CN108265027B

CN108265027B - Tumor specific T cell based on non-natural glycometabolism engineering and construction method thereof

Info

Publication number: CN108265027B
Application number: CN201710000893.3A
Authority: CN
Inventors: 周德敏; 王伟玲
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2017-01-03
Filing date: 2017-01-03
Publication date: 2021-06-29
Anticipated expiration: 2037-01-03
Also published as: CN108265027A

Abstract

The invention discloses a T cell with a sialic acid derivative modified on the surface. Wherein the sialic acid derivative contains an azide group, and the sialic acid derivative is Ac₄Mannaz. Specifically, the invention discloses Ac₄Mannaz-DIBO-c (RGDFK) or Ac₄The ManNAz-DIBO-platelet modified T cell also discloses a method for preparing the T cell and application of the modified T cell in preparing a medicament for treating and/or preventing tumor cells.

Description

Tumor specific T cell based on non-natural glycometabolism engineering and construction method thereof

Technical Field

The invention belongs to the field of immunology and cell biology, and relates to a T cell for constructing a specific target tumor and killing the tumor, a construction method and application thereof.

Background

There are several methods currently used to treat and prevent tumors, but tumors remain a significant cause of death worldwide. Traditional methods of chemoradiotherapy directed to cells in the mitotic phase also affect normal cells, causing non-negligible side effects to tumor patients. Immunotherapy is a treatment method that can utilize a specific immune system to kill tumors in a targeted manner without affecting normal cells. The passive immunotherapy strategy of adoptive cell reinfusion is a method for adoptively reinfusing in vitro stimulated cultured lymphocytes into a tumor patient to treat tumors. The adoptive back lymphocyte can directly kill the tumor without activating the immune system in vivo, thereby overcoming the important limitation that the immune system needs to be activated in vivo to play a role in vaccine treatment. Two methods for obtaining tumor-specific cells are currently mainly involved: one is the in vitro culture, amplification and tumor specificity screening of infiltrating lymphocytes existing in tumor tissues of patients; another is to modify peripheral blood cells to obtain tumor specificity and expand rapidly in vitro. Because it is relatively difficult to obtain tumor-specific T cells, tumor-specific T cells can be obtained by genetic modification to specifically kill tumors. Among them, a method for specifically recognizing a tumor-associated antigen by modifying a T cell to express a Chimeric Antigen Receptor (CAR) using genetic engineering is a promising strategy for tumor immunotherapy.

CARs are composed primarily of three parts, an extracytoplasmic antigen binding region, a hinge region, and an intracellular signaling region. The extracellular region is a single chain variable domain (scFv) and has the function of specifically recognizing and binding Tumor Associated Antigen (TAA). The hinge region is usually composed of the immunoglobulin superfamily, and the intracellular signaling region is mainly composed of the zeta chain in the CD3 complex. In recent years, intensive research shows that the addition of costimulatory molecules such as CD28 or CD137 to the intracellular signaling region can enhance the anti-tumor immune response activity of cells, so that the second generation and third generation of CAR are developed. The CAR-expressing cell can directly recognize and combine with TAA on the surface of the tumor cell, transmit signals into the cell, activate cells to secrete cytokines including perforin, granzyme, IFN-gamma, TNF-alpha and the like, and thus, the CAR-expressing cell plays a role in killing the tumor cell. CAR-T therefore integrates antibody-antigen specific binding capacity and T cell killing function. In addition, CARs have other advantages, such as the antigens recognized by CARs do not require APC for processing and presentation; CARs can recognize not only short antigenic peptides, but also carbohydrate and glycolipid non-protein antigens; the high affinity scFv can make T cells sensitive to low concentrations of antigen, and exciting results have been obtained in clinical application cases.

At present, many early clinical trials on CAR-T cells for tumor treatment are ongoing, and some have already completed the assessment of safety and feasibility of adoptive reinfusion of CAR-T cells. For example, CD19 CAR-T cells have entered clinical trials for treating lymphomas and leukemias, GD2 CAR-T cells for treating gliomas, PSMA CAR-T cells for treating prostate cancer, HER2/neu CAR-T cells for treating lung cancer, etc., with good results.

The CAR vector system is an important factor influencing the quality of CAR-T cells, different vector systems have different advantages and disadvantages in the aspects of cost, safety and CAR expression level, and the gamma-retrovirus vector and lentivirus vector systems which are successfully applied in current clinical trials are used for CAR gene transduction, but virus vectors have many defects, such as carcinogenicity, limited target gene packaging capacity, complex production process, high cost and the like. Its carcinogenesis was found in one clinical trial, and 1 patient developed T cell leukemia after 30 months of treatment. Researchers have therefore attempted to use non-viral vectors for gene editing, including RNA-based electrotransformation and, more recently, hotter transposon systems, and while these approaches offer feasibility for T cell modification, there are still a number of issues to consider: the size and number of packaged gene fragments are limited and protein expression levels are unpredictable. Meanwhile, because of the technical limit of lentivirus transfection, the CAR-T is prepared in a longer period and is expensive. Meanwhile, the quality of the viral vector seriously influences the function of the CAR-T cell, and becomes a big problem of GMP of CAR-T. On the other hand, the existing CAR-T cells (CD 19-targeted CAR-T, CTL019) are permanently present in patients after gene editing, resulting in the side effect of B cell depletion. Therefore, the search and development of non-gene-editing T cell modification methods become a hot spot for tumor immunotherapy.

Bispecific antibodies, another technology of great interest in the field of tumor immunotherapy, can act as a vehicle to redirect immune effector cells and enhance the killing function of tumor cells by recognizing two targets, such as CD19 and CD3, simultaneously. Meanwhile, by targeting two different receptors on the same cell, bispecific antibodies can induce changes in cell signals such as cell proliferation, cell migration, and inflammatory responses. The use of bispecific antibodies for T cell activation is also a method for non-genetic editing of T cells.

However, bispecific antibodies also have the following drawbacks in application: 1. and (3) immunogenicity. The antibody drug is obtained by fusing 2 hybridomas expressing different monoclonal antibodies, and with the development of molecular biology, particularly the application of phage display technology of transgenic mice and antibody libraries, people modify the traditional murine antibodies to obtain various types of antibodies, but the antibodies can cause allergic reactions and anti-antibody reactions of different degrees, including human anti-mouse antibody reactions, human anti-chimeric antibody reactions, human anti-humanized antibody reactions and the like, and the reactions sometimes cause serious clinical consequences. 2. The molecular weight is too large. Even if the bispecific antibody with small molecular weight is used, the molecular weight of the bispecific antibody reaches 50kDa, and the bispecific antibody is not easy to pass through the blood vessel wall or tissue barrier so as to achieve the target effect; meanwhile, low-temperature storage is required, and the requirements on storage and transportation conditions of the medicine are high. 3. The production process is complex. The production of the antibody involves many links, including high expression cell strain screening, culture medium research and development, purification technology research and development and other aspects, so that the research and development period of the antibody drug is as long as tens of years, the cost is billions of dollars, how to shorten the research and development period, and reducing the production cost is an important factor for restricting the research and development of the antibody drug.

The T cells are modified by the small molecules, so that the T cells can be combined with tumor cells in a targeted mode, and signal paths in the cells can be activated to play a killing function. Sialic acid (sialic acid) is a nine-carbon sugar, which is one of important monosaccharides constituting sugar chains, widely present in various biological tissues, is an important component constituting cell-surface glycoconjugates, and is one of the most important components of glycans in the human immune system. Studies have shown that sialic acid is directly involved in immune cell migration, immune signaling and immune cell activation, making sialic acid and its derivatives candidate compounds for the modification of T cells.

Disclosure of Invention

The invention provides a T cell, the surface of which is modified with a sialic acid derivative, wherein the sialic acid derivative contains an azide group;

in a preferred embodiment of the invention, said saliva is salivaThe liquid acid derivative is Ac₄ManNAz。

In one embodiment of the invention, said Ac₄Mannaz is linked to DIBO-c (RGDFK) or DIBO-foil;

the invention also provides a method for preparing a sialic acid derivative modified T cell, comprising incubating a T cell with a sialic acid derivative to produce a modified T cell and subsequently washing; in a preferred embodiment of the invention, the sialic acid derivative is Ac₄ManNAz；

Further, said Ac₄The concentration of the Mannaz is 10-25 mu M, and the incubation time is 72-120 hours; preferably, said Ac₄Mannaz was incubated at 20. mu.M for 96 hours, washed 3 times with PBS and freed Ac was removed from the culture₄Mannaz; further, adding Ac₄Prior to ManNAz, the number of activated T cells is adjusted, preferably to 1X 10⁶/ml；

Furthermore, the preparation method also comprises the steps of adding DIBO-c (RGDFK) or DIBO-Folate into the modified T cells, incubating and washing; the specific operation method comprises adding DIBO-c (RGDFK) or DIBO-foil with final concentration of 100 μ M, incubating at room temperature for 2h, and washing with PBS for 3 times;

the invention also provides a pharmaceutical composition, which comprises the T cell and optional pharmaceutically acceptable auxiliary materials.

The invention also provides the application of the T cell in preparing a medicament for treating and/or preventing and/or adjunctively treating tumor cells;

in a preferred embodiment of the present invention, wherein said tumor cell is a tumor cell highly expressing integrin α v β 3; further, the tumor cells with high expression of integrin α v β 3 are U87 cells.

In another preferred embodiment of the present invention, wherein said tumor cell is a folate receptor high expressing tumor cell; further, the tumor cells with high expression of the folate receptor are SKOV3 cells.

The invention is based on the non-natural sugar metabolism engineering strategy to modify T cells, modifies the surface of the T cells with non-natural sugar sialic acid derivatives, simulates the method of a bispecific antibody (CD3 targeting) to activate the T cells, and constructs a CAR-T-like T cell, so that the CAR-T-like T cell can be combined with tumor specific antigen and activated to play a role in tumor killing. The principle of the invention will be described in detail below:

the invention selects sialic acid derivatives to modify the surface of T cells, preferably Ac₄ManNAz, in which azide and terminal alkyne (DIBO) are bioreactive groups, can undergo bio-orthogonal reactions to couple a series of functional groups.

In selecting the target, in one embodiment of the present invention, the present invention selects the integrin α v β 3 highly expressed in glioma cells, wherein the α -chain extracellular region of the integrin α v β 3 can specifically recognize the polypeptide containing the RGD sequence, thereby being used for diagnosis and treatment of tumors. The invention selects DIBO-c (RGDFK) polypeptide, wherein the DIBO-group can be connected with the azide group of sialic acid derivative through bioorthogonal reaction, and the-c (RGDFK) can be specifically combined with integrin alpha V beta 3 with high affinity, thereby being capable of targeting U87 glioma cells with high expression of integrin alpha V beta 3.

In selecting a target, in another embodiment of the invention, the invention selects folate receptor alpha, wherein folate receptor alpha specifically binds folate (folate) and is therefore useful for the diagnosis and treatment of tumors. The invention selects DIBO-Folate, wherein a DIBO-group can be connected with an azide group of a sialic acid derivative through a bioorthogonal reaction, and-Folate can be specifically combined with a Folate receptor alpha with high affinity, so that SKOV3 tumor cells with high expression of the Folate receptor alpha can be specifically targeted.

The T cell constructed by the invention can specifically recognize and combine with tumor antigen. Compared with CAR-T, the preparation process is simpler, the CAR-T construction process needs about 21 days, and in the technology, the labeling of the non-natural sugar only needs 4 days; whereas the second step requires only 2h for targeting molecule attachment. Meanwhile, the immune cells modified by the non-natural sugar can be metabolized into normal immune cells in about 7 days without antigen stimulation, which indicates that the technology can not permanently transform the immune cells.

Compared with the prior art, the invention has the following characteristics:

1. the non-viral vector is used for modifying the T cell in a non-gene editing manner, so that the application safety is improved;

the quantity of the target molecules borne on the surface of the T cells can be regulated, namely the killing capacity of the T cells can be regulated, so that the application flexibility is improved;

3. the compounds used for T cell marking are small molecules, but not antibodies or polypeptides, so that the T cell targeting modification is more possible;

4. sialic acid derivatives metabolized to the surface of T cells can metabolize the lower membranes over a period of time without permanent changes to the T cells, reducing side effects (e.g., B cell regeneration failure by CAR-T);

5. the sialic acid on the surface of the T cell is modified to promote the activation of the T cell, and a new mechanism for activating a T cell signal channel is discovered.

Drawings

FIG. 1 is Ac₄Metabolic characteristics of ManNAz in Jurkat cells, wherein A is Ac detected by fluorescence₄ManNAz metabolic upper membrane time curve, B is fluorescence detection Ac₄Membrane time curve under Mannaz metabolism, C is fluorescence detection Ac₄ManNAz metabolic upper membrane dose curve;

FIG. 2 is Ac₄Metabolic characteristics of ManNAz in T cells, wherein A is Ac for fluorescence detection₄Picture and time curve of anabolic membrane of Mannaz, B is fluorescence detection Ac₄Picture and time curve of membrane under Mannaz metabolism, C is fluorescence detection Ac₄Pictures and dose curves of the membrane on ManNAz metabolism; d is different dosage Ac₄ManNAz cytotoxicity assay;

FIG. 3 is Ac₄The Mannaz-DIBO-c (RGDFK) -modified Jurkat cells have specific targeting binding to the Integrin alpha v beta 3 high-expression U87 tumor cells.

FIG. 4 is Ac₄The Mannaz-DIBO-c (RGDfK) modified T cells have specific target killing on U87 tumor cells with high expression of Integrin alpha v beta 3, wherein A is an optical microscope photograph of the T cells modified in different modes after being incubated with target cells for 18h, and B is detectionThe cell killing effect of the T cells modified in different modes after being incubated with the target cells for 18 hours, C is the cell factor secretion amount in the cell supernatant after the T cells modified in different modes and the target cells are incubated for 2, 4, 8, 12 and 24 hours, and D is Ac with different effect-target ratios₄The optical microscope photograph of the Mannaz-DIBO-c (RGDFK) -T cells after 18h of co-incubation with the target cells, E is Ac for detecting different effective target ratios₄The cell killing effect of the ManNAz-DIBO-c (RGDFK) -T cell after 18h of co-incubation with the target cell is realized, and F is Ac for detecting different effect-target ratios₄Cell supernatant cytokine secretion after the ManNAz-DIBO-c (RGDFK) -T cells and target cells are incubated for 18 h;

FIG. 5 is Ac₄ManNAz-DIBO-c (rgdfk) modification promotes T cell activation and is dependent on expression of Integrin α v β 3; detecting the expression level of CD25 (late activation marker) and CD69 (early activation marker) of T cells after co-culture with target cells by flow cytometry;

FIG. 6 is Ac₄ManNAz-DIBO-c (rgdfk) modification promotes T cell immune synapse formation; fluorescence photographs of CD3 ζ (T cell receptor activation indicator) localization of Jurkat cells modified by different methods;

FIG. 7 is Ac₄The Mannaz-DIBO-c (RGDfK) modified T cells have specificity on the combination and the killing of Integrin alpha v beta 3 high expression cells, wherein A is Ac₄Fluorescent microscope pictures of Mannaz-DIBO-c (RGDFK) -modified T cells (green fluorescent marker) co-cultured with U87 cells (red fluorescent marker) and Hela cells (red fluorescent marker) for 18h, respectively, and B is Ac₄Photographs of ManNAz-DIBO-c (rgdfk) -modified T cells were taken with a fluorescence microscope co-cultured with U87 cells (red fluorescent label) and Hela cells (green fluorescent label) at a ratio of E: T of 2:1 for 18 h; c is Ac₄Photographs of Mannaz-DIBO-c (RGDFK) -modified T cells were taken with a fluorescent microscope co-cultured with U87 cells and Hela cells at a ratio of E: T of 5:1 for 18 h;

FIG. 8 is Ac₄The ManNAz-DIBO-Folate modified T cells have specific targeted killing on SKOV3 tumor cells with high expression of Folate receptor alpha. Wherein, A is an optical microscope photo of the T cells modified in different modes after being incubated with the target cells for 24 hours, B is a photo of the T cells modified in different modes after being incubated with the target cells for 24 hours,c is used for detecting the secretion amount of cytokines in cell supernatant after the T cells modified in different modes and target cells are incubated for 2, 4, 8, 12 and 24 hours, and D is Ac with different effective target ratios₄An optical microscope photograph of the Mannaz-DIBO-platelet-T cells after being incubated with the target cells for 24h, wherein E is Ac for detecting different effective target ratios₄After the ManNAz-DIBO-Folate-T cells and the target cells are incubated for 24h, the cell killing effect is achieved, and F is Ac for detecting different effect-target ratios₄After the Mannaz-DIBO-platelet-T cells and the target cells are incubated for 24h, the secretion amount of cytokines in cell supernatant is increased;

Detailed Description

The present invention is further illustrated by the following specific examples.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The unnatural sugar involved in the invention is Ac₄Mannaz; the immune cells are T cells; targeting molecules are DIBO-c (RGDFK) and DIBO-Folate, which target integrin α v β 3 and Folate Receptor (Folate Receptor) α, respectively; to carry out Ac₄In a killing experiment of the ManNAz-DIBO-RGD modified T cells, U87 cells are an integrin alpha v beta 3 high-expression cell line, and Hela cells are an integrin alpha v beta 3 low-expression cell line; to carry out Ac₄In a killing experiment of the ManNAz-DIBO-Folate modified T cells, SKOV3 cells are Folate receptor alpha high-expression cell lines, and A549 cells are Folate receptor alpha low-expression cell lines.

Tumor cells Jurkat cells, U87 cells, Hela cells, SKOV3 cells and A549 cells used by the invention are purchased from Shanghai Zhongzhou academy of sciences cell banks; human Peripheral Blood Mononuclear Cells (PBMCs) were purchased from the beijing blood center.

The sources of the reagents used in the invention are as follows:

the RPMI 1640 culture solution manufacturer is Gibco, and the model is A1049101; DMEM medium manufacturer museum 15019; the FBS manufacturer is PAN, P30-3302; hIL-2 is R & D; CD3/CD28 magnetic bead manufacturer Invitrogen, 11161D; ac4ManNAz from Thermo, 88904; DIBO-Alex 488 from Invitrogen, C10405; DIBO-RGD is available from Gill Biochemical Co., Ltd, and in the present invention DIBO-RGD is equivalent to DIBO-c (RGDfK); the LDH cytotoxic assay kit is from Promega, G1708; the Human IL-2ELISA kit was from Biolegend, 431804; the Human IFN-. gamma.ELISA kit was from Biolegend, 430104; the Human IL-6 ELISA kit was from Biolegend, 430504; the Human TNF-. alpha.ELISA kit was obtained from Biolegend, 430204; anti-human CD69 PE was from eBioscience, 12-0699; anti-human CD25 PE was from eBioscience, 12-0259; anti-human CD3 FITC was from eBioscience, 11-0039; CD3 ζ (6B10.2) was from Santa Cruz, sc-1239.

EXAMPLE 1 cell culture

1. Cell culture

Adherent cells: u87 cells, Hela cells and A549 cells were cultured in DMEM (containing 10% fetal calf serum; 100U/ml penicillin; 100. mu.g/ml streptomycin) at 37 ℃ and SKOV3 cells in RPIM-1640 medium at 2X 10⁴One/ml was plated on 60-mm cell culture dishes, and passaged 1 time every 3 days.

Suspension of cells: jurkat cells were cultured in RPMI-1640 medium (containing 10% fetal bovine serum; 100U/ml penicillin; 100. mu.g/ml streptomycin) at 37 ℃ at 2X 10⁴One/ml was plated on 60-mm cell culture dishes, and passaged 1 time every 3 days.

2. Flow cytometry detection of tumor cell tumor antigen (integrin alpha v beta 3 and folate receptor alpha) expression

Collecting U87 and Hela cells (detecting integrin alpha v beta 3), SKOV3 and A549 cells (detecting folate receptor alpha) in the logarithmic growth phase, and adjusting the cell number to be 1 × 10⁶Fixing 4% paraformaldehyde at room temperature for 15min, washing with PBS, and blocking with 5% BSA at room temperature for 1 h; anti-integrin α v β 3 (or folate receptor α) antibody was incubated for 2h at 37 ℃ with PBS as negative control; and (3) washing with PBS, incubating with FITC-labeled secondary antibody for 1h at room temperature, washing, and detecting with a flow cytometer.

3. Isolation and activation of peripheral blood mononuclear cells

Collecting peripheral blood of healthy volunteers, diluting the blood with PBS at a ratio of 1:1, slowly adding into a 15ml centrifuge tube filled with lymphocyte separation liquid along the wall of the centrifuge tube, centrifuging at 2000rpm for 20min, sucking leukocyte membrane layer as PBMCs, washing cells with PBS once, resuspending cells in RPMI-1640 culture medium, transferring to 10cm cell culture dish, 37 deg.C, and 5% CO₂Culturing for 2h to make the mononuclear cells adhere to the wall, and collecting the suspension cells, namely the peripheral blood lymphocytes.

T cell activation in vitro: will be 1 × 10⁶Placing the peripheral blood lymphocytes obtained above in 10% FBS RPMI-1640 complete culture medium, adding appropriate amount of CD3/CD28 magnetic beads according to the ratio of cell to magnetic beads of 1:3, adding 100IU/ml IL-2 every 2 days, 37 deg.C, 5% CO₂Culturing under the condition. After 3d stimulation, cell clumping was seen, indicating that the cells had been activated, and subsequent experiments were performed with cells in this state.

Example 2 detection of Ac₄Metabolic characterization of ManNAz in cells

To detect Ac₄The marking efficiency of the ManNAz on the surfaces of Jurkat cells and T cell membranes is realized by adopting the following method:

1×10⁶the Jurkat cells or T cells/ml were seeded in 60-mm cell culture dishes and different doses (final concentrations 0. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 40. mu.M, 80. mu.M, 160. mu.M and 320. mu.M) of Ac were added₄Mannaz incubation for 48h (for dose curves) or 20. mu.M Ac₄ManNAz were incubated for different times (0h, 24h, 48h, 72h and 96h for the time curves). The cells were washed 3 times with PBS to remove free Ac from the culture medium₄Mannaz, add Alexa Fluor488-DIBO at a final concentration of 200. mu.M, incubate for 2h at room temperature, wash 3 times with PBS, take pictures with a fluorescence microscope or detect with a flow cytometer.

Ac₄The metabolic characteristics of Mannaz in Jurkat cells are shown in FIG. 1, where A is Ac detected by fluorescence₄Pictures and time curves of the membrane on ManNAz metabolism; b is fluorescence detection Ac₄Pictures and time curves of the membrane under ManNAz metabolism; c is fluorescence detection Ac₄ManNAz images of membranes metabolically and dose curves.

This test shows Ac₄Mannaz is metabolized in Jurkat cells with dose-and time-dependent characteristics, Ac when the dose reaches 40. mu.M₄The membrane amount on the Mannaz metabolism reaches a platform; the incubation time reaches 48When h is, Ac₄The membrane amount on the Mannaz metabolism reaches a platform; ac that has been metabolized to the cell membrane of Jurkat cells₄Mannaz in culture (without Ac)₄Culture medium of ManNAz) for 48h, almost all membranes were metabolized.

Ac₄The metabolic characteristics of ManNAz in T cells are shown in fig. 2, where a is fluorescence detected Ac₄Pictures and time curves of the membrane on ManNAz metabolism; b is fluorescence detection Ac₄Pictures and time curves of the membrane under ManNAz metabolism; c is fluorescence detection Ac₄Pictures and dose curves of the membrane on ManNAz metabolism; d is different dosage Ac₄Cytotoxicity experiments of ManNAz.

This test shows Ac₄ManNAz metabolism has dose-and time-dependent characteristics in T cells, Ac when the dose reaches 80. mu.M₄The membrane amount on the Mannaz metabolism reaches a platform, and the labeling efficiency is about 55%; when the incubation time reaches 96h, Ac₄The membrane amount on the Mannaz metabolism reaches a platform; similarly, the film removal time was about 96 hours. The results indicate low doses of Ac₄ManNAz (less than or equal to 300 mu M) has no obvious cytotoxicity and is safer.

EXAMPLE 3 preparation of Ac₄Mannaz-DIBO-c (RGDfK) -modified T lymphocytes

The activated T lymphocytes obtained in example 1 were collected and the number of the cells was adjusted to 1X 10⁶Perml, add Ac to a final concentration of 20. mu.M₄Mannaz was incubated for 96 hours, cells were washed 3 times with PBS, and free Ac was removed from the culture broth₄ManNAz, to obtain surface modified with Ac₄T cells of Mannaz.

Then, DIBO-c (RGDFK) with a final concentration of 200 μ M was added to the modified T cells, incubated at room temperature for 2 hours, and washed with PBS 3 times to obtain Ac₄Mannaz-DIBO-c (RGDFK) -modified T lymphocytes for subsequent functional assays.

EXAMPLE 4 preparation of Ac₄Mannaz-DIBO-platelet modified T lymphocytes

The activated T lymphocytes obtained in example 1 were collected and the number of the cells was adjusted to 1X 10⁶Perml, add Ac to a final concentration of 20. mu.M₄Mannaz was incubated for 96 hours, cells were washed 3 times with PBS, and the culture medium was removedOf free Ac₄ManNAz, to obtain surface modified with Ac₄T cells of Mannaz.

Subsequently, DIBO-Folate was added to the modified T cells at a final concentration of 200. mu.M, incubated at room temperature for 2 hours, and washed with PBS 3 times to obtain Ac₄Mannaz-DIBO-platelet modified T lymphocytes for subsequent functional assays.

Example 5 immunofluorescence detection of Ac₄Binding of Mannaz-DIBO-c (RGDFK) -modified Jurkat cells to target cells

Jurkat cells engineered to Ac according to the method described in example 3₄Mannaz-DIBO-c (RGDFK) -Jurkat cells, CFSE (Green) fluorescent labeling, U87 cells or Hela cells (negative control) Mito Tracker Red (Red) fluorescent labeling, Ac₄Mannaz-DIBO-c (RGDFK) -Jurkat cells were CO-incubated with U87 cells or HeLa cells at a ratio of 1:1 at 37 ℃ with 5% CO₂Culturing for 4h in an incubator, gently blowing and beating 3 times by PBS, observing by a fluorescence microscope, and connecting after green and red fluorescence are superposed, namely, the Jurkat cells and the U87 cells are considered to be combined in a targeted way. As shown in FIG. 3, Ac₄The Mannaz-DIBO-c (RGDFK) -Jurkat cells aggregated with the U87 cells to form a cell mass, and Ac₄No significant cell aggregation was observed between Mannaz-DIBO-c (RGDFK) -Jurkat cells and Hela cells.

Experimental example 1 Ac₄Mannaz-DIBO-c (RGDfK) modified T cells have specific targeted killing on U87 tumor cells with high expression of integrin alpha v beta 3

To detect Ac₄The ManNAz-DIBO-c (RGDfK) modified T cells have a targeted killing function on U87 tumor cells highly expressing integrin alpha v beta 3, and the death condition of the tumor cells is detected by using a CytoTox 96 Non-Radioactive cytoxicity method, which comprises the following steps:

100. mu.l each of U87 cells and Ac4Mannaz-DIBO-c (RGDfK) modified T cells prepared in example 3 (effective target ratio: 10:1) were added to a 96-well plate; u87 cells and culture solution are added into a U87 cell natural release hole, and each 100 mu l of the culture solution is added; u87 cells and lysate are added into a U87 cell maximum release pore, and each 100 mu l of the lysate is added; adding T cells and culture solution into the natural T cell release hole by 100 mu L each; all the above-mentioned materials are equipped with three composite holes, at 37 deg.C and 5% CO₂Culturing for 18h in an incubator, centrifuging the 96-well culture plate at 1500r/min for 5min, sucking 50 μ l of supernatant per well, placing in a flat-bottomed 96-well culture plate, adding 50 μ l of detection solution, reacting for 30min, adding 50 μ l of reaction stop solution per well, and measuring Optical Density (OD) at 490nm with a microplate reader.

And (3) calculating:

incubation of Ac alone with untreated T cells₄ManNAz's T cells and DIBO-c (RGDfK) -only incubated T cell groups as controls, Ac₄Mannaz-DIBO-c (RGDFK) -modified T cells were used as experimental groups. After 18 hours of co-culture of each group of cells, photographs were taken with an optical microscope, and as a result, Ac was found in FIG. 4A₄Significant clones were formed from ManNAz-DIBO-c (rgdfk) -modified T cells and significant U87 cell debris was observed, which was not seen in the control groups (including Hela cells). The result of the detection of the CytoTox 96 Non-Radioactive Cytoxicity method proves that Ac is₄The killing effect of the Mannaz-DIBO-c (RGDFK) modified T cells on U87 cells reaches 80%, as shown in FIG. 4B.

The amount of cytokine secretion in the supernatant was measured using an ELISA kit, and IFN-. gamma.IL-2, TNF-. alpha.and IL-6 concentration values were calculated. The results show that Ac₄After the T cells modified by Mannaz-DIBO-c (RGDFK) and U87 cells are cultured together, IFN-gamma and IL-2 are greatly increased, and the statistical difference with each control group (including Hela cells) is obvious; and Ac with₄Compared with the co-culture group of the ManNAz-DIBO-c (RGDFK) modified T cells and the Hela cells, the secretion amount of the TNF-alpha is not obviously different; since U87 cells were able to synthesize large amounts of IL-6, there was no significant difference in the amount of IL-6 secreted compared to the co-cultured group of untreated T cells and U87 cells, as shown in FIG. 4C.

Ac of different effective target ratio₄Results of the experiment for co-culturing ManNAz-DIBO-c (RGDFK) -modified T cells and U87 cells show that Ac is shown in FIG. 4D₄When the Mannaz-DIBO-c (RGDFK) modified T cells and U87 cells are incubated at an effective target ratio of 2:1, a certain killing effect occurs, and Ac has high effective target ratio₄Mannaz-DIBO-c (RGDfK) -modified T cell pairsKilling ability of U87 cells was also increased; and Ac₄When the Mannaz-DIBO-c (RGDfK) modified T cells and Hela cells are incubated at the effective target ratio of 2:1, no obvious killing effect is generated, and when the effective target ratio is increased to 10:1, Ac₄The killing of Hela cells by the ManNAz-DIBO-c (RGDfK) modified T cells was only 40%. Meanwhile, the secretion of IFN-gamma is also increased, but other cytokines are not obviously changed, which is shown in the figure 4E and the figure 4F. This result indicates Ac₄The killing function of the T cells modified by the ManNAz-DIBO-c (RGDFK) depends on the expression of the integrin alpha v beta 3, is realized by increasing the secretion of IFN-gamma and IL-2, has smaller influence on the secretion of TNF-alpha and IL-6, and indicates that the T cell modification method causes weaker cytokine storm.

EXAMPLE 2 Ac₄Mannaz-DIBO-c (RGDfK) modified T cells promote T cell activation

Untreated T cells, Ac from example 3₄The T cells modified by Mannaz-DIBO-c (RGDFK) were co-cultured with U87 cells or Hela cells, and the cells were collected and stained with CD3/CD25 (middle and late stage activation index) or CD3/CD69 (early stage activation index) in duplicate, and subjected to flow assay, the results of which are shown in FIG. 5. Ac was found to be comparable to untreated T cells₄The expression of CD25 and CD69 of the T cells modified by Mannaz-DIBO-c (RGDfK) is obviously up-regulated, and the Ac is proved₄The ManNAz-DIBO-c (RGDFK) modification method can promote the activation of T cells. Ac co-cultured with U87 compared to Hela group₄Expression of both CD25 and CD69 was up-regulated in Mannaz-DIBO-c (RGDfK) -modified T cells, demonstrating that Ac₄Mannaz-DIBO-c (RGDfK) -modified T cell activation is integrin α v β 3 dependent.

Experimental example 3 Ac₄Modification of T cells by Mannaz-DIBO-c (RGDfK) to promote T cell immune synapse formation

Immune synapses serve as a specialized contact surface between T cells and APC, and their formation is a prerequisite for the recognition of membrane surface molecules and the transmission of signals between immune cells, and play an important role in the activation of T cells. To detect Ac₄Whether the ManNAz-DIBO-c (rgdfk) modification method promotes immune synapse formation, we performed the following experiment:

u87 cells and Hela cellsPancreatin digestion, serum-free DMEM culture solution heavy suspension, counting, taking 3X 10⁵U87 and HeLa cells, 5. mu.M CFSE dye was added and incubated at 37 ℃ for 30 minutes. Wash and resuspend U87 and Hela cells in 25. mu.l RPMI-1640 with 10% serum for use. 3X 10⁵Jurkat cells, resuspended in 25. mu.l RPMI-1640 containing 10% serum; jurkat cells and Ac₄Mannaz-DIBO-c (RGDFK) -modified Jurkat cells were mixed with U87 cells and Hela cells, respectively, and placed in a 1.5ml EP tube, and incubated at 37 ℃ for 10 min. The co-cultured cells were spread evenly on a confocal laser culture dish and incubated at 37 ℃ for 30 min. Fixing formaldehyde at room temperature for 15min, and sealing sheep serum at room temperature for 30 min. Add 200. mu.l CD3 ζ mab overnight at 4 ℃. PBS washing, adding 200. mu.l of Goat anti mouse Alex 488 dye, and dyeing for 1h at room temperature in the dark. After washing with PBS, formation of cellular immune synapses in the DISH was observed under a laser confocal microscope and photographed.

The results are shown in FIG. 6, and Ac was found to be comparable to untreated Jurkat cells₄Mannaz-DIBO-c (RGDFK) -modified Jurkat cell CD3 ζ was predominantly localized at the site of interaction with U87 cells, demonstrating that Ac₄The ManNAz-DIBO-c (RGDfK) modification method promotes the formation of T cell immune synapses. Whereas, the CD3 zeta localization was not significantly altered after co-culturing unmodified T cells with U87 cells, demonstrating that Ac₄The ManNAz-DIBO-c (rgdfk) modification promotes T cell immune synapse formation.

EXAMPLE 4 Ac₄Mannaz-DIBO-c (RGDfK) -modified T cells specifically bind to and kill integrin alpha v beta 3 high expression cells

To verify Ac₄Specificity of Targeted killing by Mannaz-DIBO-c (RGDfK) -modified T cells, we labeled U87 cells (Red) and Hela cells (Green) with different fluorescence, respectively, from Ac of example 3₄Mannaz-DIBO-c (RGDFK) -modified T cells were co-cultured for 18h and photographed by fluorescence microscope, and the results are shown in FIG. 7B. It was found that when the ratio of effective targets was 2:1, the red fluorescence decreased, and when the ratio of effective targets was increased to 5:1, the red fluorescence almost disappeared, indicating that the U87 cells were lysed, while the green fluorescence did not significantly change, indicating that the Hela cells were not affected, which proved that Ac was present₄Mannaz-DIBO-c (RGDfK) -modified T cells highly express Integrin alpha v beta 3The killing of the cells has obvious specificity.

Experimental example 5 Ac₄The ManNAz-DIBO-folate modified T cell has specific targeted killing to SKOV3 tumor cells with high expression of folate receptor alpha

The specific procedure is as above, and the results are shown in FIG. 8, Ac from example 4₄The killing effect of the T cell modified by the ManNAz-DIBO-Folate on SKOV3 cells (with high expression of Folate receptor alpha) reaches 60%, while the killing effect on A549 cells (with low expression of Folate receptor alpha) is only 14%. Ac of₄After the ManNAz-DIBO-Folate modified T cells and SKOV3 cells are co-cultured, IFN-gamma and IL-2 in the supernatant are greatly increased, and the results are obviously different from those of each control group (including A549 cells) in statistics, and the results are shown in a figure 8B; ac of different effective target ratio₄Results of the experiment for co-culturing Mannaz-DIBO-Folate modified T cells and SKOV3 cells showed that Ac was shown in FIG. 8C₄When the Mannaz-DIBO-Folate modified T cells and SKOV3 cells are incubated at an effective target ratio of 2:1, a certain killing effect occurs, and Ac has an effect of increasing the effective target ratio₄Killing capacity of Mannaz-DIBO-Folate modified T cells and SKOV3 cells is increased, and secretion of IFN-gamma and IL-2 in supernatant is increased; and Ac₄No increase in killing activity was observed when the Mannaz-DIBO-c (RGDFK) -modified T cells and A549 cells were co-cultured at an effective-to-target ratio of 10: 1. This result indicates Ac₄The killing function of the ManNAz-DIBO-Folate modified T cells and SKOV3 cells is dependent on the expression of Folate receptors and is realized by the increased secretion of IFN-gamma and IL-2.

The result shows that the T cell modified by the non-natural sugar-targeting molecule has obviously enhanced killing effect on tumor cells with high expression of tumor antigens, and the killing effect depends on the expression of the tumor antigens.

It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. Not all embodiments are exhaustive. All obvious changes and modifications which are obvious to the technical scheme of the invention are covered by the protection scope of the invention.

Claims

1. A healthy human peripheral blood T cell, wherein the surface of the T cell is modified with a sialic acid derivative, the sialic acid derivative comprises an azide group, and the sialic acid derivative is Ac₄Mannaz, said Ac₄Mannaz is linked to DIBO-c (RGDFK) or DIBO-foil.

2. A method for modifying T cells in peripheral blood of a healthy person, which is characterized in that the T cells and sialic acid derivatives are incubated together and washed; the sialic acid derivative is Ac₄ManNAz，Ac₄The concentration of the Mannaz is 10-25 mu M, and the incubation time is 72-120 hours; then washed 3 times with PBS to remove free Ac from the culture broth₄Mannaz; also includes adding DIBO-c (RGDFK) or DIBO-Folate to the modified T cells, incubating and washing.

3. The method of claim 2, wherein Ac₄The concentration of ManNAz was 20 μ M and the incubation time was 96 hours.

4. A pharmaceutical composition comprising the healthy human peripheral blood T cells of claim 1, and optionally a pharmaceutically acceptable excipient.

5. Use of the healthy human peripheral blood T cells according to claim 1 for the preparation of a medicament for the therapeutic and/or prophylactic and/or adjunctive treatment of tumor cells; wherein the tumor cell is a tumor cell with high expression of integrin α v β 3; or the tumor cell is a tumor cell with high expression of folate receptor.

6. The use according to claim 5, characterized in that said tumor cells highly expressing integrin α v β 3 are U87 cells and said tumor cells highly expressing folate receptor are SKOV3 cells.