T2A and E2A sequences.

[0014] In one or more embodiments, the polypeptide further comprises an intracellular effector region located C-terminal to the transmembrane region. The intracellular effector region comprises the intracellular region of the chimeric antigen receptor or the signal intracellular region of the signal switching receptor.

[0015] In one or more embodiments, the polypeptide is a membrane surface protein or a transmembrane protein.

[0016] In one or more embodiments, the BCMA extracellular domain is set forth in SEQ ID NO: 11.

[0017] In one or more embodiments, the variant is a truncated variant.

[0018] In one or more embodiments, the truncated variant of the BCMA extracellular domain is set forth in SEQ ID NO: 13.

[0019] In one or more embodiments, the polypeptide further comprises a signal peptide.

[0020] In one or more embodiments, the polypeptide is a chimeric antigen receptor, wherein the membrane surface domain is located N-terminal to the transmembrane region of the chimeric antigen receptor. The chimeric antigen receptor comprises an antigen binding region targeting an antigen, a hinge region, the membrane surface domain, a transmembrane region and an intracellular domain. Preferably, the chimeric antigen receptor further comprises a signal peptide. Preferably, the signal peptide is located N-terminal to the chimeric antigen receptor.

[0021] In one or more embodiments, the polypeptide is a chimeric antigen receptor against HER2, comprising an antigen binding region which is an antibody or antigen binding fragment thereof targeting HER2, a membrane surface domain which is the BCMA extracellular domain or a truncated variant thereof, a hinge region which is a CD28 hinge region, a transmembrane region which is a CD28 transmembrane region, and an intracellular region comprising a signaling region and a costimulatory region. Preferably, the signaling region is a CD3zeta intracellular signaling region. Preferably, the costimulatory region is a CD28 intracellular costimulatory region. Preferably, the chimeric antigen receptor against HER2 further comprises a signal peptide. Preferably, the signal peptide is a CD8 signal peptide. Preferably, the signal peptide is located at the N-terminus of the chimeric antigen receptor against HER2.

[0022] In one or more embodiments, the polypeptide is a membrane surface cytokine, comprising a cytokine region and the membrane surface domain. Preferably, the cytokine region comprises a cytokine protein sequence or a fragment thereof, such as the sequence of IL-7. Preferably, the cytokine region is located at the N-terminus of the membrane surface domain; the membrane surface domain and the cytokine region are connected by a soft linker, a rigid linker, or a multiple expression linker. In one or more embodiments, the membrane surface cytokine comprises: a cytokine protein sequence or a fragment thereof, the membrane surface domain, and a transmembrane region, or the membrane surface cytokine comprises: a cytokine protein sequence or a fragment thereof, a hinge region, a transmembrane region, a multiple expression linker, the membrane surface domain, a soft linker or a rigid linker, a transmembrane region.

[0023] In one or more embodiments, the membrane surface cytokine is a membrane surface IL-7, comprising IL-7, a membrane surface domain, and a transmembrane region. Preferably, the membrane surface domain is the BCMA extracellular domain or a truncated variant thereof, and the transmembrane region is a CD8 transmembrane region. Preferably, the membrane surface IL-7 further comprises a signal peptide. Preferably, the signal peptide is an IL-7 signal peptide. Preferably, the signal peptide is located at the N-terminus of the membrane surface IL-7.

[0024] In one or more embodiments, the membrane surface cytokine comprises: IL-7, a hinge region, a transmembrane region, 2A, a membrane surface domain, a linker, a transmembrane region. In one or more embodiments, the membrane surface domain is the BCMA ectodomain or a truncated variant of the BCMA ectodomain; the hinge region is a CD8a ecto-hinge region; the transmembrane region is a CD8a transmembrane region; the 2A is selected from F2A, P2A, T2A and E2A; the amino acid sequence of the linker is set forth in any one of SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 and SEQ ID NO: 53.

[0025] In one or more embodiments, the polypeptide comprises the membrane surface domain, a linker and a transmembrane region; preferably, the linker is a rigid linker. In one or more embodiments, the membrane surface domain is the BCMA ectodomain or a truncated variant of the BCMA ectodomain; the transmembrane region is a CD8a transmembrane region; the amino acid sequence of the linker is set forth in any one of SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 and SEQ ID NO: 53.

[0026] In one or more embodiments, the polypeptide is a signal switching receptor, wherein the membrane surface domain is located in the N-terminal direction of the transmembrane region of the signal switching receptor. The signal switching receptor comprises a signal ectodomain, a hinge region or a linker, the membrane surface domain, a transmembrane region and a signal endodomain. The signal ectodomain is an ectodomain that recognizes an extracellular signal. The signal endodomain is an endodomain of a receptor that recognizes a signal different from the extracellular signal.

[0027] In one or more embodiments, the hinge region is a CD8a ecto-hinge region. Preferably, the linker is a soft linker or a rigid linker; more preferably, a rigid linker. Preferably, the linker is located N-terminal to the membrane surface domain, between the signal ectodomain and the membrane surface domain, or C-terminal to the membrane surface domain, between the membrane surface domain and the transmembrane region.

[0028] In one or more embodiments, the signal-converting receptor is a PD-1 signal-converting receptor comprising a PD-1 extracellular region, a membrane surface domain, a transmembrane region and an intracellular region. The membrane surface domain is the BCMA extracellular domain or a truncated variant of the BCMA extracellular domain, and the transmembrane region is an IL-7Ra transmembrane region or a mutant of the IL-7Ra transmembrane region. Preferably, the mutant of the IL-7Ra transmembrane region has an amino acid sequence as set forth in SEQ ID NO: 101. Preferably, the mutant of the IL-7Ra transmembrane region has a nucleotide sequence as set forth in SEQ ID NO: 102.

[0029] In one or more embodiments, the PD-1 signal-converting receptor further comprises a linker. Preferably, the linker is a rigid linker. More preferably, the rigid linker has an amino acid sequence as set forth in any one of SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 and SEQ ID NO: 53. Preferably, the linker is located at the N-terminus of the BCMA extracellular domain or the truncated variant of the BCMA extracellular domain, between the PD-1 extracellular region and the BCMA extracellular domain or the truncated variant of the BCMA extracellular domain, or at the C-terminus of the BCMA extracellular domain or the truncated variant of the BCMA extracellular domain, between the BCMA extracellular domain or the truncated variant of the BCMA extracellular domain and the IL-7Ra transmembrane region or the mutant of the IL-7Ra transmembrane region.

[0030] The present application also provides a nucleic acid molecule comprising a sequence selected from the group consisting of:

[0031] (1) a coding sequence of the polypeptide described in any one of the embodiments herein or a fragment thereof as an amplification primer or a detection probe,

[0032] (2) a variant having at least 80% sequence identity to (1),

[0033] (3) a complementary sequence of (1) or (2);

[0034] (4) a nucleic acid comprising an expression cassette of an exogenous coding sequence and a membrane surface domain linked to the exogenous coding sequence via a multiple expression linker.

[0035] In one or more embodiments, the multiple expression linker comprises a coding sequence of a polypeptide linker sequence that can be cleaved in vivo, and / or a ribosome internal entry site (IRES) sequence.

[0036] In one or more embodiments, the exogenous coding sequence comprises any one or more selected from the group consisting of: a chimeric antigen receptor coding sequence, a cytokine region coding sequence, and a signal conversion receptor coding sequence.

[0037] In one or more embodiments, the nucleic acid molecule encoding a polypeptide is a fusion sequence containing the membrane surface domain coding sequence and the chimeric antigen receptor coding sequence. The C-terminal region of the membrane surface domain includes a transmembrane region.

[0038] In one or more embodiments, the membrane surface domain is a BCMA extracellular domain or a truncated variant thereof.

[0039] In one or more embodiments, the transmembrane region of the membrane surface domain C-terminal region includes any one or more selected from the group consisting of a BCMA transmembrane region, a transmembrane domain of alpha, beta, or zeta of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C.

[0040] In one or more embodiments, the membrane surface domain comprises a BCMA extracellular domain, the transmembrane region of the C-terminal region of the membrane surface domain is the transmembrane region of BCMA.

[0041] In one or more embodiments, the membrane surface domain comprises a truncated variant of a BCMA extracellular domain, the transmembrane region of the C-terminal region of the membrane surface domain is the transmembrane region of BCMA.

[0042] In one or more embodiments, the membrane surface domain comprises a BCMA extracellular domain, the transmembrane region of the C-terminal region of the membrane surface domain is the transmembrane region of CD8a.

[0043] In one or more embodiments, the membrane surface domain comprises a truncated variant of a BCMA extracellular domain, the transmembrane region of the C-terminal region of the membrane surface domain is the transmembrane region of CD8a.

[0044] In one or more embodiments, the membrane surface domain further comprises a linker.

[0045] In one or more embodiments, the linker is located at the N-terminus of the BCMA extracellular domain or the truncated variant of the BCMA extracellular domain. In one or more embodiments, the linker is located at the C-terminus of the BCMA extracellular domain or the truncated variant of the BCMA extracellular domain, between the BCMA extracellular domain or the truncated variant of the BCMA extracellular domain and the transmembrane region.

[0046] In one or more embodiments, the linker is a soft linker or a rigid linker. Preferably, the amino acid sequence of the rigid linker is set forth in any one of SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, and SEQ ID NO: 53.

[0047] In one or more embodiments, the CAR comprises, in order, an antigen binding region that targets an antigen, a hinge region, a transmembrane region, and an intracellular domain. The antigen binding region that targets an antigen, the hinge region, the transmembrane region, and the intracellular domain are as described elsewhere herein.

[0048] In one or more embodiments, the membrane surface domain-encoding sequence and the chimeric antigen receptor-encoding sequence can be linked by a coding sequence of a polypeptide linker sequence that is cleavable in vivo. Preferably, the polypeptide linker sequence that is cleavable in vivo comprises a F2A, P2A, T2A, and E2A sequence.

[0049] In one or more embodiments, the membrane surface domain coding sequence and the chimeric antigen receptor coding sequence can be linked by a DNA sequence of an IRES. Preferably, the IRES comprises any one or more selected from the group consisting of HCV IRES, EMCV IRES, FMDV IRES, retroviral IRES, and picornaviral IRES.

[0050] In one or more embodiments, the nucleic acid molecule encoding the polypeptide is a fusion sequence containing the membrane surface domain coding sequence and the cytokine region coding sequence. The C-terminal region of the membrane surface domain includes a transmembrane region.

[0051] In one or more embodiments, the membrane surface domain is a BCMA extracellular domain or a truncated variant thereof.

[0052] In one or more embodiments, the transmembrane region of the C-terminal region of the membrane surface domain comprises any one or more selected from the group consisting of a BCMA transmembrane region, a transmembrane domain of alpha, beta, or zeta of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C.

[0053] In one or more embodiments, the membrane surface domain comprises a BCMA extracellular domain, the transmembrane region of the C-terminal region of the membrane surface domain is the transmembrane region of BCMA.

[0054] In one or more embodiments, the membrane surface domain comprises a truncated variant of a BCMA extracellular domain, the transmembrane region of the C-terminal region of the surface domain is the transmembrane region of BCMA.

[0055] In one or more embodiments, the membrane surface cytokine comprises, in order, a cytokine region, a hinge region, and a transmembrane region. The cytokine region, hinge region, and transmembrane region are as described elsewhere herein.

[0056] In one or more embodiments, the membrane surface domain coding sequence and the cytokine region coding sequence can be linked by a coding sequence for a polypeptide linker sequence that can be cleaved in vivo. In exemplary embodiments, the polypeptide linker sequence that can be cleaved in vivo comprises a F2A, P2A, T2A, and E2A sequence.

[0057] In one or more embodiments, the membrane surface domain coding sequence and the cytokine region coding sequence can be linked by a DNA sequence for an IRES. In exemplary embodiments, the IRES comprises any one or more selected from the group consisting of an HCV IRES, an EMCV IRES, an FMDV IRES, a retroviral IRES, and a picornavirus IRES.

[0058] In one or more embodiments, the nucleic acid molecule encoding the polypeptide is a fusion sequence comprising the membrane surface domain coding sequence and the signal switching receptor coding sequence. The C-terminal region of the membrane surface domain comprises a transmembrane region.

[0059] In one or more embodiments, the membrane surface domain is a BCMA extracellular domain or a truncated variant thereof.

[0060] In one or more embodiments, the transmembrane region of the C-terminal region of the membrane surface domain comprises any one or more of a transmembrane region of BCMA, a transmembrane domain of alpha, beta, or zeta of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C.

[0061] In one or more embodiments, the membrane surface domain comprises a BCMA extracellular domain, the transmembrane region of the C-terminal region of the surface domain is the transmembrane region of BCMA.

[0062] In one or more embodiments, the membrane surface domain comprises a truncated variant of a BCMA extracellular domain, the transmembrane region of the C-terminal region of the surface domain is the transmembrane region of BCMA.

[0063] In one or more embodiments, the signal-converting receptor comprises, in order, a first signal extracellular region, a transmembrane region, and a signal intracellular region. The first signal extracellular region, transmembrane region, and signal intracellular region are as described elsewhere herein.

[0064] In one or more embodiments, the signal-converting receptor comprises, in order, a PD-1 extracellular domain, a mutant IL-7Rα transmembrane domain, and an IL-7Rα intracellular domain.

[0065] In one or more embodiments, the membrane surface domain-encoding sequence and the signal-converting receptor-encoding sequence are linked by a coding sequence for a polypeptide sequence that is cleavable in vivo. Preferably, the polypeptide sequence that is cleavable in vivo comprises a F2A, P2A, T2A, and E2A sequence.

[0066] In one or more embodiments, the membrane surface domain-encoding sequence and the signal-converting receptor-encoding sequence are linked by a DNA sequence for an IRES. Preferably, the IRES is selected from any one or more of a HCV IRES, an EMCV IRES, an FMDV IRES, a retroviral IRES, and a picornaviral IRES.

[0067] Another aspect of the present application provides a nucleic acid construct, the nucleic acid construct:

[0068] (1) expressing a polypeptide as described in any one of the embodiments herein, and / or

[0069] (2) comprising a nucleic acid molecule as described herein.

[0070] In one or more embodiments, the nucleic acid construct is a cloning vector or an expression vector.

[0071] Another aspect of the present application provides a host cell comprising, expressing, and / or secreting a polypeptide as described herein.

[0072] In one or more embodiments, the host cell comprises a nucleic acid molecule, a nucleic acid construct as described in any one of the embodiments herein.

[0073] In one or more embodiments, the host cell expresses: (1) a polypeptide comprising a cytokine protein sequence or fragment thereof, a hinge region, and a transmembrane region, and (2) a polypeptide comprising the membrane surface domain, a linker, and a transmembrane region.

[0074] In one or more embodiments, (1) is a polypeptide comprising IL-7, a hinge region, and a transmembrane region. Preferably, (1) is a polypeptide comprising IL-7, a CD8α extracellular hinge region, and a CD8α transmembrane region.

[0075] In one or more embodiments, (2) is a polypeptide comprising the BCMA extracellular domain or a truncated variant thereof, a linker, and a CD8a transmembrane region, the amino acid sequence of the linker being as set forth in any one of SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, and SEQ ID NO: 53.

[0076] In another aspect, the present application provides a pharmaceutical composition comprising any one or more of the polypeptides, nucleic acid molecules, nucleic acid constructs, and host cells described in any of the embodiments herein and a pharmaceutically acceptable carrier.

[0077] In another aspect, the present application provides the use of a membrane surface domain as an immune brake element, a molecular linker, and / or a recognition element, inducing ADCC, ADCP, and / or CDC effect.

[0078] In one or more embodiments, the membrane surface domain is located in the extracellular region of the aforementioned engineered polypeptide.

[0079] In one or more embodiments, the membrane surface domain comprises a BCMA extracellular domain or a variant thereof.

[0080] In one or more embodiments, the recognition element is a detection tag or a sorting tag.

[0081] In one or more embodiments, the membrane surface domain is connected to a transmembrane region at its C-terminus.

[0082] In one or more embodiments, the membrane surface domain is anchored to the cell membrane by a transmembrane region at its C-terminus.

[0083] In one or more embodiments, the membrane surface domain is connected to the exogenous gene expression protein by a polypeptide linker sequence that can be cleaved in vivo. The polypeptide linker sequence can be any polypeptide sequence that can be cleaved in vivo, including but not limited to F2A, P2A, T2A, and E2A sequences.

[0084] In one or more embodiments, the membrane surface domain is covalently linked to the exogenous gene expression protein at the DNA level and the RNA level, forming a fusion gene expression frame encoding the membrane surface domain gene and the exogenous gene. Preferably, the covalent linkage is by a DNA sequence or an RNA sequence of the polypeptide linker that can be cleaved in vivo, or by a DNA sequence or an RNA sequence of an internal ribosome entry site (IRES).

[0085] In one or more embodiments, the membrane surface domain further comprises a linker.

[0086] In one or more embodiments, the linker is a soft linker.

[0087] In one or more embodiments, the linker is a rigid linker.

[0088] In one or more embodiments, the amino acid sequence of the rigid linker is set forth in any one of SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, and SEQ ID NO: 53.

[0089] In one or more embodiments, the linker is located at the C-terminus of the BCMA extracellular domain or variant thereof, between the BCMA extracellular domain or variant thereof and the transmembrane region.

[0090] In one or more embodiments, the N-terminus of the membrane surface domain is linked to an extracellular recognition region. Preferably, through a hinge region or a linker. The extracellular recognition region is selected from one or more of: an antigen binding region of a chimeric antigen receptor, a cytokine region of a membrane surface cytokine, or a signal extracellular region of a signal switching receptor.

[0091] In one or more embodiments, the C-terminus of the transmembrane region is linked to an intracellular effector region. The intracellular effector region comprises an intracellular region of a chimeric antigen receptor or a second signal intracellular region of a signal switching receptor.

[0092] In one or more embodiments, the BCMA extracellular domain is set forth in SEQ ID NO: 11.

[0093] In one or more embodiments, the variant is a truncated variant.

[0094] In one or more embodiments, the variant of the BCMA extracellular domain is set forth in SEQ ID NO: 13.

[0095] The present application also provides the use of the polypeptide, nucleic acid molecule or nucleic acid construct of any one of the embodiments herein for inducing ADCC, ADCP or CDC effect of a cell, inducing immune brake, or recognizing a cell.

[0096] In one or more embodiments, the cell is a host cell of any one of the embodiments herein.

[0097] The present application also provides a method of inducing ADCC, ADCP or CDC effect, or inducing immune brake, comprising: contacting a cell having a membrane surface domain on the cell surface with an antibody (e.g., IgG) against the membrane surface domain that mediates ADCC, ADCP or CDC effect of a cell, the membrane surface domain comprising a BCMA extracellular domain or variant thereof.

[0098] In one or more embodiments, the membrane surface domain is anchored at the cell surface by a transmembrane region at its C-terminus.

[0099] In one or more embodiments, the cell expresses a polypeptide described herein.

[0100] In one or more embodiments, the cell is a host cell described herein.

[0101] In one or more embodiments, the BCMA extracellular domain is set forth in SEQ ID NO: 11.

[0102] In one or more embodiments, the membrane surface domain further comprises a linker.

[0103] In one or more embodiments, the linker is a soft linker.

[0104] In one or more embodiments, the linker is a rigid linker.

[0105] In one or more embodiments, the rigid linker has an amino acid sequence set forth in any one of SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, and SEQ ID NO: 53.

[0106] In one or more embodiments, the linker is located at the C-terminus of the BCMA extracellular domain or variant thereof, between the BCMA extracellular domain or variant thereof and the transmembrane region.

[0107] In one or more embodiments, the variant is a truncated variant.

[0108] In one or more embodiments, the variant of BCMA extracellular domain is set forth in SEQ ID NO: 13.

[0109] In one or more embodiments, the heavy chain sequence of the antibody is set forth in SEQ ID NO: 29 and the light chain sequence is set forth in SEQ ID NO: 31 in Chinese patent CN103562225B.

[0110] The present application also provides a method of identifying, detecting, or sorting a cell, comprising: contacting a cell having a membrane surface domain on its cell surface with an antibody against the membrane surface domain, the membrane surface domain comprising a BCMA extracellular domain or a variant thereof.

[0111] In one or more embodiments, the membrane surface domain is anchored at the cell surface by a transmembrane region at its C-terminus.

[0112] In one or more embodiments, the membrane surface domain further comprises a linker.

[0113] In one or more embodiments, the linker is a soft linker.

[0114] In one or more embodiments, the linker is a rigid linker.

[0115] In one or more embodiments, the amino acid sequence of the rigid linker is set forth in any one of SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, and SEQ ID NO: 53.

[0116] In one or more embodiments, the linker is located at the C-terminus of the BCMA extracellular domain or variant thereof, between the BCMA extracellular domain or variant thereof and the transmembrane region. In one or more embodiments, the cell expresses the polypeptide described herein.

[0117] In one or more embodiments, the variant is a truncated variant.

[0118] In one or more embodiments, the cell is the host cell described herein.

[0119] Advantages of the present application: the BCMA extracellular region fragment is short in length and has less impact on the exogenous gene, and is an excellent detection and sorting tag. Meanwhile, the BCMA extracellular region or variant thereof can act as a linker fragment when present alone or connected with a linker sequence, connecting different regions in the extracellular domain of the exogenous gene or connecting the extracellular domain and the transmembrane region of the exogenous gene without affecting the structure and function of the exogenous gene. In addition, in the presence of antibodies, it can effectively mediate ADCC and CDC effects, timely kill cells containing the BCMA extracellular region, and play the role of a molecular brake. BRIEF DESCRIPTION OF DRAWINGS

[0120] Figure 1 CAR expression positive rate was detected by indirect labeling method and direct labeling method.

[0121] Figure 2 Killing effect of HER2-CAR1-T and HER2-CAR3-T on target cells SKOV-3.

[0122] Figure 3 Proliferation level of T cells expressing IL-7 on the membrane surface.

[0123] Figure 4 Positive rate of BCMA detection of T cells expressing IL-7 on the membrane surface. DETAILED DESCRIPTION

[0124] The inventors have found that the extracellular domain of some immune cell antigens (e.g., B cell surface antigens) or variants thereof can induce ADCC, ADCP, and / or CDC effects, enabling molecular immune braking, while also serving as a recognition tag for the cell, and as a linker fragment to connect different regions in the extracellular domain or to connect the extracellular domain and the transmembrane region of an exogenous gene without affecting the structure and function of the exogenous gene. The B cell surface antigen is preferably BCMA.

[0125] Accordingly, the present application provides the use of a membrane surface domain as an immune braking element and / or a recognition element, a linker, to induce ADCC, ADCP, and / or CDC effects. In the process of achieving the effects of inducing immune braking, detection / sorting tag, and linker, the membrane surface domain is usually present as part of an engineered polypeptide on the cell surface. The membrane surface domain has a transmembrane region in the C-terminal direction for anchoring the polypeptide on the cell membrane. One engineered polypeptide can have multiple membrane surface domains which are the same or different.

[0126] The skilled person can select a suitable transmembrane region as needed (e.g., according to the structure or composition of the membrane). The transmembrane region includes, but is not limited to, the transmembrane region of any one of CD28, CD8, CD134 (OX40), CD137 (4-1BB), LCK, ICOS, DAP10, siglec-9, siglec-10, siglec-15, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2Rβ, IL-2Rγ, IL-4Rα, IL-7Rα, IL-10R, IL-12R, IL-15R, IL-21R, CD27, and CD40. Exemplary transmembrane regions include CD28 transmembrane region, CD8 transmembrane region, IL-7R transmembrane region. The transmembrane region is connected to its adjacent structure (e.g., membrane surface domain) by a hinge region or linker. Thus, the polypeptide can be a membrane surface protein or a transmembrane protein.

[0127] In addition, the polypeptide further includes an extracellular recognition region covalently connected to the N-terminal of the membrane surface domain, for interacting with extracellular signals. The membrane surface domain and the extracellular recognition region are connected by a hinge region or linker. In some embodiments, the polypeptide can further include an intracellular effector region at the C-terminal of the transmembrane region, for transmitting the interaction information of the extracellular signal into the cell to produce a corresponding response. The polypeptide can also include a signal peptide, and the skilled person can select a suitable signal peptide according to the desired expression form. The signal peptide can be cleaved off during the secretion of the polypeptide.

[0128] The present application also provides a use of a membrane surface domain as an immune brake element and / or a recognition element to induce ADCC, ADCP and / or CDC effect. The C-terminal region of the membrane surface domain comprises a transmembrane region. The transmembrane region can be the transmembrane region of the membrane surface domain protein itself, or the transmembrane region of another transmembrane protein, as long as it can fix the membrane surface domain on the cell membrane surface and does not negatively affect the activity and function of the membrane surface domain. The membrane surface domain is covalently linked with the exogenous gene expression protein at the DNA level in the fusion expression, in the same expression frame, and separated at the final protein expression level. In the process of achieving the function of the immune brake and detection / sorting tag, the membrane surface domain exists on the cell membrane in the form of being fixed on the membrane surface through the transmembrane region contained in the C-terminal region. In some embodiments, the exogenous gene comprises an extracellular recognition region.

[0129] In the present application, the membrane surface domain and the exogenous gene expression protein can be connected by a polypeptide linker sequence that can be cleaved in vivo. The DNA expression frame of the fusion protein formed by the connection of the membrane surface domain and the exogenous gene expression protein through the polypeptide linker is translated into a fusion polypeptide in vivo, and the fusion polypeptide is cleaved in vivo at the same time as it is translated in vivo, and the membrane surface domain and the exogenous gene expression protein are released separately. The membrane surface domain is transported to the extracellular and fixed on the cell membrane surface through the transmembrane region of the C-terminal region.

[0130] In the present application, the polypeptide linker sequence can be any polypeptide sequence that can be cleaved in vivo, for example, including but not limited to F2A, P2A, T2A and E2A sequences.

[0131] In the present application, the membrane surface domain and the exogenous gene expression protein are covalently linked at the DNA level and the RNA level to form a fusion gene expression frame encoding the membrane surface domain gene and the exogenous gene. In the fusion gene expression frame, the membrane surface domain gene can be located at the N-terminal of the exogenous gene, or at the C-terminal of the exogenous gene expression frame, as long as the expression of the membrane surface domain gene and the exogenous gene is not affected. The covalent connection can be achieved by the DNA sequence or RNA sequence of the polypeptide linker that can be cleaved in vivo, or by the DNA sequence or RNA sequence of the internal ribosome entry site (IRES). The IRES can be any known or unknown IRES in the art, as long as it can bind to the ribosome and initiate the translation of the downstream sequence in vivo. When the membrane surface domain gene and the exogenous gene are covalently linked at the DNA level and the RNA level by IRES, the membrane surface domain and the exogenous gene expression protein are respectively and independently translated to form respective and independent polypeptide products.

[0132] Depending on the function and structure of the polypeptide, the extracellular recognition region can be one or more of the following: an antigen binding region of a chimeric antigen receptor, a cytokine region (e.g., a cytokine protein expressed on the membrane surface), or a signal extracellular region of a signal switching receptor. Likewise, depending on the function and structure of the polypeptide, the intracellular effector region can be an intracellular region of a chimeric antigen receptor or a signal intracellular region of a signal switching receptor. The polypeptides described herein can be used to induce cell ADCC, ADCP, and / or CDC effects, induce immune brakes, recognize cells in.

[0133] Chimeric antigen receptors containing membrane surface domains

[0134] In the present application, the polypeptide can be a chimeric antigen receptor (CAR) containing a membrane surface domain, wherein the membrane surface domain is located in the N-terminal direction of the transmembrane region of the chimeric antigen receptor. The position of the membrane surface domain is not limited by the elements of the chimeric antigen receptor, as long as the membrane surface domain can be recognized by its ligand (e.g., an antibody).

[0135] Therefore, the chimeric antigen receptor comprises, in order: an antigen binding region targeting an antigen, a hinge region, the membrane surface domain, a transmembrane region, and an intracellular domain, or comprises, in order: an antigen binding region targeting an antigen, the membrane surface domain, a hinge region, a transmembrane region, and an intracellular domain, or comprises, in order: the membrane surface domain, an antigen binding region targeting an antigen, a hinge region, a transmembrane region, and an intracellular domain.

[0136] In embodiments of multiple membrane surface domains, the multiple membrane surface domains can be in series or dispersed at any position in the extracellular region of the chimeric antigen receptor, as long as they are all located in the N-terminal of the transmembrane region and do not affect the function of the membrane surface domain and the chimeric antigen receptor.

[0137] In the chimeric antigen receptor containing a membrane surface domain described herein, the elements other than the membrane surface domain (including but not limited to an antigen binding region targeting an antigen, a hinge region, a transmembrane region, and an intracellular domain) can be any suitable corresponding element of the chimeric antigen receptor known in the art. Therefore, the membrane surface domain can be inserted into the first to fourth generation CAR known in the art: the first generation CAR comprises an extracellular single-chain variable fragment (scFv), a transmembrane region, and a single intracellular activation signal; the second generation CAR introduces one co-stimulatory molecule on the basis of the first generation, improving the tumor killing efficiency; the third generation CAR carries multiple co-stimulatory factors such as CD28, CD134 (OX40), and CD137 (4-1BB), etc.; the fourth generation CAR increases the optional markers and promoters encoding CAR amplification on the basis of the third generation.

[0138] In a chimeric antigen receptor, the antigen to which the antigen binding region is directed can be a tumor-associated antigen. The tumor-associated antigen includes, but is not limited to, TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, ephrinB2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / Galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGFR1, AFP, and IGLL1.

[0139] The hinge region includes, but is not limited to, a CD4 extracellular hinge region, a CD8 extracellular hinge region, a CD28 extracellular hinge region, an IgGl Fc hinge region, and an IgG4 Fc hinge region.

[0140] The transmembrane region includes, but is not limited to, the transmembrane domain of alpha, beta, or zeta of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CDl la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C.

[0141] The intracellular domain includes an intracellular stimulatory region. Preferably, it also includes an intracellular costimulatory region.

[0142] The intracellular stimulatory region includes, but is not limited to, the functional signaling domain of a protein of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, FcR gamma (FCER1G), FcR beta (FcEpsilon R1b), CD79a, CD79b, Fc gamma RIIa, DAP10, DAP12.

[0143] the functional signaling domain of a protein selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8a, CD8b, IL2Rb, IL2Ry, IL7Ra, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D.

[0144] In exemplary embodiments, the chimeric antigen receptor comprising the membrane surface domain is a chimeric antigen receptor comprising a BCMA extracellular domain described herein, an antigen binding region is an antibody or antigen binding fragment thereof targeting HER2, a hinge region is a human IgGl Fc hinge region, a transmembrane region is a CD28 transmembrane region, and an intracellular domain comprises a CD28 intracellular signaling region and a CD3 zeta signaling region.

[0145] In exemplary embodiments, the chimeric antigen receptor comprising the membrane surface domain is a chimeric antigen receptor comprising a BCMA extracellular domain described herein, an antigen binding region is an antibody or antigen binding fragment thereof targeting HER2, a hinge region is a CD28 hinge region, a transmembrane region is a CD28 transmembrane region, and an intracellular domain comprises a CD28 intracellular signaling region and a CD3 zeta signaling region.

[0146] In exemplary embodiments, in the chimeric antigen receptor containing the membrane surface domain, the membrane surface domain is a truncated variant of the BCMA extracellular domain described herein, the antigen binding region is an antibody or antigen binding fragment thereof targeting HER2, the hinge region is a CD28 hinge region, the transmembrane region is a CD28 transmembrane region, and the intracellular domain comprises a CD28 intracellular stimulatory region and a CD3 zeta stimulatory region.

[0147] In exemplary embodiments, in the chimeric antigen receptor containing the membrane surface domain, the membrane surface domain is a truncated variant of the BCMA extracellular domain described herein, the antigen binding region is an antibody or antigen binding fragment thereof targeting HER2, the hinge region is an IgG4 Fc hinge region, the transmembrane region is a CD8 transmembrane region, and the intracellular domain comprises a 4-1BB intracellular stimulatory region and a CD3 zeta stimulatory region.

[0148] In the present application, the nucleic acid encoding the polypeptide can be a fusion gene containing a gene encoding the membrane surface domain and a gene encoding the chimeric antigen receptor (CAR). The gene encoding the membrane surface domain can be located at the N-terminus of the gene encoding the CAR, or at the C-terminus thereof. The C-terminal region of the membrane surface domain includes the transmembrane region. The gene encoding the membrane surface domain and the gene encoding the chimeric antigen receptor (CAR) are connected by a coding DNA sequence of the polypeptide linker that can be cleaved in vivo or a DNA sequence of the internal ribosome entry site (IRES).

[0149] In exemplary embodiments, the membrane surface domain is a BCMA extracellular domain or a truncated variant thereof as described herein, the C-terminal region of which includes a transmembrane region selected from the group consisting of a BCMA transmembrane region, a transmembrane domain of alpha, beta, or zeta of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CDl la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C.

[0150] In exemplary embodiments, the membrane surface domain includes a BCMA extracellular domain, the C-terminal region of which includes a transmembrane region of BCMA or a CD8 alpha transmembrane region. An exemplary amino acid sequence of the membrane surface domain is set forth in SEQ ID NO: 103; an exemplary coding sequence of the membrane surface domain is set forth in SEQ ID NO: 104.

[0151] In exemplary embodiments, the membrane surface domain comprises a truncated variant of a BCMA extracellular domain, and the transmembrane region comprised in the C-terminal region of the membrane surface domain is a transmembrane region of BCMA or a CD8a transmembrane region. An exemplary amino acid sequence of the membrane surface domain is set forth in SEQ ID NO: 105; an exemplary coding sequence of the membrane surface domain is set forth in SEQ ID NO: 106.

[0152] In exemplary embodiments, the membrane surface domain comprises a BCMA extracellular domain, and the transmembrane region comprised in the C-terminal region of the membrane surface domain is a transmembrane region of CD8a. In exemplary embodiments, the membrane surface domain further comprises a linker, which is located at the C-terminus of the BCMA extracellular domain, between the BCMA extracellular domain and the transmembrane region. An exemplary sequence of the linker is a rigid linker. An exemplary amino acid sequence of the rigid linker is set forth in SEQ ID NO: 51 or 53; an exemplary amino acid sequence of the membrane surface domain is set forth in SEQ ID NO: 107. An exemplary coding sequence of the membrane surface domain is set forth in SEQ ID NO: 108.

[0153] In exemplary embodiments, the membrane surface domain comprises a truncated variant of a BCMA extracellular domain, and the transmembrane region comprised in the C-terminal region of the membrane surface domain is a transmembrane region of CD8a. In exemplary embodiments, the membrane surface domain further comprises a linker, which is located at the C-terminus of the truncated variant of the BCMA extracellular domain, between the truncated variant of the BCMA extracellular domain and the transmembrane region. An exemplary sequence of the linker is a rigid linker. An exemplary amino acid sequence of the rigid linker is set forth in SEQ ID NO: 51 or 53. An exemplary amino acid sequence of the membrane surface domain is set forth in SEQ ID NO: 109. An exemplary coding sequence of the membrane surface domain is set forth in SEQ ID NO: 110.

[0154] In exemplary embodiments, the CAR comprises, in order, an antigen binding region targeting an antigen, a hinge region, a transmembrane region, and an intracellular domain. The antigen binding region targeting an antigen, the hinge region, the transmembrane region, and the intracellular domain are as described above.

[0155] In exemplary embodiments, the membrane surface domain gene and the chimeric antigen receptor gene can be linked by a coding sequence of a polypeptide linker sequence that is cleavable in vivo. In exemplary embodiments, the polypeptide linker sequence that is cleavable in vivo comprises a F2A, P2A, T2A, and E2A sequence.

[0156] In exemplary embodiments, the membrane surface domain gene and the chimeric antigen receptor gene can be linked by a DNA sequence of an IRES. In exemplary embodiments, the IRES is selected from any one of the group consisting of HCV IRES, EMCV IRES, FMDV IRES, retroviral IRES, and picornaviral IRES.

[0157] Membrane surface cytokines containing membrane surface domains

[0158] In the present application, the polypeptide can be a membrane surface cytokine containing a membrane surface domain described herein, whose extracellular region comprises a membrane surface domain and one or more cytokine region or cytokine fragment region.

[0159] The membrane surface domain is located at the N-terminal or C-terminal of the cytokine region or cytokine fragment region. Thus, the membrane surface cytokine can have the structure of: membrane surface domain-cytokine region (or cytokine fragment region)-transmembrane region, or cytokine region (or cytokine fragment region)-membrane surface domain-transmembrane region. In embodiments of multiple cytokine regions (or cytokine fragment regions), the membrane surface domain can be located at the N-terminal and C-terminal of any cytokine region (or cytokine fragment region). For example, in the case of two cytokine regions (or cytokine fragment regions), the membrane surface cytokine can have the structure of: membrane surface domain-first cytokine region (or cytokine fragment region)-second cytokine region (or cytokine fragment region)-transmembrane region, or first cytokine region (or cytokine fragment region)-membrane surface domain-second cytokine region (or cytokine fragment region)-transmembrane region, or first cytokine region (or cytokine fragment region)-second cytokine region (or cytokine fragment region)-membrane surface domain-transmembrane region. And so on. Preferably, the cytokine region (or cytokine fragment region) is located at the N-terminal of the membrane surface domain.

[0160] In embodiments of multiple membrane surface domains, the multiple membrane surface domains can be in series or dispersed at any position in the extracellular region of the membrane surface cytokine (e.g. at the N-terminal and C-terminal of any cytokine region or cytokine fragment region), as long as they are all located at the N-terminal of the transmembrane region and do not affect the function of the membrane surface domain and the membrane surface cytokine.

[0161] At the N-terminal of the membrane surface domain, the C-terminal of the transmembrane region, and between the membrane surface domain, the recognition region, and the transmembrane region, they can be directly connected by a hinge region or a linker, or can have any sequence that does not affect the function of the membrane surface cytokine, such as a signal peptide, or other polypeptides that need to be expressed on the membrane surface in addition to the recognition region. The membrane surface cytokine generally does not contain an intracellular region.

[0162] The recognition region is any polypeptide that needs to be expressed on the surface of a membrane, such as a cytokine protein. In some embodiments, the polypeptide comprises the sequence of a cytokine protein at the N-terminus. Cytokine proteins include, but are not limited to, interleukins, tumor necrosis factors (TNF), interferons (IFN), colony stimulating factors (CSF), and tumor growth factors (TGF). The interleukins include, but are not limited to, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-18, and IL-21. The tumor necrosis factors include, but are not limited to, TNF-a and TNF-β. The interferons include, but are not limited to, IFN-a, IFN-β, and IFN-γ. The colony stimulating factors include, but are not limited to, M-CSF, G-CSF, and GM-CSF. The tumor growth factors include, but are not limited to, TGF-a, TGF-β1, TGF-β2, and TGF-β3.

[0163] In exemplary embodiments, the membrane surface cytokine comprising a membrane surface domain comprises, in order, the amino acid sequence of IL-7, a BCMA extracellular domain, and a CD8 transmembrane region. An exemplary amino acid sequence of a BCMA extracellular domain is set forth in SEQ ID NO: 11; an exemplary coding sequence of a BCMA extracellular domain is set forth in SEQ ID NO: 12.

[0164] In exemplary embodiments, the membrane surface cytokine comprising a membrane surface domain comprises, in order, the amino acid sequence of IL-7, a truncated variant of a BCMA extracellular domain, and a CD8 transmembrane region. An exemplary amino acid sequence of a truncated variant of a BCMA extracellular domain is set forth in SEQ ID NO: 13; an exemplary coding sequence of a BCMA extracellular domain is set forth in SEQ ID NO: 14.

[0165] In the present application, the nucleic acid encoding the polypeptide can be a fusion gene comprising a gene encoding the membrane surface domain and a gene encoding the membrane surface cytokine. The gene encoding the membrane surface domain can be located at the N-terminus of the gene encoding the membrane surface cytokine, or at the C-terminus thereof. The C-terminal region of the membrane surface domain includes a transmembrane region. The gene encoding the membrane surface domain and the gene encoding the membrane surface cytokine are connected by a coding DNA sequence of the polypeptide linker that can be cleaved in vivo or a DNA sequence of an internal ribosome entry site (IRES).

[0166] In exemplary embodiments, the membrane surface domain is a BCMA extracellular domain or a truncated variant thereof as described herein, the C-terminal region of which comprises a transmembrane region selected from the group consisting of a transmembrane domain of BCMA, alpha, beta, or zeta of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C.

[0167] In exemplary embodiments, the membrane surface domain comprises a BCMA extracellular domain, the C-terminal region of which comprises a transmembrane region of BCMA. An exemplary amino acid sequence of the membrane surface domain is set forth in SEQ ID NO: 103; an exemplary coding sequence of the membrane surface domain is set forth in SEQ ID NO: 104.

[0168] In exemplary embodiments, the membrane surface domain comprises a truncated variant of a BCMA extracellular domain, the transmembrane region of the C-terminal region of the surface domain is the transmembrane region of BCMA. An exemplary amino acid sequence of the membrane surface domain is set forth in SEQ ID NO: 105; an exemplary coding sequence of the membrane surface domain is set forth in SEQ ID NO: 106.

[0169] In exemplary embodiments, the membrane surface domain comprises a BCMA extracellular domain, the transmembrane region comprised in the C-terminal region of the membrane surface domain is the transmembrane region of CD8a. In exemplary embodiments, the membrane surface domain further comprises a linker, the linker is located at the C-terminus of the BCMA extracellular structure, between the BCMA extracellular domain and the transmembrane region. Exemplarily, the amino acid sequence of the linker is selected from any one of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, and SEQ ID NO: 53. Exemplarily, the coding nucleic acid sequence of the linker is selected from any one of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, and SEQ ID NO: 54. Exemplarily, the sequence of the linker is a rigid linker. Exemplarily, the amino acid sequence of the rigid linker is set forth in SEQ ID NO: 51 or 53; an exemplary amino acid sequence of the membrane surface domain is set forth in SEQ ID NO: 107. An exemplary coding sequence of the membrane surface domain is set forth in SEQ ID NO: 108.

[0170] In exemplary embodiments, the membrane surface domain comprises a truncated variant of a BCMA extracellular domain, and the transmembrane region comprised in the C-terminal region of the membrane surface domain is a transmembrane region of CD8a. In exemplary embodiments, the membrane surface domain further comprises a linker, which is located at the C-terminus of the truncated variant of the BCMA extracellular domain, between the truncated variant of the BCMA extracellular domain and the transmembrane region. Exemplarily, the amino acid sequence of the linker is selected from any one of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, and SEQ ID NO: 53. Exemplarily, the nucleic acid sequence encoding the linker is selected from any one of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, and SEQ ID NO: 54. Exemplarily, the sequence of the linker is a rigid linker. Exemplarily, the amino acid sequence of the rigid linker is set forth in SEQ ID NO: 51 or 53. Exemplarily, the amino acid sequence of the membrane surface domain is set forth in SEQ ID NO: 109. Exemplarily, the nucleic acid sequence encoding the membrane surface domain is set forth in SEQ ID NO: 110.

[0171] In exemplary embodiments, the membrane surface cytokine comprises, in order, a cytokine region, a hinge region, and a transmembrane region. The cytokine region, hinge region, and transmembrane region are as described above.

[0172] In exemplary embodiments, the membrane surface domain gene and the membrane surface cytokine gene can be linked by a coding sequence of a polypeptide linker sequence that can be cleaved in vivo. In exemplary embodiments, the polypeptide linker sequence that can be cleaved in vivo comprises F2A, P2A, T2A, and E2A sequences.

[0173] In exemplary embodiments, the membrane surface domain gene and the membrane surface cytokine gene can be linked by a DNA sequence of an IRES. In exemplary embodiments, the IRES is selected from any one of the group consisting of HCV IRES, EMCV IRES, FMDV IRES, retroviral IRES, and picornaviral IRES.

[0174] Signal conversion receptors containing membrane surface domains

[0175] The polypeptide can be a signal-converting receptor, wherein the membrane surface domain is located at the N-terminal direction of the transmembrane region of the signal- converting receptor. The signal-converting receptor can convert the extracellular recognition of one or more signals into intracellular stimulation of other signals. For example, the signal- converting receptor can be obtained by replacing the intracellular region comprising ITIM with other intracellular region comprising ITAM, converting the original inhibitory signal into an activating signal, or vice versa, replacing the intracellular region comprising ITAM with other intracellular region comprising ITIM, converting the original activating signal into an inhibitory signal. Thus, due to the competition effect and the like, the signal-converting receptor can block or weaken the stimulation of the signal corresponding to the signal extracellular region, and at the same time induce or enhance the stimulation of the signal corresponding to the signal intracellular region.

[0176] Generally, the signal-converting receptor comprises one or more signal extracellular regions, a membrane surface domain, a transmembrane region, and one or more signal intracellular regions. The signal extracellular region and the signal intracellular region are from transmembrane proteins of different signal pathways. The membrane surface domain can be located at the N-terminal or C-terminal of the signal extracellular region. Thus, the signal-converting receptor can have a structure of, for example, first signal extracellular region-membrane surface domain-transmembrane region-second signal intracellular region, or membrane surface domain-first signal extracellular region-transmembrane region-second signal intracellular region. Among them, the first signal extracellular region is the extracellular region that recognizes the first signal, which is usually the extracellular region of the first signal receptor; the second signal intracellular region is the intracellular region of the second signal receptor.

[0177] In embodiments of multiple signal extracellular regions, the membrane surface domain can be located at the N-terminal and C-terminal of any signal extracellular region. In embodiments of multiple membrane surface domains, the multiple membrane surface domains can be in series or dispersed at any position in the extracellular region of the signal-converting receptor (such as the N-terminal and C-terminal of any signal extracellular region), as long as they are all located at the N-terminal of the transmembrane region and do not affect the function of the membrane surface domain and the signal-converting receptor.

[0178] Preferably, the signal transduction receptor may further include a linker. Preferably, the linker is located between the extracellular signaling region (e.g., a first extracellular signaling region) and the membrane surface domain. Preferably, the amino acid sequence of the linker is selected from any one of SEQ ID NO:15 (GSG), SEQ ID NO:17 (GGS), SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, and SEQ ID NO:53. Preferably, the coding nucleic acid sequence of the adapter is selected from any one of SEQ ID NO:16 (ggcagcggc), SEQ ID NO:18 (ggcggcagc), SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, and SEQ ID NO:54. Preferably, the adapter can be a rigid adapter or a flexible adapter. Preferably, the rigid adapter has helical forming capability. Preferably, the amino acid sequence of the rigid linker is any one of SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, and SEQ ID NO:53. Preferably, the coding nucleic acid sequence of the rigid linker is any one of SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, and SEQ ID NO:54. Preferably, the amino acid sequence of the rigid linker is SEQ ID NO:51 or SEQ ID NO:53.

[0179] The signal extracellular region (e.g., the first signal extracellular region) can be an extracellular region of a transmembrane protein in any signaling pathway that has an interaction with an extracellular signal, including but not limited to an extracellular region of any one or more of PD-1, CTLA-4, TGF, TIM3, LAG3, and TIGIT.

[0180] The transmembrane region can be any transmembrane region capable of transmitting recognition information for an extracellular signal into the cell, such as a transmembrane region of a transmembrane protein in a signaling pathway. Suitable transmembrane regions include but are not limited to a transmembrane region of any one of CD28, CD8, CD134 (OX40), CD137 (4-1BB), GITR, LCK, ICOS, DAP10, siglec-9, siglec-10, siglec-15, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, CD40, CD226, NKG2C, NKG2D, NKp30, NKp44, NKp46, 2B4, NKp80, CD94 / NKG2A, KLRB1, KLRC3, KLRD1, KLRF1, KLRK1, KLRG1, CD161, siglec-7, and siglec-9. Preferably, the IL-7R transmembrane region; preferably, the amino acid sequence of the IL-7R transmembrane region is SEQ ID NO: 111; preferably, the encoding nucleic acid sequence of the IL-7R transmembrane region is SEQ ID NO: 112. More preferably, the mutant IL-7R transmembrane region, the amino acid sequence of the mutant IL-7R transmembrane region is SEQ ID NO: 101; preferably, the encoding nucleic acid sequence of the mutant IL-7R transmembrane region is SEQ ID NO: 102.

[0181] The intracellular region of a signaling cell (e.g., the second signaling cell intracellular region) can be an intracellular region of a transmembrane protein in any signaling pathway capable of responding to an extracellular signal to cause an intracellular effect, including but not limited to: CD28, CD8, CD134 (OX40), CD137 (4-1BB), GITR, LCK, ICOS, DAP10, siglec-9, siglec-10, siglec-15, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2Rβ, IL-2Rγ, IL-4Rα, IL-7Rα, IL-10R, IL-12R, IL-15R, IL-21R, CD27, CD40, CD226, NKG2C, NKG2D, NKp30, NKp44, NKp46, 2B4, NKp80, CD94 / NKG2A, KLRB1, KLRC3, KLRD1, KLRF1, KLRK1, KLRG1, CD161, siglec-7, and siglec-9.

[0182] In exemplary embodiments, the signal-converting receptor comprising a membrane surface domain comprises, in order: a PD-1 extracellular region, a linker, a BCMA extracellular domain described herein, a mutant IL-7Rα transmembrane region, and an IL-7Rα intracellular region.

[0183] In exemplary embodiments, the signal-converting receptor comprising a membrane surface domain comprises, in order: a PD-1 extracellular region, a linker, a truncated variant of a BCMA extracellular domain described herein, a mutant IL-7Rα transmembrane region, and an IL-7Rα intracellular region.

[0184] In the present application, the nucleic acid encoding the polypeptide can be a fusion gene comprising a gene encoding the signal-converting receptor and a gene encoding the membrane surface domain. The gene encoding the membrane surface domain can be located at the N-terminus of the gene encoding the signal-converting receptor, or at the C-terminus thereof. The C-terminal region of the membrane surface domain includes a transmembrane region. The gene encoding the membrane surface domain and the gene encoding the signal-converting receptor are connected by a DNA sequence encoding the polypeptide linker cleavable in vivo or a DNA sequence of an internal ribosome entry site (IRES).

[0185] In exemplary embodiments, the membrane surface domain is a BCMA extracellular domain or a truncated variant thereof as described herein, the C-terminal region of which comprises a transmembrane region selected from the group consisting of a transmembrane domain of BCMA, alpha, beta, or zeta of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C.

[0186] In exemplary embodiments, the membrane surface domain comprises a BCMA extracellular domain, the C-terminal region of which comprises a transmembrane region of BCMA. An exemplary amino acid sequence of the membrane surface domain is set forth in SEQ ID NO: 103; an exemplary coding sequence of the membrane surface domain is set forth in SEQ ID NO: 104.

[0187] In exemplary embodiments, the membrane surface domain comprises a truncated variant of a BCMA extracellular domain, and the transmembrane region of the C-terminal region of the surface domain is the transmembrane region of BCMA. An exemplary amino acid sequence of the membrane surface domain is set forth in SEQ ID NO: 105; an exemplary coding sequence of the membrane surface domain is set forth in SEQ ID NO: 106.

[0188] In exemplary embodiments, the signal-converting receptor comprises, in order, a first signal extracellular region, a transmembrane region, and a signal intracellular region. The first signal extracellular region, transmembrane region, and signal intracellular region are as described above.

[0189] In exemplary embodiments, the signal-converting receptor comprises, in order, a PD-1 extracellular region, a mutant IL-7Ra transmembrane region, and an IL-7Ra intracellular region.

[0190] In exemplary embodiments, the membrane surface domain gene and the signal- converting receptor gene can be linked by a coding sequence of a polypeptide linker sequence that can be cleaved in vivo. In exemplary embodiments, the polypeptide linker sequence that can be cleaved in vivo comprises a F2A, P2A, T2A, and E2A sequence.

[0191] In exemplary embodiments, the membrane surface domain gene and the signal- converting receptor gene can be linked by a DNA sequence of an IRES. In exemplary embodiments, the IRES is selected from any one of the group consisting of an HCV IRES, an EMCV IRES, an FMDV IRES, a retroviral IRES, and a picornaviral IRES.

[0192] In particular embodiments, the polypeptide comprises: 1) a signal peptide, a single chain antibody (scFv) targeting HER2, a human IgGl hinge region, a BCMA extracellular domain or a truncated BCMA extracellular domain, a CD28 transmembrane region, a CD28 intracellular stimulatory region, and a CD3 zeta stimulatory region, or 2) a signal peptide, IL-7, a BCMA extracellular domain or a truncated BCMA extracellular domain, and a CD8 transmembrane region, or 3) a PD-1 signal peptide, a PD-1 extracellular region, a linker, a BCMA extracellular domain or a truncated BCMA extracellular domain, a mutant IL-7Ra transmembrane region, and an IL-7Ra intracellular region.

[0193] An exemplary amino acid sequence of a BCMA extracellular domain is set forth in SEQ ID NO: 11; an exemplary coding sequence of a BCMA extracellular domain is set forth in SEQ ID NO: 12.

[0194] The amino acid sequence of an exemplary truncated BCMA extracellular domain is set forth in SEQ ID NO: 13; the coding sequence of an exemplary truncated BCMA extracellular domain is set forth in SEQ ID NO: 14.

[0195] The polypeptides of the application can be recombinant polypeptides, synthetic polypeptides. The polypeptides of the application can be the product of chemical synthesis, or produced using recombinant technology from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants). Depending on the host used for recombinant production, the polypeptides of the application can be glycosylated, or can be non-glycosylated. The polypeptides of the application can or can not include the initiating methionine residue.

[0196] The application also includes fragments, derivatives, and analogs of the polypeptides or domains. As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially retain the same biological function or activity of the polypeptides.

[0197] The fragments, derivatives, or analogs of the polypeptides or domains can be (i) polypeptides or domains having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues can or can not be encoded by the genetic code, or (ii) polypeptides or domains having a substituent group at one or more amino acid residues, or (iii) polypeptides formed by fusing the mature polypeptide or domain to another compound, such as a compound that increases the half-life of the polypeptide, for example, a polyethylene glycol, or (iv) polypeptides formed by adding additional amino acid sequences to the polypeptide or domain sequence (such as leader sequences or secretion sequences or sequences for purification of the polypeptide or prosequences, or fusion proteins formed with antigen IgG fragments). These fragments, derivatives, and analogs are within the scope of one skilled in the art based on the teachings herein.

[0198] The term "variant" or "mutant" refers to a peptide or polypeptide whose amino acid sequence has been changed by the insertion, deletion, or substitution of one or more amino acids as compared to a reference sequence, while retaining at least one biological activity. Mutants described in any of the embodiments herein include an amino acid sequence that has at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity to a reference sequence (such as the amino acid sequence encoded by SEQ ID NO: 2, 4, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 12, or 14 as described herein) and retains the biological activity of the reference sequence (such as as a chimeric antigen receptor, a surface cytokine expressing protein, a signal-switching receptor). Sequence identity between two aligned sequences can be calculated using, for example, BLASTp of NCBI. Mutants also include an amino acid sequence that has one or more mutations (insertions, deletions, or substitutions) in the amino acid sequence of a reference sequence, while still retaining the biological activity of the reference sequence. The number of mutations typically refers to 1-10, for example, 1-8, 1-5, or 1-3. Substitutions are preferably conservative substitutions. For example, in the art, a conservative substitution is typically made when an amino acid with similar or similar properties is used, which does not change the function of the protein or polypeptide. "Amino acids with similar or similar properties" include, for example, families of amino acid residues with similar side chains, which include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, replacing one or more sites in a polypeptide of the present application with another amino acid residue from the same side chain class will not substantially affect its activity.

[0199] Truncated variants of the polypeptides or domains can also be used in the present application, as long as the variant substantially maintains the same biological function or activity as the polypeptide or domain, such as a truncated BCMA extracellular domain variant that maintains the antigen-antibody reactivity of the BCMA extracellular domain with an antibody. An exemplary truncated BCMA extracellular domain amino acid sequence is set forth in SEQ ID NO: 13; an exemplary truncated BCMA extracellular domain coding sequence is set forth in SEQ ID NO: 14.

[0200] The polypeptides described herein can be modified polypeptides. Modifications, which generally do not alter the primary structure, include: chemical derivatization of the polypeptide in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those polypeptides that have been glycosylationally modified during synthesis and processing or further processing steps. Such modifications can be accomplished by exposing the polypeptide to enzymes that glycosylate (such as mammalian glycosylation enzymes or deglycosylation enzymes). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their proteolytic resistance or to optimize solubility.

[0201] Nucleic acid molecules encoding the polypeptides or domains are also within the scope of the present disclosure. The nucleic acid molecules can be in the form of DNA or RNA. The nucleic acid molecules described herein include sequences that have been altered by codon optimization, provided that the amino acid sequence encoded by the nucleic acid molecule is unchanged. Codon-optimized sequences can exhibit more suitable expression in a particular species. Methods for codon-optimizing nucleic acid molecule sequences are well known in the art. The coding region sequence encoding the mature polypeptide can be a degenerate variant. As used herein, "degenerate variant" refers to a nucleic acid sequence that encodes the amino acid sequence encoded by SEQ ID NO: 2, 4, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, and 100, or a domain fragment thereof, but differs from the sequence set forth in SEQ ID NO: 2, 4, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, and 100.

[0202] Nucleic acid molecules encoding the mature polypeptide include: a coding sequence that encodes only the mature polypeptide; a coding sequence that encodes the mature polypeptide and various additional coding sequences; a coding sequence that encodes the mature polypeptide (and optional additional coding sequences) and non-coding sequences. The term "nucleic acid molecule encoding a polypeptide" can be a nucleic acid molecule that includes a sequence encoding the polypeptide, or a nucleic acid molecule that includes an additional coding and / or non-coding sequence.

[0203] The nucleic acid molecules of the present application can be a coding sequence for a polypeptide or domain, or an expression cassette for a polypeptide or domain. As used herein, a coding sequence refers to a portion of a nucleic acid sequence that directly specifies the amino acid sequence of its protein product (e.g., a CAR, a single chain antibody, a hinge region, a transmembrane region, an intracellular signaling region, or a cytokine protein). The boundaries of the coding sequence are typically determined by a ribosome binding site (for prokaryotes) just upstream of the open reading frame at the 5' end of the mRNA and a transcription terminator sequence just downstream of the open reading frame at the 3' end of the mRNA. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences. As used herein, an expression cassette refers to the complete elements required for expression of a gene of interest, including a promoter, a gene coding sequence, and a PolyA tailing signal sequence. In certain embodiments, the coding sequence or expression cassette is integrated into the genome of a cell. Thus, in these embodiments, the genome of the cells described herein stably incorporates an expression cassette comprising a coding sequence for a polypeptide described herein.

[0204] The present application also relates to variants of the above-described nucleic acid molecules that encode polypeptides or fragments, analogs, and derivatives of polypeptides having the same amino acid sequence as the present application. Such variants of the polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide, which can result from natural

[0205] The present application also relates to polynucleotides that hybridize to the above-described sequences and have at least 50%, preferably at least 70%, more preferably at least 80%, 85%, 90%, 95% identity between the two sequences. The present application particularly relates to polynucleotides that hybridize to the polynucleotides described herein under stringent conditions (or stringency conditions). In the present application, "stringent conditions" refer to: (1) hybridization and washing under low ionic strength and high temperature, such as 0.2 x SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization only when the identity between the two sequences is at least 90%, more preferably 95%.

[0206] This invention also relates to complementary sequences of the above-described sequences or nucleic acid fragments that hybridize with the above-described sequences. As used herein, a "nucleic acid fragment" is at least 15 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more. Nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding desired polypeptides or domains. "Hybridization" as used herein primarily refers to nucleic acid sequence pairing under stringent conditions. An exemplary stringent condition is hybridization and washing at 65°C in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS.

[0207] In some embodiments, the nucleic acid molecule is a nucleic acid construct containing the coding sequence of the polypeptide described herein, and one or more regulatory sequences operatively linked to these sequences. The polynucleotides described herein can be manipulated in various ways to ensure polypeptide expression. The nucleic acid constructs can be manipulated before insertion into a vector, depending on the expression vector or requirements. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0208] The regulatory sequence can be a suitable promoter sequence. The promoter sequence is typically operatively linked to the coding sequence of the protein to be expressed. The promoter can be any nucleotide sequence exhibiting transcriptional activity in the selected host cell, including mutant, truncated, and heterozygous promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide homologous or heterologous to that host cell. The regulatory sequence can also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operatively linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used herein. The regulatory sequence can also be a suitable leader sequence, the untranslated region of mRNA important for translation in the host cell. The leader sequence is operatively linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used in this invention.

[0209] In certain embodiments, the nucleic acid construct is a vector. The vector can be a cloning vector, an expression vector, or a homologous recombination vector. Specifically, the coding sequence of the polypeptides herein can be cloned into a number of types of vectors, including but not limited to plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Cloning vectors can be used to provide the coding sequence of the polypeptides of the application. Expression vectors can be provided to cells in the form of vectors. Expression of the polynucleotides of the application is typically achieved by operably linking the coding sequence of the application to a promoter, and incorporating the construct into an expression vector. The vector can be suitable for replication and integration in eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of expression of the desired nucleic acid sequences. The vector can be a virus, and viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses, among others. Vector technology is well known in the art and described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Homologous recombination vectors are used to integrate the expression cassettes described herein into the host genome.

[0210] Generally, a suitable vector will comprise an origin of replication functional in at least one organism, a promoter sequence, convenient restriction sites, and one or more selectable markers.

[0211] Suitable promoters include, but are not limited to, the immediate early cytomegalovirus (CMV) promoter sequence, elongation factor-1 alpha (EF-1 alpha), the simian virus 40 (SV40) early promoter, the mouse mammary tumor virus (MMTV), the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukosis virus promoter, the Epstein-Barr virus immediate early promoter, the Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter, among others.

[0212] To assess expression of the polypeptides, the expression vectors introduced into the cells can also comprise a selectable marker gene to facilitate identification and selection of expressing cells from a population of cells sought to be transfected or infected by the viral vector. The selectable marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable markers can be flanked by appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.

[0213] The polynucleotides described herein can generally be obtained by PCR amplification. Specifically, primers can be designed based on the nucleotide sequences disclosed herein, particularly the open reading frame sequences, and the relevant sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art as templates. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified in each amplification in the correct order. Alternatively, the nucleic acid molecules described herein can be synthesized directly.

[0214] Methods of introducing nucleic acid molecules into cells and expressing genes into cells are known in the art. Nucleic acid molecules, such as vectors, can be readily introduced into host cells, e.g., mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means. Physical methods include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Biological methods include the use of DNA and RNA vectors. Chemical means include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, micelles, beads; and lipid- based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0215] As described herein, host cells express polypeptides described herein or contain nucleic acid molecules described herein. Host cells include both the final cell of interest (e.g., a T cell) and various cells used in the process of producing the cell of interest, such as E. coli cells, for example, to provide a coding sequence for a protein of the application or to provide a vector described herein. In certain embodiments, provided herein is a T cell that stably expresses a polypeptide described herein. T cells suitable for use in the application can be various types of T cells of various origins, such as PBMC derived from the peripheral blood of a healthy adult human.

[0216] Also provided herein are pharmaceutical compositions containing a polypeptide described herein and a pharmaceutically acceptable excipient, and a CAR-T cell described herein and a pharmaceutically acceptable excipient. In this context, a pharmaceutically acceptable excipient refers to a carrier and / or excipient that is compatible, in a pharmacological and / or physiological sense, with the subject and the active ingredient, including but not limited to: pH adjusters, surfactants, adjuvants, ion strength enhancers. More specifically, suitable pharmaceutically acceptable excipients can be those commonly used in the art for polypeptide or T cell administration.

[0217] Generally, a pharmaceutical composition contains a therapeutically effective amount of a polypeptide or cell. A therapeutically effective amount refers to a dosage that can achieve treatment, prevention, reduction, and / or alleviation of a disease or condition in a subject. A therapeutically effective amount can be determined according to the age, sex, condition being treated, severity of the condition, other physical conditions of the patient, and the like. In this context, a subject or patient generally refers to a mammal, particularly a human.

[0218] The present application also includes a cell therapy in which T cells are genetically modified to express a polypeptide described herein, and administering the T cells to a subject. The administered cells are capable of killing tumor cells of the recipient. The immune response elicited by the T cells can be an active or passive immune response. For example, the CAR-mediated immune response can be part of an adoptive immunotherapy procedure in which the CAR-T cells induce an immune response specific to the antigen-binding portion in the CAR.

[0219] Herein, the diseases suitable for treatment using the pharmaceutical compositions described herein are related to the single-chain antibody in the chimeric antigen receptor, the cytokine in the polypeptide, or the extracellular region in the signal transducing receptor. The diseases described herein include solid tumors and hematological tumors, such as adenocarcinoma, lung cancer, colon cancer, large intestine cancer, breast cancer, ovarian cancer, cervical cancer, stomach cancer, bile duct cancer, gallbladder cancer, esophageal cancer, pancreatic cancer, and prostate cancer, etc. solid tumors, and leukemia and lymphoma, such as B-cell lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, and acute myeloid leukemia, etc. In embodiments in which the tumor antigen is HER2, the diseases that can be treated using the chimeric antigen receptor polypeptides described herein comprising an anti-HER2 single-chain antibody are HER2 expression-related diseases; for example, HER2 expression-positive tumors, such as breast cancer, cervical cancer, and ovarian cancer.

[0220] In embodiments of the membrane surface cytokine, the cells expressing the membrane surface cytokine described herein comprising a membrane surface domain and a signal recognition region can be contacted with the corresponding signal, thereby achieving the corresponding function of the signal to the cells. For example, in the case where the signal recognition region is a cytokine protein (e.g., IL-7), the polypeptides described herein comprising a membrane surface domain and a cytokine protein sequence can activate immune cells such as T cells, NK cells, thereby enhancing the therapeutic effect of the cellular immune drugs.

[0221] In embodiments of the signal transducing receptor in which the extracellular first signal recognition region is a PD-1 extracellular region, the diseases that can be treated using the polypeptides described herein comprising a PD-1 extracellular region are PD-1-mediated immune suppression-related diseases; for example, various types of tumors expressing PD-1 ligands, including but not limited to breast cancer, colon cancer, prostate cancer, head and neck cancer, skin cancer; genito-urinary tract cancer, such as ovarian cancer, endometrial cancer, cervical cancer; kidney cancer, lung cancer, stomach cancer, small intestine cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, and thyroid cancer, etc.

[0222] The polypeptides or cells of the application can be administered alone or as a pharmaceutical composition in a manner appropriate for treating (or preventing) a disease. The amount and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease. Administration of the composition can be carried out in any convenient way, including by aerosol, injection, ingestion, infusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous injection or intraperitoneally. In one embodiment, the polypeptides or cells or pharmaceutical compositions of the application are administered to a patient by intradermal or subcutaneous injection (e.g., intravenous injection). Alternatively, the polypeptides or cells or pharmaceutical compositions can be injected directly into a tumor, lymph node or site of infection.

[0223] In some embodiments of the application, the polypeptides or cells of the application can be combined with other therapies known in the art. Such therapies include, but are not limited to, chemotherapy, radiation therapy, and immunosuppressive agents. For example, treatment can be combined with a radiation or chemotherapy agent known in the art for treating a tumor antigen-mediated disease.

[0224] In certain embodiments, a kit is also provided herein, which contains a nucleic acid construct, e.g., a vector, described herein. The kit can also contain various reagents suitable for transfecting the nucleic acid construct into a cell, and optionally, instructions for a person of skill in the art to transfect the nucleic acid construct into a cell.

[0225] In certain embodiments, the application also provides a method of inducing ADCC, ADCP or CDC effect, or inducing immune brake, comprising: contacting a cell having a membrane surface domain on its surface with an antibody (e.g., IgG) against a membrane surface domain described herein, which antibody is capable of mediating ADCC, ADCP or CDC effect of the cell. The application also provides a method of recognizing, detecting, sorting cells, comprising: contacting a cell having a membrane surface domain on its surface with an antibody against a membrane surface domain described herein, which membrane surface domain comprises a BCMA extracellular domain or a variant thereof. The membrane surface domain is anchored on the cell surface by a transmembrane region at its C-terminus. Preferably, the cell expresses a polypeptide described herein.

[0226] In a specific embodiment, the polypeptide is a chimeric antigen receptor comprising an amino acid sequence encoded by the sequence set forth in SEQ ID NO: 4, a T cell expressing the polypeptide can induce higher cell killing compared to a T cell expressing the same polypeptide without the BCMA extracellular domain, and can achieve cell lysis and molecular brake under the action of a BCMA antibody, while a T cell expressing the polypeptide can be accurately and easily detected and sorted by a BCMA flow antibody.

[0227] In specific embodiments, the polypeptide is an IL-7 expression protein comprising an amino acid sequence encoded by a sequence set forth in SEQ ID NO: 58, and a T cell expressing the polypeptide can achieve cell lysis and molecular brake under the action of a BCMA antibody compared to a T cell expressing the same polypeptide without the BCMA extracellular domain, while the T cell expressing the polypeptide can be accurately and easily detected and sorted by a BCMA flow antibody.

[0228] In specific embodiments, the polypeptide is a signal conversion receptor comprising an amino acid sequence encoded by a sequence selected from any one of the sequences set forth in SEQ ID NO: 90, 92, 94, 96, 98 and 100, and a T cell expressing the polypeptide can achieve cell lysis and molecular brake under the action of a BCMA antibody compared to a T cell expressing the same polypeptide without the BCMA extracellular domain, while the T cell expressing the polypeptide can be accurately and easily detected and sorted by a BCMA flow antibody.

[0229] The embodiments related to the present application will be described in detail below with reference to the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. If the specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art (for example, refer to J. Sambrook et al., Huang Peitang et al. Translated from Molecular Cloning Experiment Guide, Third Edition, Science Press) or according to the product manual are used. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be obtained by purchase.

[0230] Examples

[0231] Part of the experimental materials and instruments

[0232] OKT3 was purchased from ThermoFisher, item number: 14-0037-82

[0233] CD28 antibody was purchased from ThermoFisher, item number: 14-0281-82

[0234] The electrotransformation instrument was Lonza Nucleofector 2b

[0235] Example 1, construction of expression vector expressing chimeric antigen receptor

[0236] Two genes encoding chimeric antigen receptors (CARs) targeting HER2 were synthesized, named HER2-CAR1 and HER2-CAR3. HER2-CAR1 comprises, from 5' to 3', a signal peptide, a single-chain antibody (scFv) targeting HER2, a CD28 extracellular region, a CD28 transmembrane region, a CD28 intracellular stimulation region, and a CD3 zeta stimulation region, the amino acid sequence of which is shown as SEQ ID NO: 1, and the coding sequence of which is shown as SEQ ID NO: 2. HER2-CAR3 comprises, from 5' to 3', a signal peptide, a single-chain antibody (scFv) targeting HER2, a BCMA extracellular region, a CD28 transmembrane region, a CD28 intracellular stimulation region, and a CD3 zeta stimulation region, the amino acid sequence of which is shown as SEQ ID NO: 3, and the coding sequence of which is shown as SEQ ID NO: 4. The HER2 scFv sequence used in the present application is derived from the single-chain antibody portion in the 4D5-5-BBZ CAR described in SEQ ID NO: 43 of Chinese patent application CN106163547A, which is incorporated herein by reference in its entirety. The 4D5-5 in CN106163547A is a mutant HER2 scFv, which has significantly reduced affinity for HER2 compared to the wild-type scFv 4D5 (see Table 3 on pages 194 / 237 of CN106163547A).

[0237] The pKB20 vector was constructed according to the method described in Example 1 on page 21 of the specification of PCT application WO2022078310A1. The pKB20 vectors containing the HER2-CAR1 and HER2-CAR3 genes were constructed according to the method described in the example, specifically, the DNA sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4 were commissioned to be synthesized commercially, and after adding a linker containing the corresponding enzyme digestion site to both ends of the sequence with a terminal ligase, they were cloned into the prepared pKB20 vector according to the method described in Example 1 on page 21 of the specification of WO2022078310A1, and were named pKB20-HER2-CAR1 and pKB20-HER2-CAR3, respectively. The recombinant plasmids obtained above were transformed into E. coli (DH5c), and after sequencing, the high-quality plasmids of the respective recombinant expression vectors were obtained by extraction and purification of the plasmids using the Qiagen plasmid purification kit.

[0238] Example 2, Construction of an expression vector expressing the membrane surface cytokine IL-7

[0239] A membrane surface cytokine IL-7 encoding sequence having the structure and sequence shown in the following table was synthesized, wherein the amino acid sequence of the Myc tag included in mbIL-7-1 is shown as SEQ ID NO: 5, and the encoding nucleic acid sequence thereof is shown as SEQ ID NO: 6.

[0240] Table 1: Membrane surface IL-7 gene structure

[0241]

[0242]

[0243] A pKB20 vector containing the mbIL-7-1 to mbIL-7-16 genes was constructed by synthesizing the DNA sequences shown in SEQ ID NOs: 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, and 86, and referring to the method described in Example 1, and was named pKB20-mbIL-7-1 to pKB20-mbIL-7-16, respectively. The recombinant plasmids obtained as described above were transformed into E. coli (DH5c), and after sequencing, the plasmids were extracted and purified using a plasmid purification kit of Qiagen, and high-quality plasmids of each recombinant expression vector were obtained.

[0244] Example 3, Construction of an expression vector expressing a PD-1 signal conversion receptor

[0245] A PD-1 signal conversion receptor encoding sequence having the structure and sequence shown in the following table was synthesized

[0246] Table 2: PD-1 signal conversion receptor gene structure

[0247]

[0248]

[0249] The DNA sequences shown in SEQ ID NO: 88, 90, 92, 94, 96, 98, and 100 were synthesized by a commercial synthesis, and pKB20 vectors containing the PD-1-m7R, PD-1-m7R1, PD-1-m7R2, PD-1-m7R3, PD-1-m7R4, PD-1-m7R5, and PD-1-m7R6 genes were constructed by referring to the method described in Example 1, and were designated as pKB20-PD-1-m7R, pKB20-PD-1-m7R1, pKB20-PD-1-m7R2, pKB20-PD-1-m7R3, pKB20-PD-1-m7R4, pKB20-PD-1-m7R5, and pKB20-PD-1-m7R6, respectively. The recombinant plasmids thus obtained were transformed into E. coli (DH5c), and after being confirmed to be correct by sequencing, the plasmids were extracted and purified using a plasmid purification kit from Qiagen, and high-quality plasmids of each recombinant expression vector were obtained.

[0250] Example 4, Construction of an expression vector expressing EGFP

[0251] A pKB20 vector containing an EGFP-encoding gene was constructed by referring to the method described in Example 1 of WO2022078310A1, and was designated as pKB20-EGFP. The recombinant plasmids thus obtained were transformed into E. coli (DH5c), and after being confirmed to be correct by sequencing, the plasmids were extracted and purified using a plasmid purification kit from Qiagen, and high-quality plasmids of each recombinant expression vector were obtained.

[0252] Example 5, Preparation of HER2-CAR-T cells

[0253] PBMCs were electroporated with the expression vectors in Example 1 to prepare HER2-targeting CAR-T cells, and the PBMCs used were purchased from AllCells and derived from the peripheral blood of a healthy adult.

[0254] 1) The suspended cells were collected in a 50-ml centrifuge tube, centrifuged at 1200 rpm for 3 min;

[0255] 2) The supernatant was discarded, physiological saline was added to resuspend, centrifuged at 1200 rpm for 3 min, the physiological saline was discarded, and this step was repeated, and the cells were counted;

[0256] 3) Three 1.5-ml centrifuge tubes were taken, 5 x 10 6 cells were added to each tube, centrifuged at 1200 rpm for 3 min;

[0257] 4) Discard the supernatant, take the electroporation kit (purchased from Lonza Company), add 18 μL of solution I reagent and 82 μL of solution II reagent, add 6 μg of pKB20 empty plasmid as a control in the first tube, add 6 μg of pKB20-HER2-CAR1 plasmid in the second tube, and add 6 μg of pKB20-HER2-CAR3 plasmid in the third tube;

[0258] 5) Transfer the cell suspension mixed with the plasmid in the centrifuge tube to the electroporation cup, place it in the electroporation instrument, select the T020 program, and perform the electric shock;

[0259] 6) Use the micropipette in the kit to transfer the electroporated cell suspension to a twelve-well plate containing AIM-V culture solution (containing 2% FBS AIM-V culture solution), mix well, and place it in a 37°C, 5% CO2 incubator for culture; at the same time, coat 6 wells of a 6-well plate with 5 μg / mL OKT-3 and 5 μg / mL CD28 antibodies, add 1 mL per well, and place the 6-well plate in a 37°C incubator.

[0260] 7) After 6 hours, transfer the cells cultured in the 37°C, 5% CO2 incubator after electroporation into the 6-well plate coated with OKT-3 and CD28 antibodies, and add IL-2 to a final concentration of 100 IU / mL, add culture solution to 3 mL, and culture for 4-5 days. Observe the growth of T cells, and obtain HER2-CAR1-T cells and HER2-CAR3-T cells, respectively; the cells transfected with pKB20 empty plasmid are Mock-T cells as a control.

[0261] Example 6, indirect labeling method and direct labeling method for detecting CAR-expressing positive cells

[0262] 1. Indirect labeling method

[0263] 1) Collect Mock-T, HER2-CAR1-T, and HER2-CAR3-T, respectively, and collect 1 x 10 6 cells for each type of cell, centrifuge at 1000 rpm for 3 min;

[0264] 2) Discard the supernatant, resuspend the cells in physiological saline, centrifuge at 1000 rpm for 3 min;

[0265] 3) Discard the supernatant, resuspend the cells in 100 μL of physiological saline, add 1 μL of biotin-labeled HER2 antigen (purchased from Kaikai Biological, item number: HER-HM402) to each tube, and incubate at 4°C for 30 minutes;

[0266] 4) Add an appropriate amount of physiological saline, centrifuge at 1000 rpm for 3 min, wash twice, and discard the supernatant;

[0267] 5) each add 100 μL of physiological saline to resuspend the cells, add 1 μL of PE-labeled streptavidin (purchased from ThermoFisher, item number: S20982) to each tube, mix, and incubate at 4°C for 30 min;

[0268] 6) each add an appropriate amount of physiological saline, centrifuge at 1000 rpm for 3 min, wash twice, and discard the supernatant;

[0269] 7) resuspend with 400 μL of physiological saline, and detect by flow cytometry.

[0270] 2, direct labeling method

[0271] 1) collect HER2-CAR1-T and HER2-CAR3-T respectively, collect 1 x 10 6 cells of each type, centrifuge at 1000 rpm for 3 min;

[0272] 2) discard the supernatant, resuspend the cells with physiological saline, centrifuge at 1000 rpm for 3 min;

[0273] 3) discard the supernatant, resuspend the cells with 100 μL of physiological saline, add 2 μL of BCMA flow cytometry antibody (purchased from Biolegend, item number: 357504) to each tube, and incubate at room temperature for 30 min;

[0274] 4) each add an appropriate amount of physiological saline, centrifuge at 1000 rpm for 3 min, wash twice, and discard the supernatant;

[0275] 5) resuspend with 400 μL of physiological saline, and detect by flow cytometry.

[0276] The results are shown in Figure 1 . Figure 1The results show that the ratio of HER2-CAR1-T to HER2-CAR3-T cells with CAR expression detected by indirect labeling method is 5.00% and 5.53% respectively, while the ratio of HER2-CAR1-T to HER2-CAR3-T cells with CAR expression detected by direct labeling method is 0.64% and 19.97% respectively. Since the extracellular part of HER2-CAR3 adopts low-affinity single-chain antibody 4D5-5, the affinity of the extracellular region of CAR to the antigen is significantly lower than that of the normal single-chain antibody to the antigen. Therefore, when the indirect labeling method commonly used in the art to detect CAR-expressing cells is used, the ratio of CAR-expressing cells obtained is significantly lower than that detected by direct labeling method. Direct labeling method uses the introduced BCMA extracellular region in the extracellular structure of CAR as a label, so it can more accurately detect all CAR-expressing cells, without the secondary labeling dependent on the interaction between antigen and single-chain antibody in indirect labeling method. Accordingly, HER2-CAR1 without introducing BCMA extracellular region as a label for the extracellular domain of CAR is not detected by direct labeling method. According to the positive rate value obtained by indirect labeling method, it is speculated that the real ratio of CAR-expressing cells of HER2-CAR1 should be close to that of HER2-CAR3.

[0277] The above results show that introducing the BCMA extracellular region as a detection label in the extracellular domain of the CAR structure can overcome the error in the detection of the ratio of CAR-expressing cells by indirect labeling method due to the difference in affinity between antigen and antigen-binding fragments (such as single-chain antibody), and effectively improve the accuracy of the detection of the positive rate of CAR-T cells.

[0278] Example 7, testing of cell killing function of HER2-CAR1-T and HER2-CAR3-T

[0279] The real-time label-free cell function analyzer (RTCA) of ACEA Biosciences was used to detect the in vitro killing activity of HER2-CAR1-T cells and HER2-CAR3-T cells obtained in Example 4, and the specific steps are as follows:

[0280] (1) Zero setting: add 50 μL of DMEM culture solution to each well, put it into the instrument, select step 1, and set zero;

[0281] (2) Target cell plating: human ovarian cancer cells SKOV-3 (purchased from American Type Culture Collection, ATCC) were plated at 10 4 cells / 50 μL per well in a plate containing a detection electrode, and placed for a few minutes. After the cells were stable, the instrument was put into the instrument, and step 2 was started to culture the cells;

[0282] (3) Add effector cells: after 18h of culturing target cells, observe cell index, when the cell index is 1, add effector cells Mock-T, HER2-CAR1-T and HER2-CAR3-T respectively, 50ul per well, effector to target ratio is 8:1, start step 3, after more than 72h of co-culturing, observe cell proliferation curve.

[0283] Results are shown in Table 2. Figure 2 As shown in Table 2, HER2-CAR1-T and HER2-CAR3-T both showed very obvious killing effect on target cells SKOV-3. Mock-T cells also had certain background killing effect on target cells, but far lower than the killing effect of HER2-CAR1-T and HER2-CAR3-T on target cells. And compared with HER2-CAR1-T, HER2-CAR3-T containing BCMA extracellular region had obviously stronger killing effect on target cells. The above results showed that introducing BCMA extracellular region into the extracellular domain of CAR as a tag did not affect the target cell killing function of CAR-T cells. And the introduction of BCMA extracellular domain in HER2-CAR3 further improved the killing effect of CAR-T cells on target cells.

[0284] Example 8, Preparation of T cells expressing membrane surface IL-7

[0285] PBMCs were electroporated with the expression vectors in Example 2 to prepare T cells expressing membrane surface IL-7 according to the following steps, and the PBMCs were purchased from AllCells company and from peripheral blood of healthy adult.

[0286] 1) Collect the suspended cells into a 50ml centrifuge tube, centrifuge at 1200rpm for 3min;

[0287] 2) Discard the supernatant, resuspend with physiological saline, centrifuge at 1200rpm for 3min, discard the physiological saline, and repeat this step, and count the cells;

[0288] 3) Take 17 1.5ml centrifuge tubes, add 5x10 6 cells to each tube, centrifuge at 1200rpm for 3min;

[0289] 4) Discard the supernatant, take the electroporation kit (purchased from Lonza company), add 18ul of solution I reagent and 82ul of solution II reagent to each tube, then add 6ug of pKB20-mbIL-7-1, pKB20-mbIL-7-2, pKB20-mbIL-7-3…….pKB20-mbIL-7-16 plasmids prepared in Example 2 and pKB20-EGFP plasmids prepared in Example 4 to each of the 17 centrifuge tubes respectively;

[0290] 5) Transfer the cell suspension containing plasmids from the centrifuge tube to the electroporation cuvette, place it in the electroporator, select program T020, and perform electroporation;

[0291] 6) Using the micropipettes provided in the kit, transfer the electroporated cell suspension to the wells of a G-REX 24-well plate containing AIM-V medium (AIM-V medium containing 2% FBS), mix well, and incubate at 37°C in a 5% CO2 incubator. Add IL-2 to a final concentration of 100 IU / mL. Simultaneously, coat 17 wells of a 6-well plate with 1 mL of antibody containing 5 μg / mL OKT-3 and 5 μg / mL CD28, and incubate the 6-well plate at 37°C.

[0292] 7) After 6 hours, the cells that had been electroporated and cultured in a 37°C, 5% CO2 incubator were transferred into a six-well plate coated with OKT-3 and CD28 antibodies, and IL-2 was added to a final concentration of 100 IU / mL. 3 ml of culture medium was added and the cells were cultured to obtain T-mbIL-7-1 cells, T-mbIL-7-2 cells, T-mbIL-7-3 cells...T-mbIL-7-16 cells and T-EGFP cells, respectively. The T-EGFP cells were used as control cells.

[0293] Example 9, Proliferation of T cells expressing membrane-surface IL-7

[0294] T cells overexpressing IL-7 on the membrane surface, prepared in Example 8, were selected and their proliferation was observed.

[0295] The results are as follows Figure 3 As shown in Table 3, at day 13 (D13), both T-EGFP and T-mbIL-7-1-T-mbIL-7-16 showed significant proliferation, while the proliferation levels of T-mbIL-7-1 to T-mbIL-7-15, which express IL-7 on the membrane surface, were significantly higher than those of T-EGFP.

[0296] Table 3: Proliferation of T cells overexpressing membrane-surface IL-7

[0297] Cell name D13 cell number (x 10 7 )]]> T-EGFP 1.35 T-mbIL-7-1 2.11 T-mbIL-7-2 3.99 T-mbIL-7-3 3.88 T-mbIL-7-4 4.02 T-mbIL-7-5 3.79 T-mbIL-7-6 3.56 T-mbIL-7-7 3.87 T-mbIL-7-8 3.63 T-mbIL-7-9 3.51 T-mbIL-7-10 3.39 T-mbIL-7-11 3.43 T-mbIL-7-12 3.21 T-mbIL-7-13 4.18 T-mbIL-7-14 3.63 T-mbIL-7-15 3.96 T-mbIL-7-16 3.91

[0298] Example 10: Detection of IL-7-positive cells on membrane surface

[0299] The cells T-EGFP and T-mbIL-7-1-T-mbIL-7-16 obtained from Example 8 were selected to detect the proportion of cells positive for exogenous gene expression by flow cytometry. The method was in accordance with the direct labeling method described in Example 6. T-mbIL-7-1 was labeled with anti-Myc flow cytometry antibody (purchased from Biolegend, item number: 626805), and T-mbIL-7-2-T-mbIL-7-16 was labeled with anti-BCMA flow cytometry antibody (purchased from Biolegend, item number: 357504). T-EGFP cells were directly detected by flow cytometry.

[0300] The results are shown in Table 4 and Table 4. Figure 4 Figure 4 The flow cytometry detection cell subpopulation chart of T-EGFP, T-mbIL-7-1, T-mbIL-7-2, T-mbIL-7-4, T-mbIL-7-6 and T-mbIL-7-12. Table 4 is the flow cytometry detection positive rate results of T-EGFP and T-mbIL-7-1-T-mbIL-7-16.

[0301] Table 4: Proportion of T cells overexpressing membrane surface IL-7 positive cells

[0302] Cell name Proportion of positive cells detected by flow cytometry (%) T-EGFP 37.24 T-mbIL-7-1 12.49 T-mbIL-7-2 29.54 T-mbIL-7-3 9.63 T-mbIL-7-4 11.56 T-mbIL-7-5 13.01 T-mbIL-7-6 8.63 T-mbIL-7-7 19.24 T-mbIL-7-8 15.73 T-mbIL-7-9 18.76 T-mbIL-7-10 28.77 T-mbIL-7-11 30.23 T-mbIL-7-12 32.50 T-mbIL-7-13 29.89 T-mbIL-7-14 29.19 T-mbIL-7-15 10.31 T-mbIL-7-16 33.79

[0303] Figure 4 In the results of Table 4, the proportion of EGFP-expressing cells in T-EGFP was 37.24%. The proportion of T-EGFP cells positive for exogenous gene EGFP expression can be considered as an experimental reference for the efficiency of electroporation of exogenous genes. The proportion of cells positive for Myc expression in T-mbIL-7-1 cells was 12.49%, which was quite different from the experimental reference result, while the proportion of cells positive for BCMA expression in T-mbIL-7-2 was 29.54%, which was closer to the experimental reference result. This indicates that as a fusion tag, the Myc tag in the extracellular structure of membrane surface IL-7 may not be able to bind the Myc antibody well, while the truncated BCMA extracellular region can bind its antibody better than Myc, thus more accurately and more closely reflecting the proportion of cells positive for exogenous gene expression.

[0304] ​T-mbIL-7-3~T-mbIL-7-16 are cells expressing membrane surface IL-7 factor and BCMA extracellular region (or truncated BCMA extracellular region) non-fusion membrane surface tag. Among them, the positive cell proportion detected by flow cytometry of several cells of T-mbIL-7-3~T-mbIL-7-9 and T-mbIL-7-15 is significantly lower than that of the experimental reference group T-EGFP. Among them, in T-mbIL-7-3, T-mbIL-7-4, T-mbIL-7-5 and T-mbIL-7-15, the truncated BCMA extracellular region as a tag is connected with the CD8 transmembrane region through a soft linker (linker 3, linker 6, linker 11) and linker 14 (CD8α extracellular hinge region) respectively; the BCMA extracellular region (full length) tag in T-mbIL-7-6, T-mbIL-7-7, T-mbIL-7-8 and T-mbIL-7-9 is connected with the CD8 transmembrane region through linker 14 (CD8α extracellular hinge region), linker 7, linker 13 (CD34 antigen epitope) and linker 13+linker 14 respectively. Correspondingly, the positive cell proportion detected by flow cytometry of T-mbIL-7-10~T-mbIL-7-14 and T-mbIL-7-16 is close to that of the T-EGFP group, which basically truly reflects the proportion of cells with positive expression of exogenous genes. The truncated BCMA extracellular region in T-mbIL-7-10~T-mbIL-7-14 and T-mbIL-7-16 is connected with the CD8 transmembrane region through a rigid linker (linker 15, linker 16, linker 17, linker 18, linker 19) and a linker with rigid properties (linker 20, CD52 extracellular stem).

[0305] The above results show that when the BCMA extracellular region or the truncated BCMA extracellular region as an extracellular tag is connected with the transmembrane region through a soft linker, a CD8α extracellular hinge region linker or a CD34 antigen epitope linker, it may be affected by antibody recognition and cannot truly function as an extracellular tag. However, when a rigid linker or a linker with rigid properties (such as CD52 stem) is used to connect the truncated BCMA extracellular region with the transmembrane region, the truncated BCMA extracellular region can basically normally bind to its antibody and better play its extracellular tag role.

[0306] Example 11, in vitro molecular brake function detection of T cells expressing membrane surface IL-7 containing BCMA extracellular region

[0307] Antibody synthesis

[0308] BCMA antibody was synthesized by a commercial company, the antibody heavy chain sequence is SEQ ID NO: 29 of Chinese patent CN103562225B, and the light chain sequence is SEQ ID NO: 31 of the patent, which is incorporated by reference in its entirety into the present application.

[0309] Antibody-dependent cytotoxicity (ADCC) assay

[0310] NK cells were obtained from the blood of healthy volunteers using an NK cell culture kit (purchased from Tongli Haiyuan, catalog number AS-01) according to the manufacturer's instructions. The T-EGFP and T-mbIL-7-1-T-mbIL-7-16 cells prepared in Example 8 were used as target cells and resuspended in AIM-V medium at 1.0 × 10⁻⁶. 4 Add 50 μL of cells / well to each well of a 96-well plate. Simultaneously, resuspend the synthesized BCMA antibody in AIM-V to a final concentration of 500 μg / mL, then add 10 μL to each well of the 96-well plate and incubate with the target cells at room temperature for 40 minutes. NK cells, serving as effector cells, are resuspended in AIM-V medium containing 2% FCS to a concentration of 2.5 × 10⁻⁶. 6 After incubating the target cells with BCMA antibody for 40 minutes, 40 μL of the above NK cell suspension was added to the target cell and BCMA antibody mixture to achieve an effector-to-target ratio of 10:1. The final concentration of BCMA antibody in the system was 50 μg / mL, and the total system volume was 100 μL. The system was gently mixed by pipetting, and the wells were incubated in a 5% CO2 incubator at 37°C for 3 hours. The antibody-dependent cytotoxicity (ADCC) effect of BCMA antibody on the above target cells was evaluated using the LDH-Cytotoxicity Colorimetric Assay Kit (Biovision, catalog number K313-500).

[0311] Complement-dependent cytotoxicity (CDC) assay

[0312] The T-EGFP and T-mbIL-7-1-T-mbIL-7-16 cells prepared in Example 8 were used as target cells and resuspended in AIM-V medium at 1.0 × 10⁻⁶. 4Cells / well / 50 μΐ were added to the wells of a 96-well plate. 10 μΐ of the aforementioned synthesized BCMA antibody, which was also resuspended in AIM-V to a final concentration of 500 μg / mL, was added to the wells of a 96-well plate and incubated with the target cells in the wells at room temperature for 40 minutes. Normal Human Serum Complement (Quidel, Cat. No. A112) was diluted 3-fold with AIM-V medium. After the 40-minute incubation of the target cells with the BCMA antibody, 40 μΐ of the diluted human serum complement was added to the wells to give a final BCMA antibody concentration of 50 μg / mL and a total volume of 100 μΐ. The system was mixed gently by pipetting and the plate was incubated in a 5% CO2 37°C incubator for 3 hours. The complement-dependent cytotoxicity (CDC) effect of the complement on the above target cells was evaluated using an LDH Lactate Dehydrogenase-Cytotoxicity Colorimetric Assay Kit (Biovision, Cat. No. K313-500). According to the instructions of the LDH Lactate Dehydrogenase-Cytotoxicity Colorimetric Assay Kit, the cytotoxicity calculation formula is as follows:

[0313]

[0314] wherein the low control value is the absorbance value corresponding to the LDH content in the supernatant under the condition of complete non-lysis of the cells, and the high control value is the absorbance value of the supernatant under the condition of complete lysis of the cells.

[0315] The ADCC and CDC toxicity results of each group of T cells expressing membrane surface IL-7 and BCMA extracellular region tag are shown in Table 5.

[0316] Table 5: ADCC and CDC toxicity of T cells overexpressing membrane surface IL-7

[0317]

[0318]

[0319] The results in Table 5 show that in T-EGFP and T-mbIL-7-1 cells without the extracellular region of BCMA, no ADCC and CDC toxicity was observed when BCMA antibody was added, while in T-mbIL-7-2 cells with the fused truncated extracellular region of BCMA tag, obvious ADCC and CDC toxicity was observed when BCMA antibody was added, indicating that for cells without the epitope of the extracellular region of BCMA, such as T-mbIL-7-1 cells, the addition of BCMA antibody does not cause ADCC to recruit NK cells or CDC to recruit complement to kill them, showing that the molecular brake element based on the mechanism of the extracellular region of BCMA has high safety and effectiveness.

[0320] Under the premise that the positive rate of T-EGFP cells is 37.24% as the reference value of all cell positive rates, in cells expressing the membrane surface IL-7 factor and the non-fused membrane surface tag of the extracellular region of BCMA (or truncated extracellular region of BCMA), T-mbIL-7-3 to T-mbIL-7-9 and T-mbIL-7-15 cells have low ADCC and CDC toxicity in the presence of anti-BCMA antibody. Correspondingly, T-mbIL-7-10 to T-mbIL-7-14 and T-mbIL-7-16 cells show higher ADCC and CDC toxicity relative to the reference cell positive rate. The above results are consistent with the results of flow detection of the proportion of positive cells, i.e. when the extracellular region of BCMA (or truncated extracellular region of BCMA) is connected to the transmembrane region through a soft linker, a CD8α extracellular hinge region linker or a CD34 epitope linker, its recognition by the BCMA antibody may be affected, thereby affecting antibody-mediated ADCC and CDC toxicity, resulting in its inability to fully exert the function of a suicide gene. When the extracellular region of BCMA (or truncated extracellular region of BCMA) is connected to the transmembrane region through a rigid linker, it can be normally recognized by the anti-BCMA antibody, and also can exert the molecular brake function of a suicide gene through the antibody, effectively eliminating cells positive for the expression of the extracellular region of BCMA (or truncated extracellular region of BCMA) through ADCC or CDC.

[0321] Example 12, Preparation of T cells expressing PD-1 signal conversion receptor

[0322] The T cells expressing PD-1 signal conversion receptor were prepared by electroporation of PBMC with the expression vector in Example 3, and the PBMC used was purchased from AllCells Company and was from the peripheral blood of a healthy adult.

[0323] 1) Collect the suspended cells into a 50 ml centrifuge tube, centrifuge at 1200 rpm for 3 min;

[0324] 2) Discard the supernatant, resuspend with physiological saline, centrifuge at 1200 rpm for 3 min, discard the physiological saline, and repeat this step, and count the cells;

[0325] 3) Take 8 1.5 ml centrifuge tubes, add 5 x 10 6 cells to each tube, centrifuge at 1200 rpm for 3 min;

[0326] 4) Discard the supernatant, take the electroporation kit (purchased from Lonza Company), add 18 μL of solution I reagent and 82 μL of solution II reagent, add 6 μg of pKB20-EGFP plasmid to the first tube as a control, and add 6 μg of pKB20-PD-1-m7R, pKB20-PD-1-m7R1, pKB20-PD-1-m7R2, pKB20-PD-1-m7R3, pKB20-PD-1-m7R4, pKB20-PD-1-m7R5 and pKB20-PD-1-m7R6 plasmids to the second to eighth tubes, respectively;

[0327] 5) Transfer the cell suspension mixed with the plasmid in the centrifuge tube to the electroporation cup, put it into the electroporation instrument, select the T020 program, and perform electroporation;

[0328] 6) Use the micropipette in the kit to transfer the electroporated cell suspension to the wells of the G-REX 24-well plate with added AIM-V culture solution (AIM-V culture solution containing 2% FBS), mix well, and place it in a 37°C, 5% CO2 incubator for culture, and add IL-2 at a final concentration of 100 IU / mL; At the same time, coat 3 wells of a 6-well plate with 5 μg / mL OKT-3 and 5 μg / mL CD28 antibodies, add 1 mL to each well, and place the 6-well plate in a 37°C incubator;

[0329] 7) After 6 hours, transfer the cells cultured in the 37°C, 5% CO2 incubator after electroporation to the 6-well plate coated with OKT-3 and CD28 antibodies, and add IL-2 at a final concentration of 100 IU / mL, add culture solution to 3 ml, and culture to obtain T-EGFP cells, T-PD-1-m7R cells, T-PD-1-m7R1 cells, T-PD-1-m7R2 cells, T-PD-1-m7R3 cells, T-PD-1-m7R4 cells, T-PD-1-m7R5 cells and T-PD-1-m7R6 cells, wherein the T-EGFP cells are control cells.

[0330] Example 13 Proliferation of T cells expressing PD-1 signal transduction receptors

[0331] The T-EGFP cells, T-PD-1-m7R cells, T-PD-1-m7R1 cells, T-PD-1-m7R2 cells, T-PD-1-m7R3 cells, T-PD-1-m7R4 cells, T-PD-1-m7R5 cells, and T-PD-1-m7R6 cells prepared in Example 11 were cultured in both ligand-stimulated and non-ligand-stimulated groups. The ligand-stimulated cell treatment method was as follows: Recombinant protein of the extracellular region of PD-1 ligand (purchased from: Sinocare, catalog number: 10084-HNAH) was diluted with PBS to a final concentration of 5 μg / mL, coated with wells at 4°C overnight, and the wells were washed three times with PBS to remove unadsorbed residual free PD-L1. Then, the culture medium from step 7) of Example 11 was added to the PD-L1-coated wells, and the T-EGFP cells, T-PD-1-m7R6 cells, and T-PD-1-m7R6 cells were cultured separately. m7R cells, T-PD-1-m7R1 cells, T-PD-1-m7R2 cells, T-PD-1-m7R3 cells, T-PD-1-m7R4 cells, T-PD-1-m7R5 cells, and T-PD-1-m7R6 cells were cultured. In the ligand-free stimulation group, the above cells were directly cultured without being coated with the PD-L1 extracellular recombinant protein. After 13 days (D13), the proliferation of each T cell under different treatment conditions was observed.

[0332] The results are shown in Table 6 below. The growth rate of T-EGFP cells in the ligand-stimulated group was significantly slower than that in the ligand-free group. This indicates that at least some T cells have a background natural PD-1 receptor on their surface, and the binding of the ligand PD-L1 to it inhibits proliferation. Compared with the ligand-free group, the D13 cell proliferation levels in the ligand-stimulated groups of T-PD-1-m7R, T-PD-1-m7R2, T-PD-1-m7R3, T-PD-1-m7R4, T-PD-1-m7R5, and T-PD-1-m7R6 cells were significantly higher. This indicates that the expression of PD-1 signal transduction receptors in these cells activated cell proliferation under the stimulation of the ligand PD-L1, and that the activation effect of the PD-1 signal transduction receptor by the PD-L1 ligand was greater than the inhibitory effect produced by its binding to the natural PD-1 receptor on T cells.

[0333] The proliferation level of the ligand stimulation group of T-PD-1-m7R1 cells was significantly lower than that of the non-ligand stimulation group, indicating that the PD-L1 ligand did not effectively activate the PD-1-m7R1 signal conversion receptor. The possible reason is that there is no linker sequence between the truncated BCMA extracellular region and the PD-1 extracellular region in the structure of PD-1-m7R1, which affects the structure of the truncated BCMA extracellular region and the PD-1 extracellular region, and further hinders the binding of PD-L1 to PD-1-m7R1 and the activation of the downstream signal.

[0334] Table 6: T cell proliferation overexpressing PD-1 signal conversion receptor

[0335]

[0336] Example 14: Detection of PD-1 signal conversion receptor expression positive cells

[0337] The cells obtained in Example 12 were detected by flow cytometry to determine the proportion of cells with exogenous gene expression. The method was based on the direct labeling method described in Example 10, and the cells were labeled with anti-BCMA flow cytometry antibody (purchased from Biolegend, item number: 357504). T-EGFP cells were directly detected by flow cytometry. The results are shown in Table 7.

[0338] Table 7: Proportion of T cells overexpressing PD-1 signal conversion receptor positive cells

[0339] Cell name Proportion of positive cells detected by flow cytometry (%) T-EGFP 35.79 T-PD-1-m7R 0.13 T-PD-1-m7R1 13.06 T-PD-1-m7R2 10.45 T-PD-1-m7R3 12.17 T-PD-1-m7R4 31.77 T-PD-1-m7R5 28.92 T-PD-1-m7R6 33.84

[0340] In the results shown in Table 7, the proportion of EGFP expression positive cells in T-EGFP was 35.79%, which can be considered as an experimental reference for the efficiency of exogenous gene electroporation based on the analysis of the results of Example 10. T-PD-1-m7R does not contain the BCMA extracellular region (or truncated BCMA extracellular region), and the anti-BCMA flow cytometry antibody basically detects no positive cells. This indicates that the proportion of BCMA antibody positive cells detected has good specificity and does not produce non-specific binding.

[0341] The positive cell ratio of T-PD-1-m7R1-T-PD-1-m7R3 cells was significantly lower than that of T-EGFP as a reference, and the positive cell ratio of T-PD-1-m7R4-T-PD-1-m7R6 cells was very close to the reference value. In T-PD-1-m7R1, the truncated BCMA extracellular region is directly connected to the PD-1 extracellular domain without any linker sequence, in T-PD-1-m7R2 and T-PD-1-m7R3, the truncated BCMA extracellular region is connected to the PD-1 extracellular domain through a soft linker, and in T-PD-1-m7R4-T-PD-1-m7R6, the truncated BCMA extracellular region is connected to the PD-1 extracellular domain through a rigid linker or a linker with rigid properties.

[0342] The above results show that when the BCMA extracellular region or the truncated BCMA extracellular region as an extracellular tag is connected to the PD-1 extracellular domain through a soft linker, it may affect its recognition by antibodies and cannot truly function as an extracellular tag. However, when the truncated BCMA extracellular region is connected to the PD-1 extracellular domain using a rigid linker or a linker with rigid properties (such as the CD52 stalk), the truncated BCMA extracellular region can bind to its antibody normally and better play its role as an extracellular tag.

[0343] Example 16, Detection of the Molecular Brake Function of T Cells Expressing PD-1 Signal Conversion Receptors Containing BCMA Extracellular Region

[0344] According to the method described in Example 11, the molecular brake function of the T cells expressing PD-1 signal conversion receptors prepared in Example 12 was detected, and the results are shown in Table 8.

[0345] Table 8: ADCC and CDC toxicity of T cells overexpressing PD-1 signal conversion receptors

[0346]

[0347] The results in Table 8 show that when BCMA antibodies are added to T-EGFP and T-PD-1-m7R cells without BCMA extracellular regions in the extracellular, there is essentially no ADCC and CDC toxicity, indicating that for cells without BCMA extracellular region epitopes, such as T-PD-1-m7R cells, the addition of BCMA antibodies will not cause ADCC to recruit NK cells or CDC to recruit complement to kill them, showing that the molecular brake element based on the BCMA extracellular region mechanism has high safety and effectiveness.

[0348] Under the premise that the T-EGFP cell positive rate of 35.79% is the reference value of all cell positive rates, in the cells expressing the PD-1 signal transduction receptor fused with the membrane surface tag of the BCMA extracellular region (or truncated BCMA extracellular region), the T-PD-1-m7R1-T-PD-1-m7R3 cells are at a lower level of ADCC and CDC toxicity in the presence of anti-BCMA antibody. Correspondingly, the T-PD-1-m7R4-T-PD-1-m7R6 cells show higher ADCC and CDC toxicity relative to the reference cell positive rate. The above results are consistent with the flow detection positive cell proportion results, that is, when the BCMA extracellular region (or truncated BCMA extracellular region) is connected with the transmembrane region without any linker or through a soft linker, its recognition by the BCMA antibody may be affected, thereby affecting the antibody-mediated ADCC and CDC toxicity, resulting in that it cannot fully exert the function of the suicide gene. When the BCMA extracellular region (or truncated BCMA extracellular region) is connected with the transmembrane region through a rigid linker, it can be normally recognized by the anti-BCMA antibody, and also can better exert the molecular brake function of the suicide gene through the antibody, effectively eliminating the cells positive for the expression of the BCMA extracellular region (or truncated BCMA extracellular region) through ADCC or CDC.

[0349] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed herein, and such changes are within the scope of protection of the present application. The entire scope of the present application is given by the appended claims and any equivalents thereof.

[0350] Sequences herein

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Claims

1. An engineered polypeptide for enhancing tumor cell killing, the amino acid sequence of said polypeptide being shown in SEQ ID NO:

3.

2. A nucleic acid molecule comprising the coding sequence of the polypeptide of claim 1.

3. A nucleic acid construct, wherein the nucleic acid construct: (1) Expressing the polypeptide of claim 1, and / or (2) It comprises the nucleic acid molecule as described in claim 2.

4. The nucleic acid construct as described in claim 3, characterized in that, The nucleic acid construct is a cloning vector or an expression vector.

5. A host cell, wherein the host cell: (1) Containing, expressing, and / or secreting the polypeptide of claim 1, and / or (2) It comprises the nucleic acid molecule of claim 2 and / or the nucleic acid construct of claim 3 or 4.

6. A pharmaceutical composition comprising the polypeptide of claim 1, the nucleic acid molecule of claim 2, the nucleic acid construct of claim 3 or 4, and any one or more of the host cell and pharmaceutically acceptable carrier of claim 5.

7. Use of the polypeptide of claim 1, the nucleic acid molecule of claim 2, or the nucleic acid construct of claim 3 or 4 in the preparation of a medicament for treating a disease selected from HER2-positive breast cancer, ovarian cancer, gastric cancer, and prostate cancer.

8. Use of the polypeptide of claim 1, the nucleic acid molecule of claim 2, or the nucleic acid construct of claim 3 or 4 in the preparation of a reagent for recognizing cells, said cells expressing the polypeptide of claim 1, said recognition comprising contacting said cells with an antibody targeting a membrane surface domain, said membrane surface domain being the BCMA extracellular domain.

9. A non-diagnostic, non-therapeutic method for detecting or sorting cells, comprising: The cell is contacted with an antibody targeting a membrane surface domain, wherein the membrane surface domain is the BCMA extracellular domain, and the cell expresses the polypeptide of claim 1.

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