Gated system

Abstract

The present application relates to a chimeric polypeptide, comprising, from an N-terminus to a C-terminus, an extracellular domain, a Notch receptor polypeptide and an intracellular domain, wherein the extracellular domain comprises a PD1 (programmed death receptor-1) extracellular segment or a functionally active fragment thereof, the PD1 extracellular segment or the functionally active fragment thereof is capable of binding to a PD1 ligand, and the Notch receptor polypeptide comprises a linker polypeptide and a transmembrane domain.

Description

A gating system Technical Field

[0001] This application relates to the field of biomedicine, specifically to a gating system. Background Technology

[0002] Cellular function is influenced by external and internal stimuli, and responses to these stimuli are encoded in the genome. Controlling the transcriptional response of cells to specific stimuli can lead to the development of live cell therapies with programmed therapeutic capabilities. The innate Notch receptor in mammals interacts with its ligand via its extracellular portion, triggering the release of intracellular transcription factors to regulate gene expression. Notch receptors can be engineered and synthesized by replacing their extracellular, transmembrane, and intracellular domains with other alternative domains.

[0003] Currently, existing Notch receptor synthetic derivatives are generally referred to as "SynNotch." However, SynNotch receptors developed based on the Notch negative regulatory domain (NRR) have low expression efficiency. Iowis Zhu et al. (Cell 185, 1431–1443, April 14, 2022) provided a class of hybrid SynNotch receptors that combine juxtamembrane domains and intracellular signal transduction domains (e.g., stimulatory and co-stimulatory domains from 4-1BB, CD28, and CD3zeta). These domains can initiate T cell activation and are accompanied by customized transcriptional regulation.

[0004] The choice of extracellular and intracellular domains for SynNotch receptors is crucial to their functional activity. Different selections of the extracellular domain in these SynNotch receptors may prevent T cell activation and transcriptional regulation. Therefore, there is a need for a SynNotch receptor with higher targeting and transcriptional activity. Summary of the Invention

[0005] Current research on SynNotch receptors still requires precise modular engineering, optimization and combination of multiple key domains (such as ECD, TMD, JMD). Different combinations of SynNotch receptors exhibit varying degrees of precision, targeting, immunogenicity, safety, and stability in signal transduction. This application provides a novel hybrid SynNotch receptor that integrates extracellular domains, using the extracellular segment of PD1 or its functionally active fragment as a gating mechanism to regulate cellular expression. Furthermore, it can be used to regulate the cellular transcriptional expression of chimeric antigen receptors (CARs). This novel SynNotch receptor has the following effects: (1) It activates T cells to secrete IL-2 and IFNγ, and the activation capacity increases with the increase of PD-L1 expression level in tumor cells; (2) It is highly sensitive to PD-L1 expression; (3) SynNotch receptor has high transcriptional expression efficiency, and its transcriptional expression capacity increases with the increase of PD-L1 expression level in tumor cells, exhibiting a good gating effect; (4) It selectively kills tumor cells expressing PD-L1 with high clinical safety; (5) It increases the safety of ROR1 target, as normal tissue cells also express low levels of ROR1. Through dual recognition of PD1 and ROR1, off-target toxicity is significantly reduced; (6) It improves the survival rate of transfected T cells, facilitating large-scale production.

[0006] This application provides a chimeric polypeptide comprising, from the N-terminus to the C-terminus:

[0007] a) an extracellular domain, wherein the extracellular domain includes an extracellular segment of PD1 (programmed death receptor-1) or a functionally active fragment thereof, the extracellular segment of PD1 or the functionally active fragment thereof being capable of binding to a PD1 ligand;

[0008] b) Notch receptor polypeptide, wherein the Notch receptor polypeptide comprises a linker polypeptide and a transmembrane domain;

[0009] c) Intracellular domains.

[0010] In some embodiments, the extracellular segment of PD1 or its functionally active fragment includes at least an immunoglobulin variable region (IgV)-like structure.

[0011] In some embodiments, the PD1 extracellular segment or its functionally active fragment further includes at least one N-glycosylation site. In some embodiments, the PD1 extracellular segment or its functionally active fragment includes four N-glycosylation sites. In some embodiments, the N-glycosylation sites are selected from one or more of the following group: N49, N58, N74, and N116.

[0012] In some embodiments, the extracellular segment of PD1 or its functionally active fragment further includes an N-terminal loop region (N-loop).

[0013] In some embodiments, the extracellular segment of PD1 or its functionally active fragment comprises soluble PD1 (sPD1). In some embodiments, the PD1 ligand comprises PD-L1 and / or PD-L2. In some embodiments, the extracellular segment of PD1 or its functionally active fragment comprises wild-type PD1 or a variant thereof.

[0014] In some embodiments, the PD1 extracellular segment or its functionally active fragment is a mammalian PD1 extracellular segment or its functionally active fragment. In some embodiments, the mammal includes humans, mice, dogs, cats, cattle, rabbits, or sheep.

[0015] In some embodiments, the PD1 extracellular segment or its functionally active fragment is the human PD1 extracellular segment or its functionally active fragment. In some embodiments, the PD1 extracellular segment or its functionally active fragment includes amino acid mutations, and the sites of the amino acid mutations include V39, L40, N41, Y43, M45, N49, K53, L97, A100, A107, A132, G124, K131, F19, I65, and / or A66. In some embodiments, the amino acid mutations include V39H, L40V, N41V, Y43H, M45E, N49G, K53T, L97V, A100V, A107I, A132I, A132F, A132T, A132V, G124S, K131Y, F19W, I65F, and / or Q66S.

[0016] In some embodiments, the extracellular segment of PD1 or its functionally active fragment comprises the amino acid sequence shown in SEQ ID NO:1.

[0017] In some embodiments, the Notch receptor peptide is derived from a type I transmembrane protein. In some embodiments, the Notch receptor peptide is derived from the Notch receptor family, the advanced glycation end product receptor family, the cadherin superfamily of cell adhesion molecules, the tyrosine phosphatase receptor family, or the epidermal growth factor (EGF) family.

[0018] In some embodiments, the linker polypeptide includes a juxtamembrane domain derived from the Notch receptor family, the advanced glycation end product receptor family, the cadherin superfamily of cell adhesion molecules, the tyrosine phosphatase receptor family, or the epidermal growth factor (EGF) family. In some embodiments, the linker polypeptide includes a juxtamembrane domain derived from Robo1, Notch1, Notch2, Notch3, Notch4, CLSTN2, AGER, PTPRF, or NRG1.

[0019] In some embodiments, the linker polypeptide includes a juxtamembrane domain derived from Notch2. In some embodiments, the linker polypeptide includes a sequence having at least about 80%, about 85%, about 90%, or about 95% homology to the amino acid sequence shown in SEQ ID NO:6. In some embodiments, the linker polypeptide includes the amino acid sequence shown in SEQ ID NO:6.

[0020] In some embodiments, the transmembrane domain includes a transmembrane domain derived from the Notch receptor family, the advanced glycation end product receptor family, the cadherin superfamily of cell adhesion molecules, the tyrosine phosphatase receptor family, or the epidermal growth factor (EGF) family. In some embodiments, the transmembrane domain includes a transmembrane domain derived from Robo1, Notch1, Notch2, Notch3, Notch4, CLSTN2, AGER, PTPRF, or NRG1.

[0021] In some embodiments, the transmembrane domain is derived from the transmembrane domain of Notch1. In some embodiments, the transmembrane domain further includes one or more proteolytic cleavage sites. In some embodiments, the transmembrane domain includes a sequence having at least about 80%, about 85%, about 90%, or about 95% homology to the amino acid sequence shown in SEQ ID NO:2. In some embodiments, the transmembrane domain further includes mutations in G318 and / or V319. In some embodiments, the transmembrane domain further includes mutations in G318A and / or V319A. In some embodiments, the transmembrane domain includes the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

[0022] In some embodiments, the Notch receptor polypeptide includes the transmembrane domain and the linker polypeptide from the N-terminus to the C-terminus.

[0023] In some embodiments, the intracellular domain includes transcription factors.

[0024] In some implementations, the transcription factor is capable of activating a second portion that specifically targets the tumor antigen.

[0025] In some embodiments, the transcription factor is selected from the group consisting of Gal4-VP16, Gal4-VP64, tetR-VP64, ZFHD1-VP64, Gal4-KRAB, and HAP1-VP16. In some embodiments, the transcription factor includes Gal4-VP64.

[0026] In some embodiments, the tumor antigen is selected from the group consisting of: ROR1, AXL, CD19, CD20, CD22, ROR1, mesothelin, CD33, IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRVIII, GD-2, BCMA, CD123, CD7, Her2, B7-H3, CLDN18, EGFR, Nectin-4, CD90, CD133, DLL3, CEA, CD4, CD5, CD123, SIRP alpha, CD70, FORR1, NY-ESO-1, MAGE A3, GP100, MART-1, EBV, and p53. In some embodiments, the tumor antigen is ROR1.

[0027] In some embodiments, the second portion comprises at least one CDR in the heavy chain variable region VH targeting ROR1, the heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO:10.

[0028] In some embodiments, the second portion comprises HCDR3, HCDR2 and HCDR1 targeting ROR1, wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO:11, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:12 and HCDR1 comprises the amino acid sequence shown in SEQ ID NO:13.

[0029] In some embodiments, the transcription factor activates a second portion via an activation sequence. In some embodiments, the second portion includes a CAR and / or a TCR.

[0030] In some embodiments, the CAR comprises ROR1VHH, and the ROR1VHH comprises the amino acid sequence shown in SEQ ID NO:10.

[0031] In some implementations, the activation sequence is UAS.

[0032] This application also provides a nucleic acid molecule that encodes the chimeric polypeptide described above.

[0033] This application also provides a cell comprising the chimeric polypeptide described above.

[0034] In some embodiments, the cell includes the following structure:

[0035] a) an extracellular domain, wherein the extracellular domain includes an extracellular segment of PD1 (programmed death receptor-1) or a functionally active fragment thereof, the extracellular segment of PD1 or the functionally active fragment thereof being capable of binding to a PD1 ligand;

[0036] b) Notch receptor polypeptide, wherein the Notch receptor polypeptide comprises a linker polypeptide and a transmembrane domain;

[0037] c) Intracellular domains;

[0038] d) Exogenous nucleic acid sequence, wherein the exogenous nucleic acid sequence includes a second nucleic acid portion that specifically targets tumor antigens.

[0039] In some embodiments, the second nucleic acid moiety encodes a CAR and / or TCR that specifically targets the tumor antigen.

[0040] In some embodiments, the second nucleic acid portion includes a nucleic acid sequence encoding an antigen-binding region capable of specifically targeting tumor antigens.

[0041] In some embodiments, the tumor antigen is selected from the group consisting of: ROR1, AXL, CD19, CD20, CD22, mesothelin, CD33, IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRVIII, GD-2, BCMA, CD123, CD7, Her2, B7-H3, CLDN18, EGFR, Nectin-4, CD90, CD133, DLL3, CEA, CD4, CD5, CD123, SIRP alpha, CD70, FORR1, NY-ESO-1, MAGE A3, GP100, MART-1, EBV, and p53. In some embodiments, the tumor antigen is ROR1.

[0042] In some embodiments, the antigen-binding region includes at least one CDR in the heavy chain variable region VH that targets ROR1, the heavy chain variable region VH containing the amino acid sequence shown in SEQ ID NO:10.

[0043] In some embodiments, the antigen-binding region comprises HCDR3, HCDR2, and HCDR1 targeting ROR1, wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO:11, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:12, and HCDR1 comprises the amino acid sequence shown in SEQ ID NO:13.

[0044] In some embodiments, the antigen-binding region includes an antibody or an antigen-binding fragment thereof.

[0045] In some embodiments, the antibody includes monoclonal antibodies, chimeric antibodies, humanized antibodies, and / or fully human antibodies.

[0046] In some embodiments, the antigen-binding fragment includes Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH, and / or dAb. In some embodiments, the antigen-binding fragment is VHH.

[0047] In some embodiments, the antigen-binding fragment is ROR1VHH, and ROR1VHH contains the amino acid sequence shown in SEQ ID NO:10.

[0048] In some embodiments, the second nucleic acid portion includes a nucleic acid sequence encoding a co-stimulatory domain. In some embodiments, the co-stimulatory signaling domain comprises an intracellular co-stimulatory signaling domain derived from one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88. In some embodiments, the co-stimulatory signaling domain is a 4-1BB co-stimulatory signaling domain. In some embodiments, the co-stimulatory signaling domain comprises the amino acid sequence shown in SEQ ID NO:14.

[0049] In some embodiments, the second nucleic acid portion includes a nucleic acid sequence encoding an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from one or more proteins selected from the group consisting of: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM. In some embodiments, the intracellular signaling domain is the intracellular signaling domain of CD3ζ. In some embodiments, the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:15.

[0050] In some embodiments, the second nucleic acid portion further includes a nucleic acid sequence encoding a transmembrane region. In some embodiments, the transmembrane region is derived from the transmembrane region of one or more proteins selected from the group consisting of: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM. In some embodiments, the transmembrane region is the transmembrane region of CD28. In some embodiments, the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:16.

[0051] In some embodiments, the second nucleic acid portion further includes a nucleic acid sequence encoding a hinge region. In some embodiments, the hinge region comprises a hinge region derived from one or more proteins selected from the group consisting of: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT. In some embodiments, the hinge region is the hinge region of CD28. In some embodiments, the hinge region comprises the amino acid sequence shown in SEQ ID NO:17.

[0052] In some embodiments, the cells include immune effector cells. In some embodiments, the cells include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral blood mononuclear cells, embryonic stem cells, lymphoprogenitor cells and / or kinetic stem cells, or variants thereof. In some embodiments, the cells are T cells.

[0053] This application also provides a nucleic acid molecule comprising a first nucleic acid portion and a second nucleic acid portion; wherein the first nucleic acid portion comprises a nucleic acid sequence encoding an extracellular domain, the extracellular domain comprising the PD1 extracellular segment or a functionally active fragment thereof; the first nucleic acid portion further comprises a nucleic acid sequence encoding a Notch receptor polypeptide, the Notch receptor polypeptide comprising a linker polypeptide and a transmembrane domain; and the first nucleic acid portion further comprises a nucleic acid sequence encoding an intracellular domain, the intracellular domain comprising a transcription factor.

[0054] In some embodiments, the extracellular segment of PD1 or its functionally active fragment includes at least one immunoglobulin variable region (IgV)-like structure.

[0055] In some embodiments, the PD1 extracellular segment or its functionally active fragment further includes at least one N-glycosylation site. In some embodiments, the PD1 extracellular segment or its functionally active fragment includes four N-glycosylation sites. In some embodiments, the N-glycosylation sites are selected from one or more of the following group: N49, N58, N74, and N116.

[0056] In some embodiments, the extracellular segment of PD1 or its functionally active fragment further includes an N-terminal loop region (N-loop).

[0057] In some embodiments, the extracellular segment of PD1 or its functionally active fragment comprises soluble PD1 (sPD1). In some embodiments, the PD1 ligand comprises PD-L1 and / or PD-L2. In some embodiments, the extracellular segment of PD1 or its functionally active fragment comprises wild-type PD1 or a variant thereof.

[0058] In some embodiments, the PD1 extracellular segment or its functionally active fragment is a mammalian PD1 extracellular segment or its functionally active fragment. In some embodiments, the mammal includes humans, mice, dogs, cats, cattle, rabbits, or sheep.

[0059] In some embodiments, the PD1 extracellular segment or its functionally active fragment is the human PD1 extracellular segment or its functionally active fragment.

[0060] In some embodiments, the extracellular segment of PD1 or its functionally active fragment includes V39, L40, N41, Y43, M45, N49, K53, L97, A100, A107, A132, G124, K131, F19, I65, and / or A66. In some embodiments, the extracellular segment of PD1 or its functionally active fragment includes V39H, L40V, N41V, Y43H, M45E, N49G, K53T, L97V, A100V, A107I, A132I, A132F, A132T, A132V, G124S, K131Y, F19W, I65F, and / or Q66S.

[0061] In some embodiments, the extracellular segment of PD1 or its functionally active fragment comprises the amino acid sequence shown in SEQ ID NO:1.

[0062] In some embodiments, the Notch receptor peptide is derived from a type I transmembrane protein. In some embodiments, the Notch receptor peptide is derived from the Notch receptor family, the advanced glycation end product receptor family, the cadherin superfamily of cell adhesion molecules, the tyrosine phosphatase receptor family, or the epidermal growth factor (EGF) family.

[0063] In some embodiments, the linker polypeptide includes a juxtamembrane domain derived from the Notch receptor family, the advanced glycation end product receptor family, the cadherin superfamily of cell adhesion molecules, the tyrosine phosphatase receptor family, or the epidermal growth factor (EGF) family. In some embodiments, the linker polypeptide includes a juxtamembrane domain derived from Robo1, Notch1, Notch2, Notch3, Notch4, CLSTN2, AGER, PTPRF, or NRG1.

[0064] In some embodiments, the linker polypeptide includes a juxtamembrane domain derived from Notch2. In some embodiments, the linker polypeptide includes a sequence having at least about 80%, about 85%, about 90%, or about 95% homology to the amino acid sequence shown in SEQ ID NO:6. In some embodiments, the linker polypeptide includes the amino acid sequence shown in SEQ ID NO:6.

[0065] In some embodiments, the transmembrane domain includes a transmembrane domain derived from the Notch receptor family, the advanced glycation end product receptor family, the cadherin superfamily of cell adhesion molecules, the tyrosine phosphatase receptor family, or the epidermal growth factor (EGF) family. In some embodiments, the transmembrane domain includes a transmembrane domain derived from Robo1, Notch1, Notch2, Notch3, Notch4, CLSTN2, AGER, PTPRF, or NRG1.

[0066] In some embodiments, the transmembrane domain is derived from the transmembrane domain of Notch1. In some embodiments, the transmembrane domain further includes one or more proteolytic cleavage sites. In some embodiments, the transmembrane domain includes a sequence having at least about 80%, about 85%, about 90%, or about 95% homology to the amino acid sequence shown in SEQ ID NO:2. In some embodiments, the transmembrane domain further includes mutations in G318 and / or V319. In some embodiments, the transmembrane domain further includes mutations in G318A and / or V319A. In some embodiments, the transmembrane domain includes the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

[0067] In some embodiments, the Notch receptor polypeptide includes the transmembrane domain and the linker polypeptide from the N-terminus to the C-terminus.

[0068] In some embodiments, the transcription factor is selected from the group consisting of Gal4-VP16, Gal4-VP64, tetR-VP64, ZFHD1-VP64, Gal4-KRAB, and HAP1-VP16. In some embodiments, the transcription factor includes Gal4-VP64.

[0069] In some embodiments, the second nucleic acid portion includes a CAR and / or TCR encoding a tumor antigen that can specifically target tumor antigens.

[0070] In some embodiments, the second nucleic acid portion includes a nucleic acid sequence encoding an antigen-binding region capable of specifically targeting tumor antigens.

[0071] In some embodiments, the tumor antigen is selected from the group consisting of: ROR1, AXL, CD19, CD20, CD22, mesothelin, CD33, IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRVIII, GD-2, BCMA, CD123, CD7, Her2, B7-H3, CLDN18, EGFR, Nectin-4, CD90, CD133, DLL3, CEA, CD4, CD5, CD123, SIRP alpha, CD70, FORR1, NY-ESO-1, MAGE A3, GP100, MART-1, EBV, and p53. In some embodiments, the tumor antigen is ROR1.

[0072] In some embodiments, the antigen-binding region includes at least one CDR in the heavy chain variable region VH that targets ROR1, the heavy chain variable region VH containing the amino acid sequence shown in SEQ ID NO:10.

[0073] In some embodiments, the antigen-binding region comprises HCDR3, HCDR2, and HCDR1 targeting ROR1, wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO:11, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:12, and HCDR1 comprises the amino acid sequence shown in SEQ ID NO:13.

[0074] In some embodiments, the antigen-binding region includes an antibody or an antigen-binding fragment thereof.

[0075] In some embodiments, the antibody includes monoclonal antibodies, chimeric antibodies, humanized antibodies, and / or fully human antibodies.

[0076] In some embodiments, the antigen-binding fragment includes Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH, and / or dAb. In some embodiments, the antigen-binding fragment is VHH.

[0077] In some embodiments, the antigen-binding fragment is ROR1VHH, and ROR1VHH contains the amino acid sequence shown in SEQ ID NO:10.

[0078] In some embodiments, the second nucleic acid portion includes a nucleic acid sequence encoding a co-stimulatory domain. In some embodiments, the co-stimulatory signaling domain comprises an intracellular co-stimulatory signaling domain derived from one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88. In some embodiments, the co-stimulatory signaling domain is a 4-1BB co-stimulatory signaling domain. In some embodiments, the co-stimulatory signaling domain comprises the amino acid sequence shown in SEQ ID NO:14.

[0079] In some embodiments, the second nucleic acid portion includes a nucleic acid sequence encoding an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from one or more proteins selected from the group consisting of: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM. In some embodiments, the intracellular signaling domain is the intracellular signaling domain of CD3ζ. In some embodiments, the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:15.

[0080] In some embodiments, the second nucleic acid portion further includes a nucleic acid sequence encoding a transmembrane region. In some embodiments, the transmembrane region is derived from the transmembrane region of one or more proteins selected from the group consisting of: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM. In some embodiments, the transmembrane region is the transmembrane region of CD28. In some embodiments, the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:16.

[0081] In some embodiments, the second nucleic acid portion further includes a nucleic acid sequence encoding a hinge region. In some embodiments, the hinge region comprises a hinge region derived from one or more proteins selected from the group consisting of: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT. In some embodiments, the hinge region is the hinge region of CD28. In some embodiments, the hinge region comprises the amino acid sequence shown in SEQ ID NO:17.

[0082] In some implementations, the first nucleic acid portion is linked to a promoter sequence.

[0083] In some embodiments, the boot sequence is selected from the group consisting of: CMV boot sequence, SFFV boot sequence, EF1α boot sequence, and PGK boot sequence. In some embodiments, the boot sequence is an SFFV boot sequence.

[0084] In some embodiments, the second nucleic acid portion is linked to an activation sequence. In some embodiments, the activation sequence is a UAS sequence.

[0085] In some embodiments, it comprises, from the 5' end to the 3' end, the following sequences in sequence: a second nucleic acid moiety activation sequence, a nucleic acid sequence encoding the antigen-binding region, a nucleic acid sequence encoding the hinge region, a nucleic acid sequence encoding the transmembrane region, a nucleic acid sequence encoding the co-stimulatory domain, a nucleic acid sequence encoding the intracellular signal transduction domain, a first nucleic acid moiety initiation sequence, a nucleic acid sequence encoding the extracellular domain, a nucleic acid sequence encoding the Notch receptor polypeptide, and a nucleic acid sequence encoding the intracellular domain.

[0086] This application also provides an expression vector comprising the aforementioned nucleic acid molecule.

[0087] In some implementations, it includes a viral vector or a non-viral vector.

[0088] In some embodiments, the viral vector includes a lentiviral vector, an adenovirus vector, an adeno-associated virus vector, or a retroviral vector. In some embodiments, the viral vector is a lentiviral vector.

[0089] This application also provides a pharmaceutical composition comprising the chimeric polypeptide, the nucleic acid molecule, the cell, the nucleic acid molecule, and / or the expression vector, and optionally a pharmaceutically acceptable carrier.

[0090] This application also provides a system for regulating cell activity, the system comprising the chimeric polypeptide, the nucleic acid molecule, the cell, the nucleic acid molecule, the expression vector, and / or the pharmaceutical composition.

[0091] This application also provides a method for regulating cell activity, the method comprising:

[0092] a) Provide cells capable of expressing the chimeric polypeptide;

[0093] b) Contact the cells with the nucleic acid molecules described above;

[0094] c) Obtain the cells and bring the cells into contact with tumor cells;

[0095] In the cells described in d), PD1 binds to ligands in tumor cells, inducing transmembrane domain cleavage and releasing intracellular domains;

[0096] e) The intracellular domains described regulate the antitumor activity of cells.

[0097] In some implementations, the intracellular domain modulates cellular antitumor activity by activating CAR and / or TCR expression.

[0098] In some embodiments, the CAR and / or TCR can specifically target tumor antigens selected from the group consisting of: ROR1, AXL, CD19, CD20, CD22, ROR1, mesothelin, CD33, IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRVIII, GD-2, BCMA, CD123, CD7, Her2, B7-H3, CLDN18, EGFR, Nectin-4, CD90, CD133, DLL3, CEA, CD4, CD5, CD123, SIRP alpha, CD70, FORR1, NY-ESO-1, MAGE A3, GP100, MART-1, EBV, and p53. In some embodiments, the tumor antigen is ROR1.

[0099] In some embodiments, the method is an in vitro method or an ex vivo method.

[0100] In some embodiments, the cells include immune effector cells. In some embodiments, the cells include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral blood mononuclear cells, embryonic stem cells, lymphoprogenitor cells and / or kinetic stem cells, or variants thereof. In some embodiments, the cells are T cells.

[0101] The use of the chimeric polypeptide, the nucleic acid molecule, the cell, the expression vector, the pharmaceutical composition, and / or the system described in this application in the preparation of medicaments for the prevention, treatment, and / or relief of diseases and / or symptoms.

[0102] The chimeric polypeptide, the nucleic acid molecule, the cell, the expression vector, the pharmaceutical composition, and / or the system described in this application are for the prevention, treatment, and / or relief of diseases and / or symptoms.

[0103] This application provides a method for preventing, treating, and / or alleviating diseases and / or symptoms, the method comprising administering to a subject in need the chimeric polypeptide, the nucleic acid molecule, the cell described in any one of the above, the nucleic acid molecule, the expression vector, the pharmaceutical composition, and / or the system described.

[0104] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0105] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0106] Figure 1 shows the structural design of the PD1 ECD chimeric peptide gating system.

[0107] Figure 2 shows the structural design of the TGFβRII ECD chimeric peptide gating system.

[0108] Figure 3 shows the PD1ECD(HA tag) gated expression assay after transfection. The upper part represents untransfected T cells, and the lower part represents PD1SNIPR ROR1CAR-T cells on the second day after transfection.

[0109] Figure 4 shows the TGFβRII ECD-gated expression assay after transfection. The HA tag is the CD22 CAR expression tag. The upper part represents untransfected T cells, and the lower part represents TGFβRII SNIPR CD22 CAR-T cells on the second day after transfection.

[0110] Figure 5 shows the PD1SNIPR ROR1CAR-T cell induction expression assay. MEC cells do not express ROR1 but express low levels of PDL1, while K562 cells do not express either PDL1 or ROR1.

[0111] Figure 6 shows the TGFβRII SNIPR-CD22 CAR-T cell induction expression assay. The HA tag is the CD22 CAR-T expression tag.

[0112] Figure 7A shows the IL2 cytokine secretion assay after co-culturing the PD1 ECD chimeric peptide-gated system with target cells. The left panel shows an effector-to-target ratio of 1:1; the right panel shows an effector-to-target ratio of 1:3. Figure 7B shows the IFNγ cytokine secretion assay after co-culturing the PD1 ECD chimeric peptide-gated system with target cells. MEC cells do not express ROR1 but express low levels of PDL1; MEC-ROR1 cells express both ROR1 and low levels of PDL1; MEC-ROR1-PDL1 cells express both ROR1 and high levels of PDL1; K562 cells do not express either PDL1 or ROR1; K562-PDL1 cells express both ROR1 and high levels of PDL1; Nalm6 cells express low levels of PDL1; MB231 cells express both ROR1 and PDL1; A549-PDL1 cells express ROR1 and overexpress PDL1; HEP3B-PDL1 cells express ROR1 and overexpress PDL1; the Mock-T group represents untransfected T cells; and the 8151v2 group represents ROR1VHH CARs modified using the PD1 ECD gating system.

[0113] Figure 8 shows the five-day real-time cell killing and re-killing assays (Incucyte Assay) of the PD1 ECD chimeric peptide gating system, where the Mock-T group represents untransfected T cells and the 8151v2 group represents ROR1VHH CARs modified using the PD1 ECD gating system. Figure 8A shows the tumor killing assay of the PD1 ECD chimeric peptide gating system on MEC target cells (ROR1- / PDL1+); Figure 8B shows the tumor killing assay of the PD1 ECD chimeric peptide gating system on MB231 target cells (ROR1+ / PDL1+); Figure 8C shows the tumor killing assay of the PD1 ECD chimeric peptide gating system on MEC-ROR1 target cells (ROR1+ / PDL1 low-level expression); Figure 8D shows the tumor killing assay of the PD1 ECD chimeric multi-gating system peptide on A549-PDL1 target cells (ROR1+ / PDL1 overexpression); Figure 8E shows the tumor killing assay of the PD1 ECD chimeric peptide gating system on HEP3B-PDL1 target cells (ROR1+ / PDL1 overexpression).

[0114] Figure 9 shows real-time monitoring of tumor cell growth using RTCA. In the figure, 8020-Cart is a CAR-T cell (#8020v2) with an scfv sequence derived from UC-961, 8030-Cart is a ROR1VHH CAR, and 8151V2-Cart represents a ROR1VHH CAR modified using a PD1 ECD gating system. The Blank group is a tumor cell control group without any cells. Figure 9A shows the tumor cells compared to natural MB231 target cells (ROR1...). + / PDL1 +Co-culture (20kJ) with an effector-target ratio of 1:5 and reculture (20kJ); Figure 9B shows co-culture with MDA-MB-231 cells (ROR1) overexpressing PDL1. + / PDL1 +++ Co-culture (20kJ) with an effector-to-target ratio of 1:5 and re-culture (20kJ); Figure 9C shows the interaction with Huh7 target cells overexpressing PDL1 (ROR1). - / PDL1 +++ Co-culture (20k), with an effect-to-target ratio of 1:5 and re-culture (20k).

[0115] Figure 10 shows the in vivo efficacy evaluation experiment of PD1SNIPR ROR1CAR-T. The scfv sequence in the 8020v2 CAR element is derived from UC-961, 8151v2 is a ROR1VHH CAR modified using the PD1 ECD gating system, and Mock-T represents untransfected T cells. Detailed Implementation

[0116] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0117] Terminology Definition

[0118] As used herein, the term "Notch receptor" is a large transmembrane protein that binds to surface ligands expressed on contacting cells, thereby transmitting signals. Natural Notch receptors include at least an extracellular domain (ECD), a Notch negative regulatory domain (NRR), a transmembrane domain (TMD), and a transcription factor (TF). In this application, the Notch receptor polypeptide is synthetically produced and contains a linker polypeptide and a transmembrane domain, but does not contain an NRR. Activation of the Notch receptor depends on cell-cell contact, where one cell is the "receiving" cell and the other is the "sending" cell. The Notch receptor is expressed on the surface of the receiving cell, and its activation is a sequential process: the extracellular domain recognizes the ligand expressed on the sending cell, leading to the hydrolysis of the transmembrane domain, releasing the intracellular domain from the membrane into the cytoplasm of the receiving cell. This intracellular domain contains a transcription factor that acts on the receiving cell for signal transduction.

[0119] As used herein, the term "PD1 extracellular segment or functionally active fragment thereof" refers to a fragment capable of binding to PD-L1 and / or PD-L2. "PD1 extracellular segment" refers to a segment containing one or more immunoglobulin (Ig)-like domains, such as at least an IgV-like structure, or at least one or more IgV-like and IgC-like structures. These domains are interconnected by disulfide bonds, enabling them to be correctly positioned on the cell surface and bind to their ligands PD-L1 and PD-L2. "Functionally active fragment" refers to a portion of or a modified fragment of the PD-1 extracellular segment, which can be any peptide or domain capable of binding to PD-L1 / PD-L2. In this application, the PD1 extracellular segment or its functionally active fragment may also be engineered, for example, by mutating one or more amino acid sites. In one embodiment, to enhance its stability, prolong its half-life, or improve its affinity, the extracellular segment of PD1 or its functionally active fragment includes amino acid mutations, the sites of which include, but are not limited to, V39, L40, N41, Y43, M45, N49, K53, L97, A100, A107, A132, G124, K131, F19, I65, and / or A66.

[0120] As used herein, the term "soluble" refers to a receptor polypeptide that does not bind to the cell membrane, and in this application, it refers to a PD1 receptor or its functionally active fragment that does not bind to the cell membrane. Soluble PD1 receptors lack the most common ligand-binding receptor polypeptides with transmembrane and cytoplasmic domains. Soluble receptors may include additional amino acid residues, such as affinity tags for polypeptide purification or providing sites for polypeptide adhesion to substrates, or constant regions targeting immunoglobulin sequences. Many cell surface receptors have naturally occurring soluble counterparts derived from proteolysis. When soluble receptor polypeptides lack sufficient transmembrane and intracellular polypeptide segments, respectively, to provide membrane anchoring or signal transduction, they are considered substantially devoid of transmembrane and intracellular polypeptide segments.

[0121] As used herein, the term "transmembrane domain" refers to a domain located on the cell membrane that contains one or more ligand-induced proteolytic cleavage sites. After binding to the extracellular receptor and ligand, specific proteases recognize and cleave these sites, simultaneously transmitting signals into the cell to activate intracellular signaling pathways. Transmembrane domains can originate from various proteins, including but not limited to members of the Notch receptor family or other types of transmembrane proteins. In this application, the transmembrane domain is derived from members of the Notch receptor family, such as Notch1, Notch2, Notch3, and Notch4.

[0122] As used herein, the term "connecting polypeptide" is located between the transmembrane domain and the intracellular domain of the Notch receptor and typically contains one or more cleavage sites. In this application, connecting polypeptide refers to the juxtamembrane domain (JMD) containing multiple LIN-12 / Notch repeats (LNRs) derived from members of the Notch receptor family, such as Notch1, Notch2, Notch3, and Notch4.

[0123] As used herein, the term "intracellular domain" refers to an intracellular domain capable of activating intracellular signal transduction. Activation of intracellular signal transduction can lead to alterations in cellular behavior, such as regulation of gene expression, cell proliferation, and cell differentiation. In this application, an intracellular domain refers to a domain containing transcriptional activators that, upon receptor activation, can enter the nucleus and regulate the transcription of specific genes. Intracellular domains may also contain co-stimulatory signaling domains, such as CD28, 4-1BB, and OX40. After binding to the extracellular receptor, the intracellular domain is cleaved, thereby releasing a fragment with transcriptional regulatory function.

[0124] As used herein, the term "antibody" generally refers to an immunoglobulin that can specifically bind to a corresponding antigen. The antibody may be secreted by immune cells (e.g., effector B cells). The antibody may be a monoclonal antibody (including full-length monoclonal antibodies comprising two light chains and two heavy chains), a polyclonal antibody, a multispecific antibody (e.g., a bispecific antibody), a humanized antibody, a fully human antibody, a chimeric antibody, and / or a camelified single-domain antibody. An "antibody" may generally comprise a protein, or an antigen-binding fragment thereof, of at least two heavy chains (HC) and two light chains (LC) linked together by disulfide bonds. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region. In some naturally occurring IgG, IgD, and IgA antibodies, the heavy chain constant region comprises three domains, CH1, CH2, and CH3. In some naturally occurring antibodies, each light chain comprises a light chain variable region (VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which alternate with more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four framework regions (FRs), arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable domains of the natural heavy and light chains each contain four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4), mostly in a β-sheet configuration, linked by three CDRs to form loops, and in some cases forming part of a β-sheet structure. The CDRs in each chain are closely clustered together by the FR regions and, together with CDRs from the other chain, form the antigen-binding site of the antibody. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0125] As used herein, the term "VHH" generally refers to an antibody containing a variable antigen-binding domain of a heavy chain antibody. VHHs may also be referred to as nanobodies (Nb) and / or single-domain antibodies. For example, a VHH may bind to ROR1. For example, a VHH may be specific for ROR1.

[0126] As used herein, the term "variable" generally refers to the fact that certain portions of the sequence of the variable domain of an antibody vary strongly, resulting in the binding and specificity of a particular antibody to its specific antigen. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, referred to as complementarity-determining regions (CDRs) or hypervariable regions (HVRs). More conserved portions within the variable domain are referred to as frames (FRs). In the art, antibody CDRs can be defined by a variety of approaches, such as the Kabat definition rule based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, 5th Edition, NIH, Bethesda, MD (1991)), the Chothia definition rule based on the location of the structural loop region (see, Al-Lazikani et al., JMol Biol 273:927-48, 1997), and the KABAT definition rule based on the concepts of IMGT ontology and the IMGT Scientific diagram rules. IMGT refers to the International ImMunoGeneTics Information System, a global reference database for immunogenetics and immunoinformatics (http: / / www.imgt.org). IMGT specifically studies immunoglobulins (IG) or antibodies, T cell receptors (TR), major histocompatibility (MH) from humans and other vertebrates, as well as immunoglobulin superfamily (IgSF), MH superfamily (MhSF), and immune system-associated proteins (RPI) from vertebrates and invertebrates. The CDRs described in this application are classified according to the IMGT definition.

[0127] As used herein, the term "chimeric antigen receptor" (CAR) generally refers to a recombinant polypeptide comprising at least an antigen-binding region, a transmembrane region, and an intracellular signaling domain that specifically binds to tumor antigens. For example, a hinge region may be included between the antigen-binding region and the transmembrane region. For example, the chimeric antigen receptor may include a signal peptide. For example, the chimeric antigen receptor may include a co-stimulatory signaling domain. The binding of the antigen-binding region of the CAR to a target antigen on the surface of a target cell leads to CAR clustering and delivers activation stimulation to CAR-containing cells. The CAR redirects the specificity of immune effector cells and triggers proliferation, cytokine production, phagocytosis, and / or production of molecules capable of mediating cell death expressing the target antigen in a major histocompatibility (MHC)-independent manner. For example, the antigen-binding region may specifically bind to ROR1. In this application, when the ECD in the constructed PD1 ECD chimeric polypeptide gating system binds to a ligand on a target cell, it triggers cleavage of the transmembrane domain, thereby releasing the intracellular domain to activate intracellular CAR expression. For example, the CAR may be ROR1CAR.

[0128] As used herein, the term "T-cell receptor" (TCR) refers to a specific receptor polypeptide on the surface of T cells that recognizes and binds to antigenic peptides presented by the major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs). For example, the antigenic peptide may be the ROR1 antigenic peptide.

[0129] As used herein, the term "PBMC" refers to mononuclear cells in peripheral blood, primarily including lymphocytes (such as T cells, B cells, and natural killer (NK) cells), monocytes, dendritic cells, and a small number of other cell types. In this application, PBMC refers to T cells. The PBMC transducing the PD1 ECD chimeric peptide described in this application refers to PD1SNIPR ROR1CAR-T, also known as: PD1 ECD-gated ROR1VHH CAR, PD1SINPR, PD1ECD ROR1CAR-T, 8151v2-CART, or 8151v2. The PBMC transducing the TGFβRII ECD chimeric peptide described in this application refers to TGFβRII SNIPR ROR1CAR-T.

[0130] As used herein, the term "tumor" generally refers to a growth formed by the proliferation of localized tissue cells. For example, the tumor may include a solid tumor. For example, the tumor may be selected from the following groups: ROR1, AXL, CD19, CD20, CD22, mesothelin, CD33, IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRVIII, GD-2, BCMA, CD123, CD7, Her2, B7-H3, CLDN18, EGFR, Nectin-4, CD90, CD133, DLL3, CEA, CD4, CD5, CD123, SIRP alpha, CD70, FOLR1, NY-ESO-1, MAGE A3, GP100, MART-1, EBV, and p53.

[0131] As used herein, the term “nucleic acid molecule” generally refers to any length of isolated nucleotide, deoxyribonucleotide, or ribonucleotide or analogue, whether isolated from its natural environment or synthesized artificially.

[0132] As used herein, the term “expression vector” generally refers to a recombinant polynucleotide construct designed for transfer between host cells and can be used for transformation purposes, such as introducing heterologous DNA into host cells.

[0133] As used herein, the term "cell" refers to a cell to which nucleic acids can be transfected, and includes prokaryotic cells used for plasmid propagation and eukaryotic cells used for nucleic acid expression and polypeptide encoding. For example, a cell may include the antigen-binding protein, the nucleic acid molecule, and / or the vector. For example, the cell may be an immune cell. The term "immune cell" generally refers to an immune cell that participates in an immune response and performs effector functions. For example, performing effector functions may include clearing foreign antigens or promoting immune effector responses. For example, immune cells may include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral blood mononuclear cells, embryonic stem cells, lymphoprogenitor cells, and / or pluripotent stem cells. For example, an immune cell may be a T cell.

[0134] As used herein, the term "specific binding" or "specific" generally refers to a highly selective biomolecular interaction process in which one molecule (such as an antibody, receptor, or ligand) can bind to another molecule (such as an antigen, ligand, or receptor) at a specific structural site. This binding is typically achieved through non-covalent bonds (such as hydrogen bonds, electrostatic interactions, hydrophobic interactions, etc.) and exhibits high specificity and affinity. In this application, the extracellular domain of PD1 (PD1 ECD) specifically binds to specific ligands (PD-L1 and PD-L2), and the antigen-binding region in the CAR structure specifically binds to tumor antigens (e.g., ROR1VHH specifically binds to the ROR1 antigen). The gating system constructed in this application enables precise targeted therapy.

[0135] As used herein, the terms “homology” or “identity” are used interchangeably and generally refer to the degree of similarity between protein or nucleic acid sequences. Homology information is used to understand the genetic relevance of certain types of proteins or nucleic acids. Homology can be determined by aligning and comparing sequences. Typically, to determine amino acid homology, a protein sequence is compared to a database of known protein sequences. Homologous sequences share common functional similarity at some point in their sequence. While a lower degree of similarity or identity does not necessarily indicate a lack of homology, a higher degree of similarity or identity usually indicates the presence of homology.

[0136] As used in this article, the term “UC-961” is a humanized anti-ROR1 monoclonal antibody product that can effectively kill ROR1 tumor cells (Clin Lymphoma Myeloma Leuk. 2015 Jun; 15 Suppl(0): S167-9.).

[0137] As used in this article, the term "RTCA" refers to Real-Time Cellular Analysis, a technique used to monitor the physiological state and behavioral changes of cells in real time and dynamically.

[0138] As used in this article, the term "IncuCyte" refers to a real-time live-cell analysis system that supports high-resolution fluorescence and bright-field image acquisition, enabling real-time recording of changes in cells within an incubator.

[0139] Invention Details

[0140] On the one hand, this application provides a chimeric receptor polypeptide, which is constructed based on the mechanism of action of the Notch receptor and includes an extracellular domain, a transmembrane domain, a juxtamembrane domain and an intracellular domain from the N segment to the C segment, wherein the extracellular domain includes the extracellular segment of PD1 or its functionally active fragment.

[0141] Notch receptor

[0142] Notch receptors are type I transmembrane proteins activated by regulated intramembrane proteolysis (RIP). Mammalians possess four distinct Notch receptors, designated Notch1, Notch2, Notch3, and Notch4. The classic mammalian Notch signaling pathway lacks intermediates; the receptor undergoes three cleavages before being transported to the nucleus (Zhu et al., Signal Transduct Target Ther. 2022; 7:95.). In signal-receiving cells, Notch receptors are first generated in the endoplasmic reticulum and then transported to the Golgi apparatus. During transport, the Notch receptor is glycosylated at an epidermal growth factor (EGF)-like repeat domain. Then, in the Golgi apparatus, the Notch receptor is cleaved into a heterodimer (S1 cleavage) and transported to the cell membrane. With the aid of ubiquitin ligases, some Notch receptors on the cell membrane are endocytosed into endosomes. Endosomals contain an acidic environment with deintegrins and metalloproteinases (ADAMs) and γ-secretase. Notch receptors in endosomes can be reclaimed onto the cell membrane, cleaved into the Notch intracellular domain (NICD), or transported to lysosomes for degradation. During signal transduction, Notch ligands are distributed on the cell membrane and can bind to Notch receptors on receiver cells. However, the ligands are inactive until ubiquitinated by Neur or Mib. After ubiquitination, the ligands can be endocytosed, thereby generating traction on the binding receptor. Without traction, the S2 site of the Notch receptor is hidden by the NRR domain, and the Notch receptor cannot be cleaved by ADAMs. Under the influence of traction, the NRR domain is extended, exposing the S2 site for cleavage. Both ADAMs and traction are necessary conditions for S2 site cleavage. After S2 cleavage, the remaining portion of the Notch receptor can be further cleaved on the cell membrane by γ-secretase or endocytosed into endosomes. In the former mode, the NICD is released on the cell membrane. In the latter mode, the remaining portion of the Notch receptor can be cleaved into the NICD or transported to lysosomes for degradation. The classic model posits that, in the absence of NICD, transcription factors (CSLs) bind to co-repressors, inhibiting the transcription of target genes. Once NICD enters the nucleus, it can bind to CSLs and recruit MAMLs, releasing co-repressors and recruiting co-activators, thereby promoting the transcription of Notch target genes.

[0143] In simple terms, the Notch signaling pathway is a continuous process. The ECD binds to ligands on target cells, triggering the cleavage of the transmembrane domain and releasing the intracellular domain to activate intracellular signaling pathways. Based on this principle, the artificial synthesis of Notch derivatives has shown great promise in the treatment of tumors (Leonardo Morsut et al., Cell. 2016 February 11; 164(4):780–791.).

[0144] In the design of SynNotch receptors, the selection of ECD, TMD, and linker peptides is crucial. Choosing a suitable combination of ECD, linker peptide, and TMD is essential for achieving optimal signal transduction. For example, the SynNotch-gated CAR designed by Kole T. Royal et al. (Cell. 2016 October 6; 167(2):419–432.e16.) can achieve "AND" logic gate control for tumor killing, meaning that tumor killing can only be activated when two tumor antigens are present simultaneously. However, its linker peptide contains an NRR domain, which contains approximately 160 amino acids, resulting in a large SynNotch receptor size. This necessitates the use of two lentiviral vectors to construct a gated plasmid containing only fusion protein transcriptional regulatory elements (e.g., Gal4-VP64) and a CAR effector plasmid containing an activation sequence (e.g., UAS), respectively. In practical applications, the need for two lentiviral transfections of the same T cell leads to a large number of transfected T cells dying, resulting in low viability and hindering large-scale production. Meanwhile, the system uses non-human components, which can easily induce immune rejection. TMDs are sites for γ-secretase-mediated intracellular domain cleavage and release into the cytosol. Although γ-secretases are known to cleave various peptides, certain TMD mutations are known to negatively impact cleavage efficiency. Therefore, Iowis Zhu et al. (Cell 185, 1431–1443, April 14, 2022) optimized the linker peptide and TMD domain, replacing NRR with Notch2JMD and combining it with human Notch1TMD to construct a novel SynNotch receptor (also known as Synthetic intramembrane proteolysis receptor, SNIPR). However, this new SNIPR is not applicable to all ECDs. On the one hand, Gordon et al. (BLOOD, 30 APRIL 2009, VOLUME 113, NUMBER 18) reported that ECD mutations affect ADAM protease-mediated detachment sites; Iowis Zhu et al.'s research showed that ECD selection affects Notch receptor activity and T cell activation sensitivity. On the other hand, the inventors found that selecting other ECDs that can bind to known expressed ligands (e.g., TGFβ ligands) cannot achieve the gating effect. Therefore, SynNotch activity is affected by its extracellular domain, linker polypeptide, and intracellular domain; only by selecting specific SynNotch combinations can signal transduction functions be better realized.

[0145] The components of the chimeric polypeptide are described below.

[0146] PD1 extracellular segment or its functionally active fragment

[0147] This application selects the extracellular domain of PD1 or its functionally active fragment as the extracellular domain of the chimeric polypeptide.

[0148] In some embodiments, the extracellular domain of PD1 or its functionally active fragment may contain one or more immunoglobulin (Ig)-like domains, such as at least an IgV-like structure. The study by Krzysztof M. Zak et al. (Structure 25, August 1, 2017) showed that in the structure of the chimeric mouse / human and human PD-1 / PD-L1 complex, both proteins utilize the large hydrophobic surface interaction of their respective Ig-like V-shaped domains, indicating that this modular structure of the extracellular region is crucial for its binding to ligands.

[0149] In some embodiments, the extracellular segment of PD1 or its functionally active fragment may also be a part of or a modified fragment of the extracellular segment of PD-1, which may be any peptide or domain capable of binding to PD-L1 / PD-L2.

[0150] In some embodiments, the extracellular domain of PD1 or its functionally active fragment may further include at least one N-glycosylation site. PD-1 glycosylation can enhance the binding affinity of PD-1 / PD-L1. In one embodiment, the N-glycosylation site may be selected from one or more of the following groups: N49, N58, N74, and N116. Blocking any glycosylation site leads to decreased PD-1 stability and downregulation of expression levels, suggesting that PD-1 glycosylation is closely related to maintaining its stability and expression levels (SUN L et al., Cancer Res, 2020, 80(11):2298-2310.).

[0151] In some embodiments, the extracellular domain of PD1 or its functionally active fragment may also include an N-terminal loop region (N-loop). PD1 lacking the N-loop completely loses its ability to bind to nivolumab (a PD1-targeting antibody drug), indicating that the N-loop plays an important role in the function of PD-1 (Shuguang Tan et al., Nature Communications. 2016 Dec 21).

[0152] In some embodiments, the extracellular segment of PD-1 or its functionally active fragment includes soluble PD-1 (sPD-1). The soluble PD-1 extracellular segment is expressed in vitro using genetic engineering techniques and does not contain transmembrane or intracellular regions. It can bind to PD-1 ligands in vitro, thereby potentially affecting the PD-1 / PD-L1 interaction. For example, a study (He Yufei et al., Chinese Journal of Biotechnology, 2004, 20(5)) constructed a eukaryotic plasmid expression vector encoding mouse PD-1 extracellular cDNA and expressed it in eukaryotic cells. The expression product was mainly the soluble product (sPD-1) secreted extracellularly. This soluble PD-1 extracellular segment can block the PD-L / PD-1 interaction and enhance the tumor cell killing effect of splenic lymphocytes in tumor cell killing experiments.

[0153] In some implementations, the extracellular segment of PD1 or its functionally active fragment may also be engineered, for example by mutating one or more amino acid sites. In some embodiments, to enhance stability, prolong half-life, or improve affinity, the extracellular segment of PD1 or its functionally active fragment includes amino acid mutations. The sites of these amino acid mutations may include, but are not limited to, V39, L40, N41, Y43, M45, N49, K53, L97, A100, A107, A132, G124, K131, F19, I65, and / or A66 (Maute RL et al., Proceedings of the National Academy of Sciences of the United States of America 112, no. 47 (2015): E6506-E6514.; Lázár-Molnár E et al., EBioMedicine 17 (2017): 30-44.; Li Y et al., Cancer Science 109, no. 8 (2018): 2435-2445.; Liang, Z et al., Cancer Letters, 447, 164-173. In one embodiment, the amino acid mutation sites may include, but are not limited to, V39H, L40V, N41V, Y43H, M45E, N49G, K53T, L97V, A100V, A107I, A132I, A132F, A132T, A132V, G124S, K131Y, F19W, I65F, and / or Q66S. The amino acid numbering above begins with the starting amino acid (i.e., methionine) of the natural human PD1 protein, labeled as residue 1.

[0154] In some embodiments, the PD1 extracellular segment or its functionally active fragment comprises a sequence having at least 80% sequence identity with SEQ ID NO:1, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity. In some embodiments, the PD1 extracellular segment or its functionally active fragment comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:1. In some embodiments, the PD1 extracellular segment or its functionally active fragment comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:1. In some embodiments, the PD1 extracellular segment or its functionally active fragment comprises an amino acid sequence having at least 100% sequence identity with SEQ ID NO:1. In some embodiments, the PD1 extracellular segment or its functionally active fragment comprises the amino acid sequence shown in SEQ ID NO:1, wherein the 1st, 2nd, 3rd, 4th, or 5th amino acid residues in SEQ ID NO:1 may be substituted with different amino acid residues.

[0155] Linking peptides

[0156] The linker polypeptide described in this application is located between the transmembrane domain and the intracellular domain, and can be selected from the juxtamembrane domain derived from the Notch receptor family, the glycosylation advanced product receptor family, the cadherin superfamily of cell adhesion molecules, the tyrosine phosphatase receptor family, the epidermal growth factor (EGF) family, or the regulatory transmembrane proteolytic protein (RIP) family.

[0157] For example, the linker polypeptide includes a juxtamembrane domain derived from Robo1, Notch1, Notch2, Notch3, Notch4, CLSTN2, AGER, PTPRF, or NRG1.

[0158] In some implementations, the linker peptide may be selected from Notch JMD sequences. For example, the linker peptide may be human Notch JMD.

[0159] In some embodiments, the linker polypeptide may also include a γ-secretase cleavage site, wherein the γ-secretase cleavage site contains one or more specific amino acid sequences, such as sequences containing aromatic amino acids (e.g., phenylalanine, tyrosine, tryptophan).

[0160] In some embodiments, the linker polypeptide is rich in basic amino acids (R / K / H). For example, the linker polypeptide may be an amino acid sequence selected from any of the following groups: SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. In one embodiment, the linker polypeptide is a protein tyrosine phosphatase receptor type F. For example, the linker polypeptide may be an amino acid sequence as shown in SEQ ID NO:6.

[0161] In some embodiments, the linker polypeptide comprises a sequence having at least 80% sequence identity with SEQ ID NO:6, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity. In some embodiments, the linker polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:6. In some embodiments, the linker polypeptide comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:6. In some embodiments, the linker polypeptide comprises an amino acid sequence having at least 100% sequence identity with SEQ ID NO:6. In some embodiments, the linker polypeptide comprises the amino acid sequence shown in SEQ ID NO:6, wherein one, two, three, four, or five amino acid residues in one or more of SEQ ID NO:6 may be substituted with different amino acid residues.

[0162] Transmembrane domain

[0163] The transmembrane domain (TMD) described in this application may be selected from transmembrane domains derived from the Notch receptor family, the glycation end product receptor family, the cadherin superfamily of cell adhesion molecules, the tyrosine phosphatase receptor family, the epidermal growth factor (EGF) family, or the RIP family. For example, the transmembrane domain may be selected from transmembrane domains derived from Notch1, Notch2, Notch3, Notch4, Robo1, Robo4, CLSTN1, CLSTN2, AGER, APLP1, APLP2, LRP8, APP, BTC, TGBR3, SPN, CD44, CSF1R, CXCL16, CX3CL1, DCC, DLL1, DSG2, DAG1, CDH1, EPCAM, EPHA4, EPHB2, EFNB1, EFNB2, ErbB4, GHR, ELLA-A, PTPRF, NRG1, and IFNAR2.

[0164] The transmembrane domain contains at least one or more protease cleavage sites, including γ-secretase cleavage sites and / or metalloproteinase cleavage sites. The γ-secretase cleavage site contains one or more specific amino acid sequences, such as sequences containing aromatic amino acids (e.g., phenylalanine, tyrosine, tryptophan). The metalloproteinase cleavage site typically contains a sequence recognizable by metalloproteinases, such as a sequence containing RXKR (R represents arginine, X represents any amino acid, K represents lysine, and R represents arginine). The transmembrane domain may also contain plasminogen activator cleavage sites, such as urokinase plasminogen activator (uPA) or tissue plasminogen activator (tPA) cleavage sites.

[0165] In some embodiments, the TMD may be selected from TMDs derived from Notch1, and may further include Notch1 TMD variants, such as those involving one or more amino acid mutations, which may be G318A and / or V319A. For example, the amino acid sequence of the TMD may be as shown in SEQ ID NO:3 or SEQ ID NO:4. Numbering begins at the C-terminus of the natural transmembrane domain of the Notch receptor.

[0166] In some embodiments, the TMD includes a sequence having at least 80% sequence identity with SEQ ID NO:2, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity. In some embodiments, the TMD includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO:2. In some embodiments, the TMD includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO:2. In some embodiments, the TMD includes an amino acid sequence having at least 100% sequence identity with SEQ ID NO:2. In some embodiments, the TMD includes the amino acid sequence shown in SEQ ID NO:2, wherein one, two, three, four, or five amino acid residues in one or more of SEQ ID NO:2 may be substituted with different amino acid residues; for example, the TMD may also include the amino acid sequences shown in SEQ ID NO:3 or SEQ ID NO:4.

[0167] Intracellular domains

[0168] The intracellular domain (ICD) described in this application includes at least a transcription factor that activates or inhibits a promoter-driven transcriptional element of a DNA sequence. For example, it can be used to activate chimeric antigen receptor (CAR) expression or to activate T cells.

[0169] The transcription factors applicable to this application may be naturally occurring transcription factors or may be engineered, designed, or modified to provide desired and / or improved properties, such as regulation of transcription. In some embodiments, the transcription factor may directly regulate the expression of one or more genes involved in cell differentiation. In some embodiments, the transcription factor may indirectly regulate the expression of one or more genes involved in cell differentiation by regulating the expression of a second transcription factor, which in turn regulates the expression of one or more genes involved in cell differentiation. Those skilled in the art will understand that the transcription factor may be a transcription activator or a transcription repressor. In some embodiments, the transcription factor is a transcription repressor. In some embodiments, the transcription factor is a transcription activator. In some embodiments, the transcription factor may also include a nuclear localization signal. In some embodiments, the transcription factor is selected from Gal4-VP16, Gal4-VP64, tetR-VP64, ZFHD1-VP64, Gal4-KRAB, and HAP 1-VP 16. In some embodiments, the transcription factor is Gal4-VP64.

[0170] For example, transcription factors have an amino acid sequence as shown in SEQ ID NO:9.

[0171] In some embodiments, the intracellular domains described in this application may further include intracellular signal transduction domains. The intracellular signal transduction domain may have at least two distinct domains: at least one co-stimulatory domain and an activation domain.

[0172] In some embodiments, the co-stimulatory domain contains a sequence derived from a signaling molecule. The signaling molecule may be a protein selected from class 1 or class 3 human membrane proteins. In some embodiments, the signaling molecule is selected from CD28, 4-IBB, OX40, ICOS, CTLA4, PD1, PD1H, BTLA, B71, B7H1, CD226, and CRT. AM, TIGIT, CD96, TIM1, TIM2, TIM3, TIM4, CD2, SLAM, 2B4, Lyl08, CD84, Ly9, CRACC, BTN1, BTN2, BTN3, LAIR1, LAG3, CD160,, CD27, GITR, CD 30. TNFR1, TNFR2, HVEM, LT_R, DR3, DCR3, FAS, CD40, RANK, OPG, TRAILR1, TACI, BAFFR, BCMA, TWEAKR, EDAR, XEDAR, RELT, DR6, TROY, NGFR, C D22, SIGLEC-3, SIGLEC-5, SIGLEC-7, KLRG1, NKR-P1A, ILT2, KIR2DL1, KIR3DL1, CD94-NKG2A, CD300b, CD300e, TREM1, TREM2, ILT7, ILT3, ILT4, TLT-1, CD200R, CD300a, CD300f, DC-SIGN, B7-2, Allergen-1, LAT, BLNK, LAYN, SLP76, EMB-LMP1, HIV-NEF, HVS-TIP, HVS-ORF5 and HVS-stpC.

[0173] In some embodiments, the activation domain includes one or more immune receptor tyrosine-based activation motifs (ITAMs). In some embodiments, the activation domain is derived from CD3ζ, CD3σ, CD3 / , and CD3ε.

[0174] In some embodiments, the ICD comprises a sequence having at least 80% sequence identity with SEQ ID NO:9, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity. In some embodiments, the ICD comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:9. In some embodiments, the ICD comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:9. In some embodiments, the ICD comprises an amino acid sequence having at least 100% sequence identity with SEQ ID NO:9. In some embodiments, the ICD comprises the amino acid sequence shown in SEQ ID NO:9, wherein one, two, three, four, or five amino acid residues in one or more of SEQ ID NO:9 may be substituted with different amino acid residues.

[0175] Chimeric Peptides

[0176] In some embodiments, the chimeric polypeptide described in this application may include:

[0177] (1) An extracellular domain having at least 80% sequence homology with the amino acid sequence shown in SEQ ID NO:1;

[0178] (2) A linker polypeptide having at least 80% sequence homology with the amino acid sequence shown in SEQ ID NO:6;

[0179] (3) A transmembrane domain having at least 80% sequence homology with the amino acid sequence shown in SEQ ID NO:2; and

[0180] (4) An intracellular domain having at least 80% sequence homology with the amino acid sequence shown in SEQ ID NO:9.

[0181] In some embodiments, the chimeric polypeptide described in this application may further include:

[0182] (1) An extracellular domain having the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence in SEQ ID NO:1 including the mutations of V39H, L40V, N41V, Y43H, M45E, N49G, K53T, L97V, A100V, A107I, A132I, A132F, A132T, A132V, G124S, K131Y, F19W, I65F and / or Q66S;

[0183] (2) A linking polypeptide having the amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8;

[0184] (3) A transmembrane domain having the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4; and

[0185] (4) Intracellular domain, which has the amino acid sequence shown in SEQ ID NO:9.

[0186] In some embodiments, the chimeric polypeptide described in this application may further include:

[0187] (1) An extracellular domain having the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence in SEQ ID NO:1 including the mutations of V39H, L40V, N41V, Y43H, M45E, N49G, K53T, L97V, A100V, A107I, A132I, A132F, A132T, A132V, G124S, K131Y, F19W, I65F and / or Q66S;

[0188] (2) A linking polypeptide having the amino acid sequence shown in SEQ ID NO:6;

[0189] (3) A transmembrane domain having the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4; and

[0190] (4) Intracellular domain, which has the amino acid sequence shown in SEQ ID NO:9.

[0191] In some embodiments, the chimeric polypeptide described in this application may further include:

[0192] (1) An extracellular domain having the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence in SEQ ID NO:1 including the mutations of V39H, L40V, N41V, Y43H, M45E, N49G, K53T, L97V, A100V, A107I, A132I, A132F, A132T, A132V, G124S, K131Y, F19W, I65F and / or Q66S;

[0193] (2) A linking polypeptide having the amino acid sequence shown in SEQ ID NO:6;

[0194] (3) A transmembrane domain having the amino acid sequence shown in SEQ ID NO:2; and

[0195] (4) Intracellular domain, which has the amino acid sequence shown in SEQ ID NO:9.

[0196] In some embodiments, the chimeric polypeptide described in this application may further include:

[0197] (1) An extracellular domain having the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence in SEQ ID NO:1 including the mutations of V39H, L40V, N41V, Y43H, M45E, N49G, K53T, L97V, A100V, A107I, A132I, A132F, A132T, A132V, G124S, K131Y, F19W, I65F and / or Q66S;

[0198] (2) A linking polypeptide having the amino acid sequence shown in SEQ ID NO:6;

[0199] (3) A transmembrane domain having the amino acid sequence shown in SEQ ID NO:3; and

[0200] (4) Intracellular domain, which has the amino acid sequence shown in SEQ ID NO:9.

[0201] In some embodiments, the chimeric polypeptide described in this application may further include:

[0202] (1) An extracellular domain having the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence in SEQ ID NO:1 including the mutations of V39H, L40V, N41V, Y43H, M45E, N49G, K53T, L97V, A100V, A107I, A132I, A132F, A132T, A132V, G124S, K131Y, F19W, I65F and / or Q66S;

[0203] (2) A linking polypeptide having the amino acid sequence shown in SEQ ID NO:6;

[0204] (3) A transmembrane domain having the amino acid sequence shown in SEQ ID NO:4; and

[0205] (4) Intracellular domain, which has the amino acid sequence shown in SEQ ID NO:9.

[0206] The extracellular domain, transmembrane domain, linking polypeptide, and intracellular domain are directly linked.

[0207] In some embodiments, the chimeric polypeptide of this application comprises an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with any sequence provided in this application.

[0208] Nucleic acid molecules

[0209] On the other hand, this application provides a nucleic acid molecule comprising a first nucleic acid portion and a second nucleic acid portion; wherein the first nucleic acid portion comprises a nucleic acid sequence encoding an extracellular domain, the extracellular domain comprising the PD1 extracellular segment or a functionally active fragment thereof; the first nucleic acid portion further comprises a nucleic acid sequence encoding a Notch receptor polypeptide, the Notch receptor polypeptide comprising a linker polypeptide and a transmembrane domain; and the first nucleic acid portion further comprises a nucleic acid sequence encoding an intracellular domain, the intracellular domain comprising a transcription factor.

[0210] In some embodiments, the PD1 extracellular segment or its functionally active fragment comprises a sequence having at least 80% sequence identity with SEQ ID NO:1, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity. In some embodiments, the PD1 extracellular segment or its functionally active fragment comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:1. In some embodiments, the PD1 extracellular segment or its functionally active fragment comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:1. In some embodiments, the PD1 extracellular segment or its functionally active fragment comprises an amino acid sequence having at least 100% sequence identity with SEQ ID NO:1. In some embodiments, the PD1 extracellular segment or its functionally active fragment comprises the amino acid sequence shown in SEQ ID NO:1, wherein the 1st, 2nd, 3rd, 4th, or 5th amino acid residues in SEQ ID NO:1 are substituted with different amino acid residues. The sites where the amino acid residues are substituted may include, but are not limited to, V39, L40, N41, Y43, M45, N49, K53, L97, A100, A107, A132, G124, K131, F19, I65, and / or A66. In one embodiment, the amino acid residues may include, but are not limited to, V39H, L40V, N41V, Y43H, M45E, N49G, K53T, L97V, A100V, A107I, A132I, A132F, A132T, A132V, G124S, K131Y, F19W, I65F, and / or Q66S.

[0211] In some embodiments, the Notch receptor polypeptide includes a transmembrane domain and a linker polypeptide. The transmembrane domain and the linker polypeptide are each independently selected from the Notch receptor family or the RIP family.

[0212] For example, the transmembrane domain can be selected from Notch1TMD, and the linker peptide can be selected from Notch2JMD.

[0213] For example, the transmembrane domain can be selected from Notch1TMD, and the linker peptide can be selected from Notch1JMD.

[0214] In some embodiments, the linking polypeptide may be an amino acid sequence selected from the group consisting of: SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

[0215] In one embodiment, the linker polypeptide is a protein tyrosine phosphatase receptor type F. For example, the linker polypeptide may include the amino acid sequence shown in SEQ ID NO:6.

[0216] In some embodiments, the linker polypeptide comprises a sequence having at least 80% sequence identity with SEQ ID NO:6, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity. In some embodiments, the linker polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:6. In some embodiments, the linker polypeptide comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:6. In some embodiments, the linker polypeptide comprises an amino acid sequence having at least 100% sequence identity with SEQ ID NO:6. In some embodiments, the linker polypeptide comprises the amino acid sequence shown in SEQ ID NO:6, wherein one, two, three, four, or five amino acid residues in one or more of SEQ ID NO:6 are substituted with different amino acid residues.

[0217] In some implementations, the TMD may be selected from TMDs derived from Notch1, and may also include Notch1TMD variants, such as those with one or more amino acid mutations, which may be G318A and / or V319A.

[0218] In some embodiments, the TMD includes a sequence having at least 80% sequence identity with SEQ ID NO:2, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity. In some embodiments, the TMD includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO:2. In some embodiments, the TMD includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO:2. In some embodiments, the TMD includes an amino acid sequence having at least 100% sequence identity with SEQ ID NO:2. In some embodiments, the TMD includes the amino acid sequence shown in SEQ ID NO:2, wherein one, two, three, four, or five amino acid residues in one or more of SEQ ID NO:2 are substituted with different amino acid residues; for example, the TMD may also include the amino acid sequences shown in SEQ ID NO:3 or SEQ ID NO:4.

[0219] In some embodiments, the second nucleic acid portion includes a nucleic acid sequence encoding a cytokine, cytotoxin, chemokine, immunomodulator, pro-apoptotic factor, anti-apoptotic factor, hormone, differentiation factor, dedifferentiation factor, immune cell receptor, or reporter gene.

[0220] In some embodiments, the immune cell receptor is a TCR. A TCR typically comprises two polypeptides (e.g., polypeptide chains), such as the α-chain, β-chain, γ-chain, d-chain, or a combination thereof. Such TCR polypeptide chains and methods for their preparation are known in the art.

[0221] In some implementations, the immune cell receptor is a CAR.

[0222] In some embodiments, the structure of the CAR or TCR may include an antigen-binding domain. The antigen-binding domain contains an antibody or an antigen-binding fragment thereof. The antibody includes monoclonal antibodies, chimeric antibodies, humanized antibodies, and / or fully human antibodies. The antigen-binding fragment includes Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv, VHH, and / or dAb. The antigen-binding domain is capable of specifically targeting tumor antigens selected from the following group: ROR1, AXL, CD19, CD20, CD22, mesothelin, CD33, IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRVIII, GD-2, BCMA, CD123, CD7, Her2, B7-H3, CLDN18, EGFR, Nectin-4, CD90, CD133, DLL3, CEA, CD4, CD5, CD123, SIRP alpha, CD70, FORR1, NY-ESO-1, MAGE A3, GP100, MART-1, EBV, and p53.

[0223] For example, the antigen-binding domain of the CAR targets ROR1. As another example, the antigen-binding domain may be at least one CDR comprising a heavy chain variable region VH targeting ROR1, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO:10. As yet another example, the antigen-binding region may comprise HCDR3, HCDR2, and HCDR1 targeting ROR1, wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO:11, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:12, and HCDR1 comprises the amino acid sequence shown in SEQ ID NO:13.

[0224] For example, the antigenic domain of the CAR is a VHH targeting ROR1. As another example, the ROR1VHH may contain a sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:10, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity. As yet another example, the ROR1VHH has the amino acid sequence shown in SEQ ID NO:10.

[0225] In some embodiments, the CAR structure may further include a co-stimulatory domain. The co-stimulatory signaling domain comprises an intracellular co-stimulatory signaling domain derived from one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88.

[0226] For example, the co-stimulatory signaling domain is a 4-1BB co-stimulatory signaling domain. As another example, the co-stimulatory signaling domain contains the amino acid sequence shown in SEQ ID NO:14. As yet another example, the co-stimulatory signaling domain has the amino acid sequence shown in SEQ ID NO:14.

[0227] In some embodiments, the CAR structure may further include an intracellular signaling domain. The intracellular signaling domain comprises an intracellular signaling domain derived from one or more proteins selected from the group consisting of: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM.

[0228] For example, the intracellular signal transduction domain is the intracellular signal transduction domain of CD3ζ. As another example, the intracellular signal transduction domain comprises the amino acid sequence shown in SEQ ID NO:15. As yet another example, the intracellular signal transduction domain has the amino acid sequence shown in SEQ ID NO:15.

[0229] In some embodiments, the CAR structure may further include a transmembrane region. The transmembrane region is derived from the transmembrane region of one or more proteins selected from the group consisting of: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

[0230] For example, the transmembrane region is the transmembrane region of CD28. As another example, the transmembrane region contains the amino acid sequence shown in SEQ ID NO:16. As yet another example, the transmembrane region has the amino acid sequence shown in SEQ ID NO:16.

[0231] In some embodiments, the CAR structure may further include a hinge region. The hinge region comprises a hinge region derived from one or more proteins selected from the group consisting of: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

[0232] For example, the hinge region is the hinge region of CD28. As another example, the hinge region contains the amino acid sequence shown in SEQ ID NO:17. As yet another example, the hinge region has the amino acid sequence shown in SEQ ID NO:17.

[0233] In some implementations, the first nucleic acid portion is linked to a promoter sequence. The promoter sequence may be selected from the group consisting of: CMV promoter sequences, SFFV promoter sequences, EF1α promoter sequences, and PGK promoter sequences.

[0234] For example, the startup sequence is an SFFV startup sequence.

[0235] In some implementations, the second nucleic acid portion is linked to an activation sequence, which may be a UAS sequence.

[0236] In some embodiments, the nucleic acid molecule provided in this application comprises, from its 5' end to its 3' end, the following sequences: a second nucleic acid moiety activation sequence, a nucleic acid sequence encoding the antigen-binding region, a nucleic acid sequence encoding the hinge region, a nucleic acid sequence encoding the transmembrane region, a nucleic acid sequence encoding the co-stimulatory domain, a nucleic acid sequence encoding the intracellular signal transduction domain, a first nucleic acid moiety initiation sequence, a nucleic acid sequence encoding the extracellular domain, a nucleic acid sequence encoding the Notch receptor polypeptide, and a nucleic acid sequence encoding the intracellular domain.

[0237] The nucleic acid molecules of this application may have any length, including, for example, between about 1.5 Kb and about 50 Kb, between about 5 Kb and about 40 Kb, between about 5 Kb and about 30 Kb, between about 5 Kb and about 20 Kb, or between about 10 Kb and about 50 Kb, for example, between about 15 Kb and about 30 Kb, between about 20 Kb and about 50 Kb, between about 20 Kb and about 40 Kb, between about 5 Kb and about 25 Kb, or between about 30 Kb and about 50 Kb.

[0238] In some embodiments, the nucleic acid molecules of this application may be integrated into an expression cassette or expression vector. It should be understood that an expression cassette typically comprises a construct of genetic material containing a coding sequence and sufficient regulatory information to guide the proper transcription and / or translation of the coding sequence in recipient cells (in vivo and / or in vitro). Typically, the expression cassette may be inserted into a vector to target desired host cells and / or individuals. Therefore, in some embodiments, the expression cassette of this application includes the coding sequence of the chimeric receptor of this application, operatively linked to an expression control element (e.g., a promoter) and optionally, any other nucleic acid sequences or combinations thereof that influence the transcription or translation of the coding sequence.

[0239] The nucleic acid sequence encoding a chimeric polypeptide can be optimized for expression in a target host cell. For example, the GC content of the sequence can be adjusted to the average level of a given host cell, as calculated by referencing known genes expressed in the host cell. Codon usage optimization methods are known in the art. Codon usage in the coding sequence of the chimeric polypeptide of this application can be optimized to enhance expression in host cells, such that approximately 1%, approximately 5%, approximately 10%, approximately 25%, approximately 50%, approximately 75%, or up to 100% of the codons in the coding sequence have been optimized for expression in a specific host cell.

[0240] The nucleic acid molecules provided in this application may contain naturally occurring sequences or sequences different from naturally occurring sequences, but encode the same polypeptides, such as antibodies, due to the degeneracy of the genetic code. These nucleic acid molecules may consist of combinations or modifications of nucleotides within RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA, such as DNA produced through phosphoramide-based synthesis) or these types of nucleic acids. Furthermore, nucleic acid molecules may be double-stranded or single-stranded (e.g., sense strands or antisense strands).

[0241] The nucleic acid molecules provided in this application are not limited to sequences encoding the chimeric polypeptides; they may also include partial or complete non-coding sequences located upstream or downstream of the coding sequence (e.g., the coding sequence of the chimeric polypeptide). Those skilled in the art of molecular biology are familiar with conventional procedures for isolating nucleic acid molecules. For example, they can be generated by treating genomic DNA with restriction endonucleases or by performing polymerase chain reaction (PCR). If the nucleic acid molecule is ribonucleic acid (RNA), the molecule can be generated through in vitro transcription or other methods.

[0242] expression carrier

[0243] On the other hand, this application also provides an expression vector into which the nucleic acid molecule can be integrated. Therefore, in some embodiments, the vector can be a replicon, such as a plasmid, bacteriophage, or granule, into which another DNA fragment can be inserted to achieve replication of the inserted fragment. In some embodiments, the expression vector can be an integration vector.

[0244] In some implementations, the expression vector may be a viral vector. The term "viral vector" is widely used to refer to nucleic acid molecules comprising virus-derived nucleic acid elements (e.g., transfer plasmids) that typically facilitate the transfer or integration of nucleic acid molecules into the genome of a cell, or to viral particles mediating nucleic acid transfer. Viral particles typically comprise various viral components and sometimes host cell components other than nucleic acids. "Viral vector" can refer to a virus or viral particle capable of transferring nucleic acid into a cell, or to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements primarily derived from viruses. For example, a retroviral vector is a viral vector or plasmid containing structural and functional genetic elements or portions thereof primarily derived from retroviruses. Similarly, a lentiviral vector is a viral vector or plasmid containing structural and functional genetic elements or portions thereof, including LTRs primarily derived from lentiviruses (a type of retrovirus).

[0245] cell

[0246] On the other hand, this application provides a cell in which the nucleic acid molecule can be introduced. Upon introduction, the promoter of the first nucleic acid portion is immediately activated, and the first nucleic acid portion is expressed as a chimeric polypeptide. The second nucleic acid portion enters the cell, and when the chimeric polypeptide is activated and releases transcription factors, the second nucleic acid portion is expressed.

[0247] The cells provided in this application include the following structures:

[0248] a) an extracellular domain, wherein the extracellular domain includes an extracellular segment of PD1 (programmed death receptor-1) or a functionally active fragment thereof, the extracellular segment of PD1 or the functionally active fragment thereof being capable of binding to a PD1 ligand;

[0249] b) Notch receptor polypeptide, wherein the Notch receptor polypeptide comprises a linker polypeptide and a transmembrane domain;

[0250] c) Intracellular domains;

[0251] d) Exogenous nucleic acid sequence, wherein the exogenous nucleic acid sequence includes a second nucleic acid portion that specifically targets tumor antigens.

[0252] In some embodiments, the extracellular domain, Notch receptor polypeptide, and intracellular domain of the cells provided in this application are as described above. The exogenous nucleic acid portion can specifically target tumor antigens after expression. The tumor antigens are selected from the following group: ROR1, AXL, CD19, CD20, CD22, mesothelin, CD33, IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRVIII, GD-2, BCMA, CD123, CD7, Her2, B7-H3, CLDN18, EGFR, Nectin-4, CD90, CD133, DLL3, CEA, CD4, CD5, CD123, SIRP alpha, CD70, FORR1, NY-ESO-1, MAGE A3, GP100, MART-1, EBV, and p53.

[0253] In some embodiments, the exogenous nucleic acid portion may include a nucleic acid sequence encoding an immune cell receptor.

[0254] In some embodiments, the immune cell receptor is a TCR. A TCR typically comprises two polypeptides (e.g., polypeptide chains), such as the α-chain, β-chain, g-chain, d-chain, or a combination thereof. The TCR polypeptide chains and methods for their preparation are known in the art.

[0255] In some implementations, the immune cell receptor is a CAR.

[0256] In some embodiments, the structure of the CAR may include an antigen-binding domain. The antigen-binding domain contains an antibody or an antigen-binding fragment thereof. The antibody includes monoclonal antibodies, chimeric antibodies, humanized antibodies, and / or fully human antibodies. The antigen-binding fragment includes Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv, VHH, and / or dAb.

[0257] For example, the antigen-binding domain of the CAR targets ROR1. As another example, the antigen-binding domain may be at least one CDR comprising a heavy chain variable region VH targeting ROR1, wherein the heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO:10. As yet another example, the antigen-binding region may comprise HCDR3, HCDR2, and HCDR1 targeting ROR1, wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO:11, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:12, and HCDR1 comprises the amino acid sequence shown in SEQ ID NO:13.

[0258] For example, the antigenic domain of the CAR is a VHH targeting ROR1. As another example, the ROR1VHH may contain a sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:10, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity. As yet another example, the ROR1VHH has the amino acid sequence shown in SEQ ID NO:10.

[0259] In some embodiments, the CAR structure may further include a co-stimulatory domain. The co-stimulatory signaling domain comprises an intracellular co-stimulatory signaling domain derived from one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88.

[0260] For example, the co-stimulatory signaling domain is a 4-1BB co-stimulatory signaling domain. As another example, the co-stimulatory signaling domain contains the amino acid sequence shown in SEQ ID NO:14. As yet another example, the co-stimulatory signaling domain has the amino acid sequence shown in SEQ ID NO:14.

[0261] In some embodiments, the CAR structure may further include an intracellular signaling domain. The intracellular signaling domain comprises an intracellular signaling domain derived from one or more proteins selected from the group consisting of: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a domain containing at least one ITAM. For example, the intracellular signaling domain is the intracellular signaling domain of CD3ζ. Another example is that the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:15. Yet another example is that the intracellular signaling domain has the amino acid sequence shown in SEQ ID NO:15.

[0262] In some embodiments, the CAR structure may further include a transmembrane region. The transmembrane region is derived from the transmembrane region of one or more proteins selected from the group consisting of: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM. For example, the transmembrane region is the transmembrane region of CD28. For example, the transmembrane region contains the amino acid sequence shown in SEQ ID NO:16. For example, the transmembrane region has the amino acid sequence shown in SEQ ID NO:16.

[0263] In some embodiments, the CAR structure may further include a hinge region. The hinge region comprises a hinge region derived from one or more proteins selected from the group consisting of: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT. For example, the hinge region is the hinge region of CD28. Another example is that the hinge region comprises the amino acid sequence shown in SEQ ID NO:17. Yet another example is that the hinge region has the amino acid sequence shown in SEQ ID NO:17.

[0264] The introduction of nucleic acid molecules into cells according to this application can be achieved by methods known to those skilled in the art, such as viral infection, transfection, conjugation, protoplast fusion, lipid transfection, electroporation, nuclear transfection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, and nanoparticle-mediated nucleic acid delivery.

[0265] Therefore, in some embodiments, nucleic acid molecules can be delivered using viral or non-viral delivery vectors known in the art. For example, nucleic acid molecules can be stably integrated into the host genome, or can replicate freely, or exist in recombinant host cells as microcircular expression vectors for transient expression. Thus, in some embodiments, nucleic acid molecules are maintained and replicated as free units in recombinant host cells. In some embodiments, nucleic acid molecules are stably integrated into the genome of the recombinant cell. Stable integration can be achieved using classic random genomic recombination techniques or more precise techniques, such as guide RNA-guided CRISPR / Cas9 genome editing, or DNA-guided endonuclease genome editing using NgAgo (Natronobacterium gregoryi Argonaute), or TALENs genome editing (transcription activator-like effector nucleases). In some embodiments, nucleic acid molecules exist in recombinant host cells as small circular expression vectors for transient expression.

[0266] Nucleic acid molecules can be encapsulated in viral capsids or lipid nanoparticles, or delivered via viral or non-viral delivery methods and techniques known in the art, such as electroporation. For example, nucleic acids can be introduced into cells via viral transduction. In a non-limiting example, adeno-associated virus (AAV) is engineered to deliver nucleic acids to target cells via viral transduction. Several AAV serotypes have been described, and all known serotypes can infect cells from a wide variety of tissue types. AAV is capable of transducing multiple species and tissues in vivo, and there is no evidence of toxicity; furthermore, it elicits relatively mild innate and adaptive immune responses.

[0267] Lentiviral vector systems can also be used for nucleic acid delivery and gene therapy via viral transduction. Lentiviral vectors offer several attractive characteristics as gene delivery vectors, including: (i) stable integration into the host genome for sustained gene delivery; (ii) the ability to infect both dividing and non-dividing cells; (iii) broad tissue tropism, including important gene and cell therapy target cell types; (iv) no expression of viral proteins after transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or intronic sequences; (vi) potentially safer integration sites; and (vii) relatively simple vector handling and production systems.

[0268] In some embodiments, the host cell may be genetically engineered (e.g., transduced, transformed, or transfected) using a vector construct, such as those described in this application. The vector construct may be, for example, a viral vector or a vector for homologous recombination, comprising a nucleic acid sequence homologous to a portion of the host cell genome, or it may be an expression vector for expressing a target polypeptide. The host cell may be an untransformed cell or a cell that has been transfected with at least one nucleic acid molecule.

[0269] In some embodiments, the recombinant cells are prokaryotic or eukaryotic cells. In some embodiments, the cells are in vivo. In some embodiments, the cells are in vitro. In some embodiments, the recombinant cells are eukaryotic cells. In some embodiments, the recombinant cells are animal cells. In some embodiments, the animal cells are mammalian cells. In some embodiments, the animal cells are human cells. In some embodiments, the cells are non-human primate cells. In some embodiments, the mammalian cells are immune cells, neurons, epithelial and endothelial cells, or stem cells. In some embodiments, the recombinant cells are immune system cells, such as lymphocytes (e.g., T cells or NK cells) or dendritic cells. In some embodiments, the immune cells are B cells, monocytes, natural killer (NK) cells, basophils, eosinophils, neutrophils, dendritic cells, macrophages, regulatory T cells, helper T cells (TH cells), cytotoxic T cells (TCTLs), or other T cells. In some embodiments, the immune system cells are T lymphocytes.

[0270] In some embodiments, the cells are stem cells. In some embodiments, the cells are hematopoietic stem cells. In some embodiments, the cells are lymphocytes. In some embodiments, the cells are precursor T cells or regulatory T (Treg) cells. In some embodiments, the cells are CD34+, CD8+, or CD4+ cells. In some embodiments, the cells are CD8+ T cytotoxic lymphocytes selected from naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and abundant CD8+ T cells. In some embodiments, the cells are CD4+ T helper lymphocytes selected from naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and abundant CD4+ T cells. In some embodiments, the cells can be obtained by leukocyte ablation of a sample obtained from a subject. In some embodiments, the subject is a human patient.

[0271] Pharmaceutical Composition

[0272] On the other hand, this application also provides a pharmaceutical composition that may comprise the chimeric polypeptide, the nucleic acid molecule, and / or the cell, and any pharmaceutically acceptable excipient. The excipient refers to any suitable carrier, diluent, or excipient. The excipient includes all aqueous and non-aqueous isotonic sterile injectable solutions, which may contain antioxidants, buffers, and solutes to make the composition isotonic with the blood of the intended recipient. The pharmaceutical compositions of this application may also include other supplemental physiologically active agents.

[0273] The pharmaceutical compositions provided in this application can be sterile aqueous solutions, dispersions, or sterile powders. The pharmaceutical compositions may contain solvents or dispersion media, such as water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate flowability can be maintained by using coatings such as lecithin, maintaining the desired particle size in the dispersed state, and using surfactants (e.g., sodium lauryl sulfate). Microbial action can be prevented by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents are typically included in the composition, such as sugars, polyols (e.g., mannitol, sorbitol), and sodium chloride. The absorption of injectable compositions can be prolonged by including agents that delay absorption (e.g., aluminum monostearate and gelatin).

[0274] application

[0275] On the other hand, this application also provides a system for regulating cell activity, the system comprising the chimeric polypeptide, the nucleic acid molecule, the cell, the nucleic acid molecule, the expression vector, and / or the pharmaceutical composition.

[0276] In some implementations, the system can be used alone for the treatment, relief, and / or prevention of diseases and / or conditions.

[0277] In some implementations, the system can be used in combination with other cancer treatment drugs to jointly treat, alleviate, and / or prevent diseases and / or symptoms.

[0278] In one implementation, the disease and / or symptom can be any currently known disease and / or symptom.

[0279] In one implementation, the disease and / or condition can be any currently known CAR and / or TCR-targeted disease and / or condition.

[0280] In some implementations, the disease and / or condition may be an autoimmune disease.

[0281] In some embodiments, the disease and / or symptom may be a tumor, which may be a solid tumor and / or a non-solid tumor.

[0282] For example, the disease and / or condition may be one expressing tumor antigens ROR1, AXL, CD19, CD20, CD22, mesothelin, CD33, IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRVIII, GD-2, BCMA, CD123, CD7, Her2, B7-H3, CLDN18, EGFR, Nectin-4, CD90, CD133, DLL3, CEA, CD4, CD5, CD123, SIRP alpha, CD70, FOLR1, NY-ESO-1, MAGE A3, GP100, MART-1, EBV, and / or p53. As another example, the disease and / or condition may express tumor antigen ROR1.

[0283] For example, the diseases and / or conditions mentioned may be breast cancer, lung cancer, liver cancer, stomach cancer, prostate cancer, pancreatic cancer, ovarian cancer, esophageal cancer, colon cancer, head and neck cancer, bladder cancer, B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, acute myeloid leukemia (AML), non-Hodgkin's lymphoma, rheumatoid arthritis, mesothelioma, glioblastoma, neuroblastoma, HIV, melanoma, and / or lymphoma.

[0284] On the other hand, this application also provides a method for regulating cell activity, the method comprising:

[0285] a) Provide cells capable of expressing the chimeric polypeptide;

[0286] b) Contact the cells with the nucleic acid molecules described above;

[0287] c) Obtain the cells and bring the cells into contact with tumor cells;

[0288] In the cells described in d), PD1 binds to ligands in tumor cells, inducing transmembrane domain cleavage and releasing intracellular domains;

[0289] e) The intracellular domains described regulate the antitumor activity of cells.

[0290] In some implementations, the intracellular domain modulates cellular antitumor activity by activating CAR and / or TCR expression.

[0291] In some implementations, the CAR and / or TCR may be selected from any currently known CAR and / or TCR.

[0292] In some embodiments, the CAR and / or TCR can specifically target tumor antigens selected from the group consisting of: ROR1, AXL, CD19, CD20, CD22, ROR1, mesothelin, CD33, IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRVIII, GD-2, BCMA, CD123, CD7, Her2, B7-H3, CLDN18, EGFR, Nectin-4, CD90, CD133, DLL3, CEA, CD4, CD5, CD123, SIRP alpha, CD70, FORR1, NY-ESO-1, MAGE A3, GP100, MART-1, EBV, and p53. For example, the tumor antigen may be ROR1.

[0293] In some embodiments, the method is an in vitro method or an ex vivo method.

[0294] In some embodiments, the cells include immune effector cells.

[0295] In some embodiments, the cells include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral blood mononuclear cells, embryonic stem cells, lymphoprogenitor cells and / or kinetic stem cells, or variants thereof.

[0296] In some embodiments, the cells are T cells.

[0297] On the other hand, this application also provides methods for applying the chimeric peptide, the nucleic acid molecule, the cell, and / or the pharmaceutical composition. In some embodiments, the method includes administering to an individual a first therapy comprising one or more chimeric peptides, nucleic acids, cells, and pharmaceutical compositions disclosed herein, and a second therapy, wherein the first therapy and the second therapy together treat the individual's disease and / or condition.

[0298] On the other hand, the use of the chimeric polypeptide, the nucleic acid molecule, the cell, the nucleic acid molecule, the expression vector, the pharmaceutical composition, and / or the system described in this application in the preparation of medicaments for the prevention, treatment, and / or relief of diseases and / or symptoms.

[0299] Without being limited by any theory, the embodiments described below are merely for illustrating the various technical solutions of the present invention and are not intended to limit the scope of the present invention.

[0300] Example

[0301] Example 1: Design of a chimeric peptide gating system

[0302] 1.1. Experimental Materials

[0303] 1.2 Cell Culture

[0304] HEK293T, MEC1, HEP3B, A549, MDA-MB-231, and Huh7 cell lines were obtained from the American Test and Computing Center (ATCC). MEC-ROR1-PDL1, Hep3B-PDL1, and A549-PDL1 are all modified cell lines that stably express PDL1. These tumor cell lines were transfected to stably express GFP and used in subsequent experiments.

[0305] HEK293T cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% heat-inactivated FBS, 100 U / mL penicillin / streptomycin, and 2 mM L-glutamine. MDA-MB-231, MEC1, and MEC1-ROR1 cell lines were cultured in IMDM containing 10% heat-inactivated FBS, 100 U / mL penicillin / streptomycin, and 2 mM L-glutamine. PBMCs were isolated from whole blood of healthy donors using Ficoll-Paque and cultured at 2 × 10⁻⁶ cells / mL. 7 A concentration of 1 cell / mL was aliquoted into 1 mL samples in heat-inactivated FBS containing 10% DMSO and then frozen in a liquid nitrogen tank.

[0306] 1.3 Clone Construction

[0307] All relevant gene constructs were synthesized using GeneWiz. The PD1 ECD chimeric peptide gate (Figure 1) and the TGFbR ECD chimeric peptide gate (Figure 2) were inserted into the pALD expression plasmid via the BstBI and XhoI cloning sites. After double enzyme digestion and sequencing verification, the expression plasmid was amplified extensively for lentiviral packaging.

[0308] 1.4 Preparation of Lentiviral Viruses

[0309] All lentiviruses were prepared using HKE293T cells. First, resuspended HEK293T cells were washed with PBS buffer and then seeded into 10 cm cell culture dishes at a density of 90%–95% covering the surface of the culture dish. The following day, the lentiviral packaging plasmid, transfection plasmid, and transfection reagent Lipofectamine 3000 were mixed and transfected into the seeded HEK293T cells using a transient transfection method. After 48 hours of culture, the supernatant was carefully collected after centrifugation at 300 x g for 10 minutes, aliquoted into 1 mL tubes, and stored at -80°C.

[0310] 1.5 T cell activation and transduction

[0311] After thawing frozen PBMCs, they were resuspended in X-VIVO 15 medium containing 10% heat-inactivated FBS, 100 U / mL penicillin / streptomycin, and 2 mM L-glutamine, and cultured with 1000 IU / mL IL-2. PBMCs were then mixed with CD3 / CD28 magnetic beads at a 1:1 ratio and activated in a 37°C, 5% CO2 cell culture incubator for 1-2 days. Activated T cells were then aliquoted into 24-well plates (0.5-1.0 × 10⁶ cells / well). 6 Cells / well: Add 0.5-1.0 mL of the corresponding lentivirus and polybrene (8 g / mL) to each well. Centrifuge at 2000 g for 2 hours, then transfer to a 37°C, 5% CO2 incubator and incubate overnight. For the negative control group, replace the lentivirus with complete culture medium, but follow the same steps. The day after transduction, replace with fresh X-VIVO complete medium and 1000 IU / mL IL-2. Cells can be used for subsequent experiments and analysis 2-5 days after transduction.

[0312] 1.6 Flow Cytometry Assay

[0313] After washing once with FACS buffer (PBS containing 0.5% BSA, 0.1% NaN3, 2mM EDTA, pH 7.0), the transfected T cells were resuspended in FACS buffer to a concentration of 1-5 × 10⁻⁵. 6 Cells / mL. Subsequently, staining was performed using fluorescently labeled antibodies, and the cells were incubated at 4°C for 45 minutes in the dark. After staining, the unbound antibodies were washed with FACS buffer, and the cells were centrifuged at 300 x g for 5 minutes to remove the supernatant. After resuspending in FACS buffer, the cells were analyzed by flow cytometry (Cytoflex LX), and all flow cytometry data were analyzed using FlowJo software.

[0314] 1.7 Enzyme-linked immunosorbent assay (ELISA)

[0315] Precise counting of PBMCs and tumor target cells transduced with chimeric peptide-gated structures for 72 hours was performed using the Countess III Automatic Cell Counter (Invitrogen). Cells were mixed in 96-well plates at an effector-to-target ratio of 1:1 or 1:3 and resuspended in 200 μL of cell culture medium at a density of 50,000 cells / 100 μL. After co-culturing T cells and tumor cells for 24 hours, the cell culture plates were centrifuged at 300 x g for 5 minutes. The supernatant was then carefully aspirated and stored at -20°C for subsequent analysis. Cytokine secretion levels in the culture medium were measured using the ELISA MAX™ Deluxe Set Human IFN-γ kit (Biolegend; 430104) and the ELISA MAX™ Standard Set Human IL-2 kit (Biolegend; 431801) according to the manufacturer's instructions.

[0316] 1.8 Gating Effect Verification Experiment

[0317] PBMC cells and tumor target cells transduced with the PD1 chimeric peptide-gated structure for 72 hours were precisely counted using a Countess III Automatic Cell Counter (Invitrogen). Cells were mixed in 96-well plates at a 1:1 effector-to-target ratio and resuspended in 200 μL of cell culture medium at a density of 50,000 cells / 100 μL. For validation of the TGFbRII chimeric peptide-gated effect, different concentrations of TGFb protein (10-200 ng / μL) were added to PBMC cells. After co-culturing for 48 hours, co-cultured cells were harvested for fluorescent staining and flow cytometry analysis to determine CAR-T structural expression.

[0318] 1.9 T-cell tumor killing experiment

[0319] PBMCs transduced with chimeric peptide-gated structures for 3–5 days and GFP-labeled tumor target cells were precisely counted using the Countess III Automatic Cell Counter (Invitrogen). Cells were mixed at a 1:1 effector-to-target ratio and resuspended in 200 μL of cell culture medium at a density of 10,000 cells / 100 μL. Cells were co-cultured in 96-well plates, with two replicates for each corresponding condition. The 96-well plates were then subjected to real-time imaging (Incucyte, Sartorius) for detection and analysis. The killing assay lasted for 5 days, with 5,000 target cells added on days 2.5–3 for tumor re-challenge.

[0320] Example 2: Gated Expression After Transfection

[0321] 2.1 Gated expression of transfected PD1 ECD chimeric peptide

[0322] The structure containing the PD1 logic gate ROR1CAR was successfully cloned and packaged into a lentivirus. As shown in Figure 3, 72 hours after lentivirus infection, high levels of HA marker expression were detected in activated human PBMCs, indicating the expression of the PD1 ECD logic gate. In the basal state, the gate is not activated, and ROR1CAR is expressed at almost or very low levels.

[0323] 2.2 Gated expression of TGFbRII ECD chimeric peptide after transfection

[0324] The structure of CD22CAR containing TGFbRII logic gates was successfully cloned and packaged into a lentivirus. As shown in Figure 4, high levels of TGFbRII ECD logic gate expression were detected in activated human PBMCs 72 hours after lentivirus infection. In the basal state, the gates were not activated, and CD22CAR showed low-level expression.

[0325] Example 3: Gating effect of T cells after transfection

[0326] 3.1 Gating effect of T cells after transfection with PD1 ECD chimeric peptide

[0327] As shown in Figure 5, low levels of ROR1CAR expression (15.6% and 17.7%) were detected in PBMCs transduced with the PD1 ECD chimeric peptide in the unstimulated basal state (SNIPR only, expressing only the PD1ECD Notch receptor without CAR activation) or after culture with PDL1-negative K562 cells. When co-cultured with PDL1-positive MEC cells, ROR1CAR expression was induced, increasing to 36.7%.

[0328] 3.2 Gating effect of T cells after transfection with TGFβRII ECD chimeric peptide

[0329] As shown in Figure 6, PBMCs transduced with the TGFβRII ECD chimeric polypeptide, when co-cultured with different concentrations of TGFβ protein (10 ng / μL-200 ng / μL), showed similar CAR structural expression compared to PBMCs in an unactivated state (SNIPR only, expressing only TGFβRII ECD Notch receptor without CAR activation), indicating that TGFβRII ECD gating cannot induce CAR expression.

[0330] Example 4: PD1 ECD chimeric peptide-gated system specifically kills tumor cells that are double-positive for ROR1 and PDL1.

[0331] As shown in Figures 7A and 7B, the PD1 ECD chimeric peptide was found to have higher levels of IL2 and IFNγ cytokine secretion after being cultured with PDL1 and ROR1 double-positive tumor cells.

[0332] As shown in Figures 8A-E, in the IncuCyte real-time imaging experiment, PBMCs transduced with the PD1 ECD chimeric peptide effectively killed PDL1 and ROR1 double-positive tumor cells (MB231, A549-PDL1, MEC-ROR1, HEP3B-PDL1), essentially clearing target cells within 48 hours. Even after re-challenge with new tumor cells, sustained killing was observed. The PD1 ECD chimeric peptide gating system maintained good killing ability against tumor cells with low PDL1 expression (MEC-ROR1), indicating the high sensitivity of the PD1 ECD chimeric peptide gating system to PDL1. With increasing PD-L1 expression levels in tumor cells (A549-PDL1 and HEP3B-PDL1 overexpressing PDL1), the transcriptional activation capacity of the PD1 ECD chimeric peptide gating system increased, inducing increased ROR1-CAR expression and demonstrating sustained tumor-killing ability, indicating that the PD1 ECD chimeric peptide gating system has a good gating effect. Meanwhile, no killing effect was observed after co-culturing with PDL1-positive and ROR1-negative MECs, indicating that the structure has good specificity and safety.

[0333] As shown in Figures 9A-C, compared to both the unmodified ROR1VHH CAR-T and the ROR1CAR-T derived from UC-961, the modified PD1SNIPR ROR1CAR-T exhibited better tumor-killing and tumor-suppressing effects in tumor cells with low PDL1 expression (MB231) and tumor cells with PDL1 overexpression (MB231-PDL1). In tumor cells with low or no ROR1 expression (Huh7), PD1SNIPR ROR1CAR-T also showed an inhibitory effect on tumor growth. These findings indicate that PD1SNIPR ROR1CAR-T has better anti-cell exhaustion and anti-tumor recurrence effects.

[0334] Example 5: In vivo pharmacodynamic experiment of PD1 ECD chimeric peptide

[0335] Approximately 1 × 10⁶ MDA-MB-231 cells were resuspended in 100 μL of PBS, mixed with an equal volume of Matrigel (v / v 1:1), and subcutaneously injected into the right side of NCG mice at a volume of 200 μL. Three days after tumor cell inoculation, mice were divided into groups of six based on body weight and administered appropriate treatments. The dosage for each animal was 4E⁶ total cells (CAR+≈50% or HA tag+≈50%) per mouse, at a volume of 200 μL per mouse.

[0336] Animal experiment results (Figure 10) showed that, on day 50 after grouping, compared with MocK-T and PBS, ROR1CAR-T (scfv sequence derived from UC-961 CAR-T (#8020v2)) and PD1SNIPR ROR1CAR-T (8151v2 group) both had significant tumor-suppressive effects. In the ROR1CAR-T group, tumor volume began to decrease significantly from day 15, and by day 25 the tumor volume had shrunk to 34.26±6.85 mm3. However, starting from day 29, tumor growth showed an upward trend, and by day 50 the tumor volume had reached 303.21±42.4 mm3. Notably, in the PD1SNIPR ROR1CAR-T group, tumor growth continued to increase on day 15; by day 18, tumor volume began to decrease, but the average tumor volume in the PD1SNIPR ROR1CAR-T group (106.16±18.34 mm3) was still twice that of the ROR1CAR-T group (43.83±6.83 mm3); on day 29, the tumor volumes in the PD1SNIPR ROR1CAR-T and ROR1CAR-T groups were comparable, and the tumor size in the PD1SNIPR ROR1CAR-T group remained stable until day 50. These results indicate that the PD1SNIPR ROR1CAR-T group exerts its tumor-killing effect more slowly than the ROR1CAR-T group, but its long-term efficacy is superior.

[0337] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various variations of the embodiments listed herein will be apparent to those skilled in the art and are reserved within the scope of the appended claims and their equivalents.

Claims

1. Chimeric polypeptides, comprising, from the N-terminus to the C-terminus: a) an extracellular domain, wherein the extracellular domain includes an extracellular segment of PD1 (programmed death receptor-1) or a functionally active fragment thereof, the extracellular segment of PD1 or the functionally active fragment thereof being capable of binding to a PD1 ligand; b) Notch receptor polypeptide, wherein the Notch receptor polypeptide comprises a transmembrane domain and a linker polypeptide; c) Intracellular domains.

2. The chimeric polypeptide according to claim 1, wherein the extracellular segment of PD1 or its functionally active fragment includes at least an immunoglobulin variable region (IgV)-like structure.

3. The chimeric polypeptide according to any one of claims 1-2, wherein the extracellular segment of PD1 or its functionally active fragment further comprises at least one N-glycosylation site.

4. The chimeric polypeptide according to any one of claims 1-3, wherein the extracellular segment of PD1 or its functionally active fragment comprises four N-glycosylation sites.

5. The chimeric polypeptide according to claim 4, wherein the N-glycosylation site is selected from one or more of the following: N49, N58, N74 and N116.

6. The chimeric polypeptide according to any one of claims 1-5, wherein the extracellular segment of PD1 or its functionally active fragment further comprises an N-terminal loop region (N-loop).

7. The chimeric polypeptide according to any one of claims 1-6, wherein the extracellular segment of PD1 or its functionally active fragment comprises soluble PD1 (sPD1).

8. The chimeric polypeptide according to any one of claims 1-7, wherein the PD1 ligand comprises PD-L1 and / or PD-L2.

9. The chimeric polypeptide according to any one of claims 1-8, wherein the extracellular segment of PD1 or its functionally active fragment comprises wild-type PD1 or a variant thereof.

10. The chimeric polypeptide according to any one of claims 1-9, wherein the extracellular segment of PD1 or its functionally active fragment is the extracellular segment of mammalian PD1 or its functionally active fragment.

11. The chimeric polypeptide according to claims 1-10, wherein the PD1 extracellular segment or its functionally active fragment is a human PD1 extracellular segment or its functionally active fragment.

12. The chimeric polypeptide according to claims 1-11, wherein, compared with the corresponding wild-type PD1 extracellular segment or its functionally active fragment, the PD1 extracellular segment or its functionally active fragment includes one or more amino acid mutations, the amino acid mutation sites being selected from V39, L40, N41, Y43, M45, N49, K53, L97, A100, A107, A132, G124, K131, F19, I65 and / or A66.

13. The chimeric polypeptide according to claim 12, wherein the amino acid mutations include V39H, L40V, N41V, Y43H, M45E, N49G, K53T, L97V, A100V, A107I, A132I, A132F, A132T, A132V, G124S, K131Y, F19W, I65F and / or Q66S.

14. The chimeric polypeptide according to any one of claims 1-13, wherein the extracellular segment of PD1 or its functionally active fragment comprises the amino acid sequence shown in SEQ ID NO:

1.

15. The chimeric polypeptide according to any one of claims 1-14, wherein the Notch receptor polypeptide is derived from a type I transmembrane protein.

16. The chimeric polypeptide according to any one of claims 1-15, wherein the Notch receptor polypeptide is derived from the Notch receptor family, the advanced glycation end product receptor family, the cadherin superfamily of cell adhesion molecules, the tyrosine phosphatase receptor family, or the epidermal growth factor (EGF) family.

17. The chimeric polypeptide according to any one of claims 1-16, wherein the linker polypeptide comprises a juxtamembrane domain derived from the Notch receptor family, the advanced glycation end product receptor family, the cadherin superfamily of cell adhesion molecules, the tyrosine phosphatase receptor family, or the epidermal growth factor (EGF) family.

18. The chimeric polypeptide according to any one of claims 1-17, wherein the linking polypeptide comprises a juxtamembrane domain derived from Robo1, Notch1, Notch2, Notch3, Notch4, CLSTN2, AGER, PTPRF, or NRG1.

19. The chimeric polypeptide according to any one of claims 1-18, wherein the linking polypeptide comprises a juxtamembrane domain derived from Notch2.

20. The chimeric polypeptide according to any one of claims 1-19, wherein the linker polypeptide comprises a sequence having at least about 80% homology with the amino acid sequence shown in SEQ ID NO:

6.

21. The chimeric polypeptide according to any one of claims 1-20, wherein the linking polypeptide comprises the amino acid sequence shown in SEQ ID NO:

6.

22. The chimeric polypeptide according to any one of claims 1-21, wherein the transmembrane domain comprises a transmembrane domain derived from the Notch receptor family, the glycation end product receptor family, the cadherin superfamily of cell adhesion molecules, the tyrosine phosphatase receptor family, or the epidermal growth factor (EGF) family.

23. The chimeric polypeptide according to any one of claims 1-22, wherein the transmembrane domain comprises a transmembrane domain derived from Robo1, Notch1, Notch2, Notch3, Notch4, CLSTN2, AGER, PTPRF, or NRG1.

24. The chimeric polypeptide according to any one of claims 1-23, wherein the transmembrane domain is derived from the transmembrane domain of Notch1.

25. The chimeric polypeptide according to any one of claims 1-24, wherein the transmembrane domain further comprises one or more proteolytic cleavage sites.

26. The chimeric polypeptide according to any one of claims 1-25, wherein the transmembrane domain comprises a sequence having at least about 80% homology with the amino acid sequence shown in SEQ ID NO:

2.

27. The chimeric polypeptide according to any one of claims 1-26, wherein the transmembrane domain further comprises a mutation of G318 and / or V319.

28. The chimeric polypeptide according to any one of claims 1-27, wherein the transmembrane domain further comprises a mutation of G318A and / or V319A.

29. The chimeric polypeptide according to any one of claims 1-28, wherein the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:

4.

30. The chimeric polypeptide according to any one of claims 1-29, wherein the Notch receptor polypeptide comprises the transmembrane domain and the linker polypeptide from the N-terminus to the C-terminus.

31. The chimeric polypeptide according to any one of claims 1-30, wherein the intracellular domain comprises a transcription factor.

32. The chimeric polypeptide of claim 31, wherein the transcription factor is capable of activating a second portion that specifically targets a tumor antigen.

33. The chimeric polypeptide according to any one of claims 31-32, wherein the transcription factor is selected from the group consisting of Gal4-VP16, Gal4-VP64, tetR-VP64, ZFHD1-VP64, Gal4-KRAB and HAP1-VP16.

34. The chimeric polypeptide according to any one of claims 31-33, wherein the transcription factor comprises Gal4-VP64.

35. The chimeric polypeptide according to claim 32, wherein the tumor antigen is selected from the group consisting of: ROR1, AXL, CD19, CD20, CD22, ROR1, mesothelin, CD33, IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRVIII, GD-2, BCMA, CD123, CD7, Her2, B7-H3, CLDN18, EGFR, Nectin-4, CD90, CD133, DLL3, CEA, CD4, CD5, CD123, SIRP alpha, CD70, FORR1, NY-ESO-1, MAGE A3, GP100, MART-1, EBV, and p53.

36. The chimeric polypeptide according to claim 35, wherein the tumor antigen is ROR1.

37. The chimeric polypeptide of claim 32, wherein the second portion comprises at least one CDR in the heavy chain variable region VH targeting ROR1, the heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO:

10.

38. The chimeric polypeptide of claim 32, wherein the second portion comprises HCDR3, HCDR2 and HCDR1 targeting ROR1, wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO:11, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:12 and HCDR1 comprises the amino acid sequence shown in SEQ ID NO:

13.

39. The chimeric polypeptide according to any one of claims 31-38, wherein the transcription factor activates the second part via an activation sequence.

40. The chimeric polypeptide of claim 39, wherein the second portion comprises CAR and / or TCR.

41. The chimeric polypeptide of claim 40, wherein the CAR comprises ROR1 VHH, and the ROR1 VHH comprises the amino acid sequence shown in SEQ ID NO:

10.

42. The chimeric polypeptide according to claim 39, wherein the activation sequence is UAS.

43. A nucleic acid molecule encoding a chimeric polypeptide as described in any one of claims 1-42.

44. Cells comprising any one of the chimeric polypeptides according to claims 1-42.

45. The cell of claim 44, comprising the structure shown below: a) an extracellular domain, wherein the extracellular domain includes an extracellular segment of PD1 (programmed death receptor-1) or a functionally active fragment thereof, the extracellular segment of PD1 or the functionally active fragment thereof being capable of binding to a PD1 ligand; b) Notch receptor polypeptide, wherein the Notch receptor polypeptide comprises a linker polypeptide and a transmembrane domain; c) Intracellular domains; d) Exogenous nucleic acid sequence, wherein the exogenous nucleic acid sequence includes a second nucleic acid portion that specifically targets tumor antigens.

46. ​​The cell of claim 45, wherein the second nucleic acid moiety encodes a CAR and / or TCR that specifically targets the tumor antigen.

47. The cell according to any one of claims 45-46, wherein the second nucleic acid portion comprises a nucleic acid sequence encoding an antigen-binding region capable of specifically targeting a tumor antigen.

48. The cell according to any one of claims 45-47, wherein the tumor antigen is selected from the group consisting of: ROR1, AXL, CD19, CD20, CD22, mesothelin, CD33, IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRVIII, GD-2, BCMA, CD123, CD7, Her2, B7-H3, CLDN18, EGFR, Nectin-4, CD90, CD133, DLL3, CEA, CD4, CD5, CD123, SIRP alpha, CD70, FORR1, NY-ESO-1, MAGE A3, GP100, MART-1, EBV, and p53.

49. The cell according to claim 48, wherein the tumor antigen is ROR1.

50. The cell of claim 47, wherein the antigen-binding region comprises at least one CDR in the heavy chain variable region VH targeting ROR1, the heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO:

10.

51. The cell of claim 47, wherein the antigen-binding region comprises HCDR3, HCDR2 and HCDR1 targeting ROR1, wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO:11, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:12 and HCDR1 comprises the amino acid sequence shown in SEQ ID NO:

13.

52. The cell according to any one of claims 47-51, wherein the antigen-binding region comprises an antibody or an antigen-binding fragment thereof.

53. The cell according to claim 52, wherein the antibody comprises a monoclonal antibody, a chimeric antibody, a humanized antibody, and / or a fully human antibody.

54. The cell according to claim 52, wherein the antigen-binding fragment comprises Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

55. The cell according to claim 54, wherein the antigen-binding fragment is VHH.

56. The cell according to claim 55, wherein the antigen-binding fragment is ROR1 VHH, and the ROR1 VHH comprises the amino acid sequence shown in SEQ ID NO:

10.

57. The cell according to any one of claims 45-56, wherein the second nucleic acid portion comprises a nucleic acid sequence encoding a co-stimulatory domain.

58. The cell of claim 57, wherein the co-stimulatory signaling domain comprises an intracellular co-stimulatory signaling domain derived from one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88.

59. The cell according to claim 58, wherein the co-stimulatory signaling domain is a 4-1BB co-stimulatory signaling domain.

60. The cell according to claim 59, wherein the co-stimulatory signaling domain comprises the amino acid sequence shown in SEQ ID NO:

14.

61. The cell according to any one of claims 45-60, wherein the second nucleic acid portion comprises a nucleic acid sequence encoding an intracellular signal transduction domain.

62. The cell of claim 61, wherein the intracellular signaling domain comprises an intracellular signaling domain derived from one or more proteins selected from the group consisting of: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM.

63. The cell according to claim 562, wherein the intracellular signal transduction domain is the intracellular signal transduction domain of CD3ζ.

64. The cell of claim 63, wherein the intracellular signal transduction domain comprises the amino acid sequence shown in SEQ ID NO:

15.

65. The cell according to any one of claims 45-64, wherein the second nucleic acid portion further comprises a nucleic acid sequence encoding a transmembrane region.

66. The cell of claim 65, wherein the transmembrane region is derived from the transmembrane region of one or more proteins selected from the group consisting of: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

67. The cell according to claim 66, wherein the transmembrane region is the transmembrane region of CD28.

68. The cell of claim 67, wherein the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:

16.

69. The cell according to any one of claims 45-68, wherein the second nucleic acid portion further comprises a nucleic acid sequence encoding the hinge region.

70. The cell of claim 69, wherein the hinge region comprises a hinge region derived from one or more proteins selected from the group consisting of: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

71. The cell according to claim 70, wherein the hinge region is the hinge region of CD28.

72. The cell of claim 71, wherein the hinge region comprises the amino acid sequence shown in SEQ ID NO:

17.

73. The cell according to any one of claims 44-72, wherein the cell comprises immune effector cells.

74. The cell according to any one of claims 44-73, wherein the cell comprises T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral blood mononuclear cells, embryonic stem cells, lymphoprogenitor cells and / or kinetic stem cells, or variants thereof.

75. The cell according to any one of claims 44-74, wherein the cell is a T cell.

76. A nucleic acid molecule comprising a first nucleic acid moiety and a second nucleic acid moiety; wherein the first nucleic acid moiety includes a nucleic acid sequence encoding an extracellular domain, the extracellular domain including a PD1 extracellular segment or a functionally active fragment thereof, the PD1 extracellular segment or the functionally active fragment thereof being capable of binding to a PD1 ligand; the first nucleic acid moiety further includes a nucleic acid sequence encoding a Notch receptor polypeptide, the Notch receptor polypeptide including a linker polypeptide and a transmembrane domain; and the first nucleic acid moiety further includes a nucleic acid sequence encoding an intracellular domain, the intracellular domain including a transcription factor.

77. The nucleic acid molecule according to claim 76, wherein the extracellular segment of PD1 or its functionally active fragment comprises at least one immunoglobulin variable region (IgV)-like structure.

78. The nucleic acid molecule according to any one of claims 76-77, wherein the extracellular segment of PD1 or its functionally active fragment further comprises at least one N-glycosylation site.

79. The nucleic acid molecule according to any one of claims 76-77, wherein the extracellular segment of PD1 or its functionally active fragment comprises four N-glycosylation sites.

80. The nucleic acid molecule according to claim 79, wherein the N-glycosylation site is selected from one or more of the group consisting of N49, N58, N74 and N116.

81. The nucleic acid molecule according to any one of claims 76-80, wherein the extracellular segment of PD1 or its functionally active fragment further comprises an N-terminal loop region (N-loop).

82. The nucleic acid molecule according to any one of claims 76-81, wherein the extracellular segment of PD1 or its functionally active fragment comprises soluble PD1 (sPD1).

83. The nucleic acid molecule according to any one of claims 76-82, wherein the PD1 ligand comprises PD-L1 and / or PD-L2.

84. The nucleic acid molecule according to any one of claims 76-83, wherein the extracellular segment of PD1 or its functionally active fragment comprises wild-type PD1 or a variant thereof.

85. The nucleic acid molecule according to any one of claims 76-84, wherein the PD1 extracellular segment or its functionally active fragment is the mammalian PD1 extracellular segment or its functionally active fragment.

86. The nucleic acid molecule according to any one of claims 76-85, wherein the PD1 extracellular segment or its functionally active fragment is a human PD1 extracellular segment or its functionally active fragment.

87. The nucleic acid molecule according to any one of claims 76-86, wherein, compared with the corresponding wild-type PD1 extracellular segment or its functionally active fragment, the PD1 extracellular segment or its functionally active fragment comprises one or more amino acid mutations, the amino acid mutation sites including V39, L40, N41, Y43, M45, N49, K53, L97, A100, A107, A132, G124, K131, F19, I65 and / or A66.

88. The nucleic acid molecule according to claim 87, wherein the amino acid mutations include V39H, L40V, N41V, Y43H, M45E, N49G, K53T, L97V, A100V, A107I, A132I, A132F, A132T, A132V, G124S, K131Y, F19W, I65F and / or Q66S.

89. The nucleic acid molecule according to any one of claims 76-88, wherein the extracellular segment of PD1 or its functionally active fragment comprises the amino acid sequence shown in SEQ ID NO:

1.

90. The nucleic acid molecule according to any one of claims 76-89, wherein the Notch receptor polypeptide is derived from a type I transmembrane protein.

91. The nucleic acid molecule according to any one of claims 76-90, wherein the Notch receptor polypeptide is derived from the Notch receptor family, the advanced glycation end product receptor family, the cadherin superfamily of cell adhesion molecules, the tyrosine phosphatase receptor family, or the epidermal growth factor (EGF) family.

92. The nucleic acid molecule according to any one of claims 76-91, wherein the linker polypeptide comprises a juxtamembrane domain derived from the Notch receptor family, the advanced glycation end product receptor family, the cadherin superfamily of cell adhesion molecules, the tyrosine phosphatase receptor family, or the epidermal growth factor (EGF) family.

93. The nucleic acid molecule according to any one of claims 76-92, wherein the linker polypeptide comprises a juxtamembrane domain derived from Robo1, Notch1, Notch2, Notch3, Notch4, CLSTN2, AGER, PTPRF, or NRG1.

94. The chimeric polypeptide according to any one of claims 76-93, wherein the linking polypeptide comprises a juxtamembrane domain derived from Notch2.

95. The nucleic acid molecule according to any one of claims 76-94, wherein the linker polypeptide comprises a sequence having at least about 80% homology with the amino acid sequence shown in SEQ ID NO:

6.

96. The nucleic acid molecule according to any one of claims 76-95, wherein the linker polypeptide comprises the amino acid sequence shown in SEQ ID NO:

6.

97. The nucleic acid molecule according to any one of claims 76-96, wherein the transmembrane domain comprises a transmembrane domain derived from the Notch receptor family, the advanced glycation end product receptor family, the cadherin superfamily of cell adhesion molecules, the tyrosine phosphatase receptor family, or the epidermal growth factor (EGF) family.

98. The nucleic acid molecule according to any one of claims 76-97, wherein the transmembrane domain comprises a transmembrane domain derived from Robo1, Notch1, Notch2, Notch3, Notch4, CLSTN2, AGER, PTPRF, or NRG1.

99. The nucleic acid molecule according to any one of claims 76-98, wherein the transmembrane domain is derived from the transmembrane domain of Notch1.

100. The nucleic acid molecule according to any one of claims 76-99, wherein the transmembrane domain further comprises one or more proteolytic cleavage sites.

101. The nucleic acid molecule according to any one of claims 76-100, wherein the transmembrane domain comprises a sequence having at least about 80% homology with the amino acid sequence shown in SEQ ID NO:

2.

102. The nucleic acid molecule according to any one of claims 76-101, wherein the transmembrane domain further comprises a mutation of G318 and / or V319.

103. The nucleic acid molecule according to any one of claims 76-102, wherein the transmembrane domain further comprises mutations in G318A and / or V319A.

104. The nucleic acid molecule according to any one of claims 76-103, wherein the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:

4.

105. The nucleic acid molecule according to any one of claims 76-104, wherein the Notch receptor polypeptide comprises the transmembrane domain and the linker polypeptide from the N-terminus to the C-terminus.

106. The nucleic acid molecule according to any one of claims 76-105, wherein the transcription factor is selected from the group consisting of Gal4-VP16, Gal4-VP64, tetR-VP64, ZFHD1-VP64, Gal4-KRAB, and HAP1-VP16.

107. The nucleic acid molecule according to any one of claims 76-106, wherein the transcription factor comprises Gal4-VP64.

108. The nucleic acid molecule according to any one of claims 76-107, wherein the second nucleic acid portion comprises encoding a CAR and / or TCR capable of specifically targeting tumor antigens.

109. The nucleic acid molecule according to any one of claims 76-108, wherein the second nucleic acid portion comprises a nucleic acid sequence encoding an antigen-binding region capable of specifically targeting a tumor antigen.

110. The nucleic acid molecule according to claim 109, wherein the tumor antigen is selected from the group consisting of: ROR1, AXL, CD19, CD20, CD22, mesothelin, CD33, IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRVIII, GD-2, BCMA, CD123, CD7, Her2, B7-H3, CLDN18, EGFR, Nectin-4, CD90, CD133, DLL3, CEA, CD4, CD5, CD123, SIRP alpha, CD70, FORL1, NY-ESO-1, MAGE A3, GP100, MART-1, EBV, and p53.

111. The nucleic acid molecule according to claim 110, wherein the tumor antigen is ROR1.

112. The nucleic acid molecule of claim 109, wherein the antigen-binding region comprises at least one CDR in the heavy chain variable region VH targeting ROR1, the heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO:

10.

113. The nucleic acid molecule of claim 109, wherein the antigen-binding region comprises HCDR3, HCDR2 and HCDR1 targeting ROR1, wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO:11, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:12 and HCDR1 comprises the amino acid sequence shown in SEQ ID NO:

13.

114. The nucleic acid molecule according to any one of claims 109-113, wherein the antigen-binding region comprises an antibody or an antigen-binding fragment thereof.

115. The nucleic acid molecule according to claim 114, wherein the antibody comprises a monoclonal antibody, a chimeric antibody, a humanized antibody, and / or a fully human antibody.

116. The nucleic acid molecule according to claim 114, wherein the antigen-binding fragment comprises Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

117. The nucleic acid molecule according to claim 114, wherein the antigen-binding fragment is VHH.

118. The nucleic acid molecule according to claim 117, wherein the antigen-binding fragment is ROR1 VHH, and the ROR1 VHH contains the amino acid sequence shown in SEQ ID NO:

10.

119. The nucleic acid molecule according to any one of claims 76-118, wherein the second nucleic acid portion comprises a nucleic acid sequence encoding a co-stimulatory domain.

120. The nucleic acid molecule of claim 119, wherein the co-stimulatory signaling domain comprises an intracellular co-stimulatory signaling domain derived from one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88.

121. The nucleic acid molecule according to claim 120, wherein the co-stimulatory signaling domain is a 4-1BB co-stimulatory signaling domain.

122. The nucleic acid molecule according to claim 121, wherein the co-stimulatory signaling domain comprises the amino acid sequence shown in SEQ ID NO:

14.

123. The nucleic acid molecule according to any one of claims 76-122, wherein the second nucleic acid portion comprises a nucleic acid sequence encoding an intracellular signal transduction domain.

124. The nucleic acid molecule of claim 123, wherein the intracellular signal transduction domain comprises an intracellular signal transduction domain derived from one or more proteins selected from the group consisting of: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM.

125. The nucleic acid molecule according to claim 124, wherein the intracellular signal transduction domain is the intracellular signal transduction domain of CD3ζ.

126. The nucleic acid molecule according to claim 125, wherein the intracellular signal transduction domain comprises the amino acid sequence shown in SEQ ID NO:

15.

127. The nucleic acid molecule according to any one of claims 76-126, wherein the second nucleic acid portion further comprises a nucleic acid sequence encoding a transmembrane region.

128. The nucleic acid molecule of claim 127, wherein the transmembrane region is derived from the transmembrane region of one or more proteins selected from the group consisting of: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

129. The nucleic acid molecule according to claim 128, wherein the transmembrane region is the transmembrane region of CD28.

130. The nucleic acid molecule according to claim 129, wherein the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:

16.

131. The nucleic acid molecule according to any one of claims 76-130, wherein the second nucleic acid portion further comprises a nucleic acid sequence encoding the hinge region.

132. The nucleic acid molecule of claim 131, wherein the hinge region comprises a hinge region derived from one or more proteins selected from the group consisting of: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

133. The nucleic acid molecule according to claim 132, wherein the hinge region is the hinge region of CD28.

134. The nucleic acid molecule according to claim 133, wherein the hinge region comprises the amino acid sequence shown in SEQ ID NO:

17.

135. The nucleic acid molecule according to any one of claims 76-134, wherein the first nucleic acid portion is linked to a promoter sequence.

136. The nucleic acid molecule according to claim 135, wherein the promoter sequence is selected from the group consisting of: CMV promoter sequence, SFFV promoter sequence, EF1α promoter sequence and PGK promoter sequence.

137. The nucleic acid molecule according to claim 136, wherein the promoter sequence is an SFFV promoter sequence.

138. The nucleic acid molecule according to any one of claims 76-137, wherein the second nucleic acid portion is linked to an activation sequence.

139. The nucleic acid molecule according to claim 138, wherein the activation sequence is a UAS sequence.

140. The nucleic acid molecule according to any one of claims 76-139, comprising, from its 5' end to its 3' end, respectively: a second nucleic acid moiety activation sequence, a nucleic acid sequence encoding the antigen-binding region, a nucleic acid sequence encoding the hinge region, a nucleic acid sequence encoding the transmembrane region, a nucleic acid sequence encoding the co-stimulatory domain, a nucleic acid sequence encoding the intracellular signal transduction domain, a first nucleic acid moiety initiation sequence, a nucleic acid sequence encoding the extracellular domain, a nucleic acid sequence encoding the Notch receptor polypeptide, and a nucleic acid sequence encoding the intracellular domain.

141. An expression vector comprising the nucleic acid molecule of any one of claims 76-140.

142. The expression vector according to claim 141, comprising a viral vector or a non-viral vector.

143. The expression vector according to claim 142, wherein the viral vector comprises a lentiviral vector, an adenovirus vector, an adeno-associated virus vector, or a retroviral vector.

144. The expression vector according to any one of claims 142-143, wherein the viral vector is a lentiviral vector.

145. A pharmaceutical composition comprising a chimeric polypeptide according to any one of claims 1-42, a nucleic acid molecule according to claim 43, a cell according to any one of claims 44-75, a nucleic acid molecule according to any one of claims 76-140, and / or an expression vector according to any one of claims 142-144, and optionally a pharmaceutically acceptable carrier.

146. A system for regulating cell activity, the system comprising a chimeric polypeptide according to any one of claims 1-42, a nucleic acid molecule according to claim 43, a cell according to any one of claims 44-75, a nucleic acid molecule according to any one of claims 76-140, an expression vector according to any one of claims 141-144, and / or a pharmaceutical composition according to claim 145.

147. A method for regulating cell activity, the method comprising: a) Providing cells capable of expressing the chimeric polypeptide of any one of claims 1-42; b) Contacting the cell with the nucleic acid molecule according to any one of claims 76-140; c) Obtaining the cells according to any one of claims 44-75, and contacting the cells according to any one of claims 44-75 with tumor cells; d) In any of the cells of claims 44-75, the PD1 binds to a ligand in the tumor cell, inducing the cleavage of the transmembrane domain and releasing the intracellular domain; e) The intracellular domains described regulate the antitumor activity of cells.

148. The method of claim 147, wherein the intracellular domain modulates cellular antitumor activity by activating CAR and / or TCR expression.

149. The method of claim 148, wherein the CAR and / or TCR is specifically targeted at a tumor antigen selected from the group consisting of: ROR1, AXL, CD19, CD20, CD22, ROR1, mesothelin, CD33, IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRVIII, GD-2, BCMA, CD123, CD7, Her2, B7-H3, CLDN18, EGFR, Nectin-4, CD90, CD133, DLL3, CEA, CD4, CD5, CD123, SIRP alpha, CD70, FORL1, NY-ESO-1, MAGE A3, GP100, MART-1, EBV, and p53.

150. The method according to claim 149, wherein the tumor antigen is ROR1.

151. The method according to any one of claims 147-150, wherein the method is an in vitro method or an ex vivo method.

152. The method according to any one of claims 147-151, wherein the cells comprise immune effector cells.

153. The method according to any one of claims 147-152, wherein the cells comprise T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral blood mononuclear cells, embryonic stem cells, lymphoprogenitor cells and / or kinetic stem cells, or variants thereof.

154. The method according to any one of claims 147-153, wherein the cell is a T cell.

155. Use of the chimeric polypeptide of any one of claims 1-42, the nucleic acid molecule of claim 43, the cell of any one of claims 44-75, the nucleic acid molecule of any one of claims 76-140, the expression vector of any one of claims 141-144, the pharmaceutical composition of claim 145, and / or the system of claim 146 in the preparation of a medicament for the prevention, treatment and / or relief of diseases and / or symptoms.

156. The chimeric polypeptide of any one of claims 1-42, the nucleic acid molecule of claim 43, the cell of any one of claims 44-75, the nucleic acid molecule of any one of claims 76-140, the expression vector of any one of claims 141-144, the pharmaceutical composition of claim 145, and / or the system of claim 146, for the prevention, treatment and / or relief of diseases and / or symptoms.

157. A method for preventing, treating, and / or alleviating diseases and / or symptoms, the method comprising administering to a subject in need a chimeric polypeptide of any one of claims 1-42, a nucleic acid molecule of claim 43, a cell of any one of claims 44-75, a nucleic acid molecule of any one of claims 76-140, an expression vector of any one of claims 141-144, a pharmaceutical composition of claim 145, and / or a system of claim 146.

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