WO2021027867A1 - 嵌合抗原受体及其构建方法和应用 - Google Patents

嵌合抗原受体及其构建方法和应用 Download PDF

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WO2021027867A1
WO2021027867A1 PCT/CN2020/108850 CN2020108850W WO2021027867A1 WO 2021027867 A1 WO2021027867 A1 WO 2021027867A1 CN 2020108850 W CN2020108850 W CN 2020108850W WO 2021027867 A1 WO2021027867 A1 WO 2021027867A1
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chimeric antigen
cells
antigen receptor
cell
car
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French (fr)
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李俊
张鹏潮
徐昭
陈影
钟林茂
江雨辰
何玲
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苏州方德门达新药开发有限公司
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Priority to US17/635,170 priority Critical patent/US20230172980A1/en
Publication of WO2021027867A1 publication Critical patent/WO2021027867A1/zh

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Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to a chimeric antigen receptor and its construction method and application.
  • CAR-T Chimeric Antigen Receptor-T cellular immunotherapy
  • the CAR expressed by CAR-T cells generally contains an extracellular antigen binding domain, a transmembrane domain, a costimulatory factor domain, and an intracellular signal domain.
  • CAR-T cells are transduced and amplified from the patient's T cells through the CAR gene, and finally returned to the patient's body.
  • CAR-T cells can effectively recognize tumor antigens and cause specific anti-tumor immune responses, without being restricted by Major Histocompatibility Complex (MHC).
  • MHC Major Histocompatibility Complex
  • CAR-T has approved two autologous CAR-T cell products, namely Novartis’ Kymriah and Kate’s YesCAR-Ta, respectively for acute lymphoblastic leukemia (ALL) and refractory/relapsed non-Hodgkin Lymphoma and treatment.
  • ALL acute lymphoblastic leukemia
  • N-Hodgkin Lymphoma refractory/relapsed non-Hodgkin Lymphoma and treatment.
  • a large number of clinical trials have proved that CAR-T has great anti-tumor potential as a personalized living cell drug (Maude et al., 2018; Park et al., 2018; Schuster et al., 2017).
  • CAR-T therapy has achieved unprecedented efficacy in the treatment of hematological tumors, there are still some patients who do not respond to CAR-19-T; or even if the initial treatment has a certain efficacy, the persistence of CAR-19-T still exists Big problem.
  • Some studies have shown that part of the reason for the limited efficacy of CAR-T comes from the loss or down-regulation of tumor cell surface antigens (Grupp et al., 2013; Ruella et al., 2016).
  • non-Hodgkin’s lymphoma Neelapu reported the Phase II clinical results of their CAR-19-T product.
  • the main method to solve the loss of antigen during CAR-T treatment is to target multiple antigens to solve the negative escape of a single tumor antigen.
  • the researchers made the first attempt to treat multiple CAR-T simultaneously.
  • the dual CAR-T of HER2 and IL-23R ⁇ 2 can significantly prevent the escape of antigen and achieve a better tumor suppressing effect (Hegde et al., 2013).
  • Zah developed a CD19-CD20 tandem dual CAR-T, which can significantly inhibit the spontaneous escape of CD19-negative tumor cells in immunodeficient mice (Zah et al.
  • Dual CAR-Ts in series share a single conduction signal, while dual CAR-Ts in parallel use their own conduction signals (see Figure 1), so compared with dual CAR-Ts in series, parallel CAR-T signals are independent , Will not interfere with each other, and can most effectively exert the therapeutic effect of CAR-T cells.
  • the bispecific CAR-T in the parallel form provided by the present invention adopts two independent chimeric antigen receptors. Compared with the bispecific CAR-T in the tandem form, it has the advantages of independent signals and no mutual influence.
  • the WO2016/102965A1 patented invention is aimed at the tumor targets CD19 and CD22.
  • This patent compares the in vitro tumoricidal activity of the combination of four intracellular stimulating factors (including 41BBz-41BBz, OX40z-OX40z) on dual CAR19-CAR22 in parallel form. , 41BBz-28z and OX40z-28z), but the screening data of this patent is single, few combinations, and no relevant in vivo data.
  • the present invention conducts research on tumor targets CD19, CD20 and CD22, and performs a more systematic comparison and confirmation of tumor killing activity in vivo and in vitro on multiple combinations of parallel dual CAR19-CAR20 and parallel dual CAR19-CAR22.
  • this application provides a series of specific chimeric antigen receptors and their construction methods and applications, in particular, provides a bispecific chimeric antigen receptor containing two different antigen binding regions. ⁇ CAR-T.
  • the present inventors conducted in-depth research and repeated experiments, respectively, on the bispecific chimeric antigen receptor CAR19-CAR20 in parallel and the bispecific chimeric antigen receptor in parallel.
  • the extracellular hinge region, transmembrane region and costimulatory factor domain of CAR19-CAR22 in CAR20 structure and CAR22 structure are different from each other by system optimization and comparison, thereby completing the present invention. That is, the present invention is as follows:
  • a chimeric antigen receptor is first provided, characterized in that the chimeric antigen receptor is composed of an antigen binding region, an extracellular hinge region, a transmembrane region, a costimulatory domain, and a CD3z signal domain. composition.
  • the "Co-stimulatory Domain” can also be referred to as a costimulatory factor or a costimulatory factor domain.
  • the extracellular hinge region is selected from CD8 extracellular hinge region (CD8hinge), CD28 extracellular hinge region (CD28hinge), ICOS extracellular hinge region (ICOShinge) or IgG4mt10+N297A extracellular hinge Any one of the group consisting of IgG4mt10+N297Ahinge;
  • the transmembrane region is selected from any one of the group consisting of CD8 transmembrane region (CD8TM), CD28 transmembrane region (CD28TM) or ICOS transmembrane region (ICOSTM);
  • the costimulatory domain is selected from any of the group consisting of 4-1BB costimulatory domain (4-1BBCSD), CD28 costimulatory domain (CD28CSD), ICOS costimulatory domain (ICOSCSD) or OX40 costimulatory domain (OX40CSD) .
  • the structures comprising the extracellular hinge region, the transmembrane region and the costimulatory domain are respectively: CD8hinge-CD8TM-4-1BBCSD, CD28hinge-CD28TM-CD28CSD, ICOShinge-ICOSTM-ICOSCSD, CD28hinge -CD28TM-OX40CSD, IgG4mt10+N297Ahinge-CD8TM-4-1BBCSD, IgG4mt10+N297Ahinge-CD28TM-CD28CSD or IgG4mt10+N297Ahinge-ICOSTM-ICOSCSD.
  • - is independently a connecting peptide or peptide bond
  • hinge is the hinge region
  • TM is the transmembrane region
  • CSD is the costimulatory domain.
  • amino acid sequence of the IgG4mt10+N297Ahinge-CD8TM-4-1BBCSD is SEQ ID NO: 36
  • nucleotide sequence encoding the amino acid sequence is SEQ ID NO: 33;
  • amino acid sequence of the IgG4mt10+N297Ahinge-CD28TM-CD28CSD is SEQ ID NO: 37, and the nucleotide sequence encoding the amino acid sequence is SEQ ID NO: 34;
  • amino acid sequence of the IgG4mt10+N297Ahinge-ICOSTM-ICOSCSD is SEQ ID NO: 38, and the nucleotide sequence encoding the amino acid sequence is SEQ ID NO: 35.
  • the antigen binding region contained in the above-mentioned chimeric antigen receptor is a single chain antibody (scFv) or a single domain antibody (sdAb).
  • the scFv is formed by linking the antibody heavy chain variable region and light chain variable region through a short peptide (linker) of 15-20 amino acids.
  • the single domain antibody is also called nanobody or heavy chain antibody (hcAb), and its volume is about 1/10 of that of traditional antibodies.
  • hcAb nanobody or heavy chain antibody
  • the antigen binding region recognizes CD20 or recognizes CD22.
  • the antigen binding region is Leu16, wherein the Leu16 is a humanized scFv that recognizes CD20, and its amino acid sequence is shown in SEQ ID NO: 3.
  • the antigen binding region is M971, wherein the M971 is a scFv that recognizes CD22, and its amino acid sequence is shown in SEQ ID NO: 7.
  • chimeric antigen receptor T cells namely CAR-T cells, which can express any specific chimeric antigen receptor described in the first aspect of the present invention .
  • the CAR-T cell expresses two independent chimeric antigen receptors.
  • the two independent chimeric antigen receptors are CAR19 and CAR20, respectively; wherein the CAR19 recognizes CD19, and the CAR20 recognizes CD20.
  • the two independent chimeric antigen receptors are CAR19 and CAR22, respectively; wherein the CAR19 recognizes CD19, and the CAR22 recognizes CD22.
  • nucleic acid molecule which encodes any specific chimeric antigen receptor described in the first aspect of the present invention.
  • a vector which contains the nucleic acid molecule according to the third aspect of the present invention.
  • a host cell containing the vector according to the fourth aspect of the present invention or integrating the nucleic acid molecule according to the third aspect of the host cell into the chromosome of the host cell .
  • a pharmaceutical composition which contains a pharmaceutically acceptable carrier and any specific chimeric antigen receptor according to the first aspect of the present invention.
  • any specific chimeric antigen receptor according to the first aspect of the present invention, the nucleic acid molecule according to the third aspect of the present invention, and the fourth aspect of the present invention are provided The application of the carrier or the host cell as described in the fifth aspect of the present invention in the preparation of anti-tumor drugs or anti-tumor preparations.
  • the tumor is a hematological tumor, and preferably the hematological tumor is a B-cell malignant tumor, acute lymphatic leukemia, chronic lymphatic leukemia, lymphoma, mast cell tumor or follicular lymphoma.
  • a method for preparing CAR-T cells characterized in that the CAR-T cells express the specific chimeric antigen receptor as described in the first aspect of the present invention, and The method includes the following steps:
  • the nucleic acid molecule according to the third aspect of the present invention or the vector according to the fourth aspect of the present invention is introduced into a T cell to obtain the CAR-T cell.
  • the present invention provides CAR19-CAR20 lentiviral vectors containing bispecific chimeric antigen receptors in parallel form that can infect human T lymphocytes in vitro, and 7 CAR19-CAR20- containing bispecific chimeric antigen receptors of different structures.
  • T cells have higher killing efficiency for CD19 + K562-luc-GFP target cells and CD20 + K562-luc-GFP target cells; among them, CAR19-CAR20-T cells containing PCTL152 and PCTL153 have a lower effective target ratio. In this case, it still has a high killing efficiency for target cells.
  • the bispecific chimeric antigen receptor CAR19-CAR20-T cells provided in the present invention can maintain a high proportion of stem cell central memory T cells (TSCM).
  • TSCM stem cell central memory T cells
  • CAR19-CAR20-T cells containing PCTL152 and PCTL153 can better express CAR19 + CAR20 + double positive population.
  • the bispecific chimeric antigen receptor CAR19-CAR20-T cells provided in the present invention in parallel have good anti-tumor effects in vivo.
  • CAR19-CAR20-T cells containing PCTL153 can more significantly improve the survival rate of tumor-bearing mice;
  • dual CAR19-CAR20-T cells in parallel form containing PCTL153 can significantly improve the survival rate of tumor-bearing mice.
  • the bispecific chimeric antigen receptor CAR19-CAR22-T cells provided in the present invention have a killing efficiency of CD19 + K562-luc-GFP greater than that of the target cells when the effective target ratio is 10:1. 90%.
  • the bispecific chimeric antigen receptor CAR19-CAR22-T cell has a killing effect on the target cell CD22 + K562-luc-GFP under a series of different effect-to-target ratios, and has obvious effect targets. Ratio of dependence.
  • Figure 1 is a schematic diagram of the structure of a bispecific CAR-T in series and a bispecific CAR-T in parallel;
  • Figure 2 is a schematic diagram of the structure of seven bispecific chimeric antigen receptors CAR19-CAR20 in parallel;
  • Figure 3 shows the killing efficiency of seven CAR19-CAR20-T cells containing bispecific chimeric antigen receptors with different structures on target cells CD19 + K562-luc-GFP;
  • Figure 4 shows the killing efficiency of seven CAR19-CAR20-T cells containing bispecific chimeric antigen receptors with different structures on target cells CD20 + K562-luc-GFP;
  • Figure 5 shows the T cell phenotypes of seven CAR19-CAR20-T cells containing bispecific chimeric antigen receptors of different structures
  • Fig. 6 is the result of CAR19-CAR20-T cell expressing CAR19 + CAR20 + double positive population containing PCTL152 and PCTL153;
  • Figure 7 is a schematic diagram of the dosage regimen described in the validation test of the anti-tumor activity of CAR19-CAR20-T cells containing PCTL152 and PCTL153 in mice;
  • Figure 8 shows the survival rates of the tumor-bearing mice in groups G1, G3 and G4 after being given PBS, a carrier containing PCTL152 CAR19-CAR20-T cells, and a carrier containing PCTL153 CAR19-CAR20-T cells;
  • Figure 9 shows the dosage regimen of CAR19-CAR20-T cells containing PCTL153, single CAR19-T cells, single CAR20-T cells and CAR20-19-T cells in tandem form in the validation test of tumor suppressor activity in mice
  • Figure 10 shows the tumor-bearing mice in groups G1, G3, G4, G5 and G7 after being given PBS, a vector containing CAR-19-T cells, a vector containing CAR-20-T cells, and a parallel form containing PCTL153. Survival rate after the carrier of the bispecific chimeric antigen receptor CAR19-CAR20-T cell and the carrier containing the bispecific chimeric antigen receptor CAR20-19-T cell in tandem;
  • Figure 11 shows the killing efficiency of 7 CAR19-CAR22-T cells containing bispecific chimeric antigen receptors with different structures on target cells CD19 + K562-luc-GFP;
  • Figure 12 shows the killing efficiency of seven CAR19-CAR22-T cells containing bispecific chimeric antigen receptors with different structures on target cells CD22 + K562-luc-GFP.
  • Example 1 Design of bispecific chimeric antigen receptor CAR19-CAR20 and CAR19-CAR22 in parallel form
  • the present inventors designed 7 different bispecific chimeric antigen receptors CAR19-CAR20 in parallel, in which the structure of CAR19 is kept fixed, that is, they all have FMC63-CD8hinge-CD8TM-4-1BB-CD3z Structure of CAR19 (see CN105392888A), and combine it with 7 different CAR20 structures.
  • the amino acid sequence of the antigen binding domain FMC63 in CAR19 is shown in SEQ ID NO: 5
  • the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO: 6.
  • the amino acid sequences of CD8 hint, CD8TM, 4-1BB, and CD3z in CAR19 are shown in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively; those that encode the above amino acid sequence
  • the nucleotide sequence is shown in SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16.
  • the 7 different CAR20s include: Leu16-CD8hinge-CD8TM-4-1BBCSD-CD3z (the bispecific chimeric antigen receptor containing both the CAR20 and the above CAR19 is called PCTL126), Leu16- CD28 hinge-CD28 TM-CD28CSD-CD3z (the bispecific chimeric antigen receptor containing both the CAR20 and CAR19 mentioned above is called PCTL137), Leu16-ICOS hinge-ICOS TM-ICOSCSD-CD3z (will also contain The bispecific chimeric antigen receptor of CAR20 and CAR19 mentioned above is called PCTL138), Leu16-CD28hinge-CD28TM-OX40CSD-CD3z (will contain both CAR20 and CAR19 mentioned above) The combined antigen receptor is called PCTL139), Leu16-IgG4mt10+N297Ahinge-CD8TM-4-1BBCSD-CD3z (the bispecific chimeric antigen receptor containing both the C
  • the scFv in the seven different CAR20s described in Table 1 were tested against mouse-derived scFv (where the amino acid sequence of the mouse-derived scFv is shown in SEQ ID NO:1, which encodes the The nucleotide sequence of the amino acid sequence is shown in SEQ ID NO: 2) has been humanized, and the humanized scFv is named Leu16, and its amino acid sequence is shown in SEQ ID NO: 3, which encodes the The nucleotide sequence of the amino acid sequence is shown in SEQ ID NO: 4.
  • the hinge region of CAR20 has four different options, namely CD8hinge, CD28hinge, ICOShinge or IgG4mt10+N297Ahinge, and their amino acid sequences are as SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 23 and SEQ ID, respectively. NO: 31; the nucleotide sequence encoding the above amino acid sequence is shown in SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 24 and SEQ ID NO: 32, respectively.
  • the hinge region shown by IgG4mt10+N297Ahinge is mutated with 8 amino acid positions on the basis of natural IgG4.
  • the 228th amino acid in natural IgG4 was mutated from S to P
  • the 233rd amino acid was mutated from E to P
  • the 234th amino acid was mutated from F to V
  • the 235th amino acid was changed.
  • Mutation to K removes the binding ability of Fc ⁇ R (Fc gamma receptor), avoids antibody-dependent cell-mediated cytotoxicit (ADCC) and complement-dependent cytotoxicity (complement-dependent cytotoxicity) , CDC), thereby effectively enhancing the in vivo activity of CAR-T.
  • Fc ⁇ R Fc gamma receptor
  • the transmembrane region of CAR20 has three different options, namely CD8TM, CD28TM or ICOSTM, and their amino acid sequences are shown in SEQ ID NO: 11, SEQ ID NO: 19 and SEQ ID NO: 25, respectively; encoding the above amino acids
  • the nucleotide sequence of the sequence is shown in SEQ ID NO: 12, SEQ ID NO: 20 and SEQ ID NO: 26, respectively.
  • the costimulatory factor domain of CAR20 has four different options, namely 4-1BBCSD, CD28CSD, ICOSCSD and OX40CSD.
  • Their amino acid sequences are as shown in SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 27 and SEQ ID NO: 29; the nucleotide sequence encoding the above amino acid sequence is shown in SEQ ID NO: 14, SEQ ID NO: 22, SEQ ID NO: 28 and SEQ ID NO: 30, respectively.
  • the CAR20 part of the seven bispecific chimeric antigen receptors CAR19-CAR20 encodes the same antigen binding domain (that is, the same scFv) and the CD3z signal domain, and the amino acid sequence of the CD3z signal domain is as SEQ ID NO : Shown in 15 (the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO: 16).
  • the difference between the 7 constructs is only the different combination of hinge region, transmembrane region and costimulatory factor domain of CAR20.
  • the schematic diagram of the structure of seven bispecific chimeric antigen receptors CAR19-CAR20 in parallel is shown in Figure 2.
  • the present inventors also designed 7 different bispecific chimeric antigen receptors CAR19-CAR22 in parallel form, in which the structure of CAR19 is kept fixed, that is, they are all selected to have FMC63-CD8hinge-CD8TM-4-1BB- CD3z structure CAR19, and combine it with 7 different CAR22 structures.
  • the 7 different CAR22 include: M971-CD8hinge-CD8TM-4-1BBCSD-CD3z (will contain both the CAR22 and the above
  • the bispecific chimeric antigen receptor of CAR19 is called PCTL81), M971-CD28 hinge-CD28 TM-CD28CSD-CD3z (the bispecific chimeric antigen receptor that contains both CAR22 and CAR19 is called PCTL81).
  • PCTL103 M971-ICOShinge-ICOSTM-ICOSCSD-CD3z (the bispecific chimeric antigen receptor containing both CAR22 and CAR19 mentioned above is called PCTL105), M971-CD28hinge-CD28TM-OX40CSD- CD3z (the bispecific chimeric antigen receptor containing both the CAR22 and the aforementioned CAR19 is called PCTL124), M971-IgG4mt10+N297A hinge-CD8 TM-4-1BBCSD-CD3z (will contain both the CAR22 and the above
  • the bispecific chimeric antigen receptor of CAR19 described in the article is called PCTL148), M971-IgG4mt10+N297A hinge-CD28 TM-CD28CSD-CD3z (will contain both the CAR22 and the bispecific chimeric antigen of CAR19 described above The receptor is called PCTL149), M971-IgG4mt10+N297A hinge-ICOS
  • the amino acid sequence of the scFv in the seven different CAR22 described in Table 2 is shown in SEQ ID NO: 7, and the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO: 8.
  • the hinge region of CAR22 has four different choices, namely CD8hinge, CD28hinge, ICOShinge or IgG4mt10+N297Ahinge, and their amino acid sequences are as SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 23 and SEQ ID respectively.
  • NO: 31 the nucleotide sequence encoding the above amino acid sequence is shown in SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 24 and SEQ ID NO: 32, respectively.
  • the hinge region shown by IgG4mt10+N297A is mutated with 8 amino acid positions on the basis of natural IgG4. Specifically, the 228th amino acid in natural IgG4 was mutated from S to P, the 233rd amino acid was mutated from E to P, the 234th amino acid was mutated from F to V, and the 235th amino acid was changed.
  • the transmembrane region of CAR22 has three different options, namely CD8TM, CD28TM or ICOSTM, and their amino acid sequences are shown in SEQ ID NO: 11, SEQ ID NO: 19 and SEQ ID NO: 25, respectively; encoding the above amino acids
  • the nucleotide sequence of the sequence is shown in SEQ ID NO: 12, SEQ ID NO: 20 and SEQ ID NO: 26, respectively.
  • the costimulatory factor domain of CAR22 has four different options, namely 4-1BBCSD, CD28CSD, ICOSCSD and OX40CSD.
  • Their amino acid sequences are as shown in SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 27 and SEQ ID NO: 29; the nucleotide sequence encoding the above amino acid sequence is shown in SEQ ID NO: 14, SEQ ID NO: 22, SEQ ID NO: 28 and SEQ ID NO: 30, respectively.
  • the CAR22 part of the seven bispecific chimeric antigen receptors CAR19-CAR22 encodes the same antigen binding domain (that is, with the same scFv) and the CD3z signal domain, wherein the amino acid sequence of the CD3z signal domain is as SEQ ID NO : 15 (the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO: 16), the difference between the 7 constructs is only in the hinge region, transmembrane region and costimulatory factor of CAR22 Domain combination.
  • Example 2 Comparison of the killing effect of CAR19-CAR20-T cells prepared from bispecific chimeric antigen receptors with different structures on target cells
  • T cells not infected with lentivirus hereinafter referred to as NC-T cells
  • NC-T cells Use unmodified T cells (ie, T cells not infected with lentivirus, hereinafter referred to as NC-T cells) as a background control to construct target cells
  • the strain contains luciferase, which detects the killing effect of effector cells on target cells through the principle of chemiluminescence.
  • the specific operations are as follows:
  • CD3 + T cells are tested by Dynabeads CD3/CD28 (Lifetechnologies, catalog number: 40203D) Flow cytometric detection of CD25 + CD69 + T cell ratio (CD25 + CD69 + T cell ratio: 71%) after 24 hours of activation. After CD3 + T is activated, lentiviral transduction is performed. The 24-well plate was coated with Novonectin and incubated at 37°C for 2 hours. The cell suspension obtained after the above operation was respectively combined with various lentiviruses prepared (that is, containing PCTL126, PCTL137, PCTL138, PCTL139, PCTL151, PCTL152, PCTL153, respectively).
  • FBS X-vivo15 medium LONZA, article number: 04-418Q
  • Tscm final concentration 2U/ml
  • NC-T cells T cells not transfected with lentivirus
  • CAR-T cells of each group 2Collect NC-T cells and CAR-T cells of each group in a 15mL or 50mL centrifuge tube, and count the total number of cells (Cellometer k2 cell counter); 3Use sterile PBS (Hyclone, item number) :SH30256.01) Wash the collected cells 1-2 times, 1500rpm, 25°C, centrifugation for 5 minutes; 4T cell culture medium X-VIVO15 (LONZA, item number: 04-418Q) (without autologous serum and IL-2) ) Resuspend the washed cell pellet and adjust the cell density to 5.0E+07cells/mL.
  • NC-T cells T cells not transfected with lentivirus
  • T cell culture medium X-VIVO15 (LONZA, article number: 04-418Q) (without autologous serum and IL-2) make up the total volume to 200 ⁇ L; 2Transfer the 200 ⁇ L killing system prepared above into a 96-well V-plate for co-incubation 24 hours; 3After 24 hours, mix the cells in each well of the 96-well V-shaped plate by gently pipetting, and transfer 100 ⁇ L of cell suspension into the impermeable 96-well plate with the white wall bottom, and add 100 ⁇ L ONE-Glo TM Luciferase Assay Substrate. After incubating at room temperature for 10 minutes in the dark, the Luminoskan Ascent chemiluminescence analyzer was used to detect the chemiluminescence (Luminescence).
  • Killing efficiency (the value corresponding to NC-T cells-the value of specific CAR19-CAR20-T cells corresponding to the effective target ratio)/the value corresponding to NC-T cells
  • Test results According to the results shown in Table 3, it can be seen that when the effective target ratio is 10:1, it contains seven bispecific chimeric antigen receptors (ie PCTL126, PCTL137, PCTL138, PCTL139, PCTL151, PCTL152 and The killing efficiency of CAR19-CAR20-T cells of PCTL153) on target cells CD19+K562-luc-GFP is greater than 90%, and CAR19-CAR20-T cells containing PCTL152 and PCTL153 are more than 90% of target cells CD19 + K562-luc-GFP. The killing efficiency is close to 100% (see Table 3 and Figure 3 for details). In addition, CAR19-CAR20-T cells containing PCTL152 and PCTL153 still have a higher killing efficiency for the target cells when the effective target is relatively low.
  • Table 3 Killing efficiency of bispecific chimeric antigen receptor CAR19-CAR20-T cells on CD19 + K562-luc-GFP target cells
  • Table 4 Killing efficiency of bispecific chimeric antigen receptor CAR19-CAR20-T cells on CD20 + K562-luc-GFP target cells
  • Example 3 T cell phenotype displayed by CAR19-CAR20-T cells prepared from bispecific chimeric antigen receptors with different structures
  • Test method The seven bispecific chimeric antigen receptor CAR19-CAR20 prepared in Example 1 were selected as test materials, and the corresponding dual-target CAR-T cells were prepared according to the preparation method of effector cells described in Example 2.
  • 1 ⁇ 10 6 CAR-T cells were taken, and the CAT-T cells were washed with PBS and then incubated with CD62L-PE-Cy5 antibody (BD, catalog number: 555545) And CD45RO-FITC antibody (BD, catalog number: 555492), incubate in a refrigerator at 4°C for 30 min. After the antibody incubation, wash with PBS (Hyclone, item number: SH30256.01) for 2-3 times, resuspend with 500 ⁇ l PBS, and place it in a flow tube for testing.
  • PBS Hyclone, item number: SH30256.01
  • Test results As shown in Figure 5, the results show that the analyzed dual-target CAR-T cells can maintain a higher proportion of stem cell central memory T cells (TSCM). Studies have reported that the proportion of the central memory T cell population of stem cells is closely related to the tumor-killing activity, expansion ability and lasting immune memory ability of CAR-T cells in vivo. This suggests that the dual-target CAR-T cells provided by the present invention can have better tumoricidal activity, expansion ability and lasting immune memory ability in organisms.
  • TSCM stem cell central memory T cells
  • Example 4 Detection of CAR19-CAR20-T cells containing PCTL152 and PCTL153 expressing CAR19 and CAR20
  • Test method The CAR19-CAR20-T cells containing PCTL152 and PCTL153 with higher killing efficiency in Example 2 were selected as the test materials. For the above two CAR-T cells, 1 ⁇ 10 6 CAR-T cells were taken respectively. After washing with 4% BSA (2500rpm, 5min) 3 times, incubate the antibody: (1) Alexa Fluor 647 AffiniPure Goat Anti-Human IgG (1:100 ⁇ 1:800), incubate in the refrigerator at 4°C for 30min; after the antibody incubation, use 4 Wash with %BSA (2500rpm, 5min), incubate the antibody after 3 times: (2) PE-labeled CAR19 (iFMC63) idiotype (Qin et al.
  • Test results As shown in Figure 6, CAR19 and CAR20 proteins can be simultaneously detected on the surface of T cells. The results indicate that the constructed CAR19-CAR20-T cells containing PCTL152 and PCTL153 can better express the CAR19 + CAR20 + double positive population.
  • Example 5 Verification of the anti-tumor activity of CAR19-CAR20-T cells containing PCTL152 and PCTL153 in mice
  • Test method CAR19-CAR20-T cells containing PCTL152 and PCTL153 with higher killing efficiency in Example 2 were selected as the test group, and PBS was selected as the control group.
  • Raji-Luc cells resuspended in PBS (Biocytometer, Item No.: B-HCL-010) were inoculated into the caudal vein at a concentration of 5 ⁇ 10 5 cells/0.2 mL and a volume of 0.2 mL/cell. (B-NSG) mice. On the day of inoculation, observe whether the tumor inoculation is successful with a small animal imager. On the third day of inoculation, use a small animal imager to measure tumor growth.
  • mice with moderate tumor imaging signals were selected into the group and randomly assigned to 3 groups, 2 in G1 group, 3 in G3 and G4 groups, and mice with too strong/low fluorescence signals were eliminated.
  • the administration was started on the day of grouping. After the administration, the tumor growth (detection and recording by small animal imager) and the weight of the animals were measured on the 4th day. After that, the mouse imager was tested once a week (day 4, day 11, day 18, and day 25) and the animal body weight was measured twice a week.
  • the specific dosing scheme is shown in Figure 7, and the type of dosing, the number of mice and the dose of each group are shown in Table 5.
  • Table 5 Types of administration, number of mice and dosage of G1, G3 and G4 groups
  • mice Dosage G1 PBS 2 PBS 200 ⁇ l//only G3 PCTL152 3 Total T 0.1E+07/200 ⁇ l/piece G4 PCTL153 3 Total T 0.1E+07/200 ⁇ l/piece
  • Example 6 Verification of tumor suppressor activity in mice containing PCTL153 CAR19-CAR20-T cells, single CAR19-T cells, single CAR20-T cells and CAR20-19-T cells in tandem form
  • Test method Raji-Luc cells resuspended in PBS were inoculated by tail vein injection at a concentration of 5 ⁇ 10 5 cells/0.2 mL and a volume of 0.2 mL/cell. (B-NSG) mice, a total of 54 mice were transferred. On the day of inoculation, observe whether the tumor inoculation is successful with a small animal imager. After successful inoculation, use a small animal imager to measure tumor growth. When the average imaging signal reaches about 1 ⁇ 10 6 [(P/S)/(cm2/sr)] , Select 30 mice with moderate tumor imaging signal into the group, and randomly assign them to 5 groups, each with 6 mice. The tumor-bearing mice with too strong/low live imaging signals are eliminated.
  • the administration was started on the day of grouping. After the administration, the weight of the experimental animals and tumor growth (detected and recorded by the small animal imager) were continued to be observed. The tumor growth was measured on the 4th, 7th and 11th day after grouping, and then the tumor growth was measured once a week (detected and recorded by small animal imager). Measure the animal body weight twice a week, make clinical observations, and record the measured values.
  • the specific dosing scheme is shown in Figure 9, and the type of dosing, number of mice and dose of each group are shown in Table 6.
  • Test result It is the same as the traditional single CAR19-T cell (G3 group, the structure and sequence of CAR19 contained in it are the same as the structure and sequence of CAR19 described in Example 1 of the present invention: FMC63-CD8hinge-CD8 TM-4-1BB- CD3z), single CAR20-T cell (G4 group, the structure and sequence of CAR20 contained therein are consistent with the structure and sequence of CAR20 in PCTL153 described in Example 1 of the present invention: Leu16-IgG4mt10+N297Ahinge-ICOSTM-ICOSCSD -CD3z) and CAR20-19-T cells in tandem form (G7 group, CD20scFv-(EAAAK)3-CD19scFv-IgG4hinge-CD28TM-4-1BB-CD3z), please refer to Zahet al.CancerImmunolRes. 2016; 4(6): 498-508), parallel dual CAR19-CAR20-T cells containing PC
  • Table 6 Types of administration, number of mice and dosage of G1, G3, G4, G5 and G7 groups
  • Example 7 Comparison of the killing effect of CAR19-CAR22-T cells prepared from bispecific chimeric antigen receptors with different structures on target cells
  • CD3 + T cells are tested by Dynabeads CD3/CD28 (Lifetechnologies, catalog number: 40203D) Flow cytometric detection of CD25 + CD69 + T cell ratio (CD25 + CD69 + T cell ratio: 71%) after 24 hours of activation. After CD3 + T is activated, lentiviral transduction is performed. The 24-well plate was coated with Novonectin and incubated at 37°C for 2 hours. The cell suspension obtained after the above operation was respectively combined with various lentiviruses prepared (that is, PCTL81, PCTL103, PCTL105, PCTL124, PCTL148, PCTL149, and PCTL150.
  • FBS X-vivo15 medium LONZA, article number: 04-418Q
  • Tscm final concentration 2U/ml
  • NC-T cells T cells not transfected with lentivirus
  • CAR-T cells of each group 2Collect NC-T cells and CAR-T cells of each group in a 15mL or 50mL centrifuge tube, and count the total number of cells (Cellometer k2 cell counter); 3Use sterile PBS (Hyclone, item number) :SH30256.01) Wash the collected cells 1-2 times, 1500rpm, 25°C, centrifugation for 5 minutes; 4T cell culture medium X-VIVO15 (LONZA, item number: 04-418Q) (without autologous serum and IL-2) ) Resuspend the washed cell pellet and adjust the cell density to 5.0E+07cells/mL.
  • NC-T cells T cells not transfected with lentivirus
  • Killing efficiency (the value corresponding to NC-T cells-the value of specific CAR19-CAR20-T cells corresponding to the effective target ratio)/the value corresponding to NC-T cells
  • Test results According to the results shown in Figure 11, it can be seen that when the effective target ratio is 10:1, it contains seven bispecific chimeric antigen receptors (ie PCTL81, PCTL103, PCTL105, PCTL124, PCTL148, PCTL149, The killing efficiency of CAR19-CAR22-T cells of PCTL150) against the target cell CD19 + K562-luc-GFP is all greater than 90% (see Figure 11 for details). According to the results shown in Figure 12, it can be seen that the CAR19-CAR22-T cells of the seven bispecific chimeric antigen receptors (i.e.
  • PCTL81, PCTL103, PCTL105, PCTL124, PCTL148, PCTL149, PCTL150 have a positive effect on the target cell CD22 + K562- Both luc-GFP have killing effects and have obvious dependence on the effective target ratio (see Figure 12 for details).

Abstract

提供了嵌合抗原受体及其构建方法和应用。该嵌合抗原受体由抗原结合区、胞外铰链区、跨膜区、共刺激域和CD3z信号结构域组成。还提供了包含两种含有不同抗原结合区的嵌合抗原受体的CAR-T细胞，其具有双特异性，展现出增高的细胞杀伤效率和更好的体内抑瘤效果。

Description

嵌合抗原受体及其构建方法和应用
优先权和相关申请
本申请要求2019年8月14日提交的名称为“嵌合抗原受体及其构建方法和应用”的中国专利申请201910748321.2的优先权，该申请包括附录在内的全部内容作为参考并入本申请。
技术领域
本发明属于生物技术领域，具体涉及嵌合抗原受体及其构建方法和应用。
背景技术
随着肿瘤治疗的发展，嵌合抗原受体T(Chimeric Antigen Receptor-T，CAR-T)细胞免疫疗法逐渐成为备受关注的治疗手段。CAR-T细胞所表达的CAR一般包含胞外抗原结合域、跨膜区、共刺激因子结构域和和胞内信号结构域。通常CAR-T细胞是由患者T细胞经CAR基因转导并扩增而来，最后再回输到该患者体内。CAR-T细胞可以有效的识别肿瘤抗原，引起特异性的抗肿瘤免疫应答，而不受主要组织相容性复合体(Major Histocompatibility Complex，MHC)的限制。目前，美国FDA已经批准了两款自体CAR-T细胞产品上市，分别是诺华的Kymriah和凯特的YesCAR-Ta，分别用于急性淋巴细胞白血病(ALL)和难治性/复发性非霍奇金淋巴瘤和的治疗。大量临床试验证明，CAR-T作为个性化的活细胞药极具抗肿瘤潜力(Maude et al.，2018；Park et al.，2018；Schuster et al.，2017)。
尽管多种多样的肿瘤抗原正在被应用于临床研究中，但CD19仍是目前CAR-T研究中最为广泛的靶点。尽管CAR-T治疗在血液肿瘤治疗中达到了前所未有的疗效，但仍有部分患者对CAR-19-T并不反应；或者即使初始治疗有一定的疗效，CAR-19-T的持久性仍存在很大问题。部分研究表明CAR-T 疗效受限的一部分原因来自于肿瘤细胞表面抗原的丢失或下调(Grupp et al.，2013；Ruella et al.，2016)。在非霍奇金淋巴瘤中，Neelapu报道了他们的CAR-19-T产品的二期临床结果，108个病人CAR-T治疗后长达至少一年的跟踪，其中42％的病人得到了很好的响应，但仍有部分患者复发，其中CD19阴性复发逃逸是主要原因(Neelapu et al.，2017)。在急性淋巴瘤治疗中，CAR-19-T的总体响应率为81％，大部分的复发病人(15/22)呈现出了CD19阴性逃逸(Kantarjian et al.，2016)。因此，靶点抗原逃逸是目前CAR-T治疗的重要瓶颈之一。
解决CAR-T治疗过程中抗原丢失的主要方法是通过靶向多个抗原，从而解决单一肿瘤抗原的阴性逃逸。在胶质瘤的CAR-T临床前研究中，研究者首次进行了多CAR-T同时治疗的尝试。HER2和IL-23Rα2的双CAR-T与单CAR-T相比，能显著阻止抗原逃逸，并达到更优的抑瘤效果(Hegde et al.，2013)。在B细胞恶性肿瘤的双CAR-T临床前研究中，Zah开发了一种CD19-CD20串联形式的双CAR-T，在免疫缺陷老鼠中能显著抑制CD19阴性肿瘤细胞的自发逃逸(Zah et al.，2016)。串联形式的双CAR-T共用一个传导信号，而并联形式的双CAR-T分别采用各自的传导信号(参见图1)，因此与串联形式的双CAR-T相比，并联CAR-T信号独立，不会相互干扰，能最有效的发挥CAR-T细胞的治疗效果。Plue在并联形式的双CAR19-CAR22方面进行了相关研究(WO2016/102965A1)，发现4-1BBzeta/OX40zeta和CD28zeta的双CAR19-CAR22组合具有最优的体外杀瘤效果，表明并联双CAR-T的不同的胞内刺激因子的组合会导致差异性的体内外抑瘤效果。因此对于并联形式的双CAR-T而言，在繁多的不同的胞外抗原结合域、跨膜区、共刺激因子结构域中找寻到最合适的组合极为关键。
本发明提供的并联形式的双特异性CAR-T采用独立的两个嵌合抗原受体，与串联形式的双特异性CAR-T相比，具有信号独立、互不影响的优势。WO2016/102965A1专利发明针对肿瘤靶点CD19和CD22进行研究，该专利对并联形式的双CAR19-CAR22进行了4种胞内刺激因子组合的体外杀瘤活性 的比较(包括41BBz-41BBz、OX40z-OX40z、41BBz-28z和OX40z-28z)，但是该专利的筛选数据单一、组合较少，且无相关体内数据。本发明针对肿瘤靶点CD19、CD20和CD22进行研究，对多种组合形式的并联双CAR19-CAR20以及并联双CAR19-CAR22进行了更为系统的体内外杀瘤活性的比较确认。
发明内容
发明要解决的问题
针对现有技术中存在的问题，本申请提供了一系列特异性嵌合抗原受体及其构建方法和应用，特别是提供了包含两个含有不同抗原结合区的嵌合抗原受体的双特异性CAR-T。
用于解决问题的方案
本发明人鉴于上述现有技术中存在的问题，进行了深入的研究、反复试验，分别对并联形式的双特异性嵌合抗原受体CAR19-CAR20和并联形式的双特异性嵌合抗原受体CAR19-CAR22中的CAR20结构和CAR22结构不同的胞外铰链区、跨膜区和共刺激因子结构域进行了系统优化和比较，从而完成了本发明。即本发明如下所述：
在本发明的第一方面，首先提供了嵌合抗原受体，其特征在于，所述嵌合抗原受体由抗原结合区、胞外铰链区、跨膜区、共刺激域和CD3z信号结构域组成。
对本领域技术人员而言，所述“共刺激域”(Co-stimulatory Domain，CSD)也可以称为共刺激因子或共刺激因子结构域。
在本发明具体的实施方案中，所述胞外铰链区选自由CD8胞外铰链区(CD8hinge)、CD28胞外铰链区(CD28hinge)、ICOS胞外铰链区(ICOShinge)或IgG4mt10+N297A胞外铰链区(IgG4mt10+N297Ahinge)组成的组中的任一种；
所述跨膜区选自由CD8跨膜区(CD8TM)、CD28跨膜区(CD28TM)或ICOS跨膜区(ICOSTM)组成的组中的任一种；
所述共刺激域选自由4-1BB共刺激域(4-1BBCSD)、CD28共刺激域 (CD28CSD)、ICOS共刺激域(ICOSCSD)或OX40共刺激域(OX40CSD)组成的组中的任一种。
在本发明具体的实施方案中，所述包含胞外铰链区、跨膜区和共刺激域的结构分别为：CD8hinge-CD8TM-4-1BBCSD、CD28hinge-CD28TM-CD28CSD、ICOShinge-ICOSTM-ICOSCSD、CD28hinge-CD28TM-OX40CSD、IgG4mt10+N297Ahinge-CD8TM-4-1BBCSD、IgG4mt10+N297Ahinge-CD28 TM-CD28CSD或IgG4mt10+N297Ahinge-ICOSTM-ICOSCSD。
上式中，“-”独立地为连接肽或肽键；hinge为铰链区；TM为跨膜区；CSD为共刺激域。
其中，所述IgG4mt10+N297Ahinge-CD8TM-4-1BBCSD的氨基酸序列为SEQ ID NO：36，编码所述氨基酸序列的核苷酸序列为SEQ ID NO：33；
所述IgG4mt10+N297Ahinge-CD28TM-CD28CSD的氨基酸序列为SEQ ID NO：37，编码所述氨基酸序列的核苷酸序列为SEQ ID NO：34；
所述IgG4mt10+N297Ahinge-ICOSTM-ICOSCSD的氨基酸序列为SEQ ID NO：38，编码所述氨基酸序列的核苷酸序列为SEQ ID NO：35。
上文所述嵌合抗原受体中包含的抗原结合区为单链抗体(scFv)或单域抗体(sdAb)。
其中，所述scFv由抗体重链可变区和轻链可变区通过15～20个氨基酸的短肽(linker)连接而成。所述单域抗体又称纳米抗体(nanobody)或重链抗体(heavy chain antibody，hcAb)，其体积约为传统抗体的1/10。与传统抗体不同的是，单域抗体仅由重链构成，其抗原结合区仅是一个通过铰链区与Fc区连接的单结构域，而且这个抗原结合区自抗体上分离后仍具有结合抗原的功能。
在本发明具体的实施方案中，所述抗原结合区识别CD20或者识别CD22。
进一步的，所述抗原结合区为Leu16，其中所述Leu16为识别CD20的人源化scFv，其氨基酸序列如SEQ ID NO：3所示。
或者，所述抗原结合区为M971，其中所述M971为识别CD22的scFv，其氨基酸序列如SEQ ID NO：7所示。
在本发明的第二方面，提供了嵌合抗原受体T细胞，即CAR-T细胞，所述CAR-T细胞能够表达本发明第一方面所述的任一种特异性嵌合抗原受体。其中，所述CAR-T细胞表达两个独立的嵌合抗原受体。
在本发明一个实施方案中，所述两个独立的嵌合抗原受体分别为CAR19和CAR20；其中所述CAR19识别CD19，所述CAR20识别CD20。
在本发明另外的实施方案中，所述两个独立的嵌合抗原受体分别为CAR19和CAR22；其中所述CAR19识别CD19，所述CAR22识别CD22。
在本发明的第三方面，提供了一种核酸分子，所述核酸分子编码本发明第一方面所述的任一种特异性嵌合抗原受体。
在本发明的第四方面，提供了一种载体，所述载体含有如本发明第三方面所述的核酸分子。
在本发明的第五方面，提供了一种宿主细胞，所述宿主细胞含有本发明第四方面所述的载体或在所述宿主细胞的染色体中整合有本发明第三方面所述的核酸分子。
在本发明的第六方面，提供了一种药物组合物，所述药物组合物中含有药学上可接受的载体以及本发明第一方面所述的任一种特异性嵌合抗原受体。
在本发明的第七方面，提供了如本发明第一方面所述的任一种特异性嵌合抗原受体、如本发明第三方面所述的核酸分子、如本发明第四方面所述的载体或如本发明第五方面所述的宿主细胞在制备治疗抗肿瘤的药物或抗肿瘤的制剂中的应用。
其中，所述肿瘤为血液肿瘤，优选的所述血液肿瘤为B细胞恶性肿瘤、急性淋巴性白血病、慢性淋巴性白血病、淋巴瘤、肥大细胞瘤或滤泡性淋巴瘤。
在本发明的第八方面，提供了一种制备CAR-T细胞的方法，其特征在于， 所述CAR-T细胞表达如本发明第一方面所述的特异性嵌合抗原受体，所述方法包括以下步骤：
将本发明第三方面所述的核酸分子或者本发明第四方面所述的载体导入T细胞内，从而获得所述CAR-T细胞。
发明的效果
1，本发明提供包含并联形式的双特异性嵌合抗原受体CAR19-CAR20慢病毒载体可体外感染人T淋巴细胞，7种包含不同结构的双特异性嵌合抗原受体的CAR19-CAR20-T细胞对于CD19 +K562-luc-GFP靶细胞和CD20 +K562-luc-GFP靶细胞均具有较高的杀伤效率；其中包含PCTL152和PCTL153的CAR19-CAR20-T细胞在较低的效靶比的情况下，对于靶细胞仍然具有较高的杀伤效率。
2，本发明提供的并联形式的双特异性嵌合抗原受体CAR19-CAR20-T细胞细胞均能保持较高比例的干细胞中央记忆型T细胞(TSCM)。其中，包含PCTL152和PCTL153的CAR19-CAR20-T细胞能够较好的表达CAR19 +CAR20 +双阳性群体。
3，本发明提供的并联形式的双特异性嵌合抗原受体CAR19-CAR20-T细胞均具有较好的体内抑瘤效果。特别的，与包含PCTL152的CAR19-CAR20-T细胞相比，包含PCTL153的CAR19-CAR20-T细胞能更加明显的提高荷瘤小鼠的生存率；与传统的单CAR19-T细胞、单CAR20-T细胞以及串联形式的CAR20-19-T细胞相比，包含PCTL153的并联形式的双CAR19-CAR20-T细胞能明显提高荷瘤小鼠的生存率。
4，本发明提供的并联形式的双特异性嵌合抗原受体CAR19-CAR22-T细胞在效靶比为10∶1的情况下，对于靶细胞CD19 +K562-luc-GFP的杀伤效率均大于90％。另外，所述双特异性嵌合抗原受体CAR19-CAR22-T细胞在一系列不同的效靶比情况下，对于靶细胞CD22 +K562-luc-GFP均具有杀伤效果，且具有明显的效靶比的依赖性。
为了让本发明的上述和其他目的、特征和优点能更明显易懂，下面特举 较佳实施例，并配合说明书附图，作详细说明如下：
附图说明
图1为串联形式的双特异性CAR-T和并联形式的双特异性CAR-T的结构示意图；
图2为7种并联形式的双特异性嵌合抗原受体CAR19-CAR20的结构示意图；
图3为7种包含不同结构的双特异性嵌合抗原受体的CAR19-CAR20-T细胞对于靶细胞CD19 +K562-luc-GFP的杀伤效率；
图4为7种包含不同结构的双特异性嵌合抗原受体的CAR19-CAR20-T细胞对于靶细胞CD20 +K562-luc-GFP的杀伤效率；
图5为7种包含不同结构的双特异性嵌合抗原受体的CAR19-CAR20-T细胞的T细胞表型；
图6为包含PCTL152和PCTL153的CAR19-CAR20-T细胞表达CAR19 +CAR20 +双阳性群体的结果；
图7为包含PCTL152和PCTL153的CAR19-CAR20-T细胞在小鼠体内抑瘤活性验证试验中所述给药方案的示意图；
图8为G1、G3和G4组所述荷瘤小鼠在分别给予PBS、包含PCTL152的CAR19-CAR20-T细胞的载体、包含PCTL153的CAR19-CAR20-T细胞的载体后的生存率；
图9为包含PCTL153的CAR19-CAR20-T细胞、单CAR19-T细胞、单CAR20-T细胞以及串联形式的CAR20-19-T细胞在小鼠体内抑瘤活性验证试验中所述给药方案的示意图；
图10为G1、G3、G4、G5和G7组所述荷瘤小鼠在分别给予PBS、包含CAR-19-T细胞的载体、包含CAR-20-T细胞的载体、包含PCTL153的并联形式的双特异性嵌合抗原受体CAR19-CAR20-T细胞的载体和包含串联形式的双特异性嵌合抗原受体CAR20-19-T细胞的载体后的生存率；
图11为7种包含不同结构的双特异性嵌合抗原受体的CAR19-CAR22-T 细胞对于靶细胞CD19 +K562-luc-GFP的杀伤效率；
图12为7种包含不同结构的双特异性嵌合抗原受体的CAR19-CAR22-T细胞对于靶细胞CD22 +K562-luc-GFP的杀伤效率。
具体实施方式
下面通过具体实施方式来进一步说明本发明的技术方案。应该强调的是，本发明不限于所举例说明的具体实施方案。此外，本文使用的任何章节标题仅用于组织目的，并不被解释为限制所描述的主题。
除非在此另外定义，否则与本发明结合使用的科学和技术术语将具有本领域普通技术人员通常理解的含义。此外，除非上下文另有要求，单数形式的术语应包括复数形式，复数形式的术语应包括单数形式。更具体地，如在本说明书和所附权利要求中所使用的，除非上下文另外明确指出，否则单数形式“一”，“一个”和“该”包括复数指示物。在本申请中，除非另有说明，否则使用“或”意指“和/或”。此外，术语“包含”以及其他形式(诸如“包括”和“含有”)的使用不是限制性的。此外，说明书和所附权利要求中提供的范围包括端点和断点之间的所有值。
实施例1：并联形式的双特异性嵌合抗原受体CAR19-CAR20和CAR19-CAR22的设计
本发明人设计了7种不同的并联形式的双特异性嵌合抗原受体CAR19-CAR20，其中保持CAR19的结构固定不变，即均选择具有FMC63-CD8 hinge-CD8 TM-4-1BB-CD3z结构的CAR19(参见CN105392888A)，并将其与7种不同的CAR20结构进行组合。其中，上述CAR19中的抗原结合域FMC63的氨基酸序列如SEQ ID NO：5所示，编码该氨基酸序列的核苷酸序列如SEQ ID NO：6所示。上述CAR19中的CD8 hinge、CD8TM、4-1BB和CD3z的氨基酸序列分别如SEQ ID NO：9、SEQ ID NO：11、SEQ ID NO：13和SEQ ID NO：15所示；编码上述氨基酸序列的核苷酸序列如SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：14和SEQ ID NO： 16所示。
所述7种不同的CAR20包括：Leu16-CD8 hinge-CD8 TM-4-1BBCSD-CD3z(将同时包含该CAR20和上文所述CAR19的双特异性嵌合抗原受体称为PCTL126)，Leu16-CD28 hinge-CD28 TM-CD28CSD-CD3z(将同时包含该CAR20和上文所述CAR19的双特异性嵌合抗原受体称为PCTL137)，Leu16-ICOS hinge-ICOS TM-ICOSCSD-CD3z(将同时包含该CAR20和上文所述CAR19的双特异性嵌合抗原受体称为PCTL138)，Leu16-CD28 hinge-CD28 TM-OX40CSD-CD3z(将同时包含该CAR20和上文所述CAR19的双特异性嵌合抗原受体称为PCTL139)，Leu16-IgG4mt10+N297A hinge-CD8 TM-4-1BBCSD-CD3z(将同时包含该CAR20和上文所述CAR19的双特异性嵌合抗原受体称为PCTL151)，Leu16-IgG4mt10+N297A hinge-CD28 TM-CD28CSD-CD3z(将同时包含该CAR20和上文所述CAR19的双特异性嵌合抗原受体称为PCTL152)，Leu16-IgG4mt10+N297A hinge-ICOS TM-ICOSCSD-CD3z(将同时包含该CAR20和上文所述CAR19的双特异性嵌合抗原受体称为PCTL153)。所述7种不同的CAR20的组成如表1所示：
表1：7种不同CAR20的组成
Figure PCTCN2020108850-appb-000001
具体来说，表1中所述7种不同CAR20中的scFv均通过常规的分子生物学手段对鼠源的scFv(其中所述鼠源scFv的氨基酸序列如SEQ ID NO：1所示，编码该氨基酸序列的核苷酸序列如SEQ ID NO：2所示)做了人源化处理，并将人源化处理后的scFv命名为Leu16，其氨基酸序列如SEQ ID NO：3所示，编码该氨基酸序列的核苷酸序列如SEQ ID NO：4所示。
所述CAR20的铰链区具有四种不同的选择，即CD8hinge、CD28hinge、ICOShinge或IgG4mt10+N297Ahinge，它们的氨基酸序列分别如SEQ ID NO：9、SEQ ID NO：17、SEQ ID NO：23和SEQ ID NO：31所示；编码上述氨基酸序列的核苷酸序列分别如SEQ ID NO：10、SEQ ID NO：18、SEQ ID NO：24和SEQ ID NO：32所示。其中，所述IgG4mt10+N297Ahinge所示的铰链区是在天然的IgG4的基础上突变了8个氨基酸位点。具体来说，是将天然的IgG4中第228位氨基酸由S突变成P、将第233位氨基酸由E突变成P、将第234位氨基酸由F突变成V、将第235位氨基酸由L突变成A、将第265位氨基酸由D突变成A、将第297位氨基酸由N突变成A、将第309位氨基酸由L突变成V、将第409位氨基酸由R突变成K，去除了FcγR(Fc gamma receptor)的结合能力，避免了抗体依赖性细胞介导的细胞毒性(antibody-dependent cell-mediated cytotoxicit，ADCC)和补体依赖的细胞毒性(complement-dependent cytotoxicity，CDC)，从而有效增强CAR-T的体内活性。
所述CAR20的跨膜区具有三种不同的选择，即CD8TM、CD28TM或ICOSTM，它们的氨基酸序列分别如SEQ ID NO：11、SEQ ID NO：19和SEQ ID NO：25所示；编码上述氨基酸序列的核苷酸序列分别如SEQ ID NO：12、SEQ ID NO：20和SEQ ID NO：26所示。
所述CAR20的共刺激因子结构域具有四种不同选择，即4-1BBCSD、CD28CSD、ICOSCSD和OX40CSD，它们的氨基酸序列分别如SEQ ID NO：13、SEQ ID NO：21、SEQ ID NO：27和SEQ ID NO：29所示；编码上述氨基酸序列的核苷酸序列分别如SEQ ID NO：14、SEQ ID NO：22、SEQ ID NO：28和SEQ ID NO：30所示。即7种双特异性嵌合抗原受体CAR19-CAR20中的 CAR20部分编码相同的抗原结合结构域(即具有相同的scFv)和CD3z信号域，其中所述CD3z信号域的氨基酸序列如SEQ ID NO：15所示(编码该氨基酸序列的核苷酸序列如SEQ ID NO：16所示)。所述7种构建体之间的区别仅在于CAR20部分不同的铰链区、跨膜区和共刺激因子结构域组合。7种并联形式的双特异性嵌合抗原受体CAR19-CAR20的结构示意图如图2所示。
本发明人还设计了7种不同的并联形式的双特异性嵌合抗原受体CAR19-CAR22，其中保持CAR19的结构固定不变，即均选择具有FMC63-CD8 hinge-CD8 TM-4-1BB-CD3z结构的CAR19，并将其与7种不同的CAR22结构进行组合，所述7种不同的CAR22包括：M971-CD8 hinge-CD8 TM-4-1BBCSD-CD3z(将同时包含该CAR22和上文所述CAR19的双特异性嵌合抗原受体称为PCTL81)，M971-CD28 hinge-CD28 TM-CD28CSD-CD3z(将同时包含该CAR22和上文所述CAR19的双特异性嵌合抗原受体称为PCTL103)，M971-ICOS hinge-ICOS TM-ICOSCSD-CD3z(将同时包含该CAR22和上文所述CAR19的双特异性嵌合抗原受体称为PCTL105)，M971-CD28 hinge-CD28 TM-OX40CSD-CD3z(将同时包含该CAR22和上文所述CAR19的双特异性嵌合抗原受体称为PCTL124)，M971-IgG4mt10+N297A hinge-CD8 TM-4-1BBCSD-CD3z(将同时包含该CAR22和上文所述CAR19的双特异性嵌合抗原受体称为PCTL148)，M971-IgG4mt10+N297A hinge-CD28 TM-CD28CSD-CD3z(将同时包含该CAR22和上文所述CAR19的双特异性嵌合抗原受体称为PCTL149)，M971-IgG4mt10+N297A hinge-ICOS TM-ICOSCSD-CD3z(将同时包含该CAR22和上文所述CAR19的双特异性嵌合抗原受体称为PCTL150)。所述7种不同的CAR22的组成如表2所示：
表2：7种不同CAR22的组成
Figure PCTCN2020108850-appb-000002
具体来说，表2中所述7种不同CAR22中的scFv的氨基酸序列如SEQ ID NO：7所示，编码该氨基酸序列的核苷酸序列如SEQ ID NO：8所示。所述CAR22的铰链区具有四种不同的选择，即CD8hinge、CD28hinge、ICOShinge或IgG4mt10+N297Ahinge，它们的氨基酸序列分别如SEQ ID NO：9、SEQ ID NO：17、SEQ ID NO：23和SEQ ID NO：31所示；编码上述氨基酸序列的核苷酸序列分别如SEQ ID NO：10、SEQ ID NO：18、SEQ ID NO：24和SEQ ID NO：32所示。其中，所述IgG4mt10+N297A所示的铰链区是在天然的IgG4的基础上突变了8个氨基酸位点。具体来说，是将天然的IgG4中第228位氨基酸由S突变成P、将第233位氨基酸由E突变成P、将第234位氨基酸由F突变成V、将第235位氨基酸由L突变成A、将第265位氨基酸由D突变成A、将第297位氨基酸由N突变成A、将第309位氨基酸由L突变成V、将第409位氨基酸由R突变成K，去除了FcγR(Fc gamma receptor)的结合能力，避免了抗体依赖性细胞介导的细胞毒性(antibody-dependent cell-mediated cytotoxicit，ADCC)和补体依赖的细胞毒性(complement-dependent cytotoxicity，CDC)，从而有效增强CAR-T的体内活性。
所述CAR22的跨膜区具有三种不同的选择，即CD8TM、CD28TM或 ICOSTM，它们的氨基酸序列分别如SEQ ID NO：11、SEQ ID NO：19和SEQ ID NO：25所示；编码上述氨基酸序列的核苷酸序列分别如SEQ ID NO：12、SEQ ID NO：20和SEQ ID NO：26所示。
所述CAR22的共刺激因子结构域具有四种不同选择，即4-1BBCSD、CD28CSD、ICOSCSD和OX40CSD，它们的氨基酸序列分别如SEQ ID NO：13、SEQ ID NO：21、SEQ ID NO：27和SEQ ID NO：29所示；编码上述氨基酸序列的核苷酸序列分别如SEQ ID NO：14、SEQ ID NO：22、SEQ ID NO：28和SEQ ID NO：30所示。即7种双特异性嵌合抗原受体CAR19-CAR22中的CAR22部分编码相同的抗原结合结构域(即具有相同的scFv)和CD3z信号域，其中所述CD3z信号域的氨基酸序列如SEQ ID NO：15所示(编码该氨基酸序列的核苷酸序列如SEQ ID NO：16所示)，所述7种构建体之间的区别仅在于CAR22部分不同的铰链区、跨膜区和共刺激因子结构域组合。
实施例2：由不同结构的双特异性嵌合抗原受体制备的CAR19-CAR20-T细胞对靶细胞杀伤作用的比较
利用实施例1中所述的并联形式的双特异性嵌合抗原受体CAR19-CAR20制备双靶点CAR-T细胞，然后将所述双靶点CAR-T细胞与CD19 +K562-luc-GFP、CD20 +K562-luc-GFP两种不同的靶细胞按不同效应细胞(E)：靶细胞(T)比例，即分别以E/T＝1∶1、2.5∶1、5∶1、10∶1、20∶1的比例共孵育18～24小时，用未进行基因修饰的T细胞(即未进行慢病毒感染的T细胞，下文称为NC-T细胞)作为本底对照，构建的靶细胞株上带有荧光素酶luciferase，通过化学发光的原理检测效应细胞对靶细胞的杀伤效果。具体操作如下：
(1)外周血PBMC的分离、T细胞分离活化、慢病毒转导、体外培养：
选择HBV、HCV和HIV检测阴性的健康供者，肘正中静脉抽血100ml，Ficoll密度梯度离心分离PBMC白膜层，根据全血流式检测CD3 +T细胞百分比，计算CD3 +T细胞数，按DynaBeads CD3/CD28与CD3 +T细胞比例3∶1，吸取使 用量磁珠，与白膜层细胞孵育30min，分离CD3 +T细胞，CD3 +T细胞经Dynabeads CD3/CD28(Lifetechnologies，货号：40203D)活化24小时后流式检测CD25 +CD69 +T细胞比例(CD25 +CD69 +T细胞比例：71％)。CD3 +T活化后，进行慢病毒转导。用Novonectin包被24孔板37℃孵育2小时，将上述操作后得到的细胞悬液分别与制备得到的各种慢病毒(即分别包含PCTL126，PCTL137，PCTL138，PCTL139，PCTL151，PCTL152，PCTL153的慢病毒)(MOI＝8)、
Figure PCTCN2020108850-appb-000003
F108(Sigma，货号：07579-250G-F，10μg/ml)、Tscm(2U/ml)配置成转导体系置于包被的24孔板中，细胞密度调整至1.0E+06/ml，500g离心30min，离心后37℃ CO 2培养箱静置培养48h。转染后以含5％FBS X-vivo15培养液(LONZA，货号：04-418Q)培养，隔日补充Tscm(终浓度2U/ml)，计数细胞，调整细胞密度至0.5E+06/ml，培养至第8-10天收获细胞。
(2)效应细胞双靶点CAR-T细胞的制备：①取出扩增5-7天的NC-T细胞(未进行慢病毒转染的T细胞)、各组CAR-T细胞，于显微镜下观察细胞生长状态是否正常；②将NC-T细胞和各组CAR-T细胞收集于15mL或50mL的离心管中，计数细胞总数(Cellometer k2细胞计数仪)；③用无菌PBS(Hyclone，货号：SH30256.01)洗涤收集的细胞1-2次，1500rpm，25℃，离心5分钟；④用T细胞培养液X-VIVO15(LONZA，货号：04-418Q)(不含自体血清以及IL-2)重悬洗涤后的细胞沉淀，并将细胞密度调整至5.0E+07cells/mL。
(3)靶细胞准备：①取出靶细胞CD19 +K562-luc-GFP和CD20 +K562-luc-GFP(Tsukahara et al.Biochem Biophys Res Commun.2013；438(1)：84-89)，于显微镜下观察细胞状态是否正常；②将上述两种靶细胞分别收集于15mL或50mL离心管中，并计算细胞总数；③用无菌PBS洗涤收集的细胞1-2次，1500rpm，25℃，离心5分钟；④用RPMI1640(gibco，货号：11875-093)(不含FBS)重悬洗涤后的细胞沉淀，并将细胞密度调整至5.0E+06cells/mL。
(4)体外杀伤：①杀伤体系的配制：于1.5mL的离心管中将上述调整好 密度的效应细胞NC-T细胞和各组CAR-T细胞分别与靶细胞CD19 +K562-luc-GFP，CD20 +K562-luc-GFP按不同效靶比，具体分别以1∶1、2.5∶1、5∶1、10∶1、20∶1的比例将效应细胞CAR-T与靶细胞进行混合，用T细胞培养液X-VIVO15(LONZA，货号：04-418Q)(不含自体血清以及IL-2)将总体积补足至200μL；②将上述配制的200μL杀伤体系分别移入96孔V型板中共孵育24小时；③24小时后，轻轻吹打混匀96孔板V型板中各孔细胞，并分别转移100μL细胞悬液入白壁底不透96孔板中，加入100μL ONE-Glo TM Luciferase Assay Substrate，室温避光孵育10分钟后上Luminoskan Ascent化学发光分析仪检测化学发光(Luminescence)。
杀伤效率的计算：杀伤效率＝(NC-T细胞对应的数值-特定的CAR19-CAR20-T细胞在对应效靶比的数值)/NC-T细胞对应的数值
试验结果：根据表3所示结果可以看出，在效靶比为10∶1的情况下，包含七种双特异性嵌合抗原受体(即PCTL126、PCTL137、PCTL138、PCTL139、PCTL151、PCTL152和PCTL153)的CAR19-CAR20-T细胞对于靶细胞CD19+K562-luc-GFP的杀伤效率均大于90％，其中包含PCTL152和PCTL153的CAR19-CAR20-T细胞对于靶细胞CD19 +K562-luc-GFP的杀伤效率接近100％(具体参见表3和图3)。另外，包含PCTL152和PCTL153的CAR19-CAR20-T细胞在效靶比较低的情况下，对于所述靶细胞依然具有较高的杀伤效率。
表3：双特异性嵌合抗原受体CAR19-CAR20-T细胞对CD19 +K562-luc-GFP靶细胞的杀伤效率
1∶1 2.5∶1 5∶1 10∶1 20∶1
PCTL137 -273.22％ 40.27％ 94.13％ 96.58％ 98.60％
PCTL138 -178.91％ 75.38％ 97.68％ 98.31％ 99.41％
PCTL139 -153.56％ 41.80％ 96.77％ 97.60％ 98.72％
PCTL126 -26.72％ 87.59％ 97.59％ 98.95％ 99.65％
PCTL151 -28.95％ 83.53％ 93.95％ 96.44％ 99.07％
PCTL152 40.19％ 95.92％ 99.12％ 99.58％ 99.85％
PCTL153 43.95％ 96.83％ 97.71％ 99.40％ 99.72％
根据表4所示结果可以看出，在效靶比为20∶1的情况下，包含七种双特异性嵌合抗原受体(即PCTL126、PCTL137、PCTL138、PCTL139、PCTL151、PCTL152和PCTL153)的CAR19-CAR20-T细胞对于靶细胞CD20 +K562-luc-GFP的杀伤效率均大于80％，其中包含PCTL152和PCTL153的CAR19-CAR20-T细胞对于靶细胞CD20 +K562-luc-GFP的杀伤效率接近100％(具体参见表4和图4)。
表4：双特异性嵌合抗原受体CAR19-CAR20-T细胞对CD20 +K562-luc-GFP靶细胞的杀伤效率
1∶1 2.5∶1 5∶1 10∶1 20∶1
PCTL137 -269.38％ -201.37％ -2.41％ 72.28％ 81.53％
PCTL138 -204.88％ -49.82％ 11.09％ 71.10％ 80.52％
PCTL139 -279.06％ -160.74％ 13.09％ 81.29％ 86.59％
PCTL126 -138.73％ -33.19％ 67.29％ 71.61％ 90.32％
PCTL151 -136.73％ -12.02％ 45.50％ 70.90％ 88.66％
PCTL152 -182.88％ 15.71％ 75.81％ 92.37％ 98.25％
PCTL153 -161.64％ -32.69％ 81.21％ 89.58％ 96.97％
实施例3：由不同结构的双特异性嵌合抗原受体制备的CAR19-CAR20-T细胞显示的T细胞表型
利用实施例1中所述并联形式的双特异性嵌合抗原受体CAR19-CAR20制备双靶点CAR-T细胞，并在慢病毒转染后的第7-10天，用常规的T细胞分化抗原抗体通过流式细胞仪分析细胞的分化群体。
试验方法：选择实施例1制备的7种双特异性嵌合抗原受体CAR19-CAR20为试验材料，按照实施例2中所述的效应细胞的制备方法制备相应的双靶点CAR-T细胞。针对制备得到的7种双靶点CAR-T细胞，分别取1×10 6个CAR-T细胞，用PBS洗涤所述CAT-T细胞后孵育CD62L-PE-Cy5抗体(BD，货号：555545)和CD45RO-FITC抗体(BD，货号：555492)，于4℃冰箱孵育30min。抗体孵育结束后用PBS(Hyclone，货号：SH30256.01)洗涤2-3遍，用500μl PBS重悬后置于流式管中准备上机检测。
试验结果：如图5所示，结果显示所分析的双靶点CAR-T细胞均能保持较高比例的干细胞中央记忆型T细胞(TSCM)。有研究报道，干细胞中央记忆型T细胞群体的比例与CAR-T细胞在生物体内的杀瘤活性、扩增能力和持久免疫记忆能力紧密相关。由此提示，本发明提供的双靶点CAR-T细胞能够在生物内具有较好的杀瘤活性、扩增能力和持久的免疫记忆能力。
实施例4：包含PCTL152和PCTL153的CAR19-CAR20-T细胞表达CAR19和CAR20的检测
试验方法：选择实施例2中具有较高杀伤效率的包含PCTL152和PCTL153的CAR19-CAR20-T细胞作为试验材料，针对上述两种CAR-T细胞，分别取1×10 6个CAR-T细胞，用4％BSA洗涤(2500rpm，5min)3次后孵育抗体：(1)Alexa Fluor 647 AffiniPure Goat Anti-Human IgG(1∶100～1∶800)，4℃冰箱孵育30min；抗体孵育结束后用4％BSA洗涤(2500rpm，5min)，3次后孵育抗体：(2)PE标记的CAR19(iFMC63)idiotype(Qin et al.Mol Ther  Oncolytics.2018；11：127～137)(1μg/ml)，4℃冰箱孵育30min。抗体孵育结束后用4％BSA洗涤2-3遍(2500rpm，5min)，用500μl PBS重悬后置于流式管中准备上机检测。
试验结果：如图6所示，CAR19和CAR20蛋白在T细胞表面上均可同步检测到。所述结果表明，构建的包含PCTL152和PCTL153的CAR19-CAR20-T细胞能够较好的表达CAR19 +CAR20 +双阳性群体。
实施例5：包含PCTL152和PCTL153的CAR19-CAR20-T细胞小鼠体内抑瘤活性验证
试验方法：选择实施例2中具有较高杀伤效率的包含PCTL152和PCTL153的CAR19-CAR20-T细胞作为试验组，选择PBS作为对照组。将PBS重悬的Raji-Luc细胞(百奥赛图，货号：B-HCL-010)以5×10 5个/0.2mL的浓度，0.2mL/只的体积通过尾静脉注射接种到
Figure PCTCN2020108850-appb-000004
(B-NSG)小鼠。接种当天用小动物成像仪观察肿瘤接种是否成功，接种第3天用小动物成像仪测量肿瘤生长情况，当平均成像信号达1×10 6[(P/S)/(cm2/sr)]左右时，挑选肿瘤成像信号适中的8只小鼠入组，随机分配到3个组中，G1组2只，G3、G4组各3只，将荧光信号过强/过弱的小鼠淘汰。分组当天开始给药，给药后，第4天开始检测肿瘤生长情况(小动物成像仪检测、记录)及测量动物体重。之后每周1次小鼠成像仪检测(第4天、第11天、第18天和第25天)及每周2次测量动物体重。具体给药方案见图7，各组的给药种类、小鼠数量和给药剂量具体参见表5。
表5：G1、G3和G4组的给药种类、小鼠数量和给药剂量
组别 给药种类 小鼠数量 给药剂量
G1 PBS 2 PBS 200μl//只
G3 PCTL152 3 Total T 0.1E+07/200μl/只
G4 PCTL153 3 Total T 0.1E+07/200μl/只
试验结果：截止至Day28，G1、G3组小鼠全部死亡，G4组小鼠生存率为67.7％。由此可见，与包含PCTL152的CAR19-CAR20-T细胞相比，包含PCTL153的CAR19-CAR20-T细胞能明显提高荷瘤小鼠的生存率(参见图8)。
实施例6：包含PCTL153的CAR19-CAR20-T细胞、单CAR19-T细胞、单CAR20-T细胞以及串联形式的CAR20-19-T细胞小鼠体内抑瘤活性验证
试验方法：将PBS重悬的Raji-Luc细胞以5×10 5个/0.2mL的浓度，0.2mL/只的体积通过尾静脉注射接种到
Figure PCTCN2020108850-appb-000005
(B-NSG)小鼠，共转接54只小鼠。接种当天用小动物成像仪观察肿瘤接种是否成功，接种成功后用小动物成像仪测量肿瘤生长情况，当平均成像信号达1×10 6[(P/S)/(cm2/sr)]左右时，挑选肿瘤成像信号适中的30只小鼠入组，随机分配到5个组中，每组6只，荷瘤小鼠活体成像信号过强/过弱淘汰。分组当天开始给药，给药后，对实验动物体重及肿瘤生长情况(小动物成像仪检测、记录)继续观察。在分组后第4天、第7天和第11天测量肿瘤生长情况，之后每周测量1次肿瘤生长情况(小动物成像仪检测、记录)。每周测量2次动物体重，进行临床观察，并记录测量值。具体给药方案见图9，各组的给药种类、小鼠数量和给药剂量具体参见表6。
试验结果：与传统的单CAR19-T细胞(G3组，其中包含的CAR19的结构和序列与本发明实施例1中所述CAR19的结构和序列相同：FMC63-CD8hinge-CD8 TM-4-1BB-CD3z)、单CAR20-T细胞(G4组，其中包含的CAR20的结构和序列与本发明实施例1中所述PCTL153中的CAR20的结构和序列一 致：Leu16-IgG4mt10+N297A hinge-ICOS TM-ICOSCSD-CD3z)以及串联形式的CAR20-19-T细胞(G7组，CD20 scFv-(EAAAK)3-CD19 scFv-IgG4 hinge-CD28 TM-4-1BB-CD3z，具体参见Zah et al.Cancer Immunol Res.2016；4(6)：498-508)相比，包含PCTL153的并联双CAR19-CAR20-T细胞(G5组)能明显提高荷瘤小鼠的生存率(参见图10)。
表6：G1、G3、G4、G5和G7组的给药种类、小鼠数量和给药剂量
Figure PCTCN2020108850-appb-000006
实施例7：由不同结构的双特异性嵌合抗原受体制备的CAR19-CAR22-T细胞对靶细胞杀伤作用的比较
利用实施例1中所述的并联形式的双特异性嵌合抗原受体CAR19-CAR22制备双靶点CAR-T细胞，然后将所述双靶点CAR-T细胞与CD19 +K562-luc-GFP、CD22 +K562-luc-GFP两种不同的靶细胞按不同效应细胞(E)：靶细胞(T)比例，即分别以E/T＝5∶1、10∶1或20∶1的比例共孵育18～24小时，用未进行基因修饰的T细胞(即未进行慢病毒感染的T细胞，下文称为NC-T细胞)作为本底对照，构建的靶细胞株上带有荧光素酶luciferase，通过化学发光的原理检测效应细胞对靶细胞的杀伤效果。具体操作如下：
(1)外周血PBMC的分离、T细胞分离活化、慢病毒转导、体外培养：
选择HBV、HCV和HIV检测阴性的健康供者，肘正中静脉抽血100ml，Ficoll密度梯度离心分离PBMC白膜层，根据全血流式检测CD3 +T细胞百分比， 计算CD3 +T细胞数，按DynaBeads CD3/CD28与CD3 +T细胞比例3∶1，吸取使用量磁珠，与白膜层细胞孵育30min，分离CD3 +T细胞，CD3 +T细胞经Dynabeads CD3/CD28(Lifetechnologies，货号：40203D)活化24小时后流式检测CD25 +CD69 +T细胞比例(CD25 +CD69 +T细胞比例：71％)。CD3 +T活化后，进行慢病毒转导。用Novonectin包被24孔板37℃孵育2小时，将上述操作后得到的细胞悬液分别与制备得到的各种慢病毒(即分别包含PCTL81，PCTL103，PCTL105，PCTL124，PCTL148，PCTL149，PCTL150的慢病毒)(MOI＝8)、
Figure PCTCN2020108850-appb-000007
F108(Sigma，货号：07579-250G-F，10μg/ml)、Tscm(2U/ml)配置成转导体系置于包被的24孔板中，细胞密度调整至1.0E+06/ml，500g离心30min，离心后37℃ CO 2培养箱静置培养48h。转染后以含5％FBS X-vivo15培养液(LONZA，货号：04-418Q)培养，隔日补充Tscm(终浓度2U/ml)，计数细胞，调整细胞密度至0.5E+06/ml，培养至第8-10天收获细胞。
(2)效应细胞双靶点CAR-T细胞的制备：①取出扩增5-7天的NC-T细胞(未进行慢病毒转染的T细胞)、各组CAR-T细胞，于显微镜下观察细胞生长状态是否正常；②将NC-T细胞和各组CAR-T细胞收集于15mL或50mL的离心管中，计数细胞总数(Cellometer k2细胞计数仪)；③用无菌PBS(Hyclone，货号：SH30256.01)洗涤收集的细胞1-2次，1500rpm，25℃，离心5分钟；④用T细胞培养液X-VIVO15(LONZA，货号：04-418Q)(不含自体血清以及IL-2)重悬洗涤后的细胞沉淀，并将细胞密度调整至5.0E+07cells/mL。
(3)靶细胞准备：①取出靶细胞CD19 +K562-luc-GFP和CD22 +K562-luc-GFP(Tsukahara et al.Biochem Biophys Res Commun.2013；438(1)：84～89)，于显微镜下观察细胞状态是否正常；②将上述两种靶细胞分别收集于15mL或50mL离心管中，并计算细胞总数；③用无菌PBS洗涤收集的细胞1-2次，1500rpm，25℃，离心5分钟；④用RPMI1640(gibco，货号：11875-093)(不含FBS)重悬洗涤后的细胞沉淀，并将细胞密度调整至5.0E+06cells/mL。
(4)体外杀伤：①杀伤体系的配制：于1.5mL的离心管中将上述调整好密度的效应细胞NC-T细胞和各组CAR-T细胞分别与靶细胞CD19 +K562-luc-GFP，CD22 +K562-luc-GFP按不同效靶比，具体分别以5∶1、10∶1、20∶1的比例将效应细胞CAR-T与靶细胞进行混合，用T细胞培养液X-VIVO15(LONZA，货号：04-418Q)(不含自体血清以及IL-2)将总体积补足至200μL；②将上述配制的200μL杀伤体系分别移入96孔V型板中共孵育24小时；③24小时后，轻轻吹打混匀96孔板V型板中各孔细胞，并分别转移100μL细胞悬液入白壁底不透96孔板中，加入100μL ONE-Glo TM Luciferase Assay Substrate，室温避光孵育10分钟后上Luminoskan Ascent化学发光分析仪检测化学发光(Luminescence)。
杀伤效率的计算：杀伤效率＝(NC-T细胞对应的数值-特定的CAR19-CAR20-T细胞在对应效靶比的数值)/NC-T细胞对应的数值
试验结果：根据图11所示结果可以看出，在效靶比为10∶1的情况下，包含七种双特异性嵌合抗原受体(即PCTL81，PCTL103，PCTL105，PCTL124，PCTL148，PCTL149，PCTL150)的CAR19-CAR22-T细胞对于靶细胞CD19 +K562-luc-GFP的杀伤效率均大于90％(具体参见图11)。根据图12所示结果可以看出，七种双特异性嵌合抗原受体(即PCTL81，PCTL103，PCTL105，PCTL124，PCTL148，PCTL149，PCTL150)的CAR19-CAR22-T细胞对于靶细胞CD22 +K562-luc-GFP均具有杀伤效果，且具有明显的效靶比的依赖性(具体参见图12)。

Claims (19)

  1. 一种嵌合抗原受体，其特征在于，所述嵌合抗原受体由抗原结合区、胞外铰链区、跨膜区、共刺激域和CD3z信号结构域组成。
  2. 根据权利要求1所述的嵌合抗原受体，其特征在于：
    所述胞外铰链区选自由CD8胞外铰链区(CD8hinge)、CD28胞外铰链区(CD28hinge)、ICOS胞外铰链区(ICOShinge)或IgG4mt10+N297A胞外铰链区(IgG4mt10+N297Ahinge)组成的组中的任一种；
    所述跨膜区选自由CD8跨膜区(CD8TM)、CD28跨膜区(CD28TM)或ICOS跨膜区(ICOSTM)组成的组中的任一种；
    所述共刺激域选自由4-1BB共刺激域(4-1BBCSD)、CD28共刺激域(CD28CSD)、ICOS共刺激域(ICOSCSD)或OX40共刺激域(OX40CSD)组成的组中的任一种。
  3. 根据权利要求2所述的嵌合抗原受体，其特征在于，其中包含胞外铰链区、跨膜区和共刺激域的结构分别为：CD8hinge-CD8TM-4-1BBCSD、CD28hinge-CD28TM-CD28CSD、ICOShinge-ICOSTM-ICOSCSD、CD28hinge-CD28TM-OX40CSD、IgG4mt10+N297Ahinge-CD8TM-4-1BBCSD、IgG4mt10+N297Ahinge-CD28 TM-CD28CSD或IgG4mt10+N297Ahinge-ICOSTM-ICOSCSD。
  4. 根据权利要求3所述的嵌合抗原受体，其特征在于，
    所述IgG4mt10+N297Ahinge-CD8TM-4-1BBCSD的氨基酸序列为SEQ ID NO：36；
    所述IgG4mt10+N297Ahinge-CD28TM-CD28CSD的氨基酸序列为SEQ ID NO：37；
    所述IgG4mt10+N297Ahinge-ICOSTM-ICOSCSD的氨基酸序列为SEQ ID NO：38。
  5. 根据权利要求2所述的嵌合抗原受体，其特征在于，所述抗原结合区为单链抗体(scFv)或单域抗体(sdAb)。
  6. 根据权利要求5所述的嵌合抗原受体，所述抗原结合区识别CD20或者 识别CD22。
  7. 根据权利要求6所述的嵌合抗原受体，所述抗原结合区为Leu16，其中所述Leu16为识别CD20的人源化scFv，其氨基酸序列如SEQ ID NO：3所示。
  8. 根据权利要求6所述的嵌合抗原受体，所述抗原结合区为M971，其中所述M971为识别CD22的scFv，其氨基酸序列如SEQ ID NO：7所示。
  9. 一种嵌合抗原受体T细胞(CAR-T细胞)，其特征在于，所述CAR-T细胞表达如权利要求1～8中任一项所述的嵌合抗原受体。
  10. 根据权利要求9所述的嵌合抗原受体T细胞(CAR-T细胞)，其特征在于，所述CAR-T细胞表达两个含有不同抗原结合区的嵌合抗原受体。
  11. 根据权利要求10所述的CAR-T细胞，其中所述的两个独立的嵌合抗原受体分别为CAR19和CAR20；其中所述CAR19识别CD19，所述CAR20识别CD20。
  12. 根据权利要求10所述的CAR-T细胞，其中所述的两个独立的嵌合抗原受体分别为CAR19和CAR22；其中所述CAR19识别CD19，所述CAR22识别CD22。
  13. 一种核酸分子，其特征在于，所述核酸分子编码如权利要求1～8中任一项所述的嵌合抗原受体。
  14. 一种载体，其特征在于，所述载体含有如权利要求13所述的核酸分子。
  15. 一种宿主细胞，其特征在于，所述宿主细胞含有如权利要求14中所述的载体或者在所述宿主细胞的染色体中整合有如权利要求13所述的核酸分子。
  16. 一种药物组合物，其特征在于，所述药物组合物中含有药学上可接受的载体以及如权利要求1～8任一项所述的嵌合抗原受体。
  17. 如权利要求1～8任一项中所述的嵌合抗原受体、如权利要求13所述的核酸分子、如权利要求14所述的载体或如权利要求15所述的宿主细胞在制备治疗抗肿瘤的药物或抗肿瘤的制剂中的应用。
  18. 根据权利要求17所述的应用，所述肿瘤为血液肿瘤，优选的所述血液肿瘤为B细胞恶性肿瘤、急性淋巴性白血病、慢性淋巴性白血病、淋巴瘤、肥大细胞瘤或滤泡性淋巴瘤。
  19. 一种制备CAR-T细胞的方法，其特征在于，所述CAR-T细胞表达如权利要求1～8中任一项的嵌合抗原受体，所述方法包括以下步骤：
    将权利要求13所述的核酸分子或者权利要求14所述的载体导入T细胞内，从而获得所述CAR-T细胞。
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