Use of non-porous, non-magnetic particles as kit for activation and proliferation of human T lymphocytes

Abstract

The present invention relates to a specific type of non-porous and non-magnetic composite material consisting of a solid surface having a specific diameter and an antigen recognition element for use in a method for activating and amplifying T lymphocytes.

Description

Technical Field

[0001] This invention relates to a specific type of microparticle, which consists of a solid surface with a specific diameter and an antigen recognition element, and can be used to activate and expand T lymphocytes. Background Technology

[0002] This invention generally relates to microparticles for stimulating cells and related methods. In particular, this invention relates to activating and proliferating human T lymphocytes by binding at least one capture ligand attached to the surface of the microparticle to a corresponding cell surface portion of the T lymphocyte.

[0003] For example, and not wanting to be bound by theory, it is known that anti-CD3 antibodies are key factors in T lymphocyte activation and proliferation upon contact with T lymphocyte populations. Co-stimulatory signals introduced by anti-CD28 antibodies contribute to increased proliferation and stability by preventing premature apoptosis (Li, Y. et al. (2010), J Transl Med 8, 104). Since receptor cross-linking provided by free antibodies is insufficient to activate intracellular signaling, methods using beads coated with anti-CD3 and anti-CD28 have been developed (Trickett A. et al. (2003), J Immunol Methods. 1; 275(1-2): 251-5).

[0004] Currently, the main system in use is:

[0005] (1) Dynabeads™ M-450 (Dynal Biotech) or CD3 / CD28 T cell expander kit (Dynal Biotech) containing fixed anti-CD3 and anti-CD28 antibodies, which are magnetic beads with diameters of 4.5 and 2.8 μm, respectively; or

[0006] (2) MACSiBeads™ (Miltenyi Biotech) contains anti-CD2, anti-CD3 and anti-CD28 antibodies, with magnetic particles having a diameter of approximately 4.5 μm.

[0007] One problem encountered when using these beads is related to the diameter of the beads used. In particular, these diameters of the particles allow monocytes and macrophages, which are often present in the sample, to engulf them. Berenson et al. ( EP1257632B1 This characteristic of monocytes is also mentioned, and it is used as a pretreatment step before the activation and expansion of T lymphocytes. Additionally, it is generally difficult to remove particles from a sample. Summary of the Invention

[0008] This invention eliminates the need to remove monocytes before activating and expanding T lymphocytes by using microparticles that cannot be phagocytosed by other cells. Furthermore, due to the particle diameter, cells can be easily removed by mechanical force and sample filtration.

[0009] These objectives are achieved through the microparticles and related methods of the present invention.

[0010] Therefore, the present invention provides microparticles, wherein

[0011] (1) The particles are non-magnetic;

[0012] (2) The diameter of the particles is 10 to 60 μm; and

[0013] (3) The microparticle has at least a first capture ligand capable of binding to a first cell surface portion of a human T lymphocyte, the first capture ligand being attached to the surface of the microparticle.

[0014] Preferably, these microparticles are suitable for the activation and proliferation of human T lymphocytes.

[0015] The present invention also provides a kit containing the said microparticles and a method for activating and proliferating human T lymphocytes using the said microparticles and the kit.

[0016] As further illustrated by examples below, the microparticles of the present invention eliminate the need to remove monocytes prior to T lymphocyte activation and expansion. Furthermore, due to the particle diameter, cells can be easily removed, for example, by mechanical force and sample filtration.

[0017] In these examples, non-porous, non-magnetic SiO2 microparticles (20 to 40 μm) are exemplified, which have been functionalized using silanes as surface modifiers with two distinct functional groups. These surface modifiers, introduced onto the microparticle surface, are activated either directly or with different linkers, allowing the capture ligands to covalently bind to the microparticle surface. In some instances, the capture ligands are anti-CD3 and anti-CD28 antibodies, which can be specifically used for the activation and proliferation of T lymphocytes.

[0018] Therefore, the microparticles used further as examples below are nonmagnetic and nonporous, and have irreversibly (covalently) bound antibodies (anti-CD3 and anti-CD28) on their surface. These particles can be a mixture of these antibodies on a single microparticle, or they can be used as a mixture of particles having only anti-CD3 or anti-CD28.

[0019] Therefore, in a preferred embodiment, the present invention provides microparticles, wherein

[0020] (1) The particles are non-magnetic;

[0021] (2) The particles are non-porous.

[0022] (3) The particles are made of SiO2;

[0023] (4) The diameter of the particles is 10 to 60 μm;

[0024] (5) The surface of the particles is modified with a surface modifier, wherein the surface modifier is silane; and

[0025] (6) The microparticle has a first capture ligand and a second capture ligand capable of binding to a first cell surface portion and a second cell surface portion of human T lymphocytes, the first capture ligand and the second capture ligand being attached to the surface of the microparticle via a surface modifier.

[0026] The first and second capturing ligands are covalently linked to the surface of the microparticles via surface modifiers; and

[0027] The first capture ligand that can bind to the first cell surface portion of human T lymphocytes is an anti-CD3 antibody, and the second capture ligand that can bind to the second cell surface portion of human T lymphocytes is an anti-CD28 antibody.

[0028] Alternatively, in a preferred embodiment, the invention provides microparticles, wherein

[0029] (1) The particles are non-magnetic;

[0030] (2) The particles are non-porous.

[0031] (3) The particles are made of SiO2;

[0032] (4) The diameter of the particles is 10 to 60 μm;

[0033] (5) The surface of the particles is modified with a surface modifier, wherein the surface modifier is silane; and

[0034] (6) The microparticle has a first capture ligand, a second capture ligand, and a third capture ligand capable of binding to a first cell surface portion, a second cell surface portion, and a third cell surface portion of a human T lymphocyte. The first capture ligand, the second capture ligand, and the third capture ligand are attached to the surface of the microparticle via a surface modifier.

[0035] The first, second, and third capturing ligands are covalently linked to the surface of the microparticles via surface modifiers; and

[0036] The first capture ligand that can bind to the first cell surface region of human T lymphocytes is the anti-CD3 antibody, the second capture ligand that can bind to the second cell surface region of human T lymphocytes is the anti-CD28 antibody, and the third capture ligand that can bind to the second cell surface region of human T lymphocytes is the anti-CD2 antibody.

[0037] Alternatively, the present invention provides microparticles, wherein

[0038] (1) The particles are non-magnetic;

[0039] (2) The particles are non-porous.

[0040] (3) The particles are made of SiO2;

[0041] (4) The diameter of the particles is 10 to 60 μm;

[0042] (5) The surface of the particles is modified with a surface modifier, wherein the surface modifier is silane; and

[0043] (6) The microparticle has at least a first capture ligand capable of binding to a first cell surface portion of a human T lymphocyte, the first capture ligand being attached to the surface of the microparticle by a surface modifier;

[0044] The first capture ligand is covalently linked to the surface of the microparticle via a surface modifier; and the first capture ligand capable of binding to the first cell surface portion of a human T lymphocyte is an anti-CD3 antibody or an anti-CD28 antibody, preferably an anti-CD3 antibody.

[0045] The present invention also provides the following preferred embodiments:

[0046] 1. Particles, among which

[0047] (1) The particles are non-magnetic;

[0048] (2) The diameter of the particles is 10 to 60 μm; and

[0049] (3) The microparticle has at least a first capture ligand capable of binding to a first cell surface portion of a human T lymphocyte, the first capture ligand being attached to the surface of the microparticle.

[0050] And the microparticles thereon are optionally microparticles for the activation and proliferation of human T lymphocytes.

[0051] 2. The microparticles of Item 1, wherein the diameter of the microparticles is 15 to 50 μm, more preferably 20 to 40 μm.

[0052] 3. The microparticles described in item 1 or 2, wherein the diameter of the microparticles is 40 μm.

[0053] 4. The microparticle of any one of items 1 to 3, wherein the microparticle has a second capture ligand capable of binding to a second cell surface portion of a human T lymphocyte, the second capture ligand being attached to the surface of the microparticle.

[0054] 5. The microparticle of any one of items 1 to 4, wherein the first capture ligand capable of binding to a first cell surface portion of a human T lymphocyte is an anti-CD3 antibody or an anti-CD28 antibody, preferably an anti-CD3 antibody.

[0055] 6. The microparticle of Item 4, wherein the first capture ligand capable of binding to a first cell surface portion of a human T lymphocyte is an anti-CD3 antibody, and wherein the second capture ligand capable of binding to a second cell surface portion of a human T lymphocyte is an anti-CD28 antibody.

[0056] 7. The microparticle of item 6, wherein the microparticle has a third capture ligand capable of binding to a third cell surface portion of a human T lymphocyte, the third capture ligand being attached to the surface of the microparticle, wherein the third capture ligand capable of binding to the third cell surface portion of a human T lymphocyte is an anti-CD2 antibody.

[0057] 8. The microparticles of any one of items 1 to 7, wherein the microparticles are non-porous.

[0058] 9. The microparticles of any one of items 1 to 8, wherein the microparticles are made of silicon dioxide (SiO2).

[0059] 10. The microparticles of any one of items 2 to 7 or 9, wherein the surface area of ​​the microparticles is about 1 m². 2 / g.

[0060] 11. The microparticles of any one of items 1 to 10, wherein the surface of the microparticles is modified with a surface modifier, optionally wherein the surface modifier is a silane, further optionally wherein the surface modifier is selected from (3-glycidoxypropyl)trimethoxysilane, 8-glycidoxyoctyltrimethoxysilane, (3-glycidoxypropyl)methyldimethoxysilane, (3-glycidoxypropyl)methyldiethoxysilane, (3-glycidoxypropyl)dimethylethoxysilane, (3-glycidoxypropyl)dimethylethoxysilane, (3-amino (3-Aminopropyl)triethoxysilane, (3-aminopropyl)methyldiethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)dimethylethoxysilane, (3-aminopropyl)diisopropylethoxysilane, carboxy-silanetriol, (3-mercaptopropyl)trimethoxysilane, (3-mercaptopropyl)methyldiethoxysilane, (3-mercaptopropyl)methyldiethoxysilane and (3-mercaptopropyl)triethoxysilane, preferably (3-glycidyloxypropyl)trimethoxysilane.

[0061] 12. The microparticle of any one of items 1 to 11, wherein at least one capture ligand capable of binding to the cell surface portion of a human T lymphocyte is covalently attached to the surface of the microparticle, optionally wherein the at least one capture ligand capable of binding to the cell surface portion of a human T lymphocyte is covalently attached via the surface modifier.

[0062] 13. The microparticle of any one of items 1 to 12, wherein at least one capture ligand capable of binding to the cell surface portion of a human T lymphocyte is covalently linked to the surface of the microparticle via a connector, optionally wherein the at least one capture ligand capable of binding to the cell surface portion of a human T lymphocyte is covalently linked to the surface modifier via a connector.

[0063] 14. The microparticles of item 13, wherein the connector is a bifunctional connector, optionally wherein the connector has been formed using at least one of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), DCC (N',N'-dicyclohexylcarbodiimide), NHS (N-hydroxysuccinimide), sulfon-NHS (N-hydroxysulfosuccinimide), DMA (dimethyl hexamethyleneimine), DMP (dimethyl heptamethine), DMS (dimethyl octylimine), glutaraldehyde, glutaraldehyde polymer, cyanogen bromide, cyanuric chloride, SMCC (4-[N-maleiminomethyl]cyclohexane-1-carboxylic acid succinimide), and sulfon-SMCC (4-(N-maleiminomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide).

[0064] 15. A method for activating and proliferating human T lymphocytes, the method comprising:

[0065] (a) Provide a cell population, wherein at least a portion of the cell population contains human T lymphocytes;

[0066] (b) Contact the cell population with the particles described in any one of items 1 to 14;

[0067] (c) The cell population is incubated with the microparticles to achieve activation and proliferation of human T lymphocytes;

[0068] And optionally (d) remove the particles.

[0069] 16. The method for activating and proliferating human T lymphocytes as described in item 15, wherein the cell population comprises monocytes, wherein optionally, the cell population is peripheral blood mononuclear cells (PBMCs).

[0070] 17. The method for activating and proliferating human T lymphocytes as described in item 15 or 16, wherein the method does not include a monocyte removal step.

[0071] 18. A kit comprising the microparticles described in any one of items 1 to 14.

[0072] 19. The kit described in Item 18, comprising at least:

[0073] (1) The first microparticle according to any one of items 1 to 14, wherein the first capture ligand capable of binding to a first cell surface portion of a human T lymphocyte is an anti-CD3 antibody; and

[0074] (2) The second microparticle according to any one of items 1 to 14, wherein the first capture ligand capable of binding to the first cell surface portion of a human T lymphocyte is an anti-CD28 antibody.

[0075] 20. The kit described in item 18 or 19, further comprising instructions for use with the method described in any one of items 15 to 17.

[0076] 21. Use of the microparticles according to any one of items 1 to 14 or the kits according to any one of items 18 to 20 for the activation and proliferation of human T lymphocytes. Attached Figure Description

[0077] Figure 1 ：This figure shows the percentage of CD69+ cells (gated with viable CD2+CD3+ cells) measured at 0, 4, 24, 48, and 72 hours after activation. CD69 is an early activation marker expressed within hours of activation initiation. This figure also shows a comparison between the CD3 / CD28 microparticles of this invention and the MACSiBead™ CD2 / CD3 / CD28 Kit and the Dynabeads™ CD3 / CD28 Kit. This figure shows the average results from three biological replicates.

[0078] Figure 2 ： This figure shows the percentage of CD25+ cells measured at 0, 4, 24, 48, and 72 hours after activation. CD25 is a marker of late activation, expressed several days after activation initiation. This figure shows a comparison between the CD3 / CD28 microparticles of this invention and the MACSiBead™ CD2 / CD3 / CD28 Kit and the Dynabeads™ CD3 / CD28 Kit. This figure shows the average results from three biological replicates.

[0079] Figure 3 ： Cell viability was measured at 0, 4, 24, 48, and 72 hours after activation. The figure shows a comparison between the CD3 / CD28 microparticles of this invention and the MACSiBead™ CD2 / CD3 / CD28 kit and the Dynabeads™ CD3 / CD28 kit. The data shown are averages from three biological replicates.

[0080] Figure 4 ： A graph showing the proliferation of live CD2+ cells measured at 0, 48, and 72 hours after activation. The data shown represent the average of three biological replicates. Proliferation was assessed by tracking dye dilutions during cell division, with the percentage of dividing cells shown in the graph.

[0081] Figure 5 ：A diagram illustrating the absolute number of T lymphocytes (CD2+ cells): T cells were activated and expanded using the microparticles of the present invention and the MACSiBead™ kit. An example of expansion after 14 days is shown. PBMCs in IL-2-containing activation medium were used as a negative control (dashed line). Expansion was evaluated after activation using the microparticles of the present invention and removal of the reagents after 24 or 48 hours. Optimal T cell expansion was achieved with initial activation of 600,000 peripheral blood mononuclear cells (PBMCs) and removal of the microparticles of the present invention after a 48-hour activation period, without the need for reactivation. Samples (black arrows) with removal of the microparticles of the present invention after 24 hours and reactivation on day 9 showed excellent expansion. The activation and expansion protocols of the microparticles of the present invention (see the "Activation and Proliferation Assay" section of Example 3 below) were compared with the MACSiBead™ activation and expansion protocol following the MACSiBead™ protocol (1×10⁻⁶). 6 The PBMC was compared with that after 72 hours (the activating agent was removed).

[0082] Figure 6 ： Cell viability was assessed throughout the amplification process and compared between the microparticles of this invention and the MACSiBead™ kit.

[0083] Figure 7 ： T lymphocytes were activated and expanded using the microparticles and MACSiBead™ reagent of this invention. An example of expansion over 14 days is shown. PBMCs in IL2-containing activation medium were used as a negative control (dashed line). Only PBMCs in IL2-containing medium also spontaneously activated over time, but the activation was slower and not all cells were activated. The microparticles of this invention were removed after 24 or 48 hours of activation. Samples after reagent removal after 24 hours were reactivated on day 9 (black arrow), which caused upregulation of the expression of the CD25 activation marker. The activation and expansion protocol according to this invention was compared with the MACSiBead™ activation and expansion protocol following the MACSiBead™ protocol (1×10⁻⁶). 6 The PBMC was compared with that after 72 hours (the activating agent was removed).

[0084] Figure 8 ：This figure shows microscopic images (phase contrast microscopy, 400x magnification) of cells in contact with different commercial beads and microparticles from the present invention. 1. Microparticles according to the present invention, CD3 / CD28, 40 μm; 2. Dynabeads™ CD3 / CD28; 3. MACSiBeads™ loaded with CD2 / CD3 / CD28; and 4. MACSiBeads™, unloaded. Numbered according to the figures. Dynabeads™ particles and MACSiBead™ particles (with and without antibody loading) were phagocytosed after incubation with PBMCs for 24 hours. Black arrows indicate some examples of cells ingesting the beads. White arrows indicate the microparticles of the present invention. MACSiBead™ and Dynabeads™ particles have a diameter of approximately 5 μm. Samples were thoroughly mixed by pipetting prior to imaging.

[0085] Figure 9 ： This figure shows microscopic images (phase contrast microscopy, 400x magnification) of cells in contact with different commercial beads and microparticles from the present invention. 1. Microparticles according to the invention, CD3 / CD28, 40 μm; 2. Dynabeads™ CD3 / CD28, with cells attached; 3. Dynabeads™ CD3 / CD28; 4. MACSiBeads™ loaded with CD2 / CD3 / CD28; and 5. MACSiBeads™, unloaded. Numbered according to the figures. Dynabeads™ particles, including loaded and unloaded MACSiBead™ particles, were ingested after 24 hours of incubation with PBMCs. White arrows indicate microparticles of the present invention, while black arrows point to some cell instances that have engulfed the beads. The MACSiBead™ and Dynabeads™ CD3 / CD28 beads are about 5 μm in diameter. Rigorous pipetting was used prior to imaging to remove beads that were only attached to cells. In activation experiments, thorough pipetting was described as a method for separating MACSiBead™ and Dynabeads™ from cells. Notably, samples containing Dynabeads™ exhibited a higher presence of adhesive cells. Detailed Implementation

[0086] Definitions and abbreviations

[0087] Unless the context clearly specifies otherwise, nouns without quantifiers as used herein include one / a kind and more / more. Unless otherwise clearly stated, “and / as well” as used herein may be used interchangeably with “or / or”. Unless the context clearly specifies otherwise, all embodiments of any aspect of the invention may be used in combination. Unless the context clearly requires otherwise, throughout the specification and claims, the words “comprising,” “including,” etc., have their ordinary meaning and should be interpreted as encompassing, not exclusive or exhaustive; that is, meaning “including but not limited to.” However, in the context of the invention, any occurrence of terms such as “comprising,” “including,” and variations thereof may optionally be replaced by exhaustive terms such as “consisting of,” and variations thereof. The use of singular or plural words also includes plural and singular, respectively. Furthermore, when used in this application, the words “in this document,” “above,” and “below,” and words with similar meanings, should refer to the application as a whole, and not to any particular part of the application. The full disclosure of all publications cited herein is incorporated by reference.

[0088] The description of embodiments of this disclosure is not intended to be exhaustive or to limit the disclosure to the precise forms disclosed. The following description of the invention is based primarily on the particles of the invention.

[0089] particle

[0090] In a first embodiment, the present invention provides microparticles, wherein

[0091] (1) The particles are non-magnetic;

[0092] (2) The diameter of the particles is 10 to 60 μm; and

[0093] (3) The microparticle has at least a first capture ligand capable of binding to a first cell surface portion of a human T lymphocyte, the first capture ligand being attached to the surface of the microparticle.

[0094] These microparticles are suitable for the activation and proliferation of human T lymphocytes.

[0095] In this invention, "non-magnetic" refers to materials or objects that lack magnetic properties. Non-magnetic materials are those that are unaffected by magnetic fields.

[0096] In another embodiment of the invention, the microparticles are non-porous. The term "non-porous" when referring to "microparticles" means that "non-porous microparticles" have a smooth surface that is substantially free of pores or depressions. For example, they can trap human T lymphocytes.

[0097] In another embodiment of the invention, the diameter of the particles is about 5 μm to 200 μm, preferably 10 μm to 100 μm, more preferably 10 μm to 60 μm, more preferably about 15 μm to 50 μm, and most preferably about 20 μm to 40 μm.

[0098] In another preferred embodiment of the invention, the diameter of the particles is about 20 or 40 μm. In a very preferred embodiment of the invention, the diameter of the particles is about 40 μm.

[0099] The particle diameter range referred to in this article, such as "approximately 20 μm to 40 μm," should be understood as different particles having the same diameter or different diameters. For example, the first particle and the second particle may both have a diameter of 20 μm, or both may both have a diameter of 40 μm, or the first particle and the second particle may have diameters of 20 μm and 40 μm respectively, or the first particle and the second particle may have diameters of 40 μm and 20 μm respectively.

[0100] It should also be understood that the particle diameter mentioned in this invention is not intended to be limited to a specified (precise) diameter value, but rather to include a deviation of + / - 5% from the specified diameter value.

[0101] In another preferred embodiment of the invention, the density of the particles is 2 g / cm³. 3 Up to 3 g / cm 3 .

[0102] In another embodiment of the invention, the surface area of ​​the particles is approximately 1 m². 2 / g.

[0103] The microparticles according to the invention are made of at least one inorganic material, which is preferably, but not limited to, microparticles made of SiO2 (silicon dioxide), organosilicon, gold, silver or platinum, or composite materials made of inorganic shell SiO2, organosilicon, gold or silver, and particle cores made of organic polymers or copolymers such as, but not limited to, PMMA (poly(methyl methacrylate)), PLA (polylactic acid), ABS (acrylonitrile butadiene styrene) and nylon.

[0104] In a preferred embodiment, the non-porous microparticles are made of SiO2. However, this does not preclude further functionalization of the microparticle surface using surface modifiers and trapping ligands, as described below.

[0105] Some examples of commercially available microparticles that can be used to prepare the particles of the present invention are:

[0106] Glantreo, Ltd. (Cork, Ireland): Non-porous silica particles: 10 μm (PNPP10.0NAR), 20 μm (PNPP20.0NAR), 40 μm (PNPP40.0NAR), 50 μm (PNPP50.0NAR), 70 μm (PNPP70.0NAR).

[0107] CD Bioparticles (New York, NY, USA): Non-porous silica particles: 20 μm (DNG-E009), 30 μm (DNG-E010), 40 μm (DNG-E011).

[0108] EPRUI (Wujiang Disctrict, Suzhou, China): Non-porous silica particles: 20 μm (EPRUI-Si-20), 30 μm (EPRUI-Si-30), 40 μm (EPRUI-Si-40), 50 μm (EPRUI-Si-50).

[0109] Abvigen (Newark, NJ, USA): Non-porous silica particles: 20 μm (ABM-3-362), 30 μm (ABM-3000), 40 μm (ABM-4000), 50 μm (ABM-5000), 60 μm (ABM-6000), 70 μm (ABM-7000), 80 μm (ABM-8000), 90 μm (ABM-9000), 100 μm (ABM-10000), 200 μm (ABM-20000);

[0110] VWR Chemicals (Radnor, PA, USA: Non-porous silica gel particles: 40 to 63 µm (27623.323), 40 to 63 µm (7631-86-9), 60 to 200 µm (84893.290), 63 to 200 µm (27647.325), and

[0111] MilliporeSigma Supelco, Merck (Darmstadt, Germany): Non-porous silica particles: 40 to 60 μm (1.09385), 40 to 75 μm (80442), 40 to 75 μm (53698), 75 to 200 μm (78991).

[0112] In a particularly preferred embodiment of the invention, the invention provides microparticles, wherein...

[0113] (1) The particles are non-magnetic;

[0114] (2) The particles are non-porous.

[0115] (3) The particles are made of SiO2;

[0116] (4) The diameter of the particles is 10 to 60 μm; and

[0117] (5) The microparticle has at least a first capture ligand capable of binding to a first cell surface portion of a human T lymphocyte, the first capture ligand being attached to the surface of the microparticle.

[0118] For example, non-porous, non-magnetic SiO2 microparticles with a diameter of 20 to 40 μm containing anti-CD3 and anti-CD28 or fragments thereof, co-incubated with PBMCs or leukocyte isolates, wherein the anti-CD3 and anti-CD28 or fragments thereof are covalently bound to the surface of the microparticles on the same or different microparticles, have been shown to induce cell activation and proliferation. Cells remain viable and begin to replicate and increase their number, and the expression of different activation markers has been demonstrated (…). Figure 1 , 2 3 and 4).

[0119] The particle diameter was carefully selected and offers several advantages compared to using smaller or larger particles. Particles of this diameter can be easily removed by mechanical force and sample filtration. Another advantage is that particles of this diameter are not phagocytosed by monocytes. Figure 8 and 9 It has been established in the scientific literature that when using mixed samples (PBMCs or leukocyte ablation without any pretreatment or cell separation steps), a high percentage of monocytes can interfere with the activation process because monocytes can engulf particles. This is a major problem for the activation and proliferation of T lymphocytes for genetic modification for CAR T therapy in cancer patients, as the percentage of monocytes in cancer patients can be very high. Without any pretreatment, smaller particles are not as effective at activation as larger particles because monocytes will take up all of them, leaving no more particles to activate T lymphocytes. Furthermore, monocytes can be activated and initiate proliferation, thus preventing T lymphocytes from being activated or proliferating (Wang X et al., 2021, Mol Ther MethodsClin Dev. 16;22:377-387). Currently, the only solutions are to perform a monocyte removal step, either through conventional methods of monocyte adhesion to plastic, or more expensive cell separation methods.

[0120] Therefore, by using the microparticles of the present invention, the inventors have eliminated the need for a mononuclear cell removal step, even when a high percentage of mononuclear cells is encountered in the sample.

[0121] target cells

[0122] The target cell can be any human T lymphocyte, as long as the target cell exhibits a portion on its cell surface that can bind to the capture ligand attached to the microparticle of the present invention, and the binding of the capture ligand to the portion on its cell surface causes activation and proliferation of the human T lymphocyte.

[0123] As described above, the present invention provides microparticles and methods for the activation and proliferation of human T lymphocytes.

[0124] "T lymphocytes" are known in the art. For example, they can be distinguished from other lymphocytes by the presence of T-cell receptors (TCRs) on their cell surface. Human T lymphocytes within the meaning of this invention include human T lymphocytes known in the art, and may optionally include human T lymphocyte precursors that can be used in the methods and uses of this invention to make them activated and proliferating human T lymphocytes.

[0125] The term "T lymphocyte activation," for example, is known in the art and refers to a process in which, for example, mature T lymphocytes expressing antigen-specific T lymphocyte receptors on their surface recognize their homologous antigens and respond by entering the cell cycle, secreting cytokines or lysins, and initiating cell-based functions of the immune system. According to the invention, whether a human T lymphocyte is an activated human T lymphocyte can be determined by measuring the expression of activation markers known in the art. Early and late activation markers of human T lymphocytes are present. Early markers include CD69, which begins to be expressed several hours after the onset of activation, and late markers include CD25, which begins to be expressed one day after the onset of activation. In a preferred embodiment of all other embodiments of the invention, activation is determined by measuring the expression of CD69 in a cell population (e.g., using a fluorophore-conjugated anti-CD69 antibody) and / or by measuring the expression of CD25 (e.g., using a fluorophore-conjugated anti-CD25 antibody), or as such, by measuring the expression of CD69 in a cell population (e.g., using a fluorophore-conjugated anti-CD69 antibody) and / or by measuring the expression of CD25 (e.g., using a fluorophore-conjugated anti-CD25 antibody). In this embodiment, the activated human T lymphocytes are human CD69+ T lymphocytes and / or human CD25+ T lymphocytes, respectively.

[0126] The term "T lymphocyte proliferation" is known in the art and refers to an increase in the number of T lymphocytes, for example, due to cell division or cytokinesis, the final step of the cell cycle. According to the invention, whether a human T lymphocyte is a proliferating human T lymphocyte can be determined by measuring the number of live human T lymphocytes, or as can be determined by measuring the number of live human T lymphocytes, for example by counting live CD2+ cells. If the measured number of live human T lymphocytes increases over time, the human T lymphocyte is identified as a proliferating human T lymphocyte. In the context of the invention, the term "proliferation" is used interchangeably with "amplification." That is, each occurrence of this term can be replaced by "amplification," and terms such as "proliferation" and "proliferating" can be replaced by "amplification" and "amplified," respectively.

[0127] As used herein, the terms “sample” or “cell population” can refer to any sample containing a suspension of live target cells. Samples can be prepared from tissues or organs taken from an individual or from a specimen that has been processed to release target cells.

[0128] Typically, human cells are about 5 to 100 μm in diameter.

[0129] Samples may be solutions containing live target cells derived from human, tissue, or organ extracts, and cell lines established from primary cells derived from these tissues or organs, including but not limited to heart, liver, kidney, colon, intestine, esophagus, stomach, nervous tissue (brain, spinal cord), lung, vascular tissue (arteries, veins, capillaries), lymphoid tissue (lymph nodes, adenoids, tonsils, bone marrow, and blood, including peripheral blood cells, placental / umbilical cord blood, menstrual blood), spleen, and fibroblasts; or solutions obtained by roughly separating cells from such solutions, and such solutions diluted with water, any buffers and / or culture media described herein, such as physiological buffers, physiological saline, Ringer's solution containing divalent cations such as calcium or magnesium ions, cell culture media such as RPMI, MEM, IMEM, or DMEM, and phosphate buffers such as PBS, HBSS, TBS, DPBS, EBSS, etc.

[0130] Preferred samples containing target cells for activation and proliferation using the microparticles and methods of the present invention include human samples, whole blood, single blood component samples, bone marrow aspirates, biopsy samples, liquefied tissue samples (e.g., liposuction samples that have been enzymatically degraded), cell culture samples, bioreactor cultures, single-cell suspensions, etc.

[0131] In a preferred embodiment, the sample, i.e., the cell population in contact with the particles of the present invention, comprises lymphocytes and monocytes.

[0132] In another embodiment, the sample, i.e. the cell population in contact with the particles of the present invention, is peripheral blood mononuclear cells (PBMCs).

[0133] cell surface

[0134] As used herein, "cell surface portion," "molecule on cell surface," or "cell surface molecule" can be any molecule expressed or displayed on the cell surface of a target cell such that the molecule can be recognized and bound by at least one capture ligand attached to the microparticle of the present invention. Cell surface molecules can be protein- or non-protein-based.

[0135] In a preferred embodiment of the invention, the cell surface portion is an antigen that can be recognized by an antibody or a derivative thereof attached to the microparticle of the invention.

[0136] According to the present invention, a suitable cell surface portion is one that, if captured by at least one capturing ligand on a particle, induces activation and proliferation of human T lymphocytes.

[0137] In a preferred embodiment, the cell surface portion is at least one selected from CD2, CD3, CD4, CD28, and CD45.

[0138] In a particularly preferred embodiment, the cell surface portion is at least one selected from CD2, CD3, and CD28.

[0139] Capture ligands

[0140] The microparticles of the invention used in the method according to the invention have at least one capture ligand attached to their surface and capable of binding to a first cell surface portion of a human T lymphocyte.

[0141] When referring to any capture ligand described herein, the terms “binding,” “specific binding,” or “specifically binding to” as used herein mean that the capture ligand on the particle binds to a cell surface portion of the target cell surface (as detailed above), resulting in the formation of a target cell / particle complex. The capture ligand will have an affinity for molecules on the target cell surface, causing the target cell / particle complex to induce activation and proliferation of human T lymphocytes, where the target cell surface molecules are suitable cell surface portions.

[0142] In one embodiment of the invention, the term "capture ligand" refers to a protein receptor capable of recognizing molecules on the surface of target cells as described above.

[0143] In one embodiment of the invention, the protein receptor captures the ligand and molecules at the surface of the target cell at approximately 10... -5 Or larger, about 10 -6Or larger, about 10 -7 Or larger, about 10 -8 Or larger, about 10 -9 Or larger, about 10 -10 Or larger, about 10 -11 Or larger, or about 10 -12 Or a larger dissociation constant for specific binding. In a preferred embodiment, the ligand is captured by molecules at the target cell surface at a dissociation constant of 10. -5 Up to 10 -12 , more preferably 10 -9 Up to 10 -11 or more preferably 10 -7 Up to 10 -10 The dissociation constant of the ligand is specifically bound. In a preferred embodiment, the affinity constant of the capturing ligand is on the order of nanomolar (approximately 10⁻⁶). -9 A variety of known analytical methods can be used to measure binding affinity, such as radioligand binding assays, surface plasmon resonance assays, fluorescence energy resonance transfer assays, and affinity chromatography.

[0144] In a preferred embodiment, the at least one capture ligand is an antibody or a derivative thereof that specifically binds to an antigen on the surface of a target cell.

[0145] Antibodies are molecules containing one or more polypeptides encoded primarily by immunoglobulin genes or segments of immunoglobulin genes. Recognized immunoglobulin genes include the κ, λ, α, γ, δ, ε, and μ constant region genes, as well as a large number of immunoglobulin variable region genes. Light chains are classified as κ or λ. Heavy chains are classified as γ, μ, α, δ, or ε, which in turn define the immunoglobulin classes: IgG, IgM, IgA, IgD, and IgE, respectively. The basic immunoglobulin structural unit is known to consist of a tetramer. Each tetramer consists of two pairs of identical polypeptide chains, each pair having one “light” chain (approximately 25 kDa) and one “heavy” chain (approximately 50 to 70 kDa). The N-terminus of each chain defines a variable region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively. Capture ligands can exist in a variety of forms, either as intact immunoglobulins (i.e., antibodies) or as derivatives thereof, including, for example, FabFc2, Fab, Fv, Fd, F(ab')2, Fv fragments containing only the variable regions of the light and heavy chains, Fab or F(ab')2 fragments containing the variable and partially constant regions, single-chain antibodies (e.g., scFv), CDR-grafted antibodies, dAb, nanobodies, etc. The heavy and light chains of Fv can be derived from the same antibody or different antibodies, resulting in chimeric Fv regions. Antibodies can also be modified antibodies (e.g., oligomers, reduced, oxidized, and labeled antibodies). Antibodies can be of animal (especially mouse, rabbit, or rat) or human origin, or can be chimeric or humanized. The term "antibody" as used herein encompasses all these forms.

[0146] In a preferred embodiment, the at least one capture ligand is an antibody, such as FabFc2, Fab, Fv, Fd, F(ab')2, an Fv fragment containing only the variable regions of the light and heavy chains, a Fab or F(ab')2 fragment containing the variable region and a portion of the constant region, a single-chain antibody (e.g., scFv), a CDR-grafted antibody, dAb, a nanobody, etc., which is approximately 10 -5 Or larger, about 10 -6 Or larger, about 10 -7 Or larger, about 10 -8 Or larger, about 10 -9 Or larger, about 10 -10 Or larger, about 10 -11 Or larger, or about 10 -12 Or a larger dissociation constant recognizes antigens on the surface of target cells. In a preferred embodiment, the capture ligand is at a concentration of 10... -5 Up to 10 -12 , more preferably 10 -9Up to 10 -11 or more preferably 10 -7 Up to 10 -10 The dissociation constant of the antibody is used to recognize the antigen on the surface of the target cell. In a preferred embodiment, the affinity constant of the antibody is on the order of nanomolar (approximately 10⁻⁶). -9 The capture ligands according to the invention are preferably suitable for the activation and / or proliferation of human T lymphocytes.

[0147] In a preferred embodiment of the invention, the first capture ligand capable of binding to a first cell surface portion of a human T lymphocyte is an anti-CD3 antibody or an anti-CD28 antibody, preferably an anti-CD3 antibody. It should be understood that the anti-CD3 antibody according to the invention is suitable for the activation and proliferation of human T lymphocytes. It should be understood that, according to the invention, the anti-CD28 antibody, acting as a co-stimulatory signal in conjunction with anti-CD3, is suitable for the proliferation of human T lymphocytes. Suitable anti-CD28 and anti-CD3 antibodies are known in the art and can be used in the present invention.

[0148] In another preferred embodiment of the invention, the first capture ligand capable of binding to the first cell surface portion of a human T lymphocyte is an anti-CD3 antibody, and the second capture ligand capable of binding to the second cell surface portion of a human T lymphocyte is an anti-CD28 antibody.

[0149] In another preferred embodiment of the invention, the first capture ligand capable of binding to a first cell surface portion of a human T lymphocyte is an anti-CD3 antibody, the second capture ligand capable of binding to a second cell surface portion of a human T lymphocyte is an anti-CD28 antibody, and the third capture ligand capable of binding to the second cell surface portion of a human T lymphocyte is an anti-CD2 antibody. Suitable anti-CD2 antibodies are known in the art and can be used in this invention.

[0150] Particularly preferred are anti-CD3 antibody (Biolegend, clone OKT3), anti-CD28 antibody (Invitrogen, clone CD28.2), and anti-CD2 antibody (Exbio, clone TS1 / 8).

[0151] As illustrated in the examples of this invention, anti-CD3 and anti-CD28 antibodies or fragments thereof that irreversibly bind to non-porous, non-magnetic SiO2 particles with a diameter of 20 to 40 μm can bind to the same particles or different particles. To remove the particles from the cell suspension, the mixture can be agitated and filtered through filters with pore sizes of 15 or 30 μm.

[0152] Antibodies, such as anti-CD3 and anti-CD28, are used in this application as signaling molecules having one or more polypeptides encoded substantially by an immunoglobulin gene or a fragment of an immunoglobulin gene.

[0153] Covalent connection between particles and trapping ligands

[0154] The microparticles used in the method according to the invention have one or more capture ligands that are attached to the surface of the microparticle and capable of binding to at least one cell surface portion of a human T lymphocyte, thereby transmitting signals that induce T lymphocyte activation and proliferation. The capture ligands, target cells, and surface portions have been described in detail above.

[0155] In a preferred embodiment of the invention, the capture ligand is covalently linked to the microparticle.

[0156] As a method for covalently linking the captured ligand to the microparticle, coupling reagents such as commonly used bifunctional agents can be used, or a method using spacers can be employed. The appropriate coupling reagent is selected based on the reactive groups on the support surface.

[0157] When the support surface has -OH groups, such as in materials made of SiO2, functional groups are introduced into the support surface using silane surface modifiers. Preferably, (3-glycidoxypropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, carboxyl-silanetriol, and (3-mercaptopropyl)trimethoxysilane are used as silane surface modifiers. The introduced functional groups can be, for example, amino, amide, imino, hydrazide, carboxyl, thiol, hydroxyl, azide, alkyl, phenyl, epoxy, ester, halide, and acyl halide. Amino, carboxyl, thiol, and epoxy groups are preferred. Epoxy groups are most preferred.

[0158] The covalent bond formed between the antibody and the surface can be formed directly between the functionalized surface and the capture ligand, or through a bifunctional linker that can react with two functional groups on the surface carrier and the ligand molecules. The linker may or may not have a PEG (polyethylene glycol) spacer. The linker that can be used to form a covalent bond between the nonporous microparticle and the trapping ligand can be, but is not limited to: EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), DCC (N',N'-dicyclohexylcarbodiimide), NHS (N-hydroxysuccinimide), sulfon-NHS (N-hydroxysulfosuccinimide), DMA (dimethyl hexamethyleneimine), DMP (dimethyl heptamethine), DMS (dimethyl octylimine), glutaraldehyde, glutaraldehyde polymer, cyanogen bromide, cyanuric chloride, SMCC (4-[N-maleimidemethyl]cyclohexane-1-carboxylic acid succinimide), sulfon-SMCC (4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfosuccinimide), or any other bifunctional linker or spacer that can activate the surface of the particles, which can then react with the functional groups present in the trapping ligand structure.

[0159] The size of the PEG spacers that can be used in this invention can vary from 0.4 kDa to greater than 20 kDa.

[0160] The microparticles and the capture ligands are directly interconnected or connected via linkers. Using only a covalent bonding method between the surface and the antibody, without any affinity ligands, prevents leakage of the capture ligands from the non-porous microparticles. Preferably, PEG-free (polyethylene glycol) linkers are used in this invention. Any other linkers capable of ensuring covalent bonding between functional groups on the surface of the non-porous microparticles and groups that may exist in the capture ligand structure, as well as other functional groups that may exist on the surface of the non-porous microparticles and react with antibody groups, with or without linker molecules, are not limiting factors in practicing this invention.

[0161] Reagent test kit

[0162] The present invention also provides a reagent kit comprising the microparticles of the present invention.

[0163] In a first embodiment, the present invention provides a kit comprising at least one of the aforementioned microparticles.

[0164] For example, the kit may contain microparticles, in which

[0165] (1) The particles are non-magnetic;

[0166] (2) The particles are non-porous;

[0167] (3) The particles are made of SiO2;

[0168] (4) The diameter of the particles is 10 to 60 μm;

[0169] (5) The surface of the particles is modified with a surface modifier, wherein the surface modifier is silane; and

[0170] (6) The microparticle has a first capture ligand and a second capture ligand capable of binding to a first cell surface portion and a second cell surface portion of a human T lymphocyte, the first capture ligand and the second capture ligand being attached to the surface of the microparticle via the surface modifier.

[0171] The first and second capturing ligands are covalently linked to the surface of the microparticles via the surface modifier; and

[0172] The first capture ligand that can bind to the first cell surface portion of human T lymphocytes is an anti-CD3 antibody, and the second capture ligand that can bind to the second cell surface portion of human T lymphocytes is an anti-CD28 antibody.

[0173] In another embodiment, the kit may contain microparticles, wherein

[0174] (1) The particles are non-magnetic;

[0175] (2) The particles are non-porous;

[0176] (3) The particles are made of SiO2;

[0177] (4) The diameter of the particles is 10 to 60 μm;

[0178] (5) The surface of the particles is modified with a surface modifier, wherein the surface modifier is silane; and

[0179] (6) The microparticle has a first capture ligand, a second capture ligand, and a third capture ligand capable of binding to a first cell surface portion, a second cell surface portion, and a third cell surface portion of a human T lymphocyte, wherein the first capture ligand, the second capture ligand, and the third capture ligand are attached to the surface of the microparticle via the surface modifier.

[0180] The first, second, and third capturing ligands are covalently linked to the surface of the microparticles via the surface modifier; and

[0181] The first capture ligand that can bind to the first cell surface region of human T lymphocytes is the anti-CD3 antibody, the second capture ligand that can bind to the second cell surface region of human T lymphocytes is the anti-CD28 antibody, and the third capture ligand that can bind to the third cell surface region of human T lymphocytes is the anti-CD2 antibody.

[0182] In another embodiment, the kit may contain microparticles, wherein

[0183] (1) The particles are non-magnetic;

[0184] (2) The particles are non-porous;

[0185] (3) The particles are made of SiO2;

[0186] (4) The diameter of the particles is 10 to 60 μm;

[0187] (5) The surface of the particles is modified with a surface modifier, wherein the surface modifier is silane; and

[0188] (6) The microparticle has at least a first capture ligand, the first capture ligand being capable of binding to a first cell surface portion of a human T lymphocyte, the first capture ligand being attached to the surface of the microparticle via the surface modifier;

[0189] The first capture ligand is covalently linked to the surface of the microparticle via the surface modifier; and the first capture ligand capable of binding to the first cell surface portion of a human T lymphocyte is an anti-CD3 antibody.

[0190] In another embodiment, the kit may contain at least one microparticle, wherein

[0191] (1) The particles are non-magnetic;

[0192] (2) The particles are non-porous;

[0193] (3) The particles are made of SiO2;

[0194] (4) The diameter of the particles is 10 to 60 μm;

[0195] (5) The surface of the particles is modified with a surface modifier, wherein the surface modifier is silane; and

[0196] (6) The microparticle has at least a first capture ligand, the first capture ligand being capable of binding to a first cell surface portion of a human T lymphocyte, the first capture ligand being attached to the surface of the microparticle via the surface modifier;

[0197] The first capture ligand is covalently linked to the surface of the microparticle via the surface modifier; and the first capture ligand capable of binding to the first cell surface portion of a human T lymphocyte is an anti-CD28 antibody.

[0198] In another implementation, the kit contains at least

[0199] (1) The first particle, wherein the first capture ligand capable of binding to the first cell surface portion of a human T lymphocyte is an anti-CD3 antibody; and

[0200] (1) The second particle, wherein the first capture ligand capable of binding to the first cell surface portion of a human T lymphocyte is an anti-CD28 antibody.

[0201] In this embodiment, the kit preferably further comprises at least a first vial and a second vial, wherein the first vial contains a first microparticle and the second vial contains a second microparticle.

[0202] It should be understood that the first particle and the second particle may have the same diameter or different diameters. For example, the first particle and the second particle may both have a diameter of 20 μm, or they may both have a diameter of 40 μm, or the first particle and the second particle may have diameters of 20 μm and 40 μm respectively, or the first particle and the second particle may have diameters of 40 μm and 20 μm respectively.

[0203] The kit may also include instructions for use in the method of activating and proliferating human T lymphocytes according to the present invention.

[0204] Methods and uses

[0205] This invention also provides methods and uses for activating and proliferating human T lymphocytes.

[0206] In one embodiment, the present invention provides the use of the microparticles or kits according to the invention for activating and proliferating human T lymphocytes.

[0207] In another embodiment, the present invention provides a method for activating and proliferating human T lymphocytes, comprising:

[0208] (a) Provide a cell population, wherein at least a portion thereof contains human T lymphocytes;

[0209] (b) Contact the cell population with any of the particles described above;

[0210] (c) Incubate the cell population with the microparticles to achieve activation and proliferation of human T lymphocytes;

[0211] And optionally (d) remove particles.

[0212] It should be understood that step (c), incubating the cell population with the microparticles to achieve activation and proliferation of human T lymphocytes, is performed in a suitable culture medium for activating and proliferating human T lymphocytes. Culture media for activating and proliferating human T lymphocytes are generally known in the art. In a preferred embodiment of all other embodiments of the invention, the culture medium for activating and proliferating human T lymphocytes contains interleukin-2 (IL-2), such as recombinant IL-2. In another preferred embodiment, the culture medium for activating and proliferating human T lymphocytes also contains interleukin-7 (IL-7), such as recombinant IL-2, and interleukin-15 (IL-15), such as recombinant IL-15. If step (c) is performed for more than 7 days, it is highly preferred that the culture medium for activating and proliferating human T lymphocytes contains interleukin-2 (IL-2), such as recombinant IL-2, interleukin-7 (IL-7), such as recombinant IL-7, and interleukin-15 (IL-15), such as recombinant IL-15.

[0213] In another embodiment of the invention, the cell population comprises mononuclear cells.

[0214] In another embodiment of the invention, the cell population is peripheral blood mononuclear cells (PBMCs).

[0215] In another embodiment of the invention, the method does not include a mononuclear cell removal step.

[0216] The cell culture media that can be used in the various steps of this method and / or after cell isolation depends on the cell sample. Different cells or cell types may have very specific requirements for culture media, serum, and supplements. Those skilled in the art will understand which media to use.

[0217] Exemplary cell culture media include MEM (minimum essential medium), DMEM (Dulbecco modified Eagle medium), RPMI-1640 (Roswell Park Memorial Institute medium 1640), and IMDM (Iscove modified Dulbecco medium) (Sigma / ThermoFisher).

[0218] The temperature at which any step of the method of the present invention is performed (including the optional step of removing particles) is not critical, as long as the target cells remain viable. Generally, the temperature will be equal to or lower than the temperature at which the target cells grow in their natural environment or culture conditions, and may be the same as or different from the temperature at which other steps in the method of the present invention are performed.

[0219] In a preferred embodiment of all other embodiments of the invention, the method for activating and proliferating human T lymphocytes according to the invention is performed in vitro, and the use of the invention for activating and proliferating human T lymphocytes is implemented in vitro.

[0220] cell population

[0221] As described above, the microparticles of the present invention can be used to activate and proliferate human T lymphocytes.

[0222] Therefore, in the context of the method of the present invention, the cell population can be any cell population, as long as it contains human T lymphocytes.

[0223] More specifically, in the context of the methods of this invention, "sample" or "cell population" can be any sample containing a suspension of live target cells. The sample can be prepared from bodily fluids (e.g., human blood), tissues or organs taken from an individual, or specimens processed to release target cells.

[0224] Contact between cell populations and particles

[0225] The method of the present invention also includes the step of contacting the cell population with the particles of the present invention.

[0226] According to the method of the invention, under the condition that the live target cells in the sample remain viable and are mixed with the microparticles, the microparticles of the invention having at least a first capture ligand capable of binding to a first cell surface portion of human T lymphocytes are brought into contact with a sample containing a suspension of live target cells, the live target cell suspension exhibiting the cell surface portion as described above on the cell surface, for example by mixing the sample stream and the microparticle suspension and / or by shaking, stirring and / or agitating.

[0227] In one embodiment of the invention, the method is initiated by bringing a suspension of the inventive particles in solution directly into contact with the sample.

[0228] In another embodiment, the sample is diluted with liquid after the microparticles are added as a suspension.

[0229] Before, during, or after the addition of microparticles, the sample can be diluted with any liquid that maintains the viability of the target cells and allows the nonporous microparticles to form a complex with the target cells during sample incubation, and the liquid can be selected based on the type of target cells present in the sample.

[0230] Such liquids include, but are not limited to, water, buffer solutions or cell culture media, solutions or media as described herein for cell transfection, transplantation, implantation and / or storage or freezing.

[0231] Buffer solution (culture medium)

[0232] The buffer solution for suspended cells can have different compositions, but the presence of serum (e.g., fetal bovine serum, human serum) or serum substitute compounds is necessary.

[0233] It will be apparent to those skilled in the art that the need for these compounds is essential for cell metabolism. The mixture of cells and microparticles used for activation and proliferation is maintained under standard conditions conducive to supporting cell growth, preferably at 37°C, with the gas inlet being air containing 5% CO2. The incubation step of the method of the present invention is also preferably carried out at 37°C with the gas inlet being air containing 5% CO2.

[0234] When using GMP-compliant buffers for T lymphocyte activation and expansion processes, the buffers contain human serum or serum substitutes approved for human use. This type of microparticle can be readily used to generate T lymphocyte therapeutic agents for in vivo use because the T lymphocytes can be easily separated from the microparticles after the procedure. Furthermore, due to the irreversible bond between the antibody and the microparticles, the antibody cannot contaminate the cell suspension in any way. Therefore, this system allows users to obtain T lymphocyte suspensions free of any harmful contaminants.

[0235] Particle removal

[0236] According to an optional step of the method of the present invention, live target cells (human T lymphocytes) can be obtained, for example, by mechanically dissociating the target cell / microparticle complex and eluting the live target cells through a filter while retaining larger, non-porous microparticles, wherein the capture ligand is covalently immobilized to the microparticle surface. Preferably, the live target cells are eluted into a sterile container that can be sterilely sealed.

[0237] Analysis of human T lymphocytes

[0238] The method of the present invention may optionally further include the step of determining whether human T lymphocytes are activated human T lymphocytes according to the present invention. This can be determined by measuring the expression of activation markers known in the art. Early and late activation markers of human T lymphocytes are present. Early markers include CD69, which begins to be expressed several hours after the onset of activation, and late markers include CD25, which begins to be expressed one day after the onset of activation. In a preferred embodiment of all other embodiments of the present invention, activation is determined by measuring the expression of CD69 in the cell population (e.g., by using a fluorophore-conjugated anti-CD69 antibody) and / or by measuring the expression of CD25 (e.g., by using a fluorophore-conjugated anti-CD25 antibody). In this embodiment, the activated human T lymphocytes are human CD69+ T lymphocytes and / or human CD25+ T lymphocytes, respectively.

[0239] The method of the present invention may optionally further include the step of determining whether human T lymphocytes are proliferating human T lymphocytes according to the present invention. This can be determined by measuring the number of viable human T lymphocytes, for example, by counting viable CD2+ cells. If the measured number of viable human T lymphocytes increases over time, the human T lymphocytes are determined to be proliferating human T lymphocytes. Another method for determining T lymphocyte proliferation is dye dilution, for example using cell tracer violet (CTV) dye, which tracks proliferation by binding to cellular proteins and being gradually diluted with cell division, thereby producing distinct fluorescence peaks corresponding to different cell generations. This allows researchers to monitor and quantify cell proliferation over time using flow cytometry or fluorescence microscopy.

[0240] Example

[0241] This invention provides microparticles, kits, and methods for activating and proliferating human T lymphocytes. The following examples describe the invention in detail, and these examples may represent more than one embodiment of the invention.

[0242] The scope of this invention is not limited to the exemplary embodiments intended to illustrate various aspects of the invention. In fact, many modifications to the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and drawings. Such modifications are intended to fall within the scope of the appended claims. The following embodiments are provided by way of illustration rather than limitation.

[0243] Example 1: Preparation of anti-CD3 and anti-CD28 antibodies covalently immobilized to carboxyl silica microparticles

[0244] Two vials of 20 mg carboxyl-functionalized non-porous silica microparticles (20 μm in diameter, purchased from Glantreo Ltd., Ireland), 1 μL of EDC ((1-ethyl-3-(3-dimethylamino)propylcarbodiimide, hydrochloride) (100 mg / mL), and 1 μL of NHS (N-hydroxysuccinimide) or sulfon-NHS (N-hydroxysulfosuccinimide) (60 mg / mL) were mixed in MES buffer at pH 6 in an Eppendorf tube at room temperature. The activated non-porous microparticles were centrifuged and the supernatant was discarded to remove excess reagents. 15 μg of anti-CD3 antibody (Biolegend, clone OKT3) was dissolved in 1 mL of PBS buffer, and 15 μg of anti-CD28 antibody (Invitrogen, clone CD28.2) was dissolved in 1 mL of PBS buffer. The two solutions were added separately to a vial containing activated silica microparticles in PBS buffer. After mixing for 2.5 hours at room temperature, the microparticles were washed in PBS and can be used to activate and expand T lymphocytes.

[0245] Example 2: Preparation of anti-CD3 and anti-CD28 antibodies covalently immobilized to epoxy-functionalized silica microparticles

[0246] Unfunctionalized silica-free SiO2 microparticles with a diameter of 40 μm were purchased from Glantreo Ltd., Ireland. The unfunctionalized silica microparticles were silanized with GLYMO to prepare epoxy-functionalized silica-free microparticles. Two vials containing 20 mg of epoxy-functionalized silica-free microparticles were washed in 1 M phosphate-buffered saline (pH 7) at room temperature in Eppendorf tubes. Subsequently, 15 μg of anti-CD3 antibody (Biolegend, clone OKT3) and 15 μg of anti-CD28 antibody (Invitrogen, clone CD28.2) were added to the corresponding (separate) Eppendorf tubes containing silica microparticles, respectively. The reaction was carried out overnight. The remaining active spots on the surface of the microparticles were blocked with 100 μL of 0.1 M ethanolamine. After washing in PBS, the microparticles were ready for activation and expansion of T lymphocytes.

[0247] The average surface area of ​​the 40 μm non-porous silica particles, as measured using a Micromeritics TriStar II analyzer, was 1 m². 2 / g particles.

[0248] Example 3: Activation and proliferation assay of PBMC fractions from whole blood

[0249] PBMC separation

[0250] Draw approximately 10 mL of fresh blood sample. Add 7 mL of Histopaque-1077 to an empty 15 mL tube. Carefully pipette a layer of 7 mL of whole blood onto the Histopaque-1077 in the tube. Centrifuge at 400 × g for exactly 30 minutes at room temperature.

[0251] After centrifugation, carefully transfer the opaque interface from the 15 mL tube into a clean 50 mL tube using a Pasteur pipette. Wash the cells by adding sterile DPBS to 50 mL and gently mix. Centrifuge at 250 × g for 10 min. Discard the supernatant. Resuspend the cell pellet in 1 mL of 1× erythrocyte lysis buffer (BD Pharmlyse) and incubate for 10 min. Centrifuge the tube at 300 × g for 10 min. Discard the supernatant and resuspend the cell pellet in 1 mL of culture medium (RPMI 1640 supplemented with 10% FBS or 10% AB human serum (optionally supplemented with 2 mM L-glutamine and antibiotics), and recombinant interleukin-2 (rIL-2)). Measure the cell concentration using flow cytometry and dilute the cells to a concentration of 6 × 10⁻⁶. 6 Cells / mL.

[0252] Activation and proliferation assay

[0253] Mix the activation reagent and cells according to the recommended amounts in Table 1. Pipette 200 μL of the cell / reagent mixture into each well. Vortex the vial for at least 15 seconds, mixing with a pipette before each pipette transfer. Immediately after mixing, transfer the mixture to a plate. Incubate at 37°C and 5% CO2 for up to 2 days. After 48 hours, remove the microparticles according to the invention and allow the cells to further expand for up to 14 days or according to the user's protocol.

[0254] Table 1: Activation Setup According to the Invention – Overview. According to Example 2, the microparticles according to the invention, combined with anti-CD3 or anti-CD28, are non-porous, non-magnetic SiO2 microparticles with a diameter of 40 μm and an average surface area of ​​approximately 1 m². 2 / g, the particles are functionalized with GLYMO and bind to anti-CD3 and anti-CD28 antibodies through the reaction of epoxy groups on the particles and amino groups on the antibodies.

[0255]

[0256] Forty-eight hours later, gently aspirate the suspension up and down with a pipette to break up clumps. Filter the mixture from each well using a 20 to 35 μm filter. Centrifuge at 300 × g for 10 minutes and completely aspirate the supernatant. Add 400 μL of fresh activation / expansion medium supplemented with IL-2 to each tube. Pipette 200 μL of cells into new wells on the plate. Incubate at 37°C and 5% CO2. Every 2 to 3 days, divide the cell suspension into two aliquots and add fresh medium (RPMI 1640 supplemented with 10% FBS or 10% AB human serum (2 mM L-glutamine and antibiotics may also be added), and recombinant interleukin-2 (rIL-2)).

[0257] Solution MACSibeads TM CD3 / CD2 / CD28 (magnetic microparticles with a diameter range of approximately 3 to 5 μm)

[0258] 1. Loading beads with antibodies

[0259] Add 100 μL of CD2, 100 μL of CD3, and 100 μL of CD28 to a 2 mL tube. Vortex the beads. Add 500 μL of MACSi beads to the antibody mixture. Add 200 μL of MACS buffer. Incubate in a rotary mixer at 2–8 °C for 2 hours.

[0260] 2. Wash MACSiBeads particles

[0261] Activate 7.5 μL of pre-loaded MACSibeads + 200 μL of activation medium into a 2 mL tube using a pipette. Centrifuge at 700 × g for 5 minutes. Collect the supernatant and resuspend the 7.5 μL beads in 500 μL of activation medium to create 5 wells. Add the same volume of wash beads as the prepared PBMCs to each tube. Mix thoroughly and pipette 200 μL of the particle / cell suspension into each well.

[0262] The solution is Dynabeads™ CD3 / CD28 (magnetic microparticles with a diameter range of approximately 3 to 5 μm).

[0263] Vortex in Dynabeads vials for at least 30 seconds. Transfer 5.7 μL Dynabeads + 200 μL culture medium to a 2 mL tube. Centrifuge at 700 ×g for 5 minutes. Collect the supernatant and resuspend the 5.7 μL beads in 500 μL of activation medium to establish 5 wells. Add the same volume of wash beads as the prepared PBMCs to each tube. Mix thoroughly and pipette 200 μL of the particle / cell suspension into each well.

[0264] analyze

[0265] Take 150 μL of sample from a 96-well plate, and add 2 μL of CD2 BV421, 0.5 μL of CD4 VioGreen, and 0.5 μL of LCD8 APC-VIO 770. Incubate in the dark at room temperature for 10 minutes. Add 1 mL / sample of 1× annexin binding buffer and centrifuge at 400 ×g for 5 minutes. Take 1.1 mL of supernatant, vortex the remaining 50 μL of cell suspension, and add 5 μL of annexin FITC and 5 μL of 7AAD. Incubate in the dark at room temperature for 15 minutes. Add 450 μL of 1× annexin binding buffer and analyze on a flow cytometer.

[0266] Early and late activation markers are present. Early markers (CD69, such as...) Figure 1 As shown, expression begins several hours after activation begins, and late markers (CD25, such as...) are expressed. Figure 2 (As shown) It began to express itself one day later.

[0267] To separate the cells from the beads, pipette up and down 10 times from each well. Transfer 50 μL of the cell / bead suspension to a flow cytometry tube and pass it through a filter to remove the beads. Stain the sample with fluorescently conjugated antibodies (CD2 BV421, CD25 PE, CD69 PE-Vio770, CD4 VioGreen, CD8 APC-Vio770, CD3 FITC, and viability dyes). Incubate in the dark at room temperature for 10 minutes. Add 400 μL of MACS buffer and analyze on a NovoCyte flow cytometer.

[0268] Cell proliferation staining

[0269] Before activation, some isolated PBMCs can be stained with cell-tracing violet dye. Cell-tracing violet dye is used in cell biology to track proliferation by binding to cellular proteins and gradually diluting with cell division, thereby producing distinct fluorescence peaks corresponding to different cell generations.

[0270] After isolating PBMCs, a portion of the cells were incubated with 1 μM cell tracer violet dye in DPBS at room temperature for 20 minutes. After incubation, 20 mL of RPMI medium containing 10% FBS was added, and the cells were centrifuged at 300 ×g for 10 minutes. The supernatant was collected, the pellet was resuspended in activation medium, and activation and amplification were performed according to the standard activation protocol.

[0271] Take 100 μL of cell suspension from the well labeled "Cell Tracing Purple Well". Add 0.3 μL of CD2-PE-Vio 770 and 0.3 μL of CD3-FITC antibody, and incubate in the dark at room temperature for 10 minutes. Add 400 μL of MACS buffer and 0.5 μL of PI. Incubate for another 5 minutes. Analyze the sample using a NovoCyte flow cytometer.

[0272] The results are as follows Figures 1 to 7 As shown. Activation using MACSibeads™ CD3 / CD2 / CD28 and Dynabeads™ CD3 / CD28 was performed according to their respective protocols.

[0273] Example 4: Assay of Mononuclear Cell Phagocytosis

[0274] Devouring settings

[0275] Use MACSibeads™ and Dynabeads™ CD3 / CD28 at the recommended dosage for T cell activation.

[0276] Table 2: Setup for Phagocytosis Measurement

[0277]

[0278] All beads were washed before being added to the cells. The required amount of beads was pipetted into a tube, and 2 mL of activation medium was added. The tube was centrifuged, the supernatant was collected, and the beads were resuspended in 0.5 mL of cell suspension. Each bead / cell mixture was pipetted into one well of a 24-well plate. The plate was incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0279] Microscopy

[0280] Cells were observed using an inverted microscope at 40× magnification. All samples were imaged before and after washing. The cell suspension from each well was mixed and transferred to the second well, and the remaining adherent cells were washed five times with 0.5 mL DPBS each time. The DPBS from each wash was transferred to the second well. Cells in the first and second wells were imaged immediately after washing. Figure 8 and Figure 9 These images are shown.

[0281] References

[0282]

Claims

1. Microparticles for the activation and proliferation of human T lymphocytes, wherein (1) The particles are non-magnetic; (2) The diameter of the particles is 10 to 60 μm; and (3) The microparticle has at least a first capture ligand capable of binding to a first cell surface portion of a human T lymphocyte, the first capture ligand being attached to the surface of the microparticle.

2. The microparticles of claim 1, wherein the diameter of the microparticles is 15 to 50 μm, more preferably 20 to 40 μm.

3. The microparticles of claim 1 or 2, wherein the diameter of the microparticles is 40 μm.

4. The microparticle of any one of claims 1 to 3, wherein the microparticle has a second capture ligand capable of binding to a second cell surface portion of a human T lymphocyte, the second capture ligand being attached to the surface of the microparticle.

5. The microparticle of any one of claims 1 to 4, wherein the first capture ligand capable of binding to the first cell surface portion of a human T lymphocyte is an anti-CD3 antibody or an anti-CD28 antibody, preferably an anti-CD3 antibody.

6. The microparticle of claim 4, wherein the first capture ligand capable of binding to a first cell surface portion of a human T lymphocyte is an anti-CD3 antibody, and wherein the second capture ligand capable of binding to a second cell surface portion of a human T lymphocyte is an anti-CD28 antibody.

7. The microparticle of claim 6, wherein the microparticle has a third capture ligand capable of binding to a third cell surface portion of a human T lymphocyte, the third capture ligand being attached to the surface of the microparticle, wherein the third capture ligand capable of binding to the third cell surface portion of a human T lymphocyte is an anti-CD2 antibody.

8. The microparticles of any one of claims 1 to 7, wherein the microparticles are non-porous.

9. The particles of any one of claims 1 to 8, wherein the particles are made of silicon dioxide (SiO2).

10. The particles of any one of claims 2 to 7 or 9, wherein the surface area of ​​the particles is about 1 m². 2 / g.

11. The microparticles of any one of claims 1 to 10, wherein the surface of the microparticles is modified with a surface modifier, optionally wherein the surface modifier is a silane, further optionally wherein the surface modifier is selected from (3-glycidoxypropyl)trimethoxysilane, 8-glycidoxyoctyltrimethoxysilane, (3-glycidoxypropyl)methyldimethoxysilane, (3-glycidoxypropyl)methyldiethoxysilane, (3-glycidoxypropyl)dimethyleth ... (3-aminopropyl)triethoxysilane, (3-aminopropyl)methyldiethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)dimethylethoxysilane, (3-aminopropyl)diisopropylethoxysilane, carboxy-silanetriol, (3-mercaptopropyl)trimethoxysilane, (3-mercaptopropyl)methyldiethoxysilane, (3-mercaptopropyl)methyldimethoxysilane and (3-mercaptopropyl)triethoxysilane, preferably (3-glycidoxypropyl)trimethoxysilane.

12. The microparticle of any one of claims 1 to 11, wherein at least one capture ligand capable of binding to the cell surface portion of a human T lymphocyte is covalently linked to the surface of the microparticle, optionally wherein the at least one capture ligand capable of binding to the cell surface portion of a human T lymphocyte is covalently linked via the surface modifier.

13. The microparticle of any one of claims 1 to 12, wherein at least one capture ligand capable of binding to the cell surface portion of a human T lymphocyte is covalently linked to the surface of the microparticle via a connector, optionally wherein the at least one capture ligand capable of binding to the cell surface portion of a human T lymphocyte is covalently linked to the surface modifier via a connector.

14. The microparticles of claim 13, wherein the connector is a bifunctional connector, optionally wherein the connector has been formed using at least one of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), DCC (N',N'-dicyclohexylcarbodiimide), NHS (N-hydroxysuccinimide), sulfon-NHS (N-hydroxysulfosuccinimide), DMA (dimethyl hexamethyleneimine), DMP (dimethyl heptamethine), DMS (dimethyl octylimine), glutaraldehyde, glutaraldehyde polymer, cyanogen bromide, cyanuric chloride, SMCC (4-[N-maleiminomethyl]cyclohexane-1-carboxylic acid succinimide), and sulfon-SMCC (4-(N-maleiminomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide).

15. A method for activating and proliferating human T lymphocytes, the method comprising: (a) Providing a cell population, wherein at least a portion of the cell population comprises human T lymphocytes; (b) Contact the cell population with the particles according to any one of claims 1 to 14; (c) The cell population is incubated with the microparticles to achieve activation and proliferation of human T lymphocytes; And optionally (d) remove the particles.

16. The method for activating and proliferating human T lymphocytes as described in claim 15, wherein the cell population comprises monocytes, wherein optionally, the cell population is peripheral blood mononuclear cells (PBMCs).

17. The method for activating and proliferating human T lymphocytes as described in claim 15 or 16, wherein the method does not include a monocyte removal step.

18. A kit comprising the microparticles according to any one of claims 1 to 14.

19. The kit of claim 18, comprising at least: (1) The first microparticle according to any one of claims 1 to 14, wherein the first capture ligand capable of binding to the first cell surface portion of a human T lymphocyte is an anti-CD3 antibody; and (2) The second microparticle according to any one of claims 1 to 14, wherein the first capture ligand capable of binding to the first cell surface portion of a human T lymphocyte is an anti-CD28 antibody.

20. The kit of claim 18 or 19, further comprising a description of the method of any one of claims 15 to 17.

21. Use of the microparticles according to any one of claims 1 to 14 or the kit according to any one of claims 18 to 20 for the activation and proliferation of human T lymphocytes.

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