CN113848324A

CN113848324A - Method for assessing efficacy of infusion of NK cells in a subject

Info

Publication number: CN113848324A
Application number: CN202010597934.3A
Authority: CN
Inventors: 何晓文; 臧爱萍; 郭昊; 凌有国; 杨焕凤
Original assignee: Oricell Therapeutics Co Ltd
Current assignee: Oricell Therapeutics Co Ltd
Priority date: 2020-06-28
Filing date: 2020-06-28
Publication date: 2021-12-28

Abstract

本申请涉及一种用于评估在受试者中输注NK细胞的效力的试剂盒，以及检测试剂在制备用于评估在受试者中输注NK细胞的效力的试剂和/或试剂盒中的用途，本申请还提供了评估NK细胞产品效力的方法和优化NK细胞产品的制备的方法，以及相应的系统。The present application relates to a kit for evaluating the efficacy of infusing NK cells in a subject, and detection reagents in the preparation of reagents and/or kits for evaluating the efficacy of infusing NK cells in a subject The application also provides methods for assessing the efficacy of NK cell products and methods for optimizing the preparation of NK cell products, as well as corresponding systems.

Description

Method for assessing the efficacy of infusion of NK cells in a subject

Technical Field

The present application relates to the field of biomedicine, and in particular to a kit for assessing the efficacy of infusion of NK cells in a subject.

Background

Aging is a gradual loss of physiological function and is always accompanied by a decline in the function of the immune system, particularly in the T cell response, thus leading to an increased incidence of infection and cancer, and many diseases may develop with age, including cancer, T2DM (type 2 diabetes), neurodegenerative diseases and cardiovascular diseases.

In general, aging is associated with a gradual decline in immune system function, with Natural Killer (NK) cells and T cells being key components of innate and adaptive immunity, respectively. NK cells characterized by expression of CD16 and CD56 play a crucial role in the first line of defense against viral infections and cancer cells. Furthermore, due to repeated antigenic stimulation throughout life, aging is always accompanied by an increase in the accumulation of aged and exhausted T cells, which in turn leads to an impairment of T cell-mediated responses.

Clinical studies have shown that the number of aged and depleted T cells in patients receiving antiviral therapy and chemotherapy is reversed, however, there is little understanding of whether or how T cell aging and depletion can be manipulated in healthy people.

Disclosure of Invention

In one aspect, the present application provides a kit for assessing the efficacy of infusion of NK cells in a subject, the kit comprising a detection reagent and optionally instructions for use, wherein the detection reagent comprises one or more selected from the group consisting of:

1) an agent for identifying the level and/or activity of one or more cells selected from the group consisting of: tfh cell, CD4⁺CD28^-Cell, CD4⁺CD57⁺Cell, CD4⁺CD28^-CD57⁺Cell, CD4⁺CD28^-KLRG1⁺Cell, CD8⁺CD28^-Cell, CD8⁺CD57⁺Cell, CD8⁺CD28^-CD57⁺Cell, CD8⁺CD28^-KLRG1⁺Cell, CD4⁺PD-1⁺Cell, CD4⁺TIM3⁺Cell, CD4⁺PDL1⁺Cell, CD4⁺LAG3⁺Cell, CD4⁺CTLA-4⁺Cell, CD8⁺PD-1⁺Cell, CD8⁺TIM3⁺Cell, CD8⁺PDL1⁺Cell, CD8⁺LAG3⁺Cells and CD8⁺CTLA- 4⁺A cell.

2) For detecting one or more selected from the group consisting ofAn agent for the level and/or activity of one or more characteristic expression products of a cell: tfh cell, CD4⁺CD28^-Cell, CD4⁺CD57⁺Cell, CD4⁺CD28^-CD57⁺Cell, CD4⁺CD28^-KLRG1⁺Cell, CD8⁺CD28^-Cell, CD8⁺CD57⁺Cell, CD8⁺CD28^-CD57⁺Cell, CD8⁺CD28^- KLRG1⁺Cell, CD4⁺PD-1⁺Cell, CD4⁺TIM3⁺Cell, CD4⁺PDL1⁺Cell, CD4⁺LAG3⁺Cell, CD4⁺CTLA-4⁺Cell, CD8⁺PD-1⁺Cell, CD8⁺TIM3⁺Cell, CD8⁺PDL1⁺Cell, CD8⁺LAG3⁺Cells and CD8⁺CTLA-4⁺A cell.

3) An agent for detecting the level and/or activity of one or more genes in a set of genes characteristic of one or more cells selected from the group consisting of: tfh cell, CD4⁺CD28^-Cell, CD4⁺CD57⁺Cell, CD4⁺CD28^-CD57⁺Cell, CD4⁺CD28^-KLRG1⁺Cell, CD8⁺CD28^-Cell, CD8⁺CD57⁺Cell, CD8⁺CD28^-CD57⁺Cell, CD8⁺CD28^-KLRG1⁺Cell, CD4⁺PD-1⁺Cell, CD4⁺TIM3⁺Cell, CD4⁺PDL1⁺Cell, CD4⁺LAG3⁺Cell, CD4⁺CTLA-4⁺Cell, CD8⁺PD-1⁺Cell, CD8⁺TIM3⁺Cell, CD8⁺PDL1⁺Cell, CD8⁺LAG3⁺Cells and CD8⁺CTLA-4⁺A cell.

4) An agent for detecting the level and/or activity of one or more cytokines selected from the group consisting of: IL-6, IL-1 α, IL-8, IL-7, MMP1, MIP-1 β, and MIP-1 α.

In certain embodiments, the method comprises the steps of: measuring a test indicator of a subject to obtain a pre-infusion measurement of the test indicator; 2) infusing NK cells into the subject; 3) after the infusion, measuring the detection index of the subject again to obtain a post-infusion measurement of the detection index; 4) comparing the post-infusion measurements of the detection indicators with the corresponding pre-infusion measurements to perform the assessment.

In certain embodiments, the infusion is an autologous infusion.

In certain embodiments, the number of NK cells infused per said infusion is 1 x 10⁷～1×10¹²。

In certain embodiments, the subject is infused with 1 or more NK cells.

In another aspect, the present application provides the use of the detection reagent in the preparation of a reagent and/or kit for assessing the efficacy of infusion of NK cells in a subject.

In another aspect, the present application provides a method of assessing the potency of an NK cell product, the method comprising the steps of: obtaining a post-infusion detection index measurement from a subject infused with the NK cell product; and assessing the potency of the NK cell product according to the post-infusion detection indicator measurement; wherein the detection index comprises one or more selected from the group consisting of: 1) the level and/or activity of one or more cells selected from the group consisting of: tfh cell, CD4⁺CD28^-Cell, CD4⁺CD57⁺Cell, CD4⁺CD28^-CD57⁺Cell, CD4⁺CD28^-KLRG1⁺Cell, CD8⁺CD28^-Cell, CD8⁺CD57⁺Cell, CD8⁺CD28^-CD57⁺Cell, CD8⁺CD28^-KLRG1⁺Cell, CD4⁺PD-1⁺Cell, CD4⁺TIM3⁺Cell, CD4⁺PDL1⁺Cell, CD4⁺LAG3⁺Cell, CD4⁺CTLA-4⁺Cell, CD8⁺PD-1⁺Cell, CD8⁺TIM3⁺Cell, CD8⁺PDL1⁺Cell, CD8⁺LAG3⁺Cells and CD8⁺CTLA-4⁺The cells are cultured in a medium such as water,

2) the level and/or activity of one or more characteristic expression products of one or more cells selected from the group consisting of: tfh cell, CD4⁺CD28^-Cell, CD4⁺CD57⁺Cell, CD4⁺CD28^-CD57⁺Cell, CD4⁺CD28^-KLRG1⁺Cell, CD8⁺CD28^-Cell, CD8⁺CD57⁺Cell, CD8⁺CD28^-CD57⁺Cell, CD8⁺CD28^-KLRG1⁺Cell, CD4⁺PD-1⁺Cell, CD4⁺TIM3⁺Cell, CD4⁺PDL1⁺Cell, CD4⁺LAG3⁺Cell, CD4⁺CTLA-4⁺Cell, CD8⁺PD-1⁺Cell, CD8⁺TIM3⁺Cell, CD8⁺PDL1⁺Cell, CD8⁺LAG3⁺Cells and CD8⁺CTLA- 4⁺The cells are cultured in a medium such as water,

3) the level and/or activity of one or more genes in a set of genes characteristic of one or more cells selected from the group consisting of: tfh cell, CD4⁺CD28^-Cell, CD4⁺CD57⁺Cell, CD4⁺CD28^-CD57⁺Cell, CD4⁺CD28^-KLRG1⁺Cell, CD8⁺CD28^-Cell, CD8⁺CD57⁺Cell, CD8⁺CD28^-CD57⁺Cell, CD8⁺CD28^-KLRG1⁺Cell, CD4⁺PD-1⁺Cell, CD4⁺TIM3⁺Cell, CD4⁺PDL1⁺Cell, CD4⁺LAG3⁺Cell, CD4⁺CTLA-4⁺Cell, CD8⁺PD-1⁺Cell, CD8⁺TIM3⁺Cell, CD8⁺PDL1⁺Cells, CD8⁺LAG3⁺Cells and CD8⁺CTLA-4⁺A cell, and,

4) the level and/or activity of one or more cytokines selected from the group consisting of: IL-6, IL-1 α, IL-8, IL-7, MMP1, MIP-1 β, and MIP-1 α;

wherein a decrease in the post-infusion assay indicator measurement compared to a reference result indicates the presence or increase in potency of the NK cell product.

In another aspect, the present application provides a method comprising the steps of: obtaining a sample of a subject infused with an NK cell product, determining a post-infusion detection indicator measurement in the sample, comparing the obtained post-infusion detection indicator measurement to a reference result to determine a difference between the post-infusion detection indicator measurement and the reference result; and recording the difference, wherein the detection index comprises one or more selected from the group consisting of:

1) the level and/or activity of one or more cells selected from the group consisting of: tfh cell, CD4⁺CD28^-Cell, CD4⁺CD57⁺Cell, CD4⁺CD28^-CD57⁺Cell, CD4⁺CD28^-KLRG1⁺Cell, CD8⁺CD28^-Cell, CD8⁺CD57⁺Cell, CD8⁺CD28^-CD57⁺Cell, CD8⁺CD28^-KLRG1⁺Cell, CD4⁺PD-1⁺Cell, CD4⁺TIM3⁺Cell, CD4⁺PDL1⁺Cell, CD4⁺LAG3⁺Cell, CD4⁺CTLA-4⁺Cell, CD8⁺PD-1⁺Cell, CD8⁺TIM3⁺Cell, CD8⁺PDL1⁺Cell, CD8⁺LAG3⁺Cells and CD8⁺CTLA-4⁺The cells are cultured in a medium such as water,

3) the level and/or activity of one or more genes in a set of genes characteristic of one or more cells selected from the group consisting of: tfh cell, CD4⁺CD28^-Cell, CD4⁺CD57⁺Cell, CD4⁺CD28^-CD57⁺Cell, CD4⁺CD28^-KLRG1⁺Cell, CD8⁺CD28^-Cell, CD8⁺CD57⁺Cell, CD8⁺CD28^-CD57⁺Cell, CD8⁺CD28^-KLRG1⁺Cell, CD4⁺PD-1⁺Cell, CD4⁺TIM3⁺Cell, CD4⁺PDL1⁺Cell, CD4⁺LAG3⁺Cell, CD4⁺CTLA-4⁺Cell, CD8⁺PD-1⁺Cell, CD8⁺TIM3⁺Cell, CD8⁺PDL1⁺Cell, CD8⁺LAG3⁺Cells and CD8⁺CTLA-4⁺A cell, and,

4) the level and/or activity of one or more cytokines selected from the group consisting of: IL-6, IL-1 α, IL-8, IL-7, MMP1, MIP-1 β, and MIP-1 α.

In another aspect, the present application provides a method of optimizing the production of NK cell products, said method comprising the steps of: 1) Obtaining NK cells; 2) activating NK cells in vitro; assessing the potency of the NK cells by detecting an indicator, wherein the indicator is selected from the group consisting of:

a) the level and/or activity of one or more cells selected from the group consisting of: tfh cell, CD4⁺CD28^-Cell, CD4⁺CD57⁺Cell, CD4⁺CD28^-CD57⁺Cell, CD4⁺CD28^-KLRG1⁺Cell, CD8⁺CD28^-Cell, CD8⁺CD57⁺Cell, CD8⁺CD28^-CD57⁺Cell, CD8⁺CD28^-KLRG1⁺Cell, CD4⁺PD-1⁺Cell, CD4⁺TIM3⁺Cell, CD4⁺PDL1⁺Cell, CD4⁺LAG3⁺Cell, CD4⁺CTLA-4⁺Cell, CD8⁺PD-1⁺Cell, CD8⁺TIM3⁺Cell, CD8⁺PDL1⁺Cell, CD8⁺LAG3⁺Cells and CD8⁺CTLA-4⁺The cells are cultured in a medium such as water,

b) the level and/or activity of one or more characteristic expression products of one or more cells selected from the group consisting of: tfh cell, CD4⁺CD28^-Cell, CD4⁺CD57⁺Cell, CD4⁺CD28^-CD57⁺Cell, CD4⁺CD28^-KLRG1⁺Cell, CD8⁺CD28^-Cell, CD8⁺CD57⁺Cell, CD8⁺CD28^-CD57⁺Cell, CD8⁺CD28^-KLRG1⁺Cell, CD4⁺PD-1⁺Cell, CD4⁺TIM3⁺Cell, CD4⁺PDL1⁺Cell, CD4⁺LAG3⁺Cell, CD4⁺CTLA-4⁺Cell, CD8⁺PD-1⁺Cell, CD8⁺TIM3⁺Cell, CD8⁺PDL1⁺Cell, CD8⁺LAG3⁺Cells and CD8⁺CTLA- 4⁺The cells are cultured in a medium such as water,

c) one or more of the characteristic gene sets of one or more cells selected from the group consisting ofLevels and/or activities of various genes: tfh cell, CD4⁺CD28^-Cell, CD4⁺CD57⁺Cell, CD4⁺CD28^-CD57⁺Cell, CD4⁺CD28^-KLRG1⁺Cell, CD8⁺CD28^-Cell, CD8⁺CD57⁺Cell, CD8⁺CD28^-CD57⁺Cell, CD8⁺CD28^-KLRG1⁺Cell, CD4⁺PD-1⁺Cell, CD4⁺TIM3⁺Cell, CD4⁺PDL1⁺Cell, CD4⁺LAG3⁺Cell, CD4⁺CTLA-4⁺Cell, CD8⁺PD-1⁺Cell, CD8⁺TIM3⁺Cell, CD8⁺PDL1⁺Cell, CD8⁺LAG3⁺Cells and CD8⁺CTLA-4⁺A cell, and,

d) the level and/or activity of one or more cytokines selected from the group consisting of: IL-6, IL-1 α, IL-8, IL-7, MMP1, MIP-1 β, and MIP-1 α.

In certain embodiments, the NK cell is derived from a cell selected from the group consisting of: peripheral blood cells, bone marrow cells, and cord blood cells.

Other aspects and advantages of the present application will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application have been shown and described in the following detailed description. As those skilled in the art will recognize, the disclosure of the present application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention as it is directed to the present application. Accordingly, the descriptions in the drawings and the specification of the present application are illustrative only and not limiting.

Drawings

The specific features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates will be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings. The brief description of the drawings is as follows:

FIG. 1 shows the changes in the levels of Th1, Th2, Th17, Tfh, and Treg cells in subjects following infusion of NK cells;

FIGS. 2 and 3 show the decrease of senescent T cells after infusion of NK cells;

FIG. 4 shows the trend associated with a positive baseline for senescent T cells, with donors listed as increasing baselines from left to right on the X-axis;

FIG. 5 shows a significant reduction in expression of T cell senescence indicators following infusion of NK cells;

FIG. 6 shows that NK cell infusion significantly reduces expression of T cell senescence indicators from different genders;

FIG. 7 shows that infusion of NK cells of different formulation types significantly reduced expression of T cell senescence indicators;

FIG. 8 shows depletion of T cells following infusion of NK cells;

figure 9 shows the trend associated with a positive baseline for depleted T cells, with donors listed as increasing baselines from left to right on the X-axis;

figure 10 shows a significant reduction in expression of T cell depletion indicators following infusion of NK cells;

FIG. 11 shows that infusion of NK cells significantly reduces expression of T cell depletion indicators from different sexes;

figure 12 shows that infusion of NK cells of different formulation types significantly reduced expression of T cell depletion indicators;

FIGS. 13 and 14 show a reduction in key senescence-associated secreted phenotype (SASP) factors following NK cell infusion;

FIG. 15 shows the relative trend of a positive baseline for SASP, with donors listed as increasing baselines from left to right on the X-axis;

FIG. 16 shows that NK cell infusion significantly reduces the expression level of senescence-associated secreted factors in subjects

FIG. 17 shows that NK cell infusion significantly reduces the expression levels of senescence-associated secreted factors in subjects from different sexes;

figure 18 shows that infusion of NK cells of different formulation types significantly reduced the expression levels of senescence-associated secreted factors in subjects.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification.

Definition of terms

In the present application, the term "NK cell", also known as "natural killer cell" or "natural killer cell", generally refers to a cell with large granules in the cytoplasm. NK cells are developed by bone marrow lymphoid stem cells and can be differentiated and developed depending on bone marrow or thymus microenvironment. It is mainly distributed in peripheral blood and spleen, and is also present in small amounts in lymph nodes and other tissues. NK cells may express CD16 and/or CD 56.

In the present application, the term "level and/or activity of a cell" may generally include the number of proliferations of the cell, the physiological functional activity of the cell, the level and/or activity of one or more characteristic expression products, and/or the level and/or activity of one or more genes in a characteristic gene set.

In the present application, the term "characteristic expression product" generally refers to a product that is differentially expressed (e.g., highly expressed, normally expressed, poorly expressed, non-expressed, compared to cells of different species, subpopulations) on cells of different species, subpopulations, and can be used to distinguish between cells of different species, subpopulations. The characteristic expression product may comprise a cytokine, a transcription factor, an antigen and/or a ligand, etc. For example, characteristic expression products may include, but are not limited to: CD28, CD4, CD57, KLRG1, PD-1, TIM3, PD-L1, LAG3 and/or CTLA-4.

In the present application, the term "characteristic gene set" generally refers to genes that differ in their expression levels and/or activities in different species, subpopulations of cells. By comparing the differences in expression of one or more genes in a gene group characteristic of a cell, different species, subpopulations of cells can be distinguished.

In the present application, the term "Tfh cells", also referred to as "follicular helper T cells", is typically a subpopulation of CD4+ T cells, and Tfh cell characteristic markers may include, but are not limited to, CXCR5+, PD-1+, and/or ICOS +. At the same time, the transcription factor BCL-6 can be expressed.

In the present application, the term "Th 1 cell" generally refers to a helper T cell whose primary transcription factor for immunization may include STAT4, T-beta.

In the present application, the term "Th 2 cell" generally refers to a helper T cell. The major cytokines responsible for its immune function include IL-4, IL-5 and IL-13, and the major transcription factors may include STAT6 and GATA.

In the present application, the term "Th 17 cell" generally refers to a helper T cell. Cytokines that act as immunizations may include IL-1, IL-6, and TNF α, and major transcription factors may include STAT3, ROR γ.

In the present application, the term "Treg cells", also known as "regulatory T cells", generally refers to a subset of T cells that control autoimmune reactivity in vivo. It may take many forms and may express one or more of CD4, CD25, FOXP3, IL-10 and TGF-beta.

In the present application, the term "depleted T cells" generally includes CD4 expressing PD-1⁺T-cells, CD4 expressing TIM3⁺T-cells, PD-L1 expressing CD4⁺T-cells, CD4 expressing TIM3⁺T-cell, CTLA-4 expressing CD4⁺T cells, PD-1 expressing CD8⁺T-cells, PD-L1 expressing CD8⁺T-cells, CD8 expressing TIM3⁺T-cell, CTLA-4 expressing CD8⁺T cells and/or CD8 expressing TIM3⁺T cells.

In the present application, the term "senescent T cells" generally includes CD28^-CD4⁺T cell, CD57⁺CD4⁺T cell, CD28^-CD57⁺CD4⁺T cell, CD28^-KLRG1⁺CD4⁺T cell, CD28^-CD8⁺T-cells, CD8 expressing CD57⁺T cells, CD28-CD57⁺CD8⁺T cells and/or CD28^-KLRG1⁺CD8⁺T cells.

In the present application, the term "CD 4⁺T cell' TongbaiRefers to a T cell expressing CD4 protein. CD4⁺The T cells may be helper T cells (Th cells), for example, subsets of Th-1 (helper T cell-1), Th-2 (helper T cell-2), Th-9 (helper T cell-9), Th-17 (helper T cell-17), regulatory T cells and follicular helper T cells. CD4⁺The T cells may be regulatory T cells (Treg cells), including thymic regulatory T cells and peripheral regulatory T cells.

In the present application, the term "CD 8⁺T cell "generally refers to a T cell that expresses CD8 protein. CD8⁺T cells are typically cytotoxic T cells that can kill cancer cells, virus-infected cells, and other damaged cells.

In the present application, the term "PD-1" generally refers to the programmed death receptor-1. Also known as CD279, is a protein on the cell surface that regulates the immune system's response to human cells by down-regulating the immune system and promoting autoimmune function. Can be used as a marker of exhausted cells.

In the present application, the term "PD-L1" generally refers to apoptosis-ligand 1, also known as surface antigen cluster of differentiation 274 or B7 homolog 1, encoded by the CD274 gene. PD-L1 is generally associated with suppression of the immune system. Can be used as a marker of exhausted cells.

In the present application, the term "TIM 3" generally refers to T cell immunoglobulin mucin 3, also known as HAVCR 2. TIM3 is generally in CD4⁺Th1 cell, CD8⁺Tc1 cells, Th17 cells, regulatory T cells and innate immune cells. Can be used as a marker of exhausted cells.

In the present application, the term "CD 57", also known as galactosylgalactosylxylosylprotein 3- β -glucuronidase 1(B3GAT1), HNK1 or LEU7, is commonly used to identify terminally differentiated senescent cells with reduced proliferative capacity and altered functional properties.

In the present application, the term "CD 28" generally refers to a protein expressed on T cells that provides a costimulatory signal required for T cell activation and survival. CD28 may be expressed less or not on senescent cells.

In the present application, the techniqueThe term "KLRG 1" generally refers to member 1 of the killer lectin-like receptor subfamily G, belonging to the family of killer lectin-like receptors (KLR). KLRG1 is generally a lymphocyte co-suppressor or immune checkpoint receptor, effector and effector memory CD8 that is predominantly differentiated in late stages⁺Expression on T and NK cells. Can be used as a marker of aging T cells.

In the present application, the superscript "+" in the description of a cell indicates that the cell may express a protein prior to "+" or that expression of the protein may be detected using existing techniques, and the superscript "-" indicates that the cell may not express a protein prior to "-" or that expression of the protein may not be substantially detected using existing techniques. For example, "CD 4⁺CD28^-Cell "means a cell that does not express CD28 and CD 4.

In the present application, the term "senescing-associated secreted phenotype (SASP)" generally refers to a pro-inflammatory phenotype in which senescent cells exhibit, and in which the expression levels of inflammatory cytokines may be significantly elevated. SASPs can mediate non-cell autonomous aging effects, including beneficial and deleterious effects. The SASPs may comprise cytokines, chemokines, growth factors and proteases. For example, IL-6 (Interleukin-6), IL-7 (Interleukin-7), IL-8 (Interleukin-8), IL-1 α (Interleukin 1 α), MMP1 (matrix Metalloproteinase 1), MIP-1 α (macrophage inflammatory protein 1 α), and MIP-1 β (macrophage inflammatory protein 1 β) can be included, but are not limited thereto.

Detailed Description

Detection reagent and kit

In one aspect, the present application provides an agent for identifying the level and/or activity of a Tfh cell. In certain instances, the agent for identifying the level and/or activity of a Tfh cell can comprise an agent for detecting the level and/or activity of one or more expression products characteristic of a Tfh cell and/or an agent for detecting the level and/or activity of one or more genes in a characteristic gene set of a Tfh cell. In certain instances, the agent may comprise one or more selected from the group consisting of: a reagent for detecting CD3, a reagent for detecting CD4, a reagent for detecting CD8, a reagent for detecting CXCR5, a reagent for detecting CD40, a reagent for detecting CD40L, a reagent for detecting ICOS (inductively T-cell costimulator), a reagent for detecting ICOSL, a reagent for detecting PD-1, a reagent for detecting CD200, a reagent for detecting BTLA (B-and T-lymphocyte activator), a reagent for detecting OX40, a reagent for detecting SAP (SAP amyloid P), a reagent for detecting FoxP3 and a reagent for detecting transcription factor BCL 6.

Provided herein are agents for identifying the level and/or activity of Th1 cells. In certain instances, the agent for identifying the level and/or activity of a Th1 cell may comprise an agent for detecting the level and/or activity of one or more characteristic expression products of a Th1 cell and/or an agent for detecting the level and/or activity of one or more genes in a characteristic gene set of a Th1 cell. In certain instances, the agent may comprise one or more selected from the group consisting of: a reagent for detecting CD3, a reagent for detecting CD4, a reagent for detecting CD8, a reagent for detecting IL-2, a reagent for detecting IFN-gamma, a reagent for detecting IFN-alpha, a reagent for detecting TNF-beta, a reagent for detecting GM-CSF, a reagent for detecting IL12, a reagent for detecting CXCR-3, a reagent for detecting CCR-5, a reagent for detecting STAT4, a reagent for detecting T-beta, a reagent for detecting beta 2 adrenergic receptor, a reagent for detecting lymphocyte activator gene 3, a reagent for detecting CDw150, a reagent for detecting CD162, a reagent for detecting CD49f, a reagent for detecting CD29 and a reagent for detecting TIM 3.

Provided herein are agents for identifying the level and/or activity of Th2 cells. In certain instances, the agent for identifying the level and/or activity of a Th2 cell may comprise an agent for detecting the level and/or activity of one or more characteristic expression products of a Th2 cell and/or an agent for detecting the level and/or activity of one or more genes in a characteristic gene set of a Th2 cell. In certain instances, the agent may comprise one or more selected from the group consisting of: a reagent for detecting CD3, a reagent for detecting CD4, a reagent for detecting CD8, a reagent for detecting STAT6, a reagent for detecting GATA, a reagent for detecting IL-4, a reagent for detecting IL-5, a reagent for detecting IL-10, a reagent for detecting IL-13, a reagent for detecting IL-9, a reagent for detecting CCR-3, a reagent for detecting CCR-4, a reagent for detecting CCR-7, a reagent for detecting CCR-8, a reagent for detecting CD30, a reagent for detecting CDw150 and a reagent for detecting TIM 1.

Provided herein are agents for identifying the level and/or activity of Th17 cells. In certain instances, the agent for identifying the level and/or activity of a Th17 cell may comprise an agent for detecting the level and/or activity of one or more characteristic expression products of a Th17 cell and/or an agent for detecting the level and/or activity of one or more genes in a characteristic gene set of a Th17 cell. In certain instances, the agent may comprise one or more selected from the group consisting of: a reagent for detecting CD3, a reagent for detecting CD4, a reagent for detecting CD8, a reagent for detecting IL-17a-f, a reagent for detecting IL-21, a reagent for detecting IL-22, a reagent for detecting IL-26, a reagent for detecting GM-CSF, a reagent for detecting TNF-alpha, a reagent for detecting ROR gamma, a reagent for detecting STAT3, a reagent for detecting IRF4, a reagent for detecting CCR-4, a reagent for detecting CCR-6, a reagent for detecting CCR-2, a reagent for detecting CCR-5 and a reagent for detecting CXCR 3.

Provided herein are agents for identifying the level and/or activity of Treg cells. In certain instances, the agent for identifying the level and/or activity of a Treg cell may comprise an agent for detecting the level and/or activity of one or more expression products characteristic of a Treg cell and/or an agent for detecting the level and/or activity of one or more genes in a characteristic gene set of a Treg cell. In certain instances, the agent may comprise one or more selected from the group consisting of: a reagent for detecting CD3, a reagent for detecting CD4, a reagent for detecting CD8, a reagent for detecting CD25, a reagent for detecting CD127, a reagent for detecting FOXP3, a reagent for detecting IL-10, a reagent for detecting TGF-beta, a reagent for detecting IL-4, a reagent for detecting STAT5, a reagent for detecting GITR, a reagent for detecting AITR, a reagent for detecting FR4, a reagent for detecting CD39, a reagent for detecting CD73, and a reagent for detecting CD 101.

The present application provides methods for authenticating CD4⁺CD28^-CD57⁺A cellular level and/or activity of the agent. In some cases, the method is used to authenticate CD4⁺CD28^-CD57⁺Agents for the level and/or activity of cells may be included for detecting CD4⁺CD28^-CD57⁺Agents for detecting the level and/or activity of one or more expression products characteristic of a cell and/or for detecting CD4⁺CD28^-CD57⁺The characteristic genes of the cell are agents that concentrate the level and/or activity of one or more genes. In certain instances, the agent may comprise one or more selected from the group consisting of: a reagent for detecting CD3, a reagent for detecting CD4, a reagent for detecting CD8, a reagent for detecting B7 family (for example, B7 family members B7-1, B7-2, B7-DC, B7-H1), a reagent for detecting CD27, a reagent for detecting CD28, and a reagent for detecting CD 57.

The present application provides methods for authenticating CD8⁺CD28^-CD57⁺A cellular level and/or activity of the agent. In some cases, the method is used to authenticate CD8⁺CD28^-CD57⁺Agents for the level and/or activity of cells may be included for detecting CD8⁺CD28^-CD57⁺Agents for detecting the level and/or activity of one or more expression products characteristic of a cell and/or for detecting CD8⁺CD28^-CD57⁺The characteristic genes of the cell are agents that concentrate the level and/or activity of one or more genes. In certain instances, the agent may comprise one or more selected from the group consisting of: a reagent for detecting CD3, a reagent for detecting CD4, a reagent for detecting CD8, a reagent for detecting B7, a reagent for detecting CD27, a reagent for detecting CD28 and a reagent for detecting CD 57.

The present application provides methods for authenticating CD4⁺CD28^-KLRG1⁺A cellular level and/or activity of the agent. In some cases, the method is used to authenticate CD4⁺CD28^-KLRG1⁺Agents for the level and/or activity of cells may be included for detecting CD4⁺CD28^- KLRG1⁺Agents for detecting the level and/or activity of one or more expression products characteristic of a cell and/or for detecting CD4⁺CD28^-KLRG1⁺The characteristic genes of the cell are agents that concentrate the level and/or activity of one or more genes. In some casesIn form, the agent may comprise one or more selected from the group consisting of: a reagent for detecting CD3, a reagent for detecting CD4, a reagent for detecting CD8, a reagent for detecting B7, a reagent for detecting CD27, a reagent for detecting CD28 and a reagent for detecting KLRG 1.

The present application provides methods for authenticating CD8⁺CD28^-KLRG1⁺A cellular level and/or activity of the agent. In some cases, the method is used to authenticate CD8⁺CD28^-KLRG1⁺Agents for the level and/or activity of cells may be included for detecting CD8⁺CD28^- KLRG1⁺Agents for detecting the level and/or activity of one or more expression products characteristic of a cell and/or for detecting CD8⁺CD28^-KLRG1⁺The characteristic genes of the cell are agents that concentrate the level and/or activity of one or more genes. In certain instances, the agent may comprise one or more selected from the group consisting of: a reagent for detecting CD3, a reagent for detecting CD4, a reagent for detecting CD8, a reagent for detecting B7, a reagent for detecting CD27, a reagent for detecting CD28 and a reagent for detecting KLRG 1.

The present application provides methods for authenticating CD4⁺PD-1⁺A cellular level and/or activity of the agent. In some cases, the method is used to authenticate CD4⁺PD-1⁺Agents for the level and/or activity of cells may be included for detecting CD4⁺PD-1⁺Agents for detecting the level and/or activity of one or more expression products characteristic of a cell and/or for detecting CD4⁺PD-1⁺The characteristic genes of the cell are agents that concentrate the level and/or activity of one or more genes. In certain instances, the agent may comprise one or more selected from the group consisting of: a reagent for detecting CD3, a reagent for detecting CD4, a reagent for detecting CD8, a reagent for detecting PD-1, a reagent for detecting PD-L1, a reagent for detecting TIM3, a reagent for detecting LAG3 and a reagent for detecting CLTA 4.

The present application provides methods for authenticating CD8⁺PD-1⁺A cellular level and/or activity of the agent. In some cases, the method is used to authenticate CD8⁺PD-1⁺Agents for the level and/or activity of cells may be included for detecting CD8⁺PD-1⁺Agents for detecting the level and/or activity of one or more expression products characteristic of a cell and/or for detecting CD8⁺PD-1⁺The characteristic genes of the cell are agents that concentrate the level and/or activity of one or more genes. In certain instances, the agent may comprise one or more selected from the group consisting of: a reagent for detecting CD3, a reagent for detecting CD4, a reagent for detecting CD8, a reagent for detecting PD-1, a reagent for detecting PD-L1, a reagent for detecting TIM3, a reagent for detecting LAG3 and a reagent for detecting CLTA 4.

The present application provides methods for authenticating CD4⁺TIM3⁺A cellular level and/or activity of the agent. In some cases, the method is used to authenticate CD4⁺TIM3⁺Agents for the level and/or activity of cells may be included for detecting CD4⁺TIM3⁺Agents for detecting the level and/or activity of one or more expression products characteristic of a cell and/or for detecting CD4⁺TIM3⁺The characteristic genes of the cell are agents that concentrate the level and/or activity of one or more genes. In certain instances, the agent may comprise one or more selected from the group consisting of: a reagent for detecting CD3, a reagent for detecting CD4, a reagent for detecting CD8, a reagent for detecting PD-1, a reagent for detecting PD-L1, a reagent for detecting TIM3, a reagent for detecting LAG3 and a reagent for detecting CLTA 4.

The present application provides methods for authenticating CD8⁺TIM3⁺A cellular level and/or activity of the agent. In some cases, the method is used to authenticate CD8⁺TIM3⁺Agents for the level and/or activity of cells may be included for detecting CD8⁺TIM3⁺Agents for detecting the level and/or activity of one or more expression products characteristic of a cell and/or for detecting CD8⁺TIM3⁺The characteristic genes of the cell are agents that concentrate the level and/or activity of one or more genes. In certain instances, the agent may comprise one or more selected from the group consisting of: a reagent for detecting CD3, a reagent for detecting CD4, a reagent for detecting CD8, a reagent for detecting PD-1, a reagent for detecting PD-L1, a reagent for detecting TIM3, a reagent for detecting LAG3 and a reagent for detecting CLTA 4.

The present application provides an agent for identifying IL-6 levels and/or activity, which agent may comprise one or more selected from the group consisting of: primers for amplifying IL-6 and IL-6 antibody.

The present application provides an agent for identifying IL-1 α levels and/or activity, which agent may comprise one or more selected from the group consisting of: primers for amplifying IL-1 alpha and IL-1 alpha antibody.

The present application provides an agent for identifying IL-8 levels and/or activity, which agent may comprise one or more selected from the group consisting of: primers for amplifying IL-8 and IL-8 antibody.

The present application provides an agent for identifying IL-7 levels and/or activity, which agent may comprise one or more selected from the group consisting of: primers for amplifying IL-7 and IL-7 antibody.

Provided herein are agents for identifying MMP1 levels and/or activities, which agents can comprise one or more selected from the group consisting of: primers to amplify MMP1 and MMP1 antibody.

The present application provides an agent for identifying MIP-1 β levels and/or activity, the agent may comprise one or more selected from the group consisting of: a primer for amplifying MIP-1 beta and an antibody of MIP-1 beta.

The present application provides an agent for identifying MIP-1 a levels and/or activity, the agent may comprise one or more selected from the group consisting of: a primer for amplifying MIP-1 alpha and an antibody of MIP-1 alpha.

The detection reagent described herein may further comprise one or more selected from the group consisting of: 1) for identifying CD4⁺CD28^-An agent of the level and/or activity of the cell; 2) for detecting CD4⁺CD28^-An agent that characterizes the level and/or activity of one or more expression products of the cell; 3) for detecting CD4⁺CD28^-An agent that concentrates the level and/or activity of one or more genes in one or more cell characteristic genes of the cell; 4) for identifying CD4⁺CD57⁺An agent of the level and/or activity of the cell; 5) for detecting CD4⁺CD57⁺An agent that characterizes the level and/or activity of one or more expression products of the cell; 6) for detecting CD4⁺CD57⁺One of the cellsAn agent that concentrates the levels and/or activities of one or more genes in a set of genes characteristic of one or more cells; 7) for identifying CD8⁺CD28^-An agent of the level and/or activity of the cell; 8) for detecting CD8⁺CD28^-An agent that characterizes the level and/or activity of one or more expression products of the cell; 9) for detecting CD8⁺CD28^-An agent that concentrates the level and/or activity of one or more genes in one or more cell characteristic genes of the cell; 10) for identifying CD8⁺CD57⁺An agent of the level and/or activity of the cell; 11) for detecting CD8⁺CD57⁺An agent that characterizes the level and/or activity of one or more expression products of the cell; 12) for detecting CD8⁺CD57⁺An agent that concentrates the level and/or activity of one or more genes in one or more cell characteristic genes of the cell; 13) for identifying CD8⁺CD28^-CD57⁺An agent of the level and/or activity of the cell; 14) for detecting CD8⁺CD28^-CD57⁺An agent that characterizes the level and/or activity of one or more expression products of the cell; 15) for detecting CD8⁺CD28^-CD57⁺An agent that concentrates the level and/or activity of one or more genes in one or more cell characteristic genes of the cell; 16) for identifying CD8⁺CD28^-KLRG1⁺An agent of the level and/or activity of the cell; 17) for detecting CD8⁺CD28^-KLRG1⁺An agent that characterizes the level and/or activity of one or more expression products of the cell; 18) for detecting CD8⁺CD28^-KLRG1⁺An agent that concentrates the level and/or activity of one or more genes in one or more cell characteristic genes of the cell; 19) for identifying CD4⁺PDL1⁺An agent of the level and/or activity of the cell; 20) for detecting CD4⁺PDL1⁺An agent that characterizes the level and/or activity of one or more expression products of the cell; 21) for detecting CD4⁺PDL1⁺An agent that concentrates the level and/or activity of one or more genes in one or more cell characteristic genes of the cell; 22) for identifying CD4⁺LAG3⁺An agent of the level and/or activity of the cell; 23) for detecting CD4⁺LAG3⁺An agent that characterizes the level and/or activity of one or more expression products of the cell; 24) for detecting CD4⁺LAG3⁺An agent that concentrates the level and/or activity of one or more genes in one or more cell characteristic genes of the cell; 25) for identifying CD4⁺CTLA⁺An agent of the level and/or activity of the cell; 26) for detecting CD4⁺CTLA⁺An agent that characterizes the level and/or activity of one or more expression products of the cell; 27) for detecting CD4⁺CTLA⁺An agent that concentrates the level and/or activity of one or more genes in one or more cell characteristic genes of the cell; 28) for identifying CD8⁺PDL1⁺An agent of the level and/or activity of the cell; 29) for detecting CD8⁺PDL1⁺An agent that characterizes the level and/or activity of one or more expression products of the cell; 30) for detecting CD8⁺PDL1⁺An agent that concentrates the level and/or activity of one or more genes in one or more cell characteristic genes of the cell; 31) for identifying CD8⁺LAG3⁺An agent of the level and/or activity of the cell; 32) for detecting CD8⁺LAG3⁺An agent that characterizes the level and/or activity of one or more expression products of the cell; 33) for detecting CD8⁺LAG3⁺An agent that concentrates the level and/or activity of one or more genes in one or more cell characteristic genes of the cell; 34) for identifying CD8⁺CTLA⁺An agent of the level and/or activity of the cell; 35) for detecting CD8⁺CTLA⁺An agent that characterizes the level and/or activity of one or more expression products of the cell; and/or, 36) for detecting CD8⁺CTLA⁺The genes characteristic of one or more of the cells are concentrated in the agent of the level and/or activity of the one or more genes.

The reagents described herein may include any of the reagents available in the art for detecting the above targets. For example, the agent may include a small molecule, a protein, a nucleic acid.

In some cases, the reagents may include reagents that detect protein levels and/or activity. For example, the reagent may include an ELISA detection reagent, a FACS detection reagent, and a colorimetric detection reagent.

In certain instances, the reagents described herein can include an antibody (e.g., a first antibody, a second antibody, a biotinylated antibody, a fluorescently labeled antibody), an antigen, a standard, an enzyme (e.g., peroxidase, trypsin), a chromogenic reagent, an enzyme blocker (e.g., peroxidase blocker), an enzyme conjugate, a substrate solution (e.g., TMB), a signal enhancer, a fluorescent label (e.g., a fluorescent protein, a chemiluminescent label, a bioluminescent label), a biomarker, streptavidin, a fluorescent probe, and/or a biological enzyme substrate.

In some cases, the reagent may include a reagent that detects the level and/or activity of nucleic acid.

In certain instances, the reagents can include probes that specifically bind to the target being detected, primers that specifically amplify the target being detected, standards, negative controls, positive controls, nucleotides, deoxynucleotides, polymerases, endonucleases, dnazymes, rnases, fluorescent labels, nucleic acid labeling probes, and/or cell lysates.

In certain instances, the agents described herein can include agents that detect cell proliferation, agents that detect cell activity, and/or agents that detect apoptosis. The reagents described herein can also include carriers, biological buffers, surfactants, coagulants, preservatives, protectants, chelating agents, and/or solvents (e.g., water, ethanol).

The various reagents described herein can be placed in one or more suitable containers. For example, the reagents may be pre-mixed in the same vessel. As another example, the reagents may each be held in separate and distinct containers prior to use. The container described herein may comprise at least one vial, test tube, flask, bottle, syringe, or other container means.

The present application provides a kit for assessing the efficacy of infusion of NK cells in a subject, the kit comprising a detection reagent described herein and optionally instructions for use.

The detection reagent and the kit can be used for measuring the detection index of a subject. The detection index may comprise one or more selected from the group consisting of: 1) (iv) the level and/or activity of Tfh cells; 2) the level and/or activity of one or more characteristic expression products of a Tfh cell; 3) the level and/or activity of one or more genes in a set of genes characteristic of a Tfh cell; 4) CD4⁺CD28^-CD57⁺The level and/or activity of the cell; 5) CD4⁺CD28^-CD57⁺The level and/or activity of one or more expression products characteristic of the cell; 6) CD4⁺CD28^-CD57⁺The level and/or activity of one or more genes in a set of genes characteristic of a cell; 7) CD4⁺CD28^-KLRG1⁺The level and/or activity of the cell; 8) CD4⁺CD28^-KLRG1⁺The level and/or activity of one or more expression products characteristic of the cell; 9) CD4⁺CD28^-KLRG1⁺The level and/or activity of one or more genes in a set of genes characteristic of a cell; 10) CD8⁺CD28^-CD57⁺The level and/or activity of the cell; 11) CD8⁺CD28^-CD57⁺The level and/or activity of one or more expression products characteristic of the cell; 12) CD8⁺CD28^-CD57⁺The level and/or activity of one or more genes in a set of genes characteristic of a cell; 13) CD8⁺CD28^-KLRG1⁺The level and/or activity of the cell; 14) CD8⁺CD28^-KLRG1⁺The level and/or activity of one or more expression products characteristic of the cell; 15) CD8⁺CD28^- KLRG1⁺The level and/or activity of one or more genes in a set of genes characteristic of a cell; 16) CD4⁺PD-1⁺The level and/or activity of the cell; 17) CD4⁺PD-1⁺The level and/or activity of one or more expression products characteristic of the cell; 18) CD4⁺PD- 1⁺The level and/or activity of one or more genes in a set of genes characteristic of a cell; 19) CD8⁺PD-1⁺The level or activity of the cell; 20) CD8⁺PD-1⁺The level and/or activity of one or more expression products characteristic of the cell; 21) CD8⁺PD-1⁺The level and/or activity of one or more genes in a set of genes characteristic of a cell; 22) CD4⁺TIM3⁺The level and/or activity of the cell; 23) CD4⁺TIM3⁺The level and/or activity of one or more expression products characteristic of the cell; 24) CD4⁺TIM3⁺The level and/or activity of one or more genes in a set of genes characteristic of a cell; 25) CD8⁺TIM3⁺The level or activity of the cell; 26) CD8⁺TIM3⁺The level and/or activity of one or more expression products characteristic of the cell; 27) CD8⁺TIM3⁺The level and/or activity of one or more genes in a set of genes characteristic of a cell; 28) the level and/or activity of IL-6; 29) the level and/or activity of IL-1 α; 30) the level and/or activity of IL-7; 31) the level and/or activity of IL-1 α; 32) the level and/or activity of MMP 1; 33) the level and/or activity of MIP-1 β; 34) the level and/or activity of MIP-1 a; 35) the level and/or activity of Th1 cells; 36) the level and/or activity of one or more characteristic expression products of Th1 cells; 37) the level and/or activity of one or more genes in a set of genes characteristic of Th1 cells; 38) the level and/or activity of Th2 cells; 39) the level and/or activity of one or more characteristic expression products of Th2 cells; 40) the level and/or activity of one or more genes in a set of genes characteristic of Th2 cells; 41) the level and/or activity of Th17 cells; 42) the level and/or activity of one or more characteristic expression products of Th17 cells; 43) the level and/or activity of one or more genes in a set of genes characteristic of Th17 cells; 44) the level and/or activity of Treg cells; 45) the level and/or activity of one or more expression products characteristic of Treg cells; and, 46) the level and/or activity of one or more genes in the set of genes characteristic of Treg cells.

The detection index may further include one or more selected from the group consisting of: 1) CD4⁺CD28^-The level and/or activity of the cell; 2) CD4⁺CD28^-The level and/or activity of one or more expression products characteristic of the cell; 3) CD4⁺CD28^-The level and/or activity of one or more genes in one or more sets of genes characteristic of the cells; 4) CD4⁺CD57⁺The level and/or activity of the cell; 5) CD4⁺CD57⁺The level and/or activity of one or more expression products characteristic of the cell; 6) CD4⁺CD57⁺The level and/or activity of one or more genes in one or more sets of genes characteristic of the cells; 7) CD8⁺CD28^-The level and/or activity of the cell; 8) CD8⁺CD28^-The level and/or activity of one or more expression products characteristic of the cell; 9) CD8⁺CD28^-The level and/or activity of one or more genes in one or more sets of genes characteristic of the cells; 10) CD8⁺CD57⁺The level and/or activity of the cell; 11) CD8⁺CD57⁺The level and/or activity of one or more expression products characteristic of the cell; 12) CD8⁺CD57⁺The level and/or activity of one or more genes in one or more sets of genes characteristic of the cells; 13) CD8⁺CD28^-CD57⁺The level and/or activity of the cell; 14) CD8⁺CD28^-CD57⁺The level and/or activity of one or more expression products characteristic of the cell; 15) CD8⁺CD28^-CD57⁺The level and/or activity of one or more genes in one or more sets of genes characteristic of the cells; 16) CD8⁺CD28^-KLRG1⁺The level and/or activity of the cell; 17) CD8⁺CD28^-KLRG1⁺The level and/or activity of one or more expression products characteristic of the cell; 18) CD8⁺CD28^-KLRG1⁺The level and/or activity of one or more genes in one or more sets of genes characteristic of the cells; 19) CD4⁺PDL1⁺The level and/or activity of the cell; 20) CD4⁺PDL1⁺The level and/or activity of one or more expression products characteristic of the cell; 21) CD4⁺PDL1⁺One or more of the cellsThe level and/or activity of one or more genes in a set of genes characteristic of a cell; 22) CD4⁺LAG3⁺The level and/or activity of the cell; 23) CD4⁺LAG3⁺The level and/or activity of one or more expression products characteristic of the cell; 24) CD4⁺LAG3⁺The level and/or activity of one or more genes in one or more sets of genes characteristic of the cells; 25) CD4⁺CTLA⁺The level and/or activity of the cell; 26) CD4⁺CTLA⁺The level and/or activity of one or more expression products characteristic of the cell; 27) CD4⁺CTLA⁺The level and/or activity of one or more genes in one or more sets of genes characteristic of the cells; 28) CD8⁺PDL1⁺The level and/or activity of the cell; 29) CD8⁺PDL1⁺The level and/or activity of one or more expression products characteristic of the cell; 30) CD8⁺PDL1⁺The level and/or activity of one or more genes in one or more sets of genes characteristic of the cells; 31) CD8⁺LAG3⁺The level and/or activity of the cell; 32) CD8⁺LAG3⁺The level and/or activity of one or more expression products characteristic of the cell; 33) CD8⁺LAG3⁺The level and/or activity of one or more genes in one or more sets of genes characteristic of the cells; 34) CD8⁺CTLA⁺The level and/or activity of the cell; 35) CD8⁺CTLA⁺The level and/or activity of one or more expression products characteristic of the cell; and/or, 36) CD8⁺CTLA⁺The genes characteristic of one or more of the cells are concentrated in the level and/or activity of one or more genes.

The specification of the present application describes methods of using the kits to assess the efficacy of infusion of NK cells in a subject. In some cases, the method may comprise the steps of: 1) measuring a test indicator of a subject to obtain a pre-infusion measurement of the test indicator; 2) infusing NK cells into the subject; 3) after the infusion, measuring the detection index of the subject again to obtain a post-infusion measurement of the detection index; 4) comparing the post-infusion measurements of the detection indicators with the corresponding pre-infusion measurements to perform the assessment.

In the present application, the infusion may be an autologous infusion. In certain instances, the infusion may be allogeneic. In certain instances, the number of NK cells infused per such infusion may be 1 x 10⁷-1×10¹²For example, it may be 1 × 10⁹-1×10¹⁰. In certain instances, the subject may be infused with 1 or more NK cells. For example, the subject may be infused 1 to 10 times with NK cells. For another example, the subject can be infused 2 times with NK cells. In certain instances, the subject may be infused with 1 or more NK cells, and the time interval between each 2 of the infusions may be within 12 months. For example, the time interval between 2 such infusions may be 1 day. For example, NK cells can be resuspended in a saline solution containing human serum albumin and injected intravenously at different instillation rates (e.g., 15 drops/min or 30-40 drops/min).

In certain instances, the autologous NK cells infused herein are safe. For example, body temperature and blood pressure are normal, and no safety events such as rashes, topical infections and bleeding, fever, chills, dyspnea, nausea and vomiting occur. Although insomnia, dizziness and fatigue appear, the normal state is recovered within two weeks. In addition, ALT, AST, urea and creatinine levels in serum were normal, and liver and kidney toxicity was not observed, as shown by blood routine examination, hematological examination, urine and virological examination. And abnormal C-reactive protein (CRP), anti-thyroglobulin antibody (TGAb), and anti-thyroid peroxidase autoantibody (TPOAb) were not present, indicating that no immune response and autoimmune effect were induced. After long time (one month later) of infusion, the plasma levels of alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) were also not elevated.

The value of the test indicator may be measured within 12 months after at least one of the infusions to obtain a post-infusion measurement of the test indicator. In some cases, when the place isSaid NK cell infusion can be assessed as being effective in said subject if the value of said post-infusion measurement minus said pre-infusion measurement is negative. In other cases, when the post-infusion measurement is substantially equal to the pre-infusion measurement, then the infused NK cells may be indicated as being effective in the subject.Use, method and system

In another aspect, the present application provides the use of the detection reagent in the manufacture of a reagent and/or kit for assessing the efficacy of infusion of NK cells in a subject.

In another aspect, the present application provides a method of assessing the potency of an NK cell product, the method comprising the steps of: obtaining a post-infusion detection index measurement from a subject infused with the NK cell product; and assessing the potency of the NK cell product according to the post-infusion detection index measurement; wherein a decrease in the post-infusion assay indicator measurement compared to a reference result indicates the presence or increase in potency of the NK cell product.

For the method of assessing NK cell product potency, the reference result is a measurement of the subject prior to the infusion. In some cases, the comparison may be a difference obtained by subtracting the reference from the post-infusion measurement of the test indicator. In certain instances, the potency of the NK cell product is present as a CD16 or CD56 positive rate that can be greater than about 10% (e.g., greater than about 12%, greater than about 14%, greater than about 16%, greater than about 18%, greater than about 20%, greater than about 25%, greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 59%, or more) compared to a reference NK cell. For example, quantitative detection can be performed using an anti-CD 16 antibody (e.g., B73.1) and/or a CD56 antibody (e.g., B159), such as using flow cytometry. In certain instances, the reference NK cell can comprise an isolated NK cell of the subject.

In another aspect, the present application provides a method, which may include the steps of: obtaining a sample of a subject infused with an NK cell product, determining a post-infusion detection indicator measurement in the sample, comparing the obtained post-infusion detection indicator measurement to a reference result to determine a difference between the post-infusion detection indicator measurement and the reference result; and recording the difference, the detection indicators may be those described above. In some cases, the method may further comprise the steps of: determining a subsequent treatment regimen for the subject based on the recorded differences. For example, the subsequent treatment regimen may include infusion of the NK cell product.

In another aspect, the present application provides a system for assessing the efficacy of infusion of NK cells in a subject, the system comprising a detection module, a storage module, and a processor, wherein the detection module is configured to measure a detection index and obtain a measurement. In some cases, wherein the storage module is operable to store the measurement; wherein the processor is operable to perform the evaluation based on the measurement. The detection module of the system of the present application can comprise a detection reagent as described herein.

In another aspect, the present application provides a method of optimizing the production of NK cell products, said method comprising the steps of: 1) Obtaining NK cells; 2) activating NK cells in vitro; 3) assessing the potency of said NK cells by detecting the indicator. In certain instances, the NK cells may be derived from cells selected from the group consisting of: peripheral blood cells, bone marrow cells, and cord blood cells. The detection index may be one or more of the detection indexes mentioned above. In some cases, the obtaining may include the steps of: isolating and obtaining the NK cells from the cells. For example, the method of separating may comprise centrifugation. In certain instances, the in vitro activation may comprise the steps of: (ii) incubating the NK cells after adding the activating agent. The activator may comprise a cytokine, for example, the cytokine may be selected from the group consisting of: interleukins and interferons. In certain instances, the interleukin may be selected from the group consisting of: IL12, IL5, IL-2, IL-7, IL-18 and IL-21. In some cases, the concentration of the activating agent may be 1 to 10000 ng/mL. For example, the incubation may be at 1-40 ℃. For example, incubation may be for 1h-96 h.

For example, the detection index of Treg cells may be CD4⁺CD25^high CD127^lowThe detection index of Th1 cell may be CD4⁺CXCR3⁺CCR6^-CCR4^-The detection index of Th2 cell may be CD4⁺CCR4⁺CXCR3^-CCR6^-The detection index of Th17 cell may be CD4⁺CCR4⁺CCR6⁺CXCR3^-The detection index of the Tfh cells can be CD4⁺CXCR5⁺PD-1⁺。

As another example, the detection reagents of the present application can include a reagent selected from the group consisting of: anti-PD-1 antibodies, anti-TIM 3 antibodies, anti-CD 28 antibodies, anti-CD 57 antibodies, anti-CD 4 antibodies (e.g., GK1.5), anti-CD 8 antibodies (e.g., 53-6.7), anti-CD 25 antibodies (e.g., PC61), anti-CD 45RA antibodies (e.g., HI100), anti-CXCR 3 antibodies (e.g., G025H7), anti-CCR 4 antibodies (e.g., L291H4), anti-CCR 6 antibodies (e.g., G034E3), anti-CCR 7 antibodies (e.g., g., G043H7), anti-CD 127 antibodies (e.g., a019D5, 1:100), anti-CXCR 5 antibodies (e.g., RF8B2, 1:100), anti-CD 2 antibodies (e.g., CD28.2, 1:100), anti-CD 2 antibodies (e.g., NK-1, 1: 36100), anti-KLRG 2 antibodies (E2), anti-TIM 2). The detection index can be detected by the reagent.

For example, after infusion of NK cells, the subject's level of Tfh cells decreases.

The present application finds that the infusion of NK cells can reduce senescent T cells in a subject. For example, 4 weeks after infusion of NK cells, CD4 compared to cells at baseline⁺CD28^-Cell, CD4⁺CD57⁺Cell, CD4⁺CD28^-CD57⁺Cell, CD4⁺CD28^-KLRG1⁺Cell, CD8⁺CD28^-Cell, CD8⁺CD57⁺Cell, CD8⁺CD28^-CD57⁺Cells and CD8⁺CD28^- KLRG1⁺The number of cells decreased. The present application further finds that the infusion of NK cells can reduce the depletion of T cells in a subject. For example, 4 weeks after infusion of NK cells, CD4⁺PD-1⁺T cell, CD8⁺PD-1⁺T cell, CD4⁺TIM3⁺T cells and CD8⁺TIM3 ⁺T cells were significantly reduced as detected using flow cytometry. The present application also found that NK cell infusion can reduce the levels of key senescence-associated secretory phenotype (SASP) -associated factors. For example, levels of IL-6, IL-8, IL-1 α, IL-17, MIP-1 α, m ip P-1 β and MMP1 decreased 4 weeks after infusion of NK cells as detected by ELISA using a multiplex assay kit. The changes in the assay index following infusion of NK cells are not affected by sex and NK cell preparation status (e.g., prepared from fresh PBMC or cryopreserved PBMC expansion).

Without intending to be bound by any theory, the following examples are merely intended to illustrate the fusion proteins, preparation methods, uses, etc. of the present application, and are not intended to limit the scope of the invention of the present application.

Examples

The data herein were analyzed using a 2 nd power differential analysis method and multiple comparisons were performed using the least squares mean square (LSM) t test. The association between variables and baseline was tested using a non-parametric Spearman rank correlation test. A p value of 0.05 or less is considered statistically significant.

Example 1 Baseline characteristics of subjects

The study (clinical trials. gov identifier: ChiCTR-OOh-17011878) was approved by the Long-standing Hospital ethics Committee. The study was eligible if the subjects were between 45-55 years of age and no disease. Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), epstein-barr virus (EBV), Cytomegalovirus (CMV) and syphilis are positive in serum, and subjects with two or more of abnormal functional results in tests including glutamic-pyruvic transaminase (ALT), aspartate Aminotransferase (AST), total bilirubin (TBil), indirect bilirubin (I-TBil), Direct Bilirubin (DBIL) and γ -GT are excluded. All subjects underwent physical examination and medical questionnaires to assess health status. Eligible subjects infused a dose of autologous NK cells twice within two days. Peripheral blood samples were taken as baseline prior to cell infusion and at

weeks

1 and 4 after cell infusion to assess the effect of infused NK cells on T cell senescence and exhaustion and SASP.

During the 7 th month to the 9 th month of 2017 to 2018, 35 out of 42 subjects were between 45 and 55 years of age. During the screening process, 7 volunteers were excluded, of which 3 were positive for syphilis, 3 had two or more abnormal results in the live functional test, 1 thyroiditis. Thus, 28 volunteers participated in the study and received leukapheresis and subsequent administration of NK cells, with 15 volunteers using fresh PBMC cells and 13 volunteers using cryopreserved PBMC cells. However, 4 subjects missed sample collection after administration of NK cells and therefore had to withdraw from the study. Finally, 24 subjects (nos. 1-24) successfully completed the study, including 10 males (nos. 1-10) and 14 females (nos. 11-24). Table 1 lists baseline characteristics and NK cell information for the subjects.

TABLE 1 Baseline characteristics

Example 2 in vitro Natural killer cell expansion and cell infusion

Leukocytes were collected from subjects using leukopheresis using Spectra optiterumo. NK cells were expanded in vitro using a feeder cells-free culture system and a Natural Killer (NK) cell culture kit (DAKEWE). Briefly, cells are plated at 1-2X 10⁶Cells/ml were seeded into activator-coated flasks and incubated at 37 ℃ with 5% CO₂The culture was carried out in an incubator (Thermo fisher). Fresh NK medium was added every 2-3 days until an effective amount of cells was obtained after about 14 days. Quality control was performed by evaluating samples taken throughout the culture and in the final cell product. Detection was performed under sterile conditions using a BacT/ALERT (bioMerieux, Durham, NC, USA) microbial detection system, and endotoxin was detected using a gel limulus reagent. The mycoplasma contamination test was performed by a PCR method using primers specific for mycoplasma (shanghai chromogen medical science and technology ltd). Cell number calculation of NK cells Using Trypan stainingAnd cell viability. NK cells were determined by expression of CD56 or CD16 and deletion of CD3 and were quantitatively detected using flow cytometry and anti-CD 3 antibody (HIT3, FITC), anti-CD 56 antibody (B159, APC) and anti-CD 16 antibody (B73.1, APC) purchased from BD bioscience.

The NK cells were resuspended in a saline solution containing human serum albumin and divided into two aliquots, one aliquot was first intravenously injected at a drip rate of 15 drops/min, then the second aliquot was intravenously injected at a rate of 30-40 drops/min, and the NK cells were infused into the subjects over two days. And after each transfusion, flushing the pipeline of the blood transfusion equipment by using 60-70 ml of saline solution.

Example 3 safety of autologous NK cells

After cell injection, all subjects had normal body temperature and blood pressure, and none had a rash, local infection and bleeding, fever, chills, dyspnea, nausea and vomiting. In addition, one subject developed insomnia within one week after cell infusion, and recovered thereafter. One subject showed dizziness within one week after cell infusion, which continued for two weeks before recovery. Fatigue occurred in 2 subjects, with mild fatigue in one subject and moderate fatigue in another, all recovered within two weeks (table 2).

Subsequently, blood routine examination, hematological examination, urine and virological examination were performed one month after cell infusion. ALT, AST, urea and creatinine levels were normal in serum, and no liver toxicity and no renal toxicity were observed. In addition, abnormal C-reactive protein (CRP), anti-thyroglobulin antibody (TGAb), and anti-thyroid peroxidase autoantibody (TPOAb) did not appear, indicating that immune response and autoimmune action were not induced, which sufficiently confirms the safety of autologous NK cell infusion.

TABLE 2 adverse events following autologous NK cell infusion

Example 4 immunophenotypic analysis of peripheral blood T cells

Peripheral Blood Mononuclear Cells (PBMCs) were isolated using ficoll-paque (ge healthcare) and the senescent and depleted T cells were subjected to cell phenotype analysis by flow cytometry (n-24). To analyze surface markers, cells were stained with antibody in PBS (FBS, Thermo fisher) containing 2% fetal bovine serum. Flow cytometric (BD, LSRFortessa X20) analysis was then performed.

Gating strategies for identifying T cell subsets are referenced to He, J., Zhang, X., Wei, Y., Sun, X., Chen, Y., Deng, J., Li, Z. (2016), "Low-dose interactive models CD4(+) T cell subsets in tissues with system capacity polyethylene production". Nat Med,22(9),991-993.doi: 10.1038/nm.4148. Briefly, the Treg cell detection index was CD4⁺CD25^high CD127^lowThe Th1 cell detection index is CD4⁺CXCR3⁺CCR6^-CCR4^-The Th2 cell detection index is CD4⁺CCR4⁺CXCR3^-CCR6^-The Th17 cell detection index is CD4⁺CCR4⁺CCR6⁺CXCR3^-The Tfh cell detection index is CD4⁺CXCR5⁺PD-1⁺。

In addition, markers associated with T cell senescence and depletion were also monitored using PD-1 antibody, TIM3 antibody, CD28 antibody, and CD57 antibody. Among these, the antibodies used are from BD Biosciences and ebiosciences, including anti-CD 4 antibody (GK1.5, 1:100), anti-CD 8 antibody (53-6.7, 1:100), anti-CD 25 antibody (PC61, 1:100), anti-CD 45RA antibody (HI100, 1:100), anti-CXCR 3 antibody (G025H7, 1:100), anti-CCR 4 antibody (L291H4, 1:100), anti-CCR 6 antibody (G034E3, 1:100), anti-CCR 7 antibody (G043H7, 1:100), anti-CD 127 antibody (a019D5, 1:100), anti-CXCR 5 antibody (RF8B2, 1:100), anti-CD 28 antibody (CD28.2, 1:100), anti-CD 57 antibody (NK-1, 1:100), anti-CXCR 1 (rg 2F 6862, 1: 861, anti-CD 57 antibody (TIM-1: 8653), anti-CD 592F 6951: 865, anti-CD 592, anti-CD 38).

Results as shown in fig. 1A-1E, significant reduction in Tfh cell levels in subjects following NK cell preparation reinfusion (fig. 1D) had a weaker effect on Th1 (fig. 1A), Th2 (fig. 1B), Th17 (fig. 1C) and Treg (fig. 1E) cell levels.

Example 5 depletion of senescent T cells following NK cell infusion

Flow cytometry performed according to the method of example 3 to detect senescent cells at baseline, and at1 and 4 weeks post-infusion, which were CD4⁺CD28^-，CD4⁺CD57⁺，CD4⁺CD28^-CD57⁺，CD4⁺CD28^-KLRG1⁺，CD8⁺CD28^-，CD8⁺CD57⁺，CD8⁺CD28^-CD57⁺，CD8⁺CD28^-KLRG1⁺The cell population of (1).

The results show CD4 compared to baseline at two time points after infusion⁺And CD8⁺The cell population of (a) is not significantly changed, wherein,

CD4⁺: mean ± SD: base line is 30.61 ± 7.55, 1w is 29.95 ± 7.41, 4w is 30.34 ± 7.33; p-value (1w vs base line) 0.7137, p-value (4w vs base line) 0.8728,

CD8⁺: mean ± SD: base line 27.61 ± 7.80, 1w 28.17 ± 6.38, 4w 27.11 ± 6.27; p-value (1w vs base line) 0.6282, and p-value (4w vs base line) 0.5840.

However, aged CD 41 and 4 weeks after NK cell infusion⁺And CD8⁺T cells were significantly reduced in response to infusion (fig. 2 and 3), as follows.

CD4⁺CD28^-Both time points declined in 21 subjects and one time point declined in 4 other subjects. Mean ± SD: base line 7.53 ± 6.21, 1w 6.05 ± 6.81, 4w 5.39 ± 5.23; 95% CI: the base line is 4.91-10.16, 1w is 3.18-8.93, and 4w is 3.19-7.61 (fig. 2A).

CD4⁺CD57⁺Both time points were decreased in 16 subjects and one time point in the other 7 subjects. Mean ± SD: base line 7.76 ± 5.60, 1w 6.57 ± 6.14, 4w 5.80 ± 4.72; 95% CI: base ofThe line is 5.39-10.12, 1w is 3.98-9.17, and 4w is 3.80-7.79 (fig. 2B).

CD4⁺CD28^-CD57⁺Both time points were decreased in 18 subjects and one time point in 4 other subjects. Mean ± SD: base line 2.72 ± 1.02, 1w 1.95 ± 0.60, 4w 2.09 ± 0.80; 95% CI: the base line is 2.29 to 3.15, 1w is 1.70 to 2.21, and 4w is 1.76 to 2.43 (fig. 2C).

CD4⁺CD28^-KLRG1⁺Both time points were decreased in 17 subjects and one time point in the other 4 subjects. Mean ± SD: base line 2.72 ± 1.02, 1w 1.95 ± 0.60, 4w 2.09 ± 0.80; 95% CI: the base line is 2.29 to 3.15, 1w is 1.70 to 2.21, and 4w is 1.76 to 2.43 (fig. 2D).

CD8⁺CD28^-Both time points were decreased in 16 subjects and one time point in the other 4 subjects. Mean ± SD: 43.30 + -14.49, 37.07 + -12.82 for 1w, 39.16 + -12.16 for 4 w; 95% CI: the base line is 37.18 to 49.42, 1w is 31.66 to 42.48, and 4w is 34.03 to 44.30 (fig. 3A).

CD8⁺CD57⁺Both time points were decreased in 17 subjects and one time point in 4 other subjects (mean ± SD: baseline 27.68 ± 10.53, 1w 24.00 ± 9.89, 4w 23.42 ± 9.27; 95% CI: baseline 23.23-32.12, 1w 19.83-28.17, 4w 19.50-27.33) (fig. 3B).

CD8⁺CD28^-CD57⁺Both time points were decreased in 13 subjects and one time point in 8 other subjects. Mean ± SD: base line 24.04 ± 10.27, 1w 21.04 ± 9.30, 4w 19.90 ± 8.71; 95% CI: the base line is 19.70 to 28.38, 1w is 17.11 to 24.96, and 4w is 16.22 to 23.58) (fig. 3C).

CD8⁺CD28^-KLRG1⁺Both time points were decreased in 20 subjects and one time point in the other 2 subjects. Mean ± SD: base line 3.21 ± 2.28, 1w 1.87 ± 1.27, 4w 2.04 ± 1.73; 95% CI: base line 2.25-4.18, 1w 1.34 e2.41, 4w is 1.31 ~ 2.78 (fig. 3D).

These data indicate that autologous NK cell infusion can reduce T cell senescence.

Furthermore, CD4 was compared to baseline 4 weeks after NK cell administration⁺CD28^-、CD4⁺CD57⁺、CD4⁺CD28^-CD57⁺、 CD4⁺CD28^-KLRG1⁺、CD8⁺CD28^-、CD8⁺CD57⁺、CD8⁺CD28^-CD57⁺And CD8⁺CD28^-KLRG1⁺The degree of change of (a) is increasing, and CD4 is observed at1 week⁺CD28^-CD57⁺、CD4⁺CD28^-KLRG1⁺、CD8⁺CD28^-、CD8⁺CD57 ⁺、CD8⁺CD28^-CD57⁺And CD8⁺CD28^-KLRG1⁺Also increases (fig. 4).

CD4⁺CD28^-Cell, CD4⁺CD57⁺Cell, CD4⁺CD28^-CD57⁺Cell, CD4⁺CD28^-KLRG1⁺Cell, CD8⁺CD28^-Cell, CD8⁺CD57⁺Cell, CD8⁺CD28^-CD57⁺Cells and CD4⁺CD28^-KLRG1⁺The proportion of cells in PBMCs is shown in fig. 5, and the expression of T cell senescence markers was significantly reduced after NK cell preparation reinfusion, and was consistent in both sex (fig. 6) and NK cell preparation (prepared by expansion of fresh PBMCs or cryopreserved PBMCs) (fig. 7).

Example 6 depletion of T cells depleted following NK cell infusion

Then, whether the injection of NK cells affected CD4 was examined according to the method of example 4⁺And CD8⁺Percentage of T cells depleted.

The results showed that CD4 was present at

weeks

1 and 4 after NK cell infusion⁺PD-1⁺T cell, CD8⁺PD-1⁺T cell, CD4⁺TIM3⁺T cells and CD8⁺TIM3⁺T cells are all significantly reducedLow (fig. 8). The details are as follows.

CD4⁺PD-1⁺Both time points were decreased in 18 subjects and one time point in the other 4 subjects. Mean ± SD: base line 2.75 ± 0.84, 1w 2.19 ± 0.92, 4w 2.09 ± 0.68; 95% CI: the base line is 2.40-3.11, 1w is 1.80-2.58, and 4w is 1.80-2.38.

CD4⁺TIM3⁺Both time points were decreased in 16 subjects and one time point in 4 other subjects (mean ± SD: baseline 0.89 ± 0.57, 1 w-0.46 ± 0.36, 4 w-0.51 ± 0.49; 95% CI: baseline 0.65-1.14, 1 w-0.31-0.61, 4 w-0.30-0.71).

CD8⁺PD^-1⁺Both time points were decreased in 20 subjects, and one time point in 4 other subjects. Mean ± SD: base line 2.67 ± 1.15, 1w 1.85 ± 0.63, 4w 1.82 ± 0.85; 95% CI: the base line is 2.18 to 3.15, 1w is 1.59 to 2.11, and 4w is 1.46 to 2.18.

CD8⁺TIM3⁺Both time points were decreased in 19 subjects and one time point in 1 other subject. Mean ± SD: base line 2.72 ± 1.02, 1w 1.95 ± 0.60, 4w 2.09 ± 0.80; 95% CI: the base line is 2.29 to 3.15, 1w is 1.70 to 2.21, and 4w is 1.76 to 2.43.

These data show that NK cell infusion can improve T cell function by reducing the depletion state of T cells.

Furthermore, CD4 was compared to baseline at1 week and 4 weeks after cell administration⁺PD-1⁺，CD4⁺TIM3⁺，CD8⁺PD-1⁺And CD8⁺TIM3⁺The degree of change of (a) is more likely to increase (fig. 9).

CD4⁺PD-1⁺Cell, CD4⁺TIM3⁺Cell, CD8⁺PD-1⁺Cells and CD8⁺TIM3⁺The proportion of cells in PBMC is shown in FIG. 10, which significantly reduces the expression of T cell depleting markers after NK cell infusion, and is tailored for different sexes (FIG. 11) and different NK cellsThe agents (prepared by amplification of fresh PBMC or cryopreserved PBMC) (FIG. 12) were all consistent.

Example 7 reduction of Key senescence-associated secretory phenotype (SASP) associated factors following NK cell infusion

To explore whether NK cell infusion could reduce systemic levels of SASP-associated factors, we examined cytokine levels in plasma before and after NK cell infusion. Cytokines in plasma were detected by Luminex xMAP technology using a multiplex assay kit (procartex 8Plex, Thermo Fisher, PPX-08) including MMP-1(n ═ 22), MIP-1 β (n ═ 22), MIP-1 α (n ═ 21), IL-8(n ═ 21), IL-1 α (n ═ 20), IL-6(n ═ 21), IL-17(n ═ 21), and IFN- γ. The detection method is according to the kit instructions.

The results show that by NK cell infusion, levels of key SASP components in plasma were reduced, including IL-6, IL-8, IL-1 α, IL-17, MIP-1 α, Μ Ι P-1 β and MMP1, while non-SASP related factors IFN- γ did not change significantly continuously (fig. 13 and 14). The details are as follows.

Plasma levels of IL-6 were decreased in both time points in 15 subjects, and in 4 other subjects at one time point. Mean ± standard deviation: baseline 10.66 ± 4.41(pg/ml), 1w 7.18 ± 3.37(pg/ml), 4w 6.48 ± 3.58 (pg/ml); 95% CI: the baseline is 8.66-12.67 (pg/ml), 1w is 5.65-8.71 (pg/ml), and 4w is 4.84-8.11 (pg/ml) (fig. 13B).

Plasma levels of IL-8 were decreased in both time points in 16 subjects and in one time point in 3 other subjects. Mean ± standard deviation: baseline 5.06 ± 2.28(pg/ml), 1w 3.70 ± 1.10(pg/ml), 4w 3.67 ± 1.25 (pg/ml); 95% CI:

(pg/ml), 1w is 3.20 to 4.20(pg/ml), and 4w is 3.10 to 4.24 (pg/ml) (fig. 13D).

Plasma levels of IL-1 α were decreased in 16 subjects at both time points and in 3 other subjects at one time point. Mean ± standard deviation: baseline 0.45 ± 0.24(pg/ml), 1w 0.30 ± 0.21(pg/ml), 4w 0.19 ± 0.11 (pg/ml); 95% CI: the baseline is 0.33-0.56 (pg/ml), 1w is 0.20-0.40 (pg/ml), and 4w is 0.14-0.23 (pg/ml) (fig. 13A).

Plasma levels of IL-17 were decreased in both time points in 17 subjects and in one time point in 2 other subjects. Mean ± standard deviation: baseline 4.03 ± 1.84(pg/ml), 1w 2.63 ± 1.55(pg/ml), 4w 2.07 ± 1.08 (pg/ml); 95% CI: the baseline is 3.20 to 4.87(pg/ml), 1w is 1.92 to 3.33(pg/ml), and 4w is 1.58 to 2.56 (pg/ml) (fig. 13C).

Plasma levels of MIP-1 α decreased at both time points in 15 subjects and decreased at one time point in another 6 subjects. Mean ± standard deviation: baseline 6.06 ± 4.68(pg/ml), 1w 4.73 ± 3.87(pg/ml), 4w 5.17 ± 4.53 (pg/ml); 95% CI: the baseline is 3.93-8.19 (pg/ml), 1w is 2.97-6.49 (pg/ml), and 4w is 3.11-7.23 (pg/ml) (fig. 14A).

Plasma levels of MIP-1 β decreased at both time points in 15 subjects and decreased at one time point in the other 5 subjects. Mean ± standard deviation: baseline 55.25 ± 38.80(pg/ml), 1w 38.85 ± 26.31(pg/ml), 4w 43.66 ± 29.11 (pg/ml); 95% CI: the baseline is 38.05-72.45 (pg/ml), 1w is 27.18-50.51 (pg/ml), and 4w is 30.76-56.57 (pg/ml) (fig. 14B).

Plasma levels of MMP1 decreased at both time points in 15 subjects and decreased at one time point in the other 5 subjects. Mean ± standard deviation: baseline 9.73 ± 4.71(pg/ml), 1w 7.78 ± 4.16(pg/ml), 4w 7.36 ± 3.41 (pg/ml); 95% CI: baseline 15 subjects decreased 7.64-11.81 (pg/ml), 1w 5.93-9.63 (pg/ml), and 4w 5.85-8.87 (pg/ml) at both time points (fig. 14C).

In addition, there was an increase in the baseline-related trend in the extent of changes in IL-6, IL-8, IL-1 α, IL-17, MIP-1 α, M1P-1 β and MMP1, both 1 and 4 weeks after cell infusion (FIG. 15).

The expression levels of MMP-1, MIP-1 β, MIP-1 α, IL-8, IL-1 α, IL-6, IL-17, and IFN- γ are shown in FIG. 16, and significantly reduced the expression levels of senescence-associated secreted factors in subjects following NK cell infusion, and were consistent in different sexes (FIG. 17) and different NK cell preparations (prepared by expansion of fresh PBMC or cryopreserved PBMC) (FIG. 18).

Claims

1. A test kit for assessing the efficacy of infusion of NK cells in a subject, the test kit comprising a detection reagent and optionally instructions for use, wherein the detection reagent comprises one selected from the group consisting of or Various:

1) A reagent for identifying the level and/or activity of one or more cells selected from the group consisting of Tfh cells, CD4 ⁺ CD28- cells, CD4 ⁺ CD57 ⁺ cells ^, CD4 ⁺ CD28 ^- CD57 ⁺ cells, CD4 ⁺ CD28 ^- KLRG1 ⁺ cells, CD8 ⁺ CD28 ^- cells, CD8 ⁺ CD57 ⁺ cells, CD8 ⁺ CD28 ^- CD57 ⁺ cells, CD8 ⁺ CD28 ^- KLRG1 ⁺ cells, CD4 ⁺ PD-1 ⁺ cells, CD4 ⁺ TIM3 ⁺ cells, CD4 ⁺ PDL1 ⁺ cells, CD4 ⁺ LAG3 ⁺ cells, CD4 ⁺ CTLA-4 ⁺ cells, CD8 ⁺ PD-1 ⁺ cells, CD8 ⁺ TIM3 ⁺ cells, CD8 ⁺ PDL1 ⁺ cells, CD8 ⁺ LAG3 ⁺ cells and CD8 ⁺ CTLA-4 ⁺ cell,

2) A reagent for detecting the level and/or activity of one or more characteristic expression products of one or more cells selected from the group consisting of Tfh cells, CD4 ⁺ CD28 ^- cells, CD4 ⁺ CD57 ⁺ cells, CD4 ⁺ CD28 ^- CD57 ⁺ cells, CD4 ⁺ CD28 ^- KLRG1 ⁺ cells, CD8 ⁺ CD28 ^- cells, CD8 ⁺ CD57 ⁺ cells, CD8 ⁺ CD28 ^- CD57 ⁺ cells, CD8 ⁺ CD28 ^- KLRG1 ⁺ cells, CD4 ⁺ PD-1 ⁺ cells, CD4 ⁺ TIM3 ⁺ cells, CD4 ⁺ PDL1 ⁺ cells, CD4 ⁺ LAG3 ⁺ cells, CD4 ⁺ CTLA-4 ⁺ cells, CD8 ⁺ PD-1 ⁺ cells, CD8 ⁺ TIM3 ⁺ cells, CD8 ⁺ PDL1 ⁺ cells, CD8 ⁺ LAG3 ⁺ cells and CD8 ⁺ CTLA-4 ⁺ cells,

3) A reagent for detecting the level and/or activity of one or more genes in the characteristic gene set of one or more cells selected from the group consisting of Tfh cells, CD4 ⁺ CD28- cells ^, CD4 ⁺ CD57 ⁺ cells , CD4 ⁺ CD28 ^- CD57 ⁺ cells, CD4 ⁺ CD28 ^- KLRG1 ⁺ cells, CD8 ⁺ CD28 ^- cells, CD8 ⁺ CD57 ⁺ cells, CD8 ⁺ CD28 ^- CD57 ⁺ cells, CD8 ⁺ CD28 ^- KLRG1 ⁺ cells, CD4 ⁺ PD-1 ⁺ cells, CD4 ⁺ TIM3 ⁺ cells, CD4 ⁺ PDL1 ⁺ cells, CD4 ⁺ LAG3 ⁺ cells, CD4 ⁺ CTLA-4 ⁺ cells, CD8 ⁺ PD-1 ⁺ cells, CD8 ⁺ TIM3 ⁺ cells, CD8 ⁺ PDL1 ⁺ cells, CD8 ⁺ LAG3 ⁺ cells and CD8 ⁺ CTLA-4 ⁺ cells, and,

4) Reagents for detecting the level and/or activity of one or more cytokines selected from the group consisting of IL-6, IL-1α, IL-8, IL-7, MMP1, MIP-1β and MIP- 1α.

2. The kit of claim 1, wherein the method comprises the steps of:

1) measuring the detection index of the subject to obtain the pre-infusion measurement result of the detection index;

2) infusing NK cells into the subject;

3) after the infusion, measure the detection index of the experimenter again to obtain the measurement result after the infusion of the detection index;

4) Comparing the post-infusion measurement of the detection index with the corresponding pre-infusion measurement for the assessment.

3. The kit of claim 1, wherein the infusion is an autologous infusion.

4. The kit of claim 1, wherein the number of NK cells infused in each infusion is 1×10 ⁷ to 1×10 ¹² .

5. The kit of claim 1, wherein the subject is infused with NK cells one or more times.

6. Use of the detection reagent of claim 1 in the preparation of a reagent and/or kit for evaluating the efficacy of NK cell infusion in a subject.

7. A method of assessing the efficacy of an NK cell product, the method comprising the steps of:

obtaining post-infusion assay measurements derived from a subject infused with the NK cell product; and

Assessing the efficacy of the NK cell product based on the post-infusion assay measurements;

Wherein the detection index comprises one or more selected from the following group:

1) The level and/or activity of one or more cells selected from the group consisting of Tfh cells, CD4 ⁺ CD28- cells, CD4 ⁺ CD57 ⁺ cells ^, CD4 ⁺ CD28 ^- CD57 ⁺ cells ^, CD4 ⁺ CD28- KLRG1 ⁺ cells , CD8 ⁺ CD28 ^- cells, CD8 ⁺ CD57 ⁺ cells, CD8 ⁺ CD28 ^- CD57 ⁺ cells, CD8 ⁺ CD28 ^- KLRG1 ⁺ cells, CD4 ⁺ PD-1 ⁺ cells, CD4 ⁺ TIM3 ⁺ cells, CD4 ⁺ PDL1 ⁺ cells, CD4 ⁺ LAG3 ⁺ cells, CD4 ⁺ CTLA-4 ⁺ cells, CD8 ⁺ PD-1 ⁺ cells, CD8 ⁺ TIM3 ⁺ cells, CD8 ⁺ PDL1 ⁺ cells, CD8 ⁺ LAG3 ⁺ cells and CD8 ⁺ CTLA-4 ⁺ cells,

2) The level and/or activity of one or more characteristic expression products of one or more cells selected from the group consisting of Tfh cells, CD4 ⁺ CD28 ^- cells, CD4 ⁺ CD57 ⁺ cells, CD4 ⁺ CD28 ^- CD57 ⁺ cells, CD4 ⁺ CD28 ^- KLRG1 ⁺ cells, CD8 ⁺ CD28 ^- cells, CD8 ⁺ CD57 ⁺ cells, CD8 ⁺ CD28 ^- CD57 ⁺ cells, CD8 ⁺ CD28 ^- KLRG1 ⁺ cells, CD4 ⁺ PD-1 ⁺ cells, CD4 ⁺ TIM3 ⁺ cells, CD4 ⁺ PDL1 ⁺ cells, CD4 ⁺ LAG3 ⁺ cells, CD4 ⁺ CTLA-4 ⁺ cells, CD8 ⁺ PD-1 ⁺ cells, CD8 ⁺ TIM3 ⁺ cells, CD8 ⁺ PDL1 ⁺ cells, CD8 ⁺ LAG3 ⁺ cells and CD8 ⁺ CTLA-4 ⁺ cells,

3) The level and/or activity of one or more genes in the characteristic gene set of one or more cells selected from the group consisting of Tfh cells, CD4 ⁺ CD28 ^- cells, CD4 ⁺ CD57 ⁺ cells, CD4 ⁺ CD28 ^- CD57 ⁺ cells, CD4 ⁺ CD28 ^- KLRG1 ⁺ cells, CD8 ⁺ CD28 ^- cells, CD8 ⁺ CD57 ⁺ cells, CD8 ⁺ CD28 ^- CD57 ⁺ cells, CD8 ⁺ CD28 ^- KLRG1 ⁺ cells, CD4 ⁺ PD-1 ⁺ cells, CD4 ⁺ TIM3 ⁺ cells, CD4 ⁺ PDL1 ⁺ cells, CD4 ⁺ LAG3 ⁺ cells, CD4 ⁺ CTLA-4 ⁺ cells, CD8 ⁺ PD-1 ⁺ cells, CD8 ⁺ TIM3 ⁺ cells, CD8 ⁺ PDL1 ⁺ cells, CD8 ⁺ LAG3 ⁺ cells and CD8 ⁺ CTLA-4 ⁺ cells, and,

4) the level and/or activity of one or more cytokines selected from the group consisting of IL-6, IL-1α, IL-8, IL-7, MMP1, MIP-1β and MIP-1α;

Wherein, when the measurement result of the post-infusion detection index decreases compared with the reference result, it indicates that the efficacy of the NK cell product exists or increases.

8. A method comprising the steps of:

Obtaining samples from subjects infused with NK cell products,

Determining the post-infusion detection index measurements in the sample,

comparing the obtained post-infusion assay measurements to reference results to determine differences between the post-infusion assay measurements and the reference results; and

record the difference,

2) The level and/or activity of one or more characteristic expression products of one or more cells selected from the group consisting of Tfh cells, CD4 ⁺ CD28 ^- cells, CD4 ⁺ CD57 ⁺ cells, CD4 ⁺ CD28 ^- CD57 ⁺ cells, CD4 ⁺ CD28-KLRG1 ⁺ cells, CD8 ⁺ CD28 ^- cells, CD8 ⁺ CD57 ⁺ cells, CD8 ⁺ CD28 ^- CD57 ⁺ cells, CD8 ⁺ CD28 ^- KLRG1 ⁺ cells, CD4 ⁺ PD-1 ⁺ cells, CD4 ⁺ TIM3 ⁺ cells, CD4 ⁺ PDL1 ⁺ cells, CD4 ⁺ LAG3 ⁺ cells, CD4 ⁺ CTLA-4 ⁺ cells, CD8 ⁺ PD-1 ⁺ cells, CD8 ⁺ TIM3 ⁺ cells, CD8 ⁺ PDL1 ⁺ cells, CD8 ⁺ LAG3 ⁺ cells and CD8 ⁺ CTLA-4 ⁺ cells,

4) The level and/or activity of one or more cytokines selected from the group consisting of IL-6, IL-1α, IL-8, IL-7, MMP1, MIP-1β and MIP-1α.

9. A method for optimizing the preparation of NK cell products, the method comprising the steps of:

1) Obtain NK cells;

2) Activation of NK cells in vitro;

3) Evaluate the efficacy of the NK cells by a detection index, wherein the detection index is selected to comprise one or more selected from the group consisting of:

a) Level and/or activity of one or more cells selected from the group consisting of Tfh cells, CD4 ⁺ CD28- cells, CD4 ⁺ CD57 ⁺ cells ^, CD4 ⁺ CD28 ^- CD57 ⁺ cells ^, CD4 ⁺ CD28- KLRG1 ⁺ cells , CD8 ⁺ CD28 ^- cells, CD8 ⁺ CD57 ⁺ cells, CD8 ⁺ CD28 ^- CD57 ⁺ cells, CD8 ⁺ CD28 ^- KLRG1 ⁺ cells, CD4 ⁺ PD-1 ⁺ cells, CD4 ⁺ TIM3 ⁺ cells, CD4 ⁺ PDL1 ⁺ cells, CD4 ⁺ LAG3 ⁺ cells, CD4 ⁺ CTLA-4 ⁺ cells, CD8 ⁺ PD-1 ⁺ cells, CD8 ⁺ TIM3 ⁺ cells, CD8 ⁺ PDL1 ⁺ cells, CD8 ⁺ LAG3 ⁺ cells and CD8 ⁺ CTLA-4 ⁺ cells,

b) level and/or activity of one or more characteristic expression products of one or more cells selected from the group consisting of Tfh cells, CD4 ⁺ CD28- cells, CD4 ⁺ CD57 ⁺ cells ^, CD4 ⁺ CD28 ^- CD57 ⁺ cells, CD4 ⁺ CD28 ^- KLRG1 ⁺ cells, CD8 ⁺ CD28 ^- cells, CD8 ⁺ CD57 ⁺ cells, CD8 ⁺ CD28 ^- CD57 ⁺ cells, CD8 ⁺ CD28 ^- KLRG1 ⁺ cells, CD4 ⁺ PD-1 ⁺ cells, CD4 ⁺ TIM3 ⁺ cells, CD4 ⁺ PDL1 ⁺ cells, CD4 ⁺ LAG3 ⁺ cells, CD4 ⁺ CTLA-4 ⁺ cells, CD8 ⁺ PD-1 ⁺ cells, CD8 ⁺ TIM3 ⁺ cells, CD8 ⁺ PDL1 ⁺ cells, CD8 ⁺ LAG3 ⁺ cells and CD8 ⁺ CTLA-4 ⁺ cells,

c) the level and/or activity of one or more genes in a gene set characteristic of one or more cells selected from the group consisting of Tfh cells, CD4 ⁺ CD28- cells ^, CD4 ⁺ CD57 ⁺ cells ^, CD4 ⁺ CD28- cells CD57 ⁺ cells, CD4 ⁺ CD28 ^- KLRG1 ⁺ cells, CD8 ⁺ CD28 ^- cells, CD8 ⁺ CD57 ⁺ cells, CD8 ⁺ CD28 ^- CD57 ⁺ cells, CD8 ⁺ CD28 ^- KLRG1 ⁺ cells, CD4 ⁺ PD-1 ⁺ cells, CD4 ⁺ TIM3 ⁺ cells, CD4 ⁺ PDL1 ⁺ cells, CD4 ⁺ LAG3 ⁺ cells, CD4 ⁺ CTLA-4 ⁺ cells, CD8 ⁺ PD-1 ⁺ cells, CD8 ⁺ TIM3 ⁺ cells, CD8 ⁺ PDL1 ⁺ cells, CD8 ⁺ LAG3 ⁺ cells and CD8 ⁺ CTLA-4 ⁺ cells, and,

d) The level and/or activity of one or more cytokines selected from the group consisting of IL-6, IL-1α, IL-8, IL-7, MMP1, MIP-1β and MIP-1α.

10. The method of claim 9, wherein the NK cells are derived from cells selected from the group consisting of peripheral blood cells, bone marrow cells, and umbilical cord blood cells.