Search

CN-121975731-A - Tumor-infiltrating lymphocyte amplification composition, kit, and amplification method and application thereof

CN121975731ACN 121975731 ACN121975731 ACN 121975731ACN-121975731-A

Abstract

The application belongs to the technical field of immune cell culture, and particularly relates to a tumor infiltration lymphocyte amplification composition, a kit and an amplification method thereof. The application provides a tumor-infiltrating lymphocyte amplification composition for a trace amount of tumor tissues, namely tumor puncture samples, which is used for amplifying tumor-infiltrating lymphocytes, solves the problem of large sample size of the tumor tissues required by the tumor-infiltrating lymphocytes and is suitable for patients incapable of receiving surgical excision to acquire the tumor tissues. In addition, the application establishes a process flow for amplifying tumor-infiltrating lymphocytes based on a puncture sample by optimizing the combination and working concentration of the antibody stimulant, so that the dryness of the tumor-infiltrating lymphocytes obtained by amplification is maintained, the killing power is improved, and the anti-tumor curative effect after the tumor-infiltrating lymphocytes are returned into the body is ensured.

Inventors

  • LIU MINGYU
  • LU YONG
  • ZHANG XI
  • HAN DEPING
  • let Paul Roche Thierry

Assignees

  • 广州百吉生物制药有限公司

Dates

Publication Date
20260505
Application Date
20241029

Claims (11)

  1. 1. A tumor-infiltrating lymphocyte amplification composition, which is characterized by comprising a tumor-infiltrating lymphocyte amplification medium and an activating antibody mixed solution; The tumor-infiltrating lymphocyte amplification culture medium comprises a basic culture medium and the following components with the concentration of 2 v/v% -10 v/v% SR serum replacement, 300 IU/mL-9000 IU/mL IL-2, 0 ng/mL-10 ng/mL IL-7 and 0 ng/mL-30 ng/mL IL-15; The activating antibody mixed solution comprises two or more of 0 mu g/mL-5 mu g/mL of anti-CD 3 antibody, 0 mu g/mL-5 mu g/mL of anti-CD 28 antibody and 0 mu g/mL-5 mu g/mL of anti-41 BB antibody.
  2. 2. The tumor-infiltrating lymphocyte amplification composition according to claim 1, wherein the activating antibody mixture comprises one or more of 0.11 μg/mL to 0.35 μg/mL of anti-CD 3 antibody, 0.11 μg/mL to 0.35 μg/mL of anti-CD 28 antibody, and 0.11 μg/mL to 0.35 μg/mL of anti-41 BB antibody.
  3. 3. The tumor-infiltrating lymphocyte amplification composition according to claim 1, wherein the concentration of the anti-CD 3 antibody is 0.30 μg/mL to 0.35 μg/mL; Optionally, the concentration of the anti-CD 28 antibody is 0.30-0.35 mug/mL; optionally, the concentration of the anti-41 BB antibody is 0.30-0.35 mug/mL; optionally, the basal medium comprises one or more of X-VIVO 15, RPMI-1640 and AIM-V medium.
  4. 4. A tumor-infiltrating lymphocyte amplification kit, characterized by comprising the tumor-infiltrating lymphocyte amplification composition according to any one of claims 1 to 3.
  5. 5. A method of expanding tumor-infiltrating lymphocytes in vitro comprising: taking a tumor puncture sample, culturing the tumor puncture sample by using the tumor-infiltrating lymphocyte amplification composition according to any one of claims 1-3, and preparing amplified tumor-infiltrating lymphocytes.
  6. 6. The method according to claim 5, comprising: Adding the activated antibody mixture into a culture container, incubating to prepare a coated culture container, and Inoculating the tumor puncture sample into the coated culture container, and adding the tumor-infiltrating lymphocyte amplification culture medium for culture to prepare amplified tumor-infiltrating lymphocytes.
  7. 7. The method of claim 6, wherein the method has one or more of the following conditions: (1) The tumor puncture sample has a volume of 1.130 mm 3 ~ 22.61 mm 3 ; (2) The culture vessel comprises a culture well plate; (3) The incubation treatment conditions comprise 36-38 ℃ and 3-6 h, or 2-8 ℃ and 12-16 h; (4) The conditions for adding the tumor-infiltrating lymphocyte amplification culture medium for culture include the temperature of 33-38 ℃ and the concentration of CO 2 of 4.8-v/v% to 5.2-v/v%.
  8. 8. The method according to any one of claims 5 to 7, further comprising the step of expanding the tumor-infiltrating lymphocytes; The tumor-infiltrating lymphocytes are subjected to expansion culture, wherein the expanded tumor-infiltrating lymphocytes are filtered by a filter screen with the diameter of 35-75 mu m, and are subjected to expansion inoculation culture at the cell density of 1X 10 5 /mL~5×10 5 /mL after centrifugal resuspension.
  9. 9. The tumor-infiltrating lymphocyte obtained by the method for in vitro amplification of tumor-infiltrating lymphocytes according to any one of claims 5 to 8.
  10. 10. Use of the tumor-infiltrating lymphocytes according to claim 9 in the preparation of a medicament for treating tumors.
  11. 11. A medicament for treating tumors, characterized in that the active ingredient of the pharmaceutical composition contains tumor-infiltrating lymphocytes according to claim 9 and pharmaceutically acceptable auxiliary materials.

Description

Tumor-infiltrating lymphocyte amplification composition, kit, and amplification method and application thereof Technical Field The application belongs to the technical field of immune cell culture, and particularly relates to a tumor infiltration lymphocyte amplification composition, a kit and an amplification method thereof. Background Tumor infiltrating lymphocytes (Tumor Infiltrating Lymphocytes, TILs) are heterogeneous populations of cells consisting of T cells (including cd8+ T cells, cd4+ T cells, γδ T cells), B cells, NK cells, and innate lymphocytes (Innate Lymphocytes, ILCs), and the like. After in vitro expansion culture, cd3+ T cells become the predominant cell population (typically greater than 95%). Among them, cd3+ T cells express TCR receptors on their surface and play a key role in mediating immune killing of cancerous cells. T cell antigen specificity is conferred by the recognition of different antigenic peptides presented by the molecules of the major histocompatibility complex (Major histocompatibility Complex, mhC) by different TCR receptors. Thus, once T cells specific for an antigen are stimulated with a particular antigen, these cells can promote programmed death of the target cells by releasing cytotoxins and receptor-mediated mechanisms, among other means. Unlike chimeric antigen T Cell (CAR-T, CHIMERIC ANTIGEN Receptor T-Cell)/TCR (T Cell Receptor) -T Cell therapeutic products, TILs are usually anti-tumor agents by recognizing tumor-associated antigens such as neoantigen (Neoantigen) and the like presented by tumor cells. Accordingly, TIL therapy can be used for anti-tumor therapeutic purposes by recognizing and targeting a variety of tumor-associated antigens. Cell therapy of TIL refers to a treatment method of killing tumor cells by separating, amplifying and activating lymphocytes in tumor tissues obtained by operation and then reinjecting the lymphocytes to a patient. A traditional TIL preparation method needs to obtain a sufficient amount of tumor tissue samples through a surgical excision mode, and the process flow comprises the steps of (1) obtaining a sufficient amount of tumor tissue, usually more than 1cm 3, through a tumor tissue excision mode and the like in a hospital, (2) transporting the tumor tissue to a GMP cell production workshop through a cold chain, (3) treating the tumor tissue in a GMP-grade environment, including cutting, digesting, lymphocyte separation and other steps, obtaining the TIL, (4) placing the separated TIL in a T cell selective expansion culture medium for culture, wherein the culture medium contains high-concentration IL-2, T cell activation antibodies (usually anti-CD 3/CD28 antibodies), amplifying and culturing for more than 10 10 weeks, harvesting, washing and canning the cells of the final product, and (5) transporting the qualified TIL to the hospital for patient feedback through liquid nitrogen. The TIL culture process based on the traditional technology has the following limitations that the requirement of tumor tissues is large (generally more than 1cm 3), so that a late-stage tumor patient who cannot accept surgical material cannot take TIL preparation material, the time for culturing and expanding T cells is long, the T cell stem property is poor, the killing power is low, and the anti-tumor curative effect after TIL feedback is limited. Disclosure of Invention Based on the above, an embodiment of the application provides a tumor-infiltrating lymphocyte amplification composition, and tumor-infiltrating lymphocytes obtained by in vitro amplification of the amplification composition have the effects of high dryness and strong killing power. In one aspect, the application provides a tumor-infiltrating lymphocyte amplification composition, which comprises a tumor-infiltrating lymphocyte amplification medium and an activating antibody mixed solution. The tumor-infiltrating lymphocyte amplification culture medium comprises a basal culture medium and components with the following concentration of 2 v/v% -10 v/v% SR serum replacement, 300 IU/mL-9000 IU/mL IL-2, 0 ng/mL-10 ng/mL IL-7 and 0 ng/mL-30 ng/mL IL-15. The activating antibody mixed solution comprises two or more of 0 mu g/mL-5 mu g/mL of anti-CD 3 antibody, 0 mu g/mL-5 mu g/mL of anti-CD 28 antibody and 0 mu g/mL-5 mu g/mL of anti-41 BB antibody. In one embodiment, the activating antibody mixture comprises one or more of 0.11 μg/mL-0.35 μg/mL anti-CD 3 antibody, 0.11 μg/mL-0.35 μg/mL anti-CD 28 antibody, and 0.11 μg/mL-0.35 μg/mL anti-41 BB antibody. In one embodiment, the anti-CD 3 antibody is at a concentration of 0.30 μg/mL to 0.35 μg/mL. Optionally, the concentration of the anti-CD 28 antibody is 0.30-0.35 mug/mL. Optionally, the concentration of the anti-41 BB antibody is 0.30-0.35 mug/mL. Optionally, the basal medium comprises one or more of X-VIVO15, RPMI-1640 and AIM-V medium. In another aspect, the application provides a tumor-infiltrating lymphocyte amplification kit, compr