JP2023096268A - Lipid particle useful for producing lymphocyte - Google Patents

Abstract

To provide a method for producing lymphocytes that can be applied to an automatic culture device or the like.SOLUTION: A lipid particle has a substance having a binding activity to CD3 or capable of interacting therewith and fibronectin fragments added to its surface.SELECTED DRAWING: None

Description

The present invention relates to a method for producing lymphocytes. The invention also relates to lipid particles for use in said method.

The body is mainly protected from foreign substances by immune response, and the immune system consists of various cells and soluble factors produced by them. Lymphocytes, which are one of the subtypes of leukocytes, play a particularly central role. Lymphocytes are mainly referred to as T cells (sometimes referred to as T lymphocytes), B cells (sometimes referred to as B lymphocytes), and Natural Killer cells (sometimes referred to as NK cells). They are divided into three main types. T cells further express CD (Cluster Designation) 4, mainly helper T cells involved in antibody production and induction of various immune responses, express CD8, and mainly exhibit cytotoxic activity. They are subclassified into sexual T cells (cytotoxic T lymphocytes, also called killer T cells), and other T cells.

As a method for producing lymphocytes, a method is known in which extracellular matrices such as matrigel, laminin and fibronectin, and derivatives thereof are used as substrates for lymphocyte culture.

For example, Patent Document 1 can be cited as research on a method for producing lymphocytes using fibronectin fragments. In Patent Document 1, cytotoxic T cells are efficiently induced and maintained by culturing lymphocyte progenitor cells on a solid phase on which an anti-CD3 antibody and a recombinant fibronectin fragment such as CH-296 are immobilized. , or a method of expanding the culture. However, this method uses a fibronectin fragment immobilized on a solid phase, and has the problem that it is not easy to apply to an automatic culture apparatus. In addition to the automatic culture apparatus, there is also a problem that it is not easy to apply to E-flasks, spinner flasks, shaking culture bags, bioreactors, and the like.

WO 2005/019450 Pamphlet

An object of the present invention is to solve the problems of conventional methods for producing lymphocytes, and to provide a method for producing lymphocytes that can be easily applied to automatic culture equipment and the like. do.

As a result of intensive research to solve the above problems, the present inventors have found that lymphocytes or cells capable of differentiating into lymphocytes are treated with a substance having binding activity or a substance capable of interacting with CD3, and a fibronectin fragment on the surface. The inventors have found that lymphocytes can be produced efficiently by including the step of culturing in the presence of added lipid particles, and have completed the present invention.

That is, if the present invention is outlined,
[1] A substance having a binding activity or a substance capable of interacting with CD3, and a lipid particle having a fibronectin fragment attached to its surface,
[2] The lipid particles according to [1], wherein the substance that has binding activity or can interact with CD3 is an anti-CD3 antibody;
[3] The lipid particle of [1], wherein the fibronectin fragment is a fragment having at least one domain selected from the group consisting of a cell-binding domain and a heparin-binding domain.
[4] The lipid particle of [3], wherein the fibronectin fragment is a fragment having a cell-binding domain and a heparin-binding domain.
[5] The fibronectin fragment is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 18 in the sequence listing, or an amino acid having one or more amino acid substitutions, deletions, insertions or additions in the amino acid sequence of said polypeptide. The lipid particle according to [1], which is a polypeptide having a sequence and has a function equivalent to that of the polypeptide;
[6] The lipid particle of [1], wherein the lipid particle is a liposome;
[7] A method for producing lymphocytes, comprising the step of culturing lymphocytes or cells capable of differentiating into lymphocytes in the presence of the lipid particles of any one of [1] to [6],
[8] The method for producing lymphocytes according to [7], wherein the lymphocytes or cells capable of differentiating into lymphocytes are mammal-derived lymphocytes or cells capable of differentiating into lymphocytes.
[9] The method for producing lymphocytes according to [7], further comprising the step of introducing a desired gene into lymphocytes or cells capable of differentiating into lymphocytes.
[10] The method for producing lymphocytes according to [9], wherein the desired gene is introduced using a viral vector;
[11] The method for producing lymphocytes according to [10], wherein the desired gene is introduced using a retroviral vector and a lentiviral vector;
Regarding.

The present invention provides a method for producing lymphocytes and lipid particles used in the method. According to the method of the present invention, it is possible to efficiently proliferate lymphocytes, maintain the functions of lymphocytes, and efficiently induce lymphocytes. Lymphocytes obtained by the present invention are preferably used for regenerative medicine, for example. Therefore, the method of the present invention is expected to greatly contribute to the medical field.

FIG. 4 is a diagram showing cell proliferation rate when the number of cells on day 0 of culture is set to 1. FIG. FIG. 4 shows the CD3+/CD4+/CD8+ ratio of CAR-T cells on day 10 of culture. FIG. 10 shows the gene transfer efficiency of CAR-T cells on day 10 of culture. FIG. 2 shows the immunophenotype of CAR-T cells on day 10 of culture. FIG. 1 shows the immunophenotype of T cells on day 10 of culture.

The present invention will be specifically described below.
(1) Lipid Particles of the Present Invention The present invention provides a substance having binding activity or interaction with CD3, and a lipid particle having a fibronectin fragment added to its surface.

<Lipid particles>
The lipid particles used in the present invention are particularly limited as long as a substance having binding activity or a substance capable of interacting with CD3 and a fibronectin fragment (hereinafter collectively referred to as a ligand) can be added to the surface. Examples include, but are not limited to, lipid nanoparticles or liposomes. Preferably, the lipid particles of the invention are liposomes.

A liposome is a closed vesicle composed of a lipid bilayer membrane, which was announced by Bangham et al. in 1965. Since it can be prepared relatively easily and its physical properties can be adjusted by the manufacturing method and constituent components, it is used in a wide range of fields such as the construction of biomembrane models and drug delivery systems.

Liposomes that can be used in the present invention are not particularly limited, and suitable liposomes can be selected and used from known liposomes. Lipids constituting liposomes include, for example, glycerophospholipids (phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, etc.), sphingolipids (ceramide, sphingomyelin, etc.), cationic lipids (DAP, DODAP, etc.). ). Fatty acids possessed by lipids are not particularly limited, and may be those containing saturated fatty acids or those containing unsaturated fatty acids. Additionally, these lipids may have functional groups that can react with other molecules to form covalent bonds. Examples of such functional groups include carboxyl groups, amino groups, sulfhydryl groups, succinimide groups, maleimide groups, carbodiimide groups and the like.

Liposomes can also contain components other than lipids. For example, known stabilizers for liposomes include sterols (cholesterol, sitosterol, campesterol, stigmasterol, brassicasterol, etc.), tocopherols, fatty acids (stearic acid, palmitic acid, etc.), alkanediols, and the like. ing.

Liposomes can be produced by a general method for producing liposomes using lipids that constitute the lipid bilayer membrane, lipids with reactive functional groups, stabilizers for liposomes, and the like. Methods for producing the liposome include, for example, an extrusion method, a vortex mixer method, an ultrasonic method, a surfactant removal method, a reverse phase evaporation method, an ethanol injection method, a prevesicle method, a French press method, and a W/O/W emulsion. method, annealing method, freeze-thaw method. The form of the liposome is not particularly limited, and liposomes having various sizes and forms such as multilamellar liposomes, small unilamellar liposomes, and large unilamellar liposomes can be produced by appropriately selecting the liposome production method. . Also, various liposomes are commercially available.

The size (average particle size) of the liposomes is not particularly limited, but from the viewpoint of storage stability, etc., it is 20 to 1000 nm, preferably 30 to 600 nm, more preferably 40 to 400 nm, and even more preferably 40 to 400 nm. 50-300 nm, most preferably 100-200 nm.

The liposomes used in the present invention may be liposomes composed of a simple lipid bilayer membrane, or those to which other substances have been added. Examples of such liposomes include liposomes to which sugars (oligosaccharides, polysaccharides, etc.) or polymers (polyethylene glycol, etc.) are added.

In the present invention, the lipid particles or other substances added to the lipid particles have functional groups that can directly or indirectly bind to the functional groups of the ligands. The functional group includes a hydrophobic group consisting of an amino group, a succinimide group, a maleimide group, a thiol group, a carboxyl group, a hydroxyl group, a disulfide group, a hydrocarbon group having a methylene chain (such as an alkyl group), a carboxyl group, or A maleimido group is preferred. In this specification, a carboxyl group may also be represented as a COOH group. Furthermore, the above functional groups may be attached to the lipid particles or other substances attached to the lipid particles via suitable spacers.

<Substance having binding activity or interacting with CD3>
Substances that have binding activity or can interact with CD3 used in the present invention are particularly those that have binding activity to CD3 or influence cell differentiation, proliferation, etc. via CD3. Examples include, but are not limited to, anti-CD3 antibodies, particularly preferably anti-CD3 monoclonal antibodies, such as OKT3 [J. Immunol. , Vol. 124, No. 6, 2708-2713 (1980)]. In the present specification, an anti-CD3 antibody is used as an example of a substance that has binding activity or can interact with CD3.

<Fibronectin fragment>
Fibronectin is a huge glycoprotein with a molecular weight of about 250 kDa (monomer) present in blood, cell surfaces, extracellular matrices, etc., and is known to have various functions such as cell adhesion. Fibronectin consists of a domain structure and contains three similar sequences in its amino acid sequence. Three types of similar sequences are called type I repeats, type II repeats, and type III repeats, respectively, of which the type III repeat consists of 87 to 96 amino acid residues, Amino acid sequence homology is 17-40%. There are 15 type III repeats in fibronectin, of which the 1st, 2nd and 3rd (hereinafter referred to as III-1, III-2 and III-3 respectively) are self-association domains, 4 th, 5th and 6th (hereinafter referred to as III-4, III-5 and III-6 respectively) to the DNA binding domain, 8th, 9th and 10th (hereinafter referred to as III-8 and III-9 respectively) , III-10) are contained in the cell-binding domain, and the 12th, 13th and 14th (hereinafter referred to as III-12, III-13 and III-14, respectively) are contained in the heparin binding domain. III-10 contains a region with binding activity for integrin α5β1 (also called VLA-5), the core sequence of which is RGD. In addition, a region called IIICS is present at a site closer to the C-terminal side of fibronectin. IIICS contains a 25-amino acid sequence called CS-1, which exhibits binding activity to integrin α4β1 (also called VLA-4).

The amino acid sequences of human fibronectin III-1-14 and CS-1 are shown in the sequence listing herein as SEQ ID NOs: 1-14 and 15, respectively.

As fibronectin fragments, many fragments having cell-binding domains or heparin-binding domains in the molecule are known [eg, J. Biochem., Vol. 110, pp. 284-291 (1991)].

Any fibronectin fragment can be used in the present invention as long as it has suitable properties for producing lymphocytes.

Fibronectin fragments used in the present invention include, but are not limited to, cell-binding domains (III-8, III-9, and III-10) and heparin-binding domains (III-12, III-13, and III). -14) having at least one domain selected from the group consisting of:

As used herein, a fibronectin fragment includes the functional domain of fibronectin described above, that is, the cell-binding domain or the heparin-binding domain, and consists of a portion of the continuous amino acid sequence of fibronectin. It doesn't have to be. Fibronectin fragments can be produced by enzymatic digestion of fibronectin obtained from nature or by recombinant DNA technology.

Recombinant fibronectin fragments are suitable for the present invention in terms of the effort involved in removing impurities and in that the desired fragment can be designed and obtained regardless of the enzyme recognition site. From the standpoint of recombinant production or handling, the fibronectin fragment used in the present invention preferably has a molecular weight of 150 kDa or less, 140 kDa or less, 130 kDa or less, 120 kDa or less, 110 kDa or less, or 100 kDa or less.

Examples of fibronectin fragments include the 120 kDa fibronectin fragment (120 k-fr), CH-271, CH-296, and FCH-296.

120k-fr is III-1, III-2, III-3, III-4, III-5, III-6, III-7, III-8, III-9, III-10 in order from the N-terminal side A polypeptide with a molecular weight of about 120 kDa, containing The predicted amino acid sequence of 120k-fr (932 amino acid residues) is shown in the sequence listing herein as SEQ ID NO:16. 120k-fr can be produced as a recombinant polypeptide by constructing DNA encoding the amino acid sequence of 120k-fr and combining it with an appropriate host-vector system. Also, commercially available 120k-fr may be used.

CH-271 is a recombinant polypeptide with a molecular weight of about 60 kDa (549 amino acid residues) containing III-8, III-9, III-10, III-12, III-13, and III-14 in order from the N-terminal side is. The amino acid sequence of CH-271 is shown in the sequence listing herein as SEQ ID NO:17.

CH-296 contains III-8, III-9, III-10, III-12, III-13, III-14, CS-1 in order from the N-terminal side, molecular weight about 63 kDa (574 amino acid residues) is a recombinant polypeptide of The amino acid sequence of CH-296 is shown in the sequence listing herein as SEQ ID NO:18. CH-296 is commercially available as Retronectin (registered trademark, manufactured by Takara Bio Inc.).

FCH-296 has III-1, III-2, III-3, III-8, III-9, III-10, III-12, III-13, III-14, and CS-1 in order from the N-terminal side. A recombinant polypeptide with a molecular weight of approximately 96 kDa (881 amino acid residues) containing The amino acid sequence of FCH-296 is shown in the sequence listing herein as SEQ ID NO:19.

The fibronectin fragment in the present specification also includes chemically modified proteins such as acetylation. In addition, in the production of lymphocytes in the present invention, a single molecular species of fibronectin fragment or a mixture of multiple molecular species may be used.

Substances functionally equivalent to these fibronectin fragments, such as substances having a cell-binding domain and/or a heparin-binding domain, can also be used. Derivatives of fibronectin fragments and the like can also be used.

<Method for producing lipid particles of the present invention>
The lipid particles of the present invention can be conveniently produced by binding lipid particles, such as liposomes, to substances that have binding activity or can interact with CD3, such as anti-CD3 antibodies, and fibronectin fragments. The functional groups of the lipid particles (including the functional groups of other substances added to the lipid particles) and the functional groups of the ligand may be directly linked and bonded, and the two may be combined with a divalent reactive compound (crosslinking agent).

In the lipid particles of the present invention, anti-CD3 antibodies and fibronectin fragments may be bound to lipid particles in any manner as long as they are present on the surface of the lipid particles. Although the binding mode is not particularly limited, covalent binding is preferred. Therefore, methods for adding anti-CD3 antibodies and fibronectin fragments to the surface of lipid particles may be carried out by known techniques.

As a method for directly binding lipid particles and ligands without using a cross-linking agent, for example, lipid particles having a maleimide group are reacted with thiol groups of the ligands to bind anti-CD3 antibodies and fibronectin fragments. Lipid particles can be prepared. More specifically, the two can be crosslinked by reducing the ligand with 2-mercaptoethanol and then bringing it into contact with lipid particles having a maleimide group. In this method, a thioether bond is formed by the reaction of a maleimide group and a thiol group.

Alternatively, a known condensing agent, an activating agent that activates the functional groups possessed by the lipid particles, or the like can be used to bind the lipid particles and the ligand. As condensing agents and activating agents, carbodiimides [eg, N,N'-dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), etc.], succinimides [eg, N-hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide (NHSS), etc.], those used for exchange reactions of thiol groups [e.g., 5,5′-dithiobis(2-nitrobenzoic acid), 2,2′-dithiobispyridine, etc.] etc. are exemplified. More specifically, after lipid particles having carboxyl groups are activated with EDC and NHS, both can be crosslinked by contacting with a ligand.

Any of the above cross-linking reactions should be carried out in sterile distilled water, buffered saline or saline, preferably in phosphate buffered saline (PBS) or Dulbecco's PBS (D-PBS). can be done.

The amount of the anti-CD3 antibody and fibronectin fragment used in the cross-linking reaction described above is not particularly limited and can be determined as appropriate by those skilled in the art. For example, when anti-CD3 antibody is added, the concentration of anti-CD3 antibody in the solution is less than 5 mg/mL, preferably 0.001-3 mg/mL, more preferably 0.01-2 mg/mL. Also, when retronectin is used as the fibronectin fragment, the concentration of retronectin in solution is less than 5 mg/mL, preferably 0.001-3 mg/mL, more preferably 0.01-2 mg/mL.

The mixing ratio of the lipid particles having a functional group and each ligand is not particularly limited, and can be appropriately determined by those skilled in the art. ~1:10.

The anti-CD3 antibody and the fibronectin fragment may be separately added to the surface of the lipid particles, or a mixture thereof may be added to the surface of the lipid particles as long as the lymphocyte activation activity of each is retained. good too. Alternatively, both ligands may be attached to the lipid particle surface in different ways. Furthermore, for example, it can be provided as a lipid particle to which a fusion protein of an anti-CD3 antibody and a fibronectin fragment is added.

The above reaction temperature and reaction time are not particularly limited and can be appropriately determined by those skilled in the art. For example, the anti-CD3 antibody and fibronectin fragment can be bound to the surface of the liposomes by incubating at room temperature for 3 hours while stirring on a mechanical stirrer.

Unreacted ligands, divalent reactive compounds, reaction by-products, and the like are removed from the reaction product of the binding reaction between the lipid particles and the ligand by gel filtration, dialysis, ultrafiltration, centrifugation, or the like, and the ligand is bound. It is also preferred to purify the lipid particles.

The lipid particles of the present invention are produced by the method described above. That is, the lipid particles of the present invention are obtained by adding a substance having a binding activity to CD3 or a substance capable of interacting with CD3 and a fibronectin fragment to the surface of the lipid particles.

Lipid particles with anti-CD3 antibodies and fibronectin fragments attached to their surface can be stored at a low temperature, eg, 4° C., until use.

(2) Method for producing lymphocytes of the present invention A method for producing lymphocytes, comprising the step of culturing in the presence of particles.

Lymphocytes are classified into various types according to differences in expression and/or function of marker molecules. The three major types of lymphocytes are T cells, B cells and natural killer cells (NK cells).

Most mature T cells in the periphery express either CD4 or CD8 as cell surface marker molecules. CD4-expressing T cells function as helper T cells that induce functional expression of other T cells, differentiation and maturation of B cells, and antibody production. On the other hand, CD8-positive T cells function as cytotoxic T cells that destroy virus-infected cells and the like. In addition, there are also NKT cells, which have the properties of both NK cells and T cells, and regulatory T cells (also referred to as Treg), which express CD25 molecules and act to suppress the activity of other T cells. Recently, it has also become known that there are peripheral T cells that differentiate and mature without going through the thymus.

On the other hand, each B cell has a specific type of antibody that it produces. It activates and starts producing antibodies only when pathogens that match its antibody type appear.

NK cells are a type of cytotoxic lymphocytes that act as a major factor in innate immunity, and are particularly important in rejecting tumor cells and virus-infected cells.

The "cells capable of differentiating into lymphocytes" used in the present invention are not particularly limited, but pluripotent stem cells such as iPS cells and ES cells, and further differentiated hematopoietic stem cells and lymphocyte progenitor cells. , naive cells, and specific cell populations (for example, CD3-positive cell populations, etc.). Peripheral blood mononuclear cells, bone marrow cells, and umbilical cord blood cells, which are cell populations containing cells capable of differentiating into lymphocytes, can also be used in the present invention.

The lymphocytes or cells capable of differentiating into lymphocytes used in the present invention may be any of the lymphocytes described above.

In addition, the method of the present invention can be used not only for purified lymphocytes or cells capable of differentiating into lymphocytes, but for example, a mixture of multiple types of lymphocytes or cells capable of differentiating into lymphocytes, and lymphocytes. Alternatively, it can be applied to cell populations containing cells capable of differentiating into lymphocytes and other cells. For example, as described in Examples below, the method of the present invention can be applied to a population of peripheral blood-derived mononuclear cells to produce lymphocytes.

The origin of the lymphocytes or cells capable of differentiating into lymphocytes used in the present invention is not particularly limited, and lymphocytes or cells capable of differentiating into lymphocytes derived from any organism, preferably mammals, can be used. can. The age and sex of the organism are not particularly limited. In one embodiment, cells from Primates (eg, chimpanzees, Japanese macaques, humans) are used. Most preferably, cells of human origin are used, but the invention is not so limited.

When lymphocytes are produced by the method of the present invention for the purpose of administration to humans, cells collected from a donor whose histocompatibility antigen type matches or is similar to that of the recipient are preferably used for the production of lymphocytes. be. For example, human peripheral blood mononuclear cells (PBMC) taken from the recipient himself are subjected to the production of lymphocytes.

The method for producing lymphocytes of the present invention is characterized by including the step of culturing in the presence of the lipid particles of the present invention described in (1) above (hereinafter sometimes referred to as the “culturing step of the present invention”). and

In a preferred embodiment of the present invention, lymphocytes or cells capable of differentiating into lymphocytes are cultured in a state in which the lipid particles of the present invention described in (1) are in contact with lymphocytes or cells capable of differentiating into lymphocytes. be implemented.

The cell culture equipment used in the present invention is not particularly limited, and for example, petri dishes, flasks, bags, large culture tanks, bioreactors and the like can be used. As the bag, for example, a CO ₂ gas permeable bag for cell culture can be used. Moreover, when producing a large amount of lymphocytes on an industrial scale, a large culture tank can be used. Cultivation can be carried out in either an open system or a closed system, but it is preferable to carry out the culture in a closed system from the viewpoint of the safety of the obtained lymphocytes.

The method for producing lymphocytes of the present invention comprises culturing lymphocytes or cells capable of differentiating into lymphocytes in the presence of the above-described lipid particles during the entire period or any part of the period of culture in lymphocyte production. performed by That is, the present invention encompasses any process that includes the culture step of the present invention as part of the lymphocyte manufacturing process. For example, even if the lymphocyte production process is started using a cell population that does not contain lymphocytes, lymphocytes are induced after the start, and the step of culturing the lymphocytes in the presence of the lipid particles If it does, it is included in the present invention.

The culturing step of the present invention includes induction of lymphocytes, maintenance of lymphocytes, and/or expansion of lymphocytes. In the method for producing lymphocytes of the present invention, the types of lymphocytes or cells capable of differentiating into lymphocytes to be subjected to the method, culture conditions, etc. are appropriately adjusted to culture lymphocytes or cells capable of differentiating into lymphocytes. By performing, it is possible to produce lymphocytes useful for regenerative medicine and the like. As used herein, lymphocytes or cells capable of differentiating into lymphocytes may mean lymphocytes or cell populations containing cells capable of differentiating into lymphocytes.

When the purpose is to induce lymphocytes, there is no particular limitation on the type of cells at the start of culture or the method of inducing lymphocytes in the culture step of the present invention. Cells at the start of culture may be pluripotent stem cells such as iPS cells and ES cells, further differentiated hematopoietic stem cells, lymphocyte progenitor cells, naive cells, specific cell populations (e.g., CD3-positive cell population, etc.). In addition, the lymphocyte induction method is not particularly limited as long as it is a known method, except for using the lipid particles of the present invention.

For the purpose of maintaining and expanding lymphocyte culture, the cell concentration at the start of culture in the culture step of the present invention is not particularly limited, but is preferably 0.005 to 20×10 ⁵ cells/mL, for example. is 0.02 to 5×10 ⁵ cells/mL, more preferably 0.05 to 2×10 ⁵ cells/mL.

Various media used for culturing lymphocytes can be used in the culturing step of the present invention. Preferable examples include media that do not contain heterologous components such as fetal bovine serum (FBS or fetal calf serum: FCS) and sheep serum, serum-free media, defined media that do not contain unknown components, and the like. be done. Such a xeno-free medium can be prepared as appropriate, and known or commercially available media may be used as they are or after modification. Examples of commercially available heterologous component-free media include LymphoONE (trademark) T-Cell Expansion Xeno-Free Medium (manufactured by Takara Bio Inc.) and GT-T551 Culture medium (manufactured by Takara Bio Inc.).

In a preferred aspect of the invention, the medium is further supplemented with cytokines. Examples of the cytokines include IL-2, IL-7, IL-12, IL-15, IFN-γ and the like. The cytokine concentration added to the medium used in this embodiment is, for example, 10 to 1000 IU/mL, preferably 50 to 800 IU/mL, more preferably 100 to 600 IU/mL in final concentration. Furthermore, other ingredients may be added to the medium as long as the effects of the present invention are not impaired.

Conditions for culturing lymphocytes are not particularly limited, and ordinary cell culture conditions can be employed. Examples of the culture conditions include a temperature of 37° C., a humidity of 95%, and a CO ₂ concentration of 5%, but the present invention is not limited to such conditions. For example, culturing at a temperature of 30 to 40° C., a humidity of 90 to 98%, and a _CO concentration of 3 to 7% is exemplified. , humidity and _CO2 concentration. During culturing, it is preferable to dilute the cell culture solution by adding fresh medium at appropriate time intervals, replace the medium with fresh medium, or replace the culture vessel as necessary. The medium to be used and other components to be used at the same time can be set as appropriate.

In a preferred embodiment of the present invention, when the purpose is to maintain and expand lymphocytes, lymphocytes use a medium containing the lipid particles of the present invention in an appropriate container, while performing medium exchange and passage. for example, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, or 10 days or more. This culture allows lymphocytes to proliferate.

Lymphocytes obtained by the production method of the present invention can be classified based on the expression of marker molecules. The expression of the marker molecule can be confirmed using, for example, an antibody that recognizes the marker molecule.

Marker molecules for lymphocytes include, but are not limited to, CD3, CD4, and CD8. CD3 is a glycoprotein expressed on mature T lymphocytes and is one of cell surface antigens. CD4 is expressed on helper T cells, monocytes, macrophages, dendritic cells and the like. In general, CD3-positive and CD4-positive cells (CD3+CD4+) are helper T cells. On the other hand, CD8 is expressed on cytotoxic T cells and some NK cells. In general, CD3-positive and CD8-positive cells (CD3+CD8+) are cytotoxic T cells.

Lymphocytes obtained by the production method of the present invention are not particularly limited. cells, γδT cells, NKT cells, cell populations containing at least one of these cells, and the like.

Furthermore, the lymphocytes of interest can be isolated from the cell population obtained by the production method of the present invention to obtain lymphocytes separated from other cells. Antibodies that recognize molecules characteristic of target lymphocytes are useful for isolating and purifying lymphocytes obtained by the present invention. The lymphocytes thus isolated can be established as cell lines by known methods. That is, one aspect of the present invention is a method for producing lymphocytes, which includes the steps of the method for producing a cell population containing lymphocytes of the present invention and the step of isolating lymphocytes from the obtained cell population. be done.

According to the present invention, there is provided a method for producing lymphocytes into which a desired gene has been introduced, using the aforementioned “method for producing lymphocytes”. The method includes a step of gene transfer in addition to the "culture step of the present invention" in the method for producing lymphocytes of the present invention. The gene introduction step may be performed before, during, or after the culture step of the present invention. A desired gene-introduced lymphocyte produced in this manner can be used, for example, as a gene therapy cell.

Desired genes to be introduced into lymphocytes in the present invention are not particularly limited, and any gene can be selected. As such genes, for example, in addition to those encoding proteins (eg, enzymes, cytokines, receptors, etc.), those encoding antisense nucleic acids, siRNA (small interfering RNA), or ribozymes can be used. In addition, a marker gene for selecting transfected cells or a suicide gene for selectively removing transfected cells may be introduced together with the above genes. That is, there is no limit to the number of desired genes to be introduced, and multiple genes can be introduced.

Although not particularly limited, one aspect of the present invention is the introduction of a gene encoding a receptor that recognizes a desired antigen. The gene includes a gene encoding a T cell receptor (TCR) that recognizes an antigen presented on the surface of a target cell, or an antigen recognition site of an antibody against a surface antigen of a target cell, a transmembrane domain derived from a receptor molecule, and Genes encoding chimeric receptors containing intracellular domains are exemplified. Lymphocytes into which these genes have been introduced have acquired properties such as exhibiting toxicity against target cells and/or proliferating upon contact with said cells, and are useful for medical use. .

The desired gene can be introduced, for example, by inserting it into any vector so that it is expressed under the control of an appropriate promoter. Other regulatory elements, such as enhancer or terminator sequences, which cooperate with the promoter or transcription initiation site, may also be present in the vector to achieve efficient transcription of the gene. The vector may be selected from plasmid vectors (including episomal vectors), viral vectors, transposon vectors, artificial chromosomes, etc., depending on the purpose and the gene to be introduced. There is no particular limitation on the means for gene introduction, and a method suitable for the vector to be used can be selected from known gene introduction methods.

Viral vectors that can efficiently introduce genes into cells can be used in the present invention. The viral vector is not particularly limited, and known viral vectors commonly used in gene transfer methods, such as retroviral vectors (including lentiviral vectors and pseudotype vectors), adenoviral vectors, adeno-associated viral vectors, Sendai Viral vectors and the like can be used. Particularly preferably retroviral, adenoviral or lentiviral vectors are used. As the viral vector, replication-deficient vectors are preferred so that they cannot self-replicate in infected cells. Many viral vectors have been reported in the literature and are commercially available, and can be used in the present invention.

Retroviral vectors and lentiviral vectors are capable of stably integrating a foreign gene inserted into the vector into the chromosomal DNA of cells into which the vector is introduced, and are used for purposes such as gene therapy. Since this vector can introduce genes into target cells with high efficiency in the coexistence of retronectin fragments, desired genes can be introduced into lymphocytes even in the coexistence of the lipid particles of the present invention. Therefore, both viral vectors are particularly suitable for combination with the method for producing lymphocytes of the present invention.

The lymphocytes obtained by the present invention can also be used, for example, for research on lymphocytes, drug screening for various diseases, efficacy and safety evaluation of drug candidate compounds, and the like. According to the present invention, it is possible to obtain a large number of lymphocytes in a single operation, so that it is possible to obtain reproducible research results without being affected by cell lot differences as in the past. Become.

The present invention provides lymphocytes for use in medicine, lymphocytes for use in the manufacture of medicines. Said lymphocytes are the cell population produced by the present invention. A medicament containing said lymphocytes is suitable for use in immunotherapy. For example, the lymphocytes produced by the present invention are used as an active ingredient, optionally mixed with other ingredients (organic or inorganic carriers suitable for parenteral administration, activators, stabilizers, etc.), and infusion or injection It can be used as an agent. The content of the cell population of the present invention in these therapeutic agents, the dosage of the therapeutic agents, and various conditions regarding the use of the therapeutic agents can be appropriately determined according to known immunotherapy. Furthermore, immunotherapy with the therapeutic agent can be used in combination with drug therapy treatment with administration of known drugs, radiotherapy, or treatment with surgery.

Diseases for which administration of the cell population is effective are not particularly limited. MRSA infection, VRE infection, or deep mycosis) and the like are exemplified. In particular, it is useful for treating AIDS (acquired immunodeficiency syndrome), an HIV infection caused by HIV that infects CD4-positive T cells. In addition, the lymphocytes produced by the method of the present invention can be used for bone marrow transplantation, donor lymphocyte transfusion for the purpose of prevention of infectious disease in an immunodeficient state such as after irradiation or remission of recurrent leukemia, anticancer drug treatment, It can be used in combination with conventional treatments such as radiotherapy, antibody therapy, hyperthermia, and other immunotherapies. Also, by introducing a desired exogenous gene, it is possible to produce lymphocytes that are useful for the treatment or prevention of various diseases that are efficacious due to the expression of the exogenous gene.

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited by these examples.

Example 1 Preparation of liposomes and cells (liposome-COOH)
1 mL of Dulbecco's PBS (manufactured by PromoCell, hereinafter referred to as D-PBS) was added to 40 mg of a liposome (Carboxyl Pegylated normal liposome, manufactured by Nanovex, hereinafter referred to as liposome-COOH) having a COOH group added to the surface, and Incubate for 20 minutes in a water bath at 0°C with stirring. After confirming the dissolution of liposome-COOH, the mixture was vigorously stirred by hand for 2 minutes to prepare a liposome solution.

Pierce EDC, No-Weight Format (manufactured by ThermoFisher Scientific) and NHS (manufactured by ThermoFisher Scientific) were dissolved in D-PBS to prepare 5 mg/mL EDC and 7.5 mg/mL NHS, respectively.

For each condition shown in Table 1, 450 μL of D-PBS was added to 50 μL of the liposome-COOH solution, and 5 μL each of 5 mg/mL EDC and 7.5 mg/mL NHS were added to activate the liposome-COOH.

Anti-human CD3 antibody (manufactured by Takara Bio Inc., hereinafter referred to as OKT3), RetroNectin (manufactured by Takara Bio Inc., hereinafter referred to as RN) and anti-CD28 antibody (Miltenyi Inc.) were added to 500 μL of activated liposome-COOH in the amounts shown in Table 1. (manufactured by TOMY) was added and crosslinked at room temperature for 3 hours while stirring with a tube shaker (manufactured by TOMY).

After the cross-linking reaction, the solution was centrifuged at 12,000 xg at room temperature for 15 minutes, and then the supernatant was removed. 1 mL of D-PBS was added to the precipitate, and the mixture was centrifuged at 12,000 xg and room temperature for 15 minutes, and then the supernatant was removed. This operation was repeated four times to wash the modified liposome-COOH crosslinked with each protein.

The washed modified liposome-COOH was suspended in 200 μL of D-PBS, and the amount of cholesterol contained was quantified using Lab Assay Cholesterol (manufactured by Fuji Film Wako).

In the following experiments, modified liposome-COOH for 18 μg of cholesterol per well of 48-well plate (manufactured by Corning) was used as a stimulation carrier based on the quantified amount of cholesterol.

OKT3/RN (hereinafter referred to as OKT3/RN plate) and T cell TransAct (manufactured by Miltenyi) immobilized on cultureware were used as liposome stimulation carriers for comparison. OKT3/RN is immobilized on the cultureware by adding 100 μL/well of D-PBS containing OKT3 at a final concentration of 5 μg/mL and RN at 25 μg/mL to a 48 well plate, and placing it in a 37° C., 5% CO ₂ incubator. It was carried out by leaving it at rest for 2 hours or more. The solution was removed from the OKT3/RN-immobilized plate immediately before use, and the plate was washed twice with 500 μL/well of D-PBS. T cell TransAct was used by adding 1/1000 volume of the cell suspension.

Healthy donor-derived peripheral blood mononuclear cells (PBMC) from PromoCell were suspended in a medium at 0.2×10 ⁶ cells/mL. The medium was LymphoONE T-Cell Expansion Xeno-Free Medium (manufactured by Takara Bio) with a final concentration of 600 IU/mL IL-2 (manufactured by Nipro) and a final concentration of 0.6% UltraGRO-PURE GI (manufactured by AventaCell). (hereinafter referred to as culture medium). The cell suspension was dispensed at 0.6625 mL/well (48 well plate), and modified liposome-COOH or T cell TransAct was added as described above. The same amount of cell suspension was added to the OKT3/RN-immobilized plate, and culture was started under conditions of 5% CO ₂ and 37° C. (culture day 0).

On the first day of culture, a gene encoding a chimeric antigen receptor targeting Mesothelin (hereinafter referred to as Mesothelin-CAR) was introduced into PBMC using a lentiviral vector.

Cell passaging was performed on days 4 and 7 of culture. On the fourth day of culture, the cell suspension was collected in a centrifugation tube, centrifuged, the supernatant was removed, and the cells were suspended in a culture medium. 200 μL/well of the cell suspension and 400 μL/well of the culture medium were mixed in a new 48-well plate, and the culture was continued. On day 7 of culture, 300 μL/well of the cell suspension and 300 μL/well of culture medium were mixed in a new 48-well plate, subcultured, and cultured until day 10 of culture.

Example 2 Cell Proliferation Rate of CAR-T Cells on Day 10 of Culture On day 10 of culture obtained in Example 1, the number of cells under each culture condition was counted by trypan blue staining. FIG. 1 shows the cell proliferation rate when the number of cells on day 0 of culture is set to 1.

As shown in FIG. 1, modified liposome-COOH crosslinked with OKT3 and RN or anti-CD28 antibody at the same time exhibited a higher cell proliferation rate than that crosslinked with OKT3 alone. Moreover, compared with TransAct, which stimulates T cells in a liquid phase like OKT3/RN plate and liposome, it showed a cell proliferation rate equal to or higher than that of TransAct.

Example 3 CD3+/CD4+/CD8+ Ratio of CAR-T Cells on Day 10 of Culture It was washed with 0.5% BSA/PBS hereinafter). Suspend the cell population in 0.5% BSA/PBS, PerCP-labeled mouse anti-human CD3 antibody (manufactured by BD Biosciences), PECy7-labeled mouse anti-human CD4 antibody (manufactured by BioLegend) and APC Cy7-labeled mouse anti-human CD8 antibody (BD Biosciences) was added and incubated. After that, the cell population was washed twice with 0.5% BSA/PBS and resuspended in 0.5% BSA/PBS. The CD3+/CD4+/CD8+ ratio of this cell population was measured using a flow cytometer (FACS CantoII, manufactured by Becton, Dickinson and Company) (Fig. 2).

As shown in FIG. 2, it was observed that the ratio of CD3+/CD8+ cells was higher in liposome-COOH crosslinked with RN than in liposome-COOH crosslinked only with OKT3. In contrast, simultaneous cross-linking of OKT3 and anti-CD28 antibody tended to increase the ratio of CD3+/CD4+ cells.

Example 4 Gene Transfer Efficiency of CAR-T Cells on Day 10 of Culture The cell population obtained in Example 1 on day 10 of culture was washed with 0.5% BSA/PBS. The cell population was suspended in 0.5% BSA/PBS, and incubated with a biotin-labeled goat anti-mouse IgG antibody (manufactured by Jackson ImmunoResearch) that recognizes CAR. After washing the cell population three times with 0.5% BSA/PBS, a mixture of PerCP-labeled mouse anti-human CD3 antibody and APC-labeled StreptAvidin (manufactured by BioLegend) was added and incubated. After that, the cell population was washed twice with 0.5% BSA/PBS and resuspended in 0.5% BSA/PBS. The ratio of CD3+/Mesothelin CAR+ cells in this cell population was measured using a flow cytometer to compare gene transfer efficiencies (Fig. 3).

As shown in FIG. 3, the OKT3/RN plate exhibited the highest gene transfer efficiency. On the other hand, no significant difference in gene transfer efficiency was observed between TransAct, which stimulates T cells in the liquid phase, and modified liposome-COOH.

Example 5 Immunophenotype of CAR-T Cells on Day 10 of Culture The cell population obtained in Example 1 on day 10 of culture was washed with 0.5% BSA/PBS. The cell population was suspended in 0.5% BSA/PBS, and a biotin-labeled goat anti-mouse IgG antibody was added for antibody reaction. After washing the cell population three times with 0.5% BSA/PBS, PerCP-labeled mouse anti-human CD3 antibody, APCCy7-labeled mouse anti-human CD8 antibody, APC-labeled StreptAvidin (manufactured by BioLegend), FITC-labeled mouse anti-human CCR7 antibody (BioLegend A mixed solution of PE-labeled mouse anti-human CD45RA antibody (manufactured by BioLegend) was added and incubated. After that, the cell population was washed twice with 0.5% BSA/PBS and resuspended in 0.5% BSA/PBS. Immunophenotype of CD3+/CD8+/CAR+ cells in this cell population (naive: CCR7+/CD45RA+, central memory (hereinafter referred to as CM): CCR7+/CD45RA-, effector memory (hereinafter referred to as EM): CCR7-/ CD45RA-, terminal differentiated (hereinafter referred to as TD): CCR7-/CD45RA+) was analyzed using a flow cytometer (Fig. 4).

As shown in FIG. 4, modified liposome-COOH obtained by simultaneously cross-linking OKT3 and RN or anti-CD28 antibody exhibited a higher naive ratio than that obtained by cross-linking OKT3 alone. In addition, it was confirmed that the naive ratio was comparable to that of TransAct, which stimulates T cells in a liquid phase like liposome.

Example 6 Preparation of liposomes and cells (liposome-maleimide)
2-Mercaptoethanol (manufactured by Nacalai Tesque, hereinafter referred to as 2-ME) was added to a final concentration of 50 mM to 1 mg/mL OKT3 or 0.5 mg/mL anti-CD28 antibody, and reduction treatment was performed at 37° C. for 90 minutes. After reduction treatment, ultrafiltration was performed using Amicon ULTRA-4 (manufactured by Merck Millipore) with a molecular weight cut off of 10 kDa, and HEPES buffer (final concentration 10 mM HEPES (pH 7.3, manufactured by Dojindo), final concentration 5 mM EDTA (pH 8.0, manufactured by Nippon Gene)) was substituted for the solvent.

1 mL of D-PBS was added to 1 vial of a liposome (Lipocapsulator FD-S MA, manufactured by Katayama Chemical Co., Ltd., hereinafter referred to as liposome-maleimide) having a maleimide group added to the surface, and allowed to stand at room temperature for 15 minutes.

To 100 μL of the liposome-maleimide solution thus prepared, 20 μg equivalent of OKT3 and anti-CD28 antibody reduced with 2-ME were added, and the mixture was crosslinked at room temperature for 2 hours while stirring with a tube shaker and then at 4° C. for 6 hours. let me

The solution after the cross-linking reaction was subjected to ultrafiltration using Amicon ULTRA-4 (manufactured by Merck Millipore) with a cut-off molecular weight of 100 kDa, and the solvent was replaced with D-PBS. A modified liposome-maleimide was thus prepared.

Healthy donor-derived PBMCs from StemExpress were suspended in a culture medium at 0.2×10 ⁶ cells/mL. This cell suspension was dispensed at 0.6625 mL/well (48 well plate), 1 μL/well of modified liposome-maleimide crosslinked with OKT3 and anti-CD28 antibody was added, and cultured under conditions of 5% CO ₂ and 37°C. was started (day 0 of culture). At the same time, modified liposome-COOH was prepared under the same conditions as condition 3 shown in Table 1 of Example 1 (OKT3 and anti-CD28 antibody crosslinked at a ratio of 1:1) and used as a control.

Cell passaging was performed on days 4 and 7 of culture. On the fourth day of culture, the cell suspension was collected in a centrifugation tube, centrifuged, the supernatant was removed, and the cells were suspended in a culture medium. 200 μL/well of the cell suspension and 400 μL/well of the culture medium were mixed in a new 48-well plate, and the culture was continued. On day 7 of culture, 300 μL/well of the cell suspension and 300 μL/well of culture medium were mixed in a new 48-well plate, subcultured, and cultured until day 10 of culture.

Example 7 Immunophenotype of T Cells on Day 10 of Culture The cell population obtained in Example 6 on day 10 of culture was washed with 0.5% BSA/PBS. A cell population was suspended in 0.5% BSA/PBS, and a mixture of PerCP-labeled mouse anti-human CD3 antibody, APCCy7-labeled mouse anti-human CD8 antibody, FITC-labeled mouse anti-human CCR7 antibody, and PE-labeled mouse anti-human CD45RA antibody was added. and incubated. After that, the cell population was washed twice with 0.5% BSA/PBS and resuspended in 0.5% BSA/PBS. The immunophenotype of CD3+/CD8+ cells in this cell population (naive: CCR7+/CD45RA+, CM: CCR7+/CD45RA-, EM: CCR7-/CD45RA-, TD: CCR7-/CD45RA+) was analyzed using a flow cytometer. (Fig. 5).

As shown in FIG. 5, it was confirmed that both T cells stimulated with modified liposome-maleimide and modified liposome-COOH exhibited similar immunophenotypes.

INDUSTRIAL APPLICABILITY The present invention provides a method for producing lymphocytes that can be applied to an automatic culture device. The present invention is particularly useful for medical applications.

SEQ ID NO:1 ; Partial region of fibronectin named III-1
SEQ ID NO:2 ; Partial region of fibronectin named III-2
SEQ ID NO:3 ; Partial region of fibronectin named III-3
SEQ ID NO:4 ; Partial region of fibronectin named III-4
SEQ ID NO:5 ; Partial region of fibronectin named III-5
SEQ ID NO:6 ; Partial region of fibronectin named III-6
SEQ ID NO:7 ; Partial region of fibronectin named III-7
SEQ ID NO:8 ; Partial region of fibronectin named III-8
SEQ ID NO:9 ; Partial region of fibronectin named III-9
SEQ ID NO:10 ; Partial region of fibronectin named III-10
SEQ ID NO:11 ; Partial region of fibronectin named III-11
SEQ ID NO:12 ; Partial region of fibronectin named III-12
SEQ ID NO:13 ; Partial region of fibronectin named III-13
SEQ ID NO:14 ; Partial region of fibronectin named III-14
SEQ ID NO:15 ; Partial region of fibronectin named CS-1
SEQ ID NO:16 ; Fibronectin fragment named 120k-fr
SEQ ID NO:17 ; Fibronectin fragment named CH-271
SEQ ID NO:18 ; Fibronectin fragment named CH-296 (RetroNectin)
SEQ ID NO:19 ; Fibronectin fragment named FCH-296

Claims (11)

A lipid particle having a substance having a binding activity or a substance capable of interacting with CD3 and a fibronectin fragment attached to the surface thereof. 2. The lipid particle according to claim 1, wherein the substance having binding activity or interacting with CD3 is an anti-CD3 antibody. Lipid particles according to claim 1, characterized in that the fibronectin fragment is a fragment having at least one domain selected from the group consisting of a cell-binding domain and a heparin-binding domain. Lipid particles according to claim 3, characterized in that the fibronectin fragment is a fragment with a cell-binding domain and a heparin-binding domain. The fibronectin fragment is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 18 in the sequence listing, or has an amino acid sequence having one or more amino acid substitutions, deletions, insertions or additions in the amino acid sequence of said polypeptide. 2. The lipid particle according to claim 1, which is a polypeptide and has a function equivalent to that of said polypeptide. 2. A lipid particle according to claim 1, wherein the lipid particle is a liposome. A method for producing lymphocytes, comprising the step of culturing lymphocytes or cells capable of differentiating into lymphocytes in the presence of the lipid particles according to any one of claims 1 to 6. 8. The method for producing lymphocytes according to claim 7, wherein the lymphocytes or cells capable of differentiating into lymphocytes are mammal-derived lymphocytes or lymphocyte progenitor cells. 8. The method for producing lymphocytes according to claim 7, further comprising the step of introducing a desired gene into lymphocytes or cells capable of differentiating into lymphocytes. 10. The method for producing lymphocytes according to claim 9, wherein the desired gene is introduced using a viral vector. 11. The method for producing lymphocytes according to claim 10, wherein the desired gene is introduced using a retroviral vector and a lentiviral vector.

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