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KR-102963851-B1 - Use of stimulants for testing immune cell efficacy

KR102963851B1KR 102963851 B1KR102963851 B1KR 102963851B1KR-102963851-B1

Abstract

A method for determining the efficacy of immune cells is described. The method comprises the steps of contacting immune cells with an effective amount of a stimulant (e.g., phytohemagglutinin (PHA)) and detecting the amount of cytokines produced by said immune cells. A kit for testing the efficacy of immune cells is also described. Testing efficacy is important for meeting FDA requirements for new biological agents, e.g., immunotherapy cells. A method for using potent immune cells as an immunotherapy treatment is described.

Inventors

  • 리 딘 앤소니
  • 타카르 아로히
  • 홀 마크
  • 무진스키 제니퍼

Assignees

  • 더 리서치 인스티튜트 앳 네이션와이드 칠드런스 하스피탈

Dates

Publication Date
20260513
Application Date
20200214
Priority Date
20190214

Claims (20)

  1. As a method for testing the potency of Interleukin (IL)-21 extended Natural Killer (NK) cells, (i) a step of contacting IL-21-expanded NK cells with an effective amount of stimulant; (ii) a step of detecting the amount of cytokines produced by the IL-21-expanded NK cells of step (i); (iii) a step of demonstrating the effector function of IL-21-extended NK cells by comparing the amount of cytokines produced by the IL-21-extended NK cells of step (i) with the cytokine efficacy level required for the use of IL-21-extended NK cells in immunotherapy; and (iv) a method comprising the step of selecting potent IL-21-expanded NK cells of step (iii) based on the amount of cytokine detected in step (ii).
  2. A method in which the amount of a plurality of cytokines is determined in paragraph 1.
  3. The method of claim 1 or 2, wherein the stimulant comprises phytohemagglutinin (PHA), phorbol myristate acetate (PMA)/ionomycin, concanavalin A (Con A), lipopolysaccharide (LPS), and/or pokeweed mitogen.
  4. A method according to claim 1 or 2, wherein the amount of cytokine is detected using an immunoassay.
  5. In claim 1 or 2, the cytokines are IL-2, IL-6, interferon (IFN)-γ (IFN-γ), B cell activation factor (BAFF)/tumor necrosis factor (TNF) ligand superfamily member 13B (BAFF/TNFSF13B), TNF-α, cluster of differentiation (CD) 163 (CD163), CD30/TNFRSF8, chitinase 3-like 1, gp130, IFN-α2, IL-6Ra, IL-8, IL-10, IL-11, IL-12(p40), IL-12(p70), IL-20, IL-22, IL-26, IL-29/IFN-11, IL-32, IL-34, IL-35, matrix metalloproteinase-1 (MMP-1), From the group comprising osteocalcin, osteopontin (OPN), pentraxin-3, tumor necrosis factor-receptor 1 (TNF-R1), TNF-R2, thymic stromal lymphopoetin (TSLP), granulocyte-macrophage colony-stimulating factor (GM-CSF), leukemia inhibitory factor (LIF), and chemokines macrophage inflammatory protein (MIP)-1α (MIP-1α), MIP-1β, RANTES, and/or TNF-related weak inducer of apoptosis (TWEAK)/TNF superfamily member 12 (TWEAK/TNFSF12 The selected method.
  6. In claim 1, the IL-21-expanded NK cells are in contact with an effective amount of stimulant for at least 4 hours.
  7. The method according to claim 1, wherein the stimulant is provided at a concentration of 5 μg/ml to 15 μg/ml.
  8. A kit for testing the efficacy of IL-21-extended NK cells, comprising a container containing an effective amount of stimulant and a buffer suitable for NK cells, and further comprising instructions for use according to the method of claim 1.
  9. In claim 8, the kit, wherein the stimulant is provided at a concentration of 5 μg/ml to 15 μg/ml.
  10. In paragraph 8, the container is a microcentrifuge tube, a kit.
  11. A composition for treating cancer comprising a therapeutically effective amount of potent IL-21-expanded NK cells, wherein the potent IL-21-expanded NK cells comprise TNF-α, IFN-γ, and at least one cytokine, wherein the at least one cytokine comprises IL-2, IL-6, B cell activating factor/tumor necrosis factor ligand superfamily member 13B (BAFF/TNFSF13B), differentiation cluster 163 (CD163), CD30/TNFRSF8, chitinase 3-like 1, gp130, IFN-α2, IL-6Ra, IL-8, IL-10, IL-11, IL-12(p40), IL-12(p70), IL-20, IL-22, IL-26, IL-29/IFN-11, IL-32, IL-34, IL-35, matrix metalloproteinase-1 (MMP-1), A composition selected from the group comprising osteocalcin, osteopontin (OPN), pentracin-3, tumor necrosis factor-receptor 1 (TNF-R1), TNF-R2, thymic stromal lymphopoetin (TSLP), granulocyte-macrophage colony-stimulating factor (GM-CSF), leukemia inhibitory factor (LIF), and chemokine macrophage inflammatory protein-1α (MIP-1α), MIP-1β, RANTES, or TNF-related weak inducer of apoptosis/TNF superfamily member 12 (TWEAK/TNFSF12), wherein the potent IL-21-expanded NK cells are formulated with a therapeutically acceptable carrier as an immunotherapeutic composition for treating cancer.
  12. A composition according to claim 11, wherein the potent IL-21-expanded NK cells are allogeneic or autologous NK cells.
  13. In claim 11, the composition comprises potent IL-21-expanded NK cells genetically modified to respond to a specific antigen.
  14. In paragraph 13, the composition comprises potent IL-21-expanded NK cells genetically modified to present a chimeric antigen receptor.
  15. The method of claim 1, wherein the cytokine is selected from IFN-γ, CD30, and IL-2.
  16. In paragraph 5, the method wherein the cytokine is selected from IFN-γ, CD30, and IL-2.
  17. In paragraph 6, the method comprises a stimulant containing phytohemagglutinin.
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Description

Use of stimulants for testing immune cell efficacy Related applications This application claims the benefit of U.S. Provisional Application No. 62/805,349 (filed February 14, 2019), the full text of which is incorporated herein by reference. Technology field The present invention relates to immunotherapy, more specifically to testing the effector function of immune cells. Immunotherapy is the treatment of diseases by activating or suppressing the immune system. Cells derived from the immune system can be used to improve immune functionality and characteristics. Recently, immunotherapy has garnered significant interest from researchers, physicians, and pharmaceutical companies, particularly due to its potential to treat various forms of cancer. Immunomodulatory therapies generally have fewer side effects than conventional drugs, including a lower likelihood of generating resistance when treating microbial diseases. Conventional cancer treatment focuses on killing or eliminating cancer cells using chemotherapy, surgery, and/or radiation. However, the field of therapeutic immune cells can be used in conjunction with, or in some cases instead of, conventional treatments to treat, prevent, or delay the onset of cancer. Immune effector cells, such as lymphocytes, macrophages, dendritic cells, natural killer (NK) cells, and cytotoxic T lymphocytes (CTLs), naturally work together to defend the body against cancer by targeting abnormal antigens expressed on the surface of tumor cells. Recent developments in cancer therapies focus on directing the patient's immune system to attack and destroy tumors. Various strategies are currently in use or are under research and testing. Adoptive cell transfer (ACT) is the delivery of cells to a patient and has been found to be possible for lung, melanoma, and other cancers. These cells are derived from the patient (autologous) or from another individual (allogeneic). Allogeneic therapy involves cells isolated and expanded from donor isolates from the patient receiving the cells. Alternatively, adoptive cell transfer can be used to culture and expand autologous extracted cells in vitro for subsequent transfusion. For example, autoimmune enhancement therapy involves extracting the subject's own peripheral blood-derived natural killer cells, cytotoxic T lymphocytes, epithelial cells, and other related immune cells, expanding these cells in vitro, and then re-injecting these cells into the subject's body. In some therapies, cells (e.g., T cells) are genetically modified and expanded in vitro before being returned to the same patient. Chimeric Antigen Receptor T-Cell Therapy (CAR-T) involves collecting T cells from a subject and then infecting the T cells with a retrovirus containing a copy of the TCR gene. The TCR gene is specialized to recognize tumor antigens (e.g., chimeric antigen receptors or CARs). The virus integrates the receptor into the T cell genome. The cells are nonspecifically expanded and/or stimulated. The cells are then re-injected to generate an immune response against tumor cells. With the approval of the first CAR-T therapy and the participation of numerous commercial companies in multiple clinical trials, this field has become commercially vibrant and has been identified as a promising future for immunotherapy. In a field where new clinical trials are advancing almost daily, there has been a steadily increasing need for reliable and reproducible potency testing for these therapeutic immune cells. The industry "gold standard" for testing the effector function of immune cells is the chromium release assay, which was developed in the 1960s and remains in use despite concerns regarding the use of radioactive materials and variability caused by target tumor cells. An available alternative is the calcein-based assay, which is triggered by the use of different tumor targets and still exhibits significant variability due to the capture of calcein within the apoptotic bodies of the tumor targets. Although there have been other efforts to develop different methods to visually observe the effector functions of these immune cells, these methods still utilize targeted tumor cells. Another surrogate method for verifying the effector functions of immune cells is to identify the cytokines produced by these cells; all conventional methods for this purpose involve inducing cytokine production from immune cells using targeted tumor cells. The use of targeted tumor cells adds biological variability to all of these tests due to variability between tumor cell types. Furthermore, these assays require tedious setups that introduce batch effects into the assay. Batch effects arise from target cell conditions, inter-individual variability in plate loading, plate conditions, variability of various reagents, and readout variability. There is a clear need for immune cell efficacy assays that can eliminate all of these variabilities and generate reliable and reproducible results. Reliable and