WO2020248486A1 - 以Tcm为主要效应成分的CAR-T制备方法及其应用 - Google Patents

以Tcm为主要效应成分的CAR-T制备方法及其应用 Download PDF

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WO2020248486A1
WO2020248486A1 PCT/CN2019/117100 CN2019117100W WO2020248486A1 WO 2020248486 A1 WO2020248486 A1 WO 2020248486A1 CN 2019117100 W CN2019117100 W CN 2019117100W WO 2020248486 A1 WO2020248486 A1 WO 2020248486A1
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car
cell
cells
tcm
proportion
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陈志国
赵宇
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Xuanwu Hospital
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Xuanwu Hospital
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Definitions

  • the invention relates to the technical field of gene therapy and cell therapy, in particular to a preparation method and application of CAR-T with Tcm as the main effect component.
  • Central memory T cells are a group of T cell subsets with CD45RO+/CD197+ as surface markers. This subgroup of T cells is differentiated from naive T cells after antigen or non-antigen stimulation, and mainly participates in specific memory immune responses in the body. The proportion of Tcm subpopulations in the T cell subpopulations isolated from peripheral blood is low. In the in vitro culture process, Tcm will proliferate rapidly in the initial stage of culture, and then will decrease with the extension of culture time.
  • Adoptive immune cell therapy is a new type of anti-tumor therapy in the current biomedical field.
  • the method mainly involves extracting the patient's autologous cells, activating, modifying, culturing and amplifying them in vitro, and then returning them to the patient's body to achieve the purpose of treating tumors.
  • CAR-T or chimeric antigen receptor modified T cells, is a targeted adoptive immune cell therapy method.
  • This technology uses genetic modification methods to modify the T cell genome to express CAR receptors that can recognize tumor-specific antigens, thereby breaking the MHC restriction of the original T cell anti-tumor, allowing T cells to directly kill tumors.
  • CD19-specific CAR-T cells have made breakthrough progress in the treatment of acute B lymphocytic leukemia and B cell lymphoma, and have achieved clinical applications.
  • the current CAR-T treatment technology still has many clinical bottlenecks.
  • the currently known Tcm preparation method is to isolate primary naive T cells from peripheral blood, and first activate them with the CD3 monoclonal antibody OKT-3 and IL-2. After 24 hours of activation, a medium containing IL-7, IL-15 and IL-21 was added for culture. The cells were harvested after 6-9 days of cultivation. At this time, the proportion of Tcm in the final product is about 50%, which is difficult to meet the requirements of clinical reinfusion.
  • Tcm cell subpopulations Due to the proliferation characteristics of Tcm cell subpopulations, in vitro culture, the proportion of Tcm cell subpopulations will rapidly decrease as the culture time increases. Therefore, the short-term culture process cannot guarantee to obtain a sufficient number of target cells, and increasing the number of initial seed cells in culture will increase the production cost sharply.
  • the current preparation process of CAR-T cannot guarantee high-purity Tcm cells in the infusion product under the premise that the subject must obtain a sufficient dose of cells for the infusion. Therefore, how to use cost-effective methods to obtain cell products with Tcm as the main effect component is a technical problem in the current industry.
  • the present invention provides a CAR-T preparation method with Tcm as the main effect component and its application.
  • the present invention provides a method for increasing the proportion of central memory T cell (Tcm) subsets in CAR-T cell products, wherein an artificial epitope is added to the CAR, and the artificial epitope activates the CAR -T; wherein the artificial epitope is not present in other domains or segments of the CAR.
  • Tcm central memory T cell
  • the artificial epitope has no or substantially no negative impact on the function of other domains or segments of the CAR; when the artificial epitope is bound, the CAR-T can be activated.
  • the artificial epitope is located in the extracellular domain of the CAR, between the extracellular domain and the hinge region, or between the hinge region and the transmembrane structure.
  • the length of the artificial epitope is 7-15 aa, preferably 8-12 aa.
  • amino acid sequence of the artificial epitope is SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
  • a molecule that specifically recognizes the artificial epitope is used to bind the artificial epitope to activate CAR-T, wherein the molecule that specifically recognizes the artificial epitope is preferably an antibody molecule.
  • the step of activating CAR-T through the artificial epitope is after the step of introducing CAR into T cells.
  • the step of activating CAR-T through the artificial epitope includes culturing CAR-T in a medium containing IL-7, IL-2 and IL-15.
  • the concentrations of IL-7, IL-2 and IL-15 are 10-200ng/mL, 20-1000IU/mL and 1-50ng/mL, respectively.
  • the method of the present invention is an in vitro method.
  • the CAR-T prepared by the method has a good tumor treatment effect
  • the direct purpose of the method of the present invention is to increase central memory T cells in isolated CAR-T cells ( Tcm) the proportion of subpopulations, the method itself does not involve any in vivo procedures.
  • the present invention provides a method for preparing CAR-T cells, including:
  • CAR chimeric antigen receptor
  • the CAR has an artificial epitope
  • the specific activation includes adding an anti-artificial epitope antibody during the culture process, and the anti-artificial epitope antibody can specifically recognize the artificial epitope.
  • the protein structure of CAR in turn includes the following domains: an extracellular domain, an optional hinge region, a transmembrane domain, and an intracellular domain.
  • the extracellular domain includes an optional signal peptide and an antigen-binding domain.
  • the antigen-binding domain includes an antibody or antibody fragment having antigen-binding activity, and the antibody or antibody fragment includes at least a heavy chain variable region and a light chain variable region.
  • the artificial epitope is located in the extracellular domain of the CAR, between the extracellular domain and the hinge region, or between the hinge region and the transmembrane domain. In a specific embodiment, the artificial epitope is located in the extracellular domain of the CAR. In a preferred embodiment, the artificial epitope is located between the signal peptide and the antigen binding domain. In another preferred embodiment, the artificial epitope is located in the antigen binding domain. Further, the artificial epitope is located between the variable region of the heavy chain and the variable region of the light chain.
  • the artificial epitope is one of the following (a1)-(a3):
  • the antibody against the artificial epitope is an anti-E-tag antibody
  • the anti-E-tag antibody can specifically recognize the E-tag.
  • the amino acid sequence of the anti-E-tag antibody is SEQ ID NO: 4.
  • the antibody against the artificial epitope is an anti-Strep-tag antibody
  • the anti-Strep-tag antibody can specifically recognize the Strep-tag.
  • the anti-Strep-tag antibody is purchased from Bio-Rad (Cat. No.: MCA2488).
  • the antibody against the artificial epitope is an anti-Strep-tag II antibody
  • the anti-Strep-tag II antibody can specifically recognize the Strep-tag II.
  • the anti-Strep-tag II antibody is purchased from Genscript (Cat. No.: A01732).
  • the method for preparing the T cells in step (1) includes: separating T cells from peripheral blood, and performing T cell activation on the separated T cells.
  • the T cell activation includes culturing the isolated T cells in a first medium, and the first medium includes OKT-3 and IL-2.
  • the concentration of OKT-3 is 5-200ng/mL, 10-100ng/mL, more preferably 50-80ng/mL, such as 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL
  • the concentration of IL-2 is 20-1000IU/mL, preferably 50-500IU/mL, more preferably 200-500IU/mL, such as 200IU/mL, 300IU/mL, 400IU/mL, 500IU/mL.
  • the first medium can be prepared by a medium commonly used in the art.
  • the first medium can be prepared by the following medium preparations, including but not limited to: X-VIVO15 medium (Lonza company), KBM581 serum-free medium (Corning company), containing 10% FBS RPMI1640 medium (Gbico company) or RPMI1640 medium (Gbico company) containing 10% human AB serum.
  • the culture time for T cell activation is 4-72h, preferably 24-36h, such as 24h, 28h, 32h, 36h, and in a preferred embodiment, 24h.
  • the culture medium used for the culture is a second culture medium, and the second culture medium includes IL-7, IL-2 and IL-15.
  • the concentration of IL-7 is 10-200ng/mL, preferably 20-100ng/mL, more preferably 80-100ng/mL, such as 80ng/mL, 90ng/mL, 100ng/mL mL
  • the concentration of IL-2 is 20-1000IU/mL, preferably 50-500IU/mL, more preferably 200-500IU/mL, such as 200IU/mL, 300IU/mL, 400IU/mL, 500IU/mL
  • IL- The concentration of 15 is 1-50ng/mL, preferably 5-30ng/mL, more preferably 10-20ng/mL, such as 10ng/mL, 12ng/mL, 14ng/mL, 16ng/mL, 18ng/mL, 20ng/mL mL.
  • the second medium can be prepared by a medium commonly used in the art.
  • the second medium can be prepared by the following medium, including but not limited to: X-VIVO15 medium (Lonza company), KBM581 serum-free medium (Corning company), containing 10% FBS RPMI1640 medium (Gbico company) or RPMI1640 medium (Gbico company) containing 10% human AB serum.
  • the culture time is 9-14 days, preferably 9-12 days, such as 9 days, 10 days, 11 days, 12 days; in a preferred embodiment, it is 12 days.
  • the concentration of the anti-artificial epitope antibody is 1-50 ⁇ g/mL, preferably 5-30 ⁇ g/mL, more preferably 10-25 ⁇ g/mL, such as 10 ⁇ g/mL, 15 ⁇ g/mL mL, 20 ⁇ g/mL, 25 ⁇ g/mL; in a preferred embodiment, 20 ⁇ g/mL.
  • step (2) the method of adding the anti-artificial epitope antibody is selected from the following group: directly adding the anti-artificial epitope antibody to the culture medium, adding the anti-artificial epitope antibody The antibody is coated on the culture vessel, or a combination thereof.
  • the method for introducing the CAR into T cells is to introduce the CAR into T cells through viral infection or into T cells through non-viral infection; wherein, the virus in the viral infection method is selected from the following viruses : Adenovirus, retrovirus, lentivirus, herpes virus and adeno-associated virus; the non-viral infection method is liposome method, microinjection method, calcium phosphate co-precipitation method, electrotransformation method or DEAE-dextran Transfection method.
  • the step (1) is to introduce CAR into T cells through viral infection.
  • the step (1) specifically includes the following steps:
  • S1 construct a viral expression vector of CAR
  • the CAR virus expression vector prepared in step S1 is introduced into corresponding packaging cells for virus packaging to obtain a virus encoding CAR;
  • step S4 Infect the T cells prepared in step S3 with the CAR-encoding virus prepared in step S2.
  • the virus expression vector is an adenovirus expression vector, a retrovirus expression vector, a lentivirus expression vector, a herpes virus expression vector or an adeno-associated virus expression vector. In a preferred embodiment, it is a lentiviral expression vector.
  • the present invention provides a CAR-T cell with a high proportion of central memory T cell (Tcm) subpopulation, which is prepared by the method of the present invention
  • the present invention provides the application of the CAR-T cells in the preparation of drugs or preparations for preventing and/or treating cancer or tumors.
  • the present invention also provides the application of the CAR-T cell in preventing and/or treating cancer or tumor.
  • the tumor is selected from the group consisting of hematological tumors, solid tumors, or a combination thereof.
  • the hematological tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma ( DLBCL), non-Hodgkin lymphoma (NHL), or a combination thereof.
  • AML acute myeloid leukemia
  • MM multiple myeloma
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphocytic leukemia
  • DLBCL diffuse large B-cell lymphoma
  • NHL non-Hodgkin lymphoma
  • the solid tumor is selected from the group consisting of gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, cervical cancer, ovarian cancer , Lymphoma, nasopharyngeal carcinoma, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer, mesothelioma, pancreatic cancer, multiple myeloma, or a combination thereof.
  • gastric cancer gastric cancer peritoneal metastasis
  • liver cancer leukemia, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, cervical cancer, ovarian cancer , Lymphoma, nasopharyngeal carcinoma, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma,
  • the inventors of the present invention reported a CAR molecule with artificial epitope.
  • the target cells expressing CAR positive can be efficiently sorted by a secondary sorting method to increase the positive CAR in the final product.
  • the proportion of T cells (CN108017717A).
  • the inventor unexpectedly discovered that during the preparation process, the use of antibodies against artificial epitopes to specifically activate CAR-introduced T cells can not only increase the proportion of CAR-positive T cells in the final product, but also It can significantly increase the ratio of Tcm in CAR-positive T cells, bringing unexpected technical effects.
  • the present invention has the following advantages:
  • the preparation method provided by the present invention does not require specific sorting of CAR-introduced T cells, and can increase the proportion of CAR-positive T cells in the product;
  • the preparation method provided by the present invention can realize the specific expansion of Tcm subpopulations in CAR-positive T cells in vitro, and significantly increase the proportion of Tcm in the final product;
  • the preparation method provided by the present invention overcomes the technical problem that Tcm cannot be cultured for a long time in vitro, realizes the long-term culture of Tcm in vitro, and effectively improves the yield of Tcm; in the prior art, the proportion of Tcm begins to decrease after the sixth day of culture. , And the Tcm ratio can still be maintained at a high level when the technical solution of the present invention is cultivated to the 12th day;
  • the CAR-T cell prepared by the present invention has good killing-related cytokine release activity in vitro and in vivo, and has better and longer lasting anti-tumor activity;
  • Figures 1-3 are the proportions of CAR-positive T cells in the cell products prepared in each experimental group in Examples 3-5;
  • Figures 4-6 respectively show the ratio of Tcm in CAR-positive T cells in the cell products prepared in each experimental group in Examples 3-5;
  • Figures 7-9 show the changes in the proportion of Tcm in the culture system in the process of preparing cell products in each experimental group in Examples 3-5 with the extension of the culture time;
  • Figure 10 shows the in vitro killing activity test results of the cell products prepared in each experimental group in Example 3.
  • Figure 11 shows the in vitro release levels of killing-related cytokines (IL-2, IFN- ⁇ , TNF- ⁇ ) of the cell products prepared in each experimental group in Example 3;
  • Figure 12 shows the evaluation results of in vivo anti-tumor activity of the cell products prepared in each experimental group in Example 3, where the vertical axis is the median survival time of tumor-bearing mice after injection of the cell products;
  • Figure 13 shows the release levels of killing-related cytokines (IL-2, IFN- ⁇ , TNF- ⁇ ) in vivo of the cell products prepared in each experimental group in Example 3;
  • Figures 14-18 show the detection of the amplification levels of different CAR-T products in patients.
  • Figures 14-18 show the results of patients 1-5 in turn.
  • the protein structure of the chimeric antigen receptor sequentially includes an extracellular domain, an optional hinge region, a transmembrane domain, and an intracellular domain.
  • the extracellular domain includes an optional signal peptide and an antigen-binding domain.
  • the antigen-binding domain can directly recognize tumor-associated antigen (TAA) without needing to be in the form of an MHC/antigen peptide complex Recognize antigen.
  • TAA tumor-associated antigen
  • the antigen-binding domains described herein include antibodies or fragments thereof having antigen-binding activity.
  • the antibodies or antibody fragments include at least a heavy chain variable region and a light chain variable region.
  • the antigen-binding domain includes Fab fragments, Fab' fragments, F(ab')2 fragments, or single Fv fragments with antigen-binding activity.
  • Fv antibody contains the variable region of the heavy chain and the variable region of the light chain, but does not have the constant region, and has the smallest antibody fragment with all the antigen binding sites.
  • an Fv antibody also contains a polypeptide linker between the variable region of the heavy chain and the variable region of the light chain, and can form a structure required for antigen binding.
  • the antigen binding domain is usually scFv (single-chain variable fragment).
  • the CAR has an artificial epitope, and the artificial epitope has the following characteristics:
  • the artificial epitope When the artificial epitope is in a free state or exists in the CAR, the artificial epitope can be recognized by the anti-artificial epitope antibody;
  • the artificial epitope may be located in the extracellular region of the CAR. Specifically, the artificial epitope may be located in the extracellular domain of the CAR, between the extracellular domain and the hinge region, or between the hinge region and the span. Between membrane structures. In a specific embodiment, the artificial epitope is located in the extracellular domain of the CAR. In a preferred embodiment, the artificial epitope is located between the signal peptide and the antigen binding domain. In another preferred embodiment, the artificial epitope is located in the antigen binding domain. Further, the artificial epitope is located between the variable region of the heavy chain and the variable region of the light chain.
  • the anti-artificial epitope antibody can specifically recognize the artificial epitope.
  • the anti-artificial epitope antibody can specifically recognize the artificial epitope, and the anti-artificial epitope antibody will not interact with the culture system.
  • the anti-artificial epitope antibody of the present invention can be selected from commercial antibodies, or can be prepared by using the artificial epitope as the antigen and according to the conventional technical methods for antibody preparation.
  • Fetal bovine serum FBS, DMEM medium, D-PBS medium, and RPMI1640 medium were purchased from Gbico; OKT-3 was purchased from Pepro Tech; IL-2, IL-7, and IL-15 were purchased from Thermo Fisher; X -VIVO15 was purchased from LONZA; SmAb (amino acid sequence is SEQ ID NO: 4) was prepared and provided by Beijing Yiqiao Shenzhou Technology Co., Ltd.; MCA2488 was purchased from Bio-Rad; A01732 was purchased from Genscript; human kidney cell line HEK293T, human Lymphoma cell line raji was purchased from ATCC, USA; NOD/SCID IL2R ⁇ c-/- immunodeficient mice were purchased from Beijing Weitongda Biotechnology Co., Ltd.; Raji-Luci cell line was purchased from Beijing Weitongda Biotechnology Co., Ltd.
  • an anti-human CD19 humanized single-chain antibody was used as an example to construct a CD19-targeting chimeric antigen molecule CD19sCAR-1 (see SEQ ID NO: 5 for the amino acid sequence).
  • This CAR molecule can target the CD19 antigen and contains artificial Epitope E-tag (see SEQ ID NO: 1 for amino acid sequence).
  • CD19CAR was constructed as a control CAR molecule.
  • CD19CAR can target CD19 antigen.
  • the difference between CD19CAR and CD19sCAR-1 is that CD19CAR does not contain artificial epitopes, and its amino acid sequence is shown in SEQ ID NO: 7.
  • lentiviruses encoding CD19sCAR-1, CD19CAR and EGFP were prepared. Specific steps are as follows:
  • HEK293T as a packaging cell to prepare chimeric antigen receptor-encoding virus.
  • the HEK293T cells in the logarithmic growth phase were digested, centrifuged at 800 rpm for 5 min, the medium was discarded and resuspended in DMEM medium containing 10% FBS. After counting the cells, adjust the density of the cell suspension to 3.6 ⁇ 10 6 /ml and place it in a 37°C cell incubator for later use.
  • the transfection of the virus packaging plasmid uses the Lipofectamine 3000 kit (Thermo Fisher), and the operation is performed in accordance with the kit instructions.
  • the three plasmids required for the lentivirus packaging including the lentiviral expression vector (using the CD19sCAR-1 lentiviral expression vector prepared in Example 1, respectively, the CD19CAR lentiviral expression vector and the lentiviral vector pLenti6.4-containing EGFP encoding gene CMV-EGFP), plasmid psPAX2 encoding viral nucleocapsid proteins Gag/Pol and Rev, and plasmid pVSVG encoding viral envelope protein, mixed with Lipofectamine 3000 according to the recommended ratio in the instructions to prepare a DNA liposome complex, and let it stand at room temperature 15min.
  • Ficol-Hypaque density gradient centrifugation was used to separate mononuclear cells (PBMC) in peripheral blood, PMBC was resuspended in D-PBS and the density was adjusted to 0.5 ⁇ 10 6 /mL. According to the 1:1 ratio of the absolute number of T cells to the CD3/CD28 sorting magnetic beads, add the sorting magnetic beads and mix gently at room temperature for 20 minutes. The mixed cell-magnetic bead suspension is used to sort CD3+ T cells using a sorting magnetic stand.
  • PBMC mononuclear cells
  • T cell activation on the sorted T cells: Resuspend the T cells in X-VIVO15 medium containing 50ng/mL OKT-3 and 500IU/mL IL-2 at 1 ⁇ 10 6 /mL, and inoculate them into a culture vessel The culture is carried out, and the culture condition is that it is placed in a 37°C, 5% CO 2 saturated humidity incubator for 24 hours. After T cell activation, the cells were collected in a 50mL centrifuge tube, counted, and the cell density was adjusted to 3 ⁇ 10 6 /mL.
  • the harvested cells were grouped according to the grouping method in Table 1 and cultured in vitro.
  • SmAb is a monoclonal antibody that represents an artificial epitope E-tag
  • the negative control group is a treatment group that uses a lentivirus encoding EGFP to infect T cells.
  • the activated T cells were grouped according to the grouping method in Table 1, they were respectively infected with the lentiviruses encoding CD19sCAR-1, CD19CAR and EGFP prepared in Example 2. 24h after infection, the cells of each treatment group were transferred to X-VIVO15 medium containing 80ng/mL IL-7, 500IU/mL IL-2 and 20ng/mL IL-15 for culture at 37°C, containing 5% CO 2 saturated humidity incubator. On the 6th day of culture, according to the processing method of the experimental group, the experimental group 1 and the experimental group 3 were specifically activated.
  • the processing method was: transfer the cells of the experimental group 1 and the experimental group 3 to the coating with 20 ⁇ g/mL SmAb
  • the antibody culture container is continued to be cultured with X-VIVO15 medium containing 80ng/mL IL-7, 500IU/mL IL-2 and 20ng/mL IL-15 under the same conditions as before.
  • the cells are refilled every 2-3 days according to the cell growth. After that, the cells of each group continued to be cultured to the 12th day, and the cell products were harvested.
  • a chimeric antigen molecule CD19sCAR-2 targeting CD19 was constructed (see SEQ ID NO: 9 for the amino acid sequence, and SEQ ID NO: 10 for the nucleotide sequence of the open reading frame).
  • the CAR molecule can target the CD19 antigen And it contains artificial antigen epitope Strep-tag (see SEQ ID NO: 2 for amino acid sequence).
  • SEQ ID NO: 9 for the amino acid sequence
  • SEQ ID NO: 10 for the nucleotide sequence of the open reading frame
  • the CAR molecule can target the CD19 antigen And it contains artificial antigen epitope Strep-tag (see SEQ ID NO: 2 for amino acid sequence).
  • Example 1 uses the sequence of CD19sCAR-1, while this step uses the sequence of CD19sCAR-2;
  • Example 2 uses the CD19sCAR-1 lentiviral expression vector prepared in Example 1, while this step uses the CD19sCAR-2 lentiviral expression vector prepared in step (1);
  • Example 3 The experimental procedure is the same as that of Example 3. The difference is that the grouping of Example 3 uses Table 1, and the grouping of this step uses Table 2, and the preparation is performed according to Table 2. Prepare each group of cell products separately.
  • Experimental group b1 Use MCA2488 for specific activation after infection with CD19sCAR-2 2
  • Experimental group b2 Does not use MCA2488 for specific activation after infection with CD19sCAR-2
  • Experimental group b3 Use MCA2488 for specific activation after CD19CAR infection 4
  • Experimental group b4 Same as experimental group a4
  • Experimental group b5 Same as experimental group a5
  • a chimeric antigen molecule CD19sCAR-3 targeting CD19 was constructed (see SEQ ID NO: 11 for the amino acid sequence, and SEQ ID NO: 12 for the nucleotide sequence of the open reading frame).
  • the CAR molecule can target the CD19 antigen And it contains the artificial antigen epitope Strep-tag II (see SEQ ID NO: 3 for the amino acid sequence).
  • SEQ ID NO: 3 for the amino acid sequence
  • Example 1 uses the sequence of CD19sCAR-1, while this step uses the sequence of CD19sCAR-3;
  • Example 2 uses the CD19sCAR-1 lentiviral expression vector prepared in Example 1, while this step uses the CD19sCAR-3 lentiviral expression vector prepared in step (1);
  • Example 3 The experimental procedure is the same as that of Example 3. The difference is that the grouping of Example 3 uses Table 1, and the grouping of this step uses Table 3, and the preparation is performed according to Table 3. Prepare each group of cell products separately.
  • Experimental group c1 Use A01732 for specific activation after infection with CD19sCAR-3 2
  • Experimental group c2 Do not use A01732 for specific activation after infection with CD19sCAR-3
  • Experimental group c3 Use A01732 for specific activation after infection with CD19CAR 4
  • Experimental group c4 Same as experimental group a4
  • Experimental group c5 Same as experimental group a5
  • a human lymphoma cell line as target cells, 5 ⁇ 10 4 /mL was inoculated into a U-shaped bottom 96-well plate.
  • the CAR-T cells in each experimental group prepared in Example 3 were used according to the effective target ratio (E/T ) Co-cultivation with ratios of 25:1, 12.5:1, 6.25:1 and 1:1, the culture medium is serum-free RPMI1640 medium, and the culture conditions are 37°C, 5% CO 2 , saturated humidity incubator, After 12 hours of culturing, the lactic dehydrogenase release method (LDH method) was used to detect the killing activity of CAR-T cells in different experimental groups on target cells. The results of the detection are shown in Figure 10.
  • E/T effective target ratio
  • the culture medium is serum-free RPMI1640 medium
  • the culture conditions are 37°C, 5% CO 2 , saturated humidity incubator
  • LDH method lactic dehydrogenase release method
  • the CAR-T cells of each experimental group prepared in Example 3 and Raji target cells were mixed at a ratio of 25:1, and after co-cultivation for 12 hours, the culture supernatant was collected and the CAR-T was detected by ELISA method
  • the release level of killing-related cytokines IL-2, IFN- ⁇ , TNF- ⁇ .
  • the CAR-T cells obtained by the preparation method of the present invention have better cytokine release activity during the killing process than CAR-T cells prepared by other experimental groups.
  • mice 6-8 weeks old NOD/SCID IL2R ⁇ c-/- immunodeficiency mice were selected, and the human Raji cell line (Raji-Luci) stably expressing luciferase protein was passed through the tail at a dose of 1 ⁇ 10 6 /100 ⁇ L/mouse.
  • the tumor-bearing mouse model was established by intravenous injection. Three days after the tumor cells were injected, they were grouped according to the experiment in Example 3. Each group of experimental animals were injected with the CAR-T cells prepared in Example 3 at a rate of 1 ⁇ 10 6 /100 ⁇ L/only through the tail vein.
  • the small animal imaging system was used to observe the tumor progression in the experimental animals every week after the injection until all the experimental animals died.
  • the results of the statistical analysis of the experimental data are shown in Figure 12.
  • the CAR-T cells obtained by the preparation method of the present invention can effectively prolong the median survival time of tumor-bearing mice and delay tumor progression in vivo , Has a longer-lasting anti-tumor activity in vivo.
  • the ELISA method was used to detect the levels of CAR-T-related anti-tumor cytokines in the serum of different experimental groups of animals in Example 9, including IFN- ⁇ , TNF- ⁇ , and IL-2.
  • the test results are shown in Figure 13.
  • the experimental animals receiving the CAR-T prepared by the technical scheme of the present invention have significantly higher levels of killing-related cytokines in vivo, suggesting that they have better resistance in vivo. Tumor activity.
  • Example 3 The CD9sCAR-T cell product prepared by the experimental group a1 was verified by a phase I clinical trial (registration number: ChiCTR1800017439, ChiCTR1800014761) for its clinical safety and effectiveness.
  • the first patient male, 9 years old, with acute B lymphocytic leukemia, relapsed after standard chemotherapy treatment, and received autologous mouse-derived CD19CAR-T and CD22CAR-T dual-targeted CAR-T treatment, one month after treatment relapse.
  • the patient received autologous CD19sCAR-T treatment
  • the reinfusion dose was 1x10 ⁇ 6/kg
  • the bone marrow morphological tumor load level before reinfusion was 4%
  • the flow cytometry was 2.28%.
  • patients received standard fludarabine/cyclophosphamide (F/C) regimen for lymphocyte clearance.
  • F/C fludarabine/cyclophosphamide
  • the first treatment achieved complete remission (CR) and lasted for about 3 and a half months; the second treatment was ineffective, and the patient’s tumor burden after treatment was higher than before treatment (Bone marrow morphology test before reinfusion: 0.5%, flow cytometry : 0.53%, fusion gene quantitative: 0.94%; bone marrow morphology detection on the 15th day after reinfusion: 1.5%, flow cytometry: 0.6%, fusion gene quantitative: 13.3%; bone marrow morphology detection on the 36th day after reinfusion: 30% , Flow cytometry: 46.81%, fusion gene quantitative: 119.58%.).
  • the patient was treated with CD19sCAR-T at a dose of 0.3x10 ⁇ 6/kg after standard F/C chemotherapy.
  • F/C fludarabine/cyclophosphamide
  • the fourth patient achieved complete remission after treatment, and relapsed again after one and a half years; the fifth patient relapsed one year after treatment reached complete remission.
  • the fifth patient was ineffective after the first treatment with the murine CD19CAR-T before bridging transplantation, and reached CR after the second reinfusion.
  • the intramedullary morphological load of the two patients was 29% and 6%, respectively, and the results of flow cytometry were 15.31% and 34.74%. There were no tumor cells in the peripheral and cerebrospinal fluid.
  • Two patients were treated with CD19sCAR-T from a donor at a dose of 3x10 ⁇ 6/kg after pretreatment with standard F/C chemotherapy. After reinfusion, both patients were on the seventh day after reinfusion. There was a slight CRS reaction around the day, and the main symptom was fever. After corresponding treatment, the symptoms disappeared, and two patients were evaluated to reach CR on the 30th day after reinfusion.
  • the in vivo amplification level of the CD9sCAR-T prepared by the technical solution of the present invention is significantly higher than that of the early CD19CAR-T ( Figure 14-18).
  • the research results of this example show that the CD19sCAR-T prepared by the technical scheme of the present invention has good safety and effectiveness in clinical applications, and has excellent in vivo expansion ability.
  • the method for increasing the proportion of central memory T cell (Tcm) subgroups in CAR-T cell products disclosed in the present invention is by adding artificial epitopes to the CAR, and activating the CAR-T through the artificial epitopes.
  • This method can not only increase the proportion of CAR-positive T cells in CAR-T cell products, but also realize the specific expansion of Tcm subsets in CAR-positive T cells in vitro, and significantly increase the proportion of Tcm in CAR-T cell products.
  • Clinical trials show that the expansion ability of CAR-T cells prepared by the present invention in vivo is significantly better than the prior art, and has better clinical safety and effectiveness.

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Abstract

一种提高CAR-T细胞制品中中央记忆型T细胞(Tcm)亚群比例的方法，包括，在CAR中加入人工抗原表位，以及通过所述人工抗原表位激活CAR-T；其中所述人工抗原表位不存在于所述CAR的其他结构域或区段中。提供了一种CAR-T细胞的制备方法，所述制备方法包括，(1)将所述CAR导入T细胞；(2)培养所述导入CAR的T细胞，在培养过程中进行特异性激活。该制备方法不仅可提高产品中CAR阳性T细胞的比例，还可实现CAR阳性T细胞中Tcm亚群的体外的特异性扩增，显著提高Tcm在终产品中的比例。临床试验表明，制备得到的CAR-T细胞在体内的扩增能力显著优于现有技术，具有更好的临床安全性和有效性。

Description

以Tcm为主要效应成分的CAR-T制备方法及其应用 技术领域
本发明涉及基因治疗与细胞治疗技术领域，具体涉及一种以Tcm为主要效应成分的CAR-T制备方法及其应用。
背景技术
中央记忆型T细胞(centrol memory T cell，Tcm)是一群以CD45RO+/CD197+为表面标记物的T细胞亚群。该亚群的T细胞由初始型T细胞经抗原或非抗原刺激后分化而来，在体内主要参与特异性记忆免疫反应。Tcm亚群在外周血分离获得的T细胞亚群中比例较低，在体外培养过程中，Tcm会在培养的初始阶段快速增殖，之后会随着培养时间的延长而降低。
过继性免疫细胞治疗是当前生物医药领域新型的抗肿瘤治疗手段。该方法主要通过将患者自体细胞提取后，经体外进行活化，修饰，培养和扩增后，再回输至患者体内，从而到达治疗肿瘤的目的。CAR-T，即嵌合抗原受体修饰性T细胞是一种具有靶向性的过继性免疫细胞治疗方法。该技术通过利用基因修饰的方法改造T细胞基因组，使其表达可识别肿瘤特异性抗原的CAR受体，进而突破原始T细胞抗肿瘤的MHC限制性，使T细胞直接杀伤肿瘤。例如CD19特异性CAR-T细胞已经在急性B淋巴细胞白血病和B细胞淋巴瘤的治疗方面取得了突破性进展，并实现了临床应用。但是目前的CAR-T治疗技术依然存在诸多临床瓶颈。
已有大量研究表明，现有CAR-T细胞在体内疗效的维持性不甚理想。接受治疗的患者在达到完全缓解后，1年内的复发率高达46％。通过对患者回输后的免疫细胞亚群和回输的CAR-T制品进行分析显示，产品中Tcm的比例以及回输后受试者体内Tcm的比例与缓解时间和CAR-T抗肿瘤活性的维持性呈现正相关性。患者输注的CAR-T制品中CAR阳性T细胞中Tcm的比例越高，患者获得长期缓解的可能性越大。临床前动物实验也表明，回输Tcm为主要亚群的CAR-T细胞可以显著延长小鼠的生存时间。
目前已知的Tcm制备方法是从外周血中分离原代初始型T细胞，先利用CD3单克隆抗体OKT-3和IL-2进行活化。活化24小时后，加入包括IL-7、IL-15和IL-21的培养基进行培养。培养至6-9天后收获细胞。此时终产品中的Tcm比例大约为50％左右，难以满足临床回输的要求。
由于Tcm细胞亚群的增殖特点，在体外培养中，随着培养时间的延长，Tcm细胞亚群的比例会迅速降低。因此，短期培养工艺无法保证获得足够数量的目的细胞，而增加初始种子细胞的培养数量，又会使制备成本急剧增加。目前CAR-T的制备工艺在必须保证受试者获得回输剂量充足的细胞前提下，无法保证回输产品中具有高纯度的Tcm细胞。因此，如何利用经济高效的方法获得以Tcm为主要效应成分的细胞制品是目前行业内的一个技术难题。
发明内容
为了解决上述技术问题，本发明提供一种以Tcm为主要效应成分的CAR-T制备方法及其应用。
为此，本发明提供一种提高CAR-T细胞制品中中央记忆型T细胞(Tcm)亚群比例的方法，其中，在CAR中加入人工抗原表位，以及通过所述人工抗原表位激活CAR-T；其中所述人工抗原表位不存在于所述CAR的其他结构域或区段中。
进一步，所述人工抗原表位对CAR的其它结构域或区段的功能不产生或基本不产生负面影响；当所述人工抗原表位被结合时能够激活CAR-T。
进一步，所述人工抗原表位位于CAR的胞外结构域中、胞外结构域与铰链区之间、或铰链区与跨膜结构之间。
进一步，所述人工抗原表位的长度为7-15aa，优选8-12aa。
进一步，所述人工抗原表位的氨基酸序列为SEQ ID NO：1、SEQ ID NO：2或SEQ ID NO：3。
进一步，采用特异性识别所述人工抗原表位的分子结合所述人工抗原表位激活CAR-T，其中特异性识别所述人工抗原表位的分子优选为抗体分子。
进一步，通过所述人工抗原表位激活CAR-T的步骤在将CAR导入T细胞的步骤之后。
进一步，通过所述人工抗原表位激活CAR-T的步骤包括将CAR-T在含有IL-7、IL-2和IL-15的培养基中进行培养。
其中IL-7、IL-2和IL-15的浓度分别为10-200ng/mL、20-1000IU/mL和1-50ng/mL。
本发明所述方法为体外方法，尽管所述方法制备的CAR-T具有良好的肿瘤治疗效果，但本发明所述方法的直接目的是为了提高离体CAR-T细胞中中央记忆型T细胞(Tcm)亚群的比例，所述方法本身也并不涉及任何体内操作的步骤。
具体地，第一方面，本发明提供了一种CAR-T细胞的制备方法，包括，
(1)将所述嵌合抗原受体(CAR)导入T细胞；
(2)培养所述导入CAR的T细胞，在培养过程中进行特异性激活；
所述CAR具有人工抗原表位；
所述特异性激活包括在培养过程中加入抗人工抗原表位的抗体，所述抗人工抗原表位的抗体可特异性识别所述人工抗原表位。
CAR的蛋白质结构依次包括以下结构域：胞外结构域、任选的铰链区、跨膜结构域和胞内结构域，所述胞外结构域包括任选的信号肽和抗原结合结构域，所述抗原结合结构域包括具有抗原结合活性的抗体或抗体片段，所述抗体或抗体片段至少包括重链可变区和轻链可变区。
进一步，所述人工抗原表位位于所述CAR的胞外结构域中、胞外结构域与铰链区之间、或铰链区与跨膜结构域之间。在一个具体的实施方式中，所述人工抗原表位位于所述CAR的胞外结构域中。在一个优选实施例中，所述人工抗原 表位位于所述信号肽与抗原结合结构域之间。在另一个优选实施例中，所述人工抗原表位位于所述抗原结合结构域中。进一步，所述人工抗原表位位于所述重链可变区和轻链可变区之间。
进一步，所述人工抗原表位为以下(a1)-(a3)中的一种：
(a1)E-tag，其氨基酸序列为KPLPEVTDEY(SEQ ID NO：1)；
(a2)Strep-tag，其氨基酸序列为WSHPQFEK(SEQ ID NO：2)；
(a3)Strep-tag II，其氨基酸序列为NWSHPQFEK(SEQ ID NO：3)。
当所述人工抗原表位为所述E-tag时，所述抗人工抗原表位的抗体为抗E-tag抗体，所述抗E-tag抗体可特异性识别所述E-tag。在一个具体的实施方式中，所述抗E-tag抗体的氨基酸序列为SEQ ID NO：4。
当所述人工抗原表位为Strep-tag时，所述抗人工抗原表位的抗体为抗Strep-tag抗体，所述抗Strep-tag抗体可特异性识别所述Strep-tag。在一个具体的实施方式中，所述抗Strep-tag抗体购自Bio-Rad公司(货号：MCA2488)。
当所述人工抗原表位为Strep-tag II时，所述抗人工抗原表位的抗体为抗Strep-tag II抗体，所述抗Strep-tag II抗体可特异性识别所述Strep-tag II。在一个具体的实施方式中，所述抗Strep-tag II抗体购自Genscript公司(货号：A01732)。
进一步，步骤(1)中所述T细胞的制备方法包括：从外周血中分离T细胞，对分离得到的T细胞进行T细胞活化。
进一步，步骤(1)中，所述T细胞活化包括将所述分离得到的T细胞在第一培养基中进行培养，所述第一培养基包括OKT-3和IL-2。进一步，所述第一培养基中，OKT-3的浓度为5-200ng/mL，10-100ng/mL，更优选为50-80ng/mL，例如50ng/mL，60ng/mL，70ng/mL，80ng/mL；IL-2的浓度为20-1000IU/mL，优选为50-500IU/mL，更优选为200-500IU/mL，例如200IU/mL，300IU/mL，400IU/mL，500IU/mL。
所述第一培养基可通过本领域常用的培养基配制得到。在具体的实施方式中，所述第一培养基可通过以下培养基配制得到，包括但不限于：X-VIVO15培养基(Lonza公司)、KBM581无血清培养基(Corning公司)、含有10％FBS的RPMI1640培养基(Gbico公司)或含有10％人AB血清的RPMI1640培养基(Gbico公司)。
进一步，所述T细胞活化的培养时间为4-72h，优选为24-36h，例如24h、28h、32h、36h，在一个优选的实施例中为24h。
进一步，步骤(2)中，所述培养所用的培养基为第二培养基，所述第二培养基包括IL-7、IL-2和IL-15。进一步，所述第二培养基中，IL-7的浓度为10-200ng/mL，优选为20-100ng/mL，更优选为80-100ng/mL，例如80ng/mL、90ng/mL、100ng/mL；IL-2的浓度为20-1000IU/mL，优选为50-500IU/mL，更优选为200-500IU/mL，例如200IU/mL，300IU/mL，400IU/mL，500IU/mL；IL-15的浓度为1-50ng/mL，优选为5-30ng/mL，更优选为10-20ng/mL，例如10ng/mL、 12ng/mL、14ng/mL、16ng/mL、18ng/mL、20ng/mL。
所述第二培养基可通过本领域常用的培养基配制得到。在具体的实施方式中，所述第二培养基可通过以下培养基配制得到，包括但不限于：X-VIVO15培养基(Lonza公司)、KBM581无血清培养基(Corning公司)、含有10％FBS的RPMI1640培养基(Gbico公司)或含有10％人AB血清的RPMI1640培养基(Gbico公司)。
进一步，步骤(2)中，所述培养的时间为9-14天，优选9-12天，例如9天、10天、11天、12天；在一个优选的实施例中，为12天。
进一步，步骤(2)中，所述特异性激活包括，从培养过程中的第m天至培养结束，在所述第二培养基中加入起所述抗人工抗原表位的抗体，所述m选自6-9的整数，例如6、7、8、9，在一个优选的实施例中，所述m＝6。
进一步，步骤(2)中，所述抗人工抗原表位的抗体的浓度为1-50μg/mL，优选为5-30μg/mL，更优选为10-25μg/mL，例如10μg/mL、15μg/mL、20μg/mL、25μg/mL；在一个优选的实施例中为20μg/mL。
进一步，步骤(2)中，所述抗人工抗原表位的抗体的加入方式选自下组：向培养基中直接加入所述抗人工抗原表位的抗体、将所述抗人工抗原表位的抗体包被于培养容器、或其组合。
进一步，步骤(1)将所述CAR导入T细胞的方法为通过病毒感染的方式导入T细胞或通过非病毒感染的方式导入T细胞；其中，所述病毒感染的方式中的病毒选自以下病毒：腺病毒、逆转录病毒、慢病毒、疱疹病毒和腺相关病毒；所述非病毒感染的方式为脂质体法、显微注射法、磷酸钙共沉淀法、电转法或DEAE-葡聚糖转染法。
进一步，所述步骤(1)为通过病毒感染将CAR导入T细胞。在优选的实施例中，所述步骤(1)具体包括以下步骤，
S1、构建CAR的病毒表达载体；
S2、将步骤S1制备得到的CAR病毒表达载体导入相应的包装细胞中进行病毒包装，得到编码CAR的病毒；
S3、从外周血中分离T细胞，对分离得到的T细胞进行T细胞活化；
S4、用步骤S2制备得到的编码CAR的病毒感染步骤S3制备得到的T细胞。
进一步，所述病毒表达载体为腺病毒表达载体、逆转录病毒表达载体、慢病毒表达载体、疱疹病毒表达载体或腺相关病毒表达载体。在一个优选的实施例中，为慢病毒表达载体。
第二方面，本发明提供了一种具有高比例中央记忆型T细胞(Tcm)亚群的CAR-T细胞，所述CAR-T细胞通过本发明所述的方法制备得到
第三方面，本发明提供了所述CAR-T细胞在制备预防和/或治疗癌症或肿瘤的药物或制剂中的应用。
同时，本发明还提供了所述CAR-T细胞在预防和/或治疗癌症或肿瘤方面的应用。
进一步，所述肿瘤选自下组：血液肿瘤、实体瘤、或其组合。
进一步，所述血液肿瘤选自下组：急性髓细胞白血病(AML)、多发性骨髓瘤(MM)、慢性淋巴细胞白血病(CLL)、急性淋巴白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、非霍奇金淋巴瘤(NHL)、或其组合。
进一步，所述实体瘤选自下组：胃癌、胃癌腹膜转移、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、宫颈癌、卵巢癌、淋巴癌、鼻咽癌、肾上腺肿瘤、膀胱肿瘤、非小细胞肺癌(NSCLC)、脑胶质瘤、子宫内膜癌、间皮瘤、胰腺癌、多发性骨髓瘤、或其组合。
本发明的发明人在此前研究中，报道了一种具有人工抗原表位的CAR分子，可以通过二次分选的方法将表达CAR阳性的目的细胞进行高效分选，以提高终产品中阳性CAR-T细胞的比例(CN108017717A)。在此研究基础上，发明人意外地发现，在制备过程中，利用抗人工抗原表位的抗体对导入CAR的T细胞进行特异性激活，不仅可以提高终产品中CAR阳性T细胞的比例，还可以显著提高CAR阳性T细胞中Tcm的比例，带来意料不到的技术效果。
与现有技术相比，本发明具有以下优点：
1、本发明提供的制备方法无需对导入CAR的T细胞进行特异性分选，即可提高产品中CAR阳性T细胞的比例；
2、本发明提供的制备方法可以实现CAR阳性T细胞中Tcm亚群的体外的特异性扩增，显著提高Tcm在终产品中的比例；
3、本发明提供的制备方法克服了Tcm在体外无法长期培养的技术问题，实现了Tcm在体外的长期培养，有效提高了Tcm的产量；现有技术培养到第6天左右，Tcm比例开始下降，而本发明的技术方案培养至第12天时，Tcm比例依然可以维持较高水平；
4、与现有技术相比，本发明制备得到的CAR-T细胞在体外、体内环境中均具有良好的杀伤相关细胞因子释放活性，具有更好、更持久的抗肿瘤活性；
5、临床试验表明，本发明制备得到的CAR-T细胞产品在体内的扩增能力显著优于现有技术，具有更好的临床安全性和有效性。
附图说明
通过阅读下文优选实施方式的详细描述，各种其他的优点和益处对于本领域普通技术人员将变得清楚明了。附图仅用于示出优选实施方式的目的，而并不认为是对本发明的限制。在附图中：
图1-3分别为实施例3-5中各实验组制备得到的细胞产品中CAR阳性T细胞的比例；
图4-6分别为实施例3-5中各实验组制备得到的细胞产品中CAR阳性T细胞中Tcm的比例；
图7-9分别为实施例3-5中各实验组在制备细胞产品的过程中，随着培养时间的延长，培养体系中Tcm的比例变化；
图10为实施例3中各实验组制备得到的细胞产品的体外杀伤活性检测结果；
图11为实施例3中各实验组制备得到的细胞产品在体外的杀伤相关细胞因子(IL-2、IFN-γ、TNF-α)释放水平；
图12为实施例3中各实验组制备得到的细胞产品在体内的抗肿瘤活性评价结果，其中，纵轴为经注射细胞产品后，荷瘤小鼠的中位生存时间；
图13为实施例3中各实验组制备得到的细胞产品在体内的杀伤相关细胞因子(IL-2、IFN-γ、TNF-α)释放水平；
图14-18为不同CAR-T产品在患者体内的扩增水平检测，其中，图14-18依次分别为患者1-5的检测结果。
具体实施方式
下面将参照附图更详细地描述本公开的示例性实施方式。虽然附图中显示了本公开的示例性实施方式，然而应当理解，可以以各种形式实现本公开而不应被这里阐述的实施方式所限制。相反，提供这些实施方式是为了能够更透彻地理解本公开，并且能够将本公开的范围完整的传达给本领域的技术人员。
为了可以更容易地理解本公开，首先定义某些术语。如本公开所使用的，除非本文另有明确规定，否则以下每一个术语具有其下面给出的含义。
嵌合抗原受体(Chimeric antigen receptor，CAR)的蛋白质结构依次包括胞外结构域、任选的铰链区、跨膜结构域和胞内结构域。其中，胞外结构域包括任选的信号肽和抗原结合结构域，所述抗原结合结构域可直接识别肿瘤相关抗原(tumor associated antigen，TAA)，而不需要以MHC/抗原肽复合物的形式识别抗原。
本文所述的抗原结合结构域包括具有抗原结合活性的抗体或其片段，所述抗体或抗体片段至少包括重链可变区和轻链可变区。具体地，所述抗原结合结构域包括具有抗原结合活性的Fab片段，Fab’片段，F(ab’)2片段，或单一Fv片段。Fv抗体含有抗体重链可变区、轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般地，Fv抗体还包含重链可变区和轻链可变区之间的多肽接头，且能够形成抗原结合所需的结构。抗原结合结构域通常是scFv(single-chain variable fragment)。
在本发明提供的嵌合抗原受体修饰性T细胞的制备方法中，所述CAR具有人工抗原表位，所述人工抗原表位具有以下特征：
(a)所述人工抗原表位不存在所述CAR的其他结构域或区段中；
(b)当所述人工抗原表位为游离态或存在于所述CAR时，所述人工抗原表位都能被抗人工抗原表位抗体识别；和
(c)不影响或基本不影响所述CAR与所述CAR靶向的抗原的结合。
所述人工抗原表位可位于CAR的胞外区，具体地，所述人工抗原表位可位于所述CAR的胞外结构域中、胞外结构域与铰链区之间、或铰链区与跨膜结构之间。在一个具体的实施方式中，所述人工抗原表位位于所述CAR的胞外结构域中。在一个优选实施例中，所述人工抗原表位位于信号肽与抗原结合结构域之间。在另一个优选实施例中，所述人工抗原表位位于所述抗原结合结构域中。进 一步，所述人工抗原表位位于所述重链可变区和轻链可变区之间。
在本发明中，“特异性识别”，即“特异性结合”，抗人工抗原表位抗体可特异性识别所述人工抗原表位。当人工抗原表位为游离态或存在于所述CAR时，所述抗人工抗原表位抗体均可以特异性识别所述人工抗原表位，且所述抗人工抗原表位抗体不会与培养体系中的其他分子或CAR中除所述人工抗原表位以外的结构域发生识别或结合。
本发明中的抗人工抗原表位抗体可以选自商业化抗体，也可以以所述人工抗原表位为抗原，按照抗体制备的常规技术方法制备得到。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，贺福初等译的《分子克隆实验指南》，第四版，科学出版社；J.E.科利根等著，曹雪涛等译的《精编免疫学实验指南》，科学出版社等)或者按照产品说明书进行。
胎牛血清FBS、DMEM培养基、D-PBS培养基、RPMI1640培养基购自Gbico公司；OKT-3购自Pepro Tech公司；IL-2、IL-7、IL-15购自Thermo Fisher公司；X-VIVO15购自LONZA公司；SmAb(氨基酸序列为SEQ ID NO：4)委托北京义翘神州科技有限公司制备提供；MCA2488购自Bio-Rad公司；A01732购自Genscript公司；人体肾脏细胞系HEK293T、人淋巴瘤细胞系raji购自美国ATCC；NOD/SCID IL2Rγc-/-免疫缺陷小鼠购自北京维通达生物技术有限公司；Raji-Luci细胞系购自北京维通达生物技术有限公司。
实施例1 嵌合抗原受体(CD19sCAR-1)的表达载体的构建
本实施例以抗人CD19人源化单链抗体为例，构建靶向CD19的嵌合抗原分子CD19sCAR-1(氨基酸序列见SEQ ID NO：5)，该CAR分子可靶向CD19抗原且包含人工抗原表位E-tag(氨基酸序列见SEQ ID NO：1)。
使用化学合成的方法将CD19sCAR-1的开放阅读框序列(核苷酸序列见SEQ ID NO：6)进行全序列合成，同时在其两端加入限制性酶切位点，将合成得到的序列利用基因克隆的方法插入含有CMV启动子的慢病毒表达载体pLenti6.4的CMV启动子下游。获得CD19sCAR-1的慢病毒表达载体。将所述的载体转化到Stbl3感受态工程菌种，进行保存及后续制备。
同时，构建CD19CAR作为对照用CAR分子，CD19CAR可靶向CD19抗原，其与CD19sCAR-1的区别在于，CD19CAR不包含人工抗原表位，其氨基酸序列如SEQ ID NO：7所示。
使用化学合成的方法将CD19CAR的开放阅读框序列(核苷酸序列见SEQ ID NO：8)进行全序列合成，同时在其两端加入限制性酶切位点，将合成得到的序列利用基因克隆的方法插入含有CMV启动子的慢病毒表达载体pLenti6.4的CMV启动子下游。获得CD19CAR的慢病毒表达载体。将所述的载体转化到Stbl3感受态工程菌种，进行保存及后续制备。
实施例2 嵌合抗原受体(CD19sCAR-1)编码病毒的制备
本实施例制备了分别编码CD19sCAR-1、CD19CAR和EGFP的慢病毒。具 体步骤如下：
利用HEK293T作为包装细胞进行嵌合抗原受体编码病毒的制备。将对数生长期的HEK293T细胞消化，800rpm离心5min，弃去培养基后用含有10％FBS的DMEM培养基重悬。进行细胞计数后，将细胞悬液的密度调整至3.6×10 6/ml，放置于37℃细胞培养箱中待用。
病毒包装质粒的转染使用Lipofectamine 3000试剂盒(Thermo Fisher公司)，并按照试剂盒说明书进行操作。将慢病毒包装需要的三种质粒，包括慢病毒表达载体(分别使用实施例1制备得到的CD19sCAR-1慢病毒表达载体、CD19CAR慢病毒表达载体和含有EGFP编码基因的慢病毒载体pLenti6.4-CMV-EGFP)、编码病毒核衣壳蛋白Gag/Pol和Rev的质粒psPAX2和编码病毒包膜蛋白的质粒pVSVG，与Lipofectamine 3000按照说明书推荐比例混合配制成DNA脂质体复合物，室温下静置15min。静置结束后，取一块6孔培养板，将DNA-脂质体复合物加入到6孔板中，每孔1ml，再将之前制备的HEK293T细胞悬液轻柔混匀，加入到6孔板中，与脂质体复合物混匀。培养板放入培养箱继续培养，分别在培养24h和48h收集含有病毒的培养上清。在最后一次收集上清后，将上清2000g离心10min，用0.45μm的滤膜过滤，即制备得到了分别编码CD19sCAR-1、CD19CAR和EGFP的慢病毒，分装后冻存于-80℃保存待用。
实施例3 嵌合抗原受体(CD19sCAR-1)修饰性T细胞的制备
用Ficol-Hypaque密度梯度离心法分离外周血中的单个核细胞(PBMC)，将PMBC用D-PBS重悬后将密度调整为0.5×10 6/mL。按照T细胞绝对数与CD3/CD28分选磁珠1:1的比例，加入分选磁珠，室温轻柔混匀20min。混匀后的细胞-磁珠混合悬液利用分选磁力架进行CD3+T细胞的分选。将分选后的T细胞进行T细胞活化：用含有50ng/mL OKT-3和500IU/mL IL-2的X-VIVO15培养基按照1×10 6/mL重悬T细胞，接种至培养容器中进行培养，培养条件为放入37℃，5％CO 2的饱和湿度培养箱中培养24h。T细胞活化后，将细胞收集到50mL离心管中，进行计数，将细胞密度调整至3×10 6/mL。
将收获的细胞按照表1的分组方式进行分组，进行体外培养。其中，SmAb为代表靶向人工抗原表位E-tag的单克隆抗体，阴性对照组是利用编码EGFP的慢病毒感染T细胞的处理组。
表1 实验组分组(a)
序号 分组编号 处理及培养方法
1 实验组a1 感染CD19sCAR-1后利用SmAb进行特异性激活
2 实验组a2 感染CD19sCAR-1后不利用SmAb进行特异性激活
3 实验组a3 感染CD19CAR后利用SmAb进行特异性激活
4 实验组a4 感染CD19CAR后不利用SmAb进行特异性激活
5 实验组a5 阴性对照组
活化后的T细胞按照表1的分组方式分组后，分别感染实施例2制备得到的编码CD19sCAR-1、CD19CAR和EGFP的慢病毒。感染后24h，将各处理组 的细胞转移至含有80ng/mL IL-7、500IU/mL IL-2和20ng/mL IL-15的X-VIVO15培养基中进行培养，培养条件为37℃，含有5％CO 2的饱和湿度培养箱。在培养的第6天，根据实验分组的处理方式，将实验组1和实验组3进行特异性激活，处理方式为：将实验组1和实验组3的细胞转移至包被有20μg/mL SmAb抗体的培养容器，继续用含80ng/mL IL-7、500IU/mL IL-2和20ng/mL IL-15的X-VIVO15培养基以原相同的条件进行培养。
培养期间，每2-3天根据细胞生长情况对细胞进行补液。之后各组细胞继续培养至第12天，分别收获细胞制品。
实施例4 嵌合抗原受体(CD19sCAR-2)修饰性T细胞的制备
本实施例构建了靶向CD19的嵌合抗原分子CD19sCAR-2(氨基酸序列见SEQ ID NO：9，开放阅读框的核苷酸序列见SEQ ID NO：10)，该CAR分子可靶向CD19抗原且包含人工抗原表位Strep-tag(氨基酸序列见SEQ ID NO：2)。制备嵌合抗原受体(CD19sCAR-2)修饰性T细胞，具体步骤如下：
(1)构建CD19sCAR-2的表达载体
实验步骤同实施例1，区别在于，实施例1使用CD19sCAR-1的序列，而本步骤使用CD19sCAR-2的序列；
(2)制备CD19sCAR-2编码病毒
实验步骤同实施例2，区别在于，实施例2使用实施例1制备得到的CD19sCAR-1慢病毒表达载体，而本步骤使用步骤(1)制备得到的CD19sCAR-2慢病毒表达载体；
(3)制备CDsCAR-2修饰性T细胞
实验步骤同实施例3，区别在于，实施例3的分组采用表1，本步骤的分组采用表2，并按照表2进行制备。分别制备得到各组细胞制品。
表2 实验组分组(b)
序号 分组编号 处理及培养方法
1 实验组b1 感染CD19sCAR-2后利用MCA2488进行特异性激活
2 实验组b2 感染CD19sCAR-2后不利用MCA2488进行特异性激活
3 实验组b3 感染CD19CAR后利用MCA2488进行特异性激活
4 实验组b4 同实验组a4
5 实验组b5 同实验组a5
实施例5 嵌合抗原受体(CD19sCAR-3)修饰性T细胞的制备
本实施例构建了靶向CD19的嵌合抗原分子CD19sCAR-3(氨基酸序列见SEQ ID NO：11，开放阅读框的核苷酸序列见SEQ ID NO：12)，该CAR分子可靶向CD19抗原且包含人工抗原表位Strep-tag II(氨基酸序列见SEQ ID NO：3)。制备嵌合抗原受体(CD19sCAR-3)修饰性T细胞，具体步骤如下：
(1)构建CD19sCAR-3的表达载体
实验步骤同实施例1，区别在于，实施例1使用CD19sCAR-1的序列，而本步骤使用CD19sCAR-3的序列；
(2)制备CD19sCAR-3编码病毒
实验步骤同实施例2，区别在于，实施例2使用实施例1制备得到的CD19sCAR-1慢病毒表达载体，而本步骤使用步骤(1)制备得到的CD19sCAR-3慢病毒表达载体；
(3)制备CDsCAR-3修饰性T细胞
实验步骤同实施例3，区别在于，实施例3的分组采用表1，本步骤的分组采用表3，并按照表3进行制备。分别制备得到各组细胞制品。
表3 实验组分组(c)
序号 分组编号 处理及培养方法
1 实验组c1 感染CD19sCAR-3后利用A01732进行特异性激活
2 实验组c2 感染CD19sCAR-3后不利用A01732进行特异性激活
3 实验组c3 感染CD19CAR后利用A01732进行特异性激活
4 实验组c4 同实验组a4
5 实验组c5 同实验组a5
实施例6 细胞制品中CAR阳性T细胞、Tcm比例
统计3个独立批次实验，分别对实施例3-5制备得到的各实验组的终产品进行流式检测分析，流式检测分析结果见图1-6。由图1-3可知，实验组a1、b1、c1的终产品中CAR阳性T细胞比例显著高于其他实验组，说明根据本发明提供的制备方法，可促进CAR阳性T细胞的扩增，提高其在终产品中的比例；由图4-6可知，实验组a1、b1、c1的CAR阳性T细胞中Tcm的比例约为80％，显著高于其他实验组。
对培养过程中Tcm亚群的增殖情况进行分析，分析结果见图7-9所示。由图7-9可知，实验组a1、b1、c1的CAR阳性T细胞中Tcm亚群比例在培养初期显著升高，且随着培养时间的延长，扩增比例未发生显著变化；而其他实验组培养体系中的Tcm比例，在培养初期有所升高，但是随着培养时间的延长，比例逐渐下降。这表明，本发明提供的制备方法有效克服了Tcm在体外无法长期培养的限制，延长了其体外培养的时间，保证了终产品收获时的细胞数量和Tcm亚群的比例。
实施例7 体外杀伤活性
以人淋巴瘤细胞系raji为靶细胞，按照5×10 4/mL接种至U型底96孔板，将实施例3制备得到的各实验组CAR-T细胞，按照效靶比(E/T)比例25:1、12.5:1、6.25:1和1:1的比例进行共培养，培养基为不含血清的RPMI1640培养基，培养条件为37℃，5％CO 2，饱和湿度培养箱，培养12h后，利用乳酸脱氢酶释放法(LDH法)检测不同实验组的CAR-T细胞对靶细胞的杀伤活性，检测结果见图10。由图10的实验结果可知，在相同效靶比的条件下，本发明的制备方法获得的CAR-T细胞产品的杀伤活性显著优于其他实验组，同时也说明Tcm比例较高的CAR-T细胞制品具有更好的靶细胞杀伤活性。
实施例8 杀伤相关细胞因子释放能力的检测
按照实施例7的方法，将实施例3制备得到的各实验组CAR-T细胞与Raji靶细胞按照25:1的比例混合，共培养12h后，收集培养上清，利用ELISA方法检测CAR-T杀伤相关细胞因子(IL-2、IFN-γ、TNF-α)的释放水平。如图11显示的实验结果所示，本发明的制备方法获得的CAR-T细胞与其他实验组制备的CAR-T细胞相比，在杀伤过程中具有更好的细胞因子释放活性。
实施例9 体内抗肿瘤活性评价
选择6-8周龄的NOD/SCID IL2Rγc-/-免疫缺陷小鼠，将稳定表达荧光素酶蛋白的人Raji细胞系(Raji-Luci)以1×10 6/100μL/只的剂量，经过尾静脉注射的方式建立荷瘤小鼠模型。注射肿瘤细胞3天后，按照实施例3的实验分组，每组实验动物通过尾静脉按照1×10 6/100μL/只注射实施例3制备得到的CAR-T细胞。注射后每周利用小动物成像系统观测实验动物的体内肿瘤进展，直至全部实验动物死亡。对实验数据的统计分析结果见图12所示，与其他实验组相比，本发明的制备方法获得的CAR-T细胞可以有效的延长荷瘤小鼠的中位生存时间，延缓肿瘤体内的进展，具有更持久的体内抗肿瘤活性。
实施例10 体内杀伤细胞因子释放活性的评价
利用ELISA方法检测实施例9中不同实验组动物血清中CAR-T相关抗肿瘤细胞因子的水平，包括IFN-γ、TNF-α、IL-2。检测结果见图13所示，与其他实验组相比，接受本发明技术方案制备的CAR-T的实验动物，体内的杀伤相关细胞因子的水平显著更高，提示其在体内具有更好的抗肿瘤活性。
实施例11 临床安全性及有效性评估
实施例3实验组a1制备得到的CD9sCAR-T细胞产品，通过I期临床试验(注册号：ChiCTR1800017439，ChiCTR1800014761)对其临床安全性和有效性进行了验证。
5名前期接受过鼠源性CD19CAR-T治疗后又再次复发的急性B淋巴细胞白血病患者接受了本发明实施例3实验组a1制备的CD9sCAR-T产品，每名患者均为单次输注，回输剂量范围为0.3×10 6/kg-3×10 6/kg。所有患者均在回输后每周接受安全性评估，在第15天和第30天接受了疗效评估。
第一例患者，男性，9岁，急性B淋巴细胞白血病，经标准化疗治疗后复发后，接受自体鼠源性CD19CAR-T和CD22CAR-T双靶向性CAR-T进行治疗，治疗一个月后复发。复发后，患者接受了自体CD19sCAR-T治疗，回输剂量为1x10^6/kg，回输前骨髓形态学肿瘤负荷水平4％，流式检测为2.28％。回输前患者接受标准氟达拉滨/环磷酰胺(F/C)方案进行淋巴细胞清除。回输后第7天患者出现发热，持续3天，经治疗后，体温正常。回输后第30天患者进行疗效评估，达到完全缓解。第二例患者，男性，14岁，急性B淋巴细胞白血病并伴有复杂染色体核型，曾接受标准化疗缓解后复发。接受CD19sCAR-T前，分别接受了鼠源性CD19CAR-T和鼠源性CD19CAR-T/CD22CAR-T双靶向性CAR-T的治疗。其中第一次治疗达到完全缓解(CR)，并持续约3个半月；第二次治疗无 效，且治疗后患者肿瘤负荷较治疗前升高(回输前骨髓形态检测：0.5％、流式检测：0.53％、融合基因定量：0.94％；回输后第15天骨髓形态检测：1.5％、流式检测：0.6％、融合基因定量：13.3％；回输后第36天骨髓形态检测：30％、流式检测：46.81％、融合基因定量：119.58％。)。患者经标准F/C化疗后接受了剂量为0.3x10^6/kg的CD19sCAR-T的治疗，治疗过程中未出现CRS相关副反应。第15天评估骨髓形态肿瘤比例为10.5％，流式检测结果为14.98％，比回输前的相应指标有显著降低(回输前骨髓形态符合：46％，流式检测：34.86％)；但第30天评估时，患者骨髓形态符合和流式比例均发生了反弹，分别为82％和71.84％，未达到CR。第三例患者，男性，17岁，急性B淋巴细胞白血病并伴有中枢系统白血病，曾接受标准化疗及母亲来源的半相合异基因造血干细胞移植治疗，缓解后复发。复发后接受两次鼠源性CD19CAR-T治疗，第一次回输后完全缓解，第二次回输20天后复发，并伴有中枢系统复发。复发后患者接受半相合供者来源(母亲)的CD19sCAR-T细胞治疗，回输剂量1x10^6/kg，回输前骨髓形态负荷0.5％，中枢负荷66.13％。回输前患者接受标准氟达拉滨/环磷酰胺(F/C)方案进行淋巴细胞清除。回输后第5天，患者出现发热，给予抗感染后体温恢复正常。回输后第30天评估，达到CR。第四例患者和第五例患者均为女性，年龄分别为14岁和20岁。两名患者均为急性B淋巴细胞白血病，曾经标准化疗后达到缓解，但在缓解后1个月左右复发，之后经标准化疗治疗无效后，接受了鼠源性CAR-T桥接半相合异基因造血干细胞移植的治疗方案。其中，第四例患者治疗后达到完全缓解，一年半后再次复发；第五例患者治疗达到完全缓解后一年后复发。此外，第五名患者在桥接移植前的鼠源性CD19CAR-T首次治疗后无效，第二次回输后达到CR。在接受CD19sCAR-T治疗前，两名患者髓内形态学负荷分别为29％和6％，流式检测结果分别为15.31％和34.74％，外周和脑脊液中未见肿瘤细胞。两名患者在接受标准F/C化疗预处理后，均接受了剂量为3x10^6/kg的供者来源的CD19sCAR-T的治疗，回输后，两名患者均在在回输后第7天前后出现轻微CRS反应，主要症状为发热。在给予相应处理后，症状消失，回输后第30天对两名患者进行评估达到CR。
研究结果见表4所示，5名患者中有4名患者在第30天评估为达到完全缓解，总体缓解率为80％。5名患者均未出现3级以上的毒副反应。
通过与患者前期接受CD19CAR-T治疗后监测的CAR-T体内扩增数据对比，本发明技术方案制备的CD9sCAR-T的体内扩增水平显著高于前期CD19CAR-T(图14-18)。本实施例的研究结果表明，本发明的技术方案制备的CD19sCAR-T在临床应用中具有良好的安全性和有效性，且具有优异的体内扩增能力。
表4 受试者基本信息及临床疗效评估
Figure PCTCN2019117100-appb-000001
Figure PCTCN2019117100-appb-000002
Figure PCTCN2019117100-appb-000003
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。
工业实用性
本发明公开的提高CAR-T细胞制品中中央记忆型T细胞(Tcm)亚群比例的方法，通过在CAR中加入人工抗原表位，并通过所述人工抗原表位激活CAR-T。该方法不仅可提高CAR-T细胞制品中CAR阳性T细胞的比例，还可实现CAR阳性T细胞中Tcm亚群的体外的特异性扩增，显著提高Tcm在CAR-T细胞制品中的比例。临床试验表明，本发明制备得到的CAR-T细胞在体内的扩增能力显著优于现有技术，具有更好的临床安全性和有效性。

Claims (10)

  1. 一种提高CAR-T细胞制品中中央记忆型T细胞(Tcm)亚群比例的方法，其特征在于，在CAR的胞外结构域中、胞外结构域与铰链区之间或铰链区与跨膜结构之间中加入人工抗原表位，以及通过所述人工抗原表位激活CAR-T；其中所述人工抗原表位不存在于所述CAR的其他结构域或区段中。
  2. 如权利要求1所述提高CAR-T细胞制品中中央记忆型T细胞(Tcm)亚群比例的方法，其特征在于，所述人工抗原表位对CAR的其它结构域或区段的功能不产生或基本不产生负面影响；当所述人工抗原表位被结合时能够激活CAR-T。
  3. 如权利要求1所述提高CAR-T细胞制品中中央记忆型T细胞(Tcm)亚群比例的方法，其特征在于，所述人工抗原表位的长度为7-15aa，优选8-12aa。
  4. 如权利要求1所述提高CAR-T细胞制品中中央记忆型T细胞(Tcm)亚群比例的方法，其特征在于，所述人工抗原表位的氨基酸序列为SEQ ID NO：1、SEQ ID NO：2或SEQ ID NO：3。
  5. 如权利要求1-4任一所述提高CAR-T细胞制品中中央记忆型T细胞(Tcm)亚群比例的方法，其特征在于，采用特异性识别所述人工抗原表位的分子结合所述人工抗原表位激活CAR-T，其中特异性识别所述人工抗原表位的分子优选为抗体分子。
  6. 如权利要求1-5任一所述提高CAR-T细胞制品中中央记忆型T细胞(Tcm)亚群比例的方法，其特征在于，通过所述人工抗原表位激活CAR-T的步骤在将CAR导入T细胞的步骤之后。
  7. 如权利要求1-6任一所述提高CAR-T细胞制品中中央记忆型T细胞(Tcm)亚群比例的方法，其特征在于，通过所述人工抗原表位激活CAR-T的步骤包括将CAR-T在含有IL-7、IL-2和IL-15的培养基中进行培养。
  8. 一种具有高比例中央记忆型T细胞(Tcm)亚群的CAR-T细胞，其特征在于，所述CAR-T细胞通过权利要求1-7任一项所述的方法制备得到。
  9. 如权利要求8所述的CAR-T细胞在制备预防和/或治疗癌症或肿瘤的药物或制剂中的应用。
  10. 如权利要求8所述的CAR-T细胞在预防和/或治疗癌症或肿瘤方面的应用。
PCT/CN2019/117100 2019-06-13 2019-11-11 以Tcm为主要效应成分的CAR-T制备方法及其应用 Ceased WO2020248486A1 (zh)

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