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WO2025083206A1 - Utilisation de microparticules non poreuses, non magnétiques en tant que kit d'activation et de prolifération de lymphocytes t humains - Google Patents

Utilisation de microparticules non poreuses, non magnétiques en tant que kit d'activation et de prolifération de lymphocytes t humains Download PDF

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Publication number
WO2025083206A1
WO2025083206A1 PCT/EP2024/079493 EP2024079493W WO2025083206A1 WO 2025083206 A1 WO2025083206 A1 WO 2025083206A1 EP 2024079493 W EP2024079493 W EP 2024079493W WO 2025083206 A1 WO2025083206 A1 WO 2025083206A1
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microparticle
human
capture ligand
lymphocyte
binding
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Inventor
Mojca JEZ KUSAR
Matic BARTOL
Katarina KATIC
Andrea CARDELLI
Aljaz KRAVANJA
Luka FAJS
Stephen Lewis MINGER
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Zsbcell Ltd
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Zsbcell Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/515—CD3, T-cell receptor complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2531/00—Microcarriers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/10—Mineral substrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2537/00—Supports and/or coatings for cell culture characterised by physical or chemical treatment
    • C12N2537/10—Cross-linking

Definitions

  • non-porous, non-magnetic microparticles as a kit for activation and proliferation of human T-lymphocytes
  • the present invention relates to a specific type of microparticles, composed of a solid surface of a specific diameter and an antigen recognition element that are useful for activation and expansion of T-lymphocyte cells.
  • the present invention relates generally to microparticles and related methods for stimulating cells.
  • the invention relates to activation and proliferation of human T- lymphocytes through binding of at least one capture ligand attached to the surface of the microparticle with a corresponding cell surface moiety on the T-lymphocyte.
  • anti-CD3 antibodies are known to be a key factor in T-lymphocyte activation and proliferation, when in contact with a population of these cells.
  • a co-stimulatory signal introduced with an anti-CD28 antibody is responsible for the increase and stability of the proliferation, by the means of prevention of premature cell apoptosis (Li, Y. et al. (2010), J Transl Med 8, 104). Since free antibodies provide inadequate cross-linking of the receptor to activate intracellular signalling, an approach of using beads coated with anti-CD3 and anti-CD28 has been developed (Trickett A. et al. (2003), J Immunol Methods. 1; 275(1-2): 251-5).
  • DynabeadsTM M-450 (Dynal Biotech) with immobilized anti-CD3 and anti-CD28 antibodies or CD3/CD28 T cell expander kit (Dynal Biotech), which are magnetic beads with the diameters of 4.5 and 2.8 pm, respectively; or
  • a problem that is encountered when working with these beads is related to the diameter of the beads used.
  • these diameters of microparticles allow for phagocytosis of these microparticles by monocytes and macrophages that are often present in the sample.
  • This property of monocytes is also mentioned in Berenson et al. (EP1257632B1) where this property is used as a pre-treatment step before going into the procedure of activation and expansion of T-lymphocytes. Additionally, it is often difficult to remove the particles from the sample.
  • the present invention eliminates the need for monocyte removal before the activation and expansion of T-lymphocytes by using microparticles that cannot be phagocytosed by other cells. Additionally, because of the diameter of the particles, the cells can be easily removed with mechanical force and filtration of the sample.
  • the invention provides a microparticle, wherein
  • microparticle is non-magnetic
  • the microparticle has a diameter of 10-60 pm
  • the microparticles has at least a first capture ligand that is capable of binding a first cell surface moiety of a human T-lymphocyte, the first capture ligand being attached to the surface of the microparticle.
  • these microparticles are suitable for activation and proliferation of human T- lymphocytes.
  • the invention also provides a kit comprising said microparticles and methods for activation and proliferation of human T-lymphocytes using said microparticles and kits.
  • microparticles of the present invention eliminate the need for monocyte removal before the activation and expansion of T- lymphocytes. Additionally, because of the diameter of the particles the cells can be easily removed, for example, with mechanical force and filtration of the sample.
  • non-porous, non-magnetic SiCh microparticles (20 - 40 pm) that have been functionalized using silanes as surface modifying agents with two different functional groups.
  • These surface modifying agents introduced on the surface of the microparticles are exploited and, using either the surface modifying agents directly or activating them with different linkers, the capture ligands are covalently bound to the surface of the microparticles.
  • the capture ligands are anti-CD3 and anti-CD28 antibodies that are particularly useful for activation and proliferation of T-lymphocytes.
  • the microparticles used further hereinbelow by way of example are non-magnetic and non-porous, and have irreversibly (covalently) bound antibodies (anti-CD3 and anti-CD28) on their surface.
  • the particles can have a mixture of these antibodies on one microparticle or they can be used as a mixture of particles with only anti-CD3 or anti-CD28.
  • the invention provides a microparticle, wherein
  • microparticle is non-magnetic
  • microparticle is made of SiCh
  • microparticle has a diameter of 10-60 pm
  • the surface of the microparticle is modified with a surface modifying agent, wherein the surface modifying agent is a silane;
  • the microparticle has a first and a second capture ligand capable of binding a first and a second cell surface moiety of a human T-lymphocyte, the first and second capture ligand being attached to the surface of the microparticle via the surface modifying agent, wherein the first and the second capture ligands are covalently attached to the surface of the microparticle via the surface modifying agent; and wherein the first capture ligand that is capable of binding a first cell surface moiety of a human T-lymphocyte is an anti-CD3 antibody and wherein the second capture ligand that is capable of binding a second cell surface moiety of a human T-lymphocyte is an anti-CD28 antibody.
  • the invention provides a microparticle, wherein
  • microparticle is non-magnetic
  • microparticle is made of SiCh
  • microparticle has a diameter of 10-60 pm
  • the surface of the microparticle is modified with a surface modifying agent, wherein the surface modifying agent is a silane;
  • the microparticle has a first, a second and a third capture ligand capable of binding a first, a second and a third cell surface moiety of a human T-lymphocyte, the first, the second and the third capture ligand being attached to the surface of the microparticle via the surface modifying agent, wherein the first, the second and the third capture ligands are covalently attached to the surface of the microparticle via the surface modifying agent; and wherein the first capture ligand that is capable of binding a first cell surface moiety of a human T-lymphocyte is an anti-CD3 antibody, the second capture ligand that is capable of binding a second cell surface moiety of a human T-lymphocyte is an anti-CD28 antibody and the third capture ligand that is capable of binding a second cell surface moiety of a human T-lymphocyte is an anti-CD2 antibody.
  • the invention provides a microparticle, wherein
  • microparticle is non-magnetic
  • microparticle is made of SiCh
  • microparticle has a diameter of 10-60 pm
  • the surface of the microparticle is modified with a surface modifying agent, wherein the surface modifying agent is a silane;
  • the microparticle has at least a first capture ligand that is capable of binding a first cell surface moiety of a human T-lymphocyte, the first capture ligand being attached to the surface of the microparticle via the surface modifying agent; wherein the first capture ligand is covalently attached to the surface of the microparticle via the surface modifying agent; and the first capture ligand that is capable of binding a first cell surface moiety of a human T-lymphocyte is an anti-CD3 antibody or an anti-CD28 antibody, preferably an anti-CD3 antibody.
  • microparticle is non-magnetic
  • the microparticle has a diameter of 10-60 pm
  • the microparticle has at least a first capture ligand that is capable of binding a first cell surface moiety of a human T-lymphocyte, the first capture ligand being attached to the surface of the microparticle, and wherein the microparticle is optionally a microparticle for activation and proliferation of human T-lymphocytes.
  • microparticle of item 1 wherein the diameter of the microparticle is 15-50 pm, more preferably 20 - 40 pm.
  • microparticle of any one of items 1-3 wherein the microparticle has a second capture ligand that is capable of binding a second cell surface moiety of a human T- lymphocyte, the second capture ligand being attached to the surface of the microparticle.
  • first capture ligand that is capable of binding a first cell surface moiety of a human T-lymphocyte is an anti-CD3 antibody or an anti-CD28 antibody, preferably an anti-CD3 antibody.
  • microparticle of item 6 wherein the microparticle has a third capture ligand that is capable of binding a third cell surface moiety of a human T-lymphocyte, the third capture ligand being attached to the surface of the microparticle, wherein the third capture ligand that is capable of binding a third cell surface moiety of a human T-lymphocyte is an anti-CD2 antibody.
  • Glycidoxypropyl)methyldimethoxysilane (3- Glycidoxypropyl)methyldiethoxysilane), (3- Glycidoxypropyl)dimethylethoxysilane), (3-Aminopropyl)triethoxysilane, (3-
  • Aminopropyl)dimethylethoxysilane (3-Aminopropyl)diisopropylethoxysilane, carboxy-silane triol, (3-mercaptopropyl)trimethoxysilane, (3-mercaptopropyl)methyldiethoxysilane, (3- mercaptopropyl)methyldimethoxysilane and (3-mercaptopropyl)triethoxysilane, preferably (3-Glycidyloxypropyl)trimethoxysilane.
  • linker is a bifunctional linker, optionally wherein the linker has been formed using at least one of EDC (l-ethyl-3-(3- dimethylaminopropyl) carbodiimidehydrochloride), DCC (N', N'-dicyclohexyl carbodiimide), NHS (N-Hydroxysuccinimide), sulfo-NHS (N-hydroxysulfosuccinimide), DMA (dimethyl adipimidate), DMP (dimethylpimelimidate), DMS (dimethyl suberimidate), glutaraldehyde, glutaraldehyde polymer, cyanogen bromide, cyanuric chloride, SMCC (succinimidyl 4-[N- maleimidomethyl]cyclohexane-l-carboxylate, Sulfo-SMCC (sulfosuccinimidyl 4-(N- maleimi
  • a method for activation and proliferation of human T-lymphocytes comprising:
  • PBMCs peripheral blood mononuclear cells
  • a kit comprising the microparticle of any one of items 1-14.
  • kit of item 18 comprising at least: (1) a first microparticle according to any one of items 1-14, wherein the first capture ligand that is capable of binding a first cell surface moiety of a human T-lymphocyte is an anti-CD3 antibody; and
  • kit of item 18 or 19 further comprising instructions for use in a method of any one of items 15 to 17.
  • Figure 1 This figure shows percentage of CD69+ cells (gated from viable CD2+ CD3+ cells) measured Oh, 4h, 24h, 48h, and 72h after activation.
  • CD69 is an early activation marker expressed in a few hours after the start of activation.
  • the graph shows a comparison between CD3/CD28 microparticles of the invention with MACSiBeadTM CD2/CD3/CD28 kit and DynabeadsTM CD3/CD28 kit. The graph displays the average results from three biological replicates.
  • Figure 2 This figure shows percentage of CD25+ cells measured at Oh, 4h, 24h, 48h, and 72h after activation.
  • CD25 is a late activation marker expressed a couple of days after the initiation of activation.
  • the graph shows a comparison between CD3/CD28 microparticles of the invention with MACSiBeadTM CD2/CD3/CD28 kit and DynabeadsTM CD3/CD28 kit. The graph displays the average results from three biological replicates.
  • FIG. 3 Cell viability was measured at 0 hours, 4 hours, 24 hours, 48 hours, and 72 hours after activation.
  • the graph shows a comparison between CD3/CD28 microparticles of the invention with MACSiBeadTM CD2/CD3/CD28 kit and DynabeadsTM CD3/CD28 kit.
  • the data presented is the average of three biological replicates.
  • FIG. 4 Graphical representation of proliferation of viable CD2+ cells measured Oh, 48h and 72h after activation. The data presented represents the average of three biological replicates. Proliferation was assessed by tracking dye dilution during cell divisions, with the percentage of dividing cells displayed on the graph.
  • Figure 5 Graphical representation of the absolute number of T-lymphocytes (CD2+ cells): T cells were activated and expanded using microparticles of the invention and MACSiBeadTM kits. An example of 14 days expansion is shown. PBMC in activation medium containing IL-2 were used as a negative control (dotted line). The expansion following activation using microparticles of the invention and reagent removal after 24 or 48h was accessed.
  • the optimal T cell expansion without the need for reactivation, was achieved when 0.6 million peripheral blood mononuclear cells (PBMCs) were initially activated and the microparticles of the invention were removed after a 48-hour activation period.
  • PBMCs peripheral blood mononuclear cells
  • the protocol for activation by the microparticles of the invention and the expansion protocol were compared with MACSiBeadTM activation and expansion protocol (lxlO 6 PBMC and activation reagent removal after 72h) following the MACSiBeadTM protocol.
  • Figure 6 Cell viability was assessed throughout the expansion process and compared between microparticles of the invention and MACSiBeadTM kits.
  • FIG. 7 T-lymphocytes were activated and expanded using microparticles of the invention and MACSiBeadTM reagents. An example of 14 days expansion is shown. PBMC in activation medium containing IL2 were used as a negative control (dotted line). PBMC only in medium with IL2 also get spontaneously activated over time but the activation is slower and not all cells activate. Microparticles of the invention were removed after 24 or 48h of activation. Sample after reagent removal after 24h was reactivated at day 9 (black arrow) which resulted in the upregulated expression of CD25 activation marker. Activation and expansion protocol according to the invention was compared with MACSiBeadTM activation and expansion protocol (lxlO 6 PBMC and activation reagent removal after 72h) following the MACSiBeadTM protocol.
  • FIG. 8 This figure shows microscopy images (phase contrast microscope, 400x magnification) of cells in contact with different commercial beads and the microparticles from the present invention.
  • FIG. 9 This figure shows microscopy images (phase contrast microscope, 400x magnification) of cells in contact with different commercial beads and the microparticles from the present invention.
  • DynabeadsTM particles both loaded and unloaded MACSiBeadTM particles, were ingested following a 24-hour incubation with PBMCs.
  • the white arrow indicates the microparticles of the invention, while the black arrows point at examples of cells that have engulfed the beads.
  • Both MACSiBeadTM and DynabeadsTM CD3/CD28 beads have an approximate diameter of ⁇ 5 pm.
  • Rigorous pipetting was employed prior to imaging to eliminate beads that were merely adhered to the cells. Thorough pipetting is described as a method to separate MACSiBeadTM and DynabeadsTM from the cells in activation experiments. Notably, the samples containing DynabeadsTM exhibited a higher presence of adherent cells.
  • any occurrence of terms such as “comprising”, “comprise” or “comprises” may optionally be replaced by an exhaustive term such as “consisting of”, “consist of” or “consists of”. Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words “herein,” “above,” and “below” and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application. The entire disclosure of all publications cited herein are incorporated by reference.
  • the invention provides a microparticle, wherein
  • microparticle is non-magnetic
  • the microparticle has a diameter of 10-60 pm
  • the microparticle has at least a first capture ligand that is capable of binding a first cell surface moiety of a human T-lymphocyte, the first capture ligand being attached to the surface of the microparticle.
  • microparticles are suitable for activation and proliferation of human T-lymphocytes.
  • Non-magnetic in the sense of the present invention refers to materials or objects that lack magnetic qualities. Non-magnetic materials are materials that do not have an impact on a magnetic field.
  • the microparticles are non-porous.
  • non- porous indicates that the “non-porous microparticle(s)” have a smooth surface that essentially lacks hollows or indentations on the surface. These could, for example, trap human T-lymphocytes.
  • the microparticles have a diameter of about 5 pm to 200 pm, preferably 10 pm to 100 pm, more preferable 10 pm to 60 pm, more preferably about 15 pm to 50 pm, and most preferably about 20 pm to 40 pm.
  • the microparticles have a diameter of about 20 or 40 pm. In a very preferred embodiment of the invention, the microparticles have a diameter of about 40 pm.
  • Ranges of microparticle diameters referred to herein such as "about 20 pm to 40 pm" are to be understood in a way that the different microparticles may have the same diameter or a different diameter.
  • a first and a second microparticle of the microparticles may both have a diameter of 20 pm, or a first and a second microparticle may both have a diameter of 40 pm, or a first and a second microparticle have a diameter of 20 pm and 40 pm, respectively, or a first a second microparticle have a diameter of 40 pm and 20 pm, respectively.
  • references to diameters of microparticles according to the invention are not meant to be limited to the specified (exact) diameter values but are meant to include a deviation of +/- 5% from the specified diameter value.
  • the microparticles have a density between than 2 g/cm 3 and 3 g/cm 3 .
  • the surface area of the microparticle is about 1 m 2 /g.
  • microparticles according to the invention are made of at least one inorganic material, preferably, but not limited to, microparticles made of SiC>2 (silicon dioxide), silicone, gold, silver, or platinum or composite materials made from inorganic shells SiC>2, silicone, gold, or silver and a particle core made of organic polymer or copolymer materials such as, but not limited to PMMA (poly(methyl methacrylate), PLA (polylactic acid), ABS (Acrylonitrile butadiene styrene) and nylon.
  • SiC>2 silicon dioxide
  • silicone silicone
  • gold, silver or platinum or composite materials made from inorganic shells SiC>2, silicone, gold, or silver
  • a particle core made of organic polymer or copolymer materials such as, but not limited to PMMA (poly(methyl methacrylate), PLA (polylactic acid), ABS (Acrylonitrile butadiene styrene) and nylon.
  • PMMA poly(methyl methacrylate)
  • PLA poly
  • the non-porous microparticles are made of SiO2-
  • microparticles useful in the manufacture of the particles of the present invention are:
  • Non-porous Silica particles 10 pm (PNPP10.0NAR), 20 pm (PNPP20.0NAR), 40 pm (PNPP40.0NAR), 50 pm (PNPP50.0NAR), 70 pm (PNPP70.0NAR);
  • Non-porous Silica particles 20 pm (DNG-E009), 30 pm (DNG-E010), 40 pm (DNG-E011);
  • EPRUI Wijiang Disctrict, Suzhou, China: Non-porous Silica particles: 20 pm (EPRU l-Si-20), 30 pm (EPRUI-Si-30), 40 pm (EPRUI-Si-40), 50 pm (EPRU l-Si-50);
  • Non-porous Silica gel particles 40-63 pm (27623.323), 40- 63 pm (7631-86-9), 60-200 pm (84893.290), 63-200 pm (27647.325), and MilliporeSigma Supelco, Merck (Darmstadt, Germany): Non-porous Silica gel particles: 40-60 pm (1.09385), 40-75 pm (80442), 40-75 pm (53698), 75-200 pm (78991).
  • the invention provides a microparticle, wherein
  • microparticle is non-magnetic
  • microparticle is made of SiCh
  • microparticle has a diameter of 10-60 pm
  • the microparticle has at least a first capture ligand that is capable of binding a first cell surface moiety of a human T-lymphocyte, the first capture ligand being attached to the surface of the microparticle.
  • non-porous, non-magnetic SiC>2 microparticles with the diameter of 20 to 40 pm, containing anti-CD3 and anti-CD28, or a fragment thereof, on the same or separate microparticle covalently bound to the surface of the microparticle are incubated with PBMC or leukapheresis sample.
  • the microparticles are proven to induce activation and proliferation of cells.
  • the cells remain viable and start replicating and increasing their numbers, and the expression of different activation markers is proved ( Figures 1, 2, 3 and 4).
  • the diameters of the microparticles are carefully chosen and have several advantages compared to using smaller or bigger microparticles.
  • the microparticles of this diameter can be easily removed with mechanical force and filtration of the sample.
  • Other advantage is that the particles of this diameter are not being phagocytosed by monocytes ( Figure 8 and 9).
  • PBMC peripheral blood mononuclear cell
  • leukapheresis without any pre-treatment or cell isolation step
  • the only solution is to do a monocyte removal step, either by using conventional methods as monocyte adherence to plastic, or, more expensive cell separation methods. Therefore, with the microparticles of the present invention, the inventors eliminate the need for monocyte removal steps even when encountering high monocyte percentage in the sample.
  • the target cells can be any human T-lymphocytes as long as the target cells display a moiety on their cell surface that can bind a capture ligand attached to the microparticles of the invention and binding of said capture ligand to a moiety on their cell surface results in activated and proliferating human T-Lymphocytes.
  • the present invention provides microparticles and methods for the activation and proliferation of human T-Lymphocytes.
  • T-lymphocytes are known in the art. They can be distinguished from other lymphocytes, for example, by the presence of a T-cell receptor (TCR) on their cell surface.
  • TCR T-cell receptor
  • Human T-lymphocytes within the meaning of the invention comprise the human T-lymphocytes known in the art and may optionally also comprise precursors of human T-lymphocytes which can be used in the methods and uses of the invention such that they become activated and proliferating human T-lymphocytes.
  • T-lymphocyte activation refers to a process in which, for instance, mature T-lymphocytes, which express antigen-specific T-lymphocyte receptors on their surfaces, recognize their cognate antigens and respond by entering the cell cycle, secreting cytokines or lytic enzymes, and initiating the cell-based functions of the immune system. Whether a human T-lymphocyte is an activated human T-lymphocyte in accordance with the invention can be determined by measuring the expression of activation markers known in the art. There are early and late activation markers of human T-lymphocytes.
  • an activated human T-lymphocyte is a human CD69+ T-lymphocyte and/or a human CD25+ T-lymphocyte, respectively.
  • T-lymphocyte proliferation refers to an increase in T- lymphocyte cell number, e.g., due to cell division, or cytokinesis, the final step of the cell cycle.
  • Whether human T-lymphocytes are proliferating human T-lymphocytes in accordance with the invention can be determined by, or is as determinable by, measuring the viable cell numbers of the human T-lymphocytes, e.g., by counting the viable CD2+ cells. If there is an increase in the measured viable cell numbers of the human T-lymphocytes over time, the human T-lymphocytes are determined to be proliferating human T-lymphocytes.
  • proliferation is used interchangeably with “expansion”. That is, each occurrence of this term may be replaced by “expansion”, and terms such as “proliferate” and “proliferating” may be replaced by “expand” and “expanding”, respectively.
  • sample or "cell population” can be any sample comprising a suspension of viable target cells.
  • the sample can be prepared from tissues or organs taken from individuals or specimens that have been treated to release target cells.
  • human cells typically have a diameter of about 5-lOOpm.
  • the sample may be a solution that contains viable target cells from human, tissue extract or organ extract, as well as cell lines that have been established from primary cells from such tissues or organs, including but not limited to heart, liver, kidney, colon, intestines, oesophagus, stomach, neural tissue (brain, spinal cord), lung, vascular tissue (artery, vein, capillary), lymphoid tissue (lymph gland, adenoid, tonsil, bone marrow, and blood including peripheral blood cells, placental/umbilical cord blood, menstrual blood), spleen and fibroblast, or a solution obtained by roughly separating the cells from such a solution as well as such solutions diluted with water, any of the buffers and/or culture media described herein, for example a physiological buffer described herein, physiological saline, a Ringer's solution containing a divalent cation such as a calcium ion or a magnesium ion, a cell culture media such as RPMI, MEM, IMEM, or
  • Preferred samples containing target cells that are to be activated and proliferated using the microparticles and method of the invention are human, whole blood, apheresis samples, bone marrow aspirate, biopsy samples, liquified tissue samples (such as liposuction samples that have been enzymatically degraded), cell culture samples, bioreactor cultures, single cell suspensions etc.
  • the sample i.e., the population of cells contacted with the microparticles of the invention comprises lymphocytes and monocytes.
  • the sample i.e., the population of cells contacted with the microparticles of the invention are peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • a "cell surface moiety", "molecule on the cell surface” or “cell surface molecule” can be any molecule that is expressed or displayed on the cell surface of a target cell such that the molecule is capable of being recognized and bound by the at least one capture ligand that is attached to the microparticle of the invention.
  • the molecule on the cell surface can be proteinaceous or non-proteinaceous.
  • a cell surface moiety is an antigen that is capable of being recognized by an antibody, or derivative thereof, that is attached to the microparticle of the invention.
  • any cell surface moiety which, if captured by the at least one capture ligand on the microparticle, leads to activation and proliferation of human T- lymphocytes is a suitable cell surface moiety.
  • the cell surface moiety is at least one selected from CD2, CD3, CD4, CD28 and CD45.
  • the cell surface moiety is at least one selected from CD2, CD3 and CD28.
  • microparticles if the present invention which are used in the methods according to the invention have at least one capture ligand that is attached to their surface and is capable of binding a first cell surface moiety of a human T-lymphocyte.
  • bind(s) means that a capture ligand on the microparticle binds to a cell surface moiety on the surface of the target cell (described in detail above) such that a target cell/microparticle complex is formed.
  • the capture ligand will have an affinity to a molecule at the surface of the target cell such that the target cell/microparticle complex leads to activation and proliferation of human T-lymphocytes is a suitable cell surface moiety.
  • the term “capture ligand” refers to a proteinaceous receptor that is capable of recognizing a molecule on the surface of the target cell of interest as described above.
  • the proteinaceous receptor capture ligand specifically binds to the molecule at the surface of the target cell with a dissociation constant of about 10’5 or greater, about 10'6 or greater, about 10’? or greater, about 10'8 or greater, about 10’ 9 or greater, about 10’10 or greater, about 10’H or greater, or about 10’12 or greater.
  • the capture ligand specifically binds to the molecule at the surface of the target cell with a dissociation constant between 10’5 and 10’12, more preferably between 10’0 and 10’11 or more preferably between 10’2 and 10’10.
  • the at least one capture ligand is an antibody or derivative thereof that specifically binds to an antigen on the surface of the target cell.
  • Antibodies are molecules that have one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad of immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • the basic immunoglobulin structural unit is known to comprise a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy” chain (about 50-70 kDa).
  • the N-terminus of each chain defines a variable region of about 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.
  • a capture ligand may exist as an intact immunoglobulin, i.e.
  • antibody or as a derivative thereof, in a variety of forms including, for example, FabFc2, Fab, Fv, Fd, F(ab')2, an Fv fragment containing only the light and heavy chain variable regions, a Fab or F(ab')2 fragment containing the variable regions and parts of the constant regions, a single-chain antibody, e.g. scFv, CDR-grafted antibody, dAb, nanobody and the like.
  • the heavy and light chain of a Fv may be derived from the same antibody or different antibodies thereby producing a chimeric Fv region.
  • the antibodies may also be modified antibodies (e.g. oligomers, reduced, oxidized and labelled antibodies).
  • the antibody may be of animal (especially mouse, rabbit or rat) or human origin or may be chimeric or humanized.
  • the term "antibody" includes these various forms.
  • the at least one capture ligand is an antibody, for example, FabFc2, Fab, Fv, Fd, F(ab')2, an Fv fragment containing only the light and heavy chain variable regions, a Fab or F(ab')2 fragment containing the variable regions and parts of the constant regions, a single-chain antibody, e.g., scFv, CDR-grafted antibody, dAb, nanobody and the like, that recognizes an antigen at the surface of a target cell with a dissociation constant of about 10’5 or greater, about 10'6 or greater, about 10’?
  • the capture ligand is an antibody that recognizes an antigen at the surface of a target cell with a dissociation constant between 10’5 and 10’12, more preferably between 10’9 and 10’H or more preferably between 10’2 and 10’10.
  • a capture ligand according to the invention are preferably suitable for activation and/or proliferation of human T-lymphocytes.
  • the first capture ligand that is capable of binding a first cell surface moiety of a human T-lymphocyte is an anti-CD3 antibody or an anti- CD28 antibody, preferably an anti-CD3 antibody. It is understood that an anti-CD3 antibody in accordance with the invention is suitable for activation and proliferation of human T- lymphocytes. It is understood that an anti-CD28 antibody, as a co-stimulatory signal that works together with anti-CD3, in accordance with the invention is suitable for proliferation of human T-lymphocytes. Suitable anti-CD28 and anti-CD3 antibodies are known in the art and can be used in the invention.
  • the first capture ligand that is capable of binding a first cell surface moiety of a human T-lymphocyte is an anti-CD3 antibody and the second capture ligand that is capable of binding a second cell surface moiety of a human T-lymphocyte is an anti-CD28 antibody.
  • the first capture ligand that is capable of binding a first cell surface moiety of a human T-lymphocyte is an anti-CD3 antibody
  • the second capture ligand that is capable of binding a second cell surface moiety of a human T-lymphocyte is an anti-CD28 antibody
  • the third capture ligand that is capable of binding a second cell surface moiety of a human T-lymphocyte is an anti-CD2 antibody.
  • Suitable anti- CD2 antibodies are known in the art and can be used in the invention.
  • Anti-CD3 antibody Biolegend, clone OKT3
  • anti-CD28 antibody Invitrogen, clone CD28.2
  • Anti-CD2 antibody Exbio, clone TS1/8 are particularly preferred.
  • anti-CD3 and anti-CD28 antibodies, or fragments thereof, irreversibly bound to non-porous, non-magnetic SiCh microparticles with the diameter of 20 to 40 pm can be bound to the same microparticle or to a separate microparticle.
  • the mixture can be agitated and strained through a strainer with the pore size of 15 or 30 pm, respectively.
  • Antibodies such as anti-CD3 and anti-CD28 are in this application used as signalling molecules that have one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
  • the microparticles used in the methods according to the invention have one or more capture ligands that are attached to the microparticle surface and are capable of binding at least one cell surface moiety of a human T-lymphocyte and thus transferring signals that induce activation and proliferation of T-lymphocytes.
  • the capture ligand, target cells and surface moieties are described in detail above.
  • the capture ligand is covalently attached to the microparticle.
  • a coupling reagent such as common bifunctional agent or a method of attaching via spacers may also be used.
  • a suitable coupling reagent is used depending on the reactive groups on the carrier surface.
  • Silane surface modifying agents are used to introduce the functional groups to the surface of the carrier, when the surface has -OH groups such as materials made of SiO2-
  • -OH groups such as materials made of SiO2-
  • 3- Glycidyloxypropyl)trimethoxysilane, (3-Aminopropyl)triethoxysilane, carboxy-silane triol, and (3-mercaptopropyl)trimethoxysilane are used as silane surface modifying agents.
  • the functional groups introduced can for example be amino, amido, imido, hydrazido, carboxyl, thiol, hydroxyl, azido, alkyl, phenyl, epoxy, ester, halide and acyl halide.
  • amino, carboxyl, thiol, and epoxy groups are used. Most preferably epoxy groups are used.
  • the covalent bonds formed between antibodies and surfaces can be formed directly between the functionalized surface and the capture ligand or through a bifunctional linker that can react with both functional groups on the surface carrier and on the ligand molecule.
  • the linker can be with or without a PEG (polyethylene glycol) spacer.
  • the linker that can be used for the formation of covalent bonds between the non-porous microparticle and the capture ligand can be, but is not limited to: EDC (l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride), DCC (N', N'-dicyclohexyl carbodiimide), NHS (N-Hydroxysuccinimide), sulfo- NHS (N-hydroxysulfosuccinimide), DMA (dimethyl adipimidate), DMP (dimethyl pimelimidate), DMS (dimethyl suberimidate), glutaraldehyde, glutaraldehyde polymer, cyanogen bromide, cyanuric chloride, SMCC (succinimidyl 4-[N- maleimidomethyl]cyclohexane-l-carboxylate, Sulfo-SMCC (sulfosuccinimidyl 4-(N
  • the size of the PEG spacer that might be used in this invention can vary from 0.4 kDa to more than 20 kDa.
  • microparticles and capture ligands are attached directly to one another or via a linker. Solely using methods of covalent bonding between the surface and the antibody, without the use of any affinity ligands, prevents the capture ligands from leaking from the non-porous microparticles.
  • linkers without PEG polyethylene glycol are used in this invention.
  • linker that is able to ensure the formation of covalent bonds between the functional groups on the surface of the non-porous microparticles and groups that can be found in the structure of the capture ligand as well as other functional groups that can be found on the surface of the non-porous microparticles that can react with the groups of the antibody, with or without the linker molecule, are not a limiting factor in practice of the present invention.
  • the invention also provides kits comprising the microparticles of the invention.
  • the invention provides a kit comprising at least one of the microparticles described above.
  • the kit may comprise a microparticle, wherein
  • microparticle is non-magnetic
  • microparticle is made of SiCh
  • the microparticle has a diameter of 10-60 pm; (5) the surface of the microparticle is modified with a surface modifying agent, wherein the surface modifying agent is a silane; and
  • the microparticle has a first and a second capture ligand capable of binding a first and a second cell surface moiety of a human T-lymphocyte, the first and second capture ligand being attached to the surface of the microparticle via the surface modifying agent, wherein the first and the second capture ligands are covalently attached to the surface of the microparticle via the surface modifying agent; and wherein the first capture ligand that is capable of binding a first cell surface moiety of a human T-lymphocyte is an anti-CD3 antibody and wherein the second capture ligand that is capable of binding a second cell surface moiety of a human T-lymphocyte is an anti-CD28 antibody.
  • the kit may comprise a microparticle, wherein
  • microparticle is non-magnetic
  • microparticle is made of SiCh
  • microparticle has a diameter of 10-60 pm
  • the surface of the microparticle is modified with a surface modifying agent, wherein the surface modifying agent is a silane;
  • the microparticle has a first, a second and a third capture ligand capable of binding a first, a second and a third cell surface moiety of a human T-lymphocyte, the first, the second and the third capture ligand being attached to the surface of the microparticle via the surface modifying agent, wherein the first, the second and the third capture ligands are covalently attached to the surface of the microparticle via the surface modifying agent; and wherein the first capture ligand that is capable of binding a first cell surface moiety of a human T-lymphocyte is an anti-CD3 antibody, the second capture ligand that is capable of binding a second cell surface moiety of a human T-lymphocyte is an anti-CD28 antibody and the third capture ligand that is capable of binding a third cell surface moiety of a human T-lymphocyte is an anti-CD2 antibody.
  • the kit may comprise a microparticle, wherein
  • microparticle is made of SiCh
  • microparticle has a diameter of 10-60 pm
  • the surface of the microparticle is modified with a surface modifying agent, wherein the surface modifying agent is a silane; and (6) the microparticle has at least a first capture ligand that is capable of binding a first cell surface moiety of a human T-lymphocyte, the first capture ligand being attached to the surface of the microparticle via the surface modifying agent; wherein the first capture ligand is covalently attached to the surface of the microparticle via the surface modifying agent; and the first capture ligand that is capable of binding a first cell surface moiety of a human T-lymphocyte is an anti-CD3 antibody.
  • the kit may comprise at least a microparticle, wherein
  • microparticle is non-magnetic
  • microparticle is made of SiCh
  • microparticle has a diameter of 10-60 pm
  • the surface of the microparticle is modified with a surface modifying agent, wherein the surface modifying agent is a silane;
  • the microparticle has at least a first capture ligand that is capable of binding a first cell surface moiety of a human T-lymphocyte, the first capture ligand being attached to the surface of the microparticle via the surface modifying agent; wherein the first capture ligand is covalently attached to the surface of the microparticle via the surface modifying agent; and the first capture ligand that is capable of binding a first cell surface moiety of a human T-lymphocyte is an anti-CD28 antibody.
  • the kit comprises at least
  • the kit preferably further comprises at least a first vial and a second vial, wherein the first vial contains the first microparticle and the second vial contains the second microparticle.
  • first microparticle and the second microparticle may have the same diameter or a different diameter.
  • first and the second microparticle may both have a diameter of 20 pm, or they may both have a diameter of 40 pm, or the first and second microparticle have a diameter of 20 pm and 40 pm, respectively, or the first and second microparticle have a diameter of 40 pm and 20 pm, respectively.
  • the kit may further comprise instructions for use in a method for activation and proliferation of human T-lymphocytes according to the invention.
  • the present invention also provides methods and uses for activation and proliferation of human T-lymphocytes.
  • the invention provides a use of a microparticle according to the invention or a kit according to the invention for activation and proliferation of human T-lymphocytes.
  • the invention provides a method for activation and proliferation of human T-lymphocytes, comprising:
  • the step of (c) incubating said population of cells together with the microparticles to effect activation and proliferation of the human T-lymphocytes is carried out in a suitable medium for activation and proliferation of the human T-lymphocytes.
  • Media for activation and proliferation of the human T-lymphocytes are generally known in the art.
  • the medium for activation and proliferation of the human T-lymphocytes contains interleukin-2 (IL-2), e.g., recombinant IL-2.
  • the medium for activation and proliferation of human T-lymphocytes also contains interleukin-7 (IL-7), e.g., recombinant IL-2 and interleukin-15 (IL-15), e.g., recombinant IL-15.
  • IL-7 interleukin-7
  • IL-15 interleukin-15
  • the step of (c) incubating is carried out for more than 7 days, it is very preferred that the medium for activation and proliferation of the human T-lymphocytes contains interleukin-2 (IL-2), e.g., recombinant IL-2, interleukin- 7 (IL-7), e.g., recombinant IL-7 and interleukin-15 (IL-15), e.g., recombinant IL-15.
  • IL-2 interleukin-2
  • IL-7 interleukin- 7
  • IL-15 interleukin-15
  • the population of cells comprises monocytes.
  • the population of cells are peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • the method does not comprise a monocyte removal step.
  • Cell culture media that can used in the various steps of the method and/or following cell separation depends on the cell sample. Different cells or cell types can have very specific medium, serum and supplement requirements. A person skilled in the art will know what media to use.
  • Exemplified cell culture media include MEM (Minimum Essential Medium), DMEM (Dulbecco's Modified Eagle's Medium), RPMI-1640 (Roswell Park Memorial Institute Medium 1640), and IMDM (Iscove's Modified Dulbecco's Media) (Sigma/ThermoFisher).
  • MEM Minimum Essential Medium
  • DMEM Dulbecco's Modified Eagle's Medium
  • RPMI-1640 Roswell Park Memorial Institute Medium 1640
  • IMDM Iscove's Modified Dulbecco's Media
  • the temperature at which any of the steps for carrying out the method of the invention occur including the (optional) step of removing the microparticles is not critical as long as the target cells remain viable. Typically, the temperature will be at or below the temperature at which the target cells grow in their natural environment or culture conditions and can be the same temperature or a different temperature from the temperature at which other steps in the method according to the invention are performed.
  • the method for activation and proliferation of human T-lymphocytes according to the invention is carried out in vitro, and the use for activation and proliferation of human T-lymphocytes according to the invention is carried out in vitro.
  • microparticles of the invention are useful for activation and proliferation of human T-lymphocytes.
  • the population of cells in the context of the methods of the invention may be any population of cells, as long as it comprises human T-lymphocytes.
  • sample or "cell population” in the context of the method of the invention can be any sample comprising a suspension of viable target cells.
  • the sample can be prepared from body fluids (e.g., human blood), tissues or organs taken from individuals or specimens that have been treated to release target cells.
  • the method of the present invention also comprises a step of contacting the population of cells with the microparticles of the invention.
  • the microparticles of the invention that have at least a first capture ligand that is capable of binding a first cell surface moiety of a human T- lymphocyte are contacted with the sample comprising a suspension of viable target cells that display a cell surface moiety as described above on the cell surface under conditions such that said target cells in the sample remain viable and are mixed with the microparticles, for example by causing the flow of the sample and the suspension of microparticles to mix and/or by shaking, stirring and/or agitating.
  • the method is initiated by contacting the suspension of microparticles of the invention in a solution directly with the sample.
  • the sample is diluted with a liquid after addition of the microparticles as a suspension.
  • the sample can be diluted with any liquid prior to, during or after the addition of the microparticles that maintains the viability of the target cells and allows the non-porous microparticles to form a complex with the target cells during the incubation of the sample and can be chosen based on the type of cells of interest, i.e., target cells, present in the sample.
  • Such liquids include but are not limited to water, buffered solutions or cell culture media, solutions or media used for cell transfection, transplantation, implantation and/or storage or freezing as described herein.
  • the buffer that the cells are suspended in can be of different composition, but the presence of serum (e.g., fetal bovine serum, human serum) or serum replacement compounds is necessary.
  • serum e.g., fetal bovine serum, human serum
  • the mixture of cells and microparticles for activation and proliferation is kept at standard conditions that are useful for supporting cell growth, preferably at 37 °C, with a gas inlet of air with 5% CO2.
  • the step of incubating of the methods of the invention is also preferably carried out at 37 °C, with a gas inlet of air with 5% CO2.
  • this type of microparticles can be easily applied for generating T-lymphocyte therapeutics for in vivo use, because of the fact that the T-lymphocytes after the procedure with the microparticles can be easily separated from the microparticles. Additionally, because of the irreversible bond between the antibodies and the microparticle the antibodies cannot in any way contaminate the cell suspension. Therefore, this system allows the user to obtain a T-lymphocyte cell suspension without any contaminants that would be in any way harmful.
  • viable target cells human T- lymphocytes
  • viable target cells may be obtained, for instance, by mechanically dissociating the target cell/microparticle complex and eluting said viable target cells through a filter while retaining the larger non-porous microparticles with said capture ligand covalently immobilized to the microparticle surface.
  • the viable target cells will be preferably eluted into a sterile container that can be aseptically closed.
  • the methods of the invention may optionally further comprise a step of determining whether the human T-lymphocyte is an activated human T-lymphocyte in accordance with the invention. This can be determined by measuring the expression of activation markers known in the art. There are early and late activation markers of human T-lymphocytes. Early markers include CD69, which starts expressing a few hours after the start of activation, and late markers, which include CD25, which starts expressing a day after the start of activation.
  • activation is determined by measuring the expression of CD69 (e.g., by using fluorophore-conjugated anti-CD69 antibody) and/or by measuring the expression of CD25 (e.g., by using fluorophore- conjugated anti-CD25 antibody) in the population of cells.
  • an activated human T-lymphocyte is a human CD69+ T-lymphocyte and/or a human CD25+ T-lymphocyte, respectively.
  • the methods of the invention may optionally further comprise a step of determining whether the human T-lymphocytes are proliferating human T-lymphocytes in accordance with the invention. This can be determined by measuring the viable cell numbers of the human T- lymphocytes, e.g., by counting the viable CD2+ cells. If there is an increase in the measured viable cell numbers of the human T-lymphocytes over time, the human T-lymphocytes are determined to be proliferating human T-lymphocytes.
  • Another method of T-lymphocyte proliferation determination is dye dilution, for example, Cell Trace Violet (CTV) dye to track proliferation by binding to cellular proteins and gradually diluting as the cells divide, creating distinct fluorescence peaks that correspond to different generations of cells. This allows researchers to monitor and quantify cell proliferation over time through flow cytometry or fluorescence microscopy.
  • CTV Cell Trace Violet
  • the invention provides microparticles, kits and methods for activation and proliferation of human T-lymphocytes.
  • the present invention is described in detail in the following examples which may represent more than one embodiment of the present invention.
  • Example 1 Preparation of anti-CD3 and anti-CD28 antibody covalently immobilized to carboxyl silica microparticles
  • the activated non-porous microparticles are centrifuged and the supernatant is discarded in order to remove excess reagents.
  • 15 pg of anti-CD3 antibody (Biolegend, clone OKT3) is dissolved in 1 mL of PBS buffer and 15 pg of anti-CD28 antibody (Invitrogen, clone CD28.2) is dissolved in 1 mL of PBS buffer and both solutions are added to the respective (separate) vials of activated silica microparticles. After 2.5 h of mixing at room temperature the microparticles are washed in PBS and may be used for activation and expansion of T-lymphocytes.
  • Non functionalized silica non-porous SiCh microparticles with the diameter of 40 pm were purchased from Glantreo Ltd, Ireland.
  • the non-functionalized silica microparticles are silanized with GLYMO in order to prepare silica non-porous microparticles functionalized with epoxy groups.
  • the average surface area of the 40 pm nonporous silica particles is 1 m 2 /g particles, measured with Micromeritics TriStar II analyser.
  • Example 3 Activation and proliferation assay using PBMC fraction of whole blood
  • RPMI 1640 supplemented with 10 % FBS or 10 % AB human serum (2 mM L- Glutamine and antibiotics can also be added
  • rlL-2 recombinant interleukin-2
  • Microparticles according to the invention bound to either anti-CD3 or anti-CD28 are non-porous, non-magnetic SiCh microparticles with the diameter of 40 pm, with the average surface area of about 1 m 2 /g particles functionalized with GLYMO and bound to anti-CD3 and anti-CD28 antibodies via the reaction of epoxy group on the particles and amino groups on the antibodies, according to example 2.
  • a part of isolated PBMCs can be stained with Cell Trace Violet dye before performing activation.
  • Cell Trace Violet dye is employed in cell biology to track proliferation by binding to cellular proteins and gradually diluting as the cells divide, creating distinct fluorescence peaks that correspond to different generations of cells.
  • PBMCs isolation part of the cells is incubated in 1 pM Cell Trace Violet dye in DPBS for 20 minutes at room temperature. After incubation 20 mL of RPMI medium with 10% FBS is added and cells are centrifuged at 300xg for 10 min. Supernatant is removed and pellet is resuspended in the activation medium and the standard activation protocol is followed to set up activation and expansion.
  • the MACSiBeadsTM and DynabeadsTM CD3/CD28 were used in the amounts recommended for T cell activation.

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Abstract

La présente invention concerne un type spécifique de composites non poreux et non magnétiques, constitué d'une surface solide d'un diamètre spécifique et d'un élément de reconnaissance d'antigène qui sont utilisés dans un procédé d'activation et de multiplication de cellules de lymphocytes T.
PCT/EP2024/079493 2023-10-19 2024-10-18 Utilisation de microparticules non poreuses, non magnétiques en tant que kit d'activation et de prolifération de lymphocytes t humains Pending WO2025083206A1 (fr)

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