WO2022127882A1 - 筛选靶向CD47-SIRPα免疫检查点的候选药物的方法和试剂盒 - Google Patents

筛选靶向CD47-SIRPα免疫检查点的候选药物的方法和试剂盒 Download PDF

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WO2022127882A1
WO2022127882A1 PCT/CN2021/138975 CN2021138975W WO2022127882A1 WO 2022127882 A1 WO2022127882 A1 WO 2022127882A1 CN 2021138975 W CN2021138975 W CN 2021138975W WO 2022127882 A1 WO2022127882 A1 WO 2022127882A1
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sirpα
cells
tcr
cell
expression
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PCT/CN2021/138975
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French (fr)
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贾文双
李恋曲
韩宇
刘传鑫
殷刘松
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南京蓬勃生物科技有限公司
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Publication of WO2022127882A1 publication Critical patent/WO2022127882A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

Definitions

  • the application relates to the field of medicine, in particular, the application relates to methods and kits for screening candidate drugs targeting the CD47-SIRP ⁇ immune checkpoint.
  • T cells are the main target of cancer immunotherapy today, and a variety of immunosuppressants have been developed based on CTLA-4 and PD-1 on their cell membranes. Such inhibitors activate T cells and kill cancer cells by blocking ligands such as PD-L1 on the cancer cell membrane from binding to immunosuppressive sites.
  • T-cell-dependent immunotherapies have demonstrated durable and significant therapeutic effects in some patients, there are no responses or transient responses in many patients, and some patients even experience significant side effects, including severe autoimmunity. This casts a shadow over the field of immunotherapy in cancer treatment, and also forces people to develop immunotherapies with greater likelihood of response and fewer side effects, but this direction is currently a huge obstacle and challenge for immune cancer treatment.
  • anti-CD47 antibody can also synergize with rituximab, a commercialized therapeutic antibody that recognizes CD20 on human B cells, to kill human B lymphoma cells transplanted in tumor-bearing mice.
  • the present invention provides a method for screening candidate drugs targeting CD47-SIRP ⁇ immune checkpoint, comprising:
  • a target cell mimic that provides a CD47 protein and a T cell receptor (TCR) agonist
  • a drug candidate targeting the CD47-SIRP ⁇ immune checkpoint and an effector cell
  • the effector cell comprising a reporter gene and Stably expressed SIRP ⁇ and TCR
  • the expression of the reporter gene or its expression intensity is detected to determine the blocking of the CD47-SIRP ⁇ immune checkpoint by the candidate drug.
  • the application also provides a method for quality control of a drug targeting the CD47-SIRP ⁇ immune checkpoint, comprising:
  • a target cell mimic that provides a CD47 protein and a TCR agonist, a drug targeting the CD47-SIRP ⁇ immune checkpoint, and an effector cell comprising a reporter gene and stably expressed SIRP ⁇ and TCR;
  • the expression intensity of the reporter gene is detected for quality control of the drug.
  • the methods of the present application utilize target cell mimics that mimic target cells (eg, tumor cells) and effector cells comprising SIRP ⁇ , T cell receptor (TCR), and a reporter gene for in vitro assays for the efficacy of CD47-SIRP ⁇ binding inhibitor drugs and Evaluation.
  • target cell mimic is used to activate effector cells that can utilize the CD47-SIRP ⁇ -TCR signaling pathway.
  • the TCR activation signal is inhibited by CD47-SIRP ⁇ -activated cytoplasmic phosphatase SHP (Src homology region 2 domain-containing phosphatase)-1 or SHP-2, unable to activate reporter genes such as luciferase (e.g.
  • the invention utilizes an effector cell line of the CD47-SIRP ⁇ -SHP-TCR-NFAT signaling pathway, and the binding of CD47 on the target cell to SIRP ⁇ on the surface of the effector cell triggers SIRP ⁇ to interact with recruited SHP-1 or SHP
  • the coupling of -2 phosphatase and activation of the phosphatase inhibit the signal activated by the TCR, resulting in the inability to express the luciferase driven by the NFAT response element, and thus the luciferase signal cannot be detected.
  • the applicant also constructed an anti-CD3scFv and CD47 dual expression cell line as target cells for activating effector function cell lines that depend on the CD47-SIRP ⁇ -SHP-TCR-NFAT signaling pathway.
  • the TCR activation signal is inhibited by SHP-1 or SHP-2 phosphatase activated by CD47-SIRP ⁇ , and cannot drive the expression of downstream NFAT response elements to activate luciferase;
  • CD47-SIRP ⁇ immunosuppressant the CD47-SIRP ⁇ interaction is blocked, and SHP-1 or SHP-2 phosphatase cannot be recruited and activated, and the TCR activation signal is transduced to drive the downstream NFAT response element, which in turn activates luciferase.
  • CD47 Cluster of Differentiation 47, which belongs to the membrane glycoprotein and is expressed in both normal and diseased tissues. Expression in tumor cells can lead to immune escape. This glycoprotein was named ovarian cancer antigen (OA3) because it was first found to be overexpressed on ovarian cancer. Also known as integrin-associated protein (IAP) because of its relationship with beta integrin. Later, the protein was also found on the surface of red blood cells (lacking integrins), so it was named CD47. CD47 also belongs to the immunoglobulin superfamily (IgSF) and has an N-terminal extracellular Ig-like domain, five transmembrane helices and a C-terminal cytoplasmic tail.
  • IgSF immunoglobulin superfamily
  • the cytoplasmic tail According to its cytoplasmic tail, it is divided into four subtypes, type 1, type 2, type 3 and type 4, and the amino acid length varies from four amino acids (type 1) to 34 amino acids (type 4), but 16 amino acids
  • the tail isoform (type 2) is the most common and the most abundant isoform in human cells and mice.
  • SIRP ⁇ signal regulatory protein alpha
  • signal regulatory protein alpha signal regulatory protein alpha
  • ITIMs Immunoreceptor Tyrosine-Based Inhibitory Motifs
  • SH2 Src homology 2 domain protein tyrosine phosphatase
  • CD47-SIRP ⁇ binding In the absence of CD47-SIRP ⁇ binding, lack of recruitment of SHP-1 and SHP-2 is able to activate phagocytic receptors to trigger phagocytosis.
  • a "(candidate) drug targeting the CD47-SIRP ⁇ immune checkpoint” is also referred to as a "CD47-SIRP ⁇ binding inhibitor", which is any biological or biological agent capable of inhibiting the interaction of CD47 with SIRP ⁇ .
  • Chemical agents, used to block the CD47-SIRP ⁇ macrophage immune checkpoint pathway can be used to treat tumors such as leukemia, breast cancer, colorectal cancer, and more.
  • Examples of such (candidate) drugs can be antibodies targeting CD47, antibodies targeting SIRP ⁇ , recombinant SIRP ⁇ proteins or fusion proteins (such as SIRP ⁇ -Fc), or small molecules that block the interaction between CD47 and SIRP ⁇ ( Such as compounds, interfering RNA) and so on.
  • the term "antibody” covers not only complete polyclonal or monoclonal antibodies, but also any antigen-binding fragments or single chains thereof, fusion proteins comprising antibodies, and immunoglobulin molecules comprising antigen-recognition sites Any other modified configuration, such as but not limited to: scFv, single domain antibody, F(ab') 2 , etc.
  • the antibody may also be a bispecific antibody, eg, may be specific for both CD47 and other targets (eg, tumor targets such as CD20, PD-1/PD-L1, EGFR, etc.).
  • the drug or drug candidate targeting the CD47-SIRP ⁇ immune checkpoint may be selected from any one or more of the following: anti-CD47 monoclonal antibody, anti-CD47 single domain antibody, anti-CD47 Bispecific antibodies (eg, antibodies bispecific for CD47 and targets other than CD47 (eg, tumor targets)), anti-CD47 multispecific antibodies, anti-CD47 single chain antibodies, antibody conjugates comprising anti-CD47 antibodies compound (ADC), anti-SIRP ⁇ monoclonal antibody, anti-SIRP ⁇ single-chain antibody, anti-SIRP ⁇ multispecific antibody, antibody conjugate comprising anti-SIRP ⁇ antibody, SIRP ⁇ recombinant or fusion protein, and blocking the interaction between CD47 and SIRP ⁇ interacting small molecules.
  • anti-CD47 monoclonal antibody anti-CD47 single domain antibody
  • anti-CD47 Bispecific antibodies eg, antibodies bispecific for CD47 and targets other than CD47 (eg, tumor targets)
  • anti-CD47 multispecific antibodies anti-CD47 single chain antibodies
  • target cell mimetic as described herein is used to provide a CD47 protein and a TCR agonist that interact with effector cells, wherein the CD47 and TCR agonist may be contained in the same cell or target (eg, microparticles), or CD47 and TCR agonists can also exist independently of each other.
  • target cell mimics can be in the form of cells stably expressing CD47 and a TCR agonist, a TCR agonist and cells stably expressing CD47, or a TCR agonist and a microparticle coupled to the CD47 protein.
  • the cells stably expressing CD47 or stably expressing CD47 and TCR agonists can be tumor cell lines that originally express CD47 or CD47 and TCR agonists, or cells that have been genetically engineered to stably express CD47 or CD47 and TCR agonists.
  • the genetic modification can be carried out by any means known to those skilled in the art, for example by transfection of a viral vector.
  • the type of cells in the target cell mimic can be any suitable cells known in the art, such as CHO-K1, Raji and other mammalian cells.
  • the microparticles may be microspheres coated with a TCR agonist such as anti-CD3 antibody and/or CD47 protein.
  • the diameter of the microparticles or microspheres may be, for example, 5-10 ⁇ m.
  • the target cell mimic is a cell stably expressing CD47 and a TCR agonist.
  • TCR agonist is any biological or chemical agent that can activate the TCR.
  • TCR agonists can achieve specific TCR activation mediated by the major histocompatibility complex (MHC), anti-CD3 antibody-mediated activation of the TCR complex, or superantigen-mediated activation of non-specific TCR activation.
  • MHC major histocompatibility complex
  • anti-CD3 antibody-mediated activation of the TCR complex or superantigen-mediated activation of non-specific TCR activation.
  • the TCR agonist is selected from any one or more of the following: an antigen presented by the major histocompatibility complex (MHC), an anti-CD3 antibody or antibody fragment thereof, and a superantigen.
  • MHC major histocompatibility complex
  • the TCR agonist is an anti-CD3 antibody.
  • the target cell mimic is an anti-CD3 antibody and CD47 dual expressing cell line. In a preferred embodiment, the target cell mimic is an anti-CD3 scFv and CD47 dual expressing cell line.
  • step "contacting a target cell mimic that provides CD47 protein and T cell receptor (TCR) agonism, a (candidate) drug targeting the CD47-SIRP ⁇ immune checkpoint, and effector cells” the step of the methods of the present application agent, the effector cells comprise a reporter gene and stably expressed SIRP ⁇ and TCR"
  • the order of addition of target cell mimics, (candidate) drugs and effector cells is not particularly limited.
  • this step includes providing a target cell mimic, and adding a (candidate) drug and effector cells targeting the CD47-SIRP ⁇ immune checkpoint to the target cell mimic.
  • Said "providing a target cell mimic” may comprise plating and culturing cells stably expressing CD47 and a TCR agonist, or comprising plating and culturing cells stably expressing CD47 and adding a TCR agonist to said cells, or comprising adding TCR Agonists and microparticles conjugated to CD47 protein.
  • effector cell as used herein comprises a reporter gene as well as stably expressed SIRP ⁇ and TCR.
  • the effector cells may be cells genetically engineered to express SIRP ⁇ , T cell receptor (TCR) and reporter genes, or may be genetically engineered to partially or completely delete endogenous CD47 gene expression, stably express SIRP ⁇ , TCR and reporter gene cells.
  • the genetic modification can be carried out by any means known to those skilled in the art, for example by transfection of a viral vector.
  • the type of effector cells can be any suitable cells known in the art, such as Jurkat or other human-derived T cells, such as HuT-78, CEM, and the like. In some embodiments, the effector cells are cells with partial or complete deletion of CD47 gene expression.
  • the effector cells are genetically engineered to partially or completely delete the CD47 gene expression of the effector cells. In some preferred embodiments, the effector cells are gene-edited to partially or completely delete the expression of the CD47 gene. In one embodiment, the effector cells are CD47 knockout cells. In some embodiments, the CD47 gene of the effector cell is endogenous.
  • Gene engineering also known as Genetic Engineering
  • Genetic Engineering is a technique used to alter the genetic composition of an organism, including deletion of genetic material, and introduction of DNA directly prepared in vitro into a host or The cells are then fused or hybridized with the host.
  • genetic engineering includes transgenic technology and gene editing technology.
  • the "gene editing” or “gene editing technology” mentioned in the method of the present application can be a partial or complete technology such as CRISPR-Cas9 technology, zinc finger endonuclease (ZFN) technology and transcription activator-like effector nuclease (TALEN) technology. Techniques for inactivating genes.
  • the effector cell expresses the receptor for CD47, SIRP ⁇ , upon contact with CD47, the formation of a CD47-SIRP ⁇ complex inhibits TCR pathway-dependent signaling pathway activation.
  • the effector cells express SIRP ⁇ after knocking out their endogenously expressed CD47, so as to avoid the spontaneous formation of CD47-SIRP ⁇ complexes between effector cells and inhibit TCR pathway-dependent signaling pathway activation.
  • the amount of the effector cells added may be any suitable amount amount.
  • the ratio of the effector cells to the cells stably expressing CD47 or stably expressing CD47 and a TCR agonist can be, for example, 8:1, 7:1, 6:1, 5:1, 4:1 , 3:1, 2:1 or 1:1 etc.
  • the ratio of the effector cells to the cells stably expressing CD47 or CD47 and a TCR agonist may be 3:1.
  • the amount of the drug (drug candidate) added may be one or more amounts, eg, multiple amounts in serial dilution.
  • T cell receptor may be the original TCR expressed by T cells or a modified TCR, eg, a TCR modified to have a higher affinity.
  • reporter gene can be any reporter gene known in the art that is suitable for effector cells, for example, the reporter gene can be the luciferase (Luciferase) gene, secreted embryonic alkaline phosphatase (Secreted embryonic alkaline phosphatase [SEAP]) gene, ⁇ -galactosidase (Beta-galactosidase) gene, fluorescent proteins such as green fluorescent protein (Green fluorescent protein, [GFP]) gene, etc.
  • the reporter gene is driven by an activated T cell nuclear factor (NFAT) response element (RE), an activating protein 1 (AP-1) response element, or a nuclear factor kappa B (NF- ⁇ B) response element.
  • NFAT nuclear factor
  • AP-1 activating protein 1
  • NF- ⁇ B nuclear factor kappa B
  • the expression of the reporter gene is TCR pathway dependent.
  • the CD47 protein provided by the target cell mimic forms a complex upon contact with SIRP ⁇ expressed by an effector cell resulting in repressed expression of a TCR pathway-dependent reporter gene.
  • the drug candidate or drug targeting the CD47-SIRP ⁇ immune checkpoint inhibits the complex formed by the CD47 protein and SIRP ⁇ , resulting in increased expression of a TCR pathway-dependent reporter gene.
  • the step of "incubating the mixture of the target cell mimic, the drug candidate and the effector cells" described in this application can be carried out at any suitable temperature for a suitable time, such as incubation at 37°C for 2, 3, 4, 5, 6, 7, 8, 9 or 10 hours, or 12 or 24 hours.
  • the “quality control detection” step can be performed using any qualitative or quantitative method known in the art for detecting reporter genes, eg, chemiluminescence detection, flow cytometry, radioimmunoassay, fluorescence microscopy, and the like.
  • the detection is chemiluminescence detection using a microplate reader. Therefore, in some embodiments, the step of "detecting the expression or expression intensity of the reporter gene” comprises: adding the substrate of the reporter gene to the incubated mixture, and chemiluminescent method (such as a microplate reader) ) to check.
  • determining that the candidate drug blocks the CD47-SIRP ⁇ immune checkpoint includes: if the expression of the reporter gene is not detected or observed, it is determined that the candidate drug does not have the ability to block the CD47-SIRP ⁇ immune checkpoint.
  • the role of the CD47-SIRP ⁇ immune checkpoint if the expression of the reporter gene is detected or observed, it is determined that the drug candidate has the effect of blocking the CD47-SIRP ⁇ immune checkpoint, and can potentially be used as a CD47-SIRP ⁇ binding inhibitor, and , the stronger the expression intensity of the reporter gene, the higher the activity of the candidate drug as a CD47-SIRP ⁇ binding inhibitor.
  • the intensity of reporter gene expression increases with increasing antibody concentration.
  • quality control of the drug includes: if the expression intensity of the drug is reduced compared with the control drug, it is indicated that the drug is of unqualified quality or loses stability.
  • the drug candidates or drugs targeting the CD47-SIRP ⁇ immune checkpoint in the present invention inhibit the complex formed by the CD47 protein and SIRP ⁇ , resulting in increased expression of TCR pathway-dependent reporter genes.
  • the increased expression is at least 2-fold (eg, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold times, 14 times, 15 times, 20 times, 40 times, 50 times, 100 times, etc.).
  • the present application also provides a kit for screening or quality control of a candidate drug or drug targeting the CD47-SIRP ⁇ immune checkpoint, comprising: a target cell mimic, the target cell mimic
  • the drug provides CD47 protein and a TCR agonist; and effector cells comprising a reporter gene and stably expressed SIRP ⁇ and T cell receptor (TCR).
  • kits of the present application can be used according to the methods described in the present application.
  • the definitions, descriptions and preferences listed in the first and second aspects of the present application relating to the method apply equally to the third aspect.
  • the cell ratio of the effector cells to the target cell mimic is 8:1 to 1:1, preferably 3:1.
  • one microsphere corresponds to one target cell.
  • the target cell mimic is a TCR agonist and a microparticle coupled with CD47 protein, it corresponds to a cell that stably expresses TCR agonist and CD47 protein.
  • the kit may also include one or more selected from the group consisting of positive controls (eg, an antibody targeting CD47, an antibody targeting SIRP ⁇ , recombinant SIRP ⁇ protein, or fusion proteins, or small molecules that block the interaction between CD47 and SIRP ⁇ ); substrates for reporter genes; consumables (eg, multi-well plates); instructions describing methods of use (eg, the methods of the present application).
  • positive controls eg, an antibody targeting CD47, an antibody targeting SIRP ⁇ , recombinant SIRP ⁇ protein, or fusion proteins, or small molecules that block the interaction between CD47 and SIRP ⁇
  • substrates for reporter genes eg, consumables (eg, multi-well plates); instructions describing methods of use (eg, the methods of the present application).
  • the application also provides a system comprising: a target cell mimic that provides a CD47 protein and a TCR agonist; and an effector cell comprising a TCR pathway-dependent Reporter genes and stably expressed SIRP ⁇ and TCR.
  • system further comprises a drug candidate or drug targeting the CD47-SIRP ⁇ immune checkpoint.
  • the system of the present application can be used in accordance with the methods described herein.
  • the definitions, descriptions and preferences set out in the first, second and third aspects of the present application relating to the method apply equally to the fourth aspect.
  • the present application provides a cell comprising a TCR pathway-dependent reporter gene, stably expressed SIRP ⁇ and TCR, characterized in that the expression of the CD47 gene in the cell is partially or completely absent.
  • the cell is genetically engineered such that the cell has a partial or complete deletion of CD47 gene expression.
  • the genetic engineering is gene editing, preferably gene knockout.
  • the cell is a CD47 knockout cell.
  • the cell is any T cell with a TCR activation pathway.
  • the cells can be used as effector cells, such as Jurkat cells and the like.
  • the method of the present application is a cell-based high-throughput in vitro method capable of simulating drug inhibition of the interaction between CD47 and SIRP ⁇ in vitro for antibody screening, identification, stability evaluation, potency and potency determination, and QC batches Sub-releases, etc., provide a stable and inexpensive tool for biological activity detection.
  • the method of the present application is fast, sensitive, simple and easy to operate.
  • the method of the present application eliminates the need for the differentiation and culture of primary cells (needing the differentiation and culture of macrophages, which requires complicated experimental operations and huge time costs, and such a method relies heavily on the isolation and culture of PBMCs, resulting in poor reproducibility and high sensitivity of experimental results.
  • the evaluation index is more comprehensive and perfect.
  • the method of the present application utilizes stable cell lines such as cells with partial or complete loss of CD47 gene expression as effector cells, and the evaluation results have high reproducibility and high sensitivity.
  • effector cells utilizing the CD47-SIRP ⁇ -SHP-FCGR2A (Fc Fragment of IgG Receptor IIa)-NFAT signaling pathway (see for example section 1.5 of the present application).
  • binding of CD47 to SIRP ⁇ on target cells triggers the coupling of SIRP ⁇ to recruited SHP-1 or SHP-2 phosphatases and activates the phosphatase, inhibiting the signal that activates FCGR2A dependent on the Fc region of the antibody (Fragment crystallizable region).
  • the luciferase driven by the NFAT response element cannot be expressed and the luciferase signal cannot be detected.
  • CD47-SIRP ⁇ -SHP-FCGR2A Fragment of IgG Receptor IIa
  • the Fc region of the antibody can independently FCGR2A signaling pathway without blocking the action pathway of CD47-SIRP ⁇ , thereby activating the NFAT signaling pathway and causing background interference signals.
  • SEQ ID NO: 1 is the PGK promoter + hygromycin B gene fragment.
  • SEQ ID NO: 2 is the NFAT-RE nucleotide sequence.
  • SEQ ID NO:3 is the anti-CD3 mAb OKT3 light chain sequence.
  • SEQ ID NO:4 is the anti-CD3 mAb OKT3 heavy chain sequence.
  • SEQ ID NO:5 is the human CD47 ORF DNA sequence.
  • SEQ ID NO: 6 is the human SIRP ⁇ sequence.
  • SEQ ID NO:7 is the neomycin resistance gene sequence.
  • SEQ ID NO: 8 is the human FCGR2A sequence.
  • SEQ ID NO: 9 is the guide RNA sequence.
  • SEQ ID NO: 10 is the upstream primer sequence F.
  • SEQ ID NO: 11 is the downstream primer sequence R.
  • SEQ ID NO: 12 is the luciferase protein sequence.
  • Figure 2 shows that CHO-K1/anti-CD3-scFv activates the TCR of Jurkat/NFAT-Luc, driving the expression of NFAT-regulated luciferase.
  • Figure 3 shows the detection of CD47 expression levels in the CHO-K1/anti-CD3-scFv/CD47 cell pool.
  • Figure 4 shows the detection of SIRP ⁇ expression levels in the Jurkat/NFAT-Luc/SIRP ⁇ cell pool.
  • FIG. 5 shows that Rituxan activates the FCGR2A-NFAT-Luc signaling pathway in Jurkat/NFAT-Luc/SIRP ⁇ /FCGR2A effector cells after binding to Raji cells, and activates the expression of luciferase.
  • the binding of the Fc region of Rituxan to FCGR2A causes activation of effector cells independent of CD47-SIRP ⁇ blockade, introducing background interference signals.
  • Figure 6 shows that anti-CD47 antibodies restore activation signals inhibited by CD47-SIRP ⁇ .
  • FIG. 7 shows that anti-SIRP ⁇ antibody (OSE172) restores anti-CD3 activation signal inhibited by CD47-SIRP ⁇ .
  • FIG. 8 shows that anti-CD47 antibody (Hu5F9) restores Raji-activated Jurkat signaling inhibited by CD47-SIRP ⁇ .
  • FIG. 9 Sequencing results showing the CD47 gene editing efficiency of the Jurkat (CD47 knockout)/NFAT-Luc cell pool.
  • FIG. 10 Sequencing results show that 4 bases of gene editing site are deleted after CD47 gene knockout in Jurkat (CD47 knockout)/NFAT-Luc monoclonal cell line.
  • FIG. 11 shows the detection of CD47 expression levels in Jurkat/NFAT-Luc cells and Jurkat(CD47 knockout)/NFAT-Luc cells.
  • Figure 12 shows that anti-CD47 antibody restores the activation signal of Jurkat cells inhibited by CD47-SIRP ⁇ after CD47 gene knockout and the fold ratio of the signal to the background value.
  • Figure 13 shows serial passage stability data for cell function assays for the CHO-K1/anti-CD3-scFv/CD47 and Jurkat/NFAT-Luc/SIRP ⁇ pair.
  • Figure 14 shows serial passage stability data for cell function assays for CHO-K1/anti-CD3-scFv/CD47 and Jurkat (CD47 knockout)/NFAT-Luc/SIRP ⁇ pair.
  • HEK293T cells were trypsinized, resuspended in DMEM supplemented with 10% FBS, and plated at 6-10 x 106 HEK293T/plate (10 cm). The cell density was observed the next day, and the transfection was carried out at 80-90% confluence, and the medium was replaced with fresh medium before transfection.
  • Each plate was transfected with 7.5 ⁇ g psPAX2 (in-house synthesis by GenScript, sequence derived from Addgene plasmid #12260), 5 ⁇ g PMD2.G (in-house synthesis by GenScript, sequence derived from Addgene plasmid #12259), 12.5 ⁇ g pLVX-NFAT-Luc-Hygro .
  • Lipofectamine 3000 (Thermo Fisher, Cat. No. L3000001) was mixed with the plasmid and added to the plate. Change to fresh medium 6-8 hours after transfection. The viral supernatant was collected 48-56 hours after transfection, filtered through a 0.45 ⁇ m filter, and ultracentrifuged. The virus pellet was resuspended in 500 ⁇ l of fresh medium and stored at -80°C.
  • Infected cells Jurkat cells were plated into 6-well plates, 3 mL of medium was added, and cultured overnight. Before infection, take out from the refrigerator and quickly thaw the virus in a 37°C water bath, aspirate the original medium of the cells, add 1/2 volume of fresh medium, and then add the virus (NFAT-Luc) stock solution to the cells and mix well. After 2-3 hours of infection by centrifugation, place the culture plate in a 37°C, 5% CO2 incubator for 24 hours. On the second day (about 24 hours) after infection, the virus-containing culture medium was aspirated, replaced with fresh complete culture medium, and cultured at 37°C was continued.
  • Hygromycin resistance screening add 200 ⁇ g/ml hygromycin to the cell culture medium, and replace the complete medium containing hygromycin every 2-3 days until the cells in the uninfected screening control group are killed by hygromycin . Continuous screening until a stable cell line is obtained.
  • Detection of NFAT-mediated luciferase expression by chemiluminescence Take an appropriate amount of Jurkat/NFAT-Luc cells and spread them in a 384-well plate. 4X PMA (phorbol 12-myristate 13-acetate) and 4X ionomycin (Iono) working solutions were prepared and added to 384-well plates plated with Jurkat/NFAT-Luc cells for finalization. Concentrations reached 10 ⁇ g/mL PMA and 1 ⁇ M Iono. An equal volume of cell culture medium was added to the negative control group. The 384-well plate was then placed in a 37 °C, 5% CO2 incubator for 6 h.
  • 4X PMA phorbol 12-myristate 13-acetate
  • Iono ionomycin
  • Bio-Glo TM Promega, #G7940
  • the assay was performed using PHERAstar FSX (BMGLABTEC) to detect NFAT response element-driven luciferase signal upon Jurkat activation.
  • Jurkat/NFAT-Luc effector cells were limitedly diluted into a 96-well plate, and the 96-well plate was observed under a microscope after 2-3 days, and the wells with the monoclonal were marked. Monoclonal cells were transferred to 24-well plates and subsequently expanded to 6-well plates. After monoclonal expansion, the NFAT-mediated luciferase expression was re-verified with the above steps.
  • Lentiviral (anti-CD3-scFv) production HEK293T cells were trypsinized, resuspended in DMEM supplemented with 10% FBS, and plated at 6-10 x 106 HEK293T/plate (10 cm). The cell density was observed the next day, and the transfection was carried out at 80-90% confluence, and fresh medium was replaced before transfection. Each plate was transfected with 7.5 ⁇ g psPAX2, 5 ⁇ g PMD2.G and 12.5 ⁇ g pLVX-anti-CD3-scFv-RE-Hygro. Lipofectamine 3000 (Thermo Fisher, Cat. No. L3000001) was mixed with the plasmid and added to the plate.
  • the viral supernatant was collected 48-56 hours after transfection, filtered through a 0.45 ⁇ m filter, and ultracentrifuged. The virus pellet was resuspended in 500 ⁇ l of fresh medium and stored at -80°C.
  • Infected cells Plate CHO-K1 cells into 6-well plates, add 3 mL of medium, and culture overnight. Before infection, take out from the refrigerator and rapidly thaw the virus (anti-CD3-scFv) in a 37°C water bath, aspirate the original medium of the cells, add 1/2 volume of fresh medium, and then add the virus stock solution to the cells and mix well. After centrifuging the infection for 0.5-1 h, place the plate into a 37°C, 5% CO2 incubator for 24 h. On the second day (about 24 hours) after infection, the virus-containing medium was aspirated, replaced with fresh complete medium, and the culture was continued at 37°C.
  • virus anti-CD3-scFv
  • Hygromycin resistance screening add 400 ⁇ g/ml hygromycin to the cell culture medium, and replace the complete medium containing hygromycin every 2-3 days until the cells in the uninfected screening control group are killed by hygromycin . Continuous screening to obtain stable cell lines.
  • Anti-CD3-scFv function test to verify CHO-K1/anti-CD3-scFv cell line Take 10,000 target cells CHO-K1/anti-CD3-scFv cells and spread them in 384-well plates, and add an equal amount of CHO- K1 cells were cultured overnight. The medium in the wells was discarded the next day, and effector cells Jurkat/NFAT-Luc were added to 384-well plates plated with CHO-K1/anti-CD3-scFv cells or negative control CHO-K1 to make effector cells:target cells ( E:T) ratio is 4:1. The 384-well plate was then placed in a 37 °C, 5% CO2 incubator for 6 h.
  • Bio-Glo TM Promega, #G7940
  • PHERAstar was used for detection, and the detection index was that anti-CD3-scFv activates the TCR signaling pathway on the surface of Jurkat, and then activates the luciferase signal driven by the NFAT response element.
  • Monoclonal selection Dilute the cell pool to a 96-well plate, observe the 96-well plate under a microscope after 7 days, and mark the wells with monoclonal. Monoclonal cells were transferred to 24-well plates and subsequently expanded to 6-well plates. After monoclonal expansion, the above steps were followed to verify that CHO-K1/anti-CD3-scFv activates NFAT-mediated luciferase expression in Jurkat/NFAT-Luc effector cells.
  • the plasmid vector pLVX-Puro was digested with the same restriction endonuclease at the same time.
  • the plasmid vector fragment was ligated and transformed into E. coli competent cells to obtain plasmid pLVX-CD47-Puro.
  • Lentiviral (CD47) production HEK293T cells were trypsinized, resuspended in DMEM supplemented with 10% FBS, and plated at 6-10 x 106 HEK293T/plate (10 cm). The cell density was observed the next day, and the transfection was carried out at 80-90% confluence, and fresh medium was replaced before transfection. Each plate was transfected with 7.5 ⁇ g psPAX2, 5 ⁇ g PMD2.G and 12.5 ⁇ g pLVX-CD47-Puro. Lipofectamine 3000 (Thermo Fisher, Cat. No. L3000001) was mixed with the plasmid and added to the plate. Replace with fresh medium 6-8 hours after transfection. The viral supernatant was collected 48-56 hours after transfection, filtered through a 0.45 ⁇ m filter, and ultracentrifuged. The virus pellet was resuspended in 500 ⁇ l of fresh medium and stored at -80°C.
  • Infected cells CHO-K1/anti-CD3-scFv cells were plated into 6-well plates, 3 mL of medium was added, and cultured overnight. Before infection, take out from the refrigerator and quickly thaw the virus (CD47) in a 37°C water bath, aspirate the original medium of the cells, add 1/2 volume of fresh medium, and then add the virus stock solution to the cells and mix well. After centrifuging the infection for 0.5-1 h, place the plate into a 37°C, 5% CO2 incubator for 24 h. On the second day (about 24 hours) after infection, the virus-containing medium was aspirated, replaced with fresh complete medium, and the culture was continued at 37°C.
  • Detection of CD47 expression by FACS Take a part of the obtained stable cells into a FACS tube, and centrifuge to remove the supernatant. PE anti-human CD47 antibody (BioLegend, Cat#323108) was added and incubated at 4°C for 30 minutes. After 30 minutes, the supernatant was washed off, resuspended in FACS buffer, and the CD47 expression level was detected on the machine (BD FACSCelesta TM ). As shown in Figure 3, FACS detected that the CHO-K1/C2/CD47 cell pool overexpressed CD47.
  • Puromycin and hygromycin dual resistance screening add 8 ⁇ g/ml puromycin and 400 ⁇ g/ml hygromycin to the cell culture medium, and replace the complete medium containing puromycin and hygromycin every 2-3 days , to the uninfected selection control cells were killed by puromycin and hygromycin. Continuous screening to obtain stable cell lines.
  • Monoclonal selection Dilute the cell pool to a 96-well plate, observe the 96-well plate under a microscope after 7 days, and mark the wells with monoclonal. Monoclonal cells were transferred to 24-well plates and subsequently expanded to 6-well plates. After monoclonal expansion, CD47 expression was re-verified by the above steps.
  • the SIRP ⁇ ORF DNA fragment was combined with the vector backbone pLVX-Puro (Clontech, Cat. No. 632164) using Clone EZ (GenScript) technology. Connected and transformed into E. coli competent cells to obtain plasmid pLVX-SIRP ⁇ -Puro.
  • HEK293T cells were trypsinized, resuspended in DMEM supplemented with 10% FBS, and plated at 6-10 x 106 HEK293T/plate (10 cm). The cell density was observed the next day, and the transfection was carried out at 80-90% confluence, and fresh medium was replaced before transfection. Each plate was transfected with 7.5 ⁇ g psPAX2, 5 ⁇ g PMD2.G and 12.5 ⁇ g pLVX-SIRP ⁇ -Puro. Lipofectamine 3000 (Thermo Fisher, Cat. No. L3000001) was mixed with the plasmid and added to the plate. Replace with fresh medium 6-8 hours after transfection. The viral supernatant was collected 48-56 hours after transfection, filtered through a 0.45 ⁇ m filter, and ultracentrifuged. The virus pellet was resuspended in 500 ⁇ l of fresh medium and stored at -80°C.
  • Infected cells The Jurkat/NFAT-Luc cells prepared in Example 1.1 were plated into a 6-well plate, 3 mL of medium was added, and cultured overnight. Before infection, take out from the refrigerator and quickly thaw the virus (SIRP ⁇ ) in a 37°C water bath, aspirate the original medium of the cells, add 1/2 volume of fresh medium, and then add the virus stock solution to the cells and mix well. After 2-3 hours of infection by centrifugation, place the culture plate in a 37°C, 5% CO2 incubator for 24 hours. On the second day (about 24 hours) after infection, the virus-containing medium was aspirated, replaced with a fresh complete medium, and the culture at 37°C was continued.
  • SIRP ⁇ virus
  • Puromycin and hygromycin dual resistance screening add 1 ⁇ g/ml puromycin and 200 ⁇ g/ml hygromycin to the cell culture medium, and replace the complete medium containing puromycin and hygromycin every 2-3 days , to the uninfected selection control cells were killed by puromycin and hygromycin. Continuous screening to obtain stable cell lines.
  • FACS detection of SIRP ⁇ expression take a part of the obtained stable cells into a FACS tube, and centrifuge to remove the supernatant.
  • FITC anti-human CD172a/b (SIRP ⁇ lpha/beta) (BioLegend, Cat#337304) was added and incubated at 4°C for 30 minutes. After 30 minutes, the supernatant was washed off, resuspended in FACS buffer, and the expression level of SIRP ⁇ was detected on the machine (BD FACSCelesta TM ). As shown in Figure 4, FACS detected that the Jurkat/NFAT-Luc/SIRPa cell pool overexpressed SIRP ⁇ .
  • Monoclonal selection Dilute the cell pool to a 96-well plate, observe the 96-well plate under a microscope after 7 days, and mark the wells with monoclonal. Monoclonal cells were transferred to 24-well plates and subsequently expanded to 6-well plates. After monoclonal expansion, the expression of SIRP ⁇ was verified and established by the above steps.
  • neomycin gene (shown in SEQ ID NO: 7) was synthesized and the above fragment was cloned into pLVX-Puro (Clontech, Cat. No. 632164) vector, ligated with Clone EZ (GenScript), and transformed into E. coli competent cells to obtain the vector pLVX-Neo.
  • FCRG2A the DNA sequence of the human FCRG2A protein (the encoded FCRG2A amino acid sequence is shown in SEQ ID NO: 8) was subjected to gene synthesis, the DNA fragment of the FCRG2A ORF was connected with the vector backbone pLVX-Neo using Clone EZ (GenScript) technology, and transformed into Escherichia coli Competent cells were obtained to obtain plasmid pLVX-FCRG2a-Neo.
  • FCRG2A Lentiviral (FCRG2A) production: HEK293T cells were trypsinized, resuspended in DMEM supplemented with 10% FBS, and plated at 6-10 x 106 HEK293T/plate (10 cm). The cell density was observed the next day, and the transfection was carried out at 80-90% confluence, and fresh medium was replaced before transfection. Each plate was transfected with 7.5 ⁇ g psPAX2, 5 ⁇ g PMD2.G and 12.5 ⁇ g pLVX-FCRG2A-Neo. Lipofectamine 3000 (Thermo Fisher, Cat. No. L3000001) was mixed with the plasmid and added to the plate. Change to fresh medium 6-8 hours after transfection. The viral supernatant was collected 48-56 hours after transfection, filtered through a 0.45 ⁇ m filter, and ultracentrifuged. The virus pellet was resuspended in 500 ⁇ l of fresh medium and stored at -80
  • Infected cells The Jurkat/NFAT-Luc/SIRP ⁇ cells prepared in Example 1.4 were plated on a 6-well plate, 3 mL of medium was added, and cultured overnight. Before infection, take out from the refrigerator and quickly thaw the virus (FCRG2A) in a 37°C water bath, aspirate the original medium of the cells, add 1/2 volume of fresh medium, and then add the virus stock solution to the cells and mix well. After 2-3 hours of infection by centrifugation, place the culture plate in a 37°C, 5% CO2 incubator for 24 hours. On the second day (about 24 hours) after infection, the virus-containing medium was aspirated, replaced with fresh complete medium, and the culture was continued at 37°C.
  • FCRG2A virus
  • Neomycin, puromycin and hygromycin triple resistance screening add 400 ⁇ g/ml G418, 1 ⁇ g/ml puromycin and 200 ⁇ g/ml hygromycin to the cell culture medium, replace the complete G418-containing cell culture medium every 2-3 days The medium was cultured once until the cells of the uninfected selection control group were killed by G418, puromycin and hygromycin. Continuous screening to obtain stable cell lines.
  • the TCR activation signal is inhibited, but after the addition of Rituxan, it can still bind to CD20 on the surface of Raji cells through the variable region of the antibody It binds to the effector cell FCGR2A with the Fc region of the antibody, thereby activating the ADCP downstream signal NFAT, resulting in the expression of the luciferase gene downstream of the NFAT response element.
  • Monoclonal selection Dilute the cell pool to a 96-well plate, observe the 96-well plate under a microscope after 7 days, and mark the wells with monoclonal. Monoclonal cells were transferred to 24-well plates and subsequently expanded to 6-well plates. After monoclonal expansion, the above steps were used to verify the expression of FCGR2Aa and colonize.
  • CRISPR guide RNA plasmid According to the sequence information of CD47 genome (Gene ID: 961), a guide RNA plasmid targeting CD47 was designed and synthesized.
  • the guide RNA sequence is atcgagctaaaatatcgtgt (SEQ ID NO: 9).
  • the guide RNA plasmid (the plasmid backbone was purchased from Zhang Feng's laboratory with authorization) carries Cas9 and GFP sequences, and the GFP fluorescent protein is the marker for subsequent positive cell pool sorting.
  • Primer synthesis Design and synthesize primers at 300-500bp upstream and downstream of the CD47 genomic sequence corresponding to the guide RNA sequence.
  • the upstream and downstream primer sequences are F: 5'GCCGCTGTTTATCTTAAGGATTTG 3' (SEQ ID NO:10), R: 5'CGGTAACAGCCTGCTTTTTGATTC 3' (SEQ ID NO:11).
  • Transfection revive and culture the Jurkat NFAT-Luc effector cell line.
  • aspirate 3 ⁇ 10 6 cell suspension, centrifuge at 800 rpm for 4 minutes, and remove the supernatant; resuspend the cell pellet with 5 ml of PBS and centrifuge at 800 rpm 4 min to remove supernatant.
  • the cell pellet was resuspended with 100 ⁇ l of electroporation buffer (Celetrix), and 5 ⁇ g of guide RNA plasmid was added to the cell suspension and mixed.
  • the guide RNA plasmid-cell suspension was transferred into an electroporation tube, and the electroporation tube was placed in an electroporator (Celetrix) for electroporation.
  • a GFP-positive cell population (BD FACSMelody TM ) was flow-sorted and cultured for 3 days after sorting to restore the Jurkat (CD47 knockout)/NFAT-Luc cell pool state.
  • BD FACSMelody TM GFP-positive cell population
  • about 0.5 ⁇ 10 5 of sorted cells were taken, and the cell genome was extracted with rapid genomic extraction reagent (LUCIGEN), and then 1 ⁇ l of the genome was aspirated for PCR amplification, and the PCR products were recovered for Sanger sequencing.
  • the sequencing results showed that the cell pool The CD47 genome editing efficiency was 82%.
  • the Sanger sequencing results showed multiple peaks at the gRNA-Cas9 cleavage site (dotted line), indicating that the cell pool CD47 genome was cleaved to generate multiple genotypes.
  • Analysis of Sanger sequencing results by online software https://ice.synthego.com/#/) showed that the cell pool CD47 genome editing efficiency was 82% (Fig. 9 bottom panel).
  • the Jurkat (CD47 knockout)/NFAT-Luc cell pool was plated into a 96-well plate by limiting dilution with one cell per well. After 2-3 days, the 96-well plate was observed under a microscope, and the monoclonal cells were marked. wells, followed by continuous culture for two weeks.
  • Monoclonal identification When the monoclonal cells in the 96-well plate expand to 60% of the bottom area of the well plate, the monoclonal cells are transferred to a 24-well plate for expanded culture. At the same time, about 0.5 ⁇ 10 5 cells were taken, and the cell genome was extracted with the rapid extraction genome reagent (LUCIGEN), and then 1 ⁇ l of the genome was aspirated for PCR amplification, and the PCR products were recovered for Sanger sequencing. The results of Sanger sequencing showed that Jurkat (CD47 The CD47 gene of knockout)/NFAT-Luc clone64 was deleted by 4 bp.
  • LUCIGEN rapid extraction genome reagent
  • the Sanger sequencing results showed a 4 bp (ATCG) deletion before the gRNA-Cas9 cleavage site (dotted line).
  • the Sanger sequencing results were analyzed by online software (https://ice.synthego.com/#/), and the CD47 gene of Jurkat (CD47 knockout)/NFAT-Luc clone64 was deleted by 4 bp ( Figure 10, bottom panel)
  • Detection of CD47 expression by FACS Continue to expand the culture of Jurkat (CD47 knockout)/NFAT-Luc clone64. At the same time, a portion of the monoclonal cells was taken into a FACS tube, and the supernatant was removed by centrifugation. PE anti-human CD47 antibody (BioLegend, Cat#323108) was added and incubated at 4°C for 30 minutes. After 30 minutes, the supernatant was washed off, resuspended in FACS buffer, and the CD47 expression level was detected on the machine (BD FACSCelesta TM ). As shown in Figure 11, FACS did not detect the surface of Jurkat (CD47 knockout)/NFAT-Luc cells CD47, indicating that CD47 knockout was successful.
  • Plasmid pLVX-SIRP ⁇ -Puro was prepared as in Example 1.4, and lentivirus (SIRP ⁇ ) was produced.
  • Infected cells The Jurkat (CD47 knockout)/NFAT-Luc cells prepared in Example 1.6 were plated into a 6-well plate, 3 mL of medium was added, and cultured overnight. Before infection, take out from the refrigerator and quickly thaw the virus (SIRP ⁇ ) in a 37°C water bath, aspirate the original medium of the cells, add 1/2 volume of fresh medium, and then add the virus stock solution to the cells and mix well. After 2-3 hours of infection by centrifugation, place the culture plate in a 37°C, 5% CO2 incubator for 24 hours. On the second day (about 24 hours) after infection, the virus-containing medium was aspirated, replaced with a fresh complete medium, and the culture at 37°C was continued.
  • SIRP ⁇ virus
  • Puromycin and hygromycin dual resistance screening add 1 ⁇ g/ml puromycin and 200 ⁇ g/ml hygromycin to the cell culture medium, and replace the complete medium containing puromycin and hygromycin every 2-3 days , to the uninfected selection control cells were killed by puromycin and hygromycin. Continuous screening to obtain stable cell lines.
  • FACS detection of SIRP ⁇ expression take a part of the obtained stable cells into a FACS tube, and centrifuge to remove the supernatant.
  • FITC anti-human CD172a/b (SIRP ⁇ lpha/beta) (BioLegend, Cat#337304) was added and incubated at 4°C for 30 minutes. After 30 minutes, the supernatant was washed off, resuspended in FACS buffer, and the expression level of SIRP ⁇ was detected by computer (BD FACSCelesta TM ).
  • Monoclonal selection Dilute the cell pool to a 96-well plate, observe the 96-well plate under a microscope after 7 days, and mark the wells with monoclonal. Monoclonal cells were transferred to 24-well plates and subsequently expanded to 6-well plates. After monoclonal expansion, the expression of SIRP ⁇ was verified and established by the above steps.
  • Anti-CD47 antibody blocks CD47-SIRP ⁇ interaction pathway to restore Jurkat TCR activation signal
  • Bio-Glo TM Promega, #G7940
  • PHERAstar FSX BMGLABTEC
  • Anti-SIRP ⁇ antibody blocks CD47-SIRP ⁇ interaction pathway to restore Jurkat TCR activation signal
  • Anti-CD47 antibody blocks the CD47-SIRP ⁇ interaction pathway and restores Raji cells to activate Jurkat TCR signaling
  • Anti-CD47 antibody blocks CD47-SIRP ⁇ interaction pathway to restore Jurkat (CD47 knockout) TCR activation signal
  • Bio-Glo TM Promega, #G7940
  • PHERAstar FSX BMGLABTEC
  • Jurkat/NFAT-Luc/SIRP ⁇ effector cells Compared with Jurkat/NFAT-Luc/SIRP ⁇ effector cells (EC50 of 0.8789 ⁇ g/ml, as shown in Figure 6), Jurkat (CD47 knockout)/NFAT-Luc/SIRP ⁇ effector cells maintained detection sensitivity (EC50 of 0.5195 ⁇ g) /ml), the signal-fold ratio (signal value/background value) increased from 4-fold to 15-fold, indicating that knocking out the endogenously expressed CD47 in Jurkat significantly increased the detection window. At the same time, the knockout of the endogenous CD47 gene on the effector cells eliminated the CD47-SIRP ⁇ mutual inhibitory effect between the effector cells and improved the stability of long-term cell culture.

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Abstract

一种筛选靶向CD47-SIRPα免疫检查点的候选药物的方法和试剂盒。所述方法包括使靶细胞模拟物、靶向CD47-SIRPα免疫检查点的候选药物和效应细胞接触，所述靶细胞模拟物提供CD47蛋白和T细胞受体(TCR)激动剂，所述效应细胞包含报告基因以及稳定表达的SIRPα和TCR；孵育所述靶细胞模拟物、所述候选药物和所述效应细胞的混合物；和检测所述报告基因的表达或其表达强度来判断所述候选药物对CD47-SIRPα免疫检查点的阻断。所述方法和试剂盒能够在体外定性或定量地高通量评价所述药物的药效指标，评价结果具有高重复性和高灵敏性。

Description

筛选靶向CD47-SIRPα免疫检查点的候选药物的方法和试剂盒
相关申请的交叉引用
本申请要求2020年12月17日提交中国专利局的申请号为202011497221.6、名称为“筛选靶向CD47-SIRPα免疫检查点的候选药物的方法和试剂盒”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本申请涉及医药领域，尤其地，本申请涉及筛选靶向CD47-SIRPα免疫检查点的候选药物的方法和试剂盒。
背景技术
癌症免疫疗法在过去十年中已经成为治疗肿瘤的明星疗法。T细胞是现今癌症免疫治疗的主要靶标，根据其细胞膜上CTLA-4和PD-1已开发了多种免疫抑制剂。此类抑制剂通过阻断癌细胞膜上的配体如PD-L1与免疫抑制点结合来活化T细胞，杀伤癌细胞。虽然T细胞依赖的免疫疗法在某些患者中表现持久和显著的治疗效果，但在许多患者中无反应或反应短暂，有些患者甚至出现明显的副作用，包括严重的自身免疫。这为免疫疗法在癌症治疗领域蒙上了阴影，同时也迫使人们去开发反应可能性更大，副作用小的免疫疗法，但目前该方向是免疫癌症治疗的一个巨大障碍与挑战。
通过阻断SIRPα-CD47相互作用来促进髓样细胞(特别是巨噬细胞)吞噬和消除肿瘤细胞是继T细胞癌症免疫疗法后的一个新的热点。巨噬细胞是先天免疫反应的一部分，在实体瘤中含量丰富，通过吞噬作用清除外来病原物。巨噬细胞的吞噬作用依赖于其细胞膜表面的信号调节蛋白α(signal regulatory protein alpha，SIRPα)与靶细胞表面的糖蛋白CD47之间的相互作用来调节。大量研究发现阻断CD47与SIRPα的相互作用能显著增强巨噬细胞在体外吞噬肿瘤细胞的能力。在免疫缺陷的鼠体内，在无T细胞、B细胞和NK细胞的情况下，单 纯激活CD47-SIRPα调节的吞噬作用能迅速清除移植的肿瘤。该治疗效果同样在包括造血和非造血细胞在内的多种肿瘤细胞类型中被证实。除此之外，抗CD47抗体也可与利妥昔单抗(识别人B细胞上的CD20的商业化治疗性抗体)产生协同作用，杀伤荷瘤小鼠体内移植的人B淋巴瘤细胞。
为加速靶向CD47-SIRPα免疫检查点的候选药物开发，急需建立一种基于高通量筛选或鉴定的高效且方便的方法。
发明内容
在第一方面，本发明提供了一种筛选靶向CD47-SIRPα免疫检查点的候选药物的方法，其包括：
使靶细胞模拟物、靶向CD47-SIRPα免疫检查点的候选药物和效应细胞接触，所述靶细胞模拟物提供CD47蛋白和T细胞受体(TCR)激动剂，所述效应细胞包含报告基因以及稳定表达的SIRPα和TCR；
孵育所述靶细胞模拟物、所述候选药物和所述效应细胞的混合物；和
检测所述报告基因的表达或其表达强度来判断所述候选药物对CD47-SIRPα免疫检查点的阻断。
在第二方面，本申请还提供了一种对靶向CD47-SIRPα免疫检查点的药物进行质量控制的方法，其包括：
使靶细胞模拟物、靶向CD47-SIRPα免疫检查点的药物和效应细胞接触，所述靶细胞模拟物提供CD47蛋白和TCR激动剂，所述效应细胞包含报告基因以及稳定表达的SIRPα和TCR；
孵育所述靶细胞模拟物、所述药物和所述效应细胞的混合物；和
检测所述报告基因的表达强度来对所述药物进行质量控制。
本申请的方法利用模拟靶细胞(例如肿瘤细胞)的靶细胞模拟物和包含SIRPα、T细胞受体(TCR)和报告基因的效应细胞对CD47-SIRPα结合抑制剂药物的药效进行体外检测和评价。其中，所述靶细胞模拟物用于激活能够利用CD47-SIRPα-TCR信号通路的效应细胞，当TCR激动剂激活效应细胞上的TCR通路，在没有CD47-SIRPα结合抑制剂的存在的情况下，TCR激活信号被CD47-SIRPα激活的细胞质磷酸酶(cytoplasmic phosphatase)SHP(Src  homology region 2 domain-containing phosphatase)-1或SHP-2抑制，无法激活报告基因例如荧光素酶(例如由活化T细胞核因子(NFAT)反应元件驱动)的表达；而在存在CD47-SIRPα结合抑制剂的情况下，CD47与SIRPα之间的相互作用被阻断，无法募集并激活SHP-1或SHP-2磷酸酶，TCR激活信号被传导，从而激活报告基因例如荧光素酶的表达。通过测定报告基因例如荧光素酶的表达或信号强度，可以推断出药物或候选药物的效价和效力。
在一个实施方案中，本发明利用CD47-SIRPα-SHP-TCR-NFAT信号通路的效应功能细胞系，靶细胞上CD47与效应功能细胞表面的SIRPα的结合会触发SIRPα与募集的SHP-1或SHP-2磷酸酶的偶联并激活磷酸酶，抑制TCR激活的信号，致使NFAT反应元件驱动的荧光素酶无法表达，因而无法检测荧光素酶信号。当CD47-SIRPα相互作用被免疫抑制剂破坏后，SHP-1或SHP-2无法被募集到SIRPα胞质尾巴并被激活，从而TCR激活信号得以传导并激活下游NFAT反应元件，因此荧光素酶表达出来。通过测定荧光素酶的信号强度，可以推断出免疫抑制剂的效价和效力。
申请人也构建一种anti-CD3scFv和CD47双表达细胞系作为靶细胞，用于激活依赖于CD47-SIRPα-SHP-TCR-NFAT信号通路的效应功能细胞系，当anti-CD3scFv激活效应细胞上的TCR通路，在无CD47-SIRPα免疫抑制剂的情况，TCR激活信号被CD47-SIRPα激活的SHP-1或SHP-2磷酸酶抑制，无法驱动下游NFAT反应元件激活荧光素酶的表达；而在有CD47-SIRPα免疫抑制剂的情况，CD47-SIRPα相互作用被阻断，无法募集并激活SHP-1或SHP-2磷酸酶，TCR激活信号被传导下去驱动下游NFAT反应元件，进而激活荧光素酶的表达。
本申请中所述的“CD47”的全称是Cluster of Differentiation 47，其属于膜糖蛋白，在正常和患病组织中均有表达。在肿瘤细胞中的表达可导致免疫逃逸。该糖蛋白因其首先被发现在卵巢癌上过度表达被命名为卵巢癌抗原(OA3)。又因其与β整联蛋白的关系，也被称为整联蛋白相关蛋白(IAP)。后来在红细胞(缺乏整合素)的表面也发现了该蛋白，故而规范其命名为CD47。CD47也属于免疫球蛋白超家族(IgSF)，具有一个N端胞外Ig样结构域，五个跨膜螺旋和一个C端胞质尾。根据其胞质尾巴，又分为四个亚型1型、2型、3型和4型，氨基酸长度从四个氨基酸(1型)到34个氨基酸(4型)不等，但是16个 氨基酸的尾巴亚型(2型)是最常见的，是在人类细胞和小鼠中丰度最高的亚型。
本申请中所述的“SIRPα”的全称是信号调节蛋白α(signal regulatory protein alpha)，其是CD47的受体，是在髓样细胞(包括巨噬细胞、树突状细胞和嗜中性粒细胞)上表达的抑制性受体。SIRPα的胞质结构域中包含一段酪氨酸的模体，称为ITIM(基于免疫受体酪氨酸的抑制模体)，这些模体被磷酸化并募集抑制分子，尤其是包含Src同源2(SH2)域的蛋白质酪氨酸磷酸酶(SHP)-1和SHP-2。CD47与SIRPα的结合会触发SIRPα与这些磷酸酶的偶联，在抑制吞噬作用中起着关键作用。在没有CD47-SIRPα结合的情况下，缺乏募集的SHP-1和SHP-2能够激活吞噬细胞受体以触发吞噬作用。
在本申请中，“靶向CD47-SIRPα免疫检查点(immune checkpoint)的(候选)药物”也称为“CD47-SIRPα结合抑制剂”，其是能够抑制CD47与SIRPα的相互作用的任何生物或化学药剂，用于阻断CD47-SIRPα巨噬细胞免疫检查点通路，可用于治疗肿瘤，例如白血病、乳腺癌、结直肠癌等等。所述(候选)药物的例子可以是靶向CD47的抗体，靶向SIRPα的抗体、SIRPα重组蛋白或融合蛋白(如SIRPα-Fc)，或阻断CD47与SIRPα之间的相互作用的小分子(如化合物、干扰RNA)等。
在本发明中，术语“抗体”不仅涵盖了完整的多克隆或单克隆抗体，也包含任何抗原结合片段或者其单链，包含抗体的融合蛋白，以及包含抗原识别位点的免疫球蛋白分子的任何其它修饰的构型，例如但不限于：scFv，单域抗体，F(ab’) 2等。抗体还可以是双特异性抗体，例如可以是对CD47和其他靶标(例如肿瘤靶标，例如CD20、PD-1/PD-L1、EGFR等等)均具有特异性的抗体。
在一些实施方式中，所述靶向CD47-SIRPα免疫检查点的药物或候选药物可以选自以下中的任一种或更多种：抗CD47单克隆抗体、抗CD47单域抗体、抗CD47的双特异性抗体(例如对CD47和除CD47之外的其他靶标(例如肿瘤靶标)具有双特异性的抗体)、抗CD47多特异性的抗体、抗CD47单链抗体、包含抗CD47抗体的抗体缀合物(ADC)、抗SIRPα单克隆抗体、抗SIRPα单链抗体、抗SIRPα多特异性抗体、包含抗SIRPα抗体的抗体缀合物、SIRPα重组或融合蛋白，和阻断CD47与SIRPa之间的相互作用的小分子。
本申请所述的“靶细胞模拟物”用于提供与效应细胞相互作用的提供CD47蛋白和TCR激动剂，其中CD47和TCR激动剂可以包含在同一细胞或靶标(例如微型颗粒)中，或者CD47和TCR激动剂也可以独立于彼此存在。例如，靶细胞模拟物的形式可以是：稳定表达CD47和TCR激动剂的细胞、TCR激动剂和稳定表达CD47的细胞，或者TCR激动剂和偶联CD47蛋白的微型颗粒。其中，稳定表达CD47或者稳定表达CD47和TCR激动剂的细胞可以是原本就表达CD47或者CD47和TCR激动剂的肿瘤细胞系，或者是经遗传改造以稳定表达CD47或者CD47和TCR激动剂的细胞。所述遗传改造可以通过本领域技术人员已知的任何方式进行，例如通过病毒载体的转染。所述靶细胞模拟物中的细胞的种类可以是本领域已知的任何合适细胞，例如CHO-K1、Raji等哺乳动物细胞。所述微型颗粒可以是用TCR激动剂如抗CD3抗体和/或CD47蛋白包被的微球。所述微型颗粒或微球的直径可以为例如5-10μm。
在一个实施方式中，所述靶细胞模拟物是稳定表达CD47和TCR激动剂的细胞。
本申请所述的“TCR激动剂”是任何可以激活TCR的生物或化学剂。例如，TCR激动剂可以实现通过主要组织相容性复合体(major histocompatibility complex，MHC)介导的特异性TCR激活、抗CD3抗体介导的TCR复合体的激活，或超抗原(superantigen)介导的非特异性TCR激活。
在一些实施方式中，所述TCR激动剂选自以下中的任一种或多种：通过主要组织相容性复合体(MHC)呈递的抗原、抗CD3抗体或其抗体片段，和超抗原。在一个优选的实施方式中，所述TCR激动剂是抗CD3抗体。
在一个优选的实施方式中，所述靶细胞模拟物是抗CD3抗体和CD47双表达细胞系。在一个优选的实施方式中，所述靶细胞模拟物是抗CD3scFv和CD47双表达细胞系。
在本申请的方法的步骤“使靶细胞模拟物、靶向CD47-SIRPα免疫检查点的(候选)药物和效应细胞接触，所述靶细胞模拟物提供CD47蛋白和T细胞受体(TCR)激动剂，所述效应细胞包含报告基因以及稳定表达的SIRPα和TCR”中，靶细胞模拟物、(候选)药物和效应细胞的添加顺序没有特别的限制。例如，在一些实施方式中，该步骤包括：提供靶细胞模拟物，以及向所述靶细胞模拟物添加靶向CD47-SIRPα免疫检查点的(候选)药物和效应细胞。所述“提 供靶细胞模拟物”可以包括将稳定表达CD47和TCR激动剂的细胞铺板并培养，或者包括将稳定表达CD47的细胞铺板并培养以及向所述细胞加入TCR激动剂，或者包括加入TCR激动剂和偶联CD47蛋白的微型颗粒。
本申请所述的“效应细胞”包含报告基因以及稳定表达的SIRPα和TCR。所述效应细胞可以是经遗传改造以表达SIRPα、T细胞受体(TCR)和报告基因的细胞，也可以是经基因工程改造以使内源性CD47基因表达部分或完全缺失、稳定表达SIRPα、TCR和报告基因的细胞。所述遗传改造可以通过本领域技术人员已知的任何方式进行，例如通过病毒载体的转染。所述效应细胞的种类可以是本领域已知的任何合适细胞，例如Jurkat或其他人源T细胞，如HuT-78、CEM等。在一些实施方案中，所述效应细胞为CD47基因表达部分或完全缺失的细胞。在另一些实施方案中，所述效应细胞通过基因工程改造使所述效应细胞的CD47基因表达部分或完全缺失。在一些优选的实施方案中，所述效应细胞通过基因编辑使所述CD47基因表达部分或完全缺失。在一个实施方案中，所述效应细胞为CD47基因敲除的细胞。在一些实施方案中，所述效应细胞的CD47基因为内源性的。
本申请方法中所述“基因工程(Gene engineering)”又称遗传工程(Genetic engineering)是改变生物的遗传组成使用的技术，包括了删除可遗传材料，和将生物体外直接制备的DNA导入宿主或细胞，然后与宿主融合或杂交。在本申请中，基因工程改造包括转基因技术和基因编辑技术。本申请方法中所述“基因编辑”或“基因编辑技术”可以是CRISPR-Cas9技术、锌指核酸内切酶(ZFN)技术和转录激活子样效应因子核酸酶(TALEN)技术等部分或完全失活基因的技术。
在一些实施方式中，其中所述效应细胞表达CD47的受体SIRPα，与CD47接触后形成CD47-SIRPα复合物抑制TCR途径依赖性的信号通路激活。在一些实施方式中，所述效应细胞在敲除其内源性表达的CD47后再表达SIRPα，以避免效应细胞间自发形成的CD47-SIRPα复合物，抑制TCR途径依赖性的信号通路激活。
在本申请方法所述的“将所述靶细胞模拟物与靶向CD47-SIRPα免疫检查点的(候选)药物和效应细胞接触”步骤中，所添加的所述效应细胞的量可以是任何合适的量。在一些实施方式中，所述效应细胞与所述稳定表达CD47或者 稳定表达CD47和TCR激动剂的细胞的比例可以是例如8:1、7:1、6:1、5:1、4:1、3:1、2:1或1:1等。在一些优选实施方案中，所述效应细胞与所述稳定表达CD47或者CD47和TCR激动剂的细胞的比例可以是3:1。所添加的所述药物(候选药物)的量可以一个或更多个量，例如经系列稀释的多个量。
本申请所述的“T细胞受体(TCR)”可以是T细胞所表达的原始TCR或者经修饰的TCR，例如经修饰以具有较高亲和力的TCR。
本申请所述的“报告基因”可以是本领域已知的适用于效应细胞的任何报告基因，例如，报告基因的可以是荧光素酶(Luciferase)基因，分泌胚胎碱性磷酸酶(Secreted embryonic alkaline phosphatase[SEAP])基因，β-半乳糖苷酶(Beta-galactosidase)基因，荧光蛋白如绿色荧光蛋白(Green fluorescent protein，[GFP])基因等。在一些实施方式中，所述报告基因为活化T细胞核因子(NFAT)的反应元件(RE)、激活蛋白1(AP-1)反应元件或核因子κB(NF-κB)反应元件驱动的。例如由NFAT的反应元件驱动的荧光素酶基因。在一些实施方式中，所述报告基因的表达是TCR途径依赖性的。
在一些实施方式中，其中所述靶细胞模拟物提供的CD47蛋白与效应细胞表达的SIRPα接触后形成复合物导致TCR途径依赖性的报告基因的抑制表达。在一些实施方式中，所述靶向CD47-SIRPα免疫检查点的候选药物或药物抑制所述CD47蛋白与SIRPα形成的复合物，导致TCR途径依赖性的报告基因的增加表达。
本申请中所述的“孵育所述靶细胞模拟物、所述候选药物和所述效应细胞的混合物”步骤可以在任何合适的温度下进行合适的时间，例如在37℃下孵育2、3、4、5、6、7、8、9或10小时，或者12或24小时。
本申请中所述的“检测所述报告基因的表达或表达强度来判断所述候选药物对CD47-SIRPα免疫检查点的阻断”或“检测所述报告基因的表达强度来对所述药物进行质量控制检测”步骤可以采用本领域已知的用于将测报告基因的任何定性或定量方法进行，例如，化学发光检测法、流式细胞术、放射免疫分析、荧光显微镜检测等。在一个优选的实施方式中，所述检测是采用酶标仪的化学发光检测。因此，在一些实施方式中，所述的“检测所述报告基因的表达或表达强度”步骤包括：向孵育后的所述混合物加入报告基因的底物，并通过化学发光法(例如酶标仪)进行检测。
在本申请的候选药物筛选方法中，“判断所述候选药物对CD47-SIRPα免疫检查点的阻断”包括：若未检测或观察到报告基因的表达，则判定所述候选药物不具备阻断CD47-SIRPα免疫检查点的作用；若检测或观察到报告基因的表达，则判定所述候选药物具备阻断CD47-SIRPα免疫检查点的作用，可潜在地用作CD47-SIRPα结合抑制剂，而且，报告基因的表达强度越强，则指示所述候选药物作为CD47-SIRPα结合抑制剂的活性越高。通常情况下，对于具有抑制活性的候选药物，报告基因表达的强度会随着抗体浓度的升高而提高。
在本申请的质量控制方法，比如稳定性检测方法中，“对所述药物进行质量控制”包括：若药物与对照药物相比表达强度降低，则指示所述药物质量不合格或失去稳定性。
本发明中所述靶向CD47-SIRPα免疫检查点的候选药物或药物抑制所述CD47蛋白与SIRPα形成的复合物，导致TCR途径依赖性的报告基因的增加表达。在一些实施方案中，增加的表达为至少2倍(例如，2倍，3倍，4倍，5倍，6倍，7倍，8倍，9倍，10倍，11倍，12倍，13倍，14倍，15倍，20倍，40倍，50倍，100倍等)。
在第三方面，本申请还提供了一种用于对靶向CD47-SIRPα免疫检查点的候选药物或药物进行筛选或质量控制的试剂盒，其包含：靶细胞模拟物，所述靶细胞模拟物提供CD47蛋白和TCR激动剂；和效应细胞，所述效应细胞包含报告基因和稳定表达的SIRPα和T细胞受体(TCR)。
本申请的试剂盒可以按照本申请所述的方法使用。在本申请涉及方法的第一和第二方面中列出的定义、描述和优选项同样适用于第三方面。
本申请所述的试剂盒，其中所述效应细胞与所述靶细胞模拟物的细胞比例为8:1～1:1，优选为3:1。靶细胞模拟物中涉及微球的，一个微球相当于一个靶细胞。具体地，如靶细胞模拟物为TCR激动剂和偶联CD47蛋白的微型颗粒，则相当于稳定表达TCR激动剂和CD47蛋白的细胞。
除靶细胞模拟物和效应细胞外，所述试剂盒还可以包含选自以下中的一种或更多种：阳性对照品(例如靶向CD47的抗体，靶向SIRPα的抗体、SIRPα重组蛋白或融合蛋白，或阻断CD47与SIRPa之间的相互作用的小分子)；用于报告基因的底物；耗材(例如多孔板)；记载使用方法(例如本申请方法)的说明书。
在第四方面，本申请还提供了一种系统，其包含：靶细胞模拟物，所述靶细胞模拟物提供CD47蛋白和TCR激动剂；和效应细胞，所述效应细胞包含TCR途径依赖性的报告基因以及稳定表达的SIRPα和TCR。
在一些实施方案中，所述系统还包括靶向CD47-SIRPα免疫检查点的候选药物或药物。
本申请的系统可以按照本申请所述的方法使用。在本申请涉及方法的第一第二和第三方面中列出的定义、描述和优选项同样适用于第四方面。
在第五方面，本申请提供一种细胞，其包含TCR途径依赖性的报告基因，稳定表达的SIRPα和TCR，其特征在于，所述细胞的CD47基因的表达部分或完全缺失。
在一些实施方案中，通过对所述细胞进行基因工程改造使得所述细胞的CD47基因表达部分或完全缺失。在一些优选实施方案中，所述基因工程改造为基因编辑，优选为基因敲除。在一个实施方案中，所述细胞为CD47基因敲除的细胞。
在一些实施方案中，所述细胞为任何具有TCR激活通路的T细胞。在本申请中，所述细胞可以作为效应细胞，如Jurkat细胞等。
本申请的方法是基于细胞的高通量体外方法，能够在体外模拟药物对CD47与SIRPα之间的相互作用的抑制，用于抗体筛选、鉴定、稳定性评价、效价和效力测定以及QC批次放行等，提供生物活性检测的稳定而廉价的工具。本申请方法快速、灵敏，简便，操作简单。本申请方法消除对原代细胞(需要巨噬细胞的分化培养，需要繁复的实验操作和巨大的时间成本，而且这样的方法严重依赖于PBMC的分离与培养，实验结果重复性较差和灵敏度较低)培养的要求；不涉及动物，从而显著缩减了药物筛选或评价的成本和时间；较基于ELISA或蛋白相互作用的方法(受限于简化的模型，仅能衡量阻断蛋白互作的分子，对于联用抗体疗法或双特异性抗体的评价则无能为力，因为无法真实模拟效应细胞与靶细胞之间的相互作用，无法做到功能验证，更无法对针对作用于细胞内的靶点进行测量)，评价指标更加全面完善。此外，本申请方法利用稳定的细胞系如CD47基因表达部分或完全缺失的细胞作为效应细胞，评价结果具有高重复性和高灵敏性。
与本申请的效应细胞系形成对照的是利用CD47-SIRPα-SHP-FCGR2A(Fc Fragment of IgG Receptor IIa)-NFAT信号通路的效应细胞(例如参见本申请实施例1.5部分)。在其应用中，靶细胞上CD47与SIRPα的结合触发SIRPα与募集的SHP-1或SHP-2磷酸酶的偶联并激活磷酸酶，抑制依赖于抗体Fc区域(Fragment crystallizable region)激活FCGR2A的信号，致使NFAT反应元件驱动的荧光素酶无法表达，因而无法检测荧光素酶信号。当CD47-SIRPα相互作用被药物破坏后，SHP-1或SHP-2无法被募集到SIRPα胞质尾巴并被激活，从而FCGR2A激活信号得以传导并激活下游NFAT反应元件，荧光素酶因此得到表达。因为利用这种CD47-SIRPα-SHP-FCGR2A(Fc Fragment of IgG Receptor IIa)-NFAT信号通路的效应细胞检测和验证CD47-SIRPα通路抑制剂依赖于抗体的Fc区域，对于全长类抗体的检测，抗体的Fc区域可在不阻断CD47-SIRPα的作用途径的条件下，独立FCGR2A信号通路，从而激活NFAT信号通路，引起背景干扰的信号。
对序列表的描述
SEQ ID NO:1是PGK启动子+潮霉素B基因片段。
SEQ ID NO:2是NFAT-RE核苷酸序列。
SEQ ID NO:3是抗CD3单抗OKT3轻链序列。
SEQ ID NO:4是抗CD3单抗OKT3重链序列。
SEQ ID NO:5是人CD47ORF DNA序列。
SEQ ID NO:6是人SIRPa序列。
SEQ ID NO:7是新霉素抗性基因序列。
SEQ ID NO:8是人FCGR2A序列。
SEQ ID NO:9是guide RNA序列。
SEQ ID NO:10是上游引物序列F。
SEQ ID NO:11是下游引物序列R。
SEQ ID NO:12是荧光素酶蛋白序列。
附图说明
图1显示了联用PMA和离子霉素(Iono)刺激Jurkat/NFAT-Luc细胞后激活其胞内NFAT信号通路驱动荧光素酶的表达，S/B＝212。
图2显示了CHO-K1/抗CD3-scFv激活Jurkat/NFAT-Luc的TCR，驱动NFAT调控的荧光素酶的表达。
图3显示了CHO-K1/抗CD3-scFv/CD47细胞池CD47表达水平的检测。
图4显示了Jurkat/NFAT-Luc/SIRPα细胞池SIRPα表达水平的检测。
图5显示了Rituxan结合Raji细胞后激活Jurkat/NFAT-Luc/SIRPα/FCGR2A效应细胞的FCGR2A-NFAT-Luc信号通路，激活荧光素酶的表达。在有Raji表面的CD47与效应细胞表面的SIRPα相互作用的信号通路情况下，Rituxan的Fc区域与FCGR2A结合引起非CD47-SIRPα阻断作用依赖的效应细胞激活，引入了背景干扰信号。
图6显示了抗CD47抗体恢复被CD47-SIRPα抑制的激活信号。
图7显示了抗SIRPα抗体(OSE172)恢复被CD47-SIRPα抑制的抗CD3激活信号。
图8显示了抗CD47抗体(Hu5F9)恢复被CD47-SIRPα抑制的Raji激活Jurkat信号。
图9测序结果显示Jurkat(CD47敲除)/NFAT-Luc细胞池的CD47基因编辑效率。
图10测序结果显示Jurkat(CD47敲除)/NFAT-Luc单克隆细胞株CD47基因敲除后基因编辑位点缺失4个碱基。
图11显示了Jurkat/NFAT-Luc细胞和Jurkat(CD47敲除)/NFAT-Luc细胞CD47表达水平的检测。
图12显示了抗CD47抗体恢复CD47基因敲除后Jurkat细胞被CD47-SIRPα抑制的激活信号及其信号比上背景值的倍比。
图13显示了CHO-K1/抗CD3-scFv/CD47和Jurkat/NFAT-Luc/SIRPα一对细胞功能检测的连续传代稳定性数据。
图14显示了CHO-K1/抗CD3-scFv/CD47和Jurkat(CD47敲除)/NFAT-Luc/SIRPα一对细胞功能检测的连续传代稳定性数据。
具体实施方式
实施例
实施例1：细胞系构建
1.Jurkat/NFAT-Luc效应细胞系的构建
合成PGK启动子+潮霉素B基因片段(如SEQ ID NO:1所示)并将上述片段克隆到pLVX-Puro(Clontech，Cat.No.632164)载体中，使用的酶切位点为xbaI和MscI，得到载体pLVX-Hygro。
合成NFAT的反应元件(RE)驱动的荧光素酶(luciferase,[Luc])的序列(如SEQ ID NO:2和SEQ ID NO:12所示)，使用Clone EZ(GenScript,https://www.genscript.com/genbuilder-dna-assembly.html)技术将该片段与载体骨架pLVX-Hygro连接，转化大肠杆菌感受态细胞，获得质粒pLVX-NFAT-Luc-Hygro。
慢病毒(NFAT-Luc)生产：将HEK293T细胞用胰酶消化，重悬在加入10％FBS的DMEM中，以6-10×10 6HEK293T/平皿(10cm)铺板。第二天观察细胞密度，80-90％汇合即可进行转染，转染前更换成新鲜培养基。每个平皿按照7.5μg psPAX2(GenScript自主合成，序列来源自Addgene plasmid#12260),5μg PMD2.G(GenScript自主合成，序列来源自Addgene plasmid#12259)，12.5μg pLVX-NFAT-Luc-Hygro转染。将Lipofectamine 3000(Thermo Fisher,Cat.No.L3000001)与质粒混合加入平皿中。转染后6-8小时更换成新鲜培养液。转染后48-56小时收集病毒上清，收集后以0.45μm滤器过滤，超速离心。将病毒沉淀用500μl新鲜培养基重悬，置于-80℃保存。
感染细胞：将Jurkat细胞铺至6孔板，加入3mL培养基，过夜培养。感染前，从冰箱取出并在37℃水浴中快速融化病毒，吸去细胞原有培养基，加入1/2体积新鲜培养基，再将病毒(NFAT-Luc)原液加入细胞中混匀。离心感染2-3小时后，将培养板放入37℃,5％CO 2培养箱中培养24小时。感染后第二天(约24小时)，吸去含病毒的培养液，更换成新鲜的完全培养液，继续37℃培养。
潮霉素抗性筛选：细胞培养基中加入200μg/ml潮霉素，每2-3天更换含潮霉素的完全培养液一次，至未感染的筛选对照组的细胞被潮霉素杀光。连续筛选至获得稳定的细胞株。
利用化学发光法检测NFAT介导的荧光素酶表达：取适量Jurkat/NFAT-Luc细胞铺在384孔板中。配制4X的PMA(佛波醇12-十四酸酯13-乙酸酯)和4X离子霉素(Iono)工作液，加入至铺有Jurkat/NFAT-Luc细胞的384孔板中，使其终浓度达到10μg/mL PMA和1μM Iono。阴性对照组加入等体积的细胞培养基。然后将384孔板置于37℃，5％CO 2培养箱中孵育6小时。孵育结束后，取出384孔板，分别向384孔板每孔中加入40μL Bio-Glo TM(Promega,#G7940)，室温小心混匀5分钟。使用PHERAstar FSX(BMGLABTEC)进行检测，检测Jurkat激活后NFAT反应元件驱动的荧光素酶信号。如图1所示，PMA和Iono刺激Jurkat细胞后，其胞内NFAT的信号通路被激活，处于NFAT反应元件下游的荧光素酶基因得以表达，其信号较背景值高212倍，说明Jurkat细胞中成功导入了荧光素酶基因并构建出Jurkat/NFAT-Luc效应细胞。
单克隆挑选：将Jurkat/NFAT-Luc效应细胞极限稀释至96孔板中，2-3天后显微镜下观察96孔板，并标记出有单克隆的孔。将单克隆细胞转移至24孔板中，随后扩大至6孔板。单克隆扩大后以上述步骤再验证NFAT介导的荧光素酶表达。
2.CHO-K1/抗CD3-scFv靶细胞系构建
合成抗CD3-scFv DNA ORF序列(来自OKT3单抗的scFv序列并经内部优化，OKT3轻链氨基酸和重链氨基酸序列如SEQ ID NO:3和4所示)后，使用Clone EZ(GenScript)技术将抗CD3-scFv ORF的DNA片段与载体骨架pLVX-Hygro连接，转化大肠杆菌感受态细胞，获得质粒pLVX-抗CD3-scFv-Hygro。
慢病毒(抗CD3-scFv)生产：将HEK293T细胞用胰酶消化，重悬在加入10％FBS的DMEM中，以6-10×10 6HEK293T/平皿(10cm)铺板。第二天观察细胞密度，80-90％汇合即可进行转染，转染前更换新鲜培养基。每个平皿按照7.5μg psPAX2,5μg PMD2.G和12.5μg pLVX-抗CD3-scFv-RE-Hygro转染。将Lipofectamine 3000(Thermo Fisher,Cat.No.L3000001)与质粒混合加入平皿中。转染后6-8小时更换新鲜培养液。转染后48-56小时收集病毒上清，收集后以0.45μm滤器过滤，超速离心。将病毒沉淀用500μl新鲜培养基重悬，置于-80℃保存。
感染细胞：将CHO-K1细胞铺至6孔板，加入3mL培养基，过夜培养。感染前，从冰箱取出并在37℃水浴中快速融化病毒(抗CD3-scFv)，吸去细胞原有培养基，加入1/2体积新鲜培养基，再将病毒原液加入细胞中混匀。离心感染0.5-1小时后，将培养板放入37℃,5％CO 2培养箱中培养24小时。感染后第二天(约24小时)，吸去含病毒的培养液，换上新鲜的完全培养液，继续37℃培养。
潮霉素抗性筛选：细胞培养基中加入400μg/ml潮霉素，每2-3天更换含潮霉素的完全培养液一次，至未感染的筛选对照组的细胞被潮霉素杀光。连续筛选至获得稳定细胞株。
抗CD3-scFv功能检测验证CHO-K1/抗CD3-scFv细胞系：取10000个靶细胞CHO-K1/抗CD3-scFv细胞铺在384孔板中，并在阴性对照组加入等数量的CHO-K1细胞，过夜培养。第二天弃去孔内培养基，将效应细胞Jurkat/NFAT-Luc加入至铺有CHO-K1/抗CD3-scFv细胞或阴性对照CHO-K1的384孔板中，使效应细胞:靶细胞(E:T)比例为4:1。然后将384孔板置于37℃，5％CO 2培养箱中孵育6小时。孵育结束后，取出384孔板，分别向384孔板每孔中加入40μL Bio-Glo TM(Promega,#G7940)，室温小心混匀5分钟。使用PHERAstar进行检测，检测指标为抗CD3-scFv激活Jurkat表面的TCR信号通路的情况下，进而激活NFAT反应元件驱动的荧光素酶信号。如图2所示，靶细胞刺激Jurkat细胞后，其胞内NFAT的信号通路被激活，处于NFAT反应元件下游的荧光素酶基因得以表达，说明靶细胞CHO-K1/抗CD3-scFv成功表达功能性的抗CD3-scFv目标蛋白，靶细胞构建成功。
单克隆挑选：将细胞池极限稀释至96孔板中，7天后显微镜下观察96孔板，并标记出有单克隆的孔。将单克隆细胞转移至24孔板中，随后扩大至6孔板。单克隆扩大后以上述步骤再验证CHO-K1/抗CD3-scFv激活Jurkat/NFAT-Luc效应细胞中NFAT介导的荧光素酶表达。
3.CHO-K1/抗CD3-scFv/CD47靶细胞系构建
人CD47ORF DNA序列(如SEQ ID NO:5所示)进行基因合成后，同时利用相同限制性内切酶酶切质粒载体pLVX-Puro，酶切后获得的CD47蛋白ORF  DNA片段和带有粘性末端的质粒载体片段连接，转化大肠杆菌感受态细胞，获得质粒pLVX-CD47-Puro。
慢病毒(CD47)生产：将HEK293T细胞用胰酶消化，重悬在加入10％FBS的DMEM中，以6-10×10 6HEK293T/平皿(10cm)铺板。第二天观察细胞密度，80-90％汇合即可进行转染，转染前更换新鲜培养基。每个平皿按照7.5μg psPAX2,5μg PMD2.G和12.5μg pLVX-CD47-Puro转染。将Lipofectamine 3000(Thermo Fisher,Cat.No.L3000001)与质粒混合加入平皿中。转染后6-8小时更换新鲜培养液。转染后48-56小时收集病毒上清，收集后以0.45μm滤器过滤，超速离心。将病毒沉淀用500μl新鲜培养基重悬，置于-80℃保存。
感染细胞：将CHO-K1/抗CD3-scFv细胞铺至6孔板，加入3mL培养基，过夜培养。感染前，从冰箱取出并在37℃水浴中快速融化病毒(CD47)，吸去细胞原有培养基，加入1/2体积新鲜培养基，再将病毒原液加入细胞中混匀。离心感染0.5-1小时后，将培养板放入37℃,5％CO 2培养箱中培养24小时。感染后第二天(约24小时)，吸去含病毒的培养液，更换上新鲜的完全培养液，继续37℃培养。
FACS检测CD47表达：取一部分获得的稳定细胞至FACS管中，离心去掉上清。加入PE抗人CD47抗体(BioLegend,Cat#323108)4℃孵育30分钟。30分钟后洗去上清，重悬在FACS缓冲液中，上机(BD FACSCelesta TM)检测CD47表达水平，如图3所示，FACS检测到CHO-K1/C2/CD47细胞池过表达CD47。
嘌呤霉素和潮霉素双抗性筛选：细胞培养基中加入8μg/ml嘌呤霉素和400μg/ml潮霉素，每2-3天更换含嘌呤霉素和潮霉素的完全培养液一次，至未感染的筛选对照组的细胞被嘌呤霉素和潮霉素杀光。连续筛选至获得稳定细胞株。
单克隆挑选：将细胞池极限稀释至96孔板中，7天后显微镜下观察96孔板，并标记出有单克隆的孔。单克隆细胞转移至24孔板中，随后扩大至6孔板。单克隆扩大后以上述步骤再验证CD47表达。
4.Jurkat/NFAT-Luc/SIRPα效应细胞系的构建
合成人SIRPα蛋白的DNA序列(编码的SIRPα氨基酸序列如SEQ ID NO:6所示)后，使用Clone EZ(GenScript)技术将SIRPαORF DNA片段与载体骨架 pLVX-Puro(Clontech，Cat.No.632164)连接，转化大肠杆菌感受态细胞，获得质粒pLVX-SIRPα-Puro。
慢病毒(SIRPα)生产：将HEK293T细胞用胰酶消化，重悬在加入10％FBS的DMEM中，以6-10×10 6HEK293T/平皿(10cm)铺板。第二天观察细胞密度，80-90％汇合即可进行转染，转染前更换新鲜培养基。每个平皿按照7.5μg psPAX2,5μg PMD2.G和12.5μg pLVX-SIRPα-Puro转染。将Lipofectamine 3000(Thermo Fisher,Cat.No.L3000001)与质粒混合加入平皿中。转染后6-8小时更换新鲜培养液。转染后48-56小时收集病毒上清，收集后以0.45μm滤器过滤，超速离心。将病毒沉淀用500μl新鲜培养基重悬，置于-80℃保存。
感染细胞：将实施例1.1制备的Jurkat/NFAT-Luc细胞铺至6孔板，加入3mL培养基，过夜培养。感染前，从冰箱取出并在37℃水浴中快速融化病毒(SIRPα)，吸去细胞原有培养基，加入1/2体积新鲜培养基，再将病毒原液加入细胞中混匀。离心感染2-3小时后，将培养板放入37℃,5％CO 2培养箱中培养24小时。感染后第二天(约24小时)，吸去含病毒的培养液，更换新鲜的完全培养液，继续37℃培养。
嘌呤霉素和潮霉素双抗性筛选：细胞培养基中加入1μg/ml嘌呤霉素和200μg/ml潮霉素，每2-3天更换含嘌呤霉素和潮霉素的完全培养液一次，至未感染的筛选对照组的细胞被嘌呤霉素和潮霉素杀光。连续筛选至获得稳定细胞株。
FACS检测SIRPα表达：取一部分获得的稳定细胞至FACS管中，离心去掉上清。加入FITC抗-人CD172a/b(SIRPαlpha/beta)(BioLegend,Cat#337304)在4℃下孵育30分钟。30分钟后洗去上清，重悬在FACS缓冲液，上机(BD FACSCelesta TM)检测SIRPα表达水平，如图4所示，FACS检测到Jurkat/NFAT-Luc/SIRPa细胞池过表达SIRPα。
单克隆挑选：将细胞池极限稀释至96孔板中，7天后显微镜下观察96孔板，并标记出有单克隆的孔。将单克隆细胞转移至24孔板中，随后扩大至6孔板。单克隆扩大后以上述步骤再验证SIRPα的表达并定株。
5.Jurkat/NFAT-Luc/SIRPα/FCRG2A效应细胞系的构建
将新霉素基因(如SEQ ID NO:7所示)合成并将上述片段克隆到pLVX-Puro(Clontech，Cat.No.632164)载体中，使用Clone EZ(GenScript)连接，转化大肠杆菌感受态细胞，获得载体pLVX-Neo。
人FCRG2A蛋白的DNA序列(编码的FCRG2A氨基酸序列如SEQ ID NO:8所示)进行基因合成后，使用Clone EZ(GenScript)技术将FCRG2A ORF的DNA片段与载体骨架pLVX-Neo连接，转化大肠杆菌感受态细胞，获得质粒pLVX-FCRG2a-Neo。
慢病毒(FCRG2A)生产：将HEK293T细胞用胰酶消化，重悬在加入10％FBS的DMEM中，以6-10×10 6HEK293T/平皿(10cm)铺板。第二天观察细胞密度，80-90％汇合即可进行转染，转染前更换新鲜培养基。每个平皿按照7.5μg psPAX2,5μg PMD2.G和12.5μg pLVX-FCRG2A-Neo转染。将Lipofectamine 3000(Thermo Fisher,Cat.No.L3000001)与质粒混合加入平皿中。转染后6-8小时换新鲜培养液。转染后48-56小时收集病毒上清，收集后以0.45μm滤器过滤，超速离心。将病毒沉淀用500μl新鲜培养基重悬，置于-80℃保存。
感染细胞：将实施例1.4中制备的Jurkat/NFAT-Luc/SIRPα细胞铺至6孔板，加入3mL培养基，过夜培养。感染前，从冰箱取出并在37℃水浴中快速融化病毒(FCRG2A)，吸去细胞原有培养基，加入1/2体积新鲜培养基，再将病毒原液加入细胞中混匀。离心感染2-3小时后，将培养板放入37℃,5％CO 2培养箱中培养24小时。感染后第二天(约24小时)，吸去含病毒的培养液，换上新鲜的完全培养液，继续37℃培养。
新霉素、嘌呤霉素和潮霉素三抗性筛选：细胞培养基中加入400μg/ml G418、1μg/ml嘌呤霉素和200μg/ml潮霉素，每2-3天更换含G418的完全培养液一次，至未感染的筛选对照组的细胞被G418、嘌呤霉素和潮霉素杀光。连续筛选至获得稳定细胞株。
FCGR2A功能检测验证Jurkat/NFAT-Luc/SIRPa/FCGR2A细胞系：准备Raji靶细胞和梯度稀释的抗CD20的抗体Rituxan(Roche)，将10000个Raji细胞铺至96孔板后，加入梯度稀释的Rituxan抗体，加入适量Jurkat/NFAT-Luc/SIRPa/FCGR2A细胞，使其效应细胞:靶细胞(E:T)比例为6:1。然后将96孔板置于37℃，5％CO 2培养箱中孵育6小时。孵育结束后，取出96孔板，分别 向96孔板每孔中加入100μL Bio-Glo TM(Promega,#G7940)，室温小心混匀5分钟。使用PHERAstar进行检测，检测指标为Rituxan介导的抗体依赖性细胞吞噬信号激活Jurkat表面的FCGR2A及其下游NFAT，从而驱动荧光素酶表达。如图5所示，在有Raji表面的CD47与效应细胞表面的SIRPα相互作用的信号通路情况下，该TCR激活信号被抑制，但在加入Rituxan后仍可通过抗体可变区域结合Raji细胞表面CD20和抗体Fc区域结合效应细胞FCGR2A，进而激活ADCP下游信号NFAT，致使处于NFAT反应元件下游的荧光素酶基因得以表达。
该功能实验证实Jurkat/NFAT-Luc/SIRPa/FCGR2A效应细胞表面表达功能性的FCGR2A及全长类抗体Fc区域会造成该功能细胞系非CD47-SIRPa信号通路特异性的激活。
单克隆挑选：将细胞池极限稀释至96孔板中，7天后显微镜下观察96孔板，并标记出有单克隆的孔。单克隆细胞转移至24孔板中，随后扩大至6孔板。单克隆扩大后以上述步骤再验证FCGR2Aa表达并定株。
6.Jurkat(CD47敲除)/NFAT-Luc效应细胞系的构建
合成CRISPR guide RNA质粒：根据CD47基因组(Gene ID:961)序列信息，设计并合成靶向CD47的guide RNA质粒，guide RNA序列为atcgagctaaaatatcgtgt(SEQ ID NO:9)。guide RNA质粒(该质粒骨架购自张峰实验室，并获得授权)中带有Cas9和GFP序列，其中GFP荧光蛋白是后续阳性细胞池分选的标记。
引物合成：在guide RNA序列对应的CD47基因组序列的上下游300-500bp处，设计并合成引物。上下游引物序列为F:5'GCCGCTGTTTATCTTAAGGATTTG 3'(SEQ ID NO:10)，R:5'CGGTAACAGCCTGCTTTTTGATTC 3'(SEQ ID NO:11)。
转染：复苏并培养Jurkat NFAT-Luc效应细胞系，待细胞状态调制最佳时，吸取3×10 6细胞悬液，800rpm离心4分钟，去上清；用5ml PBS重悬细胞沉淀，800rpm离心4分钟，去上清。用100微升电转缓冲液(Celetrix)重悬细胞沉淀，吸取5微克guide RNA质粒加入细胞悬液中，混匀。将guide RNA质粒-细胞悬液转入电击管中，将电击管放入电转仪(Celetrix)中进行电转。电转72小时后，流式分选GFP阳性细胞群体(BD FACSMelody TM)，分选后持 续培养3天，以恢复Jurkat(CD47敲除)/NFAT-Luc细胞池状态。同时取约0.5×10 5分选后的细胞，用快速抽提基因组试剂(LUCIGEN)提取细胞基因组，随后吸取1微升基因组进行PCR扩增，回收PCR产物进行桑格测序，测序结果显示细胞池CD47基因组编辑效率为82％。如图9上图所示，与对照样本相比，桑格测序结果在gRNA-Cas9切割位点(虚线处)出现多峰，表明细胞池CD47基因组被切割后产生多种基因型。通过在线软件(https://ice.synthego.com/#/)分析桑格测序结果，表明细胞池CD47基因组编辑效率为82％(图9下图)。
单克隆形成：将Jurkat(CD47敲除)/NFAT-Luc细胞池通过极限稀释法按每孔一个细胞铺进96孔板中，2-3天后显微镜下观察96孔板，并标记出有单克隆的孔，随后连续培养两周。
单克隆鉴定：待96孔板中的单克隆细胞扩增至孔板底面积的60％时，将单克隆细胞转移至24孔板中扩大培养。同时取约0.5×10 5个细胞，用快速抽提基因组试剂(LUCIGEN)提取细胞基因组，随后吸取1微升基因组进行PCR扩增，回收PCR产物进行桑格测序，桑格测序结果显示Jurkat(CD47敲除)/NFAT-Luc clone64的CD47基因缺失4bp。如图10上图所示，与对照样本相比，桑格测序结果显示在gRNA-Cas9切割位点(虚线处)前缺失4bp(A-T-C-G)。通过在线软件(https://ice.synthego.com/#/)分析桑格测序结果，Jurkat(CD47敲除)/NFAT-Luc clone64的CD47基因缺失4bp(图10下图)
FACS检测CD47表达：继续扩大培养Jurkat(CD47敲除)/NFAT-Luc clone64。同时取一部分单克隆细胞至FACS管中，离心去掉上清。加入PE抗人CD47抗体(BioLegend,Cat#323108)4℃孵育30分钟。30分钟后洗去上清，重悬在FACS缓冲液中，上机(BD FACSCelesta TM)检测CD47表达水平，如图11所示，FACS未检测到Jurkat(CD47敲除)/NFAT-Luc细胞表面的CD47，表明CD47敲除成功。
7.Jurkat(CD47敲除)/NFAT-Luc/SIRPα效应细胞系的构建
同实施例1.4制备质粒pLVX-SIRPα-Puro，并生产慢病毒(SIRPα)。
感染细胞：将实施例1.6制备的Jurkat(CD47敲除)/NFAT-Luc细胞铺至6孔板，加入3mL培养基，过夜培养。感染前，从冰箱取出并在37℃水浴中快速融化病毒(SIRPα)，吸去细胞原有培养基，加入1/2体积新鲜培养基，再将病毒 原液加入细胞中混匀。离心感染2-3小时后，将培养板放入37℃,5％CO 2培养箱中培养24小时。感染后第二天(约24小时)，吸去含病毒的培养液，更换新鲜的完全培养液，继续37℃培养。
嘌呤霉素和潮霉素双抗性筛选：细胞培养基中加入1μg/ml嘌呤霉素和200μg/ml潮霉素，每2-3天更换含嘌呤霉素和潮霉素的完全培养液一次，至未感染的筛选对照组的细胞被嘌呤霉素和潮霉素杀光。连续筛选至获得稳定细胞株。
FACS检测SIRPα表达：取一部分获得的稳定细胞至FACS管中，离心去掉上清。加入FITC抗-人CD172a/b(SIRPαlpha/beta)(BioLegend,Cat#337304)在4℃下孵育30分钟。30分钟后洗去上清，重悬在FACS缓冲液，上机(BD FACSCelesta TM)检测SIRPα表达水平，结果显示SIRPα过表达，与Jurkat/NFAT-Luc/SIRPα基本一致。
单克隆挑选：将细胞池极限稀释至96孔板中，7天后显微镜下观察96孔板，并标记出有单克隆的孔。将单克隆细胞转移至24孔板中，随后扩大至6孔板。单克隆扩大后以上述步骤再验证SIRPα的表达并定株。
实施例2：TCR介导的CD47-SIRPα阻断生物实验
1.抗CD47抗体阻断CD47-SIRPα相互作用通路恢复Jurkat TCR激活信号
取10000个靶细胞CHO-K1/抗CD3-scFv/CD47细胞铺在384孔板中，过夜培养。第二天准备梯度稀释的抗CD47抗体Hu5F9(由GenScript根据商品化Hu5F9抗体的公开序列合成)，弃去原有培养基，并加入至铺有靶细胞的384孔板中，将适量效应细胞Jurkat/NFAT-Luc/SIRPα加入至对应的孔中，使其效应细胞:靶细胞(E:T)比例为3:1。然后将384孔板置于37℃，5％CO 2培养箱中孵育6小时。孵育结束后，取出384孔板，分别向384孔板每孔中加入40μL Bio-Glo TM(Promega,#G7940)，室温小心混匀5分钟。使用PHERAstar FSX(BMGLABTEC)进行检测，检测指标为抗CD3-scFv激活Jurkat表面的TCR信号通路的情况下，进而激活NFAT反应元件驱动的荧光素酶信号。如图6所示，在有CD47-SIRPα信号通路情况下，该激活信号被抑制，加入抗CD47抗体阻断了CD47-SIRPa的结合，从而恢复荧光信号，并且随着抗体浓度的提高，荧光信号逐步加强。
2.抗SIRPα抗体阻断CD47-SIRPα相互作用通路恢复Jurkat TCR激活信号
取10000个靶细胞CHO-K1/抗CD3-scFv/CD47细胞铺在384孔板中，过夜培养。第二天准备梯度稀释的抗SIRPα抗体OSE172(由GenScript根据商品化OSE172抗体的已公开序列合成)，弃去原有培养基，并加入至铺有靶细胞的384孔板中，将适量效应细胞Jurkat/NFAT-Luc/SIRPα加入至对应的孔中，使其效应细胞:靶细胞(E:T)比例为3:1。然后将384孔板置于37℃，5％CO 2培养箱中孵育6小时。孵育结束后，取出384孔板，分别向384孔板每孔中加入40μL Bio-Glo TM，室温小心混匀5分钟。使用PHERAstar进行检测，检测指标为抗CD3-scFv激活Jurkat表面的TCR信号通路的情况下，进而激活NFAT反应元件驱动的荧光素酶信号。如图7所示，在有CD47-SIRPα信号通路情况下，该激活信号被抑制，加入抗SIRPα抗体阻断了CD47-SIRPa的结合，从而恢复荧光信号，并且随着抗体浓度的提高，荧光信号逐步加强。
3.抗CD47抗体阻断CD47-SIRPα相互作用的通路，恢复Raji细胞激活Jurkat TCR信号
取20000个靶细胞Raji细胞铺在96孔板中，准备梯度稀释的抗CD47抗体Hu5F9(由GenScript根据商品化Hu5F9抗体的已公开序列合成)，并加入至铺有靶细胞的96孔板中，将适量的效应细胞Jurkat/NFAT-Luc/SIRPα加入至对应的孔中，使其效应细胞:靶细胞(E:T)比例为3:1。然后将96孔板置于37℃，5％CO 2培养箱中孵育6小时。孵育结束后，取出96孔板，分别向96孔板每孔中加入100μL Bio-Glo TM(Promega,#G7940)，室温小心混匀5分钟。使用PHERAstar进行检测，检测指标为Raji细胞激活Jurkat表面的TCR信号通路的情况下，进而激活NFAT反应元件驱动的荧光素酶信号。如图8所示，在有Raji表面的CD47与效应细胞表面的SIRPα相互作用的信号通路情况下，该TCR激活信号被抑制。加入抗CD47抗体阻断了CD47-SIRPa的结合，从而恢复荧光信号，并且随着抗体浓度的提高，荧光信号逐步加强。
4.抗CD47抗体阻断CD47-SIRPα相互作用通路恢复Jurkat(CD47敲除)TCR激活信号
取10000个靶细胞CHO-K1/抗CD3-scFv/CD47细胞铺在384孔板中，过夜培养。第二天准备梯度稀释的抗CD47抗体Hu5F9(由GenScript根据商品化Hu5F9抗体的公开序列合成)，弃去原有培养基，并加入至铺有靶细胞的384孔板中，将适量效应细胞Jurkat(CD47敲除)/NFAT-Luc/SIRPα加入至对应的孔中，使其效应细胞:靶细胞(E:T)比例为3:1。然后将384孔板置于37℃，5％CO 2培养箱中孵育6小时。孵育结束后，取出384孔板，分别向384孔板每孔中加入40μL Bio-Glo TM(Promega,#G7940)，室温小心混匀5分钟。使用PHERAstar FSX(BMGLABTEC)进行检测，检测指标为抗CD3-scFv激活Jurkat(CD47敲除)表面的TCR信号通路的情况下，进而激活NFAT反应元件驱动的荧光素酶信号。如图12所示，在有CD47-SIRPα信号通路情况下，该激活信号被抑制，加入抗CD47抗体阻断了CD47-SIRPa的结合，从而恢复荧光信号，并且随着抗体浓度的提高，荧光信号逐步加强。与Jurkat/NFAT-Luc/SIRPα效应细胞(EC50为0.8789μg/ml，如图6所示)相比，Jurkat(CD47敲除)/NFAT-Luc/SIRPα效应细胞在保持检测灵敏度(EC50为0.5195μg/ml)的情况下，信倍比(信号值/背景值)从4倍增加到15倍，说明敲除Jurkat内源性表达的CD47，显著提高检测的窗口。与此同时，效应细胞上内源性CD47基因的敲除，消除了效应细胞之间存在的CD47-SIRPα相互抑制的作用，提高了长久细胞培养的稳定性。
在效应细胞Jurkat(CD47敲除)/NFAT-Luc/SIRPα连续传代5次、15次和25次，按照上述方法检测抗CD47抗体Hu5F9阻断CD47-SIRPα相互作用通路恢复Jurkat(CD47敲除)TCR激活信号情况。与未敲除CD47基因的效应细胞Jurkat/NFAT-Luc/SIRPα对比(图13)，效应细胞Jurkat(CD47敲除)/NFAT-Luc/SIRPα在连续传代5次、10次和25次后，其检测窗口未见明显下降，依旧有>12倍的信倍比(信号值/背景值)，且保持了较好的检测灵敏度(EC50分别为0.5195μg/ml、0.5412μg/ml和0.5751μg/ml)，如图14所示。因此，效应细胞Jurkat(CD47敲除)/NFAT-Luc/SIRPα可作为CD47-SIRPα免疫检查点体外活性检测中可靠的原料。

Claims (34)

  1. 一种筛选靶向CD47-SIRPα免疫检查点的候选药物的方法，其包括：
    使靶细胞模拟物、靶向CD47-SIRPα免疫检查点的候选药物和效应细胞接触，所述靶细胞模拟物提供CD47蛋白和T细胞受体(TCR)激动剂，所述效应细胞包含报告基因以及稳定表达的SIRPα和TCR；
    孵育所述靶细胞模拟物、所述候选药物和所述效应细胞的混合物；和
    检测所述报告基因的表达或其表达强度来判断所述候选药物对CD47-SIRPα免疫检查点的阻断。
  2. 一种对靶向CD47-SIRPα免疫检查点的药物进行质量控制的方法，其包括：
    使靶细胞模拟物、靶向CD47-SIRPα免疫检查点的药物和效应细胞接触，所述靶细胞模拟物提供CD47蛋白和TCR激动剂，所述效应细胞包含报告基因以及稳定表达的SIRPα和TCR；
    孵育所述靶细胞模拟物、所述药物和所述效应细胞的混合物；和
    检测所述报告基因的表达强度来对所述药物进行质量控制。
  3. 根据权利要求1或2所述的方法，其中所述靶向CD47-SIRPα免疫检查点的候选药物或药物选自以下中的任一种或更多种：抗CD47单克隆抗体、抗CD47单域抗体、抗CD47双特异性的抗体、抗CD47多特异性的抗体、抗CD47单链抗体、包含抗CD47抗体的抗体缀合物、抗SIRPα单克隆抗体、抗SIRPα单链抗体、抗SIRPα多特异性抗体、包含抗SIRPα抗体的抗体缀合物、SIRPα重组或融合蛋白，和阻断CD47与SIRPα之间相互作用的小分子。
  4. 根据权利要求1-3中任一项所述的方法，其中所述靶细胞模拟物选自以下中的任一种或多种：稳定表达CD47和TCR激动剂的细胞、TCR激动剂和稳定表达CD47的细胞，和TCR激动剂和偶联CD47蛋白的微型颗粒。
  5. 根据权利要求1-4中任一项所述的方法，其中所述效应细胞为CD47基因表达部分或完全缺失的细胞。
  6. 根据权利要求5所述的方法，所述效应细胞通过基因工程改造使所述效应细胞的CD47基因表达部分或完全缺失；优选地，所述效应细胞为通过基因编辑使所述CD47基因表达部分或完全缺失的细胞。
  7. 根据权利要求1-6中任一项所述的方法，其中所述报告基因的表达为TCR途径依赖性的。
  8. 根据权利要求1-7中任一项所述的方法，其中所述报告基因的表达为活化T细胞核因子(NFAT)的反应元件、激活蛋白1(AP-1)反应元件或核因子κB(NF-κB)反应元件驱动的。
  9. 根据权利要求1-8中任一项所述的方法，其中所述TCR激动剂选自以下中的任一种或多种：通过主要组织相容性复合体(MHC)呈递的抗原、抗CD3抗体或其抗体片段，和超抗原。
  10. 根据权利要求1-9中任一项所述的方法，其中所述报告基因选自以下蛋白质的基因的一种或多种：荧光素酶、荧光蛋白、分泌胚胎碱性磷酸酶和β-半乳糖苷酶。
  11. 根据权利要求1-10中任一项所述的方法，其中所述接触步骤包括：提供靶细胞模拟物，以及向所述靶细胞模拟物添加靶向CD47-SIRPα免疫检查点的候选药物和效应细胞。
  12. 一种用于对靶向CD47-SIRPα免疫检查点的候选药物或药物进行筛选或质量控制的试剂盒，其包含：
    靶细胞模拟物，所述靶细胞模拟物提供CD47蛋白和TCR激动剂；和
    效应细胞，所述效应细胞包含报告基因以及稳定表达的SIRPα和TCR。
  13. 根据权利要求12所述的试剂盒，其中所述靶细胞模拟物选自以下中的任一种或多种：稳定表达CD47和TCR激动剂的细胞、TCR激动剂和稳定表达CD47的细胞，和TCR激动剂和偶联CD47蛋白的微型颗粒。
  14. 根据权利要求12或13所述的试剂盒，其中所述效应细胞为CD47基因表达部分或完全缺失的细胞。
  15. 根据权利要求14所述的试剂盒，所述效应细胞为通过基因工程改造使所述CD47基因表达部分或完全缺失的细胞。
  16. 根据权利要求12-15中任一项所述的试剂盒，其中所述报告基因的表达为TCR途径依赖性的。
  17. 根据权利要求12-16中任一项所述的试剂盒，所述报告基因选自活化T细胞核因子(NFAT)的反应元件、激活蛋白1(AP-1)反应元件或核因子κB(NF-κB)反应元件驱动的。
  18. 根据权利要求中12-17中任一项所述的试剂盒，其中所述TCR激动剂选自以下中的任一种或多种：主要组织相容性复合体(MHC)呈递的抗原、抗CD3抗体或其抗体片段，和超抗原。
  19. 根据权利要求12-18中任一项所述的试剂盒，其中所述报告基因选自以下蛋白质的基因的一种或多种：荧光素酶、荧光蛋白、分泌胚胎碱性磷酸酶和β-半乳糖苷酶。
  20. 根据权利要求12-19中任一项所述的试剂盒，其中所述效应细胞与所述靶细胞模拟物的细胞比例为8:1～1:1，优选为3：1。
  21. 一种系统，其包含：
    靶细胞模拟物，所述靶细胞模拟物提供CD47蛋白和TCR激动剂；和
    效应细胞，所述效应细胞包含TCR途径依赖性的报告基因以及稳定表达的SIRPα和TCR。
  22. 根据权利要求21所述的系统，其中所述靶细胞模拟物选自以下中的任一种或多种：稳定表达CD47和TCR激动剂的细胞、TCR激动剂和稳定表达CD47的细胞，和TCR激动剂和偶联CD47蛋白的微型颗粒。
  23. 根据权利要求21或22所述的系统，其中所述效应细胞为CD47基因表达部分或完全缺失的细胞。
  24. 根据权利要求23所述的系统，所述效应细胞为通过基因工程改造使所述CD47基因表达部分或完全缺失的细胞；优选地，所述效应细胞为通过基因编辑使所述CD47基因表达部分或完全缺失的细胞。
  25. 根据权利要求21-24中任一项所述的系统，其中所述报告基因的表达为活化T细胞核因子(NFAT)的反应元件、激活蛋白1(AP-1)反应元件或核因子κB(NF-κB)反应元件驱动的。
  26. 根据权利要求21-25中任一项所述的系统，其中所述TCR激动剂选自以下中的任一种或多种：主要组织相容性复合体(MHC)呈递的抗原、抗CD3抗体或其抗体片段，和超抗原。
  27. 根据权利要求21-26中任一项所述的系统，其还包括靶向CD47-SIRPα免疫检查点的候选药物或药物。
  28. 根据权利要求27所述的系统，其中所述靶向CD47-SIRPα免疫检查点的候选药物或药物选自以下中的任一种或更多种：抗CD47单克隆抗体、抗CD47单域抗体、抗CD47双特异性的抗体、抗CD47多特异性的抗体、抗CD47单链抗体、包含抗CD47抗体的抗体缀合物(ADC)、抗SIRPα单克隆抗体、抗SIRPα单链抗体、抗SIRPα多特异性抗体、包含抗SIRPα抗体的抗体缀合物、SIRPα重组或融合蛋白，和阻断CD47与SIRPα之间相互作用的小分子。
  29. 根据权利要求21-28中任一项所述的系统，所述报告基因选自以下的蛋白质的基因的一种或多种：荧光素酶、荧光蛋白、分泌胚胎碱性磷酸酶和β-半乳糖苷酶。
  30. 根据权利要求21-29中任一项所述的系统，其中所述效应细胞与所述靶细胞模拟物的细胞比例为8:1～1:1，优选为3：1。
  31. 一种细胞，其包含TCR途径依赖性的报告基因，稳定表达的SIRPα和TCR，其特征在于，所述细胞的CD47基因的表达部分或完全缺失。
  32. 根据权利要求31所述的细胞，其中，通过对所述细胞进行基因工程改造使得所述CD47基因表达部分或完全缺失。
  33. 根据权利要求32所述的细胞，所述基因工程改造为基因编辑，优选为基因敲除。
  34. 根据权利要求31-33中任一项所述的细胞，所述细胞为任何具有TCR激活通路的T细胞。
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