IDENTIFICATION, IN VITRO AMPLIFICATION, AND APPLICATION METHOD OF MEMORY CD8 T CELLS SPECIFIC FOR TUMOR ANTIGEN

Abstract

The present application provides identification, in vitro amplification, and application method of memory CD8 T cells specific for an antigen in a solid tumor-draining lymph node.

Claims

1. A tumor antigen-specific memory T cell derived from a tumor-draining lymph node, characterized in that it is a cell with the following marker combination: an activated CD62L.sup.+PD1.sup.Low/dimTim3.sup.CD8.sup.+ T cell.

2. The T cell according to claim 1, characterized in that it is a T cell with the following marker combination: for mouse tumor source: a CD44.sup.+CD62L.sup.+CD127.sup.+CD122.sup.+CCR7.sup.+PD1.sup.Low/dimTim3.sup.CD39.sup.LAG3.sup.CD8.sup.+ T cell; for human tumor source: a CD3.sup.+CD4.sup.CD45RA.sup.CD45RO.sup.+CD62L.sup.+CD127.sup.+CD122.sup.+CCR7.sup.+PD1.sup.Low/dimTim3.sup.CD39.sup.LAG3.sup.CD8.sup.+ T cell.

3. The T cell according to claim 1 or 2, wherein the T cell is obtained by flow cytometry or magnetic bead sorting.

4. The T cell according to any one of claims 1 to 3, characterized in that it highly expresses CD122, CD62L, CD127, CCR7, lacks expressing 2B4, LAG3, Tim3, CD39, expresses inhibitory receptor PD-1 at a relatively low level, and highly expresses transcription factor TCF-1.

5. The T cell according to any one of claims 1 to 4, characterized in that it is capable of continuously responding to PD-1/PD-L1 immune checkpoint blockade therapy.

6. Use of the T cell according to any one of claims 1 to 5 in the manufacture of a medicament for inhibiting tumor growth, wherein preferably, the T cell inhibits tumor growth through adoptive cell transfer therapy; preferably, the tumor is selected from melanoma and ovarian cancer.

7. Use of a combination of the T cell according to any one of claims 1 to 5 and a PD-1/PD-L1 immune checkpoint blockade therapy in the manufacture of a medicament for inhibiting tumor growth, wherein preferably, the T cell inhibits tumor growth through adoptive cell transfer therapy; preferably, the tumor is selected from melanoma and ovarian cancer.

8. The use according to claim 7, wherein the PD-1/PD-L1 immune checkpoint blockade therapy is an anti-PD-L1 antibody or an anti-PD-1 antibody.

9. A pharmaceutical composition, which comprises the T cell according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

10. A method for isolating a tumor antigen-specific memory CD8.sup.+ T cells, comprising the following steps: a. obtaining a single cell suspension from a mouse or human solid tumor draining lymph node; b. sorting an activated CD62L.sup.+PD1.sup.Low/dimTim3.sup.CD8.sup.+ T cell from the above single cell suspension, preferably, for the mouse, sorting a CD44.sup.+CD62L.sup.+CD127.sup.+CD122.sup.+CCR7.sup.+PD1.sup.Low/dimTim3.sup.CD39.sup.LAG3.sup.CD8.sup.+ T cell from the single cell suspension; for the human, sorting a CD3.sup.+CD4.sup.CD45RA.sup.CD45RO.sup.+CD62L.sup.+CD127.sup.+CD122.sup.+CCR7.sup.+PD1.sup.Low/dimTim3.sup.CD39.sup.LAG3.sup.CD8.sup.+ T cell from the single cell suspension; preferably, the T cell is obtained by flow cytometry or magnetic bead sorting; and c. performing in vitro amplification and culture in T cell culture medium containing CD3/28 antibodies, and interleukin 2, interleukin 7 and interleukin 15 at low dose.

11. A method for inhibiting tumor growth, comprising administering the T cell according to any one of claims 1 to 5, wherein the T cell inhibits tumor growth by adoptive cell transfer therapy.

12. The method according to claim 11, wherein the tumor is selected from melanoma and ovarian cancer.

13. The method according to claim 11, further comprising administering a PD-1/PD-L1 immune checkpoint blockade therapy, wherein the PD-1/PD-L1 immune checkpoint blockade therapy is an anti-PD-L1 antibody or an anti-PD-1 antibody.

14. The T cell according to any one of claims 1 to 5, for use in inhibiting tumor growth, wherein the T cell inhibits tumor growth by adoptive cell transfer therapy; preferably, the tumor is selected from melanoma and ovarian cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 shows the establishment of mouse subcutaneous transplant tumor model and the flow cytometry analysis strategy.

[0028] FIG. 2 shows the immunophenotypic analysis of tumor-draining lymph node antigen-specific memory CD8 T cells.

[0029] FIG. 3 shows the verification of the memory phenotype of tumor-draining lymph node antigen-specific memory CD8 T cells.

[0030] FIG. 4 shows the effective control of tumor growth by tumor-draining lymph node antigen-specific memory CD8 T cells.

[0031] FIG. 5 shows the important role of tumor-draining lymph node antigen-specific memory CD8 T cells in immune checkpoint blockade therapy and the synergistic effect with PD-L1 immunotherapy.

[0032] FIG. 6 shows the presence of antigen-specific memory CD8 T cells in human ovarian cancer draining lymph nodes.

SPECIFIC MODELS FOR CARRYING OUT THE PRESENT INVENTION

[0033] The technical solution of the present application is described below in detail in conjunction with the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present application, rather than to limit the scope of the present application. According to the following detailed description of the preferred examples, the various objects and advantages of the present application will become apparent to those skilled in the art.

Example 1: Establishment of Transplanted Tumor Model for Isolating Antigen-Specific Memory CD8 T Cells in Mouse Tumor-Draining Lymph Nodes

[0034] 1. Preparation of tumor cell lines: In this example, mouse melanoma cell line B16.F10 was used to establish a mouse subcutaneous transplanted tumor model. The cell line was passaged and cultured in vitro using DMEM medium containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin and 2 mM L-glutamine.

[0035] 2. Tumor cell inoculation: When the fusion density of mouse melanoma B16 cells reached about 80% to 90%, the cells were digested, and counted, and then the concentration of the tumor cell suspension was adjusted to 510.sup.6 cells/mL with 1PBS buffer; 0.1 mL of the tumor cell suspension was taken using a 1 mL BD insulin syringe; the skin between the mouse's ears and back was held with one hand to fully expose the mouse's abdomen, and the skin of abdomen was cleared with 75% alcohol cotton balls; the syringe was held with the other hand, and the needle was inserted horizontally (the angle to the mouse's abdominal plane was about) 0-15 into the skin of the mouse's groin, and the tumor cell suspension was injected subcutaneously to form a small skin mound by the negative pressure of back suction. After the injection was completed, the needle was quickly withdrawn in parallel manner to prevent the cell suspension from flowing out.

[0036] 3. The tumor growth was observed 8-10 days after the cell injection. Usually, it could be seen that the tumor grew to a size of a soybean in about 8 days. After that, the tumor size was measured by vernier caliper and recorded every two days (the tumor volume calculation formula was: tumor volume= lengthwidth.sup.2, unit: mm.sup.3). The observation endpoint was determined according to the specific experimental requirements (FIG. 1A). From an ethical point of view, the mouse was judged dead when the tumor volume reached 2000 mm.sup.3.

Example 2: Isolation, Acquisition and Immunophenotypic Identification of Tumor-Draining Lymph Node Antigen-Specific Memory CD8 T Cells

[0037] 1. After the mouse was sacrificed, its inguinal subcutaneous lymph nodes that were usually located at the intersection of three superficial subcutaneous blood vessels in the lower abdominal wall were carefully separated; the lymph nodes were carefully ground with frosted area of a slide, and then the grinding area was rinsed with RPMI-1640 culture medium containing 10% fetal bovine serum, the above operation was repeated several times, and then the cell suspension was collected, and centrifuged at 4 C. for 6 minutes, in which the centrifugal force was set at 400 g. After centrifugation, the cells were resuspended with sterile PBS, and the cell pellets were gently pipetted several times; after centrifugation again, the cells were resuspended with a certain volume of the above culture medium, and a single cell suspension was obtained for later use.

[0038] 2. Preferably: at the same time, the subcutaneous tumor tissue was separated, and ground thoroughly, and the lymphocytes infiltrating tumor tissue as a control were separated by density gradient centrifugation. The separation solution Percoll had the following density gradient: 44% for the upper layer and 67% for the lower layer. The centrifugation was performed at room temperature for 30 minutes, in which the centrifuge speed was set to reach 550 g.

[0039] 3. Immunophenotypic analysis of tumor antigen-specific CD8 T cells

[0040] 3.1 The cells separated from the tumor lymph nodes were counted to record the total number of cells, and then stained with fluorescent antibody. The steps for flow cytometry staining were referred to: Study on the role and mechanism of transcription factor TCF-1 in virus-specific T cell immune response. Huang Qizhao. 2018. Doctoral dissertation (published). In this example, we used Live/Dead (L/D) dye to distinguish dead cells, and to perform flow cytometry analysis, and the antigen-activated cells were labeled with CD3, CD8, and CD44. The number of antigen-specific CD8 T cells was determined based on their frequency in total cells and the total number of cells (FIG. 1B).

[0041] 3.2 Detection was performed on the expression of cell surface molecules, including inhibitory receptors such as PD-1, 2B4, LAG3, and Tim3; memory-related molecules such as CD122, CD62L, CD127, and CCR7, and other related cell surface molecules. The above surface molecules all could be quantitatively analyzed using flow cytometry;

[0042] 3.3 Detection was performed on the intranuclear transcription factors, mainly including important transcription factors related to T cell differentiation, such as TCF-1, TOX, and Eomes.

[0043] From the results, it could be seen that there was a population of antigen-specific CD8 T cells in the tumor-draining lymph nodes, which highly expressed memory-related molecules such as CD122, CD62L, CD127, and CCR7, and was lack of the expression of inhibitory receptors such as 2B4, LAG3, Tim3, and CD39; at the same time, this population of cells highly expressed memory cell-related transcription factor TCF-1 (represented by GFP) (see, FIG. 2), suggesting that this population of cells had memory cell characteristics.

Example 3: Phenotypic Verification of Tumor-Draining Lymph Node Antigen-Specific Memory CD8 T Cells

[0044] 1. Sorting of target cell subset by flow cytometry technology: It was determined in Example 2 that the tumor-draining lymph node antigen-specific memory CD8 T cells could highly express memory T cell-related marker molecules, lowly express the exhausted cell-related marker molecule PD-1, and were lack of the expression of inhibitory receptors such as 2B4, LAG3, Tim3, and CD39. Therefore, we defined the draining lymph node antigen-specific memory CD8 T cell as CD44.sup.+CD62L.sup.+CD127.sup.+CD122.sup.+CCR7.sup.+PD1.sup.Low/dimTim3.sup.CD39.sup.LAG3.sup.CD8.sup.+ T cell, and used this molecular marker to sort out the target cell subset from the mouse tumor-draining lymph nodes.

[0045] 2. Verification of memory characteristics of target cell subset: The sorted target cell subset and a classical memory cell-labeling cell proliferation dye (Cell trace Violet) in acute infection model were mixed in the same amount and then injected back to nave recipient mice. The spleens of the recipient mice were taken at different time points to observe the long-term maintenance ability and homeostatic proliferation ability of the cells derived from the two sources (FIGS. 3C to 3E); in addition, the same number of cells derived from the two sources were mixed and then injected into nave recipient mice, and the same antigen stimulation was given again to observe the difference in the cell response ability of the memory cells respectively formed in the tumor-draining lymph nodes and the acute viral infection when encountering the same antigen (FIGS. 3A and 3B). The results showed that the draining lymph node antigen-specific memory cells were similar to the classical memory cells and capable of performing homeostatic proliferation in an antigen-independent manner (self-renew), and survived for a long time (FIGS. 3C to 3E); when stimulated again by the same antigen, they had better amplification ability (FIGS. 3A and 3B) and effector function than the antigen-specific cells infiltrating tumor tissues, thus better controlling Listeria infection expressing the same antigen (FIGS. 3F and 3G).

Example 4: In Vitro Amplification and Application of Tumor-Draining Lymph Node Antigen-Specific Memory CD8 T Cells

[0046] 1. In vitro amplification of target subset: The cell CD44.sup.+CD62L.sup.+CD127.sup.+CD122.sup.+CCR7.sup.+PD1.sup.Low/dimTim3.sup.CD39.sup.LAG3.sup.CD8.sup.+ T cells isolated and obtained in Example 2 were cultured in vitro in an RPMI1640 medium containing 10% fetal bovine serum (FBS), to which 0.5 g/ml CD28 antibody, 1-5 ng/ml interleukin 2, and 20 ng/ml interleukin 7 and interleukin 15 (IL2/IL7/IL15) were added at the same time. After sorted, the target cells were counted and placed in a 24-well culture plate pre-coated with 2-5 g/ml CD3 antibody, 110.sup.6 cells per well. After the cells were cultured for 2-3 days, the cell amplification was observed. When the amplification was good, the form of a large number of clone colonies could be seen. Then, the cells were transferred to a 12-well plate without CD3/28 antibodies. This method was used for continuous culture for 7-10 days and the cells were counted for the next experiment;

[0047] 2. Effective control of tumor growth by tumor-draining lymph node antigen-specific memory CD8 T cells: When verifying the therapeutic effect of this population of cells from tumor-draining lymph nodes (B16 model), we selected antigen-specific CD8.sup.+ T cells infiltrating tumor tissue (tumor infiltrating lymphocytes, TIL) as a control. 210.sup.5 target cells were re-infused into tumor-bearing mice on days 8-10 of tumor inoculation, and then the tumor growth curve and the survival of tumor-bearing mice were recorded (FIG. 4A). As shown in FIG. 4, compared with the traditional tumor tissue infiltrating exhausted T cells (TME-TE.sub.X), the lymph node-derived memory cells of the present invention could more effectively control tumor growth and prolong the survival time of the tumor-bearing mice.

[0048] In addition, the tumor-bearing mice were subjected to the resection of bilateral draining lymph nodes on day 8, then re-infused with the lymph node antigen-specific memory cells, and administrated with PD-L1 blocking antibody (BioXCell, Cat #BE0101) or a control reagent (IgG) on days 10 and 13/16 of tumor inoculation, respectively, and at the same time, the group of cell re-infusion combined with anti-PD-L1 blocking antibody treatment was treated with FTY710 (FIG. 5A). The results showed that after the lymph node resection, the anti-PD-L1 blocking antibody lost its tumor control effect. After the cells were re-infused and replenished, the anti-tumor effect of the blocking antibody could be effectively restored. If the lymphocyte migration inhibitor FTY-720 was used to block the migration of the cells from the lymph nodes to the periphery, the anti-PD-L1 blocking antibody also lost its tumor control effect (FIGS. 5B and 5C). The above results further suggested that the memory cells specific for antigens located in tumor-draining lymph nodes played an important role in tumor immunotherapy.

Example 5: Isolation, Acquisition and Immunophenotypic Identification of Tumor-Draining Lymph Node Antigen-Specific Memory CD8 T Cells of Ovarian Cancer Patients

[0049] 1. Screening of ovarian cancer patients: In this example, 2 ovarian cancer patients suitable for surgical resection were selected. Before surgery, informed consent forms were signed with the patients and approved by the ethics committee.

[0050] 2. Processing of tumor specimens: [0051] (1) After the tumor tissue was resected surgically, the connective tissue attached to the surface was carefully removed, and the tumor tissue was cut into 1 cm1 cm tissue blocks, fully cut into pieces using ophthalmic scissors, and placed in 15 ml centrifuge tubes. Then an appropriate volume (5-8 ml) of type I collagenase (Collagenase I) solution was added to each tube of tumor tissue, and digestion was performed in a shaking incubator at 37 C. for 45 min to 1 h, in which the collagenase was dissolved in RPMI-1640 medium and had a working concentration of 1 mg/ml. [0052] (2) After digestion, an equal volume of RPMI-1640 medium (R2) containing 2% FBS was added to each tube and mixed thoroughly by inversion to terminate the collagenase reaction. On a 70 m nylon filter, the tumor tissue was ground with the plastic end of syringe (or a 300 to 400 mesh stainless steel filter could be used instead). [0053] (3) After the cell suspension was centrifugated (2000 rpm, 10 min, room temperature), the supernatant was discarded. 8 ml of 44% Percoll solution (volume ratio, diluted with RPMI) was added to each tube for resuspension. Then, capillary sampling needles (Pasteur tube) were inserted, and 4 ml of 67% Percoll solution was slowly added. After standing for a while, it could be seen that the liquid was divided into two layers, i.e., red layer and white layer. The capillary glass needles were taken out, and centrifugation was performed at room temperature at 550 g for 30 min, and the speed-up and speed-down was set to zero (if possible, it was better that the above liquid was added to a 15 ml round-bottomed bacterial tube or centrifuge tube and subjected to layered centrifugation). [0054] (4) After centrifugation, the flow tube was taken out, during which the removal process should be gentle to avoid damaging the layers. At this time, it could be seen that the liquid was divided into three layers, in which the middle layer was white and cloudy, which was the lymphocyte layer. First, a 1 ml pipette was used to carefully discard the brown-yellow oil of the top layer. Then, the lymphocyte layer was slowly pipetted using a 1 ml pipette that rotated during pipetting, added to a 15 ml centrifuge tube with 3-5 ml of R2 added in advance, mixed thoroughly, centrifuged (2000 rpm, 10 min, 4 C.), resuspended, and counted. [0055] (5) After centrifugation, the supernatant was discarded, the lymphocytes were resuspended by adding a certain volume of RPMI-1640 culture medium (R10) containing 10% FBS for later use. [0056] (6) If the lymph nodes were small, they were ground directly on a 70 m nylon filter. If they were large, after being cut into pieces, they were ground again, counted, and frozen. If tumor cells metastasized to the lymph nodes, digestion with type I collagenase was also required after cutting into pieces.

[0057] 3. Detection of immunophenotype of tumor antigen-specific CD8 T cells by flow cytometry [0058] (1) After the cells obtained in 2 was counted, 50 l of staining buffer per 0.510.sup.6 to 110.sup.6 cells was added; and a flow cytometry working solution FACS buffer was prepared: PBS+2% FBS+0.01% NaN.sub.3 [0059] (2) Fluorescent antibody labeling was performed according to the method described in Example 2.3, except that for human samples, the antigen-activated memory cells (corresponding to CD44 in mice) were labeled with CD45RA.sup.CD45RO.sup.+. [0060] (3) From the results, it could be seen that antigen-specific memory CD8 T cells were also present in ovarian cancer tumor-draining lymph nodes. Similarly, the antigen-specific CD8 T cells were capable of highly expressing memory-related molecules such as CD122, CD62L, CD127, and CCR7, and were lack of expression of inhibitory receptors such as 2B4, LAG3, Tim3, and CD39; at the same time, this population of cells were capable of highly expressing the memory cell-related transcription factor TCF-1 (FIG. 6). The above results further confirmed the presence of CD3.sup.+CD4.sup.CD45RA.sup.CD45RO.sup.+CD62L.sup.+CD127.sup.+CD122.sup.+CCR7.sup.+PD1.sup.Low/dimTim3.sup.CD39.sup.LAG3.sup.CD8.sup.+ T, thereby providing a new theoretical basis for clinical immunotherapy of tumors.