WO2022156786A1 - 一种嵌合抗原受体改造的nk细胞及其制备方法与应用 - Google Patents

一种嵌合抗原受体改造的nk细胞及其制备方法与应用 Download PDF

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WO2022156786A1
WO2022156786A1 PCT/CN2022/073311 CN2022073311W WO2022156786A1 WO 2022156786 A1 WO2022156786 A1 WO 2022156786A1 CN 2022073311 W CN2022073311 W CN 2022073311W WO 2022156786 A1 WO2022156786 A1 WO 2022156786A1
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cells
chimeric antigen
acid sequence
cell
antigen receptor
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李晓东
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南京助天中科科技发展有限公司
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Definitions

  • the present application relates to a chimeric antigen receptor-modified NK cell, a preparation method and application thereof, and belongs to the field of biomedicine.
  • Cancer immunotherapy which has been rapidly increasing in number in recent years, works by unleashing the power of a patient's immune system to fight cancer, in a way the immune system fights off pathogens such as the virus that causes the flu or the bacteria that cause strep throat .
  • Cancer immunotherapy especially immune checkpoint inhibitors and cell therapy, is one of the most exciting new cancer treatments that have entered clinical trials in recent years.
  • CAR-T cells Chimeric Antigen Receptor T cells
  • CAR-T cell therapy has shown exciting clinical efficacy in patients with malignant hematological cancers, and a number of CAR-T cell therapies targeting CD19 have been approved by the US FDA for the treatment of leukemia and lymphoma.
  • CAR-T cell therapy is advancing at an unprecedented rate, especially since the advent of CAR-T cell therapy, a strategy that has proven effective in B-cell malignancies and has shown promise in clinical trials in other hematological cancers It may also have a certain effect in the treatment of solid tumors.
  • CAR-T cell therapy in clinical application increase the length of hospital stay and the risk and cost of treatment.
  • Other limitations of CAR-T cell therapy include the logistical challenges of producing autologous cell products and taking into account the risk of T cell-mediated graft-versus-host reactions.
  • NK cells Natural killer cells
  • NK cells which are extremely important lymphocyte types other than T cells and B cells
  • NK cells account for approximately 5% to 15% of the total proportion of human peripheral monocytes, and are an important part of the innate immune system. Plays a vital role in our first line of defense against pathogens and cancer cells.
  • NK cells are specialized killers with a unique natural ability to eliminate abnormal cells damaged by viral infection or malignant transformation.
  • NK cells lack the expression of T cell receptors and CD3, and do not require the help of MHC-I molecules to recognize and kill abnormal cells.
  • NK cells the "innate counterparts" of killer T cells
  • NK receptors Blocking immunosuppressive signals that suppress NK cells with immune checkpoint inhibitors can overcome the limitations of T cell-based immunotherapy to some extent.
  • NK cells have a similar killing mechanism to kill target cells with CD8-positive killer T cells to a certain extent, which provides a solid foundation for engineering NK cells.
  • CAR-NK cell therapy can provide many advantages, such as but not limited to: (1) multiple mechanisms to activate killing function, (2) "off-the-shelf" highly convenient manufacturing process Advantages, (3) safety advantages, such as autologous therapy shows less or no cytokine release syndrome and neurotoxic syndrome and allogeneic therapy shows less or no graft-versus-host reaction.
  • the use of NK cells can result in off-the-shelf allogeneic products to treat patients, thereby removing the limitations of the necessity of personalized and patient-specific products in current CAR-T cell therapy.
  • NK cells can target NK cells to a variety of different antigens and enhance the proliferation, expansion and persistence of NK cells in vivo, thereby ultimately achieving effective anti-tumor functions.
  • NK cells as important immune cells of the body, are not only related to anti-tumor, anti-viral infection and immune regulation, but also participate in the occurrence of hypersensitivity reactions and autoimmune diseases in some cases, and can recognize target cells and kill and clear mediators. Therefore, it is of great significance to transform and apply NK cells.
  • a chimeric antigen receptor-engineered NK cell is provided, and the chimeric antigen receptor-engineered NK cell has both the advantages of an immune checkpoint inhibitor and a CAR-engineered NK cell therapy, Provide solutions for improving solid tumor treatment.
  • a NK cell transformed by a chimeric antigen receptor comprises: an extracellular target molecule binding domain, a transmembrane region domain and an intracellular signal transduction domain;
  • the transmembrane region domain connects the extracellular target molecule binding domain and the intracellular signaling domain, and fixes the two on the cell membrane of the NK cell;
  • the intracellular signaling domain includes an intracellular activation signaling domain and/or an intracellular detection signaling domain.
  • the chimeric antigen receptor-engineered NK cells are NK cells containing nucleic acids encoding chimeric antigen receptors.
  • the chimeric antigen receptor further comprises: an extracellular spacer domain
  • the extracellular spacer domain is located between the extracellular target molecule binding domain and the transmembrane domain.
  • the chimeric antigen receptor further comprises: an intracellular spacer domain
  • the intracellular spacer domain is located between and links the transmembrane domain and the intracellular signaling domain together.
  • the chimeric antigen receptor further comprises: an intracellular hinge domain
  • the intracellular hinge domain connects the intracellular detection signaling domain and the intracellular activation signaling domain together;
  • the intracellular hinge domain may be of any suitable length to connect the at least two domains of interest, and is preferably designed to be flexible enough to allow proper folding and/or function of the one or both domains to which it is connected and/or or activity.
  • the target molecule bound by the extracellular target molecule binding domain comprises at least one of the molecules of the following group: immunosuppressive signal-related molecules, tumor surface antigen molecular markers, cell surface specific antigen peptide-tissue Compatibility complex molecules.
  • the extracellular target molecule binding domain comprises at least one target molecule binding domain of a molecule selected from the group consisting of PD-1, PD-1 truncation, PD-1 protein mutant, Antibodies to PD-L1 and PD-L1-binding fragments.
  • the extracellular target molecule binding domain comprises the amino acid sequence containing SEQ ID NO: 1, the amino acid sequence containing SEQ ID NO: 3, the amino acid sequence containing SEQ ID NO: 5, the amino acid sequence containing SEQ ID NO: 7 , containing at least one of the amino acid sequence of SEQ ID NO: 9 and the amino acid sequence of SEQ ID NO: 11.
  • the nucleic acid fragment of the extracellular target molecule binding domain comprises a nucleic acid sequence comprising SEQ ID NO:2, a nucleic acid sequence comprising SEQ ID NO:4, a nucleic acid sequence comprising SEQ ID NO:6, a nucleic acid sequence comprising SEQ ID NO:6, At least one of the nucleic acid sequence of NO:8 and the nucleic acid sequence of SEQ ID NO:10.
  • the activation of the intracellular activation signal transduction domain at least depends on the binding of the extracellular target molecule binding domain to the target molecule; the intracellular activation signal transduction domain contains a catalytic functional group. molecules or fragments.
  • the intracellular activation signaling domain comprises at least one of a tyrosine kinase or a tyrosine kinase fragment;
  • the tyrosine kinase includes at least one of receptor-type tyrosine kinase and non-receptor-type tyrosine kinase;
  • the tyrosine kinase fragments include at least one of receptor-type tyrosine kinase fragments and non-receptor-type tyrosine kinase fragments.
  • the tyrosine kinase is selected from Ack, CSK, CTK, FAK, Abl, Arg, Tnk1, Pyk2, Fer, Fes, LTK, ALK, STYK1, JAK1, JAK2, JAK3, Tyk2, DDR1, DDR2, ROS, Blk, Fgr, FRK, Fyn, TIE1, TIE2, Hck, Lck, Srm, Yes, Syk, ZAP70, Etk, Btk, HER2, HER3, HER4, InsR, ITK, TEC, TXK, EGFR, IGF1R, IRR, PDGFR ⁇ , VEGFR-1, VEGFR-2, VEGFR-3, PDGFR ⁇ , Kit, CSFR, FLT3, FGFR1, FGFR2, FGFR3, FGFR4, CCK4, ROR1, ROR2, MuSK, MET, Lyn, Brk, Src, Ron, Axl, At least one of Tyro3, Mer, EphA1,
  • the intracellular activation signaling domain comprises the amino acid sequence comprising SEQ ID NO:42, the amino acid sequence comprising SEQ ID NO:44, the amino acid sequence comprising SEQ ID NO:46, the amino acid sequence comprising SEQ ID NO:48 At least one of the amino acid sequence of SEQ ID NO: 50, the amino acid sequence of SEQ ID NO: 52, and the amino acid sequence of SEQ ID NO: 52.
  • the nucleic acid fragment of the intracellular activation signaling domain comprises the nucleic acid sequence comprising SEQ ID NO:43, the nucleic acid sequence comprising SEQ ID NO:45, the nucleic acid sequence comprising SEQ ID NO:47, the nucleic acid sequence comprising SEQ ID NO:47, the nucleic acid sequence comprising SEQ ID NO:47 At least one of the nucleic acid sequence of NO:49, the nucleic acid sequence comprising SEQ ID NO:51, and the nucleic acid sequence comprising SEQ ID NO:53.
  • the intracellular detection signaling domain comprises at least one immunoreceptor tyrosine-based activation motif.
  • the intracellular detection signaling domain comprises at least one signaling domain of a molecule selected from the group consisting of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD28, CD31, CD72, CD84, CD229, CEACAM-19, CEACAM-20, SIRP ⁇ , SLAM, CLEC-1, CLEC-2, CRACC, CTLA-4, 2B4, CD244, BTLA, DCAR, DCIR, Dectin-1, DNAM-1, CD300a, CD300f, CEACAM- 1.
  • a molecule selected from the group consisting of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD28, CD31, CD72, CD84, CD229, CEACAM-19, CEACAM-20, SIRP ⁇ , SLAM, CLEC-1, CLEC-2, CRACC, CTLA-4, 2B4, CD244, BTLA, DCAR, DCIR, Dectin-1, DNAM-1, CD300a, CD300f, CEACAM- 1.
  • the intracellular detection signaling domain comprises the amino acid sequence comprising SEQ ID NO:20, the amino acid sequence comprising SEQ ID NO:22, the amino acid sequence comprising SEQ ID NO:24, the amino acid sequence comprising SEQ ID NO:26
  • the amino acid sequence of SEQ ID NO: 28 the amino acid sequence of SEQ ID NO: 30, the amino acid sequence of SEQ ID NO: 32, the amino acid sequence of SEQ ID NO: 34, the amino acid sequence of SEQ ID NO: 36 At least one of the amino acid sequence of , the amino acid sequence containing SEQ ID NO: 38, and the amino acid sequence containing SEQ ID NO: 40.
  • the nucleic acid fragment of the intracellular detection signaling domain comprises a nucleic acid sequence comprising SEQ ID NO:21, a nucleic acid sequence comprising SEQ ID NO:23, a nucleic acid sequence comprising SEQ ID NO:25, a nucleic acid sequence comprising SEQ ID NO:25
  • the nucleic acid sequence of NO:27, the nucleic acid sequence containing SEQ ID NO:29, the nucleic acid sequence containing SEQ ID NO:31, the nucleic acid sequence containing SEQ ID NO:33, the nucleic acid sequence containing SEQ ID NO:35, the nucleic acid sequence containing SEQ ID NO:35 At least one of the nucleic acid sequence of NO:37, the nucleic acid sequence comprising SEQ ID NO:39, and the nucleic acid sequence comprising SEQ ID NO:41.
  • the transmembrane domain is selected from the transmembrane domain of the transmembrane protein of the lower group, and the transmembrane protein comprises 4-1BB, 4-1BBL, ICOS, GITR, GITRL, VSIG-3, VISTA, SIRP ⁇ , OX40, OX40L, CD40, CD40L, CD86, CD80, PD-1, PD-L1, PD-L2, CD2, CD28, B7-DC, B7-H2, B7-H3, B7-H4, B7-H5, B7 -H6, B7-H7, Siglec-1, Siglec-2, Siglec-3, Siglec-4, LILRB5, 2B4, BTLA, CD160, LAG-3, Siglec-5, Siglec-6, Siglec-7, Siglec-8 , CD96, CD226, TIM-1, TIM-3, TIM-4, Siglec-9, Siglec-10, Siglec-11, Siglec-12, Siglec
  • the transmembrane domain comprises at least one of the amino acid sequence comprising SEQ ID NO: 12 and the amino acid sequence comprising SEQ ID NO: 14.
  • the nucleic acid fragment of the transmembrane region comprises at least one of the nucleic acid sequence comprising SEQ ID NO: 13 and the nucleic acid sequence comprising SEQ ID NO: 15.
  • the extracellular spacer domain comprises at least one of the amino acid sequence comprising SEQ ID NO: 16 and the amino acid sequence comprising SEQ ID NO: 18.
  • the nucleic acid fragment of the extracellular spacer domain comprises at least one of the nucleic acid sequence comprising SEQ ID NO: 17 and the nucleic acid sequence comprising SEQ ID NO: 19.
  • the intracellular spacer domain is an extension of the transmembrane domain domain, comprising at least one molecule selected from the group consisting of PD-1, PD-L1, PD-L2, CD8a, CD8b, CD4 , ICOS, GITR, GITRL, OX40, OX40L, B7-DC, B7-H2, B7-H3, B7-H4, B7-H5, CD40, CD40L, CD86, CD80, CD2, CD28, B7-H6, B7-H7 , VSIG-3, VISTA, SIRP ⁇ , KIR2DS1, KIR2DS3, KIR2DS4, KIR2DS5, Siglec-1, Siglec-2, Siglec-3, Siglec-4, CD155, CD112, CD113, TIGIT, CD96, CD226, Siglec-5, Siglec -6, Siglec-7, Siglec-8, Siglec-9, Siglec-10, LILRB1, LILRB2, LILRB3, LIL
  • the intracellular spacer domain comprises at least one of the amino acid sequence comprising SEQ ID NO:54 and the amino acid sequence comprising SEQ ID NO:56.
  • the intracellular spacer domain nucleic acid fragment comprises at least one of the nucleic acid sequence comprising SEQ ID NO:55 and the nucleic acid sequence comprising SEQ ID NO:57.
  • the intracellular hinge domain comprises the amino acid sequence comprising SEQ ID NO:58, the amino acid sequence comprising SEQ ID NO:60, the amino acid sequence comprising SEQ ID NO:62, the amino acid sequence comprising SEQ ID NO:64 sequence, at least one of the amino acid sequence comprising SEQ ID NO:66.
  • the intracellular hinge domain fragment comprises a nucleic acid sequence comprising SEQ ID NO:59, a nucleic acid sequence comprising SEQ ID NO:61, a nucleic acid sequence comprising SEQ ID NO:63, a nucleic acid sequence comprising SEQ ID NO:65 at least one of the nucleic acid sequences.
  • the chimeric antigen receptor comprises: an extracellular target molecule binding domain; a transmembrane domain; an extracellular spacer domain; an intracellular detection signaling domain; an intracellular activation signaling domain; Intracellular spacer domain; intracellular hinge domain.
  • the chimeric antigen receptor comprises: an extracellular target molecule binding domain; a transmembrane domain; an extracellular spacer domain; and an intracellular signaling domain.
  • the chimeric antigen receptor comprises: an extracellular target molecule binding domain; a transmembrane domain; an extracellular spacer domain; an intracellular detection signaling domain; and an intracellular activation signaling domain .
  • the chimeric antigen receptor comprises: an extracellular target molecule binding domain; a transmembrane domain; an extracellular spacer domain; and an intracellular activation signaling domain.
  • the chimeric antigen receptor comprises: an extracellular target molecule binding domain; a transmembrane domain; an extracellular spacer domain; an intracellular detection signaling domain; an intracellular activation signaling domain; and intracellular hinge domains.
  • the chimeric antigen receptor comprises: an extracellular target molecule binding domain; a transmembrane domain; an extracellular spacer domain; and an intracellular activation signaling domain; and an intracellular spacer domain .
  • the chimeric antigen receptor comprises: an extracellular target molecule binding domain; a transmembrane domain; an extracellular spacer domain; an intracellular signaling domain; and an intracellular spacer domain.
  • the chimeric antigen receptor comprises: an extracellular target molecule binding domain; a transmembrane domain; an extracellular spacer domain; an intracellular detection signaling domain; an intracellular activation signaling domain; and intracellular spacer domains.
  • the NK cells include at least one of endogenous NK cell subsets and/or exogenous NK cells.
  • the endogenous NK cell subsets include at least one of adaptive NK cells, memory NK cells, CD56 dim NK cells, and CD56 bright NK cells;
  • the exogenous NK cells include at least one of NK cell lines, embryonic stem cells or NK cells derived from induced pluripotent stem cells.
  • the NK cell line is selected from NK-92 cell line, haNK cell line, IMC-1 cell line, NK-YS cell line, KHYG-1 cell line, NKL cell line, NKG cell line, SNK-6 cell line. At least one of a cell line, a YTS cell line, and a HANK-1 cell line.
  • the haNK cell line is an overexpressing high-affinity CD16 positive NK cell line.
  • a method for preparing a chimeric antigen receptor-engineered NK cell comprising the following steps:
  • the preparation method comprises the following steps:
  • a pharmaceutical composition comprising the above-mentioned chimeric antigen receptor-engineered NK cells or the chimeric antigen receptor-engineered NK cells prepared according to the above-mentioned preparation method at least one of NK cells.
  • the pharmaceutical composition also includes a monoclonal antibody
  • the monoclonal antibody is selected from at least one of cetuximab, alemtuzumab, ipilimumab, and ofatumumab.
  • the pharmaceutical composition further includes cytokines
  • the cytokine is selected from at least one of gamma interferon and interleukin.
  • the chimeric antigen receptor-engineered NK cell according to any one of the above, or the chimeric antigen receptor-engineered NK cell prepared according to the preparation method described in any one of the above, Or the application of at least one of the pharmaceutical compositions described in any one of the above in the preparation of a medicine for the treatment of the following diseases:
  • Tumors infections, inflammatory diseases, immune diseases, neurological diseases.
  • the tumor is PD-L1 positive or a tumor whose PD-L1 expression level is upregulated in response to interferon gamma.
  • the tumor comprises solid tumor and/or hematological cancer.
  • the solid tumor includes at least one of breast cancer, skin cancer, liver cancer, ovarian cancer, prostate cancer, brain cancer, kidney cancer, and lung cancer.
  • the blood cancer comprises leukemia.
  • a method for the treatment of a disease comprising the steps of:
  • the chimeric antigen receptor-modified NK cells are selected from at least the chimeric antigen receptor-modified NK cells described above, or the chimeric antigen receptor-modified NK cells prepared according to the above-described preparation method. A sort of;
  • the pharmaceutical composition is selected from the above-mentioned pharmaceutical compositions;
  • the disease is selected from at least one of tumor, infection, inflammatory disease, immune disease, and nervous system disease.
  • the using method includes the following steps:
  • the chimeric antigen receptor-engineered NK cells are reinfused into the human body.
  • step 3) also includes:
  • the transformed immune cells are reinfused into the human body.
  • Table 1 is the amino acid and nucleic acid sequences involved in the application
  • the NK cells transformed by the chimeric antigen receptor provided in this application are transformed by using the chimeric antigen receptor. The killing effect of tumor cells.
  • the chimeric antigen receptor-modified NK cells provided in this application are NK cells that have been re-encoded and transformed by this new generation of PD-1-based chimeric antigen receptor molecular machines, based on the modified immune checkpoint PD-1/
  • the PD-L1 signaling pathway can better identify and kill specific tumor cells. Not only will it not be inhibited by tumor cells expressing the immune checkpoint inhibitory signal PD-1 molecular ligand PD-L1, but it will be further activated. Generate a specific immune response against the corresponding tumor cells, thereby recognizing and killing the corresponding tumor cells.
  • the NK cells transformed by the chimeric antigen receptor provided in this application have better identification and killing effects on specific tumor cells, including human-derived prostate cancer tumor cells, human-derived renal cancer tumor cells, and human-derived breast cancer tumors.
  • Cells human skin cancer tumor cells, human brain cancer tumor cells, human lung cancer tumor cells, human liver cancer cells, human ovarian cancer cells, etc.
  • Figure 1 shows an exemplary method of administering the natural killer cell chimeric antigen receptors of the present disclosure, wherein the natural killer cells may be individual allogeneic, allogeneic or autologous cells.
  • Figure 2 is a schematic diagram of the construction of the chimeric antigen receptor artificial molecular machine of the present application.
  • Figure 2a chimeric antigen receptor artificial molecular machine comprises domain #I, domain #II, domain #III and domain #VIII
  • Figure 2b chimeric antigen receptor artificial molecular machine comprises domain #I, structure Domain #II, Domain #III and Domain #VII
  • Figure 2c Chimeric Antigen Receptor Artificial Molecular Machine comprising Domain #I, Domain #II, Domain #III, Domain #V and Domain #VII
  • Figure 2d chimeric antigen receptor artificial molecular machine comprises domain #I, domain #II, domain #III, domain #V, domain #VI and domain #VII
  • Figure 2e chimeric antigen receptor artificial molecule The machine comprises Domain #I, Domain #II, Domain #III, Domain #IV and Domain #VIII
  • Figure 2f Chimeric Antigen Receptor Artificial Molecular Machine comprises Domain #I, Domain #II, Domain #III, Domain #IV and Domain #VII
  • Figure 3 is a schematic diagram of the signal activation of the artificial molecular machine of the present application
  • Figure 3a shows the release and activation of the activation signal of the artificial molecular machine under the input of the tyrosine kinase activation signal
  • Figure 3b shows the target molecule in the target cell
  • signal input eg, PD-L1
  • the activation signal of a chimeric antigen receptor artificial molecular machine containing domain #I eg, the extracellular portion of PD-1
  • Figure 4a shows that C#9, C#10, C#11, C#12, C#13, C#14, C#15 and C#16 activate protein casein with the tyrosine phosphatase inhibitor sodium pervanadate Expression results in human HeLa (HeLa) cells under the condition of amino acid phosphorylation signal.
  • Figure 4b shows the expression of C#9 and C#15 in human HeLa under the A condition of the tyrosine phosphatase inhibitor sodium pervanadate-activated protein tyrosine phosphorylation signal or the B condition of the epidermal growth factor-activated signal. Expression results in (HeLa) cells.
  • Figure 4c shows C#9 and C#15 in mouse embryos under the A condition of the tyrosine phosphatase inhibitor sodium pervanadate to activate the protein tyrosine phosphorylation signal or the B condition of the platelet-derived growth factor to activate the signal. Expression results in fibroblasts (MEF).
  • Figure 5a shows the expression distribution of C#17 and C#18 in human HeLa cells and the detection of their ability to respond to protein tyrosine phosphorylation signals stimulated by the tyrosine phosphatase inhibitor sodium pervanadate result.
  • Figure 5b shows the expression distribution of C#19 and C#20 in human HeLa cells and the detection of their ability to respond to protein tyrosine phosphorylation signals under the stimulation of tyrosine phosphatase inhibitor sodium pervanadate result.
  • Figure 5c shows that C#17, C#18, C#19, and C#20 in human HeLa (HeLa ) performance results in cells.
  • Figure 6 shows the expression results of C#9 and C#10 in the state of purified proteins under the condition that the non-receptor-type protein tyrosine kinase Lck provides a signal to activate protein tyrosine phosphorylation.
  • Figure 7a shows the expression distribution of C#19 and C#20 in human HeLa cells and the detection results in response to human PD-L1 signal stimulated by human PD-L1-modified microspheres.
  • Figure 7b shows the expression results of C#17, C#18, C#19 and C#20 in human HeLa (HeLa) cells under the condition of human PD-L1-modified microspheres stimulation signal.
  • Figure 8 shows a comparison of natural killer cells and natural killer cells modified with chimeric antigen receptors of the present disclosure.
  • Figure 8a shows the performance of natural killer cells facing tumor cells.
  • Figure 8b shows the performance of natural killer cells with chimeric antigen receptor modifications of the present disclosure against tumor cells.
  • the gray level of the natural killer cells corresponds to the tumor-killing ability of the natural killer cells.
  • Figure 9 shows the expression of different chimeric antigen receptors in natural killer cells NK-92.
  • Figure 10 shows the expression of PD-L1 in 8 human tumor cells and 8 human tumor cells pretreated with gamma interferon.
  • the 8 kinds of human tumor cells are the breast cancer tumor cell MBA-MB-231 in Fig. 10a, the brain cancer tumor cell U87-MG in Fig. 10b, the kidney cancer tumor cell 786-O in Fig. 10c, the skin cancer tumor cell A2058 in Fig. 10d, and Fig. 10e Lung cancer tumor cell H441, Figure 10f ovarian cancer tumor cell ES-2, Figure 10g prostate cancer tumor cell PC-3, Figure 10h liver cancer tumor cell HA-22T.
  • Figure 11 shows the in vitro co-culture cytotoxicity of chimeric antigen receptor modified human natural killer cells C#3, C#2 and PD-L1 positive human breast cancer tumor cells MDA-MB-231 cells, respectively The results of the image analysis of the effect.
  • Figure 11a shows the initial green fluorescent labeling in the in vitro co-culture of chimeric antigen receptor-modified human natural killer cells C#3 and PD-L1 positive human breast cancer tumor cells MDA-MB-231 cells The cells are healthy and complete human breast cancer tumor cells.
  • FIG. 11b shows the in vitro co-culture of chimeric antigen receptor modified human natural killer cells C#2 and PD-L1 positive human breast cancer tumor cells MDA-MB-231 cells, human breast cancer tumor Cells MDA-MB-231 cells are always healthy and intact, and have not been killed by human natural killer cells C#2 modified by chimeric antigen receptors.
  • FIG. 12a illustrates the test flow and mode setting of the in vitro co-culture cytotoxicity experiment of natural killer cells and PD-L1-positive human breast cancer tumor cells covered by the present application.
  • FIG. 13 a illustrates the test flow and mode setting of the in vitro co-culture cytotoxicity assay of natural killer cells and PD-L1 positive human breast cancer tumor cells covered by the present application.
  • Figure 14a illustrates the test flow and mode setting of the in vitro co-culture cytotoxicity assay of natural killer cells and PD-L1-positive human skin cancer tumor cells covered by the present application.
  • Figure 15a illustrates the test flow and mode setting of the in vitro co-culture cytotoxicity assay of natural killer cells and PD-L1 positive human prostate cancer tumor cells covered by the present application.
  • Figure 15b illustrates the quantitative analysis results of the in vitro co-culture cytotoxicity effect of different chimeric antigen receptor-modified human natural killer cells and PD-L1 positive human prostate cancer tumor cells PC-3 cells.
  • FIG. 16a illustrates the test flow and mode setting of the in vitro co-culture cytotoxicity assay of natural killer cells and PD-L1 positive human brain cancer tumor cells covered by the present application.
  • Figure 16b illustrates the quantitative analysis results of the in vitro co-culture cytotoxicity effect of different chimeric antigen receptor modified human natural killer cells and PD-L1 positive human brain cancer tumor cells U87-MG cells.
  • Figure 17a illustrates the test flow and mode setting of the in vitro co-culture cytotoxicity experiment of natural killer cells and PD-L1 positive human hepatoma tumor cells covered by the present application.
  • E/T effector cell/target cell
  • Figure 18a illustrates the test flow and mode setting of the in vitro co-culture cytotoxicity assay of natural killer cells and PD-L1-positive human renal cancer tumor cells covered by the present application.
  • Figure 18b illustrates the quantitative analysis results of the in vitro co-culture cytotoxicity effect of different chimeric antigen receptor-modified human natural killer cells and PD-L1 positive human renal cancer tumor cells 786-O cells.
  • Figure 19a illustrates the test flow and mode setting of the in vitro co-culture cytotoxicity experiment of natural killer cells and PD-L1-positive human lung cancer tumor cells covered by the present application.
  • E/T effector cell/target cell
  • FIG. 20a illustrates the test flow and mode setting of the in vitro co-culture cytotoxicity assay of natural killer cells and PD-L1-positive human ovarian cancer tumor cells covered by the present application.
  • Figure 20b illustrates the quantitative analysis results of the in vitro co-culture cytotoxicity of human natural killer cells modified with different chimeric antigen receptors and PD-L1 positive human ovarian cancer tumor cells ES-2 cells.
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • Figure 28a shows that the chimeric antigen receptor-modified human natural killer cell C#3 has high expression of CRTAM, PIK3R6, GZMB, and KLRF2 in the messenger ribonucleic acid compared with the control group, which is in line with the gene function classification system (Gene Ontology) GO:0002228 Gene-enriched categories of natural killer cell-mediated immunity.
  • Figure 28b shows that the chimeric antigen receptor-modified human natural killer cell C#5 has high expression of CRTAM, PIK3R6, GZMB, and KLRF2, respectively, relative to the messenger RNA of the control group, which is in line with the gene function classification system (Gene Ontology) GO:0002228 Gene-enriched categories of natural killer cell-mediated immunity.
  • Figure 28c shows that the chimeric antigen receptor modified human natural killer cell C#3 has high expression of CRTAM, PIK3R6, GZMB, and KLRF2 in the messenger ribonucleic acid relative to C#2, which is in line with the gene function classification system (Gene Ontology ) GO:0002228 Gene-enriched categories of natural killer cell-mediated immunity.
  • Figure 28d shows that the chimeric antigen receptor modified human natural killer cell C#5 has high expression of CRTAM, PIK3R6, GZMB, and KLRF2 in the messenger ribonucleic acid relative to C#2, which is in line with the gene function classification system (Gene Ontology ) GO:0002228 Gene-enriched categories of natural killer cell-mediated immunity.
  • Figure 29a shows that the messenger ribonucleic acid of chimeric antigen receptor modified human natural killer cell C#3 has FOXP3, TNF, CCR7, IL10, LTA, IL18R1, IL1RL1, SLAMF1, XCL1, EBI3, respectively, relative to the control group High expression, in line with the gene enrichment category of Gene Ontology GO:0032649 ⁇ interferon production regulation.
  • Figure 29b shows that the messenger ribonucleic acid of chimeric antigen receptor modified human natural killer cell C#5 has FOXP3, TNF, CCR7, IL10, LTA, IL18R1, IL1RL1, SLAMF1, XCL1, EBI3, respectively, relative to the control group High expression, in line with the gene enrichment category of Gene Ontology GO:0032649 ⁇ interferon production regulation.
  • Figure 29c shows that the messenger ribonucleic acids of chimeric antigen receptor modified human natural killer cell C#3 relative to C#2 have FOXP3, TNF, CCR7, IL10, LTA, IL18R1, IL1RL1, SLAMF1, XCL1, EBI3, respectively
  • the high expression of ⁇ -interferon is in line with the gene enrichment category of Gene Ontology GO: 0032649 ⁇ interferon production regulation.
  • Figure 29d shows that the messenger ribonucleic acids of chimeric antigen receptor modified human natural killer cell C#5 relative to C#2 have FOXP3, TNF, CCR7, IL10, LTA, IL18R1, IL1RL1, SLAMF1, XCL1, EBI3, respectively
  • the high expression of ⁇ -interferon is in line with the gene enrichment category of Gene Ontology GO: 0032649 ⁇ interferon production regulation.
  • Figure 30a shows that the messenger RNAs of chimeric antigen receptor-modified human natural killer cell C#3 relative to the control group have CCL3L1, CCR7, CCL4L1, CCL1, CCL22, CXCR6, CCL4, XCL1, CCL3, XCL2, respectively. High expression, in line with the gene enrichment category of the Gene Ontology GO:0070098 chemokine-mediated signaling pathway.
  • Figure 30b shows that the messenger RNAs of chimeric antigen receptor-modified human natural killer cell C#5 relative to the control group have CCL3L1, CCR7, CCL4L1, CCL1, CCL22, CXCR6, CCL4, XCL1, CCL3, XCL2, respectively. High expression, in line with the gene enrichment category of the Gene Ontology GO:0070098 chemokine-mediated signaling pathway.
  • Figure 30c shows that the messenger RNAs of chimeric antigen receptor modified human natural killer cell C#3 relative to C#2 are CCL3L1, CCR7, CCL4L1, CCL1, CCL22, CXCR6, CCL4, XCL1, CCL3, XCL2, respectively
  • the high expression of mRNA is in line with the gene enrichment category of the Gene Ontology GO:0070098 chemokine-mediated signaling pathway.
  • Figure 30d shows that the messenger RNAs of chimeric antigen receptor modified human natural killer cell C#5 relative to C#2 are CCL3L1, CCR7, CCL4L1, CCL1, CCL22, CXCR6, CCL4, XCL1, CCL3, XCL2, respectively
  • the high expression of mRNA is in line with the gene enrichment category of the Gene Ontology GO:0070098 chemokine-mediated signaling pathway.
  • Figure 31a shows that the messenger ribonucleic acid of chimeric antigen receptor modified human natural killer cell C#3 relative to the control group has FOXP3, TNF, CRTAM, IL10, PIK3R6, LTA, IFNG, SLAMF1, GZMB, KLRF2,
  • the high expression of XCL1, P2RX7, FCER1G, FAS, and C1QA is in line with the gene enrichment category of lymphocyte-mediated immunity of Gene Ontology (Gene Ontology) GO:0002449.
  • Figure 31b shows that the messenger ribonucleic acid of chimeric antigen receptor modified human natural killer cell C#5 relative to the control group has FOXP3, TNF, CRTAM, IL10, PIK3R6, LTA, IFNG, SLAMF1, GZMB, KLRF2,
  • the high expression of XCL1, P2RX7, FCER1G, FAS, and C1QA is in line with the gene enrichment category of lymphocyte-mediated immunity of Gene Ontology (Gene Ontology) GO:0002449.
  • Figure 31c shows that the messenger RNAs of chimeric antigen receptor modified human natural killer cells C#3 relative to C#2 are FOXP3, TNF, CRTAM, IL10, PIK3R6, LTA, IFNG, SLAMF1, GZMB, KLRF2, respectively , XCL1, P2RX7, FCER1G, FAS, C1QA high expression, in line with the gene function classification system (Gene Ontology) GO: 0002449 lymphocyte-mediated immunity gene enrichment category.
  • Figure 31d shows that the messenger ribonucleic acids of chimeric antigen receptor modified human natural killer cell C#5 relative to C#2 have FOXP3, TNF, CRTAM, IL10, PIK3R6, LTA, IFNG, SLAMF1, GZMB, KLRF2, respectively , XCL1, P2RX7, FCER1G, FAS, C1QA high expression, in line with the gene function classification system (Gene Ontology) GO: 0002449 lymphocyte-mediated immunity gene enrichment category.
  • Figure 32a shows that the messenger ribonucleic acids of chimeric antigen receptor modified human natural killer cell C#3 relative to the control group have FOXP3, CCR7, PIK3R6, IFNG, GPR183, SLAMF1, CTLA4, GPAM, XCL1, MYB,
  • the high expression of EBI3, TNFSF14, and CD80 is in line with the gene enrichment category of positive regulation of lymphocyte activation by Gene Ontology (Gene Ontology) GO:0051251.
  • Figure 32b shows that the messenger ribonucleic acid of human natural killer cell C#5 modified by chimeric antigen receptor compared to the control group has FOXP3, CCR7, PIK3R6, IFNG, GPR183, SLAMF1, CTLA4, GPAM, XCL1, MYB,
  • the high expression of EBI3, TNFSF14, and CD80 is in line with the gene enrichment category of positive regulation of lymphocyte activation by Gene Ontology (Gene Ontology) GO:0051251.
  • Figure 32c shows that the messenger ribonucleic acids of chimeric antigen receptor modified human natural killer cell C#3 relative to C#2 are FOXP3, CCR7, PIK3R6, IFNG, GPR183, SLAMF1, CTLA4, GPAM, XCL1, MYB, respectively , EBI3, TNFSF14, CD80 high expression, in line with the gene function classification system (Gene Ontology) GO: 0051251 lymphocyte activation positive regulation of gene enrichment category.
  • Figure 32d shows that the messenger ribonucleic acids of chimeric antigen receptor modified human natural killer cell C#5 relative to C#2 have FOXP3, CCR7, PIK3R6, IFNG, GPR183, SLAMF1, CTLA4, GPAM, XCL1, MYB, respectively , EBI3, TNFSF14, CD80 high expression, in line with the gene function classification system (Gene Ontology) GO: 0051251 lymphocyte activation positive regulation of gene enrichment category.
  • Figure 33a shows that the messenger ribonucleic acid of chimeric antigen receptor modified human natural killer cell C#3 compared to the control group has TREM1, CRTAM, S100A12, PIK3R6, IFNG, GZMB, KLRF2, XCL1, P2RX7, GNLY, respectively. High expression, in line with the gene enrichment category of Gene Ontology GO:0001906 cell killing.
  • Figure 33b shows that the messenger ribonucleic acid of chimeric antigen receptor modified human natural killer cell C#5 has TREM1, CRTAM, S100A12, PIK3R6, IFNG, GZMB, KLRF2, XCL1, P2RX7, GNLY respectively relative to the control group High expression, in line with the gene enrichment category of Gene Ontology GO:0001906 cell killing.
  • Figure 33c shows that the messenger RNAs of chimeric antigen receptor modified human natural killer cells C#3 relative to C#2 are TREM1, CRTAM, S100A12, PIK3R6, IFNG, GZMB, KLRF2, XCL1, P2RX7, GNLY, respectively The high expression of , in line with the gene enrichment category of Gene Ontology GO:0001906 cell killing.
  • Figure 33d shows that the messenger ribonucleic acids of chimeric antigen receptor modified human natural killer cell C#5 relative to C#2 have TREM1, CRTAM, S100A12, PIK3R6, IFNG, GZMB, KLRF2, XCL1, P2RX7, GNLY, respectively The high expression of , in line with the gene enrichment category of Gene Ontology GO:0001906 cell killing.
  • Figure 34 shows Table 1, containing different versions of chimeric protein constructs, showing examples of chimeric proteins according to the present disclosure, including immune checkpoint PD-1 fusion-based chimeric antigen receptors.
  • Figure 35 shows the vector map of the lentiviral vector, which contains two representative versions: (a) immune checkpoint PD-1 fusion based chimeric antigen receptor C#3 version and (b) immune check based Dot PD-1 fused chimeric antigen receptor C#5 version. See Figure 34 and the related content of this application for information on the components contained in the immune checkpoint PD-1 fusion-based chimeric antigen receptor C#3 and C#5 versions.
  • NK cells used in the specific examples of this application are NK-92 cell lines;
  • Figs. 4 to 7 use fluorescence energy resonance transfer microscopy imaging (Ishikawa-Ankerhold HC et al., Molecules. 2012 Apr; 17(4): 4047-132.) to detect different artificial molecular machines responding to different external stimuli Corresponding intracellular detection signal transduction domain #V phosphorylation expression and intracellular activation signal transduction domain #VII partial molecular conformation state change and corresponding activation state expression when sexual input signal.
  • fluorescence energy resonance transfer microscopy imaging Ishikawa-Ankerhold HC et al., Molecules. 2012 Apr; 17(4): 4047-132.
  • extracellular target molecule binding domains such as PD-1 extracellular fragments or targeting scFv
  • extracellular spacer domains transmembrane domains
  • intracellular spacer domains intracellular detection signaling structures Domain (belonging to the detection module)
  • intracellular hinge domain intracellular activation signaling domain (belonging to the activation module)
  • intracellular signaling domain is respectively numbered as domain #I to domain #VIII, unless otherwise specified , the corresponding contents in this application are applicable.
  • a chimeric antigen receptor (molecular machine) is constructed, comprising:
  • an intracellular signaling domain comprising at least one immune cell activation signaling pathway element; activation of the immune cell activation signaling pathway element is at least dependent on binding of the extracellular domain to the target molecule;
  • the target molecules recognized by the chimeric antigen receptor include at least one of target molecules such as tumor surface antigen molecule markers, specific antigen peptide-histocompatibility complex molecules on the cell surface, or immunosuppressive signal-related molecules.
  • the extracellular binding domain is selected from the monoclonal antibodies against immunosuppressive signal-related molecules commonly used in existing chimeric antigen receptors and their antigen-recognition binding fragments, monoclonal antibodies against tumor surface antigen molecular markers, monoclonal antibodies or monoclonal antibodies. Chain variable fragments and antigen-recognition-binding fragments thereof and antigen-recognition-binding fragments thereof. Preferably, it is at least one molecule that can recognize and bind to immunosuppressive signal-related molecules and tumor surface antigen molecular markers.
  • Intracellular signal transduction domain including at least one intracellular activation signal domain, preferably an immune cell activation signal pathway element; the intracellular activation signal domain contains a molecule with a catalytic functional group or a fragment thereof; the intracellular activation signal domain Activation of the activation signaling domain depends at least on the binding of the extracellular target molecule binding domain to the target molecule.
  • the intracellular signaling domain contains a molecule with a catalytic functional group or a fragment thereof, which enables the chimeric antigen receptor to break away from the confinement of a specific cell type and expand to cell types with applicability to a molecule with a catalytic functional group , that is, to expand the range of host cell types that the chimeric antigen receptor described in the present application can confer genetic modification to express the chimeric antigen receptor.
  • Transmembrane domain existing transmembrane proteins can be used for this technology, no other requirements.
  • the chimeric antigen receptor targets killer signaling molecules associated with apoptotic, dying, moribund, injured, infected, diseased, or necrotic cells. In certain embodiments, the chimeric antigen receptor targets antibody-binding cells associated with infectious microorganisms or particles. In additional embodiments, chimeric antigen receptors target abnormal cells associated with diseases, disorders or other adverse conditions, neoplastic tumor-associated antigens, antigenic signaling molecules exhibited by misfolded proteins.
  • One or more chimeric antigen receptors according to the present specification can be transduced into and expressed in NK cells.
  • the extracellular target molecule binding domain of the chimeric antigen receptor is engineered to bind to a specific target molecule.
  • the intracellular signaling domain of the chimeric antigen receptor is selected to provide the desired killing activity.
  • the intracellular signaling domain of the chimeric antigen receptor is also engineered Selected to provide the desired killing activity.
  • the intracellular signaling domain comprises at least one or more intracellular activation signaling domains.
  • the intracellular signaling domain comprises one or more of an intracellular detection signaling domain and an intracellular activation signaling domain; the intracellular detection signaling domain and the intracellular activation signaling domain Signaling Domain Connections.
  • the intracellular signaling domain comprises one or more of an intracellular detection signaling domain and an intracellular activation signaling domain; the intracellular detection signaling domain and the intracellular activation signaling domain The signaling domains are connected via an intracellular hinge domain.
  • NK cells genetically modified to express one or more of the chimeric antigen receptors can be used to specifically kill target cells or particles expressing target molecules bound by the extracellular domain of the chimeric antigen receptor.
  • the target cells or particles can be tumor cells, cancer cells, microorganisms (eg, bacteria, fungi, viruses), protozoan parasites, Abnormal cells, neoplastic antigens, or misselected foldin.
  • NK cells genetically modified to express one or more chimeric antigen receptors are used to treat cancer, infectious diseases (viruses, bacteria, fungi, protozoa) in a subject , inflammatory disease, immune disease (eg, autoimmune disease), or neurodegenerative disease (eg, Alzheimer's disease), as primary therapy or as adjunctive or combination therapy.
  • infectious diseases viruses, bacteria, fungi, protozoa
  • inflammatory disease eg, autoimmune disease
  • neurodegenerative disease eg, Alzheimer's disease
  • the chimeric antigen receptors of the present disclosure can be designed to confer a specific killing phenotype upon selection of the extracellular target molecule binding domain, depending on the target molecule and therapeutic indication, to use the chimeric antigen receptor for Improve cancer microenvironment and enhance tumor regression.
  • Phase-contrast imaging It is a technique for imaging based on the phase-contrast method.
  • Sequence homology refers to two or more nucleic acid molecules, two or more protein sequences having significant similarity in coding sequence, such as having at least Above 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% or more, at least 91% or more, at least 92% or more, at least 93% or more, at least 94% or more, at least 95% or more, at least 96% or more, at least 97% or more, at least 98% or more, at least 99% or more, at least 99% or more, at least 99% or more More than 99.5% or at least 100% sequence coding identity.
  • PD-L1 Binding Fragments In this application, it refers to molecules or molecular fragments that can specifically bind to PD-L1, such as antibody fragments and the like.
  • Catalytic function refers to the use of enzymes as catalysts in chemical reactions in the body to speed up chemical reactions.
  • tyrosine kinase is an enzyme that can catalyze the transfer of phosphate groups from ATP to tyrosine residues of proteins in cells, thereby regulating the "on” and “on” of signaling pathways in cells. "close”.
  • the tyrosine kinases used in this application include ZAP70 and SYK.
  • Tumor microenvironment refers to the surrounding microenvironment of tumor cells, including immune cells, fibroblasts, bone marrow-derived inflammatory cells, surrounding blood vessels, extracellular matrix and various signaling molecules. Tumors and the surrounding environment are constantly interacting, and the two are closely related.
  • the microenvironment (such as immune cells in it) can influence the growth and development of cancer cells, and the tumor can affect its microenvironment by releasing cell signaling molecules, such as, Promote tumor angiogenesis and induce immune tolerance.
  • the tumor microenvironment contributes to the formation of tumor heterogeneity.
  • Conformation refers to the spatial arrangement produced by the placement of atoms around a single bond without changing its covalent bond structure in a molecule.
  • the dominant conformation refers to the one with the lowest potential energy and the most stable among the different forms of conformation. Different conformations can be transformed into each other, and the covalent bond does not need to be broken and reformed in the process of changing from one conformation to another.
  • the conformation of molecules not only has an impact on the physical and chemical properties of compounds, but also has an important impact on the structure and properties of biological macromolecules (such as nucleic acids, proteins, enzymes, etc.).
  • the specific antigen peptide-histocompatibility complex molecule on the cell surface refers to the process of antigen presentation, the antigenic epitope peptide is firstly cleaved by the proteasome, and then combined with the antigen processing-related transfer protein (TAP), and then Combined with major histocompatibility complex (MHC) molecules, and finally transported to the surface of antigen-presenting molecules to form specific antigenic peptide-histocompatibility complex molecules, immune cells can recognize specific antigenic peptide-histocompatibility complexes Molecules present specific antigenic peptides on the cell surface.
  • TAP antigen processing-related transfer protein
  • MHC major histocompatibility complex
  • Immune checkpoints are stimulatory or inhibitory signaling-related molecules. Inhibitory proteins do not transmit signals, while costimulatory proteins transmit signals that promote immune responses to pathogens.
  • Truncation In this application refers to a fragment that is shortened because a sequence has been deleted.
  • Protein mutant In this application, it refers to changing the amino acid sequence of the original protein in order to obtain a mutant protein that has lost function or has function.
  • Immune checkpoints refers to molecules related to the intrinsic regulatory mechanisms of the immune system, such as the immune checkpoints PD-1 and CTLA-4, which not only maintain self-tolerance, but also avoid potential infections during a physiological immune response. Collateral damage to come. It is currently known that tumors can evade immune surveillance and attack by building a microenvironment, especially by regulating certain immune checkpoint pathways.
  • Tumor immune escape refers to the phenomenon that tumor cells can escape the recognition and attack of the body's immune system through a variety of different mechanisms, so as to achieve the purpose of survival and proliferation in the body.
  • malignant cells When malignant cells appear, the body's immune system can recognize them, and then specifically remove these malignant cells through immune mechanisms to prevent the occurrence and development of tumors.
  • malignant cells may also escape the immune surveillance of the body through different mechanisms, and continue to proliferate in the body to form tumors.
  • Immunosuppression refers to the inhibition of the immune response, that is, the body will not produce an immune response to its own tissue components in some cases, thereby maintaining its own tolerance. A state in which the specificity of some particular antigen does not produce a response.
  • NK cells Natural killer cells (NK cells) are important immune cells in the body, which are not only related to anti-tumor, anti-viral infection and immune regulation, but also participate in hypersensitivity reactions and autoimmune diseases in some cases. can recognize target cells, kill and clear mediators.
  • Nucleic acid molecule and “polynucleotide”: as used herein, include forms of RNA or DNA, specifically including genomic DNA, cDNA and synthetic DNA. Nucleic acid molecules include double-stranded nucleic acid molecules or single-stranded nucleic acid molecules, and single-stranded nucleic acid molecules include coding strands or antisense strands.
  • Chimeric refers to a protein or nucleic acid molecule comprising sequences bound or linked together that are not endogenous (not normally bound or linked together in nature).
  • a chimeric nucleic acid molecule may comprise regulatory and coding sequences from different sources, or from the same source but arranged in a manner different from that found in nature.
  • PD-L1 positive tumor cells refer to the expression of PD-L1 protein molecules in tumor cells reaching a certain level.
  • Cancer refers to a disease characterized by the uncontrolled and rapid growth of abnormal cells. These abnormal cells can form hematological malignancies or constitute solid tumors. Cancer cells spread throughout the body through the lymphatic system, the bloodstream, or only locally. Examples of various cancers include, but are not limited to, ovarian cancer, cervical cancer, breast cancer, prostate cancer, colorectal cancer, kidney cancer, skin cancer, brain cancer, lung cancer, pancreatic cancer, lymphoma, leukemia, liver cancer, and the like.
  • beneficial or desired clinical effects include, but are not limited to, one or more of the following: preventing tumor cell metastasis, inhibiting tumor or cancer cell proliferation or spread (or destroying cancer cell tumors), regressing PD - L1-related diseases (eg, cancer), alleviation of symptoms caused by PD-L1-related diseases (eg, cancer), reduction in the size of PD-L1-expressing tumors, reduction of the dose of drugs required to treat PD-L1-related diseases (eg, cancer), Improve the quality of life of patients with PD-L1-related diseases (eg, cancer), slow the progression of PD-L1-related diseases (eg, cancer), and/or prolong the survival of patients with PD-L1-related diseases (eg, cancer), cure PD-L1 Associated disease (eg, cancer).
  • beneficial or desired clinical effects include, but are not limited to, one or more of the following: preventing tumor cell metastasis, inhibiting tumor or cancer cell proliferation or spread (or destroying cancer cell tumors), re
  • High expression in this application, it means that a specific cell has a high level of expression of a specific molecular marker.
  • a tumor cell with high PD-L1 expression refers to a high expression level of PD-L1 protein molecule in the tumor cell.
  • Highly expressed tumor cell markers are generally associated with disease states, for example, in cells that form solid tumors within a particular organ or tissue and in cells of hematological malignancies, and can be determined by assays using criteria well known in the art. Solid tumors or hematological malignancies characterized by high tumor marker expression.
  • Vector refers to a nucleic acid molecule capable of transporting another nucleic acid.
  • Vectors include plasmids, viruses, phages, cosmids. Also included are non-viral and non-plasmid compounds that can facilitate transfer of nucleic acids into cells.
  • An "expression vector” refers to a vector that, when placed in a suitable environment, can direct the expression of a protein encoded by one or more genes carried by the vector.
  • Viral vectors include adeno-associated viral vectors, adenoviral vectors, retroviral vectors, lentiviral vectors, and gamma retroviral vectors.
  • Retrovirus refers to a virus having an RNA genome.
  • Lentivirus refers to a genus of retroviruses capable of infecting dividing and non-dividing cells. Lentiviruses include bovine immunodeficiency virus (BIV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV, including HIV type 1 and type 2 HIV), feline immunodeficiency virus (FIV), and equine infectious anemia virus.
  • BIV bovine immunodeficiency virus
  • SIV simian immunodeficiency virus
  • HV human immunodeficiency virus
  • FV feline immunodeficiency virus
  • equine infectious anemia virus equine infectious anemia virus.
  • Gamma retrovirus refers to a genus of the retroviridae family. Gamma retroviruses include, but are not limited to, feline leukemia virus, feline sarcoma virus, mouse leukemia virus, avian reticuloendothelial proliferation virus, mouse stem cell virus.
  • Non-viral vectors include modified mRNA (modRNA), self-amplifying mRNA, lipid-based DNA vectors, transposon-mediated gene transfer (PiggyBac, Sleeping Beauty), closed linear duplex (CELiD) DNA.
  • liposomes can be utilized as delivery vehicles. Nucleic acids are introduced into host cells in vitro, ex vivo or in vivo through the use of lipid formulations. Nucleic acids are encapsulated inside the liposomes, dispersed within the lipid bilayer of the liposomes, and bound to the lipids by the attachment of linker molecules that bind the nucleic acid and the liposomes together to the liposomes.
  • Extracellular Target Molecule Binding Domain refers to a molecule that has the ability to specifically and non-covalently bind, associate, unite, or recognize a target molecule, such as a peptide, oligopeptide, polypeptide Or protein, the bound target molecules include: IgA antibody, CD138, CD38, L1CAM, CD22, CD19, PD-1, CD79b, mesothelin, PSMA, CD33, CD123, BCMA, ROR1, MUC-16, IgG antibody, IgE antibodies, EGFRviii, VEGFR-2 or GD2.
  • a target molecule binding domain includes any semi-synthetic, synthetic, recombinantly produced, naturally occurring binding partner that can be directed against a target biomolecule or other target.
  • Target molecule binding domains can be antigen binding domains, including antibodies, functional binding domains thereof, antigen binding portions, and the like.
  • the binding domain can include a single chain antibody variable region (eg, sFv, Fab, scFv, domain antibody), ligand (eg, chemokine, cytokine), receptor extracellular domain (eg, PD-1) or Synthetic polypeptides selected for their specific binding ability to biomolecules.
  • Intracellular activation signal transduction domain refers to a cell selected from a non-receptor-type tyrosine kinase or receptor-type tyrosine kinase molecule or fragment with catalytic function that expresses an activation signal transduction domain It is capable of promoting a biological or physiological response when given an appropriate signal.
  • the activation signaling domain can be part of a protein or protein complex that receives a signal upon binding.
  • activation of a signaling domain can respond to the binding of a PD-1-fused chimeric antigen receptor to the target molecule PD-L1, thereby signaling to the interior of the host cell, triggering effector functions such as secretion of inflammatory cytokines and / Or chemokines, secrete anti-inflammatory and / or immunosuppressive cytokines, NK cells effectively kill tumor cells.
  • the activation signaling domain may also promote a cellular response indirectly by binding to one or more other proteins that directly promote the cellular response.
  • the immunoreceptor tyrosine-based activation motif is a conserved sequence consisting of more than ten amino acids.
  • the detection signaling domain of the chimeric antigen receptor molecular machine can respond to tyrosine kinase activation signal input and undergo phosphorylation modification, and then interact with the activation signaling domain based on phosphorylation site modification, and Its activation signaling domain is unwound from the autoinhibited molecular conformation state, releasing the activation signaling domain, and the activation signaling domain of the molecular machine is in open activation in the molecular conformation after the activation signaling domain is released. state.
  • the primary detection signal transduction sequence may include a signal motif known as an immunoreceptor tyrosine activation motif (ITAM).
  • ITAMs are well-defined signaling motifs found in the intracytoplasmic tails of various receptors that serve as binding sites for tyrosine kinases.
  • ITAMs used in the present invention may include: 2B4, CD244, BTLA, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD28, CD31, CD72, CD84, CD229, CD300a, CD300f, CEACAM-1, CEACAM-3, CEACAM-4, CEACAM-19, CEACAM-20, CLEC-1, CLEC-2, CRACC, CTLA-4, DAP10, DAP12, DCAR, DCIR, Dectin-1, DNAM-1, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIB, Fc ⁇ RI, Fc ⁇ RIIA, Fc ⁇ RIIB, Fc ⁇ RIIC, Fc ⁇ RIIIA, FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, FCRL6, G6b, KIR, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DL5B, KIR2DS1, KIR2DS3, KIR2DS4, KIR2DS5,
  • Intracellular signaling domain refers to the intracellular effector domain, when the extracellular target molecule binding domain of the chimeric antigen receptor molecular machine on the surface of the immune cell recognizes and binds the target molecule, thereby passing the Recognition binding provides target molecule recognition and binding signal input, and then the molecular conformation of the intracellular part changes to unwind its activating signaling domain from the auto-inhibitory molecular conformational state, and finally responds to upstream target molecule recognition and binding signal input.
  • the activation signaling domain in the lower cell is fully released and activated based on the conformational change of the chimeric antigen receptor molecular machine, and the activation signaling domain in the activated state can further activate its downstream signaling domain.
  • the signaling domain activates one or more signaling pathways that lead to the killing of target cells, microorganisms or particles by the host cell.
  • the signaling domain comprises at least one intracellular activation signaling domain.
  • the signaling domain comprises at least one intracellular detection signaling domain and at least one intracellular activation signaling domain.
  • the signaling domain comprises at least one intracellular detection signaling domain, an intracellular hinge domain, and at least one intracellular activation signaling domain.
  • Transmembrane domain refers to a polypeptide that spans the entire biological membrane once, used to connect the extracellular target molecule binding domain and the intracellular signaling domain, and fix them on the cell membrane .
  • Intracellular spacer domain In the present application, referring to and linking together the transmembrane domain and the intracellular signaling domain, may be an extension of the transmembrane domain.
  • Intracellular hinge domain refers to the connection between the intracellular detection signal transduction domain and the intracellular activation signal transduction domain, which can optionally be a flexible linker peptide fragment.
  • the hinge domain can provide the desired flexibility to allow the desired expression, activity and/or conformational positioning of the chimeric polypeptide.
  • the hinge domain may be of any suitable length to link the at least two domains of interest, and is preferably designed to be flexible enough to allow proper folding and/or function and/or activity of the one or both domains to which it is linked.
  • the hinge domains are at least 3 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more in length more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more above, 95 or more, 90 or more, 95 or more, or 100 or more amino acids.
  • the length of the hinge domain is about 0-200 amino acids; preferably, about 10-190 amino acids; preferably, about 20-180 amino acids; preferably, about 30-170 amino acids; preferably , about 40-160 amino acids; preferably, about 50-150 amino acids; preferably, about 60-140 amino acids; preferably, about 70-130 amino acids; preferably, about 80-120 amino acids; preferably , about 90 to 110 amino acids.
  • the hinge domain sequence may also comprise endogenous protein sequences.
  • the hinge domain sequence may contain glycine, alanine and/or serine residues.
  • the hinge domain may contain motifs, such as multiple or repeated motifs of GGSG, SGGG, GS, GGS or GGGGS.
  • the hinge domain sequence can include any non-naturally occurring amino acid, naturally occurring amino acid, or a combination thereof.
  • domain #I can be selected as the ligand recognition and binding part of PD-L1 receptor PD-1, and domain #II can be selected as the extracellular extension fragment of the transmembrane region of PD-1 (i.e.
  • domain #III can be selected as the transmembrane region part of PD-1
  • domain #IV can be selected as the transmembrane region of PD-1
  • the intracellular extension fragment of the region part that is, the intracellular part of Full-length PD-1 or Truncated PD-1 in Figure 34; wherein the full-length amino acid sequence of C#1Full-length PD-1 is SEQ ID NO:001+SEQ ID NO: 001+SEQ ID NO: 001 ID NO:016+SEQ ID NO:012+SEQ ID NO:056 and full length DNA nucleic acid sequence is SEQ ID NO:002+SEQ ID NO:017+SEQ ID NO:013+SEQ ID NO:057, C#2Truncated
  • the full-length amino acid sequence of PD-1 is SEQ ID NO:001+SEQ ID NO:016+SEQ ID NO:012+SEQ ID
  • detection and characterization methods including but not limited to, detection and characterization of the extracellular functional performance of chimeric antigen receptors in the form of purified proteins, and detection and characterization of chimeric antigen receptors by different means Functional performance in eukaryotic cells.
  • Figure 3 shows a schematic diagram of the signal activation of artificial molecular machines.
  • Figure 3a shows the release and activation of the activation signal of the artificial molecular machine in the case of tyrosine kinase activation signal input
  • Figure 3b shows the domain containing the target molecule in the case of target cell signal input (such as PD-L1)
  • target cell signal input such as PD-L1
  • the activation signal of the chimeric antigen receptor artificial molecular machine of #I (eg, the extracellular portion of PD-1) is released and activated.
  • the molecular machine working model of Figure 3a is a simplified model, including Domain #VII, Domain #VI, and Domain #V.
  • domain #VII can be selected as the tyrosine kinase part of SYK/ZAP70 family members, etc.
  • domain #V can be selected as the immunoreceptor tyrosine activation motif fragment of CD3 ⁇ , CD3 ⁇ , FcRIIA, FcR ⁇ , DAP12 and other molecules Parts (ie Sub1 to Sub7: CD3 ⁇ ITAM1 ⁇ 3, CD3 ⁇ ITAM, FcRIIA ITAM, FcR ⁇ ITAM, DAP12ITAM in sequence)
  • the domain #VI connecting domain #VII and domain #V can optionally be a flexible linking peptide fragment, see Figure 34 .
  • SYK or ZAP70 will be in an auto-inhibited molecular conformation state (Yan Q et al., Molecular and cellular biology.
  • domain #VII of the molecular machine in this conformation, domain #VII of the molecular machine is in a closed inactive state; when the tyrosine kinase activation signal is input, especially the phosphorylation signal of the immunoreceptor tyrosine activation motif, the molecule Domain #V of the machine will undergo phosphorylation modification in response to signal input, and then phosphorylated domain #V will interact with SYK or ZAP70 based on phosphorylation site modification, especially in domain #VI.
  • the flexible linker peptide fragment provides sufficient flexibility for the conformational change of the molecular machine to unwind its domain #VII from the autoinhibited molecular conformational state, releasing domain #VII, which is released after domain #VII is released.
  • the domain #VII of the molecular machine in the molecular conformation is in the open activation state, that is, the schematic diagram of the signal activation of the artificial molecular machine under the input of the tyrosine kinase activation signal shown in Figure 3a, and the domain # in the activated state VII can further activate various signaling pathways downstream of it.
  • the working model of the molecular machine in Fig. 3b is a model similar to the working principle of Fig. 3a, including seven parts: domain #I to domain #VII.
  • domain #I can be selected as the ligand-recognition binding portion of PD-L1 receptor PD-1
  • domain #II can be selected as an extracellular extension of the transmembrane region portion of PD-1 (i.e. Between the extracellular target molecule PD-L1 binding domain and the transmembrane region of PD-1), domain #III can be selected as the transmembrane region part of PD-1, and domain #IV can be selected as the transmembrane region of PD-1.
  • the intracellular extension fragment of the membrane region part (that is, the intracellular part of Truncated PD-1 in Figure 34), the domain #VII can be selected as a tyrosine kinase part of a SYK/ZAP70 family member, etc., and the domain #V can be selected as Immunoreceptor tyrosine activation motif fragments of CD3 ⁇ , CD3 ⁇ , FcRIIA, FcR ⁇ , DAP12 and other molecules (ie Sub1 to Sub7 in Figure 34: CD3 ⁇ ITAM1-3, CD3 ⁇ ITAM, FcRIIA ITAM, FcR ⁇ ITAM, DAP12ITAM), linking domain# Domain #VI of VII and domain #V can optionally be flexible linker peptide fragments (ie different length linker peptides in Figure 34: SL, ML, LL1, LL2), see Figure 2h and Figure 34.
  • SYK or ZAP70 will be in an auto-inhibited molecular conformation, in which domain #VII of the molecular machine is closed and inactive.
  • the domain #I of the chimeric antigen receptor molecular machine on the surface of the immune cell will recognize and bind the target molecule, thereby providing the target molecule recognition and binding signal input through the recognition and binding, and then intracellular Part of the molecular conformation will undergo similar changes as described in Figure 3a above, and finally, in response to the input of the upstream target molecule recognition and binding signal, the intracellular domain #VII is fully transformed based on the molecular conformation change of the chimeric antigen receptor molecular machine.
  • Fig. 3b is a schematic diagram showing the signal activation of the chimeric antigen receptor artificial molecular machine under the condition that the target molecule recognizes the binding signal input.
  • Human and mouse-derived mammalian cells express different molecular machine proteins by DNA transfection, and then use fluorescence microscopy imaging to detect and characterize different artificial molecular machines in human-derived HeLa cells and mouse embryonic fibroblasts.
  • Intracellular (MEF) performance in response to a variety of different external stimuli input signals.
  • Figure 4a demonstrates the excellent responsiveness of C#9 and C#15 signaling domains Sub1 and Sub4 to protein tyrosine phosphorylation signals as well as C#9 and C#15 in human HeLa cells.
  • the change of molecular conformation is very obvious and the release and activation of its own activation element, domain #VII (SYK and ZAP70), is very sufficient and significant, and is significantly better than that of C#11 and C#13.
  • C#10, C#12, C#14, and C#16 are compared with the corresponding C#9, C#11, C#13, C#15 versions have weaker near-zero responsiveness to protein tyrosine phosphorylation signals that differ significantly after statistical analysis, demonstrating that C#9, C#11, C#13 and C#15
  • the importance of domain #V (Sub1 ⁇ Sub4) of C#9 for excellent responsiveness to protein tyrosine phosphorylation signals and domain #V(Sub1) of C#9 and domain #V(Sub4) of C#15 are more Domain #V (Sub2) of #11 and domain #V (Sub3) of C#13 have significantly better responsiveness and sensitivity to protein tyrosine phosphorylation signals.
  • 20uM tyrosine phosphatase inhibitor sodium pervanadate can inhibit the dephosphorylation of intracellular proteins, thereby promoting the activation of protein tyrosine phosphorylation signals and providing the input of protein tyrosine phosphorylation signals.
  • Figure 4b shows the different artificial molecular machines in human under the A condition of 20 uM tyrosine phosphatase inhibitor sodium pervanadate to activate the protein tyrosine phosphorylation signal or the B condition of 50 ng/mL of epidermal growth factor to activate the signal.
  • the performance results in the source HeLa cells (mean ⁇ SD, all n 6)
  • the imaging readout index represents the quantification of the degree of responsiveness of the artificial molecular machine to the stimulus signal and the artificial molecular machine simultaneously triggered in response to the stimulus signal based on The extent to which a molecule's conformational changes release and activate its own activating elements.
  • Figure 4b demonstrates the excellent responsiveness of C#9 and C#15 domains #V (Sub1 and Sub4) to protein tyrosine phosphorylation signals in human HeLa cells as well as C#9 and C# 15 Corresponds to a very pronounced change in molecular conformation and a very substantial release and activation of its own activation element, domain #VII (SYK and ZAP70).
  • C#9 and C#15 had weaker near-zero responsiveness to this signal under the condition of epidermal growth factor activation signal that was significantly different after statistical analysis, demonstrating the structure of C#9 and C#15 The importance of domain #V (Sub1 and Sub4) for excellent responsiveness to protein tyrosine phosphorylation signals and to ensure the specific response of artificial molecular machines to specific protein tyrosine phosphorylation signals and not to irrelevant Signal inputs, such as epidermal growth factor activation signals.
  • domain #V Sub1 and Sub4
  • epidermal growth factor can bind to the epidermal growth factor receptor on the surface of HeLa cells to provide an epidermal growth factor activation signal, which is not involved in the phosphorylation of the immunoreceptor tyrosine activation motif, so it cannot be specifically affected by C# 9 and domain #V of C#15 were detected.
  • the artificial molecular machine is based on the degree of release and activation of its own activating elements based on changes in molecular conformation.
  • Figure 4c demonstrates the excellent responsiveness of C#9 and C#15 domains #V (Sub1 and Sub4) to protein tyrosine phosphorylation signals in mouse embryonic fibroblasts as well as C#9 and C# A very pronounced change in molecular conformation of #15 and a very substantial release and activation of its own activation element, domain #VII (SYK and ZAP70).
  • C#9 and C#15 had weaker near-zero responsiveness to this signal under the condition of platelet-derived growth factor activation signal that was significantly different after statistical analysis, demonstrating that C#9 and C#15 have The importance of domain #V (Sub1 and Sub4) for excellent responsiveness to protein tyrosine phosphorylation signals and to ensure the specific response of artificial molecular machines to specific protein tyrosine phosphorylation signals without responding to irrelevant signals signal input, such as platelet-derived growth factor activation signals.
  • domain #V Sub1 and Sub4
  • platelet-derived growth factor can bind to platelet-derived growth factor receptors on the surface of mouse embryonic fibroblasts to provide platelet-derived growth factor activation signals, which are not involved in the phosphorylation of immunoreceptor tyrosine activation motifs, so Not specifically detected by domain #V of C#9 and C#15.
  • Figure 5a shows the expression distribution of different artificial molecular machines in human HeLa cells and the detection results of their ability to respond to protein tyrosine phosphorylation signals under the stimulation of 20uM tyrosine phosphatase inhibitor sodium pervanadate.
  • the experimental group is C#17 modified human HeLa cells
  • the control group is C#18 modified human HeLa cells.
  • the responsiveness of the signal from low to high and the chimeric antigen receptor elicited simultaneously in response to the stimulatory signal is based on the molecular conformational change from low to high degree of release and activation of its self-activating element, domain #VII.
  • both C#17 and C#18 displayed correct membrane-localized expression distribution on the surface of human HeLa cells without any other wrong protein localization.
  • C#17-modified human HeLa cells showed a rapid and significant response to protein tyrosine phosphorylation signals stimulated by the tyrosine phosphatase inhibitor sodium pervanadate, about half an hour after stimulation It showed a very significant response to stimulatory signals and the release and activation of its own domain #VII based on molecular conformation changes;
  • C#18-modified human HeLa cells showed significantly weaker response to casein.
  • C#18 had a significantly weaker near-zero responsiveness to protein tyrosine phosphorylation signals than phase C#17 when the autoactivation element was disabled (inactive mutant Sub1FF), demonstrating that Importance and specificity of domain #V(Sub1) of C#17 for excellent responsiveness to protein tyrosine phosphorylation signals.
  • Figure 5b shows the expression distribution of different artificial molecular machines in human HeLa cells and the detection results of their ability to respond to protein tyrosine phosphorylation signals under the stimulation of 20uM tyrosine phosphatase inhibitor sodium pervanadate.
  • the experimental group is C#19 modified human HeLa cells
  • the control group is C#20 modified human HeLa cells.
  • the responsiveness of the signal from low to high and the chimeric antigen receptor elicited simultaneously in response to the stimulatory signal is based on the molecular conformational change from low to high degree of release and activation of its self-activating element, domain #VII.
  • both C#19 and C#20 displayed the correct membrane-localized expression distribution on the surface of human HeLa cells without any other wrong protein localization.
  • C#19-modified human HeLa cells showed a rapid and significant response to protein tyrosine phosphorylation signals stimulated by the tyrosine phosphatase inhibitor sodium pervanadate, about half an hour after stimulation It showed a very significant ability to respond to stimulatory signals and the release and activation of its own domain #VII based on molecular conformation changes;
  • C#20 modified human HeLa cells showed almost zero very weak The ability to respond to the protein tyrosine phosphorylation signal stimulated by the tyrosine phosphatase inhibitor sodium pervanadate cannot show an effective ability to respond to the stimulation signal after stimulation and its own domain based on molecular conformation changes# Release and activation of VII.
  • Figure 5b demonstrates the excellent responsiveness of domain #V(Sub1) of C#19 to protein tyrosine phosphorylation signals and the corresponding apparent molecular conformational change of C#19 and its activation in human HeLa cells Element-sufficient significant release and activation of domain #VII.
  • C#20 had a significantly weaker near-zero response to protein tyrosine phosphorylation signals than C#19 in the case of inactivated autoactivation elements (inactivating mutant Sub1FF), demonstrating that C#20 Importance and specificity of domain #V (Sub1) of #19 for excellent responsiveness to protein tyrosine phosphorylation signals.
  • Figure 5c demonstrates that domain #V(Sub1) of C#19 is very responsive to protein tyrosine phosphorylation signals in human HeLa cells (average of C#19 group is over 2.84) and C# 19 corresponds to a very obvious change in molecular conformation and a very sufficient and significant release and activation of its self-activating element-domain #VII, and the statistically significant difference is better than that of C#17 (the average value of the C#17 group is about approx. 2.48).
  • C#20 had a significantly weaker response to protein tyrosine phosphorylation signals than C#18 when the autoactivation element was disabled (inactive mutant Sub1FF) (C#18)
  • the mean of #20 group is about 0.055, and the mean of C#18 group is about 0.34), demonstrating the importance of domain #V of C#19 and C#17 for excellent responsiveness to protein tyrosine phosphorylation signals and the importance of C# 19 has significantly better specificity in response to protein tyrosine phosphorylation signals than C#17, indicating that domain #IV of C#19 has better functional performance than domain #IV of C#17.
  • Proteins C#9 and C#10 were purified from transfected 293T cells using chromatographic purification techniques and protein dialysis at 4°C, and then the purified molecular machine proteins were dissolved in kinase buffer solution (50 mM Tris hydrochloride with pH around 8). The solution, 100 mM sodium chloride, 10 mM magnesium chloride, 2 mM dithiothreitol) at a concentration of 50 nM, was added to provide the substrate required for phosphorylation 1 mM ATP and 100 nM non-receptor protein tyrosine kinase Lck protein in an activated state.
  • kinase buffer solution 50 mM Tris hydrochloride with pH around 8
  • Src family protein non-receptor-type protein tyrosine kinase Lck (Lymphocyte-specific protein tyrosine kinase) can promote the activation of protein tyrosine phosphorylation signals and provide specificity
  • the role of protein tyrosine phosphorylation signal input can provide the phosphorylation signal input of immunoreceptor tyrosine activation motif.
  • Optical signals before and after adding ATP and Lck were detected and quantified.
  • the performance results of C#9 and C#10 in the state of purified protein under the condition that Lck provides an activated protein tyrosine phosphorylation signal (mean ⁇ standard deviation, all n 3), imaging reading indicators represent quantification
  • the degree of responsiveness of the post-chimeric antigen receptor to a stimulatory signal and the degree to which the chimeric antigen receptor simultaneously elicited in response to the stimulatory signal releases and activates its own activating element based on molecular conformational changes.
  • the C#9(+) group of Figure 6 demonstrates that the intracellular detection signaling domain Sub1 of C#9 is very responsive to protein tyrosine phosphorylation signals (average about 0.8) and that C#9 is very The change of molecular conformation and the very sufficient and significant release and activation of its own activation element, the intracellular activation signaling domain ZAP70.
  • the C#10(+) group demonstrated that in the case where the self-detection element was disabled (the inactive mutant Sub1FF), C#10 had a weaker response to protein case than C#9 that was statistically significantly different Responsiveness to amino acid phosphorylation signals (average less than 0.08), demonstrating the importance of domain #V of C#9 for excellent responsiveness to protein tyrosine phosphorylation signals and the C#9 version has excellent response to protein casein Specificity of the amino acid phosphorylation signal response.
  • physiologically specific human PD-L1 signal input the used physiologically specific human PD-L1 signal is human PD-L1 modified microspheres.
  • Figure 7a shows the expression distribution of different chimeric antigen receptors in human HeLa cells and the detection results of their ability to respond to human PD-L1 signaling under the stimulation of human PD-L1-modified microspheres.
  • the experimental group is C#19 modified human HeLa cells
  • the control group is C#20 modified human HeLa cells.
  • the provided phase contrast imaging experimental pictures provide image information of the interaction between cells and microspheres.
  • the color bar heatmap below the picture represents from left to right the responsiveness of chimeric antigen receptors to stimulatory signals from low to high and the self-activation of chimeric antigen receptors based on molecular conformational changes that are simultaneously elicited in response to stimulatory signals Element-intracellular activation signaling domain ZAP70 release and activation degree from low to high.
  • both C#19 and C#20 shown in Figure 7a displayed correct membrane-localized expression profiles on the surface of human HeLa cells.
  • C#19-modified human HeLa cells showed a rapid and significant response to the stimulation signal of human PD-L1-modified microspheres, and showed a very significant response from about 18 minutes after stimulation.
  • the ability to respond to stimulation signals and the release and activation of its own domain #VII based on changes in molecular conformation, and the shown response to stimulation signals of human PD-L1-modified microspheres has a highly specific spatial characteristic, that is, only Locally demonstrated responsiveness at locations where cells interacted with microspheres in phase-contrast imaging experiments; whereas C#20-modified human HeLa cells showed significantly weaker stimulation of human PD-L1-modified microspheres
  • the ability to respond to the signal, after stimulation, cannot show the effective ability to respond to the stimulation signal and the release and activation of its own domain #VII based on the change of molecular conformation.
  • Figure 7a demonstrates the excellent responsiveness of domain #V(Sub1) of C#19 to human PD-L1 signaling in human HeLa cells and the corresponding apparent molecular conformational change of C#19 and its own activation of the element - Sufficient and significant release and activation of the intracellular activation signaling domain ZAP70.
  • C#20 has a significantly weaker response to human PD-L1 signaling than C#19 in the case where the autoactivation element is disabled (inactive mutant Sub1FF), demonstrating that the domain of C#19 Importance and specificity of #V(Sub1) for excellent responsiveness to human PD-L1 signaling.
  • Figure 7b demonstrates that domain #V(Sub1) of C#19 is very responsive to protein tyrosine phosphorylation signals in human HeLa cells (average of C#19 group is about 0.46) and C#19 #19 corresponds to a very obvious change in molecular conformation and a very sufficient and significant release and activation of its own activation element-intracellular activation signaling domain ZAP70, and the statistically significant difference is better than that of the C#17 version (C#17 The group mean is approximately 0.23).
  • C#20 had a significantly weaker response to protein tyrosine phosphorylation signals than C#18 when the autoactivation element was disabled (inactive mutant Sub1FF) (C#18)
  • the mean value of group #20 is about 0.046, and the mean value of group C#18 is about 0.126), demonstrating the importance of domain #V of C#19 and C#17 to the excellent responsiveness of human PD-L1 signal and C#19 Compared with C#17, it has significantly better specificity in response to human PD-L1 signal, indicating that the domain #IV of C#19 has better functional performance than the domain #IV of the C#17 version.
  • the chimeric antigen receptor artificial molecular machine exhibits excellent response ability to different stimulatory signal input, especially the highly specific response to human PD-L1 signal input.
  • intracellular signaling domain #VIII especially the ability of domain #VII to elicit engineered lymphocyte effector functions upon release activation.
  • the functionality of C#19 is particularly prominent, that is, the Truncated PD-1-Sub1-LL2-ZAP70 version, which also provides support and guarantee for subsequent cytotoxic killing experiments.
  • Figure 8a shows that when the immune checkpoint receptors (such as endogenous PD-1) on the surface of natural killer cells recognize and bind to target molecules (such as PD-L1) on the surface of tumor cells, natural killer cells kill the corresponding The ability of tumor cells is inhibited by inhibitory immune checkpoint signaling.
  • the immune checkpoint receptors such as endogenous PD-1
  • target molecules such as PD-L1
  • Figure 8b shows that when the chimeric antigen receptor modified and modified human natural killer cells based on the fusion of immune checkpoint PD-1 recognize and bind to the target molecule PD-L1 on the surface of tumor cells, the modified natural killer cells can effectively obtain It activates and effectively kills the corresponding tumor cells.
  • the human tumor cells used in the tumor cell in vitro killing experiments have been modified to express the reporter gene firefly luciferase.
  • the luciferase in tumor cells can accurately reflect the overall cell survival rate, that is, by detecting the fluorescence in tumor cells.
  • the level of peptase activity was used to quantify the number of viable tumor cells.
  • Packaging with lentivirus to prepare viral particles of chimeric antigen receptor artificial molecular machines fused with different immune checkpoint PD-1, which is about to reverse the chimeric antigen receptor artificial molecular machine with different immune checkpoint PD-1 fusions 293T cells were transfected with a transcriptovirus expression vector (such as pSIN plasmid, etc.) and packaging plasmid (such as pCMV delta R8.2 and pCMV-VSV-G or psPAX2 and pMD2.G, etc.), and the virus supernatant was harvested, filtered and frozen. to determine the virus titer.
  • a transcriptovirus expression vector such as pSIN plasmid, etc.
  • packaging plasmid such as pCMV delta R8.2 and pCMV-VSV-G or psPAX2 and pMD2.G, etc.
  • Different immune checkpoint PD-1 fused chimeric antigen receptors C#2, C#3 and C#5 were expressed in natural killer cells NK-92 by more than 90% compared to the control group, and were used for In co-culture experiments, the effect of different immune checkpoint PD-1 fusion-based chimeric antigen receptor modified natural killer cells NK-92 in killing tumor cells was tested.
  • Natural killer cell NK-92 expresses different immune checkpoint PD-1 fused chimeric antigen receptors C#2 and C#3 respectively. The ability to kill tumor cells was observed by imaging in Figure 11.
  • PD-L1 antibody was used to stain and detect the expression of PD-L1 on 8 kinds of human cancer tumor cells, namely human breast cancer tumor cells MBA-MB-231, human brain cancer tumor cells U87-MG, human Renal cancer tumor cell 786-O, human skin cancer tumor cell A2058, human lung cancer tumor cell H441, human ovarian cancer tumor cell ES-2, human prostate cancer tumor cell PC-3 and human liver cancer tumor cell HA- 22T.
  • Figures 10a to 10h show the expression of PD-L1 in 8 human tumor cells and in human cancer cells after pretreatment with gamma interferon, respectively.
  • the expression ratios of PD-L1 in eight human cancer cells were 90.1%, 97.1%, 91.9%, 89.5%, 99.4%, 99.9%, 93.7%, and 93.6%, respectively.
  • the expression ratio of -L1 was increased and the expression level was significantly increased, which were 97.5%, 99.7%, 99.9%, 99.9%, 99.6%, 100.0%, 99.9%, and 99.5%, respectively, further revealing that interferon- ⁇ can promote PD on tumor cells.
  • -L1 expression, and pretreated tumor cells with gamma interferon to simulate the tumor microenvironment in the body in in vitro experiments, and the 8 human-derived cancer tumor cells were used in tumor cell killing experiments.
  • Human breast cancer tumor cells MDA-MB-231 expressing the reporter gene green fluorescent protein were pretreated with interferon gamma for 24 hours to increase the expression of PD-L1 on the cell surface, and 1x 10 5 of the modified human-derived natural killing Cells NK-92 were co-cultured with 1x 10 5 tumor cells at a 1:1 E/T (effector/target) ratio in a 35mm glass dish and visualized using time-lapse intravital microscopy for immune checkpoint PD-1 fused chimeric antigens Whether human natural killer cells can effectively kill PD-L1 positive human breast cancer tumor cells MBA-MB-231 after modification of receptor C#3 version.
  • Human breast cancer tumor cells MDA-MB-231 expressing reporter gene firefly luciferase were pretreated with gamma interferon for 24 hours to increase the expression of PD - L1 on the cell surface.
  • 10 3 , 10 ⁇ 10 3 modified human natural killer cells NK-92 and 1 ⁇ 10 3 tumor cells were treated with 1:1, 2.5:1, 5:1, 10:1 E/T (effector cells/target) The ratio of cells) was co-cultured for 24 hours in a 48-well plate, and the time of co-culture was the 0th hour.
  • the luciferase activity in the cell culture system was detected at three co-culture time points of 0 hours, 4 hours and 24 hours after incubation, and then the number of human breast cancer tumor cells was quantified and the effect of human natural killer cells on human cells was calculated. Cytotoxicity of breast cancer tumor cells. See Figure 12. Among them, the tumor cell group was only human breast cancer tumor cells MDA-MB-231 cells themselves, and the human natural killer cells in the control group were human natural killer cells without chimeric antigen receptor modification.
  • the target cell survival index Represents the relative cell number of human breast cancer tumor cells expressing the reporter gene firefly luciferase in cell culture systems.
  • Figure 12a illustrates the test flow and mode setting of the in vitro co-culture cytotoxicity assay of natural killer cells and PD-L1-positive human breast cancer tumor cells covered by the present application.
  • Figure 12b shows the quantification of the cytotoxic effect of different in vitro co-culture cytotoxic effects of human natural killer cells modified by different immune checkpoint PD-1 fusion-based chimeric antigen receptor artificial molecular machines and PD-L1 positive human tumor cells To analyze the results, natural killer cells and tumor cells were subjected to the experiment at a 1:1 E/T (effector cell/target cell) ratio.
  • E/T effector cell/target cell
  • immune checkpoint PD-1 fused chimeric antigen receptor compared to human natural killer cells in control group
  • the human-derived immune natural killer cells after C#3 modification and transformation respectively showed the largest tumor cell clearance ability, and the cell number of human-derived tumor cells was 66.6% compared to the control group.
  • Quantitative analysis line graphs demonstrate the remarkable ability of chimeric antigen receptor C#3-modified immune natural killer cells to recognize and kill tumor cells with statistically significant differences when co-cultured with PD-L1-positive human tumor cells
  • the human-derived natural killer cells in the other control groups failed to show the ability to effectively recognize and kill tumor cells under the co-culture conditions of PD-L1-positive human-derived tumor cells.
  • Figure 12c shows the quantification of the cytotoxic effect of different co-cultures of human natural killer cells modified with PD-1 fusion-based chimeric antigen receptor artificial molecular machines and PD-L1 positive human tumor cells in vitro Analyzing the results, natural killer cells and tumor cells were experimented at an E/T (effector cell/target cell) ratio of 2.5:1 at 24 hours after incubation (average of C#3 group was 0.9, and the average of control group was 2.2) , compared with the human-derived natural killer cells in the control group, the human-derived immune natural killer cells after the modification of the chimeric antigen receptor C#3 showed the greatest tumor cell clearance ability respectively, and the cell number of the human-derived tumor cells was relative 40.9% in the control group.
  • E/T effector cell/target cell
  • Quantitative analysis line graphs demonstrate the remarkable ability of chimeric antigen receptor C#3-modified immune natural killer cells to recognize and kill tumor cells with statistically significant differences when co-cultured with PD-L1-positive human tumor cells
  • the human-derived natural killer cells in the other control groups failed to show the ability to effectively recognize and kill tumor cells under the co-culture conditions of PD-L1-positive human-derived tumor cells.
  • Figure 12d shows the quantitative analysis results of the in vitro co-culture cytotoxicity of human natural killer cells modified with different chimeric antigen receptors and PD-L1-positive human tumor cells.
  • Natural killer cells and tumor cells are in accordance with 5: The E/T (effector/target) ratio of 1 was tested at 24 hours post-incubation (mean 0.2 in group C#3, 0.4 in group C#5, and 1.4 in control group), compared to In the human natural killer cells in the control group, the chimeric antigen receptors C#3 and C#5 modified and transformed the human-derived immune natural killer cells showed the greatest tumor cell clearance ability, respectively, and the number of human-derived tumor cells was different. 14.3% versus 28.6% in the control group.
  • Quantitative analysis line graphs demonstrate excellent recognition and killing of chimeric antigen receptor C#3, C#5 modified immune natural killer cells with statistically significant differences in co-culture with PD-L1 positive human tumor cells
  • Figure 12e shows the quantitative analysis results of the in vitro co-culture cytotoxicity effect of different chimeric antigen receptor modified human natural killer cells and PD-L1 positive human tumor cells.
  • Natural killer cells and tumor cells are in accordance with 10: The E/T (effector cell/target cell) ratio of 1 was tested at 24 hours after incubation (0.1 in the C#3 group and 0.9 in the control group), compared to the human natural killer cells in the control group, The chimeric antigen receptors C#3 and C#5 modified and transformed human-derived immune natural killer cells showed the largest tumor cell clearance ability, respectively, and the number of human-derived tumor cells was 11.1% of that in the control group.
  • Quantitative analysis line graphs demonstrate the remarkable ability of chimeric antigen receptor C#3-modified immune natural killer cells to recognize and kill tumor cells with statistically significant differences when co-cultured with PD-L1-positive human tumor cells
  • the human natural killer cells in other control groups failed to show the ability to effectively recognize and kill tumor cells under the co-culture conditions of PD-L1 positive human tumor cells.
  • Human breast cancer tumor cells MDA-MB-231 expressing the reporter gene firefly luciferase were pretreated with gamma interferon for 24 hours to increase the expression of PD - L1 on the cell surface.
  • Killer cells NK-92 were co-cultured with 1 x 10 3 tumor cells at an E/T (effector cell/target cell) ratio of 5:1 in a 48-well plate for 60 hours, and the co-culture time started at the 0th hour. Then, the luciferase activity in the cell culture system was detected at the three co-culture time points of 0 hours, 4 hours and 60 hours after incubation, and then the number of human breast cancer tumor cells was quantified and the effect of human natural killer cells on human cells was calculated.
  • FIG. 13 a illustrates the test flow and mode setting of the in vitro co-culture cytotoxicity assay of natural killer cells and PD-L1 positive human breast cancer tumor cells covered by the present application.
  • Figure 13b and c show the cytotoxicity effect of co-culture of human natural killer cells modified by different immune checkpoint PD-1 fusion-based chimeric antigen receptor artificial molecular machines and PD-L1 positive human tumor cells in vitro
  • the quantitative analysis results of natural killer cells and tumor cells were carried out according to the ratio of E/T (effector cells/target cells) of 5:1.
  • E/T effector cells/target cells
  • Natural killer cells even under long-term co-culture with human breast cancer tumor cells, the immune checkpoint PD-1 fused chimeric antigen receptor C#3 and C#5 modified and transformed human immune natural killer cells showed The largest tumor cell clearance capacity, the cell number of human tumor cells was 21.4% and 28.6% relative to the control group.
  • the line graph of quantitative analysis demonstrated that the immune natural killer cells modified with chimeric antigen receptors C#3 and C#5 had significant differences after statistical analysis when co-cultured with PD-L1-positive human tumor cells for a long time.
  • the ability to recognize and kill tumor cells, while the human-derived natural killer cells in other control groups failed to show the ability to effectively recognize and kill tumor cells in the co-culture condition of PD-L1-positive human-derived tumor cells.
  • Human skin cancer tumor cells A2058 expressing the reporter gene firefly luciferase were pretreated with gamma interferon for 24 hours to increase the expression of PD - L1 on the cell surface.
  • the human natural killer cells NK-92 and 1 x 10 3 tumor cells were respectively at 1: 1, 2.5: 1, 5: 1, 10: 1 E/T (effector cells/target cells) ratio of 48
  • the plates were co-cultured for 24 hours, and the time of co-culture was the 0th hour.
  • the luciferase activity in the cell culture system was detected at three co-culture time points of 0 hours, 4 hours and 24 hours after incubation, and then the number of human-derived skin cancer tumor cells was quantified and the effect of human-derived natural killer cells on human-derived natural killer cells was calculated. Cytotoxicity of skin cancer tumor cells. See Figure 14. Among them, the tumor cell group was only human-derived skin cancer tumor cells A2058 cells themselves, and the human-derived natural killer cells in the control group were human-derived natural killer cells without chimeric antigen receptor modification.
  • the target cell survival index represents the cell culture Relative cell numbers of human skin cancer tumor cells expressing the reporter gene firefly luciferase in the system.
  • Figure 14a illustrates the test flow and mode setting of the in vitro co-culture cytotoxicity assay of natural killer cells and PD-L1-positive human skin cancer tumor cells covered by the present application.
  • Figure 14b shows the quantitative analysis results of the cytotoxic effect of different chimeric antigen receptor-modified human natural killer cells and PD-L1 positive human tumor cells in vitro co-culture cytotoxicity, natural killer cells and tumor cells according to 1: Experiments were performed at an E/T (effector/target) ratio of 1.
  • the modified human-derived chimeric antigen receptor C#3 Immune natural killer cells showed the greatest tumor cell clearance capacity, respectively, and the cell number of human-derived tumor cells was 60.4% relative to that in the control group.
  • Quantitative analysis line graphs demonstrate the remarkable ability of chimeric antigen receptor C#3-modified immune natural killer cells to recognize and kill tumor cells with statistically significant differences when co-cultured with PD-L1-positive human tumor cells
  • the human-derived natural killer cells in the other control groups failed to show the ability to effectively recognize and kill tumor cells under the co-culture conditions of PD-L1-positive human-derived tumor cells.
  • Figure 14c shows the results of quantitative analysis of the cytotoxic effect of in vitro co-culture of human natural killer cells modified with different chimeric antigen receptors and PD-L1 positive human tumor cells.
  • Natural killer cells and tumor cells are in accordance with 2.5: The E/T (effector/target) ratio of 1 was tested at 24 hours post-incubation (average 1.5 in C#3 group, 3.8 in control group), compared to the human-derived natural killer in the control group Cells, human-derived immune natural killer cells modified with chimeric antigen receptor C#3 showed the greatest tumor cell clearance ability respectively, and the cell number of human-derived tumor cells was 39.5% relative to the control group.
  • Quantitative analysis line graphs demonstrate the remarkable ability of chimeric antigen receptor C#3-modified immune natural killer cells to recognize and kill tumor cells with statistically significant differences when co-cultured with PD-L1-positive human tumor cells
  • the human-derived natural killer cells in the other control groups failed to show the ability to effectively recognize and kill tumor cells under the co-culture conditions of PD-L1-positive human-derived tumor cells.
  • Figure 14(d) shows the quantitative analysis results of the cytotoxicity effect of in vitro co-culture of human natural killer cells modified with different chimeric antigen receptors and PD-L1 positive human tumor cells.
  • Natural killer cells and tumor cells Experiments were performed at an E/T (effector/target) ratio of 5:1 at 24 hours post-incubation (mean 0.5 in group C#3, 0.3 in group C#5, and 3.3 in control group) , Compared with the human natural killer cells in the control group, the immune checkpoint PD-1-fused chimeric antigen receptors C#3 and C#5 modified and transformed the human immune natural killer cells showed the largest number of tumor cells, respectively Clearing ability, the cell numbers of human tumor cells were 15.0% and 9.1% relative to the control group, respectively.
  • Quantitative analysis line graphs demonstrate superior recognition and killing of chimeric antigen receptor C#3, C#5 modified immune natural killer cells with statistically significant differences in co-culture with PD-L1-positive human tumor cells
  • Figure 14e shows the quantitative analysis results of the in vitro co-culture cytotoxicity of human natural killer cells modified with different chimeric antigen receptors and PD-L1-positive human tumor cells.
  • Natural killer cells and tumor cells are in accordance with 10: The E/T (effector cell/target cell) ratio of 1 was tested at 24 hours after incubation (0.1 in the C#3 group and 2.7 in the control group), compared to the human natural killer cells in the control group, The human-derived immune natural killer cells after chimeric antigen receptor C#3 modification and transformation respectively showed the greatest tumor cell clearance ability, and the cell number of human-derived tumor cells was 3.7% compared to the control group.
  • Quantitative analysis line graphs demonstrate superior recognition and killing of chimeric antigen receptor C#3, C#5 modified immune natural killer cells with statistically significant differences in co-culture with PD-L1-positive human tumor cells
  • Human prostate cancer tumor cells PC-3 expressing the reporter gene firefly luciferase were pretreated with interferon gamma for 24 hours to increase the expression of PD-L1 on the cell surface, and 5 ⁇ 10 3 modified human natural killer cells NK -92 were co-cultured with 1 ⁇ 10 3 tumor cells in a 48-well plate at an E/T (effector cell/target cell) ratio of 5:1 for 24 hours, and the co-culture time started at the 0th hour.
  • E/T effector cell/target cell
  • the luciferase activity in the cell culture system was detected at the three co-culture time points of 0 hours, 4 hours and 24 hours after incubation, and then the number of human prostate cancer tumor cells was quantified and the effect of human natural killer cells on human Cytotoxicity of prostate cancer tumor cells.
  • the tumor cell group was only human-derived prostate cancer tumor cells PC-3 cells themselves, and the human-derived natural killer cells in the control group were human-derived natural killer cells without chimeric antigen receptor modification.
  • the target cell survival index represents Relative cell numbers of human prostate cancer tumor cells expressing the reporter gene firefly luciferase in cell culture systems.
  • Figure 15a illustrates the test flow and mode setting of the in vitro co-culture cytotoxicity assay of natural killer cells and PD-L1 positive human prostate cancer tumor cells covered by the present application.
  • Figures 15b and c show the results of quantitative analysis of the cytotoxicity effect of in vitro co-culture of human natural killer cells modified with different chimeric antigen receptors and PD-L1 positive human tumor cells. Experiments were performed with an E/T (effector/target) ratio of 5:1.
  • chimeric antigen receptors C#3 and C#5 modified and transformed human-derived immune natural killer cells showed the greatest tumor cell clearance ability, respectively, and the number of human-derived tumor cells was relative to that in the control group. 8.6% versus 25.7%.
  • Quantitative analysis line graphs demonstrate superior recognition and killing of chimeric antigen receptor C#3 and C#5 modified immune natural killer cells with statistically significant differences in co-culture with PD-L1-positive human tumor cells
  • Human-derived brain cancer tumor cells U87-MG expressing the reporter gene firefly luciferase were pretreated with ⁇ -interferon for 24 hours to increase the expression of PD-L1 on the cell surface, and 5 ⁇ 10 3 modified human natural killer cells NK -92 were co-cultured with 1 ⁇ 10 3 tumor cells in a 48-well plate at an E/T (effector cell/target cell) ratio of 5:1 for 24 hours, and the co-culture time started at the 0th hour.
  • E/T effector cell/target cell
  • the luciferase activity in the cell culture system was detected at three co-culture time points of 0 hours, 4 hours and 24 hours after incubation, and then the number of human-derived brain cancer tumor cells was quantified and the effect of human-derived natural killer cells on human-derived natural killer cells was calculated. Cytotoxicity of brain cancer tumor cells. See Figure 16. Among them, the tumor cell group was only human-derived brain cancer tumor cells U87-MG cells themselves, and the human-derived natural killer cells in the control group were human-derived natural killer cells without chimeric antigen receptor modification.
  • the target cell survival index represents Relative cell numbers of human brain cancer tumor cells expressing the reporter gene firefly luciferase in cell culture systems.
  • FIG. 16a illustrates the test flow and mode setting of the in vitro co-culture cytotoxicity assay of natural killer cells and PD-L1 positive human brain cancer tumor cells covered by the present application.
  • Figure 16b and c show the results of quantitative analysis of the cytotoxic effect of co-culture of human natural killer cells modified with different chimeric antigen receptors and PD-L1 positive human tumor cells in vitro. Experiments were performed with an E/T (effector/target) ratio of 5:1. At 24 hours post-incubation (mean 0.2 in C#3, 0.4 in C#5, 1.6 in the control group, and 3.7 in the tumor cell-only group), compared to the human in the control group.
  • Source natural killer cells chimeric antigen receptors C#3 and C#5 modified and transformed human-derived immune natural killer cells showed the greatest tumor cell clearance ability, respectively, and the number of human-derived tumor cells was relative to that in the control group. 12.5% versus 25.0%.
  • Quantitative analysis line graphs demonstrate superior recognition and killing of chimeric antigen receptor C#3 and C#5 modified immune natural killer cells with statistically significant differences in co-culture with PD-L1-positive human tumor cells The ability of human-derived natural killer cells in other control groups to face PD-L1-positive human-derived tumor cells co-culture conditions failed to show effective ability to recognize and kill tumor cells.
  • Human hepatoma tumor cells HA-22T expressing the reporter gene firefly luciferase were pretreated with interferon gamma for 24 hours to increase the expression of PD-L1 on the cell surface, and 5x 10 3 modified human natural killer cells NK -92 were co-cultured with 1 ⁇ 10 3 tumor cells in a 48-well plate at an E/T (effector cell/target cell) ratio of 5:1 for 24 hours, and the co-culture time started at the 0th hour.
  • the luciferase activity in the cell culture system was detected at three co-culture time points of 0 hours, 4 hours and 24 hours after incubation, and then the number of human-derived liver cancer tumor cells was quantified and the effect of human-derived natural killer cells on human-derived liver cancer was calculated. Cytotoxicity of tumor cells. See Figure 17. Among them, the tumor cell group was only human-derived liver cancer tumor cells HA22T cells themselves, and the human-derived natural killer cells in the control group were human-derived natural killer cells without chimeric antigen receptor modification.
  • the target cell survival index represents the cell culture system Relative cell numbers in human hepatoma tumor cells expressing the reporter gene firefly luciferase.
  • Figure 17a illustrates the test flow and mode setting of the in vitro co-culture cytotoxicity experiment of natural killer cells and PD-L1 positive human hepatoma tumor cells covered by the present application.
  • Figure 17b and c show the results of quantitative analysis of the cytotoxic effect of co-culture of human natural killer cells modified with different chimeric antigen receptors and PD-L1 positive human tumor cells in vitro. Experiments were performed with an E/T (effector/target) ratio of 5:1. At 24 hours post-incubation (mean 0.2 in C#3, 0.2 in C#5, 0.9 in the control group, and 2.7 in the tumor cell-only group), compared to humans in the control group.
  • Source natural killer cells, chimeric antigen receptors C#3 and C#5 modified and transformed human-derived immune natural killer cells showed the greatest tumor cell clearance ability, respectively, and the number of human-derived tumor cells was relative to that in the control group. 22.2% vs. 22.2%.
  • Quantitative analysis line graphs demonstrate superior recognition and killing of chimeric antigen receptor C#3 and C#5 modified immune natural killer cells with statistically significant differences in co-culture with PD-L1-positive human tumor cells The ability of human-derived natural killer cells in other control groups to face PD-L1-positive human-derived tumor cells co-culture conditions failed to show effective ability to recognize and kill tumor cells.
  • Human renal cancer tumor cells 786-O expressing the reporter gene firefly luciferase were pretreated with interferon gamma for 24 hours to increase the expression of PD-L1 on the cell surface, and 5 ⁇ 10 3 modified human natural killer cells NK-92 was co-cultured with 1 ⁇ 10 3 tumor cells in a 48-well plate at an E/T (effector cell/target cell) ratio of 5:1 for 24 hours, and the co-culture time started at the 0th hour. Then, the luciferase activity in the cell culture system was detected at three co-cultivation time points at 0 hours, 4 hours and 24 hours after incubation, and then the number of human renal cancer tumor cells was quantified and the effect of human natural killer cells on human cells was calculated.
  • E/T effector cell/target cell
  • FIG. 18 Cytotoxicity of renal carcinoma tumor cells. See Figure 18.
  • the tumor cell group was only human renal cancer tumor cells 786-O cells themselves, and the human natural killer cells in the control group were human natural killer cells without chimeric antigen receptor modification.
  • the target cell survival index represents Relative cell numbers of human renal carcinoma tumor cells expressing the reporter gene firefly luciferase in cell culture systems.
  • Figure 18a illustrates the test flow and mode setting of the in vitro co-culture cytotoxicity assay of natural killer cells and PD-L1-positive human renal cancer tumor cells covered by the present application.
  • Figure 18b and c show the results of quantitative analysis of the cytotoxic effect of co-culture of human natural killer cells modified with different chimeric antigen receptors and PD-L1 positive human tumor cells in vitro.
  • E/T effector/target ratio of 5:1.
  • Source natural killer cells, chimeric antigen receptors C#3 and C#5 modified and transformed human-derived immune natural killer cells showed the greatest tumor cell clearance ability, respectively, and the cell number of human-derived tumor cells was about 10% compared to the control group 14.3% vs. 28.6%.
  • Quantitative analysis line graphs demonstrate superior recognition and killing of chimeric antigen receptor C#3 and C#5 modified immune natural killer cells with statistically significant differences in co-culture with PD-L1-positive human tumor cells
  • Human-derived lung cancer tumor cells H441 expressing the reporter gene firefly luciferase were pretreated with interferon-gamma for 24 hours to increase the expression of PD-L1 on the cell surface, and 5x 10 3 modified human natural killer cells NK-92
  • the cells were co-cultured with 1 x 10 3 tumor cells at an E/T (effector cell/target cell) ratio of 5:1 in a 48-well plate for 24 hours, and the co-culture time started at the 0th hour.
  • the luciferase activity in the cell culture system was detected at three co-culture time points of 0 hours, 4 hours and 24 hours after incubation, and then the number of human-derived lung cancer tumor cells was quantified and the effect of human-derived natural killer cells on human-derived lung cancer was calculated. Cytotoxicity of tumor cells. See Figure 19. Among them, the tumor cell group was only human-derived lung cancer tumor cells H441 cells themselves, the human-derived natural killer cells in the control group were human-derived natural killer cells without chimeric antigen receptor modification, and the target cell survival index represented the cell culture system. Relative cell numbers in human lung cancer tumor cells expressing the reporter gene firefly luciferase.
  • Figure 19a illustrates the test flow and mode setting of the in vitro co-culture cytotoxicity experiment of natural killer cells and PD-L1-positive human lung cancer tumor cells covered by the present application.
  • Figure 19b and c show the results of quantitative analysis of the cytotoxicity effect of in vitro co-culture of human natural killer cells modified with different chimeric antigen receptors and PD-L1 positive human tumor cells. Experiments were performed with an E/T (effector/target) ratio of 5:1. At 24 hours post-incubation (mean 0.1 in C#3, 0.7 in C#5, 1.5 in the control group, and 1.8 in the tumor cell-only group), compared to humans in the control group.
  • Source natural killer cells chimeric antigen receptors C#3 and C#5 modified and transformed human-derived immune natural killer cells showed the greatest tumor cell clearance ability, respectively, and the number of human-derived tumor cells was relative to that in the control group. 6.7% vs 46.7%.
  • Quantitative analysis line graphs demonstrate superior recognition and killing of chimeric antigen receptor C#3 and C#5 modified immune natural killer cells with statistically significant differences in co-culture with PD-L1-positive human tumor cells The ability of human-derived natural killer cells in other control groups to face PD-L1-positive human-derived tumor cells co-culture conditions failed to show effective ability to recognize and kill tumor cells.
  • Human ovarian cancer tumor cells ES-2 expressing the reporter gene firefly luciferase were pretreated with gamma interferon for 24 hours to increase the expression of PD-L1 on the cell surface, and 5x 10 3 were modified into human natural killer cells.
  • NK-92 was co-cultured with 1 ⁇ 10 3 tumor cells in a 48-well plate at an E/T (effector cell/target cell) ratio of 5:1 for 24 hours, and the co-culture time started at the 0th hour.
  • the luciferase activity in the cell culture system was detected at three co-culture time points of 0 hours, 4 hours and 24 hours after incubation, and then the number of human ovarian cancer tumor cells was quantified and the effect of human natural killer cells on human cells was calculated. Cytotoxicity of lung cancer tumor cells. See Figure 20. Among them, the tumor cell group was only human ovarian cancer tumor cells ES-2 cells themselves, and the human natural killer cells in the control group were human natural killer cells without chimeric antigen receptor modification.
  • the target cell survival index represents Relative cell numbers of human lung cancer tumor cells expressing the reporter gene firefly luciferase in cell culture systems.
  • Figure 20a illustrates the test flow and mode setting of the in vitro co-culture cytotoxicity experiment of natural killer cells and PD-L1 positive human lung cancer tumor cells covered by the present application.
  • Figure 19b and c show the results of quantitative analysis of the cytotoxicity effect of in vitro co-culture of human natural killer cells modified with different chimeric antigen receptors and PD-L1 positive human tumor cells. Experiments were performed with an E/T (effector/target) ratio of 5:1. At 24 hours post-incubation (mean 0.1 in C#3, 0.1 in C#5, 1.3 in the control group, and 2.9 in the tumor cell-only group), compared to humans in the control group.
  • Source natural killer cells chimeric antigen receptors C#3 and C#5 modified and transformed human-derived immune natural killer cells showed the greatest tumor cell clearance ability, respectively, and the number of human-derived tumor cells was relative to that in the control group. 7.7% vs. 7.7%.
  • Quantitative analysis line graphs demonstrate superior recognition and killing of chimeric antigen receptor C#3 and C#5 modified immune natural killer cells with statistically significant differences in co-culture with PD-L1-positive human tumor cells The ability of human-derived natural killer cells in other control groups to face PD-L1-positive human-derived tumor cells co-culture conditions failed to show effective ability to recognize and kill tumor cells.
  • Human breast cancer tumor cells MDA-MB-231 were pretreated with gamma interferon for 24 hours to increase the expression of PD-L1 on the cell surface, and 2.5x 10 5 modified human natural killer cells NK-92 were treated with 1x 10 5 tumor cells were co-cultured in 6-well plates according to the ratio of E/T (effector cells/target cells) of 2.5:1, which was recorded as the 0th hour, and then the transformed human cells were isolated at the time point of 48 hours after incubation.
  • E/T effector cells/target cells
  • Natural killer cells NK-92 and the expression of gene transcription levels related to anti-tumor efficacy were detected, and at the same time, the expression levels of gene transcription levels related to natural killer after the transformation without co-culture with tumor cells were detected, including quantitative real-time For polymerase chain reaction (qPCR) analysis and Gene Ontology (GO, Gene Ontology) enrichment analysis, see Figures 21 to 33 .
  • qPCR quantitative real-time For polymerase chain reaction
  • GO Gene Ontology
  • the qPCR primers are: forward primer (5'-3'CGACAGTACCATTGAGTTGTGCG, as shown in SEQ ID NO:67) and reverse primer (5'-3'TTCGTCCATAGGAGACAATGCCC, as shown in SEQ ID NO:68) to detect the target Gene GZMB; forward primer (5'-3' ACTCACAGGCAGCCAACTTTGC, as shown in SEQ ID NO: 69) and reverse primer (5'-3' CCTTGAAGTCAGGGTGCAGCG, as shown in SEQ ID NO: 70) to detect the target gene PRF1; positive To the primer (5'-3' CTCTTCTGCCTGCTGCACTTTG, as shown in SEQ ID NO: 71) and the reverse primer (5'-3' ATGGGCTACAGGCTTGTCACTC, as shown in SEQ ID NO: 72) to detect the target gene TNFA; forward primer (5 '-3'GAGTGTGGAGACCATCAAGGAAG, as shown in SEQ ID NO:73) and reverse primer (5'-3'TGCTTTGCGT
  • Figure 21a Quantitative analysis results showing the transcription of effector function-related gene GZMB in different immune checkpoint PD-1 fusion-based chimeric antigen receptor-engineered human natural killer cells without co-culture with tumor cells Level of quantitative analysis results.
  • the immune checkpoint PD-1-fused chimeric antigen receptors C#3, C#5, and C#2 modified and transformed the human natural killer cells showed similar Even lower GZMB gene transcription levels (mean 0.622 in group C#3, 0.813 in group C#5, 1.000 in control group, and 1.381 in group C#2; housekeeping gene GAPDH gene as qPCR of endogenous reference genes for normalization and analysis), C#3 and C#5 were 62.2% and 81.3%, respectively, relative to the control group.
  • the quantitative analysis results of Figure 21b show the effector function-related gene GZMB of different immune checkpoint PD-1 fusion-based chimeric antigen receptor-engineered human natural killer cells at the 48-hour co-culture time point with tumor cells. Quantitative analysis of transcript levels. Among them, compared with the human-derived natural killer cells in the control group, the human-derived natural killer cells modified by the immune checkpoint PD-1 fusion chimeric antigen receptor C#3 and C#5 showed higher GZMB gene respectively.
  • the remarkable difference in GZMB gene transcription and expression level after statistical analysis means that the protein encoding Granzyme B (granzyme B is secreted by natural killer cells and can induce programmed cell death of target cells) is the natural killer cell that plays an anti-tumor immune killing effect.
  • the gene expression of important effector was up-regulated, and this protein was directly related to the anti-tumor efficacy of natural killer cells.
  • the human natural killer cells in the experimental group C#2 and the control group failed to show a significant increase in the expression level of GZMB gene transcription under the co-culture conditions of PD-L1 positive human tumor cells, and the experimental group C The transcriptional expression level of GZMB gene in #2 was lower than that in the control group.
  • Figure 22a Quantitative analysis results showing the transcription of effector function-related gene PRF1 in different immune checkpoint PD-1 fusion-based chimeric antigen receptor-engineered human natural killer cells without co-culture with tumor cells Level of quantitative analysis results.
  • the immune checkpoint PD-1-fused chimeric antigen receptors C#3, C#5, and C#2 modified and transformed the human natural killer cells showed similar Even lower PRF1 gene transcription levels (mean 1.119 in group C#3, 0.645 in group C#5, 1.000 in control group, and 1.450 in group C#2; housekeeping gene GAPDH gene as qPCR of endogenous reference genes were normalized and analyzed), C#3 and C#5 were 111.9% and 64.5%, respectively, relative to the control group.
  • Figure 22b show the effector function-related gene PRF1 of different immune checkpoint PD-1 fusion-based chimeric antigen receptor-engineered human natural killer cells at the 48-hour co-culture time point with tumor cells. Quantitative analysis of transcript levels.
  • the human-derived natural killer cells modified with the immune checkpoint PD-1 fusion chimeric antigen receptor C#3 and C#5 showed higher PRF1 gene respectively Transcript level (average of group C#3 was 1.546, average value of group C#5 was 2.702, average value of control group was 1.000, and average value of group C#2 was 0.490; the housekeeping gene GAPDH gene was used as the endogenous reference gene for qPCR. normalization and analysis), C#3 and C#5 were 1.546-fold and 2.702-fold relative to the control group, respectively.
  • Figure 23a Quantitative analysis results showing the transcription of effector function-related gene TNFA in different immune checkpoint PD-1 fusion-based chimeric antigen receptor-engineered human natural killer cells without co-culture with tumor cells Level of quantitative analysis results.
  • the immune checkpoint PD-1-fused chimeric antigen receptors C#3, C#5, and C#2 modified and transformed the human natural killer cells showed similar Or slightly higher TNFA gene transcription level (the average of C#3 group was 2.958, the average of C#5 group was 0.476, the average of control group was 1.000, and the average of C#2 group was 1.082; the housekeeping gene GAPDH gene was used as The endogenous reference gene for qPCR was normalized and analyzed), C#3 and C#5 were 2.958-fold and 47.6% relative to the control group, respectively.
  • Quantitative analysis results of Figure 23b show the effector function-related gene TNFA of different immune checkpoint PD-1 fusion-based chimeric antigen receptor-engineered human natural killer cells at the 48-hour co-culture time point with tumor cells Quantitative analysis of transcript levels.
  • the immune checkpoint PD-1 fusion chimeric antigen receptor C#3 and C#5 modified human natural killer cells showed higher TNFA gene respectively.
  • Transcript level (average of group C#3 was 3.536, average value of group C#5 was 5.240, average value of control group was 1.000, and average value of group C#2 was 0.630; the housekeeping gene GAPDH gene was used as the endogenous reference gene for qPCR.
  • C#3 and C#5 were 3.536-fold and 5.240-fold relative to the control group, respectively.
  • the above quantitative results demonstrate that human natural killer cells modified with immune checkpoint PD-1 fusion-based chimeric antigen receptor C#3 and C#5 versions have the ability to co-culture with PD-L1-positive human tumor cells.
  • the remarkable difference in TNFA gene transcription expression level after statistical analysis means that the protein encoding TNF- ⁇ (tumor necrosis factor- ⁇ is secreted by natural killer cells and can induce apoptosis to prevent tumorigenesis, which is the natural killer cell to exert anti-tumor immunity. up-regulated gene expression of an important effector of cytotoxicity), which is directly related to the antitumor efficacy of natural killer cells.
  • Figure 24a Quantitative analysis results showing the transcription of effector function-related genes IFNG in different immune checkpoint PD-1 fusion-based chimeric antigen receptor-engineered human natural killer cells without co-culture with tumor cells Level of quantitative analysis results.
  • the immune checkpoint PD-1-fused chimeric antigen receptors C#3, C#5, and C#2 modified and transformed the human natural killer cells showed similar Even lower or slightly higher IFNG gene transcription levels (mean 1.198 in group C#3, 0.339 in group C#5, 1.000 in control group, and 2.845 in group C#2;
  • the GAPDH gene was normalized and analyzed as the endogenous reference gene for qPCR), C#3 and C#5 were 119.8% and 33.9% relative to the control group, respectively.
  • the quantitative analysis results of Figure 24b show the effector function-related gene IFNG of different immune checkpoint PD-1 fusion-based chimeric antigen receptor-engineered human natural killer cells at the 48-hour co-culture time point with tumor cells. Quantitative analysis of transcript levels. Among them, compared with the human natural killer cells in the control group, the human natural killer cells modified with the immune checkpoint PD-1 fusion chimeric antigen receptor C#3 and C#5 showed higher IFNG genes respectively.
  • the remarkable difference in the transcriptional expression level of the IFNG gene after statistical analysis means that the encoded IFN- ⁇ protein ( ⁇ -interferon is secreted by natural killer cells and recruits and activates a variety of immune effector cells, which are the natural killer cells that play an anti-tumor immune killing effect.
  • the gene expression of important effector was up-regulated, and this protein was directly related to the anti-tumor efficacy of natural killer cells.
  • the human natural killer cells in the other experimental groups C#2 and the control group failed to show a significant increase in the expression level of IFNG gene transcription under the co-culture conditions of PD-L1 positive human tumor cells, and the experimental group The transcriptional expression level of IFNG gene in C#2 was lower than that in the control group.
  • Figure 25a Quantitative analysis results showing the transcription of effector function-related gene NCAM1 in different immune checkpoint PD-1 fusion-based chimeric antigen receptor-engineered human natural killer cells without co-culture with tumor cells Level of quantitative analysis results.
  • the immune checkpoint PD-1-fused chimeric antigen receptors C#3, C#5, and C#2 modified and transformed the human natural killer cells showed similar Even lower or slightly higher NCAM1 gene transcription levels (mean 1.016 in group C#3, 0.397 in group C#5, 1.000 in control group, and 1.418 in group C#2;
  • the GAPDH gene was normalized and analyzed as the endogenous reference gene for qPCR), C#3 and C#5 were 101.6% and 39.7%, respectively, relative to the control group.
  • Figure 25(b) Quantitative analysis results showing the effector function of different immune checkpoint PD-1 fusion-based chimeric antigen receptor engineered human natural killer cells at the 48-hour co-culture time point with tumor cells Quantitative analysis of the transcription level of the related gene NCAM1. Among them, compared with the human natural killer cells in the control group, the human natural killer cells modified by the immune checkpoint PD-1 fusion chimeric antigen receptor C#3 and C#5 showed higher NCAM1 gene respectively.
  • the excellent NCAM1 gene transcriptional expression level with statistically significant differences in the co-culture of the source tumor cells means that the encoding CD56 protein (neuronal adhesion molecule CD56 is a receptor protein expressed in the activated state of natural killer cells, promotes natural killer cells.
  • CD56 protein neurovascular adhesion molecule CD56 is a receptor protein expressed in the activated state of natural killer cells, promotes natural killer cells.
  • the activation and anti-tumor effect ability of natural killer cells play an important role in the anti-tumor immune killing of natural killer cells), and this protein is directly related to the anti-tumor efficacy of natural killer cells.
  • C#2 and human natural killer cells in the control group failed to show a significant increase in the expression level of NCAM1 gene transcription under the co-culture conditions of PD-L1-positive human tumor cells, and the experimental group The transcriptional expression level of NCAM1 gene in C#2 was lower than that in the control group.
  • Figure 26a Quantitative analysis results showing the transcription of effector function-related gene KLRK1 in different immune checkpoint PD-1 fusion-based chimeric antigen receptor-engineered human natural killer cells without co-culture with tumor cells Level of quantitative analysis results.
  • the immune checkpoint PD-1-fused chimeric antigen receptors C#3, C#5, and C#2 modified and transformed the human natural killer cells showed similar Even lower or slightly higher KLRK1 gene transcription levels (mean 1.426 in C#3 group, 0.275 in C#5 group, 1.000 in control group, and 1.368 in C#2 group;
  • the GAPDH gene was normalized and analyzed as the endogenous reference gene for qPCR), C#3 and C#5 were 1.426-fold and 27.5% higher than those in the control group, respectively.
  • Figure 26(b) Quantitative analysis results showing the effector function of different immune checkpoint PD-1 fusion-based chimeric antigen receptor engineered human natural killer cells at the 48-hour co-culture time point with tumor cells Quantitative analysis of the transcription level of the related gene KLRK1. Among them, compared with the human natural killer cells in the control group, the immune checkpoint PD-1 fusion chimeric antigen receptor C#3 and C#5 modified human natural killer cells showed higher KLRK1 gene respectively.
  • the remarkable KLRK1 gene transcriptional expression level with significant difference after statistical analysis means that the NKG2D protein (NKG2D protein is a strong activating receptor on the surface of natural killer cells) plays an important role in innate immunity and participates in the natural killer cells' tumor-related response. Clear and kill, and play a key role in natural killer cells to play a key role in anti-tumor immune killing) gene expression is up-regulated, and this protein is directly related to the anti-tumor efficacy of natural killer cells.
  • Figure 27a Quantitative analysis results showing the transcription of effector function-related gene NCR1 in different immune checkpoint PD-1 fusion-based chimeric antigen receptor-engineered human natural killer cells without co-culture with tumor cells Level of quantitative analysis results.
  • the immune checkpoint PD-1-fused chimeric antigen receptors C#3, C#5, and C#2 modified and transformed the human natural killer cells showed similar Even lower NCR1 gene transcription levels (mean 0.988 in group C#3, 0.448 in group C#5, 1.000 in control group, and 1.410 in group C#2; housekeeping gene GAPDH gene as qPCR normalization and analysis of endogenous reference genes), C#3 and C#5 were 98.8% and 44.8% relative to the control group, respectively.
  • Figure 27(b) Quantitative analysis results showing the effector functions of different immune checkpoint PD-1 fusion-based chimeric antigen receptor engineered human natural killer cells at the 48-hour co-culture time point with tumor cells Quantitative analysis of the transcript level of the related gene NCR1.
  • the human natural killer cells modified with the immune checkpoint PD-1 fusion chimeric antigen receptor C#3 and C#5 showed higher NCR1 gene respectively.
  • Transcript levels average of group C#3 was 1.809, average value of group C#5 was 4.114, average value of control group was 1.000, and average value of group C#2 was 0.686; the housekeeping gene GAPDH gene was used as the endogenous reference gene for qPCR.
  • C#3 and C#5 were 1.809-fold and 4.114-fold relative to the control group, respectively.
  • the above quantitative results demonstrate that human natural killer cells modified with immune checkpoint PD-1 fusion-based chimeric antigen receptor C#3 and C#5 versions have the ability to co-culture with PD-L1-positive human tumor cells.
  • the remarkable difference in the expression level of NCR1 gene transcription after statistical analysis means that it encodes the NKp46 protein (NKp46 is an activating receptor on the surface of natural killer cells, which is involved in the scavenging and killing of target cells by natural killer cells, and plays an anti-tumor effect on natural killer cells. This protein is directly related to the antitumor efficacy of natural killer cells.
  • C#2 and human natural killer cells in the control group failed to show a significant increase in the expression level of NCR1 gene transcription under the co-culture conditions of PD-L1-positive human tumor cells, and the experimental group The transcriptional expression level of NCR1 gene in C#2 was lower than that in the control group.
  • Figure 28 Quantitative analysis results showing the effector function-related GO of different immune checkpoint PD-1 fusion-based chimeric antigen receptor engineered human natural killer cells at the 48-hour co-culture time point with tumor cells Enrichment analysis results.
  • the immune checkpoint PD-1 fused chimeric antigen receptors C#3 and C#5 were modified and transformed.
  • Human natural killer cells showed significantly higher gene enrichment of GO: 0002228 (natural killer cell mediated immunity, that is, natural killer cell mediated immunity), and had stronger natural killer cell mediated immunity and anti-tumor functional effects.
  • Figure 29 Quantitative analysis results showing the effector function-related GO of different immune checkpoint PD-1 fusion-based chimeric antigen receptor engineered human natural killer cells at the 48-hour co-culture time point with tumor cells Enrichment analysis results.
  • the immune checkpoint PD-1 fused chimeric antigen receptors C#3 and C#5 were modified and transformed.
  • Human natural killer cells showed significantly higher gene enrichment of GO: 0032649 (regulation of interferon-gamma production, that is, the regulation of interferon-gamma production).
  • the chimeric antigen was further proved.
  • the modified human natural killer cells with receptors C#3 and C#5 have stronger interferon- ⁇ production and secretion and anti-tumor functions.
  • Figure 30 Quantitative analysis results showing the effector function-related GO of different immune checkpoint PD-1 fusion-based chimeric antigen receptor engineered human natural killer cells at the 48-hour co-culture time point with tumor cells Enrichment analysis results.
  • the immune checkpoint PD-1 fused chimeric antigen receptors C#3 and C#5 were modified and transformed.
  • Human natural killer cells show significantly higher gene enrichment of GO: 0070098 (chemokine-mediated signaling pathway, namely chemokine-mediated signaling pathway), and have stronger cell migration and anti-tumor functional effects.
  • Figure 31 Quantitative analysis results showing the effector function-related GO of different immune checkpoint PD-1 fusion-based chimeric antigen receptor engineered human natural killer cells at the 48-hour co-culture time point with tumor cells Enrichment analysis results.
  • the immune checkpoint PD-1 fused chimeric antigen receptors C#3 and C#5 were modified and transformed.
  • Human natural killer cells showed significantly higher gene enrichment of GO:0002449 (lymphocyte mediated immunity, that is, lymphocyte-mediated immunity), and had stronger lymphocyte-mediated immunity and anti-tumor functional effects.
  • Figure 32 Quantitative analysis results showing the effector function-related GO of different immune checkpoint PD-1 fusion-based chimeric antigen receptor engineered human natural killer cells at the 48-hour co-culture time point with tumor cells Enrichment analysis results.
  • the immune checkpoint PD-1 fused chimeric antigen receptors C#3 and C#5 were modified and transformed.
  • Human natural killer cells showed significantly higher gene enrichment of GO: 0051251 (positive regulation of lymphocyte activation, that is, positive regulation of lymphocyte activation), and had stronger lymphocyte activation and anti-tumor functional effects.
  • Figure 33 Quantitative analysis results showing the effector function-related GO of different immune checkpoint PD-1 fusion-based chimeric antigen receptor-engineered human natural killer cells at the 48-hour co-culture time point with tumor cells Enrichment analysis results.
  • the immune checkpoint PD-1 fused chimeric antigen receptors C#3 and C#5 were modified and transformed.
  • Human natural killer cells show significantly higher gene enrichment of GO: 0001906 (cell killing, cell killing), respectively, and have stronger cell killing and anti-tumor functions.
  • the natural killer cells engineered by the chimeric antigen receptor fused to the immune checkpoint PD-1 exhibited excellent anti-tumor effects as shown in Figure 8. Killing ability, especially against PD-L1 positive human tumor cells.
  • C#3 and C#5 which performed particularly well, were the Truncated PD-1-Sub1-LL1-ZAP70 version and the Truncated PD-1-Sub5-LL1-SYK version, respectively, and demonstrated multiple chimeric antigen receptors Necessity and importance of domains for fully functioning chimeric antigen receptors.
  • immune checkpoint blockade and cell therapy are the direction of major breakthroughs in the field of tumor immunity recently.
  • this application combines various methods such as tumor immunology, synthetic biology, molecular engineering and cell engineering to develop a new generation of PD-1 based on immune checkpoints Signaling pathways for solid tumor cell therapy.
  • the cell therapy uses an artificial molecular machine based on the immune checkpoint PD-1, which encodes a chimeric antigen receptor that regulates the function of natural killer cells.
  • tumor cells expressing the immune checkpoint inhibitory signal PD-1 molecular ligand PD-L1 pass When tumor cells expressing the immune checkpoint inhibitory signal PD-1 molecular ligand PD-L1 pass When the PD-1/PD-L1 immune checkpoint signaling pathway tries to inhibit the function of natural killer cells with the same brake blocking mechanism of natural killer cells, the new generation of PD-1-based artificial molecular machines re-encodes the natural killer cells. Instead of being inhibited by PD-L1-positive tumor cells, the cells are further activated to generate a specific immune response against the corresponding tumor cells, thereby recognizing and killing the corresponding tumor cells.
  • a variety of cytotoxicity and anti-tumor functional experiments in this application prove that natural killer cells modified by chimeric antigen receptors can better display activation in the face of immunosuppressive signaling molecule ligand PD-L1 inhibition
  • the ability and excellent effect of killing and clearing a variety of PD-L1 positive solid tumors including breast cancer, brain cancer, kidney cancer, skin cancer, lung cancer, ovarian cancer, prostate cancer, liver cancer, etc. Therefore, after the modification of the chimeric antigen receptor molecular machine, the natural killer cells successfully overcome the immunosuppression in the solid tumor microenvironment, that is, solve the key problems of immunosuppression and immune escape in the immunotherapy of solid tumors. Open new avenues for solid tumor treatment and provide innovative and precise therapeutic approaches for human cancer treatment.

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Abstract

提供了一种嵌合抗原受体改造的NK细胞，该嵌合抗原受体包括：胞外靶标分子结合结构域、跨膜区结构域和胞内信号传导结构域；所述跨膜区结构域将所述胞外靶标分子结合结构域和所述胞内信号传导结构域连接，并将二者固定在所述NK细胞的细胞膜上；所述胞内信号传导结构域包括胞内激活信号传导结构域和/或胞内检测信号传导结构域。所述嵌合抗原受体改造的NK细胞兼具免疫检查点抑制剂与CAR改造的NK细胞疗法两者的优势，为改善实体肿瘤治疗提供解决方案。

Description

一种嵌合抗原受体改造的NK细胞及其制备方法与应用 技术领域
本申请涉及一种嵌合抗原受体改造的NK细胞及其制备方法与应用，属于生物医药领域。
背景技术
尽管我们每年都通过预防癌症的研究取得非凡的进展，癌症依旧是一个巨大的公共卫生挑战。癌症的研究促进了以预防、检测、诊断、治疗甚至治愈癌症等多种疾病的新方法的出现。这些进步正在降低全球癌症发病率和死亡率，同时增加了接受癌症诊断后寿命更长、质量更高的儿童和成人的数量。例如，从1991年到2016年，按年龄调整的美国总体癌症死亡率下降了27％，这意味着减少了260多万癌症死亡人数。癌症免疫治疗的数量近年来在迅速增加，其工作原理是释放患者免疫系统的力量来对抗癌症，一定程度上类似免疫系统对抗引起流感的病毒或对抗引起链球菌性咽喉炎的细菌等病原体的方式。癌症免疫疗法，尤其是免疫检查点抑制剂与细胞疗法，是近年来最令人兴奋的也是进入临床试验极多的癌症治疗新方法。
癌症免疫治疗的一种是免疫检查点抑制剂，其通过在免疫细胞表面阻断一类名为免疫检查点的蛋白来工作，从而起到释放抑制免疫细胞功能的“刹车”的作用，例如针对免疫检查点PD-1及其配体蛋白PD-Ls的抑制剂，已有多款靶向PD-1/PD-Ls的免疫检查点抑制剂获美国FDA批准用于治疗不同癌症疾病。癌症免疫治疗的另一种是嵌合抗原受体修饰的T细胞(Chimeric Antigen receptor T cells,即CAR-T细胞)疗法。CAR-T细胞疗法在恶性血液癌症患者上已显示出令人振奋的临床疗效，且多款靶向CD19的CAR-T细胞疗法已被美国FDA分别批准用于治疗白血病与淋巴瘤。
肿瘤细胞疗法正以前所未有的速度发展，特别是自从CAR-T细胞疗法问世以来，这种策略已被证明对B细胞恶性肿瘤有效，并在其它血液学癌症的临床试验中显示出很有前景的活性，亦有可能在实体瘤治疗中产生一定效果。然而，CAR-T细胞治疗在临床应用中面临的细胞因子释放综合征和免疫效应细胞相关神经毒性综合征等副作用增加了患者的住院时间和治疗风险与费用。CAR-T细胞治疗的其它局限性包括生产自体细胞产品的管销物流挑战以及考虑到T细胞介导的移植物抗宿主反应的风险等。此外，CAR-T细胞治疗实体瘤的不佳结果主要归因于免疫抑制微环境和肿瘤间质形成的物理屏障所带来的独特挑战。鉴于传统CAR基因转导的αβ-T细胞的这些缺点，人们对先天性和适应性的其它类型免疫细胞亚群以及CAR工程的改进策略产生了极大的兴趣。自然杀伤细胞(Natural Killer cells,即NK细胞)因其独特的生物学特性、对肿瘤的特异性细胞毒性、安全性和作为现成细胞治疗的潜在用途而备受关注。值得一提的是，作为T细胞与B细胞以外极其重要淋巴细胞类型的NK细胞，NK细胞大致占人类外周单核细胞总体比例的5％到15％，是先天性免疫系统的重要组成部分，在我们抵御病原体和癌细胞的一线防御中起着至关重要的作用。正如它们的名字所暗示的，NK细胞是一种特殊的杀手，它具有独特的自然能力来消除因病毒感染或恶性转化而受损的异常细胞。与T细胞不同，NK细胞缺乏T细胞受体和CD3的表达，其不需要MHC-I分子的辅助去识别和杀伤异常细胞。在这种情况下，杀伤性T细胞的“先天对应物”——NK细胞的作用至关重要，其能够发现并识别缺少“自我”(non-self)的MHC-I类分子的异常细胞。在20世纪90年代，这一假说被抑制和激活性NK受体的发现进一步所证实。用免疫检查点抑制剂阻断抑制NK细胞的免疫抑制性信号，可以一定程度上克服基于T细胞的免疫治疗的局限性。另外，NK细胞在一定程度上具有与CD8阳性杀伤性T细胞去杀伤靶细胞的类似杀伤性机制，为工程化改造NK细胞提供了坚实基础。与CAR-T细胞疗法相比，CAR-NK细胞疗法可以提供诸多优势，比如但不限于：(1)多种激活杀伤性功能机制，(2)“off-the-shelf”的高度便利制造工艺优势，(3)安全性方面的优势，例如自体同源疗法呈现较少或无细胞因子释放综合征与神经毒性综合征以及异体同源疗法呈现较少或无移植物抗宿主反应等。值得注意的是，NK细胞的使用可以产生现成的同种异体产品来治疗患者，从而消除了目前CAR-T细胞疗法中个性化和患者专一性产品的必要性限制。工程化改造NK细胞可以使NK细胞靶向多种不同抗原并且增强NK细胞在体内的增殖扩增性与持久性等，从而最终实现有效的抗肿瘤功能。此外，NK细胞作为机体重要的免疫细胞不仅与抗肿瘤、抗病毒感染和免疫调节相关，还在某些情况下参与超敏反应和自身免疫性疾病的发生，能够识别靶细胞并杀伤清除介质。因此，对NK细胞进行改造加以应用具有十分重要的意义。
发明内容
根据本申请的一个方面，提供一种嵌合抗原受体改造的NK细胞，所述嵌合抗原受体改造的NK细胞兼具免疫检查点抑制剂与CAR改造的NK细胞疗法两者的优势，为改善实体肿瘤治疗提供解决方案。
一种嵌合抗原受体改造的NK细胞，所述嵌合抗原受体包括：胞外靶标分子结合结构域、跨膜区结构域和胞内信号传导结构域；
所述跨膜区结构域将所述胞外靶标分子结合结构域和所述胞内信号传导结构域连接，并将二者固定在所述NK细胞的细胞膜上；
所述胞内信号传导结构域包括胞内激活信号传导结构域和/或胞内检测信号传导结构域。
可选地，所述嵌合抗原受体改造的NK细胞为含有编码嵌合抗原受体核酸的NK细胞。
可选地，所述嵌合抗原受体还包括：胞外间隔区结构域；
所述胞外间隔区结构域位于所述胞外靶标分子结合结构域与所述跨膜区结构域之间。
可选地，所述嵌合抗原受体还包括：胞内间隔区结构域；
所述胞内间隔区结构域位于所述跨膜区结构域和所述胞内信号传导结构域之间并将这两者连接在一起。
可选地，所述嵌合抗原受体还包括：胞内铰链结构域；
所述胞内铰链结构域将所述胞内检测信号结构域和所述胞内激活信号结构域连接在一起；
所述胞内铰链结构域可以具有任何合适的长度以连接至少两个感兴趣的结构域，并且优选设计为足够柔性以便允许其连接的一个或两个结构域的正确折叠和/或功能和/或活性。
可选地，所述胞外靶标分子结合结构域结合的靶标分子包含下组的分子中的至少一种：免疫抑制性信号相关分子、肿瘤表面抗原分子标志物、细胞表面特定的抗原肽-组织相容性复合体分子。
可选地，所述胞外靶标分子结合结构域包含选自下组的分子的靶标分子结合结构域中的至少一种：PD-1、PD-1截短体、PD-1蛋白突变体、PD-L1的抗体及PD-L1结合片段。
可选地，所述胞外靶标分子结合结构域包含含有SEQ ID NO:1的氨基酸序列、含有SEQ ID NO:3的氨基酸序列、含有SEQ ID NO:5的氨基酸序列、含有SEQ ID NO:7、含有SEQ ID NO:9的氨基酸序列、含有SEQ ID NO:11的氨基酸序列中的至少一种。
可选地，所述胞外靶标分子结合结构域的核酸片段包含含有SEQ ID NO:2的核酸序列、含有SEQ ID NO:4的核酸序列、含有SEQ ID NO:6的核酸序列、含有SEQ ID NO:8的核酸序列、含有SEQ ID NO:10的核酸序列中的至少一种。
可选地，所述胞内激活信号传导结构域的激活至少依赖于所述胞外靶标分子结合结构域与所述靶标分子的结合；所述胞内激活信号传导结构域含有具有催化功能基团的分子或片段。
可选地，所述胞内激活信号传导结构域包括酪氨酸激酶或酪氨酸激酶片段中的至少一种；
所述酪氨酸激酶包括受体型酪氨酸激酶、非受体型酪氨酸激酶中的至少一种；
所述酪氨酸激酶片段包括受体型酪氨酸激酶片段、非受体型酪氨酸激酶片段中的至少一种。
可选地，所述酪氨酸激酶选自Ack、CSK、CTK、FAK、Abl、Arg、Tnk1、Pyk2、Fer、Fes、LTK、ALK、STYK1、JAK1、JAK2、JAK3、Tyk2、DDR1、DDR2、ROS、Blk、Fgr、FRK、Fyn、TIE1、TIE2、Hck、Lck、Srm、Yes、Syk、ZAP70、Etk、Btk、HER2、HER3、HER4、InsR、ITK、TEC、TXK、EGFR、IGF1R、IRR、PDGFRα、VEGFR-1、VEGFR-2、VEGFR-3、PDGFRβ、Kit、CSFR、FLT3、FGFR1、FGFR2、FGFR3、FGFR4、CCK4、ROR1、ROR2、MuSK、MET、Lyn、Brk、Src、Ron、Axl、Tyro3、Mer、EphA1、EphA2、EphA3、EphA4、EphA5、EphA6、EphA7、EphA8、EphA10、EphB1、trkA、trkB、trkC、EphB2、EphB3、EphB4、EphB6、Ret、RYK、Lmr1、Lmr2、Lmr3中的至少一种。
可选地，所述胞内激活信号传导结构域包含含有SEQ ID NO:42的氨基酸序列、含有SEQ ID NO:44的氨基酸序列、含有SEQ ID NO:46的氨基酸序列、含有SEQ ID NO:48的氨基酸序列、含有SEQ ID NO:50的氨基酸序列、含有SEQ ID NO:52的氨基酸序列中的至少一种。
可选地，所述胞内激活信号传导结构域的核酸片段包含含有SEQ ID NO:43的核酸序列、含有SEQ ID NO:45的核酸序列、含有SEQ ID NO:47的核酸序列、含有SEQ ID NO:49的核酸序列、含有SEQ ID NO:51的核酸序列、含有SEQ ID NO:53的核酸序列中的至少一种。
可选地，所述胞内检测信号传导结构域包含至少一个基于免疫受体酪氨酸的活化基序。
可选地，所述胞内检测信号传导结构域包含选自下组的分子的信号传导结构域的至少一种：CD3δ、CD3γ、CD3ε、CD3ζ、CD5、CD28、CD31、CD72、CD84、CD229、CEACAM-19、CEACAM-20、SIRPα、SLAM、CLEC-1、CLEC-2、CRACC、CTLA-4、2B4、CD244、BTLA、DCAR、DCIR、Dectin-1、DNAM-1、CD300a、CD300f、CEACAM-1、CEACAM-3、CEACAM-4、FcεRIα、FcεRIβ、FcγRIB、FcγRI、FcγRIIA、FcγRIIB、FcγRIIC、FcγRIIIA、DAP10、DAP12、G6b、KIR、KIR2DL1、KIR2DL2、KIR2DL3、KIR2DL4、KIR2DL5、FCRL1、FCRL2、FCRL3、FCRL4、FCRL5、FCRL6、KIR2DL5B、KIR2DS1、KIR2DS3、KIR2DS4、KIR2DS5、TIGIT、TREML1、TREML2、KIR3DL1、KIR3DL2、KIR3DL3、KIR3DS1、KLRG1、LAIR1、MICL、NKG2A、NKp44、NKp65、NKp80、NTB-A、PD-1、LILRB1、LILRB2、LILRB3、LILRB4、LILRB5、Siglec-2、Siglec-3、Siglec-5、Siglec-6、Siglec-7、Siglec-8、PDCD6、PILR-α、Siglec-9、Siglec-10、Siglec-11、Siglec-12、Siglec-14、Siglec-15、Siglec-16。
可选地，所述胞内检测信号传导结构域包含含有SEQ ID NO:20的氨基酸序列、含有SEQ ID NO:22的氨基酸序列、含有SEQ ID NO:24的氨基酸序列、含有SEQ ID NO:26的氨基酸序列、含有SEQ ID NO:28的氨基酸序列、含有SEQ ID NO:30的氨基酸序列、含有SEQ ID NO:32的氨基酸序列、含有SEQ ID NO:34的氨基酸序列、含有SEQ ID NO:36的氨基酸序列、含有SEQ ID NO:38的氨基酸序列、含有SEQ ID NO:40的氨基酸序列中的至少一种。
可选地，所述胞内检测信号传导结构域的核酸片段包含含有SEQ ID NO:21的核酸序列、含有SEQ ID NO:23的核酸序列、含有SEQ ID NO:25的核酸序列、含有SEQ ID NO:27的核酸序列、含有SEQ ID NO:29的核酸序列、含有SEQ ID NO:31的核酸序列、含有SEQ ID NO:33的核酸序列、含有SEQ ID NO:35的核酸序列、含有SEQ ID NO:37的核酸序列、含有SEQ ID NO:39的核酸序列、含有SEQ ID NO:41的核酸序列中的至少一种。
可选地，所述跨膜区结构域选自下组的跨膜蛋白的跨膜结构域，跨膜蛋白包含4-1BB、4-1BBL、ICOS、GITR、GITRL、VSIG-3、VISTA、SIRPα、OX40、OX40L、CD40、CD40L、CD86、CD80、PD-1、PD-L1、PD-L2、CD2、CD28、B7-DC、B7-H2、B7-H3、B7-H4、B7-H5、B7-H6、B7-H7、Siglec-1、Siglec-2、Siglec-3、Siglec-4、LILRB5、2B4、BTLA、CD160、LAG-3、Siglec-5、Siglec-6、Siglec-7、Siglec-8、CD96、CD226、TIM-1、TIM-3、TIM-4、Siglec-9、Siglec-10、Siglec-11、Siglec-12、Siglec-14、Siglec-15、Siglec-16、LIR1、KIR2DL1、KIR2DL2、KIR2DL3、KIR2DL4、KIR2DL5A、KIR2DL5B、KIR3DL1、KIR3DL2、KIR3DL3、KIR3DS1、KLRG1、KLRG2、KIR2DS1、KIR2DS3、KIR2DS4、KIR2DS5、LAIR1、LAIR2、LILRA3、LILRA4、DAP10、DAP12、NKG2A、NKG2C、NKG2D、LILRA5、LILRB1、LILRB2、LILRB3、LILRB4、CTLA-4、CD155、CD112、CD113、TIGIT、Galectin-9、CEACAM-1、CD8a、CD8b、CD4、MERTK、AXL、Tyro3、BAI1、MRC1、FcγR1、FcγR2A、FcγR2B1、FcγR2B2、FcγR3A、FcγR3B、FcεR2、FcεR1、FcRn、Fcα/μR或FcαR1中的至少一种；
可选地，所述跨膜区结构域包含含有SEQ ID NO:12的氨基酸序列、含有SEQ ID NO:14的氨基酸序列中的至少一种。
可选地，所述跨膜区结构域的核酸片段包含含有SEQ ID NO:13的核酸序列、含有SEQ ID NO:15的核酸序列中的至少一种。
可选地，所述胞外间隔区结构域包含含有SEQ ID NO:16的氨基酸序列、含有SEQ ID NO:18的氨基酸序列中的至少一种。
可选地，所述胞外间隔区结构域的核酸片段包含含有SEQ ID NO:17的核酸序列、含有SEQ ID NO:19的核酸序列中的至少一种。
可选地，所述胞内间隔区结构域为跨膜区结构域之延伸，包含选自下组的分子的至少一种：PD-1、PD-L1、PD-L2、CD8a、CD8b、CD4、ICOS、GITR、GITRL、OX40、OX40L、B7-DC、B7-H2、B7-H3、B7-H4、B7-H5、CD40、CD40L、CD86、CD80、CD2、CD28、B7-H6、B7-H7、VSIG-3、VISTA、SIRPα、KIR2DS1、KIR2DS3、KIR2DS4、KIR2DS5、Siglec-1、Siglec-2、Siglec-3、Siglec-4、CD155、CD112、CD113、TIGIT、CD96、CD226、Siglec-5、Siglec-6、Siglec-7、Siglec-8、Siglec-9、Siglec-10、LILRB1、LILRB2、LILRB3、LILRB4、LILRB5、Siglec-11、Siglec-12、Siglec-14、Siglec-15、Siglec-16、LIR1、KIR2DL1、KIR2DL2、KIR2DL3、KIR2DL4、DAP10、DAP12、NKG2A、NKG2C、NKG2D、KIR2DL5A、KIR2DL5B、KIR3DL1、KIR3DL2、KIR3DL3、KIR3DS1、TIM-1、TIM-3、TIM-4、KLRG1、KLRG2、LAIR1、LAIR2、LILRA3、LILRA4、LILRA5、2B4、BTLA、CD160、LAG-3、CTLA-4、Galectin-9、CEACAM-1、MERTK、AXL、Tyro3、BAI1、4-1BB、4-1BBL、MRC1、FcγR1、FcγR2A、FcγR2B1、FcγR2B2、FcγR3A、FcγR3B、FcεR2、FcεR1、FcRn、Fcα/μR或FcαR1。
可选地，所述胞内间隔区结构域包含含有SEQ ID NO:54的氨基酸序列、含有SEQ ID NO:56的氨基酸序列中的至少一种。
可选地，所述胞内间隔区结构域核酸片段包含含有SEQ ID NO:55的核酸序列、含有SEQ ID NO:57的核酸序列中的至少一种。
可选地，所述胞内铰链结构域包含含有SEQ ID NO:58的氨基酸序列、含有SEQ ID NO:60的氨基酸序列、含有SEQ ID NO:62的氨基酸序列、含有SEQ ID NO:64的氨基酸序列、含有SEQ ID NO:66的氨基酸序列中的至少一种。
可选地，所述胞内铰链结构域片段包含含有SEQ ID NO:59的核酸序列、含有SEQ ID NO:61的核酸序列、含有SEQ ID NO:63的核酸序列、含有SEQ ID NO:65的核酸序列中的至少一种。
可选地，所述嵌合抗原受体包括：胞外靶标分子结合结构域；跨膜区结构域；胞外间隔区结构域；胞内检测信号传导结构域；胞内激活信号传导结构域；胞内间隔区结构域；胞内铰链结构域。
可选地，所述嵌合抗原受体包括：胞外靶标分子结合结构域；跨膜区结构域；胞外间隔区结构域；和胞内信号传导结构域。
可选地，所述嵌合抗原受体包括：胞外靶标分子结合结构域；跨膜区结构域；胞外间隔区结构域；胞内检测信号传导结构域；和胞内激活信号传导结构域。
可选地，所述嵌合抗原受体包括：胞外靶标分子结合结构域；跨膜区结构域；胞外间隔区结构域；和胞内激活信号传导结构域。
可选地，所述嵌合抗原受体包括：胞外靶标分子结合结构域；跨膜区结构域；胞外间隔区结构域；胞内检测信号传导结构域；胞内激活信号传导结构域；和胞内铰链结构域。
可选地，所述嵌合抗原受体包括：胞外靶标分子结合结构域；跨膜区结构域；胞外间隔区结构域；和胞内激活信号传导结构域；和胞内间隔区结构域。
可选地，所述嵌合抗原受体包括：胞外靶标分子结合结构域；跨膜区结构域；胞外间隔区结构域；胞内信号传导结构域；和胞内间隔区结构域。
可选地，所述嵌合抗原受体包括：胞外靶标分子结合结构域；跨膜区结构域；胞外间隔区结构域；胞内检测信号传导结构域；胞内激活信号传导结构域；和胞内间隔区结构域。
可选地，所述NK细胞包括内源性NK细胞亚群和/或外源性NK细胞中的至少一种。
可选地，所述内源性NK细胞亚群包括适应性NK细胞、记忆性NK细胞、CD56 dim NK细胞、CD56 bright NK细胞中的至少一种；
所述外源性NK细胞包括NK细胞株、胚胎干细胞或诱导多能干细胞衍生的NK细胞中的至少一种。
可选地，所述NK细胞株选自NK-92细胞株、haNK细胞株、IMC-1细胞株、NK-YS细胞株、KHYG-1细胞株、NKL细胞株、NKG细胞株、SNK-6细胞株、YTS细胞株、HANK-1细胞株中的至少一种。
所述haNK细胞株为过表达高亲和力(high-affinity)CD16阳性NK细胞株。
根据本申请的另一方面，提供上述任一项所述的嵌合抗原受体改造的NK细胞的制备方法，所述制备方法包括以下步骤：
1)分别获得人的NK细胞和嵌合抗原受体；
2)利用所述嵌合抗原受体对所述人的NK细胞进行改造，以获得所述嵌合抗原受体改造的NK细胞。
可选地，所述制备方法包括以下步骤：
1)分别获得人的NK细胞和编码嵌合抗原受体的核酸；
2)将所述编码嵌合抗原受体的核酸导入所述人的NK细胞，以获得所述嵌合抗原受体改造的NK细胞。
根据本申请的另一方面，提供一种药物组合物，所述组合物包括上述所述的嵌合抗原受体改造的NK细胞或根据上述所述的制备方法制备得到的嵌合抗原受体改造的NK细胞中的至少一种。
可选地，所述药物组合物还包括单克隆抗体；
所述单克隆抗体选自西妥昔单抗、阿仑单抗、伊匹单抗、奥法木单抗中的至少一种。
可选地，所述药物组合物还包括细胞因子；
所述细胞因子选自γ干扰素、白细胞介素中的至少一种。
根据本申请的另一方面，提供上述任一项所述的嵌合抗原受体改造的NK细胞、或根据上述任一项所述的制备方法制备得到的嵌合抗原受体改造的NK细胞、或上述任一项所述的药物组合物中的至少一种在制备治疗以下疾病的药物中的应用：
肿瘤、感染、炎症疾病、免疫疾病、神经系统疾病。
可选地，所述肿瘤为PD-L1阳性或响应γ干扰素上调PD-L1表达水平的肿瘤。
可选地，所述肿瘤包括实体瘤和/或血液癌症。
可选地，所述实体瘤包括乳腺癌、皮肤癌、肝癌、卵巢癌、前列腺癌、脑癌、肾癌、肺癌中的至少一种。
可选地，所述血液癌症包括白血病。
根据本申请的另一个方面，提供一种疾病的治疗方法，所述治疗方法包括以下步骤：
向人体输入嵌合抗原受体改造的NK细胞或药物组合物；
所述嵌合抗原受体改造的NK细胞选自上述所述的嵌合抗原受体改造的NK细胞、或根据上述所述的制备方法制备得到的嵌合抗原受体改造的NK细胞中的至少一种；
所述药物组合物选自上述所述的药物组合物；
所述疾病选自肿瘤、感染、炎症疾病、免疫疾病、神经系统疾病中的至少一种。
根据本申请的另一个方面，提供上述任一项所述的药物组合物的使用方法，包括以下步骤：
1)分别获得人的NK细胞和嵌合抗原受体；
2)利用所述嵌合抗原受体对所述人的NK细胞进行改造，以获得嵌合抗原受体改造的NK细胞；
3)将所述嵌合抗原受体改造的NK细胞回输至人体内；
或所述使用方法包括以下步骤：
1)分别获得人的NK细胞和编码嵌合抗原受体的核酸；
2)将所述编码嵌合抗原受体的核酸导入所述人的NK细胞，以获得所述嵌合抗原受体改造的NK细胞；
3)将所述嵌合抗原受体改造的NK细胞回输至人体内。
可选地，步骤3)还包括：
3-1)对人体的整体或者部分施加细胞因子、单克隆抗体中的至少一种；
3-2)将所述改造后的免疫细胞回输至人体内。
序列同源性适用于本申请贯穿全文的所有提及的核酸序列与蛋白质序列相似性与同一性的鉴别。
表1为本申请所述涉及的氨基酸和核酸序列
表1
Figure PCTCN2022073311-appb-000001
Figure PCTCN2022073311-appb-000002
Figure PCTCN2022073311-appb-000003
本申请能产生的有益效果包括：
1)本申请所提供的嵌合抗原受体改造的NK细胞，通过利用嵌合抗原受体进行改造，嵌合抗原受体上具有经过特殊改造的胞内信号传导结构域，加强了NK细胞对肿瘤细胞的杀伤作用。
2)本申请所提供的嵌合抗原受体改造的NK细胞，为经过该新一代基于PD-1的嵌合抗原受体分子机器重新编码改造的NK细胞，基于改造免疫检查点PD-1/PD-L1信号通路，可以去更好地识别杀伤特定的肿瘤细胞，不但不会被表达免疫检查点抑制性信号PD-1分子配体PD-L1的肿瘤细胞所抑制，反而会被进一步激活，产生针对相应肿瘤细胞的特异性免疫反应，从而识别并杀伤相应的肿瘤细胞。
3)本申请所提供的嵌合抗原受体改造的NK细胞，对特定的肿瘤细胞具有更好地识别杀伤作用，包括人源前列腺癌肿瘤细胞、人源肾癌肿瘤细胞、人源乳腺癌肿瘤细胞、人源皮肤癌肿瘤细胞、人源脑癌肿瘤细胞、人源肺癌肿瘤细胞、人源肝癌细胞、人源卵巢癌细胞等。
附图说明
图1显示了施用本公开内容的自然杀伤细胞嵌合抗原受体的示例性方法，其中自然杀伤细胞可以是个体异源、异体同源或自体同源的细胞。
图2为本申请的嵌合抗原受体人工分子机器的构建示意图简图。其中，图2a嵌合抗原受体人工分子机器包含结构域#I、结构域#II、结构域#III与结构域#VIII，图2b嵌合抗原受体人工分子机器包含结构域#I、结构域#II、结构域#III与结构域#VII，图2c嵌合抗原受体人工分子机器包含结构域#I、结构域#II、结构域#III、结构域#V与结构域#VII，图2d嵌合抗原受体人工分子机器包含结构域#I、结构域#II、结构域#III、结构域#V、结构域#VI与结构域#VII，图2e嵌合抗原受体人工分子机器包含结构域#I、结构域#II、结构域#III、结构域#IV与结构域#VIII，图2f嵌合抗原受体人工分子机器包含结构域#I、结构域#II、结构域#III、结构域#IV与结构域#VII，图2g嵌合抗原受体人工分子机器包含结构域#I、结构域#II、结构域#III、结构域#IV、结构域#V与结构域#VII，图2h嵌合抗原受体人工分子机器包含结构域#I、结构域#II、结构域#III、结构域#IV、结构域#V、结构域#VI与结构域#VII。
图3为本申请的人工分子机器的信号激活示意图简图，图3a显示了在酪氨酸激酶活化信号输入的情况下人工分子机器的激活信号释放并激活，图3b显示了在靶细胞靶分子信号输入(如PD-L1)的情况下含有结构域#I(如PD-1胞外部分)的嵌合抗原受体人工分子机器的激活信号释放并激活。
图4a显示了C#9、C#10、C#11、C#12、C#13、C#14、C#15与C#16在酪氨酸磷酸酶抑制剂过钒酸钠激活蛋白酪氨酸磷酸化信号的条件下于人源海拉(HeLa)细胞中的表现结果。
图4b显示了C#9与C#15在酪氨酸磷酸酶抑制剂过钒酸钠激活蛋白酪氨酸磷酸化信号的A条件或在表皮生长因子激活信号的B条件下于人源海拉(HeLa)细胞中的表现结果。
图4c显示了C#9与C#15在酪氨酸磷酸酶抑制剂过钒酸钠激活蛋白酪氨酸磷酸化信号的A条件或在血小板源生长因子激活信号的B条件下于小鼠胚胎成纤维细胞(MEF)中的表现结果。
图5a显示了C#17与C#18于人源海拉(HeLa)细胞中的表达分布及在酪氨酸磷酸酶抑制剂过钒酸钠刺激下响应蛋白酪氨酸磷酸化信号能力的检测结果。
图5b显示了C#19与C#20于人源海拉(HeLa)细胞中的表达分布及在酪氨酸磷酸酶抑制剂过钒酸钠刺激下响应蛋白酪氨酸磷酸化信号能力的检测结果。
图5c显示了在酪氨酸磷酸酶抑制剂过钒酸钠激活蛋白酪氨酸磷酸化信号的条件下，C#17、C#18、C#19与C#20在人源海拉(HeLa)细胞中的表现结果。
图6显示了在非受体型蛋白酪氨酸激酶Lck提供激活蛋白酪氨酸磷酸化信号的条件下，C#9与C#10在纯化蛋白的状态下的表现结果。
图7a显示了C#19与C#20于人源海拉(HeLa)细胞中的表达分布及在人源PD-L1修饰的微球刺激下响应人源PD-L1信号的检测结果。
图7b显示了在人源PD-L1修饰的微球刺激信号的条件下，C#17、C#18、C#19与C#20于人源海拉(HeLa)细胞中的表现结果。
图8显示了对自然杀伤细胞和具有本公开内容的嵌合抗原受体修饰的自然杀伤细胞的比较。其中，图8a显示了自然杀伤细胞面对肿瘤细胞的表现。图8b显示了具有本公开内容的嵌合抗原受体修饰的自然杀伤细胞面对肿瘤细胞的表现。其中，自然杀伤细胞的灰度高低对应自然杀伤细胞的肿瘤杀伤能力高低。
图9显示了在自然杀伤细胞NK-92中不同嵌合抗原受体的表达。
图10显示了PD-L1在8种人源肿瘤细胞及经γ干扰素预处理的8种人源肿瘤细胞的表达。8种人源肿瘤细胞分别为图10a乳腺癌肿瘤细胞MBA-MB-231、图10b脑癌肿瘤细胞U87-MG、图10c肾癌肿瘤细胞786-O、图10d皮肤癌肿瘤细胞A2058、图10e肺癌肿瘤细胞H441、图10f卵巢癌肿瘤细胞ES-2、图10g前列腺癌肿瘤细胞PC-3、图10h肝癌肿瘤细胞HA-22T。
图11显示了嵌合抗原受体修饰改造的人源自然杀伤细胞C#3、C#2分别与PD-L1阳性的人源乳腺癌肿瘤细胞MDA-MB-231细胞中的体外共培养细胞毒性效果的影像分析结果。图11a显示嵌合抗原受体修饰改造的人源自然杀伤细胞C#3与PD-L1阳性的人源乳腺癌肿瘤细胞MDA-MB-231细胞中的体外共培养细胞情形中，初始绿色荧光标记的细胞为健康完整的人源乳腺癌肿瘤细胞，若经嵌合抗原受体修饰改造的人源自然杀伤细胞识别杀伤后，细胞膜破碎不完整，绿色荧光逐渐消失，红色荧光细胞核染色试剂的碘化丙啶会进入凋亡细胞内呈现为红色荧光的细胞。图11b显示嵌合抗原受体修饰改造的人源自然杀伤细胞C#2与PD-L1阳性的人源乳腺癌肿瘤细胞MDA-MB-231细胞中的体外共培养细胞情形，人源乳腺癌肿瘤细胞MDA-MB-231细胞始终维持健康完整，未受嵌合抗原受体修饰改造的人源自然杀伤细胞C#2的杀伤。
图12a说明本申请所涵盖的自然杀伤细胞与PD-L1阳性的人源乳腺癌肿瘤细胞的体外共培养细胞毒性实验分析测试流程与模式设置。
图12b说明不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源乳腺癌肿瘤细胞MDA-MB-231细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照1:1的E/T(效应细胞/靶细胞)比例进行实验(平均值±标准差，均为n＝3)。
图12c说明不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源乳腺癌肿瘤细胞MDA-MB-231细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照2.5:1的E/T(效应细胞/靶细胞)比例进行实验(平均值±标准差，均为n＝3)。
图12d说明不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源乳腺癌肿瘤细胞MDA-MB-231细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例进行实验(平均值±标准差，均为n＝3)。
图12e说明不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源乳腺癌肿瘤细胞MDA-MB-231细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照10:1的E/T(效应细胞/靶细胞)比例进行实验(均为n＝1)。
图13a说明本申请所涵盖的自然杀伤细胞与PD-L1阳性的人源乳腺癌肿瘤细胞的体外共培养细胞毒性实验分析测试流程与模式设置。
图13b说明不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源乳腺癌肿瘤细胞MDA-MB-231细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例进行实验(平均值±标准差，均为n＝3)，体外共培养细胞毒性效果的定量分析时间轴至60小时，并加入仅肿瘤细胞组别供分析参考。
图13c说明不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源乳腺癌肿瘤细胞MDA-MB-231细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例进行实验(平均值±标准差，均为n＝3)，体外共培养细胞毒性效果的定量分析时间轴至60小时。
图14a说明本申请所涵盖的自然杀伤细胞与PD-L1阳性的人源皮肤癌肿瘤细胞的体外共培养细胞毒性实验分析测试流程与模式设置。
图14b说明不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源皮肤癌肿瘤细胞A2058细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照1:1的E/T(效应细胞/靶细胞)比例进行实验(平均值±标准差，均为n＝3)。
图14c说明不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源皮肤癌肿瘤细胞A2058细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照2.5:1的E/T(效应细胞/靶细胞)比例进行实验(平均值±标准差，均为n＝3)。
图14d说明不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源皮肤癌肿瘤细胞A2058细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例进行实验(平均值±标准差，均为n＝3)。
图14e说明不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源皮肤癌肿瘤细胞A2058细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照10:1的E/T(效应细胞/靶细胞)比例进行实验(均为n＝1)。
图15a说明本申请所涵盖的自然杀伤细胞与PD-L1阳性的人源前列腺癌肿瘤细胞的体外共培养细胞毒性实验分析测试流程与模式设置。
图15b说明不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源前列腺癌肿瘤细胞PC-3细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例进行实验(平均值±标准差，均为n＝3)，并加入仅肿瘤细胞组别供分析参考。
图15c说明不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源前列腺癌肿瘤细胞PC-3细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例进行实验(平均值±标准差，均为n＝3)。
图16a说明本申请所涵盖的自然杀伤细胞与PD-L1阳性的人源脑癌肿瘤细胞的体外共培养细胞毒性实验分析测试流程与模式设置。
图16b说明不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源脑癌肿瘤细胞U87-MG细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例进行实验(平均值±标准差，均为n＝3)，并加入仅肿瘤细胞组别供分析参考。
图16c说明不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源脑癌肿瘤细胞U87-MG细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例进行实验(平均值±标准差，均为n＝3)。
图17a说明本申请所涵盖的自然杀伤细胞与PD-L1阳性的人源肝癌肿瘤细胞的体外共培养细胞毒性实验分析测试流程与模式设置。
图17b说明不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源肝癌肿瘤细胞HA-22T细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例进行实验(平均值±标准差，均为n＝3)，并加入仅肿瘤细胞组别供分析参考。
图17c说明不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源肝癌肿瘤细胞HA-22T细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例进行实验(平均值±标准差，均为n＝3)。
图18a说明本申请所涵盖的自然杀伤细胞与PD-L1阳性的人源肾癌肿瘤细胞的体外共培养细胞毒性实验分析测试流程与模式设置。
图18b说明不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源肾癌肿瘤细胞786-O细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例进行实验(平均值±标准差，均为n＝3)，并加入仅肿瘤细胞组别供分析参考。
图18c说明不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源肾癌肿瘤细胞786-O细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例进行实验(平均值±标准差，均为n＝3)。
图19a说明本申请所涵盖的自然杀伤细胞与PD-L1阳性的人源肺癌肿瘤细胞的体外共培养细胞毒性实验分析测试流程与模式设置。
图19b说明不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源肺癌肿瘤细胞H441细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例进行实验(平均值±标准差，均为n＝3)， 并加入仅肿瘤细胞组别供分析参考。
图19c说明不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源肺癌肿瘤细胞H441细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例进行实验(平均值±标准差，均为n＝3)。
图20a说明本申请所涵盖的自然杀伤细胞与PD-L1阳性的人源卵巢癌肿瘤细胞的体外共培养细胞毒性实验分析测试流程与模式设置。
图20b说明不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源卵巢癌肿瘤细胞ES-2细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例进行实验(平均值±标准差，均为n＝3)，并加入仅肿瘤细胞组别供分析参考。
图20c说明不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源卵巢癌肿瘤细胞ES-2细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例进行实验(平均值±标准差，均为n＝3)。
图21a不同的嵌合抗原受体修饰改造的人源自然杀伤细胞经细胞裂解与核糖核酸提取，以甘油醛-3-磷酸脱氢酶(GAPDH)基因作为内源性参照基因，针对信使核糖核酸GZMB的表达的定量实时聚合酶链锁反应(平均值±标准差，均为n＝3)。
图21b不同的嵌合抗原受体修饰改造的人源自然杀伤细胞先与PD-L1阳性的人源乳癌肿瘤细胞MDA-MB-231细胞的体外共培养48小时后，人源自然杀伤细胞经细胞裂解与核糖核酸提取，以甘油醛-3-磷酸脱氢酶(GAPDH)基因作为内源性参照基因，针对信使核糖核酸GZMB的表达的定量实时聚合酶链锁反应(平均值±标准差，均为n＝3)。
图22a不同的嵌合抗原受体修饰改造的人源自然杀伤细胞经细胞裂解与核糖核酸提取，以甘油醛-3-磷酸脱氢酶(GAPDH)基因作为内源性参照基因，针对信使核糖核酸PRF1的表达的定量实时聚合酶链锁反应(平均值±标准差，均为n＝3)。
图22b不同的嵌合抗原受体修饰改造的人源自然杀伤细胞先与PD-L1阳性的人源乳癌肿瘤细胞MDA-MB-231细胞的体外共培养48小时后，人源自然杀伤细胞经细胞裂解与核糖核酸提取，以甘油醛-3-磷酸脱氢酶(GAPDH)基因作为内源性参照基因，针对信使核糖核酸PRF1的表达的定量实时聚合酶链锁反应(平均值±标准差，均为n＝3)。
图23a不同的嵌合抗原受体修饰改造的人源自然杀伤细胞经细胞裂解与核糖核酸提取，以甘油醛-3-磷酸脱氢酶(GAPDH)基因作为内源性参照基因，针对信使核糖核酸TNFA的表达的定量实时聚合酶链锁反应(平均值±标准差，均为n＝3)。
图23b不同的嵌合抗原受体修饰改造的人源自然杀伤细胞先与PD-L1阳性的人源乳癌肿瘤细胞MDA-MB-231细胞的体外共培养48小时后，人源自然杀伤细胞经细胞裂解与核糖核酸提取，以甘油醛-3-磷酸脱氢酶(GAPDH)基因作为内源性参照基因，针对信使核糖核酸TNFA的表达的定量实时聚合酶链锁反应(平均值±标准差，均为n＝3)。
图24a不同的嵌合抗原受体修饰改造的人源自然杀伤细胞经细胞裂解与核糖核酸提取，以甘油醛-3-磷酸脱氢酶(GAPDH)基因作为内源性参照基因，针对信使核糖核酸IFNG的表达的定量实时聚合酶链锁反应(平均值±标准差，均为n＝3)。
图24b不同的嵌合抗原受体修饰改造的人源自然杀伤细胞先与PD-L1阳性的人源乳癌肿瘤细胞MDA-MB-231细胞的体外共培养48小时后，人源自然杀伤细胞经细胞裂解与核糖核酸提取，以甘油醛-3-磷酸脱氢酶(GAPDH)基因作为内源性参照基因，针对信使核糖核酸IFNG的表达的定量实时聚合酶链锁反应(平均值±标准差，均为n＝3)。
图25a不同的嵌合抗原受体修饰改造的人源自然杀伤细胞经细胞裂解与核糖核酸提取，以甘油醛-3-磷酸脱氢酶(GAPDH)基因作为内源性参照基因，针对信使核糖核酸NCAM1的表达的定量实时聚合酶链锁反应(平均值±标准差，均为n＝3)。
图25b不同的嵌合抗原受体修饰改造的人源自然杀伤细胞先与PD-L1阳性的人源乳癌肿瘤细胞MDA-MB-231细胞的体外共培养48小时后，人源自然杀伤细胞经细胞裂解与核糖核酸提取，以甘油醛-3-磷酸脱氢酶(GAPDH)基因作为内源性参照基因，针对信使核糖核酸NCAM1的表达的定量实时聚合酶链锁反应(平均值±标准差，均为n＝3)。
图26a不同的嵌合抗原受体修饰改造的人源自然杀伤细胞经细胞裂解与核糖核酸提取，以甘油醛-3-磷酸脱氢酶(GAPDH)基因作为内源性参照基因，针对信使核糖核酸KLRK1的表达的定量实时聚合酶链锁反应(平均值±标准差，均为n＝3)。
图26b不同的嵌合抗原受体修饰改造的人源自然杀伤细胞先与PD-L1阳性的人源乳癌肿瘤细胞MDA-MB-231细胞的体外共培养48小时后，人源自然杀伤细胞经细胞裂解与核糖核酸提取，以甘油醛-3-磷酸脱氢酶(GAPDH)基因作为内源性参照基因，针对信使核糖核酸KLRK1的表达的定量实时聚合酶链锁反应(平均值±标准差，均为n＝3)。
图27a不同的嵌合抗原受体修饰改造的人源自然杀伤细胞经细胞裂解与核糖核酸提取，以甘油醛-3-磷酸脱氢酶(GAPDH)基因作为内源性参照基因，针对信使核糖核酸NCR1的表达的定量实时聚合酶链锁反应(平均值±标准差，均为n＝3)。
图27b不同的嵌合抗原受体修饰改造的人源自然杀伤细胞先与PD-L1阳性的人源乳癌肿瘤细胞MDA-MB-231细胞的体外共培养48小时后，人源自然杀伤细胞经细胞裂解与核糖核酸提取，以甘油醛-3-磷酸脱氢酶(GAPDH)基因作为内源性参照基因，针对信使核糖核酸NCR1的表达的定量实时聚合酶链锁反应(平均值±标准差，均为n＝3)。
图28a显示了嵌合抗原受体修饰改造的人源自然杀伤细胞C#3相对于对照组的信使核糖核酸分别有CRTAM、PIK3R6、GZMB、KLRF2的高表达，符合基因功能分类体系(Gene Ontology)GO:0002228自然杀伤细胞介导免疫的基因富集范畴。
图28b显示了嵌合抗原受体修饰改造的人源自然杀伤细胞C#5相对于对照组的信使核糖核酸分别有CRTAM、PIK3R6、GZMB、KLRF2的高表达，符合基因功能分类体系(Gene Ontology)GO:0002228自然杀伤细胞介导免疫的基因富集范畴。
图28c显示了嵌合抗原受体修饰改造的人源自然杀伤细胞C#3相对于C#2的信使核糖核酸分别有CRTAM、PIK3R6、GZMB、KLRF2的高表达，符合基因功能分类体系(Gene Ontology)GO:0002228自然杀伤细胞介导免疫的基因富集范畴。
图28d显示了嵌合抗原受体修饰改造的人源自然杀伤细胞C#5相对于C#2的信使核糖核酸分别有CRTAM、PIK3R6、GZMB、KLRF2的高表达，符合基因功能分类体系(Gene Ontology)GO:0002228自然杀伤细胞介导免疫的基因富集范畴。
图29a显示了嵌合抗原受体修饰改造的人源自然杀伤细胞C#3相对于对照组的信使核糖核酸分别有FOXP3、TNF、CCR7、IL10、LTA、IL18R1、IL1RL1、SLAMF1、XCL1、EBI3的高表达，符合基因功能分类体系(Gene Ontology)GO:0032649γ干扰素产生调控的基因富集范畴。
图29b显示了嵌合抗原受体修饰改造的人源自然杀伤细胞C#5相对于对照组的信使核糖核酸分别有FOXP3、TNF、CCR7、IL10、LTA、IL18R1、IL1RL1、SLAMF1、XCL1、EBI3的高表达，符合基因功能分类体系(Gene Ontology)GO:0032649γ干扰素产生调控的基因富集范畴。
图29c显示了嵌合抗原受体修饰改造的人源自然杀伤细胞C#3相对于C#2的信使核糖核酸分别有FOXP3、TNF、CCR7、IL10、LTA、IL18R1、IL1RL1、SLAMF1、XCL1、EBI3的高表达，符合基因功能分类体系(Gene Ontology)GO:0032649γ干扰素产生调控的基因富集范畴。
图29d显示了嵌合抗原受体修饰改造的人源自然杀伤细胞C#5相对于C#2的信使核糖核酸分别有FOXP3、TNF、CCR7、IL10、LTA、IL18R1、IL1RL1、SLAMF1、XCL1、EBI3的高表达，符合基因功能分类体系(Gene Ontology)GO:0032649γ干扰素产生调控的基因富集范畴。
图30a显示了嵌合抗原受体修饰改造的人源自然杀伤细胞C#3相对于对照组的信使核糖核酸分别有CCL3L1、CCR7、CCL4L1、CCL1、CCL22、CXCR6、CCL4、XCL1、CCL3、XCL2的高表达，符合基因功能分类体系(Gene Ontology)GO:0070098趋化因子介导信号通路的基因富集范畴。
图30b显示了嵌合抗原受体修饰改造的人源自然杀伤细胞C#5相对于对照组的信使核糖核酸分别有CCL3L1、CCR7、CCL4L1、CCL1、CCL22、CXCR6、CCL4、XCL1、CCL3、XCL2的高表达，符合基因功能分类体系(Gene Ontology)GO:0070098趋化因子介导信号通路的基因富集范畴。
图30c显示了嵌合抗原受体修饰改造的人源自然杀伤细胞C#3相对于C#2的信使核糖核酸分别有CCL3L1、CCR7、CCL4L1、CCL1、CCL22、CXCR6、CCL4、XCL1、CCL3、XCL2的高表达，符合基因功能分类体系(Gene Ontology)GO:0070098趋化因子介导信号通路的基因富集范畴。
图30d显示了嵌合抗原受体修饰改造的人源自然杀伤细胞C#5相对于C#2的信使核糖核酸分别有CCL3L1、CCR7、CCL4L1、CCL1、CCL22、CXCR6、CCL4、XCL1、CCL3、XCL2的高表达，符合基因功能分类体系(Gene Ontology)GO:0070098趋化因子介导信号通路的基因富集范畴。
图31a显示了嵌合抗原受体修饰改造的人源自然杀伤细胞C#3相对于对照组的信使核糖核酸分别有FOXP3、TNF、CRTAM、IL10、PIK3R6、LTA、IFNG、SLAMF1、GZMB、KLRF2、XCL1、P2RX7、FCER1G、FAS、C1QA的高表达，符合基因功能分类体系(Gene Ontology)GO:0002449淋巴细胞介导免疫的基因富集范畴。
图31b显示了嵌合抗原受体修饰改造的人源自然杀伤细胞C#5相对于对照组的信使核糖核酸分别有FOXP3、TNF、CRTAM、IL10、PIK3R6、LTA、IFNG、SLAMF1、GZMB、KLRF2、XCL1、P2RX7、FCER1G、FAS、C1QA的高表达，符合基因功能分类体系(Gene Ontology)GO:0002449淋巴细胞介导免疫的基因富集范畴。
图31c显示了嵌合抗原受体修饰改造的人源自然杀伤细胞C#3相对于C#2的信使核糖核酸分别有FOXP3、TNF、CRTAM、IL10、PIK3R6、LTA、IFNG、SLAMF1、GZMB、KLRF2、XCL1、P2RX7、FCER1G、FAS、C1QA的高表达，符合基因功能分类体系(Gene Ontology)GO:0002449淋巴细胞介导免疫的基因富集范畴。
图31d显示了嵌合抗原受体修饰改造的人源自然杀伤细胞C#5相对于C#2的信使核糖核酸分别有FOXP3、TNF、CRTAM、IL10、PIK3R6、LTA、IFNG、SLAMF1、GZMB、KLRF2、XCL1、P2RX7、FCER1G、FAS、C1QA的高表达，符合基因功能分类体系(Gene Ontology)GO:0002449淋巴细胞介导免疫的基因富集范畴。
图32a显示了嵌合抗原受体修饰改造的人源自然杀伤细胞C#3相对于对照组的信使核糖核酸分别有FOXP3、CCR7、PIK3R6、IFNG、GPR183、SLAMF1、CTLA4、GPAM、XCL1、MYB、EBI3、TNFSF14、CD80的高表达，符合基因功能分类体系(Gene Ontology)GO:0051251淋巴细胞激活的正向调控的基因富集范畴。
图32b显示了嵌合抗原受体修饰改造的人源自然杀伤细胞C#5相对于对照组的信使核糖核酸分别有FOXP3、CCR7、PIK3R6、IFNG、GPR183、SLAMF1、CTLA4、GPAM、XCL1、MYB、EBI3、TNFSF14、CD80的高表达，符合基因功能分类体系(Gene Ontology)GO:0051251淋巴细胞激活的正向调控的基因富集范畴。
图32c显示了嵌合抗原受体修饰改造的人源自然杀伤细胞C#3相对于C#2的信使核糖核酸分别有FOXP3、CCR7、PIK3R6、IFNG、GPR183、SLAMF1、CTLA4、GPAM、XCL1、MYB、EBI3、TNFSF14、CD80的高表达，符合基因功能分类体系(Gene Ontology)GO:0051251淋巴细胞激活的正向调控的基因富集范畴。
图32d显示了嵌合抗原受体修饰改造的人源自然杀伤细胞C#5相对于C#2的信使核糖核酸分别有FOXP3、CCR7、PIK3R6、IFNG、GPR183、SLAMF1、CTLA4、GPAM、XCL1、MYB、EBI3、TNFSF14、CD80的高表达，符合基因功能分类体系(Gene Ontology)GO:0051251淋巴细胞激活的正向调控的基因富集范畴。
图33a显示了嵌合抗原受体修饰改造的人源自然杀伤细胞C#3相对于对照组的信使核糖核酸分别有TREM1、CRTAM、S100A12、PIK3R6、IFNG、GZMB、KLRF2、XCL1、P2RX7、GNLY的高表达，符合基因功能分类体系(Gene Ontology)GO:0001906细胞杀伤的基因富集范畴。
图33b显示了嵌合抗原受体修饰改造的人源自然杀伤细胞C#5相对于对照组的信使核糖核酸分别有TREM1、CRTAM、S100A12、PIK3R6、IFNG、GZMB、KLRF2、XCL1、P2RX7、GNLY的高表达，符合基因功能分类体系(Gene Ontology)GO:0001906细胞杀伤的基因富集范畴。
图33c显示了嵌合抗原受体修饰改造的人源自然杀伤细胞C#3相对于C#2的信使核糖核酸分别有TREM1、CRTAM、S100A12、PIK3R6、IFNG、GZMB、KLRF2、XCL1、P2RX7、GNLY的高表达，符合基因功能分类体系(Gene Ontology)GO:0001906细胞杀伤的基因富集范畴。
图33d显示了嵌合抗原受体修饰改造的人源自然杀伤细胞C#5相对于C#2的信使核糖核酸分别有TREM1、CRTAM、S100A12、PIK3R6、IFNG、GZMB、KLRF2、XCL1、P2RX7、GNLY的高表达，符合基因功能分类体系(Gene Ontology)GO:0001906细胞杀伤的基因富集范畴。
图34显示了表1，包含不同版本的嵌合蛋白构建体，其示出了根据本公开内容的嵌合蛋白的实例，包括基于免疫检查点PD-1融合的嵌合抗原受体。
图35显示了慢病毒载体的载体图谱，其中包含有具有代表性的两个版本：(a)基于免疫检查点PD-1融合的嵌合抗原受体C#3版本和(b)基于免疫检查点PD-1融合的嵌合抗原受体C#5版本。基于免疫检查点PD-1融合的嵌合抗原受体C#3和C#5版本所包含的各组成部分信息请见图34以及本申请相关内容。
具体实施方式
下面结合实施例详述本申请，但本申请并不局限于这些实施例。本发明决不应被解释为受限于以下实施例，而是应被解释为涵盖由于本文提供的教导而显而易见的任何和所有改动。
无进一步描述时，认为本领域的普通技术人员能够利用前文描述和下文示例性实施历来制备和应用本发明的化合物以及实践请求保护的方法。因此，下文工作实施例具体地指出了本发明的优选实施方式，而不被解释为以任何方式限制本公开的其余部分。
如无特别说明，本申请的实施例中的原料均通过商业途径购买。
本申请具体实施例中所使用的NK细胞为NK-92细胞株；
图4～图7的检测方法为使用荧光能量共振转移的显微镜成像方法(Ishikawa-Ankerhold HC等，Molecules.2012Apr；17(4):4047-132.)去检测不同人工分子机器在响应不同外界刺激性输入信号时相应的胞内检测信号传导结构域#V磷酸化表现和胞内激活信号传导结构域#VII部分分子构象的状态变化以及相应的激活状态表现。
现对这些实验中的使用的材料和方法进行描述。
本申请实施例中，“分子机器”、“嵌合抗原受体”均为嵌合蛋白，为本申请的示范例，包含不同版本的嵌合抗原受体构建体。此外，将胞外靶标分子结合结构域(如PD-1胞外片段或者靶向scFv)、胞外间隔区结构域、跨膜区结构域、胞内间隔区结构域、胞内检测信号传导结构域(属于检测模块)、胞内铰链结构域、胞内激活信号传导结构域(属于激活模块)与胞内信号传导结构域分别依次编号为结构域#I至结构域#VIII，如无特别说明，本申请中相应内容均适用。
根据本申请的一个方面，构建嵌合抗原受体(分子机器)，包括：
a)细胞外结构域，用于特异性结合靶标分子；
b)胞内信号传导结构域，包括至少一个免疫细胞激活信号通路元件；所述免疫细胞激活信号通路元件的激活至少依赖于所述细胞外结构域与所述靶标分子的结合；和
c)跨膜区结构域，用于连接所述细胞外结构域和所述胞内信号传导结构域，并将二者固定在细胞膜上。
d)胞外间隔区结构域，所述胞外靶标分子结合结构域和所述跨膜区结构域通过所述胞外间隔区结构域连接。
e)胞内间隔区结构域，所述跨膜区结构域和所述胞内信号传导结构域通过所述胞内间隔区结构域连接。
嵌合抗原受体所识别的靶标分子包括肿瘤表面抗原分子标志物、细胞表面特定的抗原肽-组织相容性复合体分子或免疫抑制信号相关分子等靶标分子中的至少一种。
细胞外结合结构域选自现有嵌合抗原受体中常用的抗免疫抑制信号相关分子单克隆抗体及其抗原识别结合片段、抗肿瘤表面抗原分子标志物的单克隆抗体、单克隆抗体或单链可变片段及其抗原识别结合片段及其抗原识别结合片段。优选为可识别结合免疫抑制信号相关分子、肿瘤表面抗原分子标志物的分子中的至少一种。也可以为可识别肿瘤表面抗原分子标志物、细胞表面特定的抗原肽-组织相容性复合体分子或结合免疫抑制信号相关分子等靶标分子的分子中的至少一种，也可以为现有嵌合抗原受体中常用的单克隆抗体或单链可变片段及其抗原识别结合片段、抗免疫抑制信号相关分子单克隆抗体及其抗原识别结合片段、抗肿瘤表面抗原分子标志物的单克隆抗体及其抗原识别结合片段或抗细胞表面特定的抗原肽-组织相容性复合体分子的单克隆抗体及其抗原识别结合片段。优选为可识别结合免疫抑制信号相关分子、肿瘤表面抗原分子标志物分子或细胞表面特定的抗原肽-组织相容性复合体分子的分子中的至少一种。
胞内信号传导结构域，包括至少一个胞内激活信号结构域，优选为免疫细胞激活信号通路元件；所述胞内激活信号结构域含有具有催化功能基团的分子或其片段；所述胞内激活信号结构域的激活至少依赖于所述胞外靶标分子结合结构域与所述靶标分子的结合。胞内信号传导结构域含有具有催化功能基团的分子或其片段，能够使得嵌合抗原受体脱离对特定细胞类型的限制，扩展到对具有催化功能基团的分子具备适用性的细胞类型中，即拓展了本申请所述的嵌合抗原受体能够赋予经基因修饰以表达所述嵌合抗原受体的宿主细胞类型的范围。
跨膜区结构域，现有的跨膜蛋白均可以用于该技术，没有其它要求。
在某些实施方式中，嵌合抗原受体靶向与凋亡、死亡、濒死、损伤、感染、病变、或坏死细胞相关的杀伤信号分子。在某些实施方式中，嵌合抗原受体靶向与感染性微生物或颗粒相关的抗体结合细胞。在另外的实施方式中，嵌合抗原受体靶向与疾病、病症或其他不利病况相关的异常细胞、新生肿瘤相关抗原、错误折叠蛋白显示出的抗原信号分子。
可以将根据本说明书的一种或多种嵌合抗原受体转导致NK细胞并在其中表达。在某些实施方式中，对嵌合抗原受体之胞外靶标分子结合结构域进行工程化改造以使其与特定靶分子结合。在某些实施方式中，对嵌合抗原受体之胞内信号传导结构域进行选择以提供所需的杀伤活性。在某些实施方式中，除了对嵌合抗原受体之胞外靶标分子结合结构域进行工程化改造以使其与特定靶分子結合，还对嵌合抗原受体之胞内信号传导结构域进行选择以提供所需的杀伤活性。在一个此类实施方式中，胞内信号传导结构域至少包含一个或多个胞内激活信号传导结构域。在一个此类实施方式中，胞内信号传导结构域包含一个或多个胞内检测信号传导结构域与胞内激活信号传导结构域；所述胞内检测信号传导结构域与所述胞内激活信号传导结构域连接。在一个此类实施方式中，胞内信号传导结构域包含一个或多个胞内检测信号传导结构域与胞内激活信号传导结构域；所述胞内检测信号传导结构域与所述胞内激活信号传导结构域经胞内铰链结构域连接。
可以将经基因修饰以表达一个或多个根据本说明书的所述嵌合抗原受体的NK细胞用于特异性杀伤表达嵌合抗原受体的胞外域结合的靶分子的靶细胞或颗粒。在某些实施方式中，所述靶细胞或颗粒可以是与感染、疾病、病症、或其他不利病况相关的肿瘤细胞、癌细胞、微生物(例如，细菌、真菌、病毒)、原生动物寄生虫、异常细胞、新生肿瘤抗原或错择叠蛋白。在进一步的实施方式中，将经基因修饰以表达一个或多个根据本说明书的嵌合抗原受体的NK细胞用于在对象中治疗癌症、感染性疾病(病毒、细菌、真菌、原生动物)、炎性疾病、免疫疾病(例如，自身免疫性疾病)或神经退行性疾病(例如，阿尔兹海默氏病)，作为主要疗法或者作为辅助或联合疗法。可以对本公开内容的嵌合抗原受体进行设计以通过选择胞外靶标分子结合结构域赋予其特异性的杀伤表型，其取决于靶分子和治疗适应症，以将嵌合抗原受体用于改善癌症的微环境和增强肿瘤消退。
定义
在更详细地阐述本公开内容之前，提供在本申请中使用的某些术语的定义，可能有助于理解本公开内容。
相位对比成像：为一种基于相位对比法进行成像的技术。
序列同源性：将在本申请中，指的是两个或两个以上的核酸分子之间、两个或两个以上的蛋白质序列之间在编码序列上具有明显的相似性，例如具有至少80％以上、至少81％以上、至少82％以上、至少83％以上、至少84％以上、至少85％以上、至少86％以上、至少87％以上、至少88％以上、至少89％以上、至少90％以上、至少91％以上、至少92％以上、至少93％以上、至少94％以上、至少95％以上、至少96％以上、至少97％以上、至少98％以上、至少99％以上、至少99.5％以上或至少100％序列编码的同一性。
PD-L1结合片段：将在本申请中，指的是可特异性结合PD-L1的分子或分子片段，比如抗体片段等。
催化功能：指的是在机体内的化学反应中，以酶作为催化剂，以加快化学反应的速度。其中，酪氨酸激酶(tyrosine kinase)是一种在细胞中可以催化磷酸基团从ATP中转移到蛋白质的酪氨酸残基上的酶，从而起到调控细胞中信号通路的“开”与“关”。在本申请中的所使用的酪氨酸激酶，包括ZAP70及SYK等。
肿瘤微环境(Tumor microenvironment)：指的是肿瘤细胞所存在的周围微环境，包括免疫细胞、成纤维细胞、骨髓源性炎性细胞、周围的血管、细胞外基质和各种信号分子。肿瘤和周围环境不断地进行交互作用，两者密切相关，微环境(如其中的免疫细胞)可影响癌细胞的增长和发育，而肿瘤可以通过释放细胞信号分子，从而影响其微环境，如，促进肿瘤的血管生成及诱导免疫耐受。肿瘤微环境有助于肿瘤异质性的形成。
构象：指在一个分子中，不改变其共价键结构，仅单键周围的原子放置所产生的空间排布。优势构象指的是在不同形式的构象中，势能最低、最稳定的构象。不同的构象之间可以相互转变，从某一种构象改变为另外一种构象过程中，共价键无需断裂和重新形成。分子的构象不仅对化合物的理化性质有影响，而且对生物大分子(如核酸、蛋白质、酶等)的结构和性能也有重要影响产生影响。
细胞表面特定的抗原肽-组织相容性复合体分子：指的是在抗原呈现的途径中，抗原决定位胜肽首先要经过蛋白酶体切割，然后与抗原加工相关传递蛋白(TAP)结合，再与主要组织相容性复合体(MHC)分子结合，最后运送到抗原呈现分子表面形成特定的抗原肽-组织相容性复合体分子，免疫细胞可以识别特定的抗原肽-组织相容性复合体分子在细胞表面所呈递特定的抗原肽。
免疫抑制性信号相关分子：免疫检查点是一种刺激性或抑制性的信号相关分子，抑制性蛋白不会传导信号，而共刺激蛋白会传导信号从而促进对病原体的免疫反应。
截短体：在本申请中指的是因为一段序列被删除而变短的片段。
蛋白突变体：在本申请中指的是改变原有蛋白的氨基酸序列，以期获得失去功能或者具有功能的突变蛋白。
免疫检查点：指的是免疫系统的内在调控机制的相关分子，如免疫检查点PD-1和CTLA-4，这些分子不仅可保持自身耐受性，而且可以避免在生理性免疫应答期间可能带来的附带损伤。目前已知，肿瘤可以通过建造微环境，从而以逃避免疫的监视和攻击，尤其是通过调节某些特定的免疫检查点通路来进行。
肿瘤免疫逃逸(Tumor immune escape)：指的是，肿瘤细胞可以通过多种不同机制来逃避机体免疫系统的识别和攻击，从而实现在体内生存和增殖的目的的现象。当出现恶变细胞时，机体的免疫系统可以识别，然后通过免疫机制特异地清除这些恶变细胞，以阻止肿瘤的发生发展。但是，恶变细胞也可能会通过不同的机制来躲过机体的免疫监视，在体内不断增殖，形成肿瘤。
免疫抑制：指的是对免疫应答的抑制作用，也就是机体在某些情况下会对自身的组织成分不产生免疫应答，从而保持自身的耐受性，简单地说，指的是免疫系统对某些特定抗原的特异性不产生应答的状态。
NK细胞：即自然杀伤细胞(Natural Killer cell，NK细胞)是机体重要的免疫细胞，不仅与抗肿瘤、抗病毒感染和免疫调节有关，而且在某些情况下参与超敏反应和自身免疫性疾病的发生，能够识别靶细胞、杀伤清除介质。
“核酸分子”和“多核苷酸”：在本申请中，包括RNA或DNA形式，具体包括基因组DNA、cDNA和合成DNA。核酸分子包括双链核酸分子或单链核酸分子，单链核酸分子包括编码链或反义链。
嵌合：在本申请中，指的是包含结合或连接在一起的序列非内源性的(在自然界中通常不会结合或连接在一起)，的蛋白或核酸分子。例如，嵌合核酸分子可以包含来自不同来源的调控序列和编码序列，或者来自相同来源但是以不同于天然存在的方式排列的调控序列和编码序列。
阳性：在本申请中，指的是特定细胞中的特定分子标记物的表达达到一定水平。例如，PD-L1阳性肿瘤细胞指的是肿瘤细胞中的PD-L1蛋白分子的表达达到一定水平。
癌症：在本申请中，指的是以异常细胞的失控和快速生长为特征的疾病。这些异常细胞可以形成恶性血液病或构成实体瘤。癌细胞通过淋巴系统、血流扩散到全身各部位，或者仅在局部扩散。各种癌症的实例包括但不限于卵巢癌、宫颈癌、乳腺癌、前列腺癌、结肠直肠癌、肾癌、皮肤癌、脑癌、肺癌、胰腺癌、淋巴瘤、白血病、肝癌等。
治疗：在本申请中，指的是获得有益或期望的临床效果的方法。出于本发明的目的，有益或期望的临床效果包括但不限于如下的一种或多种：阻止肿瘤细胞的转移，抑制肿瘤或癌细胞的增殖或扩散(或破坏癌细胞肿瘤)，消退PD-L1相关疾病(例如癌症)，减轻PD-L1相关疾病(例如癌症)引起的症状，减小表达PD-L1的肿瘤尺寸，降低治疗PD-L1相关疾病(例如癌症)所需药物的剂量，提高PD-L1相关疾病(例如癌症)患者的生活质量，减缓PD-L1相关疾病(例如癌症)的进展，和/或延长PD-L1相关疾病(例如癌症)患者的存活期，治愈PD-L1相关疾病(例如癌症)。
高表达：在本申请中，指的是特定细胞有高水平的特定分子标记物表达。例如，PD-L1高表达的肿瘤细胞指的是肿瘤细胞中的PD-L1蛋白分子的表达水平高。高表达的肿瘤细胞标记物一般是与疾病状态相关，例如，在特定器官或组织内形成实体瘤的细胞中和在恶性血液病的细胞中，可以通过利用本领域公知的标准进行测定从而确定由肿瘤标记物高表达表征的实体瘤或恶性血液病。
载体：在本申请中，指的是能够转运另一核酸的核酸分子。载体包括质粒、病毒、噬菌体、粘粒。还包括可以促进核酸转移到细胞中非病毒和非质粒化合物。“表达载体”指将其置于适宜环境中时，可以指引由载体所携带的一个或一个以上的基因编码的蛋白表达载体。病毒载体包括腺相关病毒载体、腺病毒载体、逆转录病毒载体、慢病毒载体和γ逆转录病毒载体。“逆转录病毒”指的是具有RNA基因组的病毒。“慢病毒”指的是能够感染分裂和非分裂细胞的逆转录病毒的一个属。慢病毒包括牛免疫缺陷病毒(BIV)、猿猴免疫缺陷病毒(SIV)、人类免疫缺陷病毒(HIV，包括1型HIV和2型HIV)、猫免疫缺陷病毒(FIV)、马感染性贫血病毒。“γ逆转录病毒”指的是逆转录病毒科的一个属。γ逆转录病毒包括但不限于猫白血病病毒、猫肉瘤病毒、小鼠白血病病毒、禽类网状内皮细胞增生病毒、小鼠干细胞病毒。非病毒载体包括经修饰的mRNA(modRNA)、自身扩增mRNA、基于脂质的DNA载体、转座子介导的基因转移(PiggyBac，Sleeping Beauty)、封闭式线形双链体(CELiD)DNA。当采用非病毒作为递送系统时，可以利用脂质体作为递送载剂。在体外、离体或体 内，通过使用脂质制剂将核酸引入宿主细胞。将核酸包封在脂质体内部，分散在脂质体的脂质双层内，通过将核酸和脂质体结合在一起的连接分子附接至脂质体而与脂质结合。
胞外靶标分子结合结构域：在本申请中，指的是具有特异性地和非共价地结合、缔合、联合(unite)、或识别靶分子能力的分子，如肽、寡肽、多肽或蛋白，所结合的靶分子包括：IgA抗体、CD138、CD38、L1CAM、CD22、CD19、PD-1、CD79b、间皮素、PSMA、CD33、CD123、BCMA、ROR1、MUC-16、IgG抗体、IgE抗体、EGFRviii、VEGFR-2或GD2。靶标分子结合结构域包括任何半合成、合成的、重组产生的、天然存在的，可以针对目标生物分子或其他靶点的结合配偶体。靶标分子结合结构域可以是抗原结合结构域，包括抗体、其有功能的结合结构域、抗原结合部分等。结合结构域可以包括单链抗体可变区(例如，sFv、Fab、scFv、结构域抗体)、配体(例如，趋化因子、细胞因子)、受体胞外域(例如，PD-1)或者因具有与生物分子的特异性结合能力而选择的合成多肽。
胞内激活信号传导结构域：在本申请中，指的是选自具有催化功能的非受体型酪氨酸激酶或受体型酪氨酸激酶分子或片段，表达激活信号传导结构域的细胞接受适宜信号时，其能够促进生物或生理应答。激活信号传导结构域可以是结合时接收信号的蛋白或蛋白复合物的一部分。例如，激活信号传导结构域可以对PD-1融合的嵌合抗原受体与靶分子PD-L1的结合产生应答，从而向宿主细胞的内部传导信号，激发效应功能，例如分泌炎性细胞因子和/或趋化因子、分泌抗炎性和/或免疫抑制性细胞因子、NK细胞有效杀伤肿瘤细胞作用。激活信号传导结构域也可以将通过与一个或一个以上直接促进细胞应答的其他蛋白结合来间接促进细胞应答。
检测信号传导结构域：在本申请中，指的是免疫受体酪氨酸激活基序(immunoreceptor tyrosine-based activation motif，ITAM)是一个由十多个氨基酸构成的保守序列。当时，嵌合抗原受体分子机器的检测信号传导结构域可以响应酪氨酸激酶活化信号输入并发生磷酸化修饰，进而与激活信号传导结构域发生基于磷酸化位点修饰的相互作用，并将其激活信号传导结构域从自抑制的分子构象状态下解开，释放激活信号传导结构域，在激活信号传导结构域得到释放后的分子构象下的分子机器的激活信号传导结构域处于开放的激活状态。初级检测信号转导序列可包括已知为免疫受体酪氨酸激活基序(ITAM)的信号基序。ITAM是在各种受体的胞质内尾中发现的良好定义的信号基序，其用作酪氨酸激酶的结合位点。在本发明中使用的ITAM的实例可以包括：2B4、CD244、BTLA、CD3δ、CD3γ、CD3ε、CD3ζ、CD5、CD28、CD31、CD72、CD84、CD229、CD300a、CD300f、CEACAM-1、CEACAM-3、CEACAM-4、CEACAM-19、CEACAM-20、CLEC-1、CLEC-2、CRACC、CTLA-4、DAP10、DAP12、DCAR、DCIR、Dectin-1、DNAM-1、FcεRIα、FcεRIβ、FcγRIB、FcγRI、FcγRIIA、FcγRIIB、FcγRIIC、FcγRIIIA、FCRL1、FCRL2、FCRL3、FCRL4、FCRL5、FCRL6、G6b、KIR、KIR2DL1、KIR2DL2、KIR2DL3、KIR2DL4、KIR2DL5、KIR2DL5B、KIR2DS1、KIR2DS3、KIR2DS4、KIR2DS5、KIR3DL1、KIR3DL2、KIR3DL3、KIR3DS1、KLRG1、LAIR1、LILRB1、LILRB2、LILRB3、LILRB4、LILRB5、MICL、NKG2A、NKp44、NKp65、NKp80、NTB-A、PD-1、PDCD6、PILR-α、Siglec-2、Siglec-3、Siglec-5、Siglec-6、Siglec-7、Siglec-8、Siglec-9、Siglec-10、Siglec-11、Siglec-12、Siglec-14、Siglec-15、Siglec-16、SIRPα、SLAM、TIGIT、TREML1、TREML2。
胞内信号传导结构域：在本申请中，指的是胞内效应结构域，当免疫细胞表面的嵌合抗原受体分子机器的胞外靶标分子结合结构域识别并结合靶分子，从而通过该识别结合提供靶分子识别结合信号输入，然后胞内部分的分子构象会发生改变从而将其激活信号传导结构域从自抑制的分子构象状态下解开，最终在响应上游的靶分子识别结合信号输入下胞内的激活信号传导结构域得到充分的基于嵌合抗原受体分子机器分子构象变化的激活信号传导结构域的释放与激活，且激活状态下的激活信号传导结构域可以进一步激活其下游的多种信号通路，从而是嵌合抗原受体修饰改造的免疫细胞对靶细胞行使特定的功能。在某些实施方式中，信号传导结构域激活导致宿主细胞对靶细胞、微生物或颗粒的杀伤作用的一个或多个信号传导通路。在某些实施方式中，信号传导结构域包含至少一个胞内激活信号传导结构域。在某些其他实施方式中，信号传导结构域包含至少一个胞内检测信号传导结构域与至少一个胞内激活信号传导结构域。在某些其他实施方式中，信号传导结构域包含至少一个胞内检测信号传导结构域、胞内铰链结构域与至少一个胞内激活信号传导结构域。
跨膜区结构域：在本申请中，指的是一种跨越整个生物膜一次的多肽，用于连接胞外靶标分子结合结构域和胞内信号传导结构域，并将二者固定在细胞膜上。
胞内间隔区结构域：在本申请中，指的是位于跨膜区结构域和胞内信号传导结构域之间并将这两者连接在一起，可为跨膜区结构域之延伸。
胞内铰链结构域：在本申请中，指的是连接胞内检测信号传导结构域与胞内激活信号传导结构域，可选为柔性连接肽片段。铰链结构域可提供所需的灵活性，以允许所需的嵌合多肽的表达、活性和/或构象定位。铰链结构域可以具有任何合适的长度以连接至少两个感兴趣的结构域，并且优选设计为足够柔性以便允许其连接的一个或两个结构域的正确折叠和/或功能和/或活性。铰链结构域的长度至少为3个或以上、5个或以上、10个或以上、15个或以上、20个或以上、25个或以上、30个或以上、35个或以上、40个或以上、45个或以上、50个或以上、55个或以上、60个或以上、65个或以上、70个或以上、75个或以上、80个或以上、85个或以上、90个或以上、95个或以上、90个或以上、95个或以上或100个或以上的氨基酸。在一些实施方式中，铰链结构域的长度约0～200个氨基酸；优选地，约10～190个氨基酸；优选地，约20～180个氨基酸；优选地，约30～170个氨基酸；优选地，约40～160个氨基酸；优选地，约50～150个氨基酸；优选地，约60～140个氨基酸；优选地，约70～130个氨基酸；优选地，约80～120个氨基酸；优选地，约90～110个氨基酸。铰链结构域序列也可以包含内源性蛋白序列。铰链结构域序列可以包含甘氨酸、丙氨酸和/或丝氨酸残基。铰链结构域可以含基序，例如GGSG，SGGG，GS，GGS或GGGGS的多个或重复基序。铰链结构域序列可以包括任何非天然存在的氨基酸、天然存在的氨基酸或其组合。
其它定义贯穿于本公开内容通篇之中。
实施例1构建与表达嵌合抗原受体
构建免疫检查点PD-1融合的嵌合抗原受体分子机器及载体。
(1)通过基因工程与分子生物学手段，将嵌合抗原受体的胞内部分的结构域#VIII(包括作为激活元件的结构域#VII、作为检测元件的结构域#V及作为连接元件的结构域#VI)与作为胞外识别元件的结构域#I、结构域#III以及结构域#II、结构域#IV(请见图2)使用Gibson Assembly进行连接融合，然后克隆到基因表达载体(如pCAG或pCDNA3或pMSCV逆转录病毒载体或pSIN慢病毒载体等)上进行后续体外与体内研究。
其中如图2h，结构域#I可选为PD-L1受体PD-1的配体识别结合部分，结构域#II可选为PD-1的跨膜区部分的胞外延伸片段(即胞外靶标分子PD-L1结合结构域与PD-1的跨膜区之间)，结构域#III可选为PD-1的跨膜区部分，结构域#IV可选为PD-1的跨膜区部 分的胞内延伸片段(即图34中Full-length PD-1或Truncated PD-1的胞内部分；其中C#1Full-length PD-1的全长氨基酸序列为SEQ ID NO:001+SEQ ID NO:016+SEQ ID NO:012+SEQ ID NO:056和全长DNA核酸序列为SEQ ID NO:002+SEQ ID NO:017+SEQ ID NO:013+SEQ ID NO:057，C#2Truncated PD-1的全长氨基酸序列为SEQ ID NO:001+SEQ ID NO:016+SEQ ID NO:012+SEQ ID NO:054和全长DNA核酸序列为SEQ ID NO:002+SEQ ID NO:017+SEQ ID NO:013+SEQ ID NO:055)，结构域#VII可选为SYK/ZAP70家族成员等的酪氨酸激酶部分，结构域#V可选为CD3ζ、CD3ε、FcRIIA、FcRγ、DAP12等分子的免疫受体酪氨酸活化基序片段部分(即图34中Sub1～Sub7：CD3ζITAM1～3、CD3εITAM、FcRIIA ITAM、FcRγITAM、DAP12ITAM)，连接结构域#VII与结构域#V的结构域#VI可选为柔性连接肽片段(即图34中的不同长度连接肽：SL、ML、LL1、LL2)，请见图2和图34。最终，分别构建了图34中所列举的多种不同版本的人工分子机器，包括C#1Full-length PD-1、C#2Truncated PD-1、C#3Truncated PD-1-Sub1-LL1-ZAP70、C#4Truncated PD-1-Sub1-LL1-ZAP70-ΔKD、C#5Truncated PD-1-Sub5-LL1-SYK、C#6Truncated PD-1-Sub6-LL1-SYK、C#7Truncated PD-1-Sub7-LL1-SYK、C#8Truncated PD-1-Sub4-LL1-SYK、C#9Sub1-LL2-ZAP70、C#10Sub1FF-LL2-ZAP70、C#11Sub2-LL2-ZAP70、C#12Sub2FF-LL2-ZAP70、C#13Sub3-LL2-ZAP70、C#14Sub3FF-LL2-ZAP70、C#15Sub4-LL2-SYK、C#16Sub4FF-LL2-SYK、C#17Full-length PD-1-Sub1-LL2-ZAP70、C#18Full-length PD-1-Sub1FF-LL2-ZAP70、C#19Truncated PD-1-Sub1-LL2-ZAP70以及C#20Truncated PD-1-Sub1FF-LL2-ZAP70。
(2)通过脂质体转染手段，在小鼠胚胎成纤维细胞(MEF)和人源海拉(HeLa)细胞细胞中表达不同嵌合抗原受体人工分子机器。然后，使用显微镜成像方法表征人工分子机器于小鼠胚胎成纤维细胞(MEF)和人源海拉(HeLa)细胞内的表达特征以及响应外界刺激性输入信号的表现，详见图4、图5、图6与图7。使用含10％FBS的DMEM培养基培养人源海拉细胞和小鼠胚胎成纤维细胞MEF。
另一方面，通过DNA转染在人源293T细胞中表达人工分子机器蛋白、纯化并使用纯化后的蛋白进行细胞外功能性测试与验证，尤其是比较在特异性的蛋白酪氨酸磷酸化信号输入下不同的结构域#V和结构域#VII的表现，详见图6。使用含10％FBS的DMEM培养基培养人源293T细胞。
实施例2检测与表征嵌合抗原受体
基于图2和图3采用多种检测表征手段，包括但不限于，通过纯化蛋白的形式检测并表征嵌合抗原受体在细胞外的功能表现以及通过不同手段来检测并表征嵌合抗原受体在真核细胞内的功能表现。
其中，图3显示了人工分子机器的信号激活示意图简图。其中，图3a显示了在酪氨酸激酶活化信号输入的情况下人工分子机器的激活信号释放并激活，图3b显示了在靶细胞靶分子信号输入(如PD-L1)的情况下含有结构域#I(如PD-1胞外部分)的嵌合抗原受体人工分子机器的激活信号释放并激活。
图3a的分子机器工作模型为简化模型，包括结构域#VII、结构域#VI与结构域#V。其中结构域#VII可选为SYK/ZAP70家族成员等的酪氨酸激酶部分，结构域#V可选为CD3ζ、CD3ε、FcRIIA、FcRγ、DAP12等分子的免疫受体酪氨酸活化基序片段部分(即Sub1至Sub7：依次为CD3ζITAM1～3、CD3εITAM、FcRIIA ITAM、FcRγITAM、DAP12ITAM)，连接结构域#VII与结构域#V的结构域#VI可选为柔性连接肽片段，请见图34。
基于SYK/ZAP70家族成员的分子构象的特点，在其没有激活的状态下，SYK或ZAP70会处于自抑制的分子构象状态(Yan Q等，Molecular and cellular biology.2013Jun 1；33(11):2188-201.)，此构象下分子机器的结构域#VII处于关闭的非激活状态；当酪氨酸激酶活化信号输入时，尤其是免疫受体酪氨酸激活基序的磷酸化信号输入，分子机器的结构域#V会响应信号输入并发生磷酸化修饰，进而磷酸化修饰后的结构域#V会与SYK或ZAP70发生基于磷酸化位点修饰的相互作用，尤其是在结构域#VI的柔性连接肽片段提供充足的分子机器构象改变灵活度的情况下，从而将其结构域#VII从自抑制的分子构象状态下解开，释放结构域#VII，在结构域#VII得到释放后的分子构象下的分子机器的结构域#VII处于开放的激活状态，即图3a所示的在酪氨酸激酶活化信号输入的情况下人工分子机器的信号激活示意图，且激活状态下的结构域#VII可以进一步激活其下游的多种信号通路。基于该工作原理，使用荧光能量共振转移的显微镜成像方法(Ishikawa-Ankerhold HC等，Molecules.2012Apr；17(4):4047-132.)去检测不同人工分子机器在响应不同外界刺激性输入信号时相应的结构域#V磷酸化表现和结构域#VII部分分子构象的状态变化以及相应的激活状态表现。
图3b的分子机器工作模型为与图3a工作原理相似的模型，包括七部分：结构域#I至结构域#VII。如图2h所示，结构域#I可选为PD-L1受体PD-1的配体识别结合部分，结构域#II可选为PD-1的跨膜区部分的胞外延伸片段(即胞外靶标分子PD-L1结合结构域与PD-1的跨膜区之间)，结构域#III可选为PD-1的跨膜区部分，结构域#IV可选为PD-1的跨膜区部分的胞内延伸片段(即图34中Truncated PD-1的胞内部分)，结构域#VII可选为SYK/ZAP70家族成员等的酪氨酸激酶部分，结构域#V可选为CD3ζ、CD3ε、FcRIIA、FcRγ、DAP12等分子的免疫受体酪氨酸活化基序片段部分(即图34中Sub1至Sub7：CD3ζITAM1～3、CD3εITAM、FcRIIA ITAM、FcRγITAM、DAP12ITAM)，连接结构域#VII与结构域#V的结构域#VI可选为柔性连接肽片段(即图34中的不同长度连接肽：SL、ML、LL1、LL2)，请参见图2h和图34。
再次地，基于SYK/ZAP70家族成员的分子构象的特点，在其没有激活的状态下，SYK或ZAP70会处于自抑制的分子构象状态，此构象下分子机器的结构域#VII处于关闭的非激活状态；当靶细胞的靶分子存在时，免疫细胞表面的嵌合抗原受体分子机器的结构域#I会识别并结合靶分子，从而通过该识别结合提供靶分子识别结合信号输入，然后胞内部分的分子构象会发生与上述图3a所述类似的变化，最终在响应上游的靶分子识别结合信号输入下胞内的结构域#VII得到充分的基于嵌合抗原受体分子机器分子构象变化的结构域#VII的释放与激活，且激活状态下的结构域#VII可以进一步激活其下游的多种信号通路，从而是嵌合抗原受体修饰改造的免疫细胞对靶细胞行使特定的功能，如NK细胞的细胞毒性功能等。故，图3b为所示的在靶分子识别结合信号输入的情况下嵌合抗原受体人工分子机器的信号激活示意图。同样地，类比上述图3a部分，基于该工作原理，使用显微镜成像方法(荧光能量共振转移)去检测不同设计的嵌合抗原受体人工分子机器在响应不同外界刺激性输入信号时相应的结构域#V磷酸化表现和结构域#VII部分分子构象的状态变化以及相应的激活状态表现。为了量化分析的便利，采用显微镜成像方法去表征不同人工分子机器的功能，并采用成像读数指标来代表人工分子机器对刺激信号的响应能力的程度以及响应刺激信号同时引发的人工分子机器基于分子构象改变的对其自身激活元件的释放与激活的程度。
通过DNA转染使人源及鼠源等哺乳动物细胞表达不同的分子机器蛋白，从而使用荧光显微镜成像方法去检测并表征不同人工分子机器在人源海拉(HeLa)细胞与小鼠胚胎成纤维细胞(MEF)内响应多种不同外界刺激性输入信号的表现。
图4a证明了在人源海拉细胞中C#9和C#15的胞内检测信号传导结构域Sub1和Sub4对蛋白酪氨酸磷酸化信号非常出色的响应能力以及C#9和C#15相应的非常明显分子构象的改变并对其自身激活元件——结构域#VII(SYK和ZAP70)的非常充分显著释放与激活，且显著优于C#11和C#13。此外，在自身激活元件被失能的情况下(失活性突变体Sub1FF～Sub4FF)，C#10、C#12、C#14、C#16分别较相对应的C#9、C#11、C#13、C#15版本具有统计分析后显著差异的更弱的近乎为零的对蛋白酪氨酸磷酸化信号的响应能力，证明C#9、C#11、C#13和C#15的结构域#V(Sub1～Sub4)对蛋白酪氨酸磷酸化信号出色响应能力的重要性且C#9的结构域#V(Sub1)和C#15的结构域#V(Sub4)较C#11的结构域#V(Sub2)和C#13的结构域#V(Sub3)具有显著更佳的对蛋白酪氨酸磷酸化信号响应能力及敏感性。此外，使用20uM酪氨酸磷酸酶抑制剂过钒酸钠可以抑制细胞内蛋白去磷酸化作用，从而促进蛋白酪氨酸磷酸化信号的激活，起到提供蛋白酪氨酸磷酸化信号输入的作用。
图4b显示了在20uM酪氨酸磷酸酶抑制剂过钒酸钠激活蛋白酪氨酸磷酸化信号的A条件或在50ng/mL表皮生长因子激活信号的B条件下，不同的人工分子机器在人源海拉细胞中的表现结果(平均值±标准差，均为n＝6)，成像读数指标代表量化后人工分子机器对刺激信号的响应能力的程度以及响应刺激信号同时引发的人工分子机器基于分子构象改变的对其自身激活元件的释放与激活的程度。而且，图4b证明了在人源海拉细胞中C#9和C#15的结构域#V(Sub1和Sub4)对蛋白酪氨酸磷酸化信号非常出色的响应能力以及C#9和C#15相应的非常明显分子构象的改变并对其自身激活元件—结构域#VII(SYK和ZAP70)的非常充分显著释放与激活。此外，在表皮生长因子激活信号的条件下，C#9和C#15具有统计分析后显著差异的更弱的近乎为零的对该信号的响应能力，证明C#9和C#15的结构域#V(Sub1和Sub4)对蛋白酪氨酸磷酸化信号出色响应能力的重要性且保证了人工分子机器对特定的蛋白酪氨酸磷酸化信号的特异性响应，而不会响应不相关的信号输入，比如表皮生长因子激活信号。在此，表皮生长因子可以结合HeLa细胞表面的表皮生长因子受体从而提供表皮生长因子激活信号，该信号不参与免疫受体酪氨酸激活基序的磷酸化，故无法特异性地被C#9和C#15的结构域#V所检测到。
图4c显示了在20uM酪氨酸磷酸酶抑制剂过钒酸钠激活蛋白酪氨酸磷酸化信号的A条件或在50ng/mL血小板源生长因子激活信号的B条件下，不同的人工分子机器在小鼠胚胎成纤维细胞(MEF)中的表现结果(平均值±标准差，均为n＝6)，成像读数指标代表量化后人工分子机器对刺激信号的响应能力的程度以及响应刺激信号同时引发的人工分子机器基于分子构象改变的对其自身激活元件的释放与激活的程度。而且，图4c证明了在小鼠胚胎成纤维细胞中C#9和C#15的结构域#V(Sub1和Sub4)对蛋白酪氨酸磷酸化信号非常出色的响应能力以及C#9和C#15的非常明显分子构象的改变并对其自身激活元件—结构域#VII(SYK和ZAP70)的非常充分显著释放与激活。此外，在血小板源生长因子激活信号的条件下，C#9和C#15具有统计分析后显著差异的更弱的近乎为零的对该信号的响应能力，证明C#9和C#15的结构域#V(Sub1和Sub4)对蛋白酪氨酸磷酸化信号出色响应能力的重要性且保证了人工分子机器对特定的蛋白酪氨酸磷酸化信号的特异性响应，而不会响应不相关的信号输入，比如血小板源生长因子激活信号。在此，血小板源生长因子可以结合小鼠胚胎成纤维细胞表面的血小板源生长因子受体从而提供血小板源生长因子激活信号，该信号不参与免疫受体酪氨酸激活基序的磷酸化，故无法特异性地被C#9和C#15的结构域#V所检测到。
利用DNA转染使人源细胞表达不同的嵌合抗原受体蛋白，从而使用荧光显微镜成像方法去检测并表征不同人工分子机器在人源海拉(HeLa)细胞内的表达分布及响应多种不同外界刺激性输入信号的表现。
图5a显示了不同的人工分子机器在人源海拉细胞中的表达分布及在20uM酪氨酸磷酸酶抑制剂过钒酸钠刺激下响应蛋白酪氨酸磷酸化信号能力的检测结果。其中，实验组为C#17修饰的人源海拉细胞，对照组为C#18修饰的人源海拉细胞，图片左侧的色彩条热图由下至上依次代表嵌合抗原受体对刺激信号的响应能力的由低到高以及响应刺激信号同时引发的嵌合抗原受体基于分子构象改变的对其自身激活元件—结构域#VII的释放与激活程度的由低到高。首先，如图5a所示C#17和C#18均在人源海拉细胞的表面展示出正确的膜定位表达分布，未有任何其它错误的蛋白定位。另外，C#17修饰的人源海拉细胞显示出快速且显著的对酪氨酸磷酸酶抑制剂过钒酸钠刺激的蛋白酪氨酸磷酸化信号的响应能力，在刺激后的半小时左右时间内展现出了极为显著的对刺激信号响应能力及基于分子构象改变的对其自身结构域#VII的释放与激活；而C#18修饰的人源海拉细胞显示出显著较弱的对酪氨酸磷酸酶抑制剂过钒酸钠刺激的蛋白酪氨酸磷酸化信号的响应能力，在刺激后无法展现出有效的对刺激信号响应能力及基于分子构象改变的对其自身结构域#VII的释放与激活。图5a证明了在人源海拉细胞中C#17的结构域#V(Sub1)对蛋白酪氨酸磷酸化信号出色的响应能力以及C#17相应的明显分子构象的改变并对其自身激活元件——胞内激活信号传导结构域ZAP70的充分显著释放与激活。此外，在自身激活元件被失能的情况下(失活性突变体Sub1FF)，C#18较相C#17具有显著更弱的近乎为零的对蛋白酪氨酸磷酸化信号的响应能力，证明C#17的结构域#V(Sub1)对蛋白酪氨酸磷酸化信号出色响应能力的重要性及特异性。
图5b显示了不同的人工分子机器在人源海拉细胞中的表达分布及在20uM酪氨酸磷酸酶抑制剂过钒酸钠刺激下响应蛋白酪氨酸磷酸化信号能力的检测结果。其中，实验组为C#19修饰的人源海拉细胞，对照组为C#20修饰的人源海拉细胞，图片左侧的色彩条热图由下至上依次代表嵌合抗原受体对刺激信号的响应能力的由低到高以及响应刺激信号同时引发的嵌合抗原受体基于分子构象改变的对其自身激活元件—结构域#VII的释放与激活程度的由低到高。首先，如图5b所示C#19和C#20均在人源海拉细胞的表面展示出正确的膜定位表达分布，未有任何其它错误的蛋白定位。另外，C#19修饰的人源海拉细胞显示出快速且显著的对酪氨酸磷酸酶抑制剂过钒酸钠刺激的蛋白酪氨酸磷酸化信号的响应能力，在刺激后的半小时左右时间内展现出了极为显著的对刺激信号响应能力及基于分子构象改变的对其自身结构域#VII的释放与激活；C#20修饰的人源海拉细胞显示出近乎为零的极弱的对酪氨酸磷酸酶抑制剂过钒酸钠刺激的蛋白酪氨酸磷酸化信号的响应能力，在刺激后无法展现出有效的对刺激信号响应能力及基于分子构象改变的对其自身结构域#VII的释放与激活。以上结果充分证明了图三所示的人工分子机器在人源细胞中的信号激活模式。图5b证明了在人源海拉细胞中C#19的结构域#V(Sub1)对蛋白酪氨酸磷酸化信号出色的响应能力以及C#19相应的明显分子构象的改变并对其自身激活元件—结构域#VII的充分显著释放与激活。此外，在自身激活元件被失能的情况下(失活性突变体Sub1FF)，C#20较C#19具有显著更弱的近乎为零的对蛋白酪氨酸磷酸化信号的响应能力，证明C#19的结构域#V(Sub1)对蛋白酪氨酸磷酸化信号出色响应能力的重要性及特异性。
图5c显示了在酪氨酸磷酸酶抑制剂过钒酸钠激活蛋白酪氨酸磷酸化信号的条件下，不同的人工分子机器在人源海拉细胞中的表现结果(平均值±标准差，均为n＝10)，成像读数指标代表量化后嵌合抗原受体对刺激信号的响应能力的程度以及响应刺激信号同时引发的嵌合抗原受体基于分子构象改变的对其自身激活元件的释放与激活的程度。而且，图5c证明了在人源HeLa细胞中C#19的结构域#V(Sub1)对蛋白酪氨酸磷酸化信号非常出色的响应能力(C#19组平均值为超过2.84)以及C#19相应的非常明显分子构象的改变并对其自身激活元件——结构域#VII的非常充分显著释放与激活，且统计分析后显著差异的优于C#17(C#17组平均值约为2.48)。此 外，在自身激活元件被失能的情况下(失活性突变体Sub1FF)，C#20较C#18具有统计分析后显著差异的更弱的对蛋白酪氨酸磷酸化信号的响应能力(C#20组平均值约为0.055，C#18组平均值约为0.34)，证明C#19和C#17的结构域#V对蛋白酪氨酸磷酸化信号出色响应能力的重要性且C#19较C#17具有显著更佳的对蛋白酪氨酸磷酸化信号响应的特异性，说明C#19的结构域#IV较C#17的结构域#IV具备更优异的功能表现。
利用色谱纯化技术和4℃蛋白质透析从转染的293T细胞中纯化蛋白质C#9和C#10，然后将纯化后的分子机器蛋白质溶解于激酶缓冲溶液(pH为8左右的50mM Tris盐酸盐溶液，100mM氯化钠，10mM氯化镁，2mM二硫苏糖醇)浓度可为50nM，加入提供磷酸化所需底物1mM ATP和100nM活化状态的非受体型蛋白酪氨酸激酶Lck蛋白。这里，Src家族蛋白非受体型蛋白酪氨酸激酶Lck(淋巴细胞特异的蛋白酪氨酸激酶，Lymphocyte-specific protein tyrosine kinase)可以促进蛋白酪氨酸磷酸化信号的激活，起到提供特异性的蛋白酪氨酸磷酸化信号输入的作用，可以提供免疫受体酪氨酸激活基序的磷酸化信号输入。
检测加入ATP与Lck前后的光学信号并进行量化分析。在Lck提供激活蛋白酪氨酸磷酸化信号的条件下，C#9与C#10在纯化蛋白的状态下的表现结果(平均值±标准差，均为n＝3)，成像读数指标代表量化后嵌合抗原受体对刺激信号的响应能力的程度以及响应刺激信号同时引发的嵌合抗原受体基于分子构象改变的对其自身激活元件的释放与激活的程度。
图6的C#9(+)组证明了C#9的胞内检测信号传导结构域Sub1对蛋白酪氨酸磷酸化信号非常出色的响应能力(平均值约为0.8)以及C#9非常明显分子构象的改变并对其自身激活元件——胞内激活信号传导结构域ZAP70的非常充分显著释放与激活。此外，C#10(+)组证明了，在自身检测元件被失能的情况下(失活性突变体Sub1FF)，C#10较C#9具有统计分析后显著差异的更弱的对蛋白酪氨酸磷酸化信号的响应能力(平均值不足0.08)，证明C#9的结构域#V对蛋白酪氨酸磷酸化信号出色响应能力的重要性且C#9版本具有极佳的对蛋白酪氨酸磷酸化信号响应的特异性。
利用DNA转染使人源细胞中表达不同的嵌合抗原受体蛋白，从而使用荧光显微镜成像方法去检测并表征不同嵌合抗原受体在人源海拉(HeLa)细胞内的表达分布及响应生理特异性人源PD-L1信号输入的表现，所使用的生理特异性人源PD-L1信号为人源PD-L1修饰的微球。
图7a显示了不同的嵌合抗原受体在人源海拉细胞中的表达分布及在人源PD-L1修饰的微球刺激下响应人源PD-L1信号能力的检测结果。其中，实验组为C#19修饰的人源海拉细胞，对照组为C#20修饰的人源海拉细胞，所提供的相位对比成像实验图片提供了细胞与微球相互作用的图像信息。图片下方的色彩条热图由左至右依次代表嵌合抗原受体对刺激信号的响应能力的由低到高以及响应刺激信号同时引发的嵌合抗原受体基于分子构象改变的对其自身激活元件——胞内激活信号传导结构域ZAP70的释放与激活程度的由低到高。首先，图7a所示C#19和C#20均在人源海拉细胞的表面展示出正确的膜定位表达分布。另外，C#19修饰的人源海拉细胞显示出快速且显著的对人源PD-L1修饰的微球刺激信号的响应能力，在刺激后的18分钟左右起开始展现出了极为显著的对刺激信号响应能力及基于分子构象改变的对其自身结构域#VII的释放与激活，且所示的对人源PD-L1修饰的微球刺激信号的响应具有高度特异性的空间特点，即仅局部地在相位对比成像实验图片中细胞与微球相互作用的位置展示出响应能力；而C#20修饰的人源海拉细胞显示出显著较弱的对人源PD-L1修饰的微球刺激信号的响应能力，在刺激后无法展现出有效的对刺激信号响应能力及基于分子构象改变的对其自身结构域#VII的释放与激活。图7a证明了在人源海拉细胞中C#19的结构域#V(Sub1)对人源PD-L1信号出色的响应能力以及C#19相应的明显分子构象的改变并对其自身激活元件—胞内激活信号传导结构域ZAP70的充分显著释放与激活。此外，在自身激活元件被失能的情况下(失活性突变体Sub1FF)，C#20较C#19具有显著更弱的对人源PD-L1信号的响应能力，证明C#19的结构域#V(Sub1)对人源PD-L1信号出色响应能力的重要性及特异性。
图7b显示了在人源PD-L1修饰的微球刺激信号的条件下，不同嵌合抗原受体在人源海拉细胞中的表现结果(平均值±标准差，均为n＝10)，成像读数指标代表量化后嵌合抗原受体对刺激信号的响应能力的程度以及响应刺激信号同时引发的嵌合抗原受体基于分子构象改变的对其自身激活元件的释放与激活的程度。而且，图7b证明了在人源海拉细胞中C#19的结构域#V(Sub1)对蛋白酪氨酸磷酸化信号非常出色的响应能力(C#19组平均值约为0.46)以及C#19相应的非常明显分子构象的改变并对其自身激活元件—胞内激活信号传导结构域ZAP70的非常充分显著释放与激活，且统计分析后显著差异的优于C#17版本(C#17组平均值约为0.23)。此外，在自身激活元件被失能的情况下(失活性突变体Sub1FF)，C#20较C#18具有统计分析后显著差异的更弱的对蛋白酪氨酸磷酸化信号的响应能力(C#20组平均值约为0.046，C#18组平均值约为0.126)，证明C#19和C#17的结构域#V对人源PD-L1信号出色响应能力的重要性且C#19较C#17具有显著更佳的对人源PD-L1信号响应的特异性，说明C#19的结构域#IV较C#17版本的结构域#IV具备更优异的功能表现。
综上，通过不同手段检测表征后，证明该嵌合抗原受体人工分子机器展现出了优异的对不同刺激性信号输入的响应能力，尤其是对人源PD-L1信号输入的高度特异性响应，以及胞内信号传导结构域#VIII的重要性，尤其是结构域#VII在得到释放激活后激发改造的淋巴细胞效应功能的能力。其中，如图34，C#19的功能性尤为突出，即Truncated PD-1-Sub1-LL2-ZAP70版本，也为后续的细胞毒性杀伤实验提供支撑与保障。
实施例3检测嵌合抗原受体改造的自然杀伤细胞的肿瘤杀伤能力
经由肿瘤细胞毒性杀伤实验，以理解免疫检查点PD-1融合的嵌合抗原受体修饰改造后人源自然杀伤细胞对PD-L1阳性的人源肿瘤细胞的肿瘤杀伤检测，其机理分别如图8所示。图8a显示了天然自然杀伤细胞在其表面的免疫检查点受体(如内源性的PD-1)识别并结合肿瘤细胞表面的靶标分子(如PD-L1)时，自然杀伤细胞毒杀相应肿瘤细胞的能力受到抑制性免疫检查点信号通路的抑制。图8b显示了基于免疫检查点PD-1融合的嵌合抗原受体修饰改造的人源自然杀伤细胞识别并结合肿瘤细胞表面的靶标分子PD-L1时，修饰改造的自然杀伤细胞可以有效地得到活化并对相应肿瘤细胞进行有效的杀伤。其中，所使用做肿瘤细胞体外杀伤实验的人源肿瘤细胞均经过修改表达报告基因萤火虫荧光素酶，肿瘤细胞内的荧光素酶可精确地反映整体细胞存活率，即通过检测肿瘤细胞内的荧光素酶活性高低来量化肿瘤细胞存活数量的大小。
(1)检测基于免疫检查点PD-1融合的嵌合抗原受体修饰改造的人源自然杀伤细胞的嵌合抗原受体表达水平。
以慢病毒包装以制备不同免疫检查点PD-1融合的嵌合抗原受体人工分子机器的病毒颗粒，即将携有不同免疫检查点PD-1融合的嵌合抗原受体人工分子机器的反转录病毒表达载体(如pSIN质粒等)和包装质粒(如pCMV delta R8.2与pCMV-VSV-G或psPAX2与pMD2.G等)转染293T细胞，收获病毒上清，过滤后分装冻存，测定病毒滴度。将一定量的病毒上清加入到人源自然杀伤细胞NK-92的培养皿中培养24小时，第二天弃掉病毒溶液。病毒感染自然杀伤细胞NK-92后的第2～3天，利用PD-1抗体染色筛选出细胞表面 PD-1融合的嵌合抗原受体高表达的自然杀伤细胞NK-92细胞群(请见图9)。不同的免疫检查点PD-1融合的嵌合抗原受体C#2、C#3与C#5在自然杀伤细胞NK-92中，相对于对照组，都有90％以上的表达，并用于共培养实验中检测不同的基于免疫检查点PD-1融合的嵌合抗原受体修饰改造的自然杀伤细胞NK-92的杀伤肿瘤细胞的效果。自然杀伤细胞NK-92分别表达不同的免疫检查点PD-1融合的嵌合抗原受体C#2与C#3经影像观察杀伤肿瘤细胞的能力于图11中，并与自然杀伤细胞NK-92分别表达不同的免疫检查点PD-1融合的嵌合抗原受体C#2、C#3与C#5展示杀伤肿瘤细胞的效力于图12～图20中。
(2)免疫检查点抑制性信号通路分子PD-L1在不同肿瘤细胞上表达量的检测。
利用PD-L1抗体分别染色并检测PD-L1在8种人源癌肿瘤细胞上的表达情况，分别为人源乳腺癌肿瘤细胞MBA-MB-231、人源脑癌肿瘤细胞U87-MG、人源肾癌肿瘤细胞786-O、人源皮肤癌肿瘤细胞A2058、人源肺癌肿瘤细胞H441、人源卵巢癌肿瘤细胞ES-2、人源前列腺癌肿瘤细胞PC-3与人源肝癌肿瘤细胞HA-22T。图10a～图10h显示了PD-L1分别在8种人源肿瘤癌细胞及经γ干扰素预处理后人源癌肿瘤细胞的表达情况，相对于阴性的对照组(同型对照，Isotype Control)而言PD-L1在8种人源癌细胞的表达比例分别为90.1％、97.1％、91.9％、89.5％、99.4％、99.9％、93.7％、93.6％，而经γ干扰素预处理后，PD-L1的表达比例增加且表达量显著提升，分别为97.5％、99.7％、99.9％、99.9％、99.6％、100.0％、99.9％、99.5％，进一步揭示了γ干扰素会促进肿瘤细胞上PD-L1的表现，并于体外试验中将γ干扰素预处理肿瘤细胞以模拟机体内的肿瘤微环境，该8种人源癌肿瘤细胞用于肿瘤细胞杀伤实验中。
(3)影像观察分析嵌合抗原受体改造后人源自然杀伤细胞的肿瘤杀伤能力。
基于免疫检查点PD-1融合的嵌合抗原受体C#3版本修饰改造后人源自然杀伤细胞NK-92对PD-L1阳性的人源乳腺癌肿瘤细胞MBA-MB-231的肿瘤杀伤能力经影像观察分析：
表达报告基因绿色荧光蛋白的人源乳腺癌肿瘤细胞MDA-MB-231先经γ干扰素预处理24小时以增加其细胞表面PD-L1的表达，将1x 10 5的修饰改造后人源自然杀伤细胞NK-92与1x 10 5肿瘤细胞按照1:1的E/T(效应细胞/靶细胞)比例在35mm玻璃底盘中共培养并使用延时活体显微镜观察免疫检查点PD-1融合的嵌合抗原受体C#3版本修饰改造后人源自然杀伤细胞是否可有效的杀伤PD-L1阳性的人源乳腺癌肿瘤细胞MBA-MB-231。在培养液中添加100mmol/L碘化丙啶(Propidium Iodide,PI)以及时观察乳腺癌肿瘤细胞被杀伤受损情形，共培养的时间开始即为第0分钟。请见图11，实验组为C#3修饰的人源自然杀伤细胞NK-92，对照组为C#2修饰的人源自然杀伤细胞NK-92。图11显示图像为每3分钟采集一次，显示的是绿色、红色(碘化丙啶)和亮视野通道的叠加。在实验组(图11a)，荧光图像显示在指示的时间点，表示第0分钟，修饰改造后人源自然杀伤细胞与表达绿色荧光蛋白的肿瘤靶细胞接触结合，于第36、42、81分钟显示C#3修饰改造后人源自然杀伤细胞与肿瘤靶细胞持续直接接触并促使肿瘤靶细胞的细胞膜损伤，碘化丙啶进入肿瘤靶细胞内而逐渐发红色荧光，最后于第117分钟显示是肿瘤靶细胞受损不完整死亡，整个被杀伤肿瘤靶细胞失去绿色荧光并呈现红色荧光或甚至红色荧光消失；而对照组C#2修饰的人源自然杀伤细胞(图11b)显示出显著与表达绿色荧光蛋白的人源PD-L1阳性的肿瘤靶细胞识别接触结合的效果，但是于第0、24、48、90分钟直到117分钟都未能实现对表达绿色荧光蛋白的肿瘤靶细胞有效杀伤的，也不能促使肿瘤靶细胞受损。影像观察分析结果证明了嵌合抗原受体C#3修饰的免疫自然杀伤细胞在与PD-L1阳性的人源肿瘤细胞共同培养情况下具有显著差异的卓越的识别杀伤肿瘤细胞的能力，而对照组C#2中的人源自然杀伤细胞面对PD-L1阳性的人源肿瘤细胞共培养条件下则未能显示出有效的识别杀伤肿瘤细胞的能力。
(4)检测嵌合抗原受体改造后人源自然杀伤细胞的肿瘤杀伤能力。
检测基于免疫检查点PD-1融合的嵌合抗原受体修饰改造后人源自然杀伤细胞NK-92对PD-L1阳性的人源乳腺癌肿瘤细胞MDA-MB-231的肿瘤杀伤能力：
嵌合抗原受体改造的人源自然杀伤细胞NK-92对PD-L1阳性的人源乳腺癌肿瘤细胞MDA-MB-231的肿瘤杀伤检测：
表达报告基因萤火虫荧光素酶的人源乳腺癌肿瘤细胞MDA-MB-231先经γ干扰素预处理24小时以增加其细胞表面PD-L1的表达，将1x 10 3、2.5x 10 3、5x 10 3、10x 10 3的修饰改造后人源自然杀伤细胞NK-92分别与1x 10 3肿瘤细胞按照1:1、2.5:1、5:1、10:1的E/T(效应细胞/靶细胞)比例在48孔板中共培养24小时，共培养的时间开始即为第0小时。之后分别于孵育后第0小时、4小时、24小时三个共培养时间点上检测细胞培养体系中荧光素酶活性，进而量化人源乳腺癌肿瘤细胞数量并计算人源自然杀伤细胞对人源乳腺癌肿瘤细胞的细胞毒性。请见图12。其中，仅肿瘤细胞组为仅有人源乳癌肿瘤细胞MDA-MB-231细胞本身，对照组中的人源自然杀伤细胞为未经嵌合抗原受体改造的人源自然杀伤细胞，靶细胞存活指数代表细胞培养体系中表达报告基因萤火虫荧光素酶的人源乳癌肿瘤细胞的相对细胞数量。图12a说明本申请所涵盖的自然杀伤细胞与PD-L1阳性的人源乳癌肿瘤细胞的体外共培养细胞毒性实验分析测试流程与模式设置。图12b显示了不同的基于免疫检查点PD-1融合的嵌合抗原受体人工分子机器修饰改造的人源自然杀伤细胞与PD-L1阳性的人源肿瘤细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照1:1的E/T(效应细胞/靶细胞)比例进行实验。于孵育后24小时时(C#3组平均值为2.0，对照组平均值为3.1)，相较于对照组中的人源自然杀伤细胞，免疫检查点PD-1融合的嵌合抗原受体C#3修饰改造后人源免疫自然杀伤细胞分别显示最大量的肿瘤细胞清除能力，人源肿瘤细胞的细胞数量为相对于对照组中的66.6％。定量分析线图证明了嵌合抗原受体C#3修饰的免疫自然杀伤细胞在与PD-L1阳性的人源肿瘤细胞共同培养情况下具有统计分析后显著差异的卓越的识别杀伤肿瘤细胞的能力，而其它对照组中的人源自然杀伤细胞面对PD-L1阳性的人源肿瘤细胞共培养条件下则未能显示出有效的识别杀伤肿瘤细胞的能力。图12c显示了不同的基于免疫检查点PD-1融合的嵌合抗原受体人工分子机器修饰改造的人源自然杀伤细胞与PD-L1阳性的人源肿瘤细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照2.5:1的E/T(效应细胞/靶细胞)比例进行实验于孵育后24小时时(C#3组平均值为0.9，对照组平均值为2.2)，相较于对照组中的人源自然杀伤细胞，嵌合抗原受体C#3修饰改造后人源免疫自然杀伤细胞分别显示最大量的肿瘤细胞清除能力，人源肿瘤细胞的细胞数量为相对于对照组中的40.9％。定量分析线图证明了嵌合抗原受体C#3修饰的免疫自然杀伤细胞在与PD-L1阳性的人源肿瘤细胞共同培养情况下具有统计分析后显著差异的卓越的识别杀伤肿瘤细胞的能力，而其它对照组中的人源自然杀伤细胞面对PD-L1阳性的人源肿瘤细胞共培养条件下则未能显示出有效的识别杀伤肿瘤细胞的能力。图12d显示了不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源肿瘤细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例进行实验于孵育后24小时时(C#3组平均值为0.2，C#5组平均值为0.4，对照组平均值为1.4)，相较于对照组中的人源自然杀伤细胞，嵌合抗原受体C#3、C#5修饰改造后人源免疫自然杀伤细胞分别显示最大量的肿瘤细胞清除能力，人源肿瘤细胞的细胞数量分别为相对于对照组中的14.3％与28.6％。定量分析线图证明了嵌合抗原受体C#3、C#5修饰的免疫自然杀伤细胞在与PD-L1阳性的人源肿瘤细胞共同培养情况下具有统计分析后显著差异的卓越的识别杀伤肿瘤细胞的能力，而其它对照组中的人源自然 杀伤细胞面对PD-L1阳性的人源肿瘤细胞共培养条件下则未能显示出有效的识别杀伤肿瘤细胞的能力。图12e显示了不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源肿瘤细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照10:1的E/T(效应细胞/靶细胞)比例进行实验于孵育后24小时时(C#3组数值为0.1，对照组数值为0.9)，相较于对照组中的人源自然杀伤细胞，嵌合抗原受体C#3、C#5修饰改造后人源免疫自然杀伤细胞分别显示最大量的肿瘤细胞清除能力，人源肿瘤细胞的细胞数量为相对于对照组中的11.1％。定量分析线图证明了嵌合抗原受体C#3修饰的免疫自然杀伤细胞在与PD-L1阳性的人源肿瘤细胞共同培养情况下具有统计分析后显著差异的卓越的识别杀伤肿瘤细胞的能力，而其它对照组中的人源自然杀伤细胞面对PD-L1阳性的人源肿瘤细胞共培养条件下则未能显示出有效的识别杀伤肿瘤细胞的能力。
为了解免疫检查点PD-1融合的嵌合抗原受体修饰改造后人源自然杀伤细胞是否能长时间维持杀伤肿瘤细胞的效力，进一步长时间检测基于免疫检查点PD-1融合的嵌合抗原受体修饰改造后人源自然杀伤细胞对PD-L1阳性的人源乳腺癌肿瘤细胞MDA-MB-231的肿瘤杀伤能力：
嵌合抗原受体改造的人源自然杀伤细胞NK-92对PD-L1阳性的人源乳腺癌肿瘤细胞MDA-MB-231的肿瘤杀伤长时间检测：
表达报告基因萤火虫荧光素酶的人源乳腺癌肿瘤细胞MDA-MB-231先经γ干扰素预处理24小时以增加其细胞表面PD-L1的表达，将5x 10 3的修饰改造后人源自然杀伤细胞NK-92分别与1x 10 3肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例在48孔板中共培养60小时，共培养的时间开始即为第0小时。之后分别于孵育后第0小时、4小时、60小时三个共培养时间点上检测细胞培养体系中荧光素酶活性，进而量化人源乳腺癌肿瘤细胞数量并计算人源自然杀伤细胞对人源乳腺癌肿瘤细胞的细胞毒性。请见图13。其中，仅肿瘤细胞组为仅有人源乳癌肿瘤细胞MDA-MB-231细胞本身，对照组中的人源自然杀伤细胞为未经嵌合抗原受体改造的人源自然杀伤细胞，靶细胞存活指数代表细胞培养体系中表达报告基因萤火虫荧光素酶的人源乳癌肿瘤细胞的相对细胞数量。图13a说明本申请所涵盖的自然杀伤细胞与PD-L1阳性的人源乳癌肿瘤细胞的体外共培养细胞毒性实验分析测试流程与模式设置。图13b和c显示了不同的基于免疫检查点PD-1融合的嵌合抗原受体人工分子机器修饰改造的人源自然杀伤细胞与PD-L1阳性的人源肿瘤细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例进行实验。于孵育后60小时时(C#3组平均值为0.3，C#5组平均值为0.4，对照组平均值为1.4，仅肿瘤细胞组平均值为6.8)，相较于对照组中的人源自然杀伤细胞，即使在长时间与人源乳癌肿瘤细胞共培养下，免疫检查点PD-1融合的嵌合抗原受体C#3、C#5修饰改造后人源免疫自然杀伤细胞分别显示最大量的肿瘤细胞清除能力，人源肿瘤细胞的细胞数量为相对于对照组中的21.4％与28.6％。定量分析线图证明了嵌合抗原受体C#3、C#5修饰的免疫自然杀伤细胞在与PD-L1阳性的人源肿瘤细胞长时间共同培养情况下具有统计分析后显著差异的卓越的识别杀伤肿瘤细胞的能力，而其它对照组中的人源自然杀伤细胞面对PD-L1阳性的人源肿瘤细胞共培养条件下则未能显示出有效的识别杀伤肿瘤细胞的能力。
嵌合抗原受体改造的人源自然杀伤细胞NK-92对PD-L1阳性的人源皮肤癌肿瘤细胞A2058的肿瘤杀伤检测：
表达报告基因萤火虫荧光素酶的人源皮肤癌肿瘤细胞A2058先经γ干扰素预处理24小时以增加其细胞表面PD-L1的表达，将1x10 3、2.5x 10 3、5x 10 3、10x 10 3的修饰改造后人源自然杀伤细胞NK-92分别与1x 10 3肿瘤细胞按照1:1、2.5:1、5:1、10:1的E/T(效应细胞/靶细胞)比例在48孔板中共培养24小时，共培养的时间开始即为第0小时。之后分别于孵育后第0小时、4小时、24小时三个共培养时间点上检测细胞培养体系中荧光素酶活性，进而量化人源皮肤癌肿瘤细胞数量并计算人源自然杀伤细胞对人源皮肤癌肿瘤细胞的细胞毒性。请见图14。其中，仅肿瘤细胞组为仅有人源皮肤癌肿瘤细胞A2058细胞本身，对照组中的人源自然杀伤细胞为未经嵌合抗原受体改造的人源自然杀伤细胞，靶细胞存活指数代表细胞培养体系中表达报告基因萤火虫荧光素酶的人源皮肤癌肿瘤细胞的相对细胞数量。图14a说明本申请所涵盖的自然杀伤细胞与PD-L1阳性的人源皮肤癌肿瘤细胞的体外共培养细胞毒性实验分析测试流程与模式设置。图14b显示了不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源肿瘤细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照1:1的E/T(效应细胞/靶细胞)比例进行实验。于孵育后24小时时(C#3组平均值为3.2，对照组平均值为5.3)，相较于对照组中的人源自然杀伤细胞，嵌合抗原受体C#3修饰改造后人源免疫自然杀伤细胞分别显示最大量的肿瘤细胞清除能力，人源肿瘤细胞的细胞数量为相对于对照组中的60.4％。定量分析线图证明了嵌合抗原受体C#3修饰的免疫自然杀伤细胞在与PD-L1阳性的人源肿瘤细胞共同培养情况下具有统计分析后显著差异的卓越的识别杀伤肿瘤细胞的能力，而其它对照组中的人源自然杀伤细胞面对PD-L1阳性的人源肿瘤细胞共培养条件下则未能显示出有效的识别杀伤肿瘤细胞的能力。图14c显示了不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源肿瘤细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照2.5:1的E/T(效应细胞/靶细胞)比例进行实验于孵育后24小时时(C#3组平均值为1.5，对照组平均值为3.8)，相较于对照组中的人源自然杀伤细胞，嵌合抗原受体C#3修饰改造后人源免疫自然杀伤细胞分别显示最大量的肿瘤细胞清除能力，人源肿瘤细胞的细胞数量为相对于对照组中的39.5％。定量分析线图证明了嵌合抗原受体C#3修饰的免疫自然杀伤细胞在与PD-L1阳性的人源肿瘤细胞共同培养情况下具有统计分析后显著差异的卓越的识别杀伤肿瘤细胞的能力，而其它对照组中的人源自然杀伤细胞面对PD-L1阳性的人源肿瘤细胞共培养条件下则未能显示出有效的识别杀伤肿瘤细胞的能力。图14(d)显示了不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源肿瘤细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例进行实验于孵育后24小时时(C#3组平均值为0.5，C#5组平均值为0.3，对照组平均值为3.3)，相较于对照组中的人源自然杀伤细胞，免疫检查点PD-1融合的嵌合抗原受体C#3、C#5修饰改造后人源免疫自然杀伤细胞分别显示最大量的肿瘤细胞清除能力，人源肿瘤细胞的细胞数量分别为相对于对照组中的15.0％与9.1％。定量分析线图证明了嵌合抗原受体C#3、C#5修饰的免疫自然杀伤细胞在与PD-L1阳性的人源肿瘤细胞共同培养情况下具有统计分析后显著差异的卓越的识别杀伤肿瘤细胞的能力，而其它对照组中的人源自然杀伤细胞面对PD-L1阳性的人源肿瘤细胞共培养条件下则未能显示出有效的识别杀伤肿瘤细胞的能力。图14e显示了不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源肿瘤细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照10:1的E/T(效应细胞/靶细胞)比例进行实验于孵育后24小时时(C#3组数值为0.1，对照组数值为2.7)，相较于对照组中的人源自然杀伤细胞，嵌合抗原受体C#3修饰改造后人源免疫自然杀伤细胞分别显示最大量的肿瘤细胞清除能力，人源肿瘤细胞的细胞数量为相对于对照组中的3.7％。定量分析线图证明了嵌合抗原受体C#3、C#5修饰的免疫自然杀伤细胞在与PD-L1阳性的人源肿瘤细胞共同培养情况下具有统计分析后显著差异的卓越的识别杀伤肿瘤细胞的能力，而其它对照组中的人源自然杀伤细胞面对PD-L1阳性的人源肿瘤细胞共培养条件下则未能显示出有效的识别杀伤肿瘤细胞的能力。
嵌合抗原受体改造的人源自然杀伤细胞NK-92对PD-L1阳性的人源前列腺癌肿瘤细胞PC-3的肿瘤杀伤检测：
表达报告基因萤火虫荧光素酶的人源前列腺癌肿瘤细胞PC-3先经γ干扰素预处理24小时以增加其细胞表面PD-L1的表达，将5x10 3的修饰改造后人源自然杀伤细胞NK-92分别与1x 10 3肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例在48孔板中共培养24小时，共培养的时间开始即为第0小时。之后分别于孵育后第0小时、4小时、24小时三个共培养时间点上检测细胞培养体系中荧光素酶活性，进而量化人源前列腺癌肿瘤细胞数量并计算人源自然杀伤细胞对人源前列腺癌肿瘤细胞的细胞毒性。请见图15。其中，仅肿瘤细胞组为仅有人源前列腺癌肿瘤细胞PC-3细胞本身，对照组中的人源自然杀伤细胞为未经嵌合抗原受体改造的人源自然杀伤细胞，靶细胞存活指数代表细胞培养体系中表达报告基因萤火虫荧光素酶的人源前列腺癌肿瘤细胞的相对细胞数量。图15a说明本申请所涵盖的自然杀伤细胞与PD-L1阳性的人源前列腺癌肿瘤细胞的体外共培养细胞毒性实验分析测试流程与模式设置。图15b和c显示了不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源肿瘤细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例进行实验。于孵育后24小时时(C#3组平均值为0.03，C#5组平均值为0.09，对照组平均值为0.35，仅肿瘤细胞组平均值为0.94)，相较于对照组中的人源自然杀伤细胞，嵌合抗原受体C#3与C#5修饰改造后人源免疫自然杀伤细胞分别显示最大量的肿瘤细胞清除能力，人源肿瘤细胞的细胞数量分别为相对于对照组中的8.6％与25.7％。定量分析线图证明了嵌合抗原受体C#3与C#5修饰的免疫自然杀伤细胞在与PD-L1阳性的人源肿瘤细胞共同培养情况下具有统计分析后显著差异的卓越的识别杀伤肿瘤细胞的能力，而其它对照组中的人源自然杀伤细胞面对PD-L1阳性的人源肿瘤细胞共培养条件下则未能显示出有效的识别杀伤肿瘤细胞的能力。
嵌合抗原受体改造的人源自然杀伤细胞NK-92对PD-L1阳性的人源脑癌肿瘤细胞U87-MG的肿瘤杀伤检测：
表达报告基因萤火虫荧光素酶的人源脑癌肿瘤细胞U87-MG先经γ干扰素预处理24小时以增加其细胞表面PD-L1的表达，将5x10 3的修饰改造后人源自然杀伤细胞NK-92分别与1x 10 3肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例在48孔板中共培养24小时，共培养的时间开始即为第0小时。之后分别于孵育后第0小时、4小时、24小时三个共培养时间点上检测细胞培养体系中荧光素酶活性，进而量化人源脑癌肿瘤细胞数量并计算人源自然杀伤细胞对人源脑癌肿瘤细胞的细胞毒性。请见图16。其中，仅肿瘤细胞组为仅有人源脑癌肿瘤细胞U87-MG细胞本身，对照组中的人源自然杀伤细胞为未经嵌合抗原受体改造的人源自然杀伤细胞，靶细胞存活指数代表细胞培养体系中表达报告基因萤火虫荧光素酶的人源脑癌肿瘤细胞的相对细胞数量。图16a说明本申请所涵盖的自然杀伤细胞与PD-L1阳性的人源脑癌肿瘤细胞的体外共培养细胞毒性实验分析测试流程与模式设置。图16b和c显示了不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源肿瘤细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例进行实验。于孵育后24小时时(C#3组平均值为0.2，C#5组平均值为0.4，对照组平均值为1.6，仅肿瘤细胞组平均值为3.7)，相较于对照组中的人源自然杀伤细胞，嵌合抗原受体C#3与C#5修饰改造后人源免疫自然杀伤细胞分别显示最大量的肿瘤细胞清除能力，人源肿瘤细胞的细胞数量分别为相对于对照组中的12.5％与25.0％。定量分析线图证明了嵌合抗原受体C#3与C#5修饰的免疫自然杀伤细胞在与PD-L1阳性的人源肿瘤细胞共同培养情况下具有统计分析后显著差异的卓越的识别杀伤肿瘤细胞的能力，而其它对照组中的人源自然杀伤细胞面对PD-L1阳性的人源肿瘤细胞共培养条件下则未能显示出有效的识别杀伤肿瘤细胞的能力。
嵌合抗原受体改造的人源自然杀伤细胞NK-92对PD-L1阳性的人源肝癌肿瘤细胞HA22T的肿瘤杀伤检测：
表达报告基因萤火虫荧光素酶的人源肝癌肿瘤细胞HA-22T先经γ干扰素预处理24小时以增加其细胞表面PD-L1的表达，将5x 10 3的修饰改造后人源自然杀伤细胞NK-92分别与1x 10 3肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例在48孔板中共培养24小时，共培养的时间开始即为第0小时。之后分别于孵育后第0小时、4小时、24小时三个共培养时间点上检测细胞培养体系中荧光素酶活性，进而量化人源肝癌肿瘤细胞数量并计算人源自然杀伤细胞对人源肝癌肿瘤细胞的细胞毒性。请见图17。其中，仅肿瘤细胞组为仅有人源肝癌肿瘤细胞HA22T细胞本身，对照组中的人源自然杀伤细胞为未经嵌合抗原受体改造的人源自然杀伤细胞，靶细胞存活指数代表细胞培养体系中表达报告基因萤火虫荧光素酶的人源肝癌肿瘤细胞的相对细胞数量。图17a说明本申请所涵盖的自然杀伤细胞与PD-L1阳性的人源肝癌肿瘤细胞的体外共培养细胞毒性实验分析测试流程与模式设置。图17b和c显示了不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源肿瘤细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例进行实验。于孵育后24小时时(C#3组平均值为0.2，C#5组平均值为0.2，对照组平均值为0.9，仅肿瘤细胞组平均值为2.7)，相较于对照组中的人源自然杀伤细胞，嵌合抗原受体C#3与C#5修饰改造后人源免疫自然杀伤细胞分别显示最大量的肿瘤细胞清除能力，人源肿瘤细胞的细胞数量分别为相对于对照组中的22.2％与22.2％。定量分析线图证明了嵌合抗原受体C#3与C#5修饰的免疫自然杀伤细胞在与PD-L1阳性的人源肿瘤细胞共同培养情况下具有统计分析后显著差异的卓越的识别杀伤肿瘤细胞的能力，而其它对照组中的人源自然杀伤细胞面对PD-L1阳性的人源肿瘤细胞共培养条件下则未能显示出有效的识别杀伤肿瘤细胞的能力。
嵌合抗原受体改造的人源自然杀伤细胞NK-92对PD-L1阳性的人源肾癌肿瘤细胞786-O的肿瘤杀伤检测：
表达报告基因萤火虫荧光素酶的人源肾癌肿瘤细胞786-O先经γ干扰素预处理24小时以增加其细胞表面PD-L1的表达，将5x 10 3的修饰改造后人源自然杀伤细胞NK-92分别与1x 10 3肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例在48孔板中共培养24小时，共培养的时间开始即为第0小时。之后分别于孵育后第0小时、4小时、24小时三个共培养时间点上检测细胞培养体系中荧光素酶活性，进而量化人源肾癌肿瘤细胞数量并计算人源自然杀伤细胞对人源肾癌肿瘤细胞的细胞毒性。请见图18。其中，仅肿瘤细胞组为仅有人源肾癌肿瘤细胞786-O细胞本身，对照组中的人源自然杀伤细胞为未经嵌合抗原受体改造的人源自然杀伤细胞，靶细胞存活指数代表细胞培养体系中表达报告基因萤火虫荧光素酶的人源肾癌肿瘤细胞的相对细胞数量。图18a说明本申请所涵盖的自然杀伤细胞与PD-L1阳性的人源肾癌肿瘤细胞的体外共培养细胞毒性实验分析测试流程与模式设置。图18b和c显示了不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源肿瘤细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例进行实验。于孵育后24小时时(C#3组平均值为0.1，C#5组平均值为0.2，对照组平均值为0.7，仅肿瘤细胞组平均值为7.1)，相较于对照组中的人源自然杀伤细胞，嵌合抗原受体C#3与C#5修饰改造后人源免疫自然杀伤细胞分别显示最大量的肿瘤细胞清除能力，人源肿瘤细胞的细胞数量分别约为相对于对照组中的14.3％与28.6％。定量分析线图证明了嵌合抗原受体C#3与C#5修饰的免疫自然杀伤细胞在与PD-L1阳性的人源肿瘤细胞共同培养情况下具有统计分析后显著差异的卓越的识别杀伤肿瘤细胞的能力，而其它对照组中的人源自然杀伤细胞面对PD-L1阳性的人源肿瘤细胞共培养条件下则未能显示出有效的识别杀伤肿瘤细胞的能力。
嵌合抗原受体改造的人源自然杀伤细胞NK-92对PD-L1阳性的人源肺癌肿瘤细胞H441的肿瘤杀伤检测：
表达报告基因萤火虫荧光素酶的人源肺癌肿瘤细胞H441先经γ干扰素预处理24小时以增加其细胞表面PD-L1的表达，将5x 10 3的修饰改造后人源自然杀伤细胞NK-92分别与1x 10 3肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例在48孔板中共培养24小时，共培养的时间开始即为第0小时。之后分别于孵育后第0小时、4小时、24小时三个共培养时间点上检测细胞培养体系中荧光素酶活性，进而量化人源肺癌肿瘤细胞数量并计算人源自然杀伤细胞对人源肺癌肿瘤细胞的细胞毒性。请见图19。其中，仅肿瘤细胞组为仅有人源肺癌肿瘤细胞H441细胞本身，对照组中的人源自然杀伤细胞为未经嵌合抗原受体改造的人源自然杀伤细胞，靶细胞存活指数代表细胞培养体系中表达报告基因萤火虫荧光素酶的人源肺癌肿瘤细胞的相对细胞数量。图19a说明本申请所涵盖的自然杀伤细胞与PD-L1阳性的人源肺癌肿瘤细胞的体外共培养细胞毒性实验分析测试流程与模式设置。图19b和c显示了不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源肿瘤细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例进行实验。于孵育后24小时时(C#3组平均值为0.1，C#5组平均值为0.7，对照组平均值为1.5，仅肿瘤细胞组平均值为1.8)，相较于对照组中的人源自然杀伤细胞，嵌合抗原受体C#3与C#5修饰改造后人源免疫自然杀伤细胞分别显示最大量的肿瘤细胞清除能力，人源肿瘤细胞的细胞数量分别为相对于对照组中的6.7％与46.7％。定量分析线图证明了嵌合抗原受体C#3与C#5修饰的免疫自然杀伤细胞在与PD-L1阳性的人源肿瘤细胞共同培养情况下具有统计分析后显著差异的卓越的识别杀伤肿瘤细胞的能力，而其它对照组中的人源自然杀伤细胞面对PD-L1阳性的人源肿瘤细胞共培养条件下则未能显示出有效的识别杀伤肿瘤细胞的能力。
嵌合抗原受体改造的人源自然杀伤细胞NK-92对PD-L1阳性的人源卵巢癌肿瘤细胞ES-2的肿瘤杀伤检测：
表达报告基因萤火虫荧光素酶的人源卵巢癌肿瘤细胞ES-2先经γ干扰素预处理24小时以增加其细胞表面PD-L1的表达，将5x 10 3的修饰改造后人源自然杀伤细胞NK-92分别与1x 10 3肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例在48孔板中共培养24小时，共培养的时间开始即为第0小时。之后分别于孵育后第0小时、4小时、24小时三个共培养时间点上检测细胞培养体系中荧光素酶活性，进而量化人源卵巢癌肿瘤细胞数量并计算人源自然杀伤细胞对人源肺癌肿瘤细胞的细胞毒性。请见图20。其中，仅肿瘤细胞组为仅有人源卵巢癌肿瘤细胞ES-2细胞本身，对照组中的人源自然杀伤细胞为未经嵌合抗原受体改造的人源自然杀伤细胞，靶细胞存活指数代表细胞培养体系中表达报告基因萤火虫荧光素酶的人源肺癌肿瘤细胞的相对细胞数量。图20a说明本申请所涵盖的自然杀伤细胞与PD-L1阳性的人源肺癌肿瘤细胞的体外共培养细胞毒性实验分析测试流程与模式设置。图19b和c显示了不同的嵌合抗原受体修饰改造的人源自然杀伤细胞与PD-L1阳性的人源肿瘤细胞的体外共培养细胞毒性效果的定量分析结果，自然杀伤细胞与肿瘤细胞按照5:1的E/T(效应细胞/靶细胞)比例进行实验。于孵育后24小时时(C#3组平均值为0.1，C#5组平均值为0.1，对照组平均值为1.3，仅肿瘤细胞组平均值为2.9)，相较于对照组中的人源自然杀伤细胞，嵌合抗原受体C#3与C#5修饰改造后人源免疫自然杀伤细胞分别显示最大量的肿瘤细胞清除能力，人源肿瘤细胞的细胞数量分别为相对于对照组中的7.7％与7.7％。定量分析线图证明了嵌合抗原受体C#3与C#5修饰的免疫自然杀伤细胞在与PD-L1阳性的人源肿瘤细胞共同培养情况下具有统计分析后显著差异的卓越的识别杀伤肿瘤细胞的能力，而其它对照组中的人源自然杀伤细胞面对PD-L1阳性的人源肿瘤细胞共培养条件下则未能显示出有效的识别杀伤肿瘤细胞的能力。
(5)检测嵌合抗原受体改造后人源自然杀伤细胞的抗肿瘤相关功能实验。
检测基于免疫检查点PD-1融合的嵌合抗原受体改造的人源自然杀伤细胞NK-92与肿瘤细胞共同培养条件下的多种基因转录水平表达：
人源乳腺癌肿瘤细胞MDA-MB-231先经γ干扰素预处理24小时以增加其细胞表面PD-L1的表达，将2.5x 10 5的修饰改造后人源自然杀伤细胞NK-92与1x 10 5肿瘤细胞按照2.5:1的E/T(效应细胞/靶细胞)比例在6孔板中共培养，此时记为第0小时，之后于孵育后48小时时间点上分离改造后的人源自然杀伤细胞NK-92并检测其与抗肿瘤功效相关的基因转录水平表达，并同时检测未与肿瘤细胞共培养条件下的改造后的人源自然杀伤的相关基因转录水平表达情况，包括定量实时聚合酶链锁反应(qPCR)分析与基因功能分类体系(GO，Gene Ontology)富集分析，请见图21至图33。其中，qPCR引物分别为：正向引物(5‘-3’CGACAGTACCATTGAGTTGTGCG，如SEQ ID NO:67所示)和反向引物(5‘-3’TTCGTCCATAGGAGACAATGCCC，如SEQ ID NO:68所示)检测目标基因GZMB；正向引物(5‘-3’ACTCACAGGCAGCCAACTTTGC，如SEQ ID NO:69所示)和反向引物(5‘-3’CTCTTGAAGTCAGGGTGCAGCG，如SEQ ID NO:70所示)检测目标基因PRF1；正向引物(5‘-3’CTCTTCTGCCTGCTGCACTTTG，如SEQ ID NO:71所示)和反向引物(5‘-3’ATGGGCTACAGGCTTGTCACTC，如SEQ ID NO:72所示)检测目标基因TNFA；正向引物(5‘-3’GAGTGTGGAGACCATCAAGGAAG，如SEQ ID NO:73所示)和反向引物(5‘-3’TGCTTTGCGTTGGACATTCAAGTC，如SEQ ID NO:74所示)检测目标基因IFNG；正向引物(5‘-3’CATCACCTGGAGGACTTCTACC，如SEQ ID NO:75所示)和反向引物(5‘-3’CAGTGTACTGGATGCTCTTCAGG，如SEQ ID NO:76所示)检测目标基因NCAM1；正向引物(5‘-3’GGTATGAGAGCCAGGCTTCTTG，如SEQ ID NO:77所示)和反向引物(5‘-3’GAATGGAGCCATCTTCCCACTG，如SEQ ID NO:78所示)检测目标基因KLRK1；正向引物(5‘-3’CAGCAACTTGCTGGATCTGGTG，如SEQ ID NO:79所示)和反向引物(5‘-3’AGACGGCAGTAGAAGGTCACCT，如SEQ ID NO:80所示)检测目标基因NCR1。
图21a的定量分析结果显示了，在未与肿瘤细胞共培养的条件下，不同的基于免疫检查点PD-1融合的嵌合抗原受体改造的人源自然杀伤细胞的效应功能相关基因GZMB转录水平的定量分析结果。其中，相较于对照组中的人源自然杀伤细胞，免疫检查点PD-1融合的嵌合抗原受体C#3、C#5、C#2修饰改造后人源自然杀伤细胞分别显示相近甚至较低的GZMB基因转录水平(C#3组平均值为0.622，C#5组平均值为0.813，对照组平均值为1.000，C#2组平均值为1.381；以管家基因GAPDH基因作为qPCR的内源性参照基因进行标准化与分析)，C#3和C#5分别为相对于对照组中的62.2％和81.3％。图21b的定量分析结果显示了，在与肿瘤细胞共培养48小时时间点上，不同的基于免疫检查点PD-1融合的嵌合抗原受体改造的人源自然杀伤细胞的效应功能相关基因GZMB转录水平的定量分析结果。其中，相较于对照组中的人源自然杀伤细胞，免疫检查点PD-1融合的嵌合抗原受体C#3、C#5修饰改造后人源自然杀伤细胞分别显示较高的GZMB基因转录水平(C#3组平均值为5.641，C#5组平均值为3.325，对照组平均值为1.000，C#2组平均值为0.653；以管家基因GAPDH基因作为qPCR的内源性参照基因进行标准化与分析)，C#3和C#5分别为相对于对照组中的5.641倍和3.325倍。上述定量结果证明了基于免疫检查点PD-1融合的嵌合抗原受体C#3、C#5版本修饰的人源自然杀伤细胞在与PD-L1阳性的人源肿瘤细胞共同培养情况下具有统计分析后显著差异的卓越的GZMB基因转录表达水平，意味着编码Granzyme B蛋白(颗粒酶B由自然杀伤细胞分泌并可诱导靶细胞的细胞程序性死亡，是自然杀伤细胞发挥抗肿瘤免疫杀伤作用的重要效应因子)的基因表达上调，而该蛋 白直接与自然杀伤细胞抗肿瘤功效相关。而实验组C#2与对照组中的人源自然杀伤细胞面对PD-L1阳性的人源肿瘤细胞共培养条件下则未能显示出明显的GZMB基因转录表达水平的提升，且实验组C#2中GZMB基因转录表达水平低于对照组中GZMB基因转录表达水平。
图22a的定量分析结果显示了，在未与肿瘤细胞共培养的条件下，不同的基于免疫检查点PD-1融合的嵌合抗原受体改造的人源自然杀伤细胞的效应功能相关基因PRF1转录水平的定量分析结果。其中，相较于对照组中的人源自然杀伤细胞，免疫检查点PD-1融合的嵌合抗原受体C#3、C#5、C#2修饰改造后人源自然杀伤细胞分别显示相近甚至较低的PRF1基因转录水平(C#3组平均值为1.119，C#5组平均值为0.645，对照组平均值为1.000，C#2组平均值为1.450；以管家基因GAPDH基因作为qPCR的内源性参照基因进行标准化与分析)，C#3和C#5分别为相对于对照组中的111.9％和64.5％。图22b的定量分析结果显示了，在与肿瘤细胞共培养48小时时间点上，不同的基于免疫检查点PD-1融合的嵌合抗原受体改造的人源自然杀伤细胞的效应功能相关基因PRF1转录水平的定量分析结果。其中，相较于对照组中的人源自然杀伤细胞，免疫检查点PD-1融合的嵌合抗原受体C#3、C#5修饰改造后人源自然杀伤细胞分别显示较高的PRF1基因转录水平(C#3组平均值为1.546，C#5组平均值为2.702，对照组平均值为1.000，C#2组平均值为0.490；以管家基因GAPDH基因作为qPCR的内源性参照基因进行标准化与分析)，C#3和C#5分别为相对于对照组中的1.546倍和2.702倍。上述定量结果证明了基于免疫检查点PD-1融合的嵌合抗原受体C#3、C#5版本修饰的人源自然杀伤细胞在与PD-L1阳性的人源肿瘤细胞共同培养情况下具有统计分析后显著差异的卓越的PRF1基因转录表达水平，意味着编码Perforin蛋白(穿孔素由自然杀伤细胞分泌并在自然杀伤细胞介导的细胞溶解中起到核心作用，是自然杀伤细胞发挥抗肿瘤免疫杀伤作用的重要效应因子)的基因表达上调，而该蛋白直接与自然杀伤细胞抗肿瘤功效相关。而其它实验组C#2与对照组中的人源自然杀伤细胞面对PD-L1阳性的人源肿瘤细胞共培养条件下则未能显示出明显的PRF1基因转录表达水平的提升，且实验组C#2中PRF1基因转录表达水平低于对照组中PRF1基因转录表达水平。
图23a的定量分析结果显示了，在未与肿瘤细胞共培养的条件下，不同的基于免疫检查点PD-1融合的嵌合抗原受体改造的人源自然杀伤细胞的效应功能相关基因TNFA转录水平的定量分析结果。其中，相较于对照组中的人源自然杀伤细胞，免疫检查点PD-1融合的嵌合抗原受体C#3、C#5、C#2修饰改造后人源自然杀伤细胞分别显示相近或者略微偏高的TNFA基因转录水平(C#3组平均值为2.958，C#5组平均值为0.476，对照组平均值为1.000，C#2组平均值为1.082；以管家基因GAPDH基因作为qPCR的内源性参照基因进行标准化与分析)，C#3和C#5分别为相对于对照组中的2.958倍和47.6％。图23b的定量分析结果显示了，在与肿瘤细胞共培养48小时时间点上，不同的基于免疫检查点PD-1融合的嵌合抗原受体改造的人源自然杀伤细胞的效应功能相关基因TNFA转录水平的定量分析结果。其中，相较于对照组中的人源自然杀伤细胞，免疫检查点PD-1融合的嵌合抗原受体C#3、C#5修饰改造后人源自然杀伤细胞分别显示较高的TNFA基因转录水平(C#3组平均值为3.536，C#5组平均值为5.240，对照组平均值为1.000，C#2组平均值为0.630；以管家基因GAPDH基因作为qPCR的内源性参照基因进行标准化与分析)，C#3和C#5分别为相对于对照组中的3.536倍和5.240倍。上述定量结果证明了基于免疫检查点PD-1融合的嵌合抗原受体C#3、C#5版本修饰的人源自然杀伤细胞在与PD-L1阳性的人源肿瘤细胞共同培养情况下具有统计分析后显著差异的卓越的TNFA基因转录表达水平，意味着编码TNF-α蛋白(肿瘤坏死因子-α由自然杀伤细胞分泌并可诱导细胞凋亡阻止肿瘤发生，是自然杀伤细胞发挥抗肿瘤免疫杀伤作用的重要效应因子)的基因表达上调，而该蛋白直接与自然杀伤细胞抗肿瘤功效相关。而其它实验组C#2与对照组中的人源自然杀伤细胞面对PD-L1阳性的人源肿瘤细胞共培养条件下则未能显示出明显的TNFA基因转录表达水平的提升，且实验组C#2中TNFA基因转录表达水平低于对照组中TFNA基因转录表达水平。
图24a的定量分析结果显示了，在未与肿瘤细胞共培养的条件下，不同的基于免疫检查点PD-1融合的嵌合抗原受体改造的人源自然杀伤细胞的效应功能相关基因IFNG转录水平的定量分析结果。其中，相较于对照组中的人源自然杀伤细胞，免疫检查点PD-1融合的嵌合抗原受体C#3、C#5、C#2修饰改造后人源自然杀伤细胞分别显示相近甚至较低或略微偏高的IFNG基因转录水平(C#3组平均值为1.198，C#5组平均值为0.339，对照组平均值为1.000，C#2组平均值为2.845；以管家基因GAPDH基因作为qPCR的内源性参照基因进行标准化与分析)，C#3和C#5分别为相对于对照组中的119.8％和33.9％。图24b的定量分析结果显示了，在与肿瘤细胞共培养48小时时间点上，不同的基于免疫检查点PD-1融合的嵌合抗原受体改造的人源自然杀伤细胞的效应功能相关基因IFNG转录水平的定量分析结果。其中，相较于对照组中的人源自然杀伤细胞，免疫检查点PD-1融合的嵌合抗原受体C#3、C#5修饰改造后人源自然杀伤细胞分别显示较高的IFNG基因转录水平(C#3组平均值为1.832，C#5组平均值为1.684，对照组平均值为1.000，C#2组平均值为0.649；以管家基因GAPDH基因作为qPCR的内源性参照基因进行标准化与分析)，C#3和C#5分别为相对于对照组中的1.832倍和1.684倍。上述定量结果证明了基于免疫检查点PD-1融合的嵌合抗原受体C#3、C#5版本修饰的人源自然杀伤细胞在与PD-L1阳性的人源肿瘤细胞共同培养情况下具有统计分析后显著差异的卓越的IFNG基因转录表达水平，意味着编码IFN-γ蛋白(γ-干扰素由自然杀伤细胞分泌并募集激活多种免疫效应细胞，是自然杀伤细胞发挥抗肿瘤免疫杀伤作用的重要效应因子)的基因表达上调，而该蛋白直接与自然杀伤细胞抗肿瘤功效相关。而其它实验组C#2与对照组中的人源自然杀伤细胞面对PD-L1阳性的人源肿瘤细胞共培养条件下则未能显示出明显的IFNG基因转录表达水平的提升，且实验组C#2中IFNG基因转录表达水平低于对照组中IFNG基因转录表达水平。
图25a的定量分析结果显示了，在未与肿瘤细胞共培养的条件下，不同的基于免疫检查点PD-1融合的嵌合抗原受体改造的人源自然杀伤细胞的效应功能相关基因NCAM1转录水平的定量分析结果。其中，相较于对照组中的人源自然杀伤细胞，免疫检查点PD-1融合的嵌合抗原受体C#3、C#5、C#2修饰改造后人源自然杀伤细胞分别显示相近甚至较低或者略微偏高的NCAM1基因转录水平(C#3组平均值为1.016，C#5组平均值为0.397，对照组平均值为1.000，C#2组平均值为1.418；以管家基因GAPDH基因作为qPCR的内源性参照基因进行标准化与分析)，C#3和C#5分别为相对于对照组中的101.6％和39.7％。图25(b)的定量分析结果显示了，在与肿瘤细胞共培养48小时时间点上，不同的基于免疫检查点PD-1融合的嵌合抗原受体改造的人源自然杀伤细胞的效应功能相关基因NCAM1转录水平的定量分析结果。其中，相较于对照组中的人源自然杀伤细胞，免疫检查点PD-1融合的嵌合抗原受体C#3、C#5修饰改造后人源自然杀伤细胞分别显示较高的NCAM1基因转录水平(C#3组平均值为0.929，C#5组平均值为3.331，对照组平均值为1.000，C#2组平均值为0.786；以管家基因GAPDH基因作为qPCR的内源性参照基因进行标准化与分析)，C#3和C#5分别为相对于对照组中的0.929倍和3.331倍。上述定量结果证明了基于免疫检查点PD-1融合的嵌合抗原受体C#3、C#5版本(尤其是C#5版本)修饰的人源自然杀伤细胞在与PD-L1阳性的人源肿瘤细胞共同培养情况下具有统计分析后显著差异的卓越的NCAM1基因转录表达水 平，意味着编码CD56蛋白(神经元黏附分子CD56是自然杀伤细胞活化状态下表达的受体蛋白，促进自然杀伤细胞的激活与抗肿瘤效应能力，对自然杀伤细胞发挥抗肿瘤免疫杀伤起到重要作用)的基因表达上调，而该蛋白直接与自然杀伤细胞抗肿瘤功效相关。而其它实验组C#2与对照组中的人源自然杀伤细胞面对PD-L1阳性的人源肿瘤细胞共培养条件下则未能显示出明显的NCAM1基因转录表达水平的提升，且实验组C#2中NCAM1基因转录表达水平低于对照组中NCAM1基因转录表达水平。
图26a的定量分析结果显示了，在未与肿瘤细胞共培养的条件下，不同的基于免疫检查点PD-1融合的嵌合抗原受体改造的人源自然杀伤细胞的效应功能相关基因KLRK1转录水平的定量分析结果。其中，相较于对照组中的人源自然杀伤细胞，免疫检查点PD-1融合的嵌合抗原受体C#3、C#5、C#2修饰改造后人源自然杀伤细胞分别显示相近甚至较低或者略微偏高的KLRK1基因转录水平(C#3组平均值为1.426，C#5组平均值为0.275，对照组平均值为1.000，C#2组平均值为1.368；以管家基因GAPDH基因作为qPCR的内源性参照基因进行标准化与分析)，C#3和C#5分别为相对于对照组中的1.426倍和27.5％。图26(b)的定量分析结果显示了，在与肿瘤细胞共培养48小时时间点上，不同的基于免疫检查点PD-1融合的嵌合抗原受体改造的人源自然杀伤细胞的效应功能相关基因KLRK1转录水平的定量分析结果。其中，相较于对照组中的人源自然杀伤细胞，免疫检查点PD-1融合的嵌合抗原受体C#3、C#5修饰改造后人源自然杀伤细胞分别显示较高的KLRK1基因转录水平(C#3组平均值为1.612，C#5组平均值为3.225，对照组平均值为1.000，C#2组平均值为0.664；以管家基因GAPDH基因作为qPCR的内源性参照基因进行标准化与分析)，C#3和C#5分别为相对于对照组中的1.612倍和3.225倍。上述定量结果证明了基于免疫检查点PD-1融合的嵌合抗原受体C#3、C#5版本修饰的人源自然杀伤细胞在与PD-L1阳性的人源肿瘤细胞共同培养情况下具有统计分析后显著差异的卓越的KLRK1基因转录表达水平，意味着编码NKG2D蛋白(NKG2D蛋白是自然杀伤细胞表面的强激活性受体，在天然免疫中发挥着重要作用并参与自然杀伤细胞对肿瘤的清除杀伤，对自然杀伤细胞发挥抗肿瘤免疫杀伤起到关键作用)的基因表达上调，而该蛋白直接与自然杀伤细胞抗肿瘤功效相关。而其它实验组C#2与对照组中的人源自然杀伤细胞面对PD-L1阳性的人源肿瘤细胞共培养条件下则未能显示出明显的KLRK1基因转录表达水平的提升，且实验组C#2中KLRK1基因转录表达水平低于对照组中KLRK1基因转录表达水平。
图27a的定量分析结果显示了，在未与肿瘤细胞共培养的条件下，不同的基于免疫检查点PD-1融合的嵌合抗原受体改造的人源自然杀伤细胞的效应功能相关基因NCR1转录水平的定量分析结果。其中，相较于对照组中的人源自然杀伤细胞，免疫检查点PD-1融合的嵌合抗原受体C#3、C#5、C#2修饰改造后人源自然杀伤细胞分别显示相近甚至较低的NCR1基因转录水平(C#3组平均值为0.988，C#5组平均值为0.448，对照组平均值为1.000，C#2组平均值为1.410；以管家基因GAPDH基因作为qPCR的内源性参照基因进行标准化与分析)，C#3和C#5分别为相对于对照组中的98.8％和44.8％。图27(b)的定量分析结果显示了，在与肿瘤细胞共培养48小时时间点上，不同的基于免疫检查点PD-1融合的嵌合抗原受体改造的人源自然杀伤细胞的效应功能相关基因NCR1转录水平的定量分析结果。其中，相较于对照组中的人源自然杀伤细胞，免疫检查点PD-1融合的嵌合抗原受体C#3、C#5修饰改造后人源自然杀伤细胞分别显示较高的NCR1基因转录水平(C#3组平均值为1.809，C#5组平均值为4.114，对照组平均值为1.000，C#2组平均值为0.686；以管家基因GAPDH基因作为qPCR的内源性参照基因进行标准化与分析)，C#3和C#5分别为相对于对照组中的1.809倍和4.114倍。上述定量结果证明了基于免疫检查点PD-1融合的嵌合抗原受体C#3、C#5版本修饰的人源自然杀伤细胞在与PD-L1阳性的人源肿瘤细胞共同培养情况下具有统计分析后显著差异的卓越的NCR1基因转录表达水平，意味着编码NKp46蛋白(NKp46是自然杀伤细胞表面的激活性受体，参与自然杀伤细胞对靶细胞的清除杀伤，对自然杀伤细胞发挥抗肿瘤免疫杀伤起到关键作用)的基因表达上调，而该蛋白直接与自然杀伤细胞抗肿瘤功效相关。而其它实验组C#2与对照组中的人源自然杀伤细胞面对PD-L1阳性的人源肿瘤细胞共培养条件下则未能显示出明显的NCR1基因转录表达水平的提升，且实验组C#2中NCR1基因转录表达水平低于对照组中NCR1基因转录表达水平。
图28的定量分析结果显示了，在与肿瘤细胞共培养48小时时间点上，不同的基于免疫检查点PD-1融合的嵌合抗原受体改造的人源自然杀伤细胞的效应功能相关的GO富集分析结果。其中，根据GO数据库注释分类信息，分别相较于对照组与C#2组的人源自然杀伤细胞，免疫检查点PD-1融合的嵌合抗原受体C#3、C#5修饰改造后人源自然杀伤细胞分别显示显著较高的GO:0002228(natural killer cell mediated immunity，即自然杀伤细胞介导免疫)的基因富集，具备更强的自然杀伤细胞介导免疫与抗肿瘤功能效应。
图29的定量分析结果显示了，在与肿瘤细胞共培养48小时时间点上，不同的基于免疫检查点PD-1融合的嵌合抗原受体改造的人源自然杀伤细胞的效应功能相关的GO富集分析结果。其中，根据GO数据库注释分类信息，分别相较于对照组与C#2组的人源自然杀伤细胞，免疫检查点PD-1融合的嵌合抗原受体C#3、C#5修饰改造后人源自然杀伤细胞分别显示显著较高的GO:0032649(regulation of interferon-gamma production，即γ干扰素产生调控)的基因富集，结合图24中IFNG基因的qPCR结果，更进一步证明嵌合抗原受体C#3、C#5修饰改造后人源自然杀伤细胞具备更强的γ干扰素产生分泌与抗肿瘤功能效应。
图30的定量分析结果显示了，在与肿瘤细胞共培养48小时时间点上，不同的基于免疫检查点PD-1融合的嵌合抗原受体改造的人源自然杀伤细胞的效应功能相关的GO富集分析结果。其中，根据GO数据库注释分类信息，分别相较于对照组与C#2组的人源自然杀伤细胞，免疫检查点PD-1融合的嵌合抗原受体C#3、C#5修饰改造后人源自然杀伤细胞分别显示显著较高的GO:0070098(chemokine-mediated signaling pathway，即趋化因子介导信号通路)的基因富集，具备更强的细胞迁移功能与抗肿瘤功能效应。
图31的定量分析结果显示了，在与肿瘤细胞共培养48小时时间点上，不同的基于免疫检查点PD-1融合的嵌合抗原受体改造的人源自然杀伤细胞的效应功能相关的GO富集分析结果。其中，根据GO数据库注释分类信息，分别相较于对照组与C#2组的人源自然杀伤细胞，免疫检查点PD-1融合的嵌合抗原受体C#3、C#5修饰改造后人源自然杀伤细胞分别显示显著较高的GO:0002449(lymphocyte mediated immunity，即淋巴细胞介导免疫)的基因富集，具备更强的淋巴细胞介导免疫与抗肿瘤功能效应。
图32的定量分析结果显示了，在与肿瘤细胞共培养48小时时间点上，不同的基于免疫检查点PD-1融合的嵌合抗原受体改造的人源自然杀伤细胞的效应功能相关的GO富集分析结果。其中，根据GO数据库注释分类信息，分别相较于对照组与C#2组的人源自然杀伤细胞，免疫检查点PD-1融合的嵌合抗原受体C#3、C#5修饰改造后人源自然杀伤细胞分别显示显著较高的GO:0051251(positive regulation of lymphocyte activation，即淋巴细胞激活的正向调控)的基因富集，具备更强的淋巴细胞激活与抗肿瘤功能效应。
图33的定量分析结果显示了，在与肿瘤细胞共培养48小时时间点上，不同的基于免疫检查点PD-1融合的嵌合抗原受体改造的人源自然杀伤细胞的效应功能相关的GO富集分析结果。其中，根据GO数据库注释分类信息，分别相较于对照组与C#2组的人源自然杀伤细胞，免疫检查点PD-1融合的嵌合抗原受体C#3、C#5修饰改造后人源自然杀伤细胞分别显示显著较高的GO:0001906(cell killing，即细胞杀伤)的基因富集，具备更强的细胞杀伤功能与抗肿瘤功能效应。
综上，通过多种肿瘤细胞毒性杀伤实验以及抗肿瘤功能的验证，基于免疫检查点PD-1融合的嵌合抗原受体改造的自然杀伤细胞展现出如图8所示的对肿瘤细胞优异的杀伤能力，尤其是对PD-L1阳性的人源肿瘤细胞。其中，表现尤为突出的C#3与C#5分别是Truncated PD-1-Sub1-LL1-ZAP70版本与Truncated PD-1-Sub5-LL1-SYK版本，并且证明了嵌合抗原受体的多个结构域对于嵌合抗原受体充分行使其功能的必要性与重要性。
最后，如前所述免疫检查点阻断剂与细胞疗法是最近以来肿瘤免疫领域取得重大突破的方向。综合考虑PD-1/PD-L1抗体类药物和自然杀伤细胞的特点，本申请结合肿瘤免疫学、合成生物学、分子工程与细胞工程等多种手段开发新一代的基于免疫检查点PD-1信号通路的实体肿瘤细胞疗法。该细胞疗法应用基于免疫检查点PD-1的具备编码调控自然杀伤细胞功能的嵌合抗原受体人工分子机器，当表达免疫检查点抑制性信号PD-1分子配体PD-L1的肿瘤细胞通过PD-1/PD-L1免疫检查点信号通路以同样的对自然杀伤细胞刹车阻断机制去尝试抑制自然杀伤细胞功能时，经过该新一代基于PD-1的人工分子机器重新编码改造的自然杀伤细胞，非但不会被PD-L1阳性的肿瘤细胞所抑制，反而会被进一步激活，产生针对相应肿瘤细胞的特异性免疫反应，从而识别并杀伤相应的肿瘤细胞。
本申请中的多种细胞毒性以及抗肿瘤功能性实验证明，嵌合抗原受体修饰改造后自然杀伤细胞可以更好地呈现出面对免疫抑制性信号分子配体PD-L1抑制情况下的活化能力以及杀伤清除多种PD-L1阳性实体瘤的卓越效果，包括乳腺癌、脑癌、肾癌、皮肤癌、肺癌、卵巢癌、前列腺癌、肝癌等。故而，该嵌合抗原受体分子机器修饰改造后自然杀伤细胞成功地克服了实体肿瘤微环境中的免疫抑制，即解决实体肿瘤免疫治疗中免疫抑制与免疫逃逸等关键问题，相信这类工具可为实体肿瘤治疗开辟新的途径，并为人类癌症治疗提供创新和精确的治疗方法。
以上所述，仅是本申请的几个实施例，并非对本申请做任何形式的限制，虽然本申请以较佳实施例揭示如上，然而并非用以限制本申请，任何熟悉本专业的技术人员，在不脱离本申请技术方案的范围内，利用上述揭示的技术内容做出些许的变动或修饰均等同于等效实施案例，均属于技术方案范围内。

Claims (39)

  1. 一种嵌合抗原受体改造的NK细胞，其特征在于，所述嵌合抗原受体包括：胞外靶标分子结合结构域、跨膜区结构域和胞内信号传导结构域；
    所述跨膜区结构域将所述胞外靶标分子结合结构域和所述胞内信号传导结构域连接，并将二者固定在所述NK细胞的细胞膜上；
    所述胞内信号传导结构域包括胞内激活信号传导结构域和/或胞内检测信号传导结构域。
  2. 根据权利要求1所述的嵌合抗原受体改造的NK细胞，其特征在于，所述嵌合抗原受体还包括：胞外间隔区结构域；
    所述胞外间隔区结构域位于所述胞外靶标分子结合结构域与所述跨膜区结构域之间。
  3. 根据权利要求1所述的嵌合抗原受体改造的NK细胞，其特征在于，所述嵌合抗原受体还包括：胞内间隔区结构域；
    所述胞内间隔区结构域位于所述跨膜区结构域和所述胞内信号传导结构域之间并将这两者连接在一起。
  4. 根据权利要求1所述的嵌合抗原受体改造的NK细胞，其特征在于，所述嵌合抗原受体还包括：胞内铰链结构域；
    所述胞内铰链结构域将所述胞内检测信号结构域和所述胞内激活信号结构域连接在一起。
  5. 根据权利要求1所述的嵌合抗原受体改造的NK细胞，其特征在于，所述胞外靶标分子结合结构域结合的靶标分子包含下组的分子中的至少一种：免疫抑制性信号相关分子、肿瘤表面抗原分子标志物、细胞表面特定的抗原肽-组织相容性复合体分子。
  6. 根据权利要求1所述的嵌合抗原受体改造的NK细胞，其特征在于，所述胞外靶标分子结合结构域包含选自下组的分子的靶标分子结合结构域中的至少一种：PD-1、PD-1截短体、PD-1蛋白突变体、PD-L1的抗体及PD-L1结合片段。
  7. 根据权利要求1所述的嵌合抗原受体改造的NK细胞，其特征在于，所述胞外靶标分子结合结构域包含含有SEQ ID NO:1的氨基酸序列、含有SEQ ID NO:3的氨基酸序列、含有SEQ ID NO:5的氨基酸序列、含有SEQ ID NO:7、含有SEQ ID NO:9的氨基酸序列、含有SEQ ID NO:11的氨基酸序列中的至少一种。
  8. 根据权利要求1所述的嵌合抗原受体改造的NK细胞，其特征在于，所述胞外靶标分子结合结构域的核酸片段包含含有SEQ ID NO:2的核酸序列、含有SEQ ID NO:4的核酸序列、含有SEQ ID NO:6的核酸序列、含有SEQ ID NO:8的核酸序列、含有SEQ ID NO:10的核酸序列中的至少一种。
  9. 根据权利要求1所述的嵌合抗原受体改造的NK细胞，其特征在于，所述胞内激活信号传导结构域的激活至少依赖于所述胞外靶标分子结合结构域与所述靶标分子的结合；所述胞内激活信号传导结构域含有具有催化功能基团的分子或片段。
  10. 根据权利要求1所述的嵌合抗原受体改造的NK细胞，其特征在于，所述胞内激活信号传导结构域包括酪氨酸激酶或酪氨酸激酶片段中的至少一种；
    所述酪氨酸激酶包括受体型酪氨酸激酶、非受体型酪氨酸激酶中的至少一种；
    所述酪氨酸激酶片段包括受体型酪氨酸激酶片段、非受体型酪氨酸激酶片段中的至少一种。
  11. 根据权利要求10所述的嵌合抗原受体改造的NK细胞，其特征在于，所述酪氨酸激酶选自Ack、CSK、CTK、FAK、Abl、Arg、Tnk1、Pyk2、Fer、Fes、LTK、ALK、STYK1、JAK1、JAK2、JAK3、Tyk2、DDR1、DDR2、ROS、Blk、Fgr、FRK、Fyn、TIE1、TIE2、Hck、Lck、Srm、Yes、Syk、ZAP70、Etk、Btk、HER2、HER3、HER4、InsR、ITK、TEC、TXK、EGFR、IGF1R、IRR、PDGFRα、VEGFR-1、VEGFR-2、VEGFR-3、PDGFRβ、Kit、CSFR、FLT3、FGFR1、FGFR2、FGFR3、FGFR4、CCK4、ROR1、ROR2、MuSK、MET、Lyn、Brk、Src、Ron、Axl、Tyro3、Mer、EphA1、EphA2、EphA3、EphA4、EphA5、EphA6、EphA7、EphA8、EphA10、EphB1、trkA、trkB、trkC、EphB2、EphB3、EphB4、EphB6、Ret、RYK、Lmr1、Lmr2、Lmr3中的至少一种。
  12. 根据权利要求1所述的嵌合抗原受体改造的NK细胞，其特征在于，所述胞内激活信号传导结构域包含含有SEQ ID NO:42的氨基酸序列、含有SEQ ID NO:44的氨基酸序列、含有SEQ ID NO:46的氨基酸序列、含有SEQ ID NO:48的氨基酸序列、含有SEQ ID NO:50的氨基酸序列、含有SEQ ID NO:52的氨基酸序列中的至少一种。
  13. 根据权利要求1所述的嵌合抗原受体改造的NK细胞，其特征在于，所述胞内激活信号传导结构域的核酸片段包含含有SEQ ID NO:43的核酸序列、含有SEQ ID NO:45的核酸序列、含有SEQ ID NO:47的核酸序列、含有SEQ ID NO:49的核酸序列、含有SEQ ID NO:51的核酸序列、含有SEQ ID NO:53的核酸序列中的至少一种。
  14. 根据权利要求1所述的嵌合抗原受体改造的NK细胞，其特征在于，所述胞内检测信号传导结构域包含至少一个基于免疫受体酪氨酸的活化基序。
  15. 根据权利要求1所述的嵌合抗原受体改造的NK细胞，其特征在于，所述胞内检测信号传导结构域包含选自下组的分子的信 号传导结构域的至少一种：CD3δ、CD3γ、CD3ε、CD3ζ、CD5、CD28、CD31、CD72、CD84、CD229、CEACAM-19、CEACAM-20、SIRPα、SLAM、CLEC-1、CLEC-2、CRACC、CTLA-4、2B4、CD244、BTLA、DCAR、DCIR、Dectin-1、DNAM-1、CD300a、CD300f、CEACAM-1、CEACAM-3、CEACAM-4、FcεRIα、FcεRIβ、FcγRIB、FcγRI、FcγRIIA、FcγRIIB、FcγRIIC、FcγRIIIA、DAP10、DAP12、G6b、KIR、KIR2DL1、KIR2DL2、KIR2DL3、KIR2DL4、KIR2DL5、FCRL1、FCRL2、FCRL3、FCRL4、FCRL5、FCRL6、KIR2DL5B、KIR2DS1、KIR2DS3、KIR2DS4、KIR2DS5、TIGIT、TREML1、TREML2、KIR3DL1、KIR3DL2、KIR3DL3、KIR3DS1、KLRG1、LAIR1、MICL、NKG2A、NKp44、NKp65、NKp80、NTB-A、PD-1、LILRB1、LILRB2、LILRB3、LILRB4、LILRB5、Siglec-2、Siglec-3、Siglec-5、Siglec-6、Siglec-7、Siglec-8、PDCD6、PILR-α、Siglec-9、Siglec-10、Siglec-11、Siglec-12、Siglec-14、Siglec-15、Siglec-16。
  16. 根据权利要求1所述的嵌合抗原受体改造的NK细胞，其特征在于，所述胞内检测信号传导结构域包含含有SEQ ID NO:20的氨基酸序列、含有SEQ ID NO:22的氨基酸序列、含有SEQ ID NO:24的氨基酸序列、含有SEQ ID NO:26的氨基酸序列、含有SEQ ID NO:28的氨基酸序列、含有SEQ ID NO:30的氨基酸序列、含有SEQ ID NO:32的氨基酸序列、含有SEQ ID NO:34的氨基酸序列、含有SEQ ID NO:36的氨基酸序列、含有SEQ ID NO:38的氨基酸序列、含有SEQ ID NO:40的氨基酸序列中的至少一种。
  17. 根据权利要求1所述的嵌合抗原受体改造的NK细胞，其特征在于，所述胞内检测信号传导结构域的核酸片段包含含有SEQ ID NO:21的核酸序列、含有SEQ ID NO:23的核酸序列、含有SEQ ID NO:25的核酸序列、含有SEQ ID NO:27的核酸序列、含有SEQ ID NO:29的核酸序列、含有SEQ ID NO:31的核酸序列、含有SEQ ID NO:33的核酸序列、含有SEQ ID NO:35的核酸序列、含有SEQ ID NO:37的核酸序列、含有SEQ ID NO:39的核酸序列、含有SEQ ID NO:41的核酸序列中的至少一种。
  18. 根据权利要求1所述的嵌合抗原受体改造的NK细胞，其特征在于，所述跨膜区结构域选自下组的跨膜蛋白的跨膜结构域，跨膜蛋白包含4-1BB、4-1BBL、ICOS、GITR、GITRL、VSIG-3、VISTA、SIRPα、OX40、OX40L、CD40、CD40L、CD86、CD80、PD-1、PD-L1、PD-L2、CD2、CD28、B7-DC、B7-H2、B7-H3、B7-H4、B7-H5、B7-H6、B7-H7、Siglec-1、Siglec-2、Siglec-3、Siglec-4、LILRB5、2B4、BTLA、CD160、LAG-3、Siglec-5、Siglec-6、Siglec-7、Siglec-8、CD96、CD226、TIM-1、TIM-3、TIM-4、Siglec-9、Siglec-10、Siglec-11、Siglec-12、Siglec-14、Siglec-15、Siglec-16、LIR1、KIR2DL1、KIR2DL2、KIR2DL3、KIR2DL4、KIR2DL5A、KIR2DL5B、KIR3DL1、KIR3DL2、KIR3DL3、KIR3DS1、KLRG1、KLRG2、KIR2DS1、KIR2DS3、KIR2DS4、KIR2DS5、LAIR1、LAIR2、LILRA3、LILRA4、DAP10、DAP12、NKG2A、NKG2C、NKG2D、LILRA5、LILRB1、LILRB2、LILRB3、LILRB4、CTLA-4、CD155、CD112、CD113、TIGIT、Galectin-9、CEACAM-1、CD8a、CD8b、CD4、MERTK、AXL、Tyro3、BAI1、MRC1、FcγR1、FcγR2A、FcγR2B1、FcγR2B2、FcγR3A、FcγR3B、FcεR2、FcεR1、FcRn、Fcα/μR或FcαR1中的至少一种。
  19. 根据权利要求1所述的嵌合抗原受体改造的NK细胞，其特征在于，所述跨膜区结构域包含含有SEQ ID NO:12的氨基酸序列、含有SEQ ID NO:14的氨基酸序列中的至少一种。
  20. 根据权利要求1所述的嵌合抗原受体改造的NK细胞，其特征在于，所述跨膜区结构域的核酸片段包含含有SEQ ID NO:13的核酸序列、含有SEQ ID NO:15的核酸序列中的至少一种。
  21. 根据权利要求2所述的嵌合抗原受体改造的NK细胞，其特征在于，所述胞外间隔区结构域包含含有SEQ ID NO:16的氨基酸序列、含有SEQ ID NO:18的氨基酸序列中的至少一种。
  22. 根据权利要求2所述的嵌合抗原受体改造的NK细胞，其特征在于，所述胞外间隔区结构域的核酸片段包含含有SEQ ID NO:17的核酸序列、含有SEQ ID NO:19的核酸序列中的至少一种。
  23. 根据权利要求3所述的嵌合抗原受体改造的NK细胞，其特征在于，所述胞内间隔区结构域为跨膜区结构域之延伸，包含选自下组的分子的至少一种：PD-1、PD-L1、PD-L2、CD8a、CD8b、CD4、ICOS、GITR、GITRL、OX40、OX40L、B7-DC、B7-H2、B7-H3、B7-H4、B7-H5、CD40、CD40L、CD86、CD80、CD2、CD28、B7-H6、B7-H7、VSIG-3、VISTA、SIRPα、KIR2DS1、KIR2DS3、KIR2DS4、KIR2DS5、Siglec-1、Siglec-2、Siglec-3、Siglec-4、CD155、CD112、CD113、TIGIT、CD96、CD226、Siglec-5、Siglec-6、Siglec-7、Siglec-8、Siglec-9、Siglec-10、LILRB1、LILRB2、LILRB3、LILRB4、LILRB5、Siglec-11、Siglec-12、Siglec-14、Siglec-15、Siglec-16、LIR1、KIR2DL1、KIR2DL2、KIR2DL3、KIR2DL4、DAP10、DAP12、NKG2A、NKG2C、NKG2D、KIR2DL5A、KIR2DL5B、KIR3DL1、KIR3DL2、KIR3DL3、KIR3DS1、TIM-1、TIM-3、TIM-4、KLRG1、KLRG2、LAIR1、LAIR2、LILRA3、LILRA4、LILRA5、2B4、BTLA、CD160、LAG-3、CTLA-4、Galectin-9、CEACAM-1、MERTK、AXL、Tyro3、BAI1、4-1BB、4-1BBL、MRC1、FcγR1、FcγR2A、FcγR2B1、FcγR2B2、FcγR3A、FcγR3B、FcεR2、FcεR1、FcRn、Fcα/μR或FcαR1。
  24. 根据权利要求3所述的嵌合抗原受体改造的NK细胞，其特征在于，所述胞内间隔区结构域包含含有SEQ ID NO:54的氨基酸序列、含有SEQ ID NO:56的氨基酸序列中的至少一种。
  25. 根据权利要求3所述的嵌合抗原受体改造的NK细胞，其特征在于，所述胞内间隔区结构域核酸片段包含含有SEQ ID NO:55的核酸序列、含有SEQ ID NO:57的核酸序列中的至少一种。
  26. 根据权利要求4所述的嵌合抗原受体改造的NK细胞，其特征在于，所述胞内铰链结构域包含含有SEQ ID NO:58的氨基酸序列、含有SEQ ID NO:60的氨基酸序列、含有SEQ ID NO:62的氨基酸序列、含有SEQ ID NO:64的氨基酸序列、含有SEQ ID NO:66的氨基酸序列中的至少一种。
  27. 根据权利要求4所述的嵌合抗原受体改造的NK细胞，其特征在于，所述胞内铰链结构域片段包含含有SEQ ID NO:59的核酸序列、含有SEQ ID NO:61的核酸序列、含有SEQ ID NO:63的核酸序列、含有SEQ ID NO:65的核酸序列中的至少一种。
  28. 根据权利要求1所述的嵌合抗原受体改造的NK细胞，其特征在于，所述NK细胞包括内源性NK细胞亚群和/或外源性NK细胞中的至少一种。
  29. 根据权利要求28所述的嵌合抗原受体改造的NK细胞，其特征在于，所述内源性NK细胞亚群包括适应性NK细胞、记忆性NK细胞、CD56 dimNK细胞、CD56 brightNK细胞中的至少一种；
    所述外源性NK细胞包括NK细胞株、胚胎干细胞或诱导多能干细胞衍生的NK细胞中的至少一种。
  30. 根据权利要求29所述的嵌合抗原受体改造的NK细胞，其特征在于，所述NK细胞株选自NK-92细胞株、haNK细胞株、IMC-1细胞株、NK-YS细胞株、KHYG-1细胞株、NKL细胞株、NKG细胞株、SNK-6细胞株、YTS细胞株、HANK-1细胞株中的至少一种。
  31. 权利要求1～30任一项所述的嵌合抗原受体改造的NK细胞的制备方法，其特征在于，所述制备方法包括以下步骤：
    1)分别获得人的NK细胞和嵌合抗原受体；
    2)利用所述嵌合抗原受体对所述人的NK细胞进行改造，以获得所述嵌合抗原受体改造的NK细胞。
  32. 一种药物组合物，其特征在于，所述组合物包括权利要求1～30任一项所述的嵌合抗原受体改造的NK细胞或根据权利要求31所述的制备方法制备得到的嵌合抗原受体改造的NK细胞中的至少一种。
  33. 根据权利要求32所述的组合物，其特征在于，所述药物组合物还包括单克隆抗体；
    所述单克隆抗体选自西妥昔单抗、阿仑单抗、伊匹单抗、奥法木单抗中的至少一种。
  34. 根据权利要求32所述的组合物，其特征在于，所述药物组合物还包括细胞因子；
    所述细胞因子选自γ干扰素、白细胞介素中的至少一种。
  35. 权利要求1～30任一项所述的嵌合抗原受体改造的NK细胞、或根据权利要求31所述的制备方法制备得到的嵌合抗原受体改造的NK细胞、或权利要求32～34任一项所述的药物组合物中的至少一种在制备治疗以下疾病的药物中的应用：
    肿瘤、感染、炎症疾病、免疫疾病、神经系统疾病。
  36. 根据权利要求35所述的应用，其特征在于，所述肿瘤为PD-L1阳性或响应γ干扰素上调PD-L1表达水平的肿瘤。
  37. 根据权利要求35所述的应用，其特征在于，所述肿瘤包括实体瘤和/或血液癌症。
  38. 根据权利要求37所述的应用，其特征在于，所述实体瘤包括乳腺癌、皮肤癌、肝癌、卵巢癌、前列腺癌、脑癌、肾癌、肺癌中的至少一种。
  39. 一种疾病的治疗方法，其特征在于，所述治疗方法包括以下步骤：
    向人体输入嵌合抗原受体改造的NK细胞或药物组合物；
    所述嵌合抗原受体改造的NK细胞选自权利要求1～30任一项所述的嵌合抗原受体改造的NK细胞、或根据权利要求31所述的制备方法制备得到的嵌合抗原受体改造的NK细胞中的至少一种；
    所述药物组合物选自权利要求32～34任一项所述的药物组合物；
    所述疾病选自肿瘤、感染、炎症疾病、免疫疾病、神经系统疾病中的至少一种。
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