Door control system

Abstract

The present application relates to a gating system comprising a chimeric polypeptide comprising, from N-terminus to 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 fragment or a functionally active fragment thereof, the PD1 extracellular fragment or the functionally active fragment thereof being capable of binding to a PD1 ligand, the Notch receptor polypeptide comprises a connecting polypeptide and a transmembrane domain, the intracellular domain is capable of activating a second part of a specifically targeted tumor antigen, and the tumor antigen is AXL.

Description

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] This application provides a novel hybrid SynNotch receptor that integrates an extracellular domain and uses the extracellular segment of PD1 or its functionally active fragment as a gating mechanism to regulate cell 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 has high sensitivity to PD-L1 expression; (3) the 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 PD-L1-expressing tumor cells with high clinical safety; and (5) it improves the survival rate of transfected T cells, facilitating large-scale production.

[0006] Meanwhile, existing CAR-T technology has the following defects: (1) off-target effect, existing CAR-T cell therapy often “accidentally” damages normal tissues with low expression of tumor antigens; (2) poor safety, the “off-target” killing ability will bring systemic toxicity, which may lead to irreversible organ damage; (3) the high heterogeneity of solid tumors means that single-target CAR-T can only clear some tumor cells and cannot fully cover them, resulting in a high tumor recurrence rate; (4) although dual-target or multi-target CAR-T can reduce antigen escape, the complex structure and high expression level will accelerate T cell depletion and shorten the clinical continuous remission time.

[0007] Furthermore, the chimeric polypeptide provided in this application can regulate the transcriptional expression of AXL CAR by cells, and has the following effects: (1) It is highly sensitive to PDL1 expression and can be effectively stimulated by PDL1 positive cells to express AXLCAR; (2) It has precise killing ability and releases cytotoxicity only when it recognizes double positive cells of PDL1 (activation switch) and AXL (target antigen), which is significantly better than single-target CAR-T; (3) It has strong proliferation ability and can continuously kill tumor cells; (4) It has good safety and has no killing activity on normal tissues that are single positive of PDL1, single positive of AXL, or even express low levels of PDL1, effectively reducing the risk of toxicity.

[0008] This application provides a chimeric polypeptide comprising, from the N-terminus to the C-terminus:

[0009] 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;

[0010] b) Notch receptor polypeptide, wherein the Notch receptor polypeptide comprises a linker polypeptide and a transmembrane domain;

[0011] c) An intracellular domain capable of activating a second part that specifically targets a tumor antigen, namely AXL.

[0012] In one embodiment, the extracellular segment of PD1 or its functionally active fragment includes at least an immunoglobulin variable region (IgV)-like structure.

[0013] In one embodiment, the extracellular segment of PD1 or its functionally active fragment further includes at least one N-glycosylation site.

[0014] In one embodiment, the extracellular segment of PD1 or its functionally active fragment includes four N-glycosylation sites.

[0015] In one embodiment, the N-glycosylation site is selected from one or more of the following group: N49, N58, N74, and N116.

[0016] In one embodiment, the extracellular segment of PD1 or its functionally active fragment further includes an N-terminal loop region (N-loop).

[0017] In one embodiment, the extracellular segment of PD1 or its functionally active fragment comprises soluble PD1 (sPD1).

[0018] In one embodiment, the PD1 ligand includes PD-L1 and / or PD-L2.

[0019] In one embodiment, the extracellular segment of PD1 or its functionally active fragment comprises wild-type PD1 or a variant thereof.

[0020] In one embodiment, the PD1 extracellular segment or its functionally active fragment is a mammalian PD1 extracellular segment or its functionally active fragment.

[0021] In one embodiment, the mammal includes a human, rat, dog, cat, cow, rabbit, or sheep.

[0022] In one embodiment, the PD1 extracellular segment or its functionally active fragment is a human PD1 extracellular segment or its functionally active fragment.

[0023] In one embodiment, the extracellular segment of PD1 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.

[0024] In one embodiment, 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.

[0025] In one embodiment, the extracellular segment of PD1 or its functionally active fragment comprises the amino acid sequence shown in SEQ ID NO: 1.

[0026] In one embodiment, the Notch receptor polypeptide is derived from a type I transmembrane protein.

[0027] In one embodiment, 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.

[0028] In one embodiment, 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.

[0029] In one embodiment, the linker polypeptide includes a juxtamembrane domain derived from Robo1, Notch1, Notch2, Notch3, Notch4, CLSTN2, AGER, PTPRF, or NRG1.

[0030] In one embodiment, the linker polypeptide includes a juxtamembrane domain derived from Notch2.

[0031] In one embodiment, the linker polypeptide comprises 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.

[0032] In one embodiment, the linker polypeptide comprises the amino acid sequence shown in SEQ ID NO: 6.

[0033] In one embodiment, the transmembrane domain includes transmembrane domains 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.

[0034] In one embodiment, the transmembrane domain includes a transmembrane domain derived from Robo1, Notch1, Notch2, Notch3, Notch4, CLSTN2, AGER, PTPRF, or NRG1.

[0035] In one embodiment, the transmembrane domain is derived from the transmembrane domain of Notch1.

[0036] In one embodiment, the transmembrane domain further includes one or more protein hydrolysis cleavage sites.

[0037] In one embodiment, 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.

[0038] In one embodiment, the transmembrane domain further includes mutations in G318 and / or V319.

[0039] In one embodiment, the transmembrane domain further includes mutations in G318A and / or V319A.

[0040] In one embodiment, the transmembrane domain includes the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0041] In one embodiment, the Notch receptor polypeptide includes the transmembrane domain and the linker polypeptide from the N-terminus to the C-terminus.

[0042] In one embodiment, the intracellular domain includes a transcription factor.

[0043] In one embodiment, the transcription factor is selected from the group consisting of Gal4-VP16, Gal4-VP64, tetR-VP64, ZFHD1-VP64, Gal4-KRAB, and HAP1-VP16.

[0044] In one embodiment, the transcription factor includes Gal4-VP64.

[0045] In one implementation, the transcription factor activates the second part via an activation sequence.

[0046] In one implementation, the second part includes a CAR and / or a TCR.

[0047] In one implementation, the activation sequence is UAS.

[0048] This application also provides a nucleic acid molecule that encodes the chimeric polypeptide described above.

[0049] This application also provides a cell comprising the chimeric polypeptide described above.

[0050] In one embodiment, the cell further includes an exogenous nucleic acid sequence comprising a second nucleic acid portion that specifically targets an antigen-binding region of a tumor antigen, wherein the tumor antigen is AXL.

[0051] In one embodiment, the second nucleic acid portion encodes a CAR and / or TCR that specifically targets tumor antigens.

[0052] In one embodiment, the antigen-binding region includes an antibody or an antigen-binding fragment thereof.

[0053] In one embodiment, the antibody includes monoclonal antibodies, chimeric antibodies, humanized antibodies, and / or fully human antibodies.

[0054] In one embodiment, the antigen-binding fragment includes Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

[0055] In one embodiment, the antigen-binding fragment is scFv.

[0056] In one embodiment, the second nucleic acid portion includes a nucleic acid sequence encoding a co-stimulatory domain.

[0057] In one embodiment, 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.

[0058] In one embodiment, the costimulatory signal domain is a 4-1BB costimulatory signal domain.

[0059] In one embodiment, the second nucleic acid portion includes a nucleic acid sequence encoding an intracellular signal transduction domain.

[0060] In one embodiment, 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.

[0061] In one embodiment, the intracellular signal transduction domain is the intracellular signal transduction domain of CD3ζ.

[0062] In one embodiment, the second nucleic acid portion further includes a nucleic acid sequence encoding a transmembrane region.

[0063] In one embodiment, 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.

[0064] In one embodiment, the transmembrane region is the transmembrane region of CD28.

[0065] In one embodiment, the second nucleic acid portion further includes a nucleic acid sequence encoding the hinge region.

[0066] In one embodiment, 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.

[0067] In one embodiment, the hinge area is the hinge area of ​​CD28.

[0068] In one embodiment, the cells include immune effector cells.

[0069] In one embodiment, 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.

[0070] In one embodiment, the cell is a T cell.

[0071] 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 encodes the aforementioned chimeric polypeptide, and the second nucleic acid portion includes a nucleic acid sequence encoding an antigen-binding region capable of specifically targeting a tumor antigen, wherein the tumor antigen is AXL.

[0072] In one embodiment, the second nucleic acid portion includes a nucleic acid sequence encoding an antigen-binding region capable of specifically targeting tumor antigens.

[0073] In one embodiment, 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.

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

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

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

[0077] In one embodiment, the antigen-binding fragment is scFv.

[0078] In one embodiment, the second nucleic acid portion includes a nucleic acid sequence encoding a co-stimulatory domain.

[0079] In one embodiment, 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.

[0080] In one embodiment, the costimulatory signal domain is a 4-1BB costimulatory signal domain.

[0081] In one embodiment, the second nucleic acid portion includes a nucleic acid sequence encoding an intracellular signal transduction domain.

[0082] In one embodiment, 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.

[0083] In one embodiment, the intracellular signal transduction domain is the intracellular signal transduction domain of CD3ζ.

[0084] In one embodiment, the second nucleic acid portion further includes a nucleic acid sequence encoding a transmembrane region.

[0085] In one embodiment, 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.

[0086] In one embodiment, the transmembrane region is the transmembrane region of CD28.

[0087] In one embodiment, the second nucleic acid portion further includes a nucleic acid sequence encoding the hinge region.

[0088] In one embodiment, 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.

[0089] In one embodiment, the hinge area is the hinge area of ​​CD28.

[0090] In one implementation, the first nucleic acid portion is linked to a promoter sequence.

[0091] In one embodiment, the boot sequence is selected from the group consisting of: CMV boot sequence, SFFV boot sequence, EF1α boot sequence and PGK boot sequence.

[0092] In one implementation, the startup sequence is an SFFV startup sequence.

[0093] In one embodiment, the second nucleic acid portion is linked to an activation sequence.

[0094] In one implementation, the activation sequence is a UAS sequence.

[0095] In one embodiment, it comprises, from the 5' end to the 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.

[0096] This application also provides an expression vector comprising the aforementioned nucleic acid molecule.

[0097] In one implementation, it includes a viral vector or a non-viral vector.

[0098] In one embodiment, the viral vector includes a lentiviral vector, an adenovirus vector, an adeno-associated virus vector, or a retroviral vector.

[0099] In one embodiment, the viral vector is a lentiviral vector.

[0100] 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.

[0101] 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.

[0102] This application also provides a method for regulating cell activity, the method comprising:

[0103] a) Provide cells capable of expressing the chimeric polypeptide;

[0104] b) Contact the cells with the nucleic acid molecules described above;

[0105] c) Obtain the cells and bring the cells into contact with tumor cells;

[0106] In the cells described in d), PD1 binds to ligands in tumor cells, inducing transmembrane domain cleavage and releasing intracellular domains;

[0107] e) The intracellular domains described regulate the antitumor activity of cells.

[0108] In one embodiment, the method is an in vitro method or an ex vivo method.

[0109] In one embodiment, the cells include immune effector cells.

[0110] In one embodiment, 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.

[0111] In one embodiment, the cell is a T cell.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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

[0116] 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:

[0117] Figure 1 The diagram shown is a structural design of the PD1 ECD chimeric polypeptide.

[0118] Figure 2 The diagram shown is a structural design of the TGFβRII ECD chimeric polypeptide.

[0119] Figure 3 The image shows a PD1ECD (HA tag) gated expression assay after transfection. The Mock T group represents untransfected T cells, and the PD1 SNIPR-ROR1 CAR group represents T cells on the second day after transfection.

[0120] Figure 4 The image shows the TGFβRII ECD-gated expression assay after transfection. The HA tag is the CD22 CAR expression tag. The NC group represents untransfected T cells, and the TGFβRII-SNIPR-CD22-CAR group represents T cells on the second day after transfection.

[0121] Figure 5 The results show the PD1ECD ROR1 CAR-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.

[0122] Figure 6 This shows the TGFβRII SNIPR-CD22 CAR-T cell induction expression assay. The HA tag is the CD22 CAR-T expression tag.

[0123] Figure 7A The image shows the IL2 cytokine secretion assay after co-culturing PD1 ECD chimeric peptide with target cells; the left image shows an effector-to-target ratio of 1:1; the right image shows an effector-to-target ratio of 1:3. Figure 7BThe results show an IFNγ cytokine secretion assay after co-culturing PD1 ECD chimeric peptides 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.

[0124] Figure 8A -E shows the five-day real-time cell killing assay and re-kill assay (Incucyte Assay). Figure 8A A tumor-killing assay of PD1 ECD chimeric peptide against MEC target cells (ROR1- / PDL1+); Figure 8B This study aims to investigate the tumor-killing effect of the PD1 ECD chimeric peptide on MB231 target cells (ROR1+ / PDL1+). Figure 8C This study aims to investigate the tumor-killing effect of the PD1 ECD chimeric peptide on MEC-ROR1 target cells (with low levels of ROR1+ / PDL1 expression). Figure 8D A tumor-killing assay of PD1 ECD chimeric peptide against A549-PDL1 target cells (ROR1+ / PDL1 overexpression); Figure 8E To conduct a tumor killing assay on HEP3B-PDL1 target cells (ROR1+ / PDL1 overexpression).

[0125] Figure 9 The structures shown are the PD1 logic-gated AXL CAR (8601) and the control group AXL CAR (8600).

[0126] Figure 10 The display shows the expression level of PD1 logic-gated AXL CAR after transfection. Among them, Mock-T represents untransfected T cells.

[0127] Figure 11 The figure shows the CAR expression levels of PD1-gated logic-gated AXL CAR T cells in different cell types. The NC group represents untransfected T cells, T only represents T cells expressing only the PD1ECD Notch receptor in an unactivated CAR state, HEK293T cells do not express PDL1, and K562-PDL1 cells express high levels of PDL1.

[0128] Figure 12The results show the killing effect of PD1 logic-gated AXL CAR T cells on tumor cells. Among them, MDA-MB-231 expresses high levels of AXL and PDL1, while MB453 does not express AXL and expresses low levels of PDL1. Detailed Implementation

[0129] 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.

[0130] Terminology Definition

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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 / Notchrepeats (LNRs) derived from members of the Notch receptor family, such as Notch1, Notch2, Notch3, and Notch4.

[0136] 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.

[0137] 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.

[0138] 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. 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., SignalTransduct 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 a NICD or transported to lysosomes for degradation. The classic model posits that, in the absence of a NICD, transcription factors (CSLs) bind to co-repressors, inhibiting the transcription of target genes. Once the 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. Roybal et al. (Cell. 2016 October 6; 167(2): 419–432.e16.) can achieve "AND" logic gate control of 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 Notch2 JMD and combining it with human Notch1 TMD 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 influenced 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-type 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 segment 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 PD1 or its functionally active fragment includes soluble PD1 (sPD1). 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. These mutation sites 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 starts from the starting amino acid (i.e., methionine) of the natural human PD1 protein, with the starting amino acid 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 of 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. As another example, the intracellular domain of this application can activate a chimeric antigen receptor targeting AXL.

[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 implementations, 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 implementations, the signaling molecules are selected from CD28, 4-IBB, OX40, ICOS, CTLA4, PD1, PD1H, BTLA, B71, B7H1, CD226, CRTA M, TIGIT, CD96, TIM1, TIM2, TIM3, TIM4, CD2, SLAM, 2B4, Ly108, CD84, Ly9, CRACC, BTN1, BTN2, BTN3, LAIR1, LAG3, CD160, CD27, GITR, CD30, TNFR1, TNFR2, HVEM, LT_R, DR3, DCR3, FAS, CD40, RANK, OPG, TRAILR1, TACI, BAFFR, BCMA, TWEAKR, EDAR, XEDAR, RELT, DR6, TROY, NGFR, CD22, 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 includes 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 includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 9. In some embodiments, the ICD includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 9. In some embodiments, the ICD includes an amino acid sequence having at least 100% sequence identity with SEQ ID NO: 9. In some embodiments, the ICD includes 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] In this application, the chimeric antigen receptor targeting AXL may comprise any known chimeric antigen receptor targeting AXL. In some embodiments, the chimeric antigen receptor targeting AXL may comprise an AXL-targeting scFv.

[0176] For example, an AXL-targeting scFv contains heavy chain variable regions HCDR1-3, wherein HCDR1 contains an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 20, HCDR2 contains an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 19, and HCDR3 contains an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 18. For example, an AXL-targeting scFv contains VH, wherein VH contains an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 21.

[0177] For example, an AXL-targeting scFv contains light chain variable regions LCDR1-3, wherein LCDR1 contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 24, LCDR2 contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 23, and LCDR3 contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 22. For example, an AXL-targeting scFv contains VL, wherein VL contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 25.

[0178] For example, the scFv targeting AXL contains HCDR1-3 and LCDR1-3, wherein HCDR1-3 and LCDR1-3 contain the amino acid sequences shown in the following group: HCDR1: SEQ ID NO: 20, HCDR2: SEQ ID NO: 19, HCDR3: SEQ ID NO: 18, LCDR1: SEQ ID NO: 24, LCDR2: SEQ ID NO: 23, and LCDR3: SEQ ID NO: 22.

[0179] For example, the scFv targeting AXL contains VH and VL, wherein VH contains the amino acid sequence shown in SEQ ID NO: 21 and VL contains the amino acid sequence shown in SEQ ID NO: 25.

[0180] Chimeric Peptides

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

[0182] (1) An extracellular domain having at least 80% sequence homology with the amino acid sequence shown in SEQ ID NO: 1;

[0183] (2) A linker polypeptide having at least 80% sequence homology with the amino acid sequence shown in SEQ ID NO: 6;

[0184] (3) A transmembrane domain having at least 80% sequence homology with the amino acid sequence shown in SEQ ID NO: 2; and

[0185] (4) An intracellular domain having at least 80% sequence homology with 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 mutants 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: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8;

[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 having 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 mutants 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, SEQ ID NO: 3 or SEQ ID NO: 4; and

[0195] (4) Intracellular domain having 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 mutants 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: 2; and

[0200] (4) Intracellular domain having 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 mutants 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: 3; and

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

[0206] In some embodiments, the chimeric polypeptide described in this application may further include:

[0207] (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 mutants V39H, L40V, N41V, Y43H, M45E, N49G, K53T, L97V, A100V, A107I, A132I, A132F, A132T, A132V, G124S, K131Y, F19W, I65F and / or Q66S;

[0208] (2) A linking polypeptide having the amino acid sequence shown in SEQ ID NO: 6;

[0209] (3) A transmembrane domain having the amino acid sequence shown in SEQ ID NO: 4; and

[0210] (4) Intracellular domain having the amino acid sequence shown in SEQ ID NO: 9.

[0211] The extracellular domain, transmembrane domain, linking polypeptide, and intracellular domain are directly linked.

[0212] 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.

[0213] Nucleic acid molecules

[0214] 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.

[0215] 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 1, 2, 3, 4, or 5 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.

[0216] 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.

[0217] For example, the transmembrane domain can be selected from Notch1 TMD, and the linker peptide can be selected from Notch2 JMD.

[0218] For example, the transmembrane domain can be selected from Notch1 TMD, and the linker peptide can be selected from Notch1 JMD.

[0219] 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.

[0220] In one embodiment, the linker polypeptide is a protein tyrosine phosphatase receptor of type F. For example, the linker polypeptide may include an amino acid sequence as shown in SEQ ID NO: 6.

[0221] 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.

[0222] In some implementations, the TMD may be selected from TMDs derived from Notch1, and may also include Notch1 TMD variants, such as those with one or more amino acid mutations, which may be G318A and / or V319A.

[0223] 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.

[0224] 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.

[0225] 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.

[0226] In some implementations, the immune cell receptor is a CAR.

[0227] 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. 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.

[0228] For example, the antigenic domain of the CAR targets ROR1. For example, the antigenic domain of the CAR is a VHH that targets ROR1.

[0229] For example, the antigen domain of the CAR targets AXL. For instance, the antigen domain of the CAR is an scFv that targets AXL.

[0230] For example, the antigenic domain of the CAR includes HCDR1-3, wherein HCDR1 contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20, HCDR2 contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 19, and HCDR3 contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. For example, the antigenic domain of the CAR includes VH, wherein VH contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 21.

[0231] For example, the antigenic domain of the CAR includes LCDR1-3, wherein LCDR1 contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 24, LCDR2 contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 23, and LCDR3 contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 22. For example, the antigenic domain of the CAR includes VL, wherein VL contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 25.

[0232] For example, the antigenic domain of the CAR includes HCDR1-3 and LCDR1-3, wherein HCDR1-3 and LCDR1-3 contain the amino acid sequences shown in the following group: HCDR1: SEQ ID NO: 20, HCDR2: SEQ ID NO: 19, HCDR3: SEQ ID NO: 18, LCDR1: SEQ ID NO: 24, LCDR2: SEQ ID NO: 23, and LCDR3: SEQ ID NO: 22.

[0233] For example, the antigen domain of the CAR includes VH and VL, wherein VH contains the amino acid sequence shown in SEQ ID NO: 21 and VL contains the amino acid sequence shown in SEQ ID NO: 25.

[0234] 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.

[0235] For example, the co-stimulatory signaling domain can be a 4-1BB co-stimulatory signaling domain. For example, the stimulation signaling domain can contain the amino acid sequence shown in SEQ ID NO: 10.

[0236] 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 PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM.

[0237] For example, the intracellular signal transduction domain may be the intracellular signal transduction domain of CD3ζ. For example, the intracellular signal transduction domain may contain the amino acid sequence shown in SEQ ID NO: 11.

[0238] 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.

[0239] For example, the transmembrane region can be a transmembrane region of CD28. For example, the transmembrane region can contain the amino acid sequence shown in SEQ ID NO:12.

[0240] 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.

[0241] For example, the hinge region may be a CD28 hinge region. As another example, the hinge region may be a CD8 hinge region. For example, the hinge region may contain the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 14.

[0242] In some embodiments, the structure of the CAR may further include a signal peptide. For example, the signal peptide may be a CD8 signal peptide. For example, the signal peptide may contain the amino acid sequence shown in SEQ ID NO: 15.

[0243] 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.

[0244] For example, the initiation sequence is an SFFV initiation sequence. For example, the initiation sequence may contain the amino acid sequence shown in SEQ ID NO:17.

[0245] In some embodiments, the second nucleic acid portion is linked to an activation sequence, which may be a UAS sequence. For example, the activation sequence may comprise the amino acid sequence shown in SEQ ID NO: 16.

[0246] 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 signal peptide, 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 signal peptide, a nucleic acid sequence encoding the extracellular domain, a nucleic acid sequence encoding the Notch receptor polypeptide, a nucleic acid sequence encoding the intracellular domain, and a nucleic acid sequence encoding the transcription factor.

[0247] For example, the nucleic acid molecule provided in this application includes, from the 5' end to the 3' end, the following sequences in sequence: a UAS activation sequence, a nucleic acid sequence encoding a CD8 signal peptide, a nucleic acid sequence encoding an AXL scFv, a nucleic acid sequence encoding a CD8 hinge region, a nucleic acid sequence encoding a CD28 transmembrane region, a nucleic acid sequence encoding a 4-1BB co-stimulatory domain, a nucleic acid sequence encoding a CD3ζ intracellular signal transduction domain, an SFFV initiation sequence, a nucleic acid sequence encoding a CD8 signal peptide, a nucleic acid sequence encoding a PD1 extracellular segment, a nucleic acid sequence encoding an optimized CD8 hinge region, a nucleic acid sequence encoding a Notch transmembrane region, and a nucleic acid sequence encoding a Gal4-VP64 transcription factor.

[0248] 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.

[0249] 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.

[0250] 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, such as by calculation using known genes expressed in a reference 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 a host cell, 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.

[0251] 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).

[0252] 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), it can be produced through in vitro transcription or other methods.

[0253] expression carrier

[0254] 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.

[0255] In some implementations, the expression vector may be a viral vector. Viral vector is widely used to refer to nucleic acid molecules comprising virus-derived nucleic acid elements (e.g., transfer plasmids), which 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 acids 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. 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).

[0256] cell

[0257] 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.

[0258] The cells provided in this application include the following structures:

[0259] 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;

[0260] b) Notch receptor polypeptide, wherein the Notch receptor polypeptide comprises a linker polypeptide and a transmembrane domain;

[0261] c) Intracellular domains;

[0262] d) Exogenous nucleic acid sequence, wherein the exogenous nucleic acid sequence includes a second nucleic acid portion that specifically targets tumor antigens.

[0263] 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.

[0264] For example, partial expression of the exogenous nucleic acid can specifically target the tumor antigen AXL.

[0265] In some embodiments, the exogenous nucleic acid portion may include a nucleic acid sequence encoding an immune cell receptor.

[0266] 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, δ chain, or a combination thereof of a TCR. The TCR polypeptide chains and methods for their preparation are known in the art.

[0267] In some implementations, the immune cell receptor is a CAR.

[0268] 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.

[0269] For example, the antigenic domain of the CAR targets ROR1. For example, the antigenic domain of the CAR is a VHH that targets ROR1.

[0270] For example, the antigen domain of the CAR targets AXL. For example, the antigen domain of the CAR is an scFv targeting AXL. For example, the antigen domain of the CAR may contain any of the AXL-targeting scFv sequences described in this application.

[0271] 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.

[0272] For example, the co-stimulatory signaling domain can be a 4-1BB co-stimulatory signaling domain. For example, the stimulation signaling domain can contain the amino acid sequence shown in SEQ ID NO: 10.

[0273] 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 PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a domain containing at least one ITAM. For example, the intracellular signaling domain may be an intracellular signaling domain of CD3ζ. For example, the intracellular signaling domain may contain the amino acid sequence shown in SEQ ID NO: 11.

[0274] 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 may be the transmembrane region of CD28. For example, the transmembrane region may contain the amino acid sequence shown in SEQ ID NO: 12.

[0275] 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 may be the hinge region of CD28. For example, the hinge region may be the hinge region of CD8. For example, the hinge region may contain the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 14.

[0276] In some embodiments, the structure of the CAR may further include a signal peptide. For example, the signal peptide may be a CD8 signal peptide. For example, the signal peptide may contain the amino acid sequence shown in SEQ ID NO: 15.

[0277] 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.

[0278] 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.

[0279] 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, has no evidence of toxicity, and elicits relatively mild innate and adaptive immune responses.

[0280] 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.

[0281] In some embodiments, the host cell can be genetically engineered (e.g., transduced, transformed, or transfected) using vector constructs such as those described in this application. These vector constructs can be, for example, viral vectors or vectors for homologous recombination, comprising nucleic acid sequences homologous to a portion of the host cell genome, or expression vectors for expressing a target polypeptide. The host cell can be an untransformed cell or a cell already transfected with at least one nucleic acid molecule.

[0282] 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.

[0283] 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.

[0284] Pharmaceutical Composition

[0285] 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.

[0286] 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).

[0287] application

[0288] 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.

[0289] In some implementations, the system can be used alone for the treatment, relief, and / or prevention of diseases and / or conditions.

[0290] 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.

[0291] In one implementation, the disease and / or symptom can be any currently known disease and / or symptom.

[0292] In one implementation, the disease and / or condition can be any currently known CAR and / or TCR-targeted disease and / or condition.

[0293] In some implementations, the disease and / or condition may be an autoimmune disease.

[0294] In some embodiments, the disease and / or symptom may be a tumor, which may be a solid tumor and / or a non-solid tumor.

[0295] For example, the disease and / or condition may be a disease and / or condition 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, FORR1, NY-ESO-1, MAGE A3, GP100, MART-1, EBV, and / or p53.

[0296] For example, the disease and / or condition may be a disease and / or condition that expresses the tumor antigen AXL.

[0297] For example, the diseases and / or conditions mentioned can be granulocytic leukemia, pyrethroid leukemia, megakaryocytic leukemia, endometrial cancer, gastric cancer, colon cancer, prostate cancer, thyroid cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, renal cell carcinoma, glioblastoma, melanoma, osteosarcoma, pancreatic cancer, and / or lymphoma.

[0298] On the other hand, this application also provides a method for regulating cell activity, the method comprising:

[0299] a) Provide cells capable of expressing the chimeric polypeptide;

[0300] b) Contact the cells with the nucleic acid molecules described above;

[0301] c) Obtain the cells and bring the cells into contact with tumor cells;

[0302] In the cells described in d), PD1 binds to ligands in tumor cells, inducing transmembrane domain cleavage and releasing intracellular domains;

[0303] e) The intracellular domains described regulate the antitumor activity of cells.

[0304] In some implementations, the intracellular domain modulates cellular antitumor activity by activating CAR and / or TCR expression.

[0305] In some implementations, the CAR and / or TCR may be selected from any currently known CAR and / or TCR.

[0306] 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.

[0307] In some embodiments, the method is an in vitro method or an ex vivo method.

[0308] In some embodiments, the cells include immune effector cells.

[0309] 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.

[0310] In some embodiments, the cells are T cells.

[0311] On the other hand, this application also provides methods for applying the chimeric polypeptide, the nucleic acid molecule, the cell, and / or the pharmaceutical composition.

[0312] 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 and second therapies together treat the individual’s disease and / or condition.

[0313] 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.

[0314] This application also includes the following implementation schemes:

[0315] 1. Chimeric polypeptides, comprising, from the N-terminus to the C-terminus:

[0316] 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;

[0317] b) Notch receptor polypeptide, wherein the Notch receptor polypeptide comprises a transmembrane domain and a linker polypeptide;

[0318] c) An intracellular domain capable of activating a second part that specifically targets a tumor antigen, namely AXL.

[0319] 2. The chimeric polypeptide according to embodiment 1, wherein the extracellular segment of PD1 or its functionally active fragment includes at least an immunoglobulin variable region (IgV)-like structure.

[0320] 3. The chimeric polypeptide according to any one of embodiments 1-2, wherein the extracellular segment of PD1 or its functionally active fragment further comprises at least one N-glycosylation site.

[0321] 4. The chimeric polypeptide according to any one of embodiments 1-3, wherein the extracellular segment of PD1 or its functionally active fragment comprises 4 N-glycosylation sites.

[0322] 5. The chimeric polypeptide according to embodiment 4, wherein the N-glycosylation site is selected from one or more of the following group: N49, N58, N74 and N116.

[0323] 6. The chimeric polypeptide according to any one of embodiments 1-5, wherein the extracellular segment of PD1 or its functionally active fragment further comprises an N-terminal loop region (N-loop).

[0324] 7. The chimeric polypeptide according to any one of embodiments 1-6, wherein the extracellular segment of PD1 or its functionally active fragment comprises soluble PD1 (sPD1).

[0325] 8. The chimeric polypeptide according to any one of embodiments 1-7, wherein the PD1 ligand comprises PD-L1 and / or PD-L2.

[0326] 9. The chimeric polypeptide according to any one of embodiments 1-8, wherein the extracellular segment of PD1 or its functionally active fragment comprises wild-type PD1 or a variant thereof.

[0327] 10. The chimeric polypeptide according to any one of embodiments 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.

[0328] 11. The chimeric polypeptide according to embodiments 1-10, wherein the PD1 extracellular segment or its functionally active fragment is a human PD1 extracellular segment or its functionally active fragment.

[0329] 12. The chimeric polypeptide according to embodiments 1-11, compared with the corresponding wild-type PD1 extracellular segment or its functionally active fragment, wherein the PD1 extracellular segment or its functionally active fragment includes one or more amino acid mutations, wherein the amino acid mutation sites are selected from V39, L40, N41, Y43, M45, N49, K53, L97, A100, A107, A132, G124, K131, F19, I65 and / or A66.

[0330] 13. The chimeric polypeptide according to embodiment 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.

[0331] 14. The chimeric polypeptide according to any one of embodiments 1-13, wherein the extracellular segment of PD1 or its functionally active fragment comprises the amino acid sequence shown in SEQ ID NO: 1.

[0332] 15. The chimeric polypeptide according to any one of embodiments 1-14, wherein the Notch receptor polypeptide is derived from a type I transmembrane protein.

[0333] 16. The chimeric polypeptide according to any one of embodiments 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.

[0334] 17. The chimeric polypeptide according to any one of embodiments 1-16, wherein the linker polypeptide comprises a juxtamembrane 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.

[0335] 18. The chimeric polypeptide according to any one of embodiments 1-17, wherein the linking polypeptide comprises a juxtamembrane domain derived from Robo1, Notch1, Notch2, Notch3, Notch4, CLSTN2, AGER, PTPRF, or NRG1.

[0336] 19. The chimeric polypeptide according to any one of embodiments 1-18, wherein the linking polypeptide includes a juxtamembrane domain derived from Notch2.

[0337] 20. The chimeric polypeptide according to any one of embodiments 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.

[0338] 21. The chimeric polypeptide according to any one of embodiments 1-20, wherein the linking polypeptide comprises the amino acid sequence shown in SEQ ID NO:6.

[0339] 22. The chimeric polypeptide according to any one of embodiments 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.

[0340] 23. The chimeric polypeptide according to any one of embodiments 1-22, wherein the transmembrane domain comprises a transmembrane domain derived from Robo1, Notch1, Notch2, Notch3, Notch4, CLSTN2, AGER, PTPRF, or NRG1.

[0341] 24. The chimeric polypeptide according to any one of embodiments 1-23, wherein the transmembrane domain is derived from the transmembrane domain of Notch1.

[0342] 25. The chimeric polypeptide according to any one of embodiments 1-24, wherein the transmembrane domain further comprises one or more proteolytic cleavage sites.

[0343] 26. The chimeric polypeptide according to any one of embodiments 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.

[0344] 27. The chimeric polypeptide according to any one of embodiments 1-26, wherein the transmembrane domain further comprises a mutation of G318 and / or V319.

[0345] 28. The chimeric polypeptide according to any one of embodiments 1-27, wherein the transmembrane domain further includes mutations in G318A and / or V319A.

[0346] 29. The chimeric polypeptide according to any one of embodiments 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.

[0347] 30. The chimeric polypeptide according to any one of embodiments 1-29, wherein the Notch receptor polypeptide includes the transmembrane domain and the linker polypeptide from the N-terminus to the C-terminus.

[0348] 31. The chimeric polypeptide according to any one of embodiments 1-30, wherein the intracellular domain comprises a transcription factor.

[0349] 32. The chimeric polypeptide according to embodiment 31, wherein the transcription factor is selected from the group consisting of Gal4-VP16, Gal4-VP64, tetR-VP64, ZFHD1-VP64, Gal4-KRAB and HAP1-VP16.

[0350] 33. The chimeric polypeptide according to any one of embodiments 31-32, wherein the transcription factor comprises Gal4-VP64.

[0351] 34. The chimeric polypeptide according to any one of embodiments 31-33, wherein the transcription factor activates the second part via an activation sequence.

[0352] 35. The chimeric polypeptide according to any one of embodiments 1-34, wherein the second part comprises CAR and / or TCR.

[0353] 36. The chimeric polypeptide according to embodiment 34, wherein the activation sequence is UAS.

[0354] 37. A nucleic acid molecule encoding a chimeric polypeptide as described in any one of embodiments 1-36.

[0355] 38. Cells comprising the chimeric polypeptide described in any one of embodiments 1-36.

[0356] 39. The cell according to embodiment 38 further includes an exogenous nucleic acid sequence comprising a second nucleic acid portion that specifically targets an antigen-binding region of a tumor antigen, wherein the tumor antigen is AXL.

[0357] 40. In the cell described in embodiment 39, the second nucleic acid portion encodes a CAR and / or TCR that specifically targets the tumor antigen.

[0358] 41. The cell according to any one of embodiments 39-40, wherein the antigen-binding region comprises an antibody or an antigen-binding fragment thereof.

[0359] 42. The cell according to embodiment 41, wherein the antibody comprises a monoclonal antibody, a chimeric antibody, a humanized antibody, and / or a fully human antibody.

[0360] 43. The cell according to embodiment 41, wherein the antigen-binding fragment includes Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

[0361] 44. The cell according to embodiment 43, wherein the antigen-binding fragment is scFv.

[0362] 45. The cell according to any one of embodiments 39-44, wherein the second nucleic acid portion comprises a nucleic acid sequence encoding a co-stimulatory domain.

[0363] 46. ​​The cell according to embodiment 45, 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.

[0364] 47. The cell according to embodiment 46, wherein the co-stimulatory signaling domain is a 4-1BB co-stimulatory signaling domain.

[0365] 48. The cell according to any one of embodiments 39-47, wherein the second nucleic acid portion comprises a nucleic acid sequence encoding an intracellular signal transduction domain.

[0366] 49. The cell according to embodiment 48, 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 PBj14Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM.

[0367] 50. The cell according to embodiment 49, wherein the intracellular signal transduction domain is the intracellular signal transduction domain of CD3ζ.

[0368] 51. The cell according to any one of embodiments 39-50, wherein the second nucleic acid portion further includes a nucleic acid sequence encoding a transmembrane region.

[0369] 52. The cell according to embodiment 51, 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.

[0370] 53. The cell according to embodiment 52, wherein the transmembrane region is the transmembrane region of CD28.

[0371] 54. In the cell according to any one of embodiments 39-53, the second nucleic acid portion further includes a nucleic acid sequence encoding the hinge region.

[0372] 55. The cell according to embodiment 54, 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.

[0373] 56. The cell according to embodiment 55, wherein the hinge region is the hinge region of CD8.

[0374] 57. The cell according to any one of embodiments 38-56, wherein the cell comprises immune effector cells.

[0375] 58. The cell according to any one of embodiments 38-57, said cell comprising 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.

[0376] 59. The cell according to any one of embodiments 38-58, wherein the cell is a T cell.

[0377] 60. A nucleic acid molecule comprising a first nucleic acid portion and a second nucleic acid portion; wherein the first nucleic acid portion encodes a chimeric polypeptide according to any one of embodiments 1-36, and the second nucleic acid portion comprises a nucleic acid sequence encoding an antigen-binding region capable of specifically targeting a tumor antigen, wherein the tumor antigen is AXL.

[0378] 61. The nucleic acid molecule according to embodiment 60, wherein the antigen-binding region includes an antibody or an antigen-binding fragment thereof.

[0379] 62. The nucleic acid molecule according to embodiment 61, wherein the antibody includes monoclonal antibodies, chimeric antibodies, humanized antibodies and / or fully human antibodies.

[0380] 63. The nucleic acid molecule according to embodiment 61, wherein the antigen-binding fragment includes Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

[0381] 64. The nucleic acid molecule according to embodiment 63, wherein the antigen-binding fragment is scFv.

[0382] 65. The nucleic acid molecule according to any one of embodiments 60-64, wherein the second nucleic acid portion comprises a nucleic acid sequence encoding a co-stimulatory domain.

[0383] 66. The nucleic acid molecule according to embodiment 65, 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.

[0384] 67. The nucleic acid molecule according to embodiment 66, wherein the co-stimulatory signaling domain is a 4-1BB co-stimulatory signaling domain.

[0385] 68. The nucleic acid molecule according to any one of embodiments 60-67, wherein the second nucleic acid portion comprises a nucleic acid sequence encoding an intracellular signal transduction domain.

[0386] 69. The nucleic acid molecule according to embodiment 68, 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 PBj14Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM.

[0387] 70. The nucleic acid molecule according to embodiment 69, wherein the intracellular signal transduction domain is the intracellular signal transduction domain of CD3ζ.

[0388] 71. The nucleic acid molecule according to any one of embodiments 60-70, wherein the second nucleic acid portion further comprises a nucleic acid sequence encoding a transmembrane region.

[0389] 72. The nucleic acid molecule according to embodiment 71, 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.

[0390] 73. The nucleic acid molecule according to embodiment 72, wherein the transmembrane region is the transmembrane region of CD28.

[0391] 74. The nucleic acid molecule according to any one of embodiments 60-73, wherein the second nucleic acid portion further includes a nucleic acid sequence encoding the hinge region.

[0392] 75. The nucleic acid molecule according to embodiment 74, 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.

[0393] 76. The nucleic acid molecule according to embodiment 75, wherein the hinge region is the hinge region of CD8.

[0394] 77. The nucleic acid molecule according to any one of embodiments 60-76, wherein the first nucleic acid portion is linked to a promoter sequence.

[0395] 78. The nucleic acid molecule according to embodiment 77, wherein the initiating sequence is selected from the group consisting of: CMV initiating sequence, SFFV initiating sequence, EF1α initiating sequence and PGK initiating sequence.

[0396] 79. The nucleic acid molecule according to implementation scheme 78, wherein the initiation sequence is an SFFV initiation sequence.

[0397] 80. The nucleic acid molecule according to any one of embodiments 60-79, wherein the second nucleic acid portion is linked to an activation sequence.

[0398] 81. The nucleic acid molecule according to embodiment 80, wherein the activation sequence is a UAS sequence.

[0399] 82. The nucleic acid molecule according to any one of embodiments 60-81, 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.

[0400] 83. An expression vector comprising any one of embodiments 60-82.

[0401] 84. The expression vector according to embodiment 83 includes a viral vector or a non-viral vector.

[0402] 85. The expression vector according to embodiment 84, wherein the viral vector includes a lentiviral vector, an adenovirus vector, an adeno-associated virus vector, or a retroviral vector.

[0403] 86. The expression vector according to any one of embodiments 84-85, wherein the viral vector is a lentiviral vector.

[0404] 87. A pharmaceutical composition comprising a chimeric polypeptide according to any one of embodiments 1-36, a nucleic acid molecule according to embodiment 37, a cell according to any one of embodiments 38-59, a nucleic acid molecule according to any one of embodiments 60-82, and / or an expression vector according to any one of embodiments 83-86, and optionally a pharmaceutically acceptable carrier.

[0405] 88. A system for regulating cell activity, the system comprising a chimeric polypeptide according to any one of embodiments 1-36, a nucleic acid molecule according to embodiment 38, a cell according to any one of embodiments 38-59, a nucleic acid molecule according to any one of embodiments 60-82, an expression vector according to any one of embodiments 83-86, and / or a pharmaceutical composition according to embodiment 87.

[0406] 89. A method for regulating cell activity, the method comprising:

[0407] a) Provide cells capable of expressing the chimeric polypeptide described in any one of embodiments 1-36;

[0408] b) Contact the cells with the nucleic acid molecules described in any one of embodiments 60-82;

[0409] c) Obtaining the cells described in any one of embodiments 38-59, and contacting the cells described in any one of embodiments 38-59 with tumor cells;

[0410] d) In any of the embodiments 38-59, the PD1 binds to the ligand in the tumor cell, inducing the cleavage of the transmembrane domain and releasing the intracellular domain;

[0411] e) The intracellular domains described regulate the antitumor activity of cells.

[0412] 90. The method according to embodiment 89, wherein the method is an in vitro method or an ex vivo method.

[0413] 91. The method according to any one of embodiments 89-90, wherein the cells comprise immune effector cells.

[0414] 92. The method according to any one of embodiments 89-91, wherein 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.

[0415] 93. The method according to any one of embodiments 89-92, wherein the cell is a T cell.

[0416] 94. The use of the chimeric polypeptide of any one of embodiments 1-36, the nucleic acid molecule of embodiment 37, the cell of any one of embodiments 38-59, the nucleic acid molecule of any one of embodiments 60-82, the expression vector of any one of embodiments 83-86, the pharmaceutical composition of embodiment 87, and / or the system of embodiment 88 in the preparation of a medicament for the prevention, treatment and / or relief of diseases and / or symptoms.

[0417] 95. The chimeric polypeptide of any one of embodiments 1-36, the nucleic acid molecule of embodiment 37, the cell of any one of embodiments 38-59, the nucleic acid molecule of any one of embodiments 60-82, the expression vector of any one of embodiments 83-86, the pharmaceutical composition of embodiment 87, and / or the system of embodiment 88, for the prevention, treatment and / or relief of diseases and / or symptoms.

[0418] 96. A method for preventing, treating, and / or alleviating diseases and / or symptoms, the method comprising administering to a subject in need a chimeric polypeptide according to any one of embodiments 1-36, a nucleic acid molecule according to embodiment 37, a cell according to any one of embodiments 38-59, a nucleic acid molecule according to any one of embodiments 60-82, an expression vector according to any one of embodiments 83-86, a pharmaceutical composition according to embodiment 87, and / or a system according to embodiment 88.

[0419] 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.

[0420] Example

[0421] Example 1: Design of a chimeric peptide gating system

[0422] Table 1.1. Experimental Materials

[0423]

[0424] Table 1.1 (continued). Experimental Materials

[0425]

[0426] Table 1.1 (continued). Experimental Materials

[0427]

[0428] 1.2 Cell Culture

[0429] The HEK293T, MEC1, HEP3B, A549, and MDA-MB-231 cell lines were obtained from the American Test and Computing Center (ATCC). MEC1-ROR1 was an internally constructed stable ROR1-expressing cell line. MB453, a low-positive, AXL-negative PDL1 cell line, was purchased from Procell. A549-PDL1 and HEP3B-PDL1 were both internally constructed stable PDL1-expressing cell lines. MEC-ROR1-PDL1, Hep3B-PDL1, and A549-PDL1 were all engineered cell lines that stably express PDL1. These tumor cell lines were transfected with GFP for stable expression and used in subsequent experiments.

[0430] 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 from 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.

[0431] 1.3 Clone Construction

[0432] All relevant gene constructs were synthesized using GeneWiz. PD1 ECD chimeric peptide gating ( Figure 1 ) and TGFbRECD chimeric peptide gating ( Figure 2 The gating structure was 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 use in lentiviral packaging.

[0433] 1.4 Preparation of Lentivirals

[0434] 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.

[0435] 1.5 T cell activation and transduction

[0436] 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). 6Cells / 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 are used for subsequent experiments and analysis 2-5 days after transduction.

[0437] 1.6 Flow Cytometry

[0438] 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.

[0439] 1.7 Enzyme-linked immunosorbent assay (ELISA)

[0440] 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.

[0441] 1.8 Gating Effect Verification Experiment

[0442] 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 an effector-to-target ratio of 1:1 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.

[0443] 1.9 T-cell tumor killing experiment

[0444] 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.

[0445] Example 2: Gated expression after transfection

[0446] 2.1 Gated expression of transfected PD1 ECD chimeric peptide

[0447] A structure containing a PD1 logic-gated ROR1 CAR was successfully cloned and packaged into a lentivirus. For example... Figure 3 As shown, high levels of HA marker expression were detected in activated human PBMCs 72 hours after lentiviral infection, indicating the expression of PD1 ECD logic gating. In the basal state, gating is not activated, and ROR1 CAR is expressed at almost or very low levels.

[0448] 2.2 Gated expression of TGFbRII ECD chimeric peptide after transfection

[0449] A structure containing a TGFbRII logic-gated CD22 CAR was successfully cloned and packaged into a lentivirus. For example... Figure 4As shown, high levels of TGFbRII ECD logic gating were detected in activated human PBMCs 72 hours after lentiviral infection. In the basal state, the gating was not activated, and CD22 CAR showed low-level expression.

[0450] Example 3: Gating effect of transfected T cells

[0451] 3.1 Gating effect of T cells after transfection with PD1 ECD chimeric peptide

[0452] like Figure 5 As shown, PBMCs transduced with the PD1 ECD chimeric peptide showed low levels of ROR1 CAR expression (15.6% and 17.7%) in the unstimulated basal state (SNIPR only) or after being cultured with PDL1-negative K562 cells. When co-cultured with PDL1-positive MEC cells, ROR1 CAR expression was induced, increasing to 36.7%.

[0453] 3.2 Gating effect of T cells after transfection with TGFbRII ECD chimeric peptide

[0454] like Figure 6 As shown, PBMCs transduced with TGFbRII ECD chimeric peptides, when co-cultured with different concentrations of TGFb protein (10 ng / μL-200 ng / μL), exhibited similar CAR structural expression compared to PBMCs in an unactivated state (SNIPR only), indicating that TGFbRII ECD gating cannot induce CAR expression.

[0455] Example 4: PD1 ECD chimeric peptide specifically kills tumor cells that are double-positive for ROR1 and PDL1.

[0456] like Figure 7A and 7B As shown, the PD1 ECD chimeric peptide detected higher levels of IL2 and IFNγ cytokine secretion after culturing with PDL1 and ROR1 double-positive tumor cells.

[0457] like Figure 8AAs shown in Figure -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 maintained good killing ability against tumor cells expressing low levels of PDL1 (MEC-ROR1), indicating its high sensitivity 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 increased, demonstrating sustained tumor-killing ability, indicating that the PD1 ECD chimeric peptide 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.

[0458] Example 5: PD1 ECD chimeric peptide specifically kills tumor cells that are double-positive for the targets AXL and PDL1.

[0459] 5.1 Construction and Expression of PD1 SNIPR AXL CAR

[0460] The structure includes a PD1 logic-gated AXL CAR (8601, PD1 SNIPR AXL CAR) and a control AXL CAR (8600), as shown in the figure. Figure 9 As shown, the CAR structures were successfully cloned and packaged using lentivirus. Both CAR structures were transfected into normal human PBMC cells via lentivirus. The expression results of the transfected PD1 logic-gated AXL CAR are shown below. Figure 10 As shown, 72 hours after transfection, the second-generation AXLCAR (8600) showed high AXLCAR expression but no HA marker expression. The PD1 logic-gated AXLCAR (8601) showed high levels of HA marker expression, indicating PD1 ECD logic gating. In the basal state, the gating was not activated, and AXLCAR was expressed at low levels.

[0461] 5.2 Gating effect of T cells after transfection with PD1 SNIPR AXL CAR

[0462] like Figure 11As shown, PBMCs transfected with PD1 logic-gated AXL CAR (8601) exhibited low levels of AXL CAR expression (14.7% and 17.1%) in a basal state (Tonly, expressing only PD1ECD Notch receptor without CAR activation) or after being cultured with PDL1-negative HEK293T cells. When co-cultured with PDL1-positive K562 cells, AXL CAR expression was induced, increasing to 49.3%. This result confirms that PD1 logic-gated cells can be effectively stimulated to express CAR by PDL1-positive cells.

[0463] 5.3 Specific killing assay of PD1 SNIPR AXL CAR-T cells

[0464] In the IncuCyte real-time imaging experiment, T cells and tumor cells were continuously co-cultured at a 1:1 effector-target ratio, and 2500 tumor cells were added on the third day for restimulation. Figure 12 As shown, both 8600 and 8061 effectively killed PDL1 and AXL double-positive tumor cells (MB231). Even after rechallenge with new tumor cells, they exhibited sustained killing activity. Meanwhile, after co-culturing with PDL1-low positive and AXL-negative MB453 cells, the PD1 logic-gated AXL CAR showed no killing effect, while the second-generation AXL CAR showed non-specific killing. These results indicate that the PD1 logic-gated AXL CAR has superior specific killing activity and better safety compared to the second-generation CAR structure.

[0465] 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) An intracellular domain capable of activating a second part that specifically targets a tumor antigen, namely AXL.

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. A nucleic acid molecule encoding a chimeric polypeptide as described in any one of claims 1-14.

16. Cells comprising the chimeric polypeptide according to any one of claims 1-14.

17. The cell of claim 16, further comprising an exogenous nucleic acid sequence, said exogenous nucleic acid sequence comprising a second nucleic acid portion specifically targeting an antigen-binding region of a tumor antigen, said tumor antigen being AXL.

18. A nucleic acid molecule comprising a first nucleic acid portion and a second nucleic acid portion; wherein the first nucleic acid portion encodes a chimeric polypeptide according to any one of claims 1-14, and the second nucleic acid portion comprises a nucleic acid sequence encoding an antigen-binding region capable of specifically targeting a tumor antigen, said tumor antigen being AXL.

19. An expression vector comprising the nucleic acid molecule of claim 18.

20. A pharmaceutical composition comprising the chimeric polypeptide of any one of claims 1-14, the nucleic acid molecule of claim 15, the cell of any one of claims 16-17, the nucleic acid molecule of claim 18, and / or the expression vector of claim 19, and optionally a pharmaceutically acceptable carrier.

21. A system for regulating cell activity, the system comprising the chimeric polypeptide of any one of claims 1-14, the nucleic acid molecule of claim 15, the cell of any one of claims 16-17, the nucleic acid molecule of claim 18, the expression vector of claim 19, and / or the pharmaceutical composition of claim 20.

22. A method for regulating cell activity, the method comprising: a) Providing cells capable of expressing the chimeric polypeptide of any one of claims 1-14; b) Contacting the cells with the nucleic acid molecules of claim 18; c) Obtaining the cells according to any one of claims 16-17, and contacting the cells according to any one of claims 16-17 with tumor cells; d) In any of the cells of claims 16-17, 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.

23. Use of the chimeric polypeptide of any one of claims 1-14, the nucleic acid molecule of claim 15, the cell of any one of claims 16-17, the nucleic acid molecule of claim 18, the expression vector of claim 19, the pharmaceutical composition of claim 20, and / or the system of claim 21 in the preparation of a medicament for the prevention, treatment, and / or relief of diseases and / or symptoms.

24. The chimeric polypeptide of any one of claims 1-14, the nucleic acid molecule of claim 15, the cell of any one of claims 16-17, the nucleic acid molecule of claim 18, the expression vector of claim 19, the pharmaceutical composition of claim 20, and / or the system of claim 21, for the prevention, treatment and / or relief of diseases and / or symptoms.

25. 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 1-14, a nucleic acid molecule of claim 15, a cell of any one of claims 16-17, a nucleic acid molecule of claim 18, an expression vector of claim 19, a pharmaceutical composition of claim 20, and / or a system of claim 21.

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