JPWO2008143255A1 - Method for producing cell population - Google Patents

Abstract

Step (1): removing a CD8-positive T cell from a T-cell-containing cell population or collecting a CD4-positive T cell to prepare a cell population to be cultured, and step (2): the step (1) ) In the presence of the following (A) and (B) [(A) at least one selected from the group consisting of fibronectin, a fibronectin fragment and a mixture of fibronectin fragments ( B) CD3 ligand]. A method for producing a cell population. The cell population obtained by the production method is extremely useful for the treatment of diseases caused by cell therapy.

Description

  The present invention relates to a method for producing a cell population useful in the medical field.

  The living body is protected from foreign substances mainly by the immune response, and the immune system consists of various cells and soluble factors that it produces. Among them, leukocytes, particularly lymphocytes, play a central role. These lymphocytes are divided into two main types, B lymphocytes (hereinafter referred to as B cells) and T lymphocytes (hereinafter referred to as T cells), each of which specifically recognizes an antigen, It acts on this and defends the living body.

  In the T cells, CD4 positive T cells having a CD (Cluster of Differentiation) 4 marker and CD8 positive T cells having a CD8 marker occupy most in the periphery. Most of CD4 positive T cells are called helper T cells (hereinafter referred to as Th cells), are involved in assisting antibody production and induction of various immune responses, and the types of cytokines produced by antigen stimulation are different. Differentiate into Th1 type (type 1 helper T cells) or Th2 type (type 2 helper T cells). Most of the CD8 positive T cells differentiate into cytotoxic T cells (hereinafter referred to as CTL) that show cytotoxic activity upon antigen stimulation [(cytotoxic T lymphocyte).

  In recent years, immunotherapy has attracted attention as a fourth cancer treatment method after surgery, chemotherapy, and radiation therapy. Since immunotherapy uses the immunity inherent in humans, it is said that the physical burden on the patient is lighter than other treatments. Immunotherapy includes the transfer of lymphokine-activated cells, NKT cells, γδT cells, etc. obtained by culturing by various methods from CTLs or peripheral blood mononuclear cells induced outside the body, antigen-specific CTLs in the body There are known dendritic cell transfer therapy, peptide vaccine therapy, Th1 cell therapy, and gene gene therapy in which various effects can be expected to be introduced outside the body and transferred into the body.

  Conventionally, cell therapy using CTLs exhibiting cytotoxic activity has been promoted in immunotherapy, but recently, Th cell therapy using Th cells that assist the CTL activity has attracted attention (for example, non-cell therapy). Patent Document 1, Patent Document 1). As described above, Th cells are divided into Th1 type and Th2 type. Th1 type produces cytokines such as interferon γ (IFN-γ) and interleukin (IL) -2, and acts as an effector of cellular immunity. On the other hand, the Th2 type produces cytokines such as IL-4, IL-5, and IL-13, and is responsible for regulating humoral immunity. In cell medicine, Th1 type, which is an effector of cellular immunity, is considered important.

  In recent years, in immunotherapy, naive T cells and central memory T cells in a more undifferentiated state are attracting attention rather than effector T cells that have already been terminally differentiated. Naive T cells are cells that have never been activated by an antigen, and it is clear that Th1-type and Th2-type subsets can be induced by culturing CD4-positive naive T cells in the presence of antigen and cytokine. It has become. Naive T cells are known to express cell surface antigen markers for lymphocytes such as CD45RA, CD62L, CCR7.

As described above, CD1-positive T cells such as Th1-type CD4-positive T cells and naive T cells are important in the fields of cell medicine and gene therapy. Development of a method including a process capable of obtaining many suitable cells is desired.
Chamoto K. Nine others, Cancer Research, 2006, Vol. 66 (3), P1809-1817 JP 2005-179299 A

  An object of the present invention is to provide a method for producing a cell population effective for cell therapy.

1st invention of this invention is related with the manufacturing method of the cell population characterized by including the following process.
Step (1): removing a CD8-positive T cell from a cell population containing T cells, or collecting a CD4-positive T cell to prepare a cell population to be cultured, and step (2): the step (1) (B) culturing the cell population obtained in the following (A) and (B): (A) at least one selected from the group consisting of fibronectin, a fibronectin fragment and a mixture of fibronectin fragments CD3 ligand

  The first invention of the present invention may include a step of collecting or removing a cell subpopulation before performing the step (1) or the step (2). The cell population obtained in the step (2) (A ) And / or (B) may be further included in the culture. Moreover, you may include the process of introduce | transducing a gene into the cell population obtained at the process (1) or the process (2).

The second invention of the present invention relates to a method for producing a cell population selected from the following, wherein the production method is the production method of the first invention of the present invention.
Cell population (a): Cell population containing Th1-type CD4 positive T cells Cell population (b): Cell population containing CD4 positive CD45RA positive CCR7 positive T cells (c): CD4 positive CD45RA positive CD62L positive T cells Cell populations containing

  The third invention of the present invention relates to a cell population obtained by the production method of the first invention of the present invention.

  The fourth invention of the present invention relates to a medicament containing a cell population obtained by the production method of the first invention of the present invention.

In another aspect of the present invention, a cell population containing CD8-positive CD45RA-positive CCR7-positive T cells is added to the cell population obtained by the first invention of the present invention and cultured to add CD8-positive CD45RA-positive CCR7. Positive T cells can be obtained with high efficiency. That is, this invention also provides the manufacturing method of the cell population characterized by including the following processes.
Step (2-1): Step of removing CD8 positive T cells from a cell population containing T cells or collecting CD4 positive T cells (2-2): obtained in the step (2-1) (A) At least one selected from the group consisting of a mixture of fibronectin, a fibronectin fragment and a fibronectin fragment (B) CD3 ligand step ( 2-3): a step of mixing and culturing the cell population obtained in the step (2-2) and a cell population containing CD8-positive CD45RA-positive CCR7-positive T cells. , The conditions of step (2-2), ie, (A) a mixture of fibronectin, fibronectin fragment and fibronectin fragment At least one member more selectively, and (B) is preferably carried out in the presence of a CD3 ligand.

In addition, in the culture of a cell population containing T cells, the present inventors set the number of CD4 positive T cells to 1 or more when the number of CD8 positive T cells is 2 as the ratio of CD8 positive T cells to CD4 positive T cells. Preferably, CD8-positive CD45RA-positive CCR7-positive T cells can be obtained with high efficiency by culturing using a cell population in which the number of CD4-positive T cells is 1 or more when the number of CD8-positive T cells is 1. I found out that I can. That is, another aspect of the present invention provides a method for producing a cell population characterized by including the following steps.
Step (3-1): Step of obtaining a cell population in which the ratio of CD8 positive T cells to CD4 positive T cells is 2: 1 or more Step (3-2): Cells obtained in the step (3-1) The step of culturing the population in the presence of CD3 ligand The culture in the step (3-2) preferably comprises a mixture of fibronectin, a fibronectin fragment and a fibronectin fragment in addition to the presence of CD3 ligand. It is desirable to carry out in the presence of at least one selected from the group.

  In addition, the present inventors also obtained a culture from a cell population obtained by culturing a cell population containing T cells in the presence of at least one selected from the group consisting of fibronectin, a fibronectin fragment and a mixture of fibronectin fragments. It was found that the production of IL-5 and IL-9 was suppressed. That is, as another aspect of the present invention, an IL-5 production inhibitor characterized by containing, as an active ingredient, at least one selected from the group consisting of fibronectin, a fibronectin fragment, and a mixture of fibronectin fragments. An IL-9 production inhibitor and a method for inhibiting IL-5 or IL-9 production comprising the step of administering the active ingredient are provided. Further, according to the present invention, an IL obtained by culturing a cell population containing T cells in the presence of at least one selected from the group consisting of fibronectin, a mixture of fibronectin and a mixture of fibronectin fragments, A cell population having a low production of -5 or IL-9 and a method for producing the same are provided.

  The present inventors have also found that an oligopeptide containing a VLA-4 binding oligopeptide and / or a part of the heparin binding domain of fibronectin has an inhibitory effect on the proliferation of the T cell population. That is, another aspect of the present invention provides a T cell population growth regulator comprising, as an active ingredient, a VLA-4 binding oligopeptide and / or an oligopeptide containing a part of the heparin binding domain of fibronectin. Examples of VLA-4 binding oligopeptides include oligopeptides comprising an amino acid sequence represented by LDFP or LDVP. Examples of the heparin-binding domain-derived oligopeptide include oligopeptides containing an amino acid sequence represented by LIGRKK.

In addition, as another aspect of the present invention, the present inventors provide a novel modified fibronectin fragment, a nucleic acid encoding the same, and a method for producing lymphocytes using the fragment. That is, according to the present invention, a polypeptide selected from the following (a) and (b) is provided.
(A) A group consisting of amino acid sequences represented by SEQ ID NOs: 29, 36, 38, 44, 46, 51, 53, 58, 60, 66, 68, 71, 73, 75, 79, 83 and 85 in the sequence listing A polypeptide comprising at least one amino acid sequence selected from (b) a polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, inserted, added or substituted in the amino acid sequence of the polypeptide of (a) A novel polypeptide having a function equivalent to that of the polypeptide of (a)

The present invention also provides a nucleic acid selected from the following (c) to (g).
(C) Group consisting of amino acid sequences represented by SEQ ID NOs: 29, 36, 38, 44, 46, 51, 53, 58, 60, 66, 68, 71, 73, 75, 79, 83 and 85 in the sequence listing Nucleic acid encoding a polypeptide comprising at least one amino acid sequence selected from (d) SEQ ID NO: 29, 36, 38, 44, 46, 51, 53, 58, 60, 66, 68, 71, 73 A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, inserted, added or substituted in at least one amino acid sequence selected from the group consisting of the amino acid sequences represented by, 75, 79, 83 and 85 And represented by SEQ ID NOs: 29, 36, 38, 44, 46, 51, 53, 58, 60, 66, 68, 71, 73, 75, 79, 83 and 85 in the Sequence Listing. Nucleic acid encoding a polypeptide having a function equivalent to a polypeptide comprising an amino acid sequence selected from a mino acid sequence (e) SEQ ID NOS: 30, 37, 39, 45, 47, 52, 54, 59, 61 67, 69, 72, 74, 76, 80, 84 and 86 Nucleic acid comprising a base sequence selected from the group consisting of the base sequences represented by (f) the nucleic acid of (e) above under stringent conditions A nucleic acid that hybridizes, wherein the polypeptide encoded by the nucleic acid has a function equivalent to that of the polypeptide encoded by the nucleic acid of (e) (g) in the base sequence of the nucleic acid of (e) A nucleic acid having one or more of substitution, deletion, insertion or addition of one or several bases, wherein the polypeptide encoded by the nucleic acid is Nucleic acids having a function equivalent to a polypeptide encoded by a nucleic acid of e)

  Moreover, as another aspect of the present invention, there is also provided a method for producing lymphocytes comprising a step of culturing a cell population containing T cells in the presence of the polypeptide selected from (a) and (b). The The culturing step is preferably performed in the presence of a CD3 ligand.

  Furthermore, as another aspect of the present invention, there is provided a method for producing cultured lymphocytes comprising a step of culturing a cell population containing T cells in the presence of at least the above (A) and (B). It is possible to provide a cell population with a capacity according to the conditions. In particular, the cell population provided by the present invention is clinically highly advantageous in that it has a low content of regulatory T cells, which is an obstacle in cell therapy.

The method also provides a cell population selected from:
Cell population (1): Cell population with high CD4 positive naive T cell content cell population (2): Cell population with high Th1 type CD4 positive T cell content cell population (3): Th2 type CD4 positive T cell content rate Low cell population (4): Cell population with low CD4 positive regulatory T cell content (5): Cell population with increased cytotoxic activity in the presence of cancer antigen, viral antigen, non-self antigen, etc.

  The present invention provides a method for producing a cell population containing a high amount of CD4-positive T cells. The production method has a high cell proliferation rate, and the cell population obtained by the production method includes Th1-type CD4 positive T cells, CD4 positive CD45RA positive CCR7 positive T cells, CD4 positive CD45RA positive CD62L positive T cells, CD4 positive CD45RA positive The ratio of CCR7-positive CD62L-positive T cells is high, and the ratio of Th2-type CD4-positive T cells and CD4-positive regulatory T cells is low, which is extremely useful for treatment of diseases by cellular immunotherapy.

  In the present invention, “T cell” is also called T lymphocyte, and means a cell derived from the thymus among lymphocytes involved in an immune response. T cells include helper T cells (Th1 and Th2 types), suppressor T cells, regulatory T cells, CTL, naive T cells, memory T cells, αβ T cells expressing α and β chain TCRs, γ chains. And γδ T cells that express the TCR of the δ chain. The “cell population containing T cells” used as the raw material of the present invention includes blood (peripheral blood, umbilical cord blood, etc.), bone marrow fluid, and peripheral blood mononuclear collected, isolated, purified or derived from these. Examples include cell populations including cells (PBMC), blood cells, hematopoietic stem cells, cord blood mononuclear cells, and the like. These cells may be activated in vivo (in vivo) or ex vivo (ex vivo) by cytokines such as IL-2. Any of these cells collected from a living body or those obtained through in vitro culture, for example, a cell population obtained by the method of the present invention may be used as it is or cryopreserved.

  In the present invention, “fibronectin (hereinafter referred to as FN)”, “FN fragment” and “mixture of FN fragments” are those obtained from nature or artificially synthesized, ie, non-naturally occurring. Any one of them can be used. FN and its fragments are described, for example, in Luoslati E. [Ruoslahti E. et al. , Et al. Based on the disclosure of J. Biol. Chem., Vol. 256, No. 14, pp. 7277-7281 (1981)], substantially pure from naturally occurring substances. It can be manufactured in the form. Here, substantially pure FN or fragments of FN as described herein means that they are essentially free of other proteins that are naturally present with FN. The above FN and FN fragments can be used in the present invention alone or in a mixture of plural kinds.

  In addition, although existence of many splicing variants is known for FN, as the FN used in the present invention, any variant can be used as long as it exhibits the desired effect of the present invention. . For example, in the case of plasma-derived FN, a region called ED-B existing upstream of the cell binding domain and a region called ED-A existing between the cell binding domain and the heparin binding domain may be deleted. As is known, such plasma-derived FN can also be used in the present invention.

  FN is a huge protein having a molecular weight of 250,000, and it is preferable to use a fragment of FN from the viewpoint of operation and expression of a desired effect. Useful information regarding the FN fragments that can be used in the present invention, as well as the preparation of the fragments, can be found in Kimika F. [Kimizuka F. et al. , Et al. , Journal of Biochemistry, Vol. 110, pp. 284-291 (1991)]. R. [Kornbriht A. et al. R. , Et al. , EMBO Journal (EMBO J.), Vol. 4, No. 7, 1755-1759 (1985)], and Sekiguchi K. et al. [Sekiguchi K. et al. , Et al. , Biochemistry, Vol. 25, No. 17, 4936-4941 (1986)] and the like. For the nucleic acid sequence encoding FN or the amino acid sequence of FN, Genbank Accession No. NM_002026 and NP_002017.

  The FN fragment that can be used in the present invention is preferably a fragment containing any domain of III-8, 9, 10, 11, 12, 13, 14, or CS-1, and is a fragment in which a plurality of domains are repeatedly linked. There may be. For example, a fragment containing a cell binding domain (C-domain) containing a ligand for VLA-5, a heparin binding domain (H-domain), a CS-1 domain which is a ligand for VLA-4, etc. is used in the present invention. Is done. Examples of the fragment include the above-described J.P. Biochem. 110, pp. 284-291 (1991), CH-271, CH-296, H-271, H-296, and their derivatives and modifications. The aforementioned CH-296 is commercially available under the name of RetroNectin (registered trademark).

  Examples of the FN fragment include amino acid sequences represented by SEQ ID NOs: 1 to 8 in the Sequence Listing (FN III-8, III-9, III-10, III-11, III-12, III-13, III- 14, a fragment containing CS-1 domain), preferably cell binding domain of FN (region from III-8 to III-10), heparin binding domain (region from III-12 to III-14) ), Fragments containing any of the CS-1 domains. Here, the FN fragment also includes a fragment containing the amino acid sequences represented by SEQ ID NOs: 1 to 8 in the above sequence listing. More preferably, as a fragment of FN used in the present invention, SEQ ID NOs: 9 to 23, 29, 36, 38, 44, 46, 51, 53, 58, 60, 66, 68, 71, 73 in the Sequence Listing. 1 or several in the amino acid sequence of the polypeptide comprising the fragment having the same function as the fragment of the FN exemplified above, or a fragment containing the amino acid sequence represented by 75, 79, 83 and 85, For example, it may consist of a polypeptide comprising an amino acid sequence having 1 to 20, preferably 1 to 10, more preferably 1 to 5 amino acid substitutions, deletions, insertions or additions. Here, having an equivalent function means that in the “<3> Method for producing cell population of the present invention” described later, SEQ ID NOs: 9 to 23, 29, 36, 38, 44, 46, 51, 53 in the Sequence Listing. , 58, 60, 66, 68, 71, 73, 75, 79, 83, and 85, or a naive T-like cell in the obtained T cell, having the same expansion rate as the fragment containing the amino acid sequence represented by It shows that the content rate is equivalent.

  The amino acid substitution and the like are preferably such that the physicochemical properties of the polypeptide can be changed within a range in which the function of the original polypeptide can be maintained. For example, amino acid substitution is preferably conservative within a range that does not substantially change the properties (eg, hydrophobicity, hydrophilicity, charge, pK, etc.) of the original polypeptide. For example, amino acid substitutions are: 1. glycine, alanine; 2. valine, isoleucine, leucine; 3. Aspartic acid, glutamic acid, asparagine, glutamine; 4. serine, threonine; 5. Lysine, arginine; Substitution within each group of phenylalanine and tyrosine. Deletion, addition, and insertion of amino acids substantially change the properties of the amino acid around the target site in the polypeptide with properties similar to those around the target site. Deletions, additions and insertions within the range that does not change are preferred.

Hereinafter, the production process of the present invention will be specifically described.
The present invention is a method for producing a cell population, comprising the following steps.
Step (1): removing a CD8-positive T cell from a T-cell-containing cell population or collecting a CD4-positive T cell to prepare a cell population to be cultured, and step (2): the step (1) (B) culturing the cell population obtained in the following (A) and (B): (A) at least one selected from the group consisting of fibronectin, a fibronectin fragment and a mixture of fibronectin fragments (B ) CD3 ligand In the present specification, the (A) and (B) may be described as culture components in the present invention.

<1> Step (1) of the present invention
In step (1) of the production method of the present invention, a cell population to be used for culture is prepared by removing CD8 positive T cells from a cell population containing T cells or collecting CD4 positive T cells. . The removal of CD8 positive T cells can be performed using CD8 expressed on the cell surface as an index. For example, a carrier such as a bead or culture container on which an anti-CD8 antibody is immobilized, a cell population containing T cells, And removing CD8 positive T cells bound to the carrier, or collecting a cell population that does not bind to the carrier. As the beads on which the anti-CD8 antibody is immobilized, for example, Dynabeads M-450 CD8 (manufactured by Invitrogen), Eligix anti-CD8 mAb coated nickel particles (manufactured by Biotransplant) can be suitably used. Hereinafter, in this specification, a cell population obtained by removing CD8 positive T cells from a cell population containing T cells is referred to as “CD8-removed cells”.

  CD4 positive T cells can be collected using CD4 expressed on the cell surface as an index. For example, a carrier such as a bead or a culture container on which an anti-CD4 antibody is immobilized, a cell population containing T cells, And CD4 positive T cells bound to the carrier are collected. As the carrier on which the anti-CD4 antibody is immobilized, for example, CD4 MicroBeads (manufactured by Miltenyi Biotec), Dynabeads M-450 CD4 (manufactured by Invitrogen) and the like can be suitably used.

  In the present invention, the cell population prepared by removing CD8 positive T cells from the cell population containing T cells is specifically CD4 positive T cells when PBMC is used as the cell population containing T cells. Together with monocytes and macrophages. Therefore, a cell population prepared by removing CD8 positive T cells from a cell population containing T cells is a cell population consisting only of CD4 positive T cells selected using CD4 expressed on the cell surface as an index. Different.

  Prior to performing step (1) or step (2) of the present invention, for example, any cell subpopulation having a particular cell phenotype may be collected or removed. The operation is not particularly limited, and can be performed using an appropriate marker on the cell surface, for example, a CD marker as an index. For example, as described in Example 3- (2) below, an anti-CD3 antibody and an anti-CD3 antibody and an anti-CD28 antibody are used to collect a cell subpopulation having a marker that the antibody recognizes, and use for the present invention Can do. However, from the viewpoint of operability, cell proliferation, and improvement in the ratio of Th1-type CD4-positive T cells, it is preferable not to collect CD3-positive CD28-positive T cells, but depending on the intended use of the cell population to be produced. It can also be implemented.

<2> Step (2) of the present invention
By the step (2) of the production method of the present invention, the cell population obtained in the step (1) is cultured in the presence of the following (A) and (B).
(A) At least one selected from the group consisting of FN, a fragment of FN, and a mixture of FN fragments (B) CD3 ligand In addition, “culture in the presence of (A) and (B)” means ( Means culture under the coexistence of A) and (B), meaning culture combining the culture in the presence of (A) and the culture in the presence of (B) is not.

  Step (2) in the present invention is carried out for at least 1 day, preferably 2 to 14 days, more preferably 2 to 7 days, and further preferably 3 to 7 days from the start of culture. In addition, the said process (2) may be the whole period of the culture | cultivation process of the cell population manufactured for use for immunotherapy, and may be a partial period. In the case of a partial period, the step (2) is preferably performed at the beginning of the culture, and more preferably performed at the start of the culture.

  In the present specification, hereinafter, “(A) at least one selected from the group consisting of FN, FN fragment and FN fragment” is simply referred to as “(A) component”, “(B) CD3 ligand”. May be simply referred to as “component (B)”.

  In the present invention, the total concentration of the component (A) during culture is not particularly limited, and for example, 0.001 to 500 μg / mL is preferable, and 0.01 to 500 μg / mL is more preferable. In addition, the density | concentration in culture | cultivation here applies to any of the aspect used by including the said component in a culture medium, and the aspect fixed and used for the below-mentioned appropriate solid phase.

  In the present invention, the CD3 ligand is not particularly limited as long as it is a substance having binding activity to CD3. For example, an anti-CD3 antibody is exemplified, and an anti-CD3 monoclonal antibody is preferably exemplified. For example, OKT3 [Science , 206, pp. 347-349 (1979)]. The concentration of CD3 ligand during culture is not particularly limited. For example, when an anti-CD3 monoclonal antibody is used, 0.001 to 100 μg / mL is preferable, and 0.01 to 100 μg / mL is more preferable. In addition, the density | concentration in culture | cultivation here applies to any of the aspect used by including the said component in a culture medium, and the aspect fixed and used for the below-mentioned appropriate solid phase.

The medium used in the method for producing a cell population of the present invention is not particularly limited, and a known medium prepared by mixing components necessary for T cell expansion culture can be used, for example, a commercially available medium. Can be appropriately selected and used. These media may contain cytokines, appropriate proteins, and other components in addition to the original components. Examples of cytokines include IL-2, IL-7, IL-12, IL-15, and IFN-γ, and a medium containing IL-2 is preferably used. The concentration of IL-2 in the medium is not particularly limited, but is preferably 0.01 to 1 × 10 ⁵ U / mL, more preferably 1 to 1 × 10 ⁴ U / mL, for example. Examples of suitable proteins include anti-IL-4 antibodies and CD28 ligand. In addition, a lymphocyte stimulating factor such as lectin can also be added.

  Furthermore, serum or plasma may be added to the medium. The amount added to these media is not particularly limited, but is exemplified to be more than 0% by volume to 20% by volume, and the amount of serum or plasma used can be changed depending on the culture stage. For example, the serum or plasma concentration can be decreased in stages and used. The origin of serum or plasma may be either self (meaning that the origin is the same as the cell being cultured) or non-self (meaning that the origin is different from the cell being cultured), but preferably Self-derived ones are used from the viewpoint of safety.

The number of cells at the start of the culture used in the present invention is not particularly limited, but for example, preferably 10 cells / mL to 1 × 10 ⁸ cells / mL, more preferably 1 × 10 ² cells / mL to 5 × 10 ⁷ cells / mL, more preferably 1 × 10 ³ cells / mL to 2 × 10 ⁷ cells / mL are exemplified. Moreover, there is no limitation in particular in culture conditions, The conditions used for normal cell culture can be used. For example, it can be cultured in conditions such as 37 ℃, 5% CO _2. In addition, the cell culture medium can be diluted by adding a fresh medium at an appropriate time interval, the medium can be replaced, or the cell culture equipment can be replaced.

The cell culture equipment used in the method for producing a cell population of the present invention is not particularly limited, and for example, a petri dish, a flask, a bag, a large culture tank, a bioreactor and the like can be used. As the bag, a CO ₂ gas permeable bag for cell culture can be used. Moreover, when manufacturing a large cell population industrially, a large culture tank can be used. Moreover, although culture | cultivation can be implemented by any of an open system and a closed system, it is preferable to culture | cultivate by a closed system from a viewpoint of the safety | security of the cell population obtained suitably.

  In addition, (A) component, (B) component, stimulating factor, appropriate protein contained in the above medium, other components are dissolved in the medium and coexist, and appropriate solid phase, for example, petri dish, flask, bag They may be used by immobilizing them on cell culture equipment (including both open and closed systems) or a cell culture carrier such as beads, membranes, and glass slides. In this specification, the stimulating factor is selected from the group consisting of cells capable of presenting an antigen, cells presented with an antigen, antigen, CD28 ligand, cytokine, chemokine, and cells capable of producing a cytokine. Is done. For example, when immobilizing on beads, a plurality of types of components may be mixed and immobilized on one bead, or a plurality of types of beads may be prepared by immobilizing one type of component on one bead. May be. The material of the solid phase is not particularly limited as long as it can be used for cell culture. When immobilizing the above-mentioned various components on cell culture equipment or cell culture carrier, preferably, the ratio of the amount of the component to the amount of medium used during culture is used when the component is dissolved in the medium. The ratio is adjusted to be the same as the concentration, but is not limited to this ratio as long as a desired effect is obtained.

  The method for immobilizing component (A), component (B), and other components on the solid phase is not particularly limited. For example, by bringing these substances into contact with the solid phase in an appropriate buffer solution. Can be immobilized. Further, the immobilization of the component (A) on the solid phase can also be carried out by the methods described in WO 97/18318 pamphlet and WO 00/09168 pamphlet.

  If the above-mentioned various components and the culture components used in the present invention are immobilized on a solid phase, the cell population is cultured by the method of the present invention, and then the cell population and the solid phase are separated, The above active ingredients can be removed and contamination of the active ingredients into the cell population can be prevented.

  In step (2) of the method of the present invention, a gene may be introduced into the cells obtained in step (1) before culturing. The gene introduction means is not particularly limited, and an appropriate one can be selected and used by a known gene introduction method. As the gene introduction method, either a method using a viral vector or a method not using the vector can be used in the present invention. A lot of literature has already been published on details of these methods. Of course, a step of introducing a gene may be further included after the culture in the step (2).

  The viral vector is not particularly limited, and is usually a known viral vector used for gene transfer methods, such as a retroviral vector (including lentiviral vectors and pseudotype vectors), an adenoviral vector, and an adeno-associated viral vector. Simian virus vectors, vaccinia virus vectors or Sendai virus vectors are used. Preferably, retroviral vectors, adenoviral vectors or lentiviral vectors are used. As the above-mentioned viral vector, those lacking replication ability are preferable so that they cannot self-replicate in infected cells. In addition, a substance that improves gene transfer efficiency, such as RetroNectin (registered trademark, manufactured by Takara Bio Inc.), can also be used during gene transfer.

  The gene introduction method without using a viral vector is not limited to the present invention. For example, a method using a carrier such as a liposome or a ligand-polylysine, a calcium phosphate method, an electroporation method, a particle gun method or the like is used. can do. In this case, a foreign gene incorporated into plasmid DNA, linear DNA or RNA is introduced.

  Retroviral vectors and lentiviral vectors can stably incorporate a foreign gene inserted into the chromosomal DNA of a cell into which the vector is introduced, and are used for gene therapy and other purposes.

  The foreign gene to be introduced is not particularly limited, and any gene can be selected according to the immunotherapy using the cell population obtained by the production method of the present invention and the target disease. As such a gene, for example, a gene encoding a protein (for example, enzyme, cytokines, receptors, etc.), an antisense nucleic acid, siRNA (small interfering RNA), or a ribozyme can be used. In addition, an appropriate marker gene that enables selection of a gene-introduced cell may be introduced at the same time.

  The foreign gene can be used, for example, by inserting it into a vector or plasmid so that it can be expressed under the control of an appropriate promoter. In addition, in order to achieve efficient gene transcription, other regulatory elements cooperating with the promoter and transcription initiation site, for example, enhancer sequences and terminator sequences may be present in the vector. In addition, for the purpose of inserting a foreign gene into a chromosome of a cell to be introduced by homologous recombination, for example, a base sequence having homology to base sequences on both sides of a desired target insertion site of the gene in the chromosome A foreign gene may be placed between flanking sequences consisting of The foreign gene to be introduced may be a natural gene, an artificially produced gene, or a DNA molecule having a different origin bound by a known means such as ligation. Furthermore, it may have a sequence in which a mutation is introduced into a natural sequence according to the purpose.

  About other conditions of the manufacturing method of a cell population, it can implement by a well-known method, for example, about the manufacturing method of the cell population obtained by implementing culture | cultivation in presence of the said (A) component, for example, international Publication No. 03/016511, International Publication No. 03/080817, International Publication No. 2005/018450, JP2007-061020, International Publication No. 2007/020880, or International Publication No. 2007/040105 Can be carried out with reference to the method described in.

<3> Method for producing cell population of the present invention The production method of the present invention is characterized in that the cell growth rate of the cell population is high in the course of the production. In the present specification, the cell growth rate is also referred to as the expansion culture rate. The cell growth rate is a numerical value indicating how many times the number of cells at the start of the culture grew after the culture in a certain culture period. Here, the high cell growth rate means that the cell growth rate of the cell population when cultured in the presence of the component (A) and the component (B) in the step (2) is the above (A). Means higher compared to the cell growth rate of the cell population in culture in the absence of components. Preferably, a cell population can be obtained at a cell growth rate of 5% or more, more preferably 10% or more higher than the case where the culture conditions of the present invention are not used.

  In the production method of the present invention, the cell population in step (2) may be cultured in the presence of CD28 ligand (eg, anti-CD28 antibody) or in the absence of CD28 ligand. As shown in the following Examples, in the absence of an anti-CD28 antibody, from the viewpoints of cell proliferation rate, operability, cost, improvement in content of Th1-type CD4 positive T cells, and improvement in content of CD4 positive naive T-like cells It is preferable to carry out.

  In the production method of the present invention, the content of Th1-type CD4-positive T cells can be increased by carrying out step (2) in the absence of an anti-CD28 antibody. That is, the production method of the present invention can produce a cell population having a high content of Th1-type CD4-positive T cells in the cell population. The content of Th1-type CD4-positive T cells in the obtained cell population was determined according to Examples 1- (6), 2- (2), 3- (3), 24- (4), and 28- (5) described later. ) And 29- (3), IL-4 negative and IFN-γ positive can be used as indicators. Th1-type CD4-positive T cells are effectors of cellular immunity, and a cell population having a high content of Th1-type CD4-positive T cells is extremely useful in cell immunotherapy (for example, Non-Patent Document 1 described above). Here, a cell population having a high content of Th1-type CD4-positive T cells refers to a Th1-type CD4-positive cell population obtained by carrying out step (2) in the absence of an anti-CD28 antibody in the production method of the present invention. It means a cell population with a high T cell content compared to that in the presence of an anti-CD28 antibody, preferably 3% or more, more preferably 6% or more. The content of Th1-type CD4-positive T cells in the cell population varies depending on individual differences and the physical condition of the donor and is not particularly limited, but is 10% or more, preferably 12% or more, more preferably 14% or more. is there.

  In the production method of the present invention, the content of Th2-type CD4-positive T cells can be reduced by carrying out step (2) in the absence of an anti-CD28 antibody. That is, by the production method of the present invention, a cell population having a low content of Th2-type CD4 positive T cells in the cell population can be produced. The content rate of Th2-type CD4-positive T cells in the obtained cell population is IL-4 positive as described in Examples 28- (5), (6), 29- (3) and (4) described later. , IFN-γ negative, CD4 positive CCR4 positive can be used as indicators. Th2-type CD4-positive T cells are an effector of humoral immunity, and a cell population having a low content of Th2-type CD4-positive T cells is extremely useful in cellular immunotherapy. Here, a cell population having a low content of Th2-type CD4-positive T cells means a Th2-type CD4-positive T of a cell population obtained by carrying out step (2) in the absence of anti-CD28 antibody in the production method of the present invention. It means a cell population having a low cell content compared to that carried out in the presence of an anti-CD28 antibody, preferably 3% or more, more preferably 6% or more. The content of Th2-type CD4-positive T cells in the cell population varies depending on individual differences and the physical condition of the donor and is not particularly limited, but is 40% or less, preferably 30% or less, more preferably 20% or less. is there.

  That is, in the production method of the present invention, the ratio of Th1-type CD4-positive T cells / Th2-type CD4-positive T cells in the cell population is increased by performing step (2) in the absence of anti-CD28 antibody. A method is provided.

  Moreover, in the production method of the present invention, the content of CD4 positive regulatory T cells can be lowered by carrying out the step (2) in the absence of an anti-CD28 antibody. That is, by the production method of the present invention, a cell population having a low content of CD4 positive regulatory T cells in the cell population can be produced. The content rate of CD4 positive regulatory T cells in the obtained cell population can be measured using CD4 positive CD25 positive Foxp3 positive as an index as described in Example 28- (7) described later. Regulatory T cells are known to have a function of suppressing various immune response reactions, and a cell population having a low content of regulatory T cells is extremely useful in cellular immunotherapy. Here, the cell population having a low content of CD4 positive regulatory T cells refers to a CD4 positive regulatory T of a cell population obtained by performing the above step (2) in the absence of anti-CD28 antibody in the production method of the present invention. It means a cell population having a low cell content compared to that carried out in the presence of an anti-CD28 antibody, preferably 3% or more, more preferably 6% or more. The content of CD4 positive regulatory T cells in the cell population varies depending on individual differences and the physical condition of the donor and is not particularly limited, but is 20% or less, preferably 15% or less, more preferably 10% or less. is there.

  In the production method of the present invention, a cell population containing CD4 positive CD45RA positive CCR7 positive T cells and / or CD4 positive CD45RA positive CD62L positive T is obtained by performing the above step (2) in the absence of an anti-CD28 antibody. Cell populations containing cells can be produced. That is, by the production method of the present invention, a cell population having a high content of CD4 positive CD45RA positive CCR7 positive T cells and / or CD4 positive CD45RA positive CD62L positive T cells in the cell population can be produced. CD45RA, CCR7, and CD62L are all cell surface antigen markers for lymphocytes and are known to be expressed in undifferentiated cells such as naive T cells. That is, cells contained in a high ratio in the cell population obtained by the production method of the present invention can be classified as undifferentiated cells before differentiation into memory T cells, that is, naive T cells. Hereinafter, in this specification, CD4-positive CD45RA-positive CCR7-positive T cells or CD4-positive CD45RA-positive CD62L-positive T cells may be referred to as “CD4-positive naive T-like cells”. Naive T cells have been reported to be useful in the field of cell medicine because of their high in vivo survival rate, cell proliferation effect, and tumor accumulation effect when administered to a living body. Here, the cell population having a high content of CD4 positive CD45RA positive CCR7 positive T cells and / or CD4 positive CD45RA positive CD62L positive T cells is obtained by culturing under the culture conditions used in the production method of the present invention. The cell content is higher than the content obtained by culturing under the same conditions except for the culture conditions of the present invention. Specifically, in the production method of the present invention, the cell content of the cell population obtained by carrying out step (2) in the absence of anti-CD28 antibody is compared with that in the case where the cell content is carried out in the presence of anti-CD28 antibody. A cell population that is preferably 5% or higher, more preferably 10% or higher can be obtained.

  In the production method of the present invention, a cell population with improved cytotoxic activity can be produced by culturing in the presence of (A) and (B). The cytotoxic activity of the obtained cell population can be measured as described in Example 27- (4) described later. Here, the cell population with improved cytotoxic activity refers to the cytotoxic activity of the cell population cultured in the presence of the above (A) and (B) in the production method of the present invention. It means a high cell population as compared to the case performed below, preferably 2.5% or more, more preferably 5% or more.

  Furthermore, the production method of the present invention can include a step of separating the cell population into an arbitrary cell subpopulation, a step of collecting the cell subpopulation, or a step of removing the cell subpopulation before or after each step. . For example, as described above, the cell population obtained by culturing under the culture conditions of the present invention has a high content of Th1-type CD4-positive T cells and CD4-positive naive T-like cells, and Th2-type CD4-positive T cells and CD4-positive. Since the cell population has a low content of regulatory T cells, Th1 type CD4 positive T cells, CD4 positive naive T-like cells, Th2 type CD4 can be obtained by further separating, collecting or removing cells expressing the desired surface antigen marker. Positive T cells and CD4 positive regulatory T cells may be isolated, collected or removed as cell subpopulations. By this step, it is possible to obtain a T cell population that further contains Th1-type CD4 positive T cells and / or CD4 positive naive T-like cells. Moreover, a cell population with a small number of Th2-type CD4-positive T cells and / or CD4-positive regulatory T cells can be obtained. Moreover, when this invention further includes the process of introduce | transducing a gene into the cell obtained at process (1), you may isolate | separate and acquire the cell which expresses the said gene. The separation operation is not particularly limited. For example, the separation operation can be performed by a known method using a cell sorter, a magnetic bead, a column, or the like.

  In addition, it is possible to maintain a stable T cell line by cloning T cells from the cell population produced by the method of the present invention. In addition, a new cell population can be obtained by further culturing by a known method using the cell population obtained by the method of the present invention.

  In the production method of the present invention, the cell population may be cultured in the presence of CD28 ligand (eg, anti-CD28 antibody) or in the absence of CD28 ligand. As shown in the following examples, carrying out in the absence of anti-CD28 antibody improves cell proliferation rate and operability, reduces costs, improves the content of Th1-type CD4-positive T cells, Th2-type CD4-positive T cells The addition of CD28 ligand may be selected according to the purpose, because it has characteristics such as a decreased content of CD4, an increased content of CD4 positive naive T-like cells, and a decreased content of CD4 positive regulatory T cells. The present invention is extremely useful in that an arbitrary cell population can be produced according to the purpose by combining other ligands coexisting with the component (A) and the component (B).

  In the production method of the present invention, a step of further culturing the cell population obtained in the step (2) in the absence of the component (A) and / or the component (B) may be performed. This step may be carried out by a normal cell culture method, for example, a known T cell culture method, and the same conditions as in step (2) except that the components (A) and (B) do not coexist. It may be. The medium may contain cytokines, appropriate proteins, and other components as in the above step (2). Examples of cytokines include IL-2, IL-7, IL-12, and IFN-γ.

  In the method for producing a cell population of the present invention, the total number of culture days is preferably 4 to 14 days from the viewpoint of cell quality and culture efficiency. That is, when the total number of culture days is 4 to 14 days, the obtained cell population is a cell population having a high content of Th1-type CD4-positive T cells and CD4-positive naive T-like cells, and is suitable for use in the field of cell medicine. Very suitable. When the total number of culture days is less than 4 days, it is not possible to obtain a sufficient number of cells for use in general immunotherapy. In the present invention, the total number of culture days is preferably 5 to 14 days, more preferably 6 to 14 days.

<4> Use of cell population produced according to the present invention The present invention relates to a method for producing a cell population, and more particularly to a method for producing CD4-positive T cells, Th1-type CD4-positive T cells, and CD4-positive naive T-like cells. That is, the cell population produced by the method of the present invention has a high content ratio of CD4 positive T cells, a content ratio of Th1 type CD4 positive T cells, a content ratio of CD4 positive naive T-like cells, and a Th2 type CD4 positive T cell. The content of is low. Therefore, it is useful for treatment and prevention of various diseases. The disease for which the administration of the cell population is effective is not particularly limited. For example, cancer (leukemia, solid tumor, etc.), hepatitis, infectious diseases caused by influenza, viruses, bacteria, fungi, Examples include tuberculosis, MRSA, VRE, and deep mycosis. In particular, it is useful for the treatment of AIDS (AIDS, acquired immune deficiency syndrome) caused by HIV (Human Immunodeficiency Virus) that infects CD4-positive T cells. The cell population produced by the method of the present invention can also be used for bone marrow transplantation, prevention of infection after irradiation, donor lymphocyte infusion for remission of relapsed leukemia, and the like.

Furthermore, the present invention provides a cell population having a high content of Th1-type CD4-positive T cells and / or a high content of CD4-positive naive T-like cells and a low content of Th2-type CD4-positive T cells. The present invention also provides a medicament containing the cell population as an active ingredient. Said medicament containing said cell population is suitable for use in immunotherapy. In immunotherapy, T cells suitable for patient treatment are administered into the patient's vein, artery, subcutaneous, intraperitoneal cavity, etc. by, for example, injection or infusion. The medicament is very useful for use in the above-mentioned diseases. The medicine is in accordance with a known method in the pharmaceutical field. For example, the T cell population prepared by the method of the present invention is used as an active ingredient, and known organic or inorganic carriers, excipients and stabilizers suitable for parenteral administration. Etc., and can be prepared as drops or injections. The content of the cell population of the present invention in the therapeutic agent, the dosage of the therapeutic agent, and various conditions relating to the therapeutic agent can be determined as appropriate according to known immunotherapy. For example, the content of the cell population of the present invention in medicine is not particularly limited. For example, it is preferably 1 × 10 ³ to 1 × 10 ¹¹ cells / mL, more preferably 1 × 10 ⁴ to 1 ×. 10 ¹⁰ cells / mL, more preferably 1 × 10 ⁵ to 1 × 10 ⁹ cells / mL are exemplified. The dose of the medicament of the present invention is not particularly limited. For example, it is preferably 1 × 10 ⁶ to 1 × 10 ¹² cells / day, more preferably 1 × 10 ⁷ to per adult day. Examples are 5 × 10 ¹¹ cells / day, more preferably 1 × 10 ⁸ to 2 × 10 ¹¹ cells / day. Furthermore, immunotherapy with the therapeutic agent can be used in combination with drug treatment by administration of a known drug, radiotherapy, or surgery.

  Furthermore, the present invention provides a diagnostic agent containing the cell population as an active ingredient. For example, by analyzing and screening cells obtained from a patient using the diagnostic agent of the present invention, it is possible to estimate a therapeutic effect, select an effective transgene, and the like.

  The present invention also provides a method for treating or preventing a disease comprising administering to a subject an effective amount of the cell population obtained by the above method. In the present specification, the subject is not particularly limited, but is preferably a living body (eg, human patient, non-human) suffering from a disease as described above, to which a cell population produced by the method of the present invention is administered. Animal).

  In the present specification, the effective amount refers to the T cells that exert a therapeutic or prophylactic effect when the cell population is administered to the subject as compared to a subject not administered the cell population. The amount of the population. The specific effective amount is appropriately set depending on the administration form, administration method, purpose of use and age, weight, symptom of the subject, etc., but is preferably the same as the above-mentioned medicine. There is no limitation on the administration method, and for example, it may be administered by drip, injection or the like, similar to the above-mentioned medicine.

  The present invention also provides a cell population comprising activated CD4 positive T cells by applying stimulation to at least one stimulating factor to the CD4 positive T cell population obtained by the production method of the present invention. A method of manufacturing is provided. Furthermore, the present invention provides a cell population obtained by the above production method. The cell population containing activated CD4 positive T cells obtained in this way can be used as an active ingredient of a medicament in the same manner as the CD4 positive T cell population obtained by the production method described above. Here, the stimulation by the stimulating factor is not particularly limited as long as the cell population obtained by the above-described production method of the present invention is activated by the stimulating factor. For example, CD4 positive obtained by the production method of the present invention It can be carried out by culturing in the presence of a T cell population and a stimulating factor.

  In the present specification, the cytokine as the stimulating factor is not particularly limited as long as it can act on and activate CD4-positive T cells. For example, IL-2, IFN-γ, TGF-β, IL- 15, IL-7, IFN-α, IL-12, CD40L, IL-27 and the like are exemplified, and IL-2, IFN-γ and IL-12 are preferably exemplified from the viewpoint of enhancing cellular immunity. The

  In the present specification, the chemokine is not particularly limited as long as it acts on T cells and exhibits migration activity. Examples thereof include RANTES, CCL21, MIP1α, MIP1β, CCL19, CXCL12, IP-10, and MIG. .

In addition, since the cell population produced by the method of the present invention has a high content of Th1-type CD4-positive T cells, the cell population further contains CD8-positive CD45RA-positive CCR7-positive T cells (CD8-positive naive T-like cells). The CD8-positive CD45RA-positive CCR7-positive T cells can be cultured at a high content by culturing in a mixture with the cell population. That is, the present invention provides a method for producing a cell population comprising the following steps (2-1) to (2-3).
Step (2-1): Step of removing CD8-positive T cells from a cell population containing T cells or collecting CD4-positive T cells (2-2): obtained in the step (2-1) (C) culturing a cell population in the presence of (A) and (B) below: (A) at least one selected from the group consisting of FN, a fragment of FN and a mixture of FN fragments (B) a CD3 ligand step ( 2-3): A step of mixing and culturing the cell population obtained in the step (2-2) and a cell population containing a CD8-positive CD45RA-positive CCR7-positive T cell.

  The culture in the step (2-3) is preferably performed under the conditions of the step (2-2), that is, in the presence of the component (A) and the component (B). The cell population containing CD8-positive CD45RA-positive CCR7-positive T cells is not particularly limited. For example, PBMC, umbilical cord blood mononuclear cells, or CD8-positive cells separated from these can be used. Other culture conditions and the like can be carried out by the method described in “<3> Method for producing cell population of the present invention”. In addition, since the cell population obtained by the said method contains the CD8 positive naive T-like cell in a high ratio, it is very suitable for the use for a cell medicine.

In the expansion culture of a cell population containing T cells, the present inventors stably realize a high expansion culture rate, and the content rate of CD8 positive CD45RA positive CCR7 positive T cells in the obtained cell population is stable. As a result of various studies on the method of maintaining a high level, the ratio of CD4 positive T cells and CD8 positive T cells at the start of culture was found to be extremely important. That is, as another aspect of the present invention, there is provided a method for producing a cell population comprising the following steps.
Step (3-1): Step of obtaining a cell population in which the ratio of CD8 positive T cells to CD4 positive T cells is 2: 1 or more Step (3-2): Cells obtained in the step (3-1) Culturing the population in the presence of CD3 ligand

  Here, as the ratio of CD8 positive T cells to CD4 positive T cells in the above step (3-1), “the ratio of CD8 positive T cells to CD4 positive T cells is 2: 1 or more” means the number of CD8 positive T cells. Means that the number of CD4 positive T cells is 1 or more, preferably 1: 1 or more, more preferably 1: 2 or more, and even more preferably 1: 4 or more. Illustrated. The step of obtaining a cell population of this ratio is not particularly limited, but the separation of CD8 positive T cells from the cell population containing a high amount of CD8 positive T cells obtained by the above method, and the separated CD8 positive T cells. , Separation of CD4 positive T cells from a cell population containing a high amount of CD4 positive T cells obtained by the above method, addition of separated CD4 positive T cells, etc. alone or in combination as appropriate, A cell population having a specific CD8 positive T cell and CD4 positive T cell ratio can be obtained. Moreover, before the said process (3-1), the process of measuring content of CD8 positive T cell and CD4 positive T cell in the cell population containing the said T cell, ie, T cell used for culture | cultivation A step of measuring the ratio of CD4-positive T cells to CD8-positive T cells in the blood of the population or the donor from which it is derived can also be added. In addition, from the viewpoint of realizing a higher expansion culture rate, in the above step (3-2), in addition to the T cells obtained in step (3-1), blood cells of the same origin such as monocytes, trees It is preferable that dendritic cells, NK cells, etc. are contained in the cell population to be cultured. Preferably, as shown in Example 4 described later, monocytes derived from the same donor as the cell population to be cultured are separated in advance. In addition, it is preferable to mix monocytes when the cell population containing the T cells having the above ratio is subjected to the step (3-2). In addition to the presence of CD3 ligand, the culture in the step (3-2) is preferably performed in the presence of at least one selected from the group consisting of FN, a fragment of FN, and a mixture of FN fragments. It is preferable to implement. Other culture conditions and the like can be carried out by the method described in “<3> Method for producing cell population of the present invention”.

  Moreover, as another aspect of the present invention, an IL-5 production inhibitor comprising, as an active ingredient, at least one selected from the group consisting of FN, a fragment of FN, and a mixture of FN fragments, or An IL-9 production inhibitor is provided. The IL-5 production inhibitor and IL-9 production inhibitor are useful as pharmaceuticals and functional food materials for diseases that require IL-5 or IL-9 production inhibitory action. The disease is not particularly limited, but is extremely useful for the treatment or prevention of, for example, asthma, allergic diseases, anaphylaxis, urticaria, atopic dermatitis and the like. About manufacture of the said IL-5 production inhibitor and IL-9 production inhibitor, the pharmaceutical or foodstuff containing it, it contains at least 1 selected from the group which consists of FN, the fragment of FN, and the mixture of the fragment of FN If it does, there is no limitation in particular, It can manufacture according to the manufacturing method of a well-known pharmaceutical and foodstuff. The present invention also provides a method for inhibiting IL-5 or IL-9 production, comprising administering at least one selected from the group consisting of FN, a fragment of FN, and a mixture of FN fragments. Examples of administration subjects include humans, domestic animals, pet animals, and various cells such as blood cells. The suppression method can be used as a research reagent in addition to the treatment and prevention of the above diseases.

  Further, according to the present invention, IL-obtained by performing the step of culturing a cell population containing T cells in the presence of at least one selected from the group consisting of FN, a fragment of FN and a mixture of FN fragments. A cell population having a low 5 or IL-9 production amount and a method for producing the same are provided. Since the production amount of IL-5 and IL-9 is reduced, the cell population can be administered to a patient while suppressing an increase in the cytokine in the field of cell medicine. The production method is not particularly limited with respect to conditions other than the step of culturing a cell population containing T cells in the presence of at least one selected from the group consisting of FN, a fragment of FN, and a mixture of FN fragments. For example, it can be carried out by the method described in “<3> Method for producing cell population of the present invention”.

  As another aspect of the present invention, there is provided a T cell population growth regulator comprising, as an active ingredient, a VLA-4 binding oligopeptide and / or an oligopeptide containing a part of the heparin binding domain of FN. Examples of VLA-4 binding oligopeptides include oligopeptides comprising an amino acid sequence represented by LDFP or LDVP. Moreover, as an oligopeptide containing a part of the heparin-binding domain of FN, an oligopeptide containing an amino acid sequence represented by LIGRKK is exemplified. Here, “oligopeptide” is defined as a peptide of 20 amino acids or less. Cell proliferation by using the oligopeptide for expansion culture of T cell population, particularly for expansion culture of T cell population using at least one selected from the group consisting of FN, FN fragment and FN fragment mixture Since the rate decreases, it can be used to control cell proliferation in expanded culture of T cell populations. Furthermore, any cell population can be specifically expanded by using the growth regulator and the expansion culture promoting substance in combination. The agent is also useful in studying the mechanism of lymphocyte proliferation.

  In addition, as shown in Examples 20 to 22 described later, in the expansion culture of a cell population using FN or a fragment thereof, the VLA-4 binding domain of FN is VLA-4 on the surface of cells to be cultured. It has been shown by the present invention that expansion of lymphocytes is carried out by inducing a signal by inducing signal and maintaining a high content of naive T-like cells in the resulting cell population. That is, according to the present invention, a method of expanding lymphocytes characterized by stimulating VLA-4 and inducing a signal to a cell population containing a high amount of CD4 positive T cells obtained by the present invention, CD45RA positive CCD7 A method for improving the ratio of positive cells and / or CD45RA positive CD62L positive cells is provided. The method for inducing a signal to VLA-4 is not particularly limited. For example, a polypeptide containing LDFP, which is a polypeptide capable of inducing a signal to VLA-4, such as the CS- A polypeptide having one domain (polypeptide represented by SEQ ID NO: 8 in the sequence listing) is exemplified, and the present invention provides a polypeptide having the ability. The polypeptide can be used for expansion culture of lymphocytes, and can be used properly according to the purpose to produce any cell population.

Moreover, as another aspect of the present invention, there is also provided a novel polypeptide selected from the following (a) and (b) which is very useful for use in T cell expansion culture.
(A) SEQ ID NO: 29 (H204), 36 (H296-CS1), 38 (H296-CS2), 44 (H114-HT), 46 (H114), 51 (H117-HT), 53 (H117) 58 (H206-HT), 60 (H206), 66 (H296R-HT), 68 (H296R), 71 (H105-HT), 73 (H105), 75 (CH105-HT), 79 (CH296mut2), 83 A polypeptide comprising at least one amino acid sequence selected from the group consisting of (CH296mut3) and 85 (CH296mut4) (b) deletion or insertion of one or several amino acids in the amino acid sequence of the polypeptide of (a), A polypeptide comprising an added or substituted amino acid sequence, equivalent to the polypeptide of (a) above The novel polypeptide having the function

Hereinafter, individual polypeptides will be described.
The polypeptide having the amino acid sequence represented by SEQ ID NO: 29 in the sequence listing is a polypeptide described as H204 in Example 6 described later, and FNs III-13, III-14, and CS in this order from the N-terminus. -1 is a polypeptide having a domain.

  The polypeptide having the amino acid sequence represented by SEQ ID NO: 36 in the Sequence Listing is a polypeptide described as H296-CS1 in Example 7 described later, and FN III-12, III-13, A polypeptide having III-14, CS-1 domain, and CS-1 domain.

  The polypeptide having the amino acid sequence represented by SEQ ID NO: 38 in the Sequence Listing is a polypeptide described as H296-CS2 in Example 8 described later, and FN III-12, III-13, A polypeptide having III-14, CS-1 domain, CS-1 domain, and CS-1 domain.

  The polypeptide having the amino acid sequence represented by SEQ ID NO: 44 in the sequence listing is a polypeptide described as H114-HT in Example 9 described later, and includes a His tag sequence, FN III-13, and FN in order from the N-terminus. A polypeptide having a CS-1 domain.

  The polypeptide having the amino acid sequence represented by SEQ ID NO: 46 in the sequence listing is a polypeptide described as H114 in Example 9 described later, and is a polypeptide obtained by removing the His tag sequence from the N-terminus of the above H114-HT. It is a peptide.

  The polypeptide having the amino acid sequence represented by SEQ ID NO: 51 in the Sequence Listing is a polypeptide described as H117-HT in Example 10 described later, and includes a His tag sequence, FN III-12, and FN in order from the N-terminus. A polypeptide having a CS-1 domain.

  The polypeptide having the amino acid sequence represented by SEQ ID NO: 53 in the sequence listing is a polypeptide described as H117 in Example 9 described later, and a polypeptide obtained by removing the His tag sequence from the N-terminus of the above H117-HT. It is a peptide.

  The polypeptide having the amino acid sequence represented by SEQ ID NO: 58 in the Sequence Listing is a polypeptide described as H206-HT in Example 11 described later, and includes a His tag sequence, FN III-12, It is a polypeptide having III-13 and CS-1 domains.

  The polypeptide having the amino acid sequence represented by SEQ ID NO: 60 in the sequence listing is a polypeptide described as H206 in Example 11 described later, and is a polypeptide obtained by removing the His tag sequence from the N-terminus of the above H206-HT. It is a peptide.

  The polypeptide having the amino acid sequence represented by SEQ ID NO: 66 in the sequence listing is a polypeptide described as H296R-HT in Example 12 described later, and includes a His tag sequence and an FN CS-1 domain in order from the N-terminus. , III-12, III-13 and III-14.

  The polypeptide having the amino acid sequence represented by SEQ ID NO: 68 in the sequence listing is a polypeptide described as H296R in Example 12 described later, and a polypeptide obtained by removing the His tag sequence from the N-terminus of the above H296R-HT. It is a peptide.

  The polypeptide having the amino acid sequence represented by SEQ ID NO: 71 in the sequence listing is a polypeptide described as H105-HT in Example 13 described later, and includes a His tag sequence, FN III-14 and FN in order from the N-terminus. A polypeptide having a CS-1 domain.

  The polypeptide having the amino acid sequence represented by SEQ ID NO: 73 in the sequence listing is a polypeptide described as H105 in Example 13 described later, and is a polypeptide obtained by removing the His tag sequence from the N-terminus of the above H105-HT. It is a peptide.

  The polypeptide having the amino acid sequence represented by SEQ ID NO: 75 in the sequence listing is a polypeptide described as CH105-HT in Example 14 described later, and includes a His tag sequence, FN III-8, A polypeptide having III-9, III-10, amino acid sequence “EIDKPSMA”, III-14 and CS-1 domain.

  The polypeptide having the amino acid sequence represented by SEQ ID NO: 79 in the sequence listing is a polypeptide described as CH296mut2 in Example 15 described later, and has the amino acid sequence represented by SEQ ID NO: 13 in the above sequence listing. The polypeptide is a polypeptide in which the region corresponding to amino acid numbers 543 to 548 of CH-296 is converted from “LIGRKK” to “VLAKRR”.

  The polypeptide having the amino acid sequence represented by SEQ ID NO: 83 in the sequence listing is a polypeptide described as CH296mut3 in Example 16 described later, and the region corresponding to amino acid numbers 569 to 572 of the CH-296 is “ A polypeptide converted from “LDVP” to “LELP”.

  The polypeptide having the amino acid sequence represented by SEQ ID NO: 85 in the sequence listing is a polypeptide described as CH296mut4 in Example 17 described later, and a region corresponding to amino acid numbers 543 to 548 of CH-296 described above is present. A polypeptide in which the region corresponding to amino acid numbers 569 to 572 of CH-296 has been converted from “LDVP” to “LELP” from “LIGRKK” to “VLAKRR”.

The present invention also provides a novel nucleic acid selected from the following (c) to (g).
(C) Group consisting of amino acid sequences represented by SEQ ID NOs: 29, 36, 38, 44, 46, 51, 53, 58, 60, 66, 68, 71, 73, 75, 79, 83 and 85 in the sequence listing Nucleic acid encoding a polypeptide comprising at least one amino acid sequence selected from (d) SEQ ID NO: 29, 36, 38, 44, 46, 51, 53, 58, 60, 66, 68, 71, 73 A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, inserted, added or substituted in at least one amino acid sequence selected from the group consisting of the amino acid sequences represented by 75, 79, 83 and 85 The sequences represented by SEQ ID NOs: 29, 36, 38, 44, 46, 51, 53, 58, 60, 66, 68, 71, 73, 75, 79, 83 and 85 in the Sequence Listing. Nucleic acid encoding a polypeptide having a function equivalent to a polypeptide comprising an amino acid sequence selected from a nonacid sequence (e) SEQ ID NO: 30 (base sequence of nucleic acid encoding H204), SEQ ID NO: 37 (H296) -Nucleic acid base sequence encoding CS1), SEQ ID NO: 39 (base sequence of nucleic acid encoding H296-CS2), SEQ ID NO: 45 (base sequence of nucleic acid encoding H114-HT), SEQ ID NO: 47 (encoding H114) Base sequence of nucleic acid encoding), SEQ ID NO: 52 (base sequence of nucleic acid encoding H117-HT), SEQ ID NO: 54 (base sequence of nucleic acid encoding H117), SEQ ID NO: 59 (base of nucleic acid encoding H206-HT) Sequence), SEQ ID NO: 61 (base sequence of nucleic acid encoding H206), SEQ ID NO: 67 (nucleic acid encoding H296R-HT) Base sequence), SEQ ID NO: 69 (base sequence of nucleic acid encoding H296R), SEQ ID NO: 72 (base sequence of nucleic acid encoding H105-HT), SEQ ID NO: 74 (base sequence of nucleic acid encoding H105), SEQ ID NO: 76 (base sequence of nucleic acid encoding CH105-HT), SEQ ID NO: 80 (base sequence of nucleic acid encoding CH296mut2), SEQ ID NO: 84 (base sequence of nucleic acid encoding CH296mut3) and SEQ ID NO: 86 (encoding CH296mut4) A nucleic acid having at least one base sequence selected from the group consisting of base sequences represented by (base sequence of nucleic acid) (f) a nucleic acid that hybridizes with the nucleic acid of (e) above under stringent conditions, The polypeptide encoded by the nucleic acid is a polypeptide encoded by the nucleic acid of (e) above. A nucleic acid having a function equivalent to a peptide (g) a nucleic acid in which any one or more of substitution, deletion, insertion or addition of one or several bases has occurred in the base sequence of the nucleic acid of (e), A nucleic acid having a function equivalent to that of the polypeptide encoded by the nucleic acid of (e) above, wherein the polypeptide encoded by the nucleic acid is

  The nucleic acid (c) is a nucleic acid encoding the polypeptide (a), and the nucleic acid (d) is a nucleic acid encoding the polypeptide (b). In addition, when the polypeptide (a) is used for protein expression using the nucleic acid (c), the N-terminal methionine residue may be digested.

  In the present specification, the novel polypeptide may be referred to as the polypeptide of the present invention, and the novel nucleic acid may be referred to as the nucleic acid of the present invention.

  Hereinafter, the polypeptide of the present invention, the nucleic acid (polynucleotide) of the present invention, and the production methods thereof will be described.

  The polypeptide (b) of the present invention is preferably SEQ ID NO: 29, 36, 38, 44, 46, 51, 53, 58, 60, 66, 68, 71, 73, 75, 79 in the sequence listing. More preferably, one or more of substitutions, deletions, insertions or additions of 1 to 20 amino acids have occurred in at least one amino acid sequence selected from the group consisting of amino acid sequences represented by 83, 85 In which any one or more of substitution, deletion, insertion or addition of 1 to 10 amino acids has occurred, more preferably any one of substitution, deletion, insertion or addition of 1 to 5 amino acids Examples in which the above has occurred are exemplified. Here, examples of the addition include addition of a tag sequence in peptide purification such as a His tag sequence at the N-terminal of the polypeptide, addition of a linker sequence in protein purification and the like. Examples of the deletion include removal of a tag sequence from the polypeptide having the tag sequence, removal of a methionine residue at the N-terminus of the polypeptide, and the like. The amino acid substitution and the like may be such that the physicochemical properties of the polypeptide can be changed within a range in which the original function of the polypeptide can be maintained. The amino acid substitution and the like at that time are preferably those that can change the physicochemical properties of the polypeptide within the range in which the function of the original polypeptide can be maintained.

  It is represented by SEQ ID NO: 30, 37, 39, 45, 47, 52, 54, 59, 61, 67, 69, 72, 74, 76, 80, 84 and 86 in the sequence listing encoding the polypeptide of the present invention. A nucleic acid selected from the group consisting of a base sequence is a nucleic acid encoding a natural FN or a recombinant FN fragment such as a nucleic acid encoding the above-mentioned CH-296, H-296, etc. The nucleic acid encoding each domain constituting the polypeptide can be obtained by connecting them by a known method, or by introducing a range at an appropriate location by the PCR method. For example, it can be obtained by the method described in Examples 6 to 17 described later.

A nucleotide sequence selected from the group consisting of the nucleotide sequences represented by SEQ ID NOs: 30, 37, 39, 45, 47, 52, 54, 59, 61, 67, 69, 72, 74, 76, 80, 84 and 86 The nucleic acid of the present invention also includes a nucleic acid that encodes a polypeptide that hybridizes under stringent conditions with a nucleic acid comprising a polypeptide having a function equivalent to that of the polypeptide of the present invention, that is, a desired effect in the production of the lymphocytes described above. It is. The “stringent conditions” are not particularly limited. For example, depending on the DNA to be hybridized to DNA consisting of these base sequences, the temperature and salt concentration at the time of hybridization, preferably at the time of washing, are appropriately determined. may be set by, but stringent conditions, for example, Molecular cloning A Laboratory manual, third Edition [San Bourke (Sambrook) et al, Molecular cloning, A laboratory manual 3 rd edition, 2001 years, cold spring Harbor Laboratory Conditions described in documents such as a press (published by Cold Spring Harbor Laboratory Press).

  Specifically, for example, 6 × SSC (1 × SSC is 0.15M NaCl, 0.015M sodium citrate, pH 7.0), 0.5% SDS and 5 × Denhardt [Denhardt's, 0.1 For example, conditions of incubation at 50 ° C., preferably 65 ° C., in a solution containing 100% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% Ficoll 400] and 100 μg / mL salmon sperm DNA. . When the Tm value of the DNA used is known, the temperature may be 5 to 12 ° C. lower than that value. Further, a step of removing non-specifically hybridized DNA by washing. Here, from the viewpoint of improving accuracy, lower ionic strength, for example, 2 × SSC, more stringent, 0.1 × SSC, etc. Depending on the conditions and / or higher temperatures, for example, the Tm value of the nucleic acid used, 25 ° C. or higher, more stringent 37 ° C. or higher, more stringent 42 ° C. or higher, and even more stringent In addition, a condition that washing is performed under a condition such as 50 ° C. or higher may be added.

Also encompassed by the invention are nucleic acid molecules that hybridize to the polynucleotides of the invention at lower stringency hybridization conditions. Changes in hybridization stringency and signal detection are made primarily by manipulation of formamide concentration (lower percentages of formamide result in reduced stringency), salt concentration, or temperature. For example, lower stringency conditions include: 6 × SSPE (20 × SSPE = 3 M NaCl; 0.2 M NaH ₂ PO ₄ ; 0.02 M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 μg / Incubate overnight at 37 ° C. in a solution containing mL salmon sperm blocking DNA; then wash at 50 ° C. with 1 × SSPE, 0.1% SDS. Furthermore, to achieve lower stringency, washing performed after stringent hybridization can be performed at higher salt concentrations (eg, 5 × SSC).

  The above conditions can be modified by adding and / or replacing alternative blocking reagents that are used to suppress background in hybridization experiments. Exemplary blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercial product formulations. In addition, depending on this modification, other elements of the above hybridization conditions may need to be modified.

  The nucleic acids of the present invention are represented by SEQ ID NOs: 30, 37, 39, 45, 47, 52, 54, 59, 61, 67, 69, 72, 74, 76, 80, 84 and 86 in the sequence listing. In the base sequence of a nucleic acid selected from the group consisting of a base sequence, one having one or more of substitution, deletion, insertion or addition of one to several bases is included. For example, 1 to 60 bases from the base sequence described in SEQ ID NO: 30, 37, 39, 45, 47, 52, 54, 59, 61, 67, 69, 72, 74, 76, 80, 84 or 86 in the sequence listing Those in which any one or more of substitution, deletion, insertion or addition has occurred, more preferably those in which any one or more of substitution, deletion, insertion or addition of 1 to 30 bases has occurred, and more preferably 1 Examples in which at least one of substitution, deletion, insertion or addition of ˜15 bases occurred are exemplified. The base substitution or the like may be such that the physicochemical properties of the polypeptide can be changed within a range in which the function of the polypeptide encoded by the nucleic acid can be maintained. Details of the method, such as base substitution, are the same as those described above for amino acid substitution.

  On the other hand, using the nucleic acid thus obtained, the polypeptide of the present invention, preferably SEQ ID NO: 29, 36, 38, 44, 46, 51, 53, 58, 60, 66, 68, A polypeptide having an amino acid sequence selected from the group consisting of amino acid sequences represented by 71, 73, 75, 79, 83, and 85 can be obtained by genetic engineering. That is, the nucleic acid is obtained by inserting the nucleic acid into an appropriate expression vector, for example, a pET vector, a pCold vector, etc., and by expressing the polypeptide in, for example, Escherichia coli using a known method. Can do.

  Since a high expansion culture rate is realized by performing expansion culture of a cell population containing T cells in the presence of the polypeptide of the present invention, the polypeptide is extremely useful. Further, among the polypeptides, the amino acid sequences represented by SEQ ID NOs: 79, 83 and 85 in the sequence listing are polypeptides containing mutations in the heparin binding domain and / or VLA-4 binding domain of CH-296. It is also extremely useful in functional studies of FN and FN heparin-binding domains or VLA-4 binding domains. The method for producing the polypeptide is not particularly limited, and can be produced by a known method using a nucleic acid selected from the above (c) to (g), for example, the methods of Examples 6 to 17 Can be manufactured.

  The present invention also provides a method for producing a cell population comprising the step of culturing a cell population containing T cells in the presence of the polypeptide selected from (a) and (b). The culturing step is preferably performed in the presence of a CD3 ligand. Other culture conditions are not particularly limited, and can be carried out, for example, by the method described in <3> the method for producing a cell population of the present invention. Furthermore, the polypeptides represented by the above (a) and (b) can also be used for gene transfer into cells using viral vectors. That is, a gene can be introduced into a cell with high efficiency by carrying out gene introduction into a cell using a viral vector in the presence of the polypeptide. Here, the viral vector is not particularly limited, and examples thereof include the various viral vectors described above.

  In this specification, the production of a cell or a cell population refers to a process including each step of induction (activation), maintenance, and expansion of the cell or cell population, or a combination of these.

  The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to the following examples. In the following examples, cells are cultured using culture equipment in which the stimulating factor used in each example is immobilized, and the stimulating factor is soluble and cultured in a culture solution added to the culture solution. In addition, culturing using magnetic beads or the like on which a stimulating factor is immobilized may be referred to as “stimulation”.

Example 1 Expansion of CD4-positive T cells using FN fragment CH-296-1
(1) Isolation and storage of PBMC After collecting blood components from a healthy human donor with informed consent, the collected blood was phosphate buffered saline (Baxter, hereinafter referred to as PBS) or Dulbeccoline. After diluting 2 times with acid buffered saline (Invitrogen or Sigma, hereinafter referred to as DPBS) and overlaying on Ficoll-paque (Amersham Biosciences or GE Healthcare Biosciences), 700 Centrifuged at xg for 20 minutes. After centrifugation, peripheral blood mononuclear cells (PBMC) in the intermediate layer were collected with a pipette and washed. The collected PBMC was suspended in RPMI 1640 medium (manufactured by Sigma, hereinafter referred to as RPMI 1640) to 5 × 10 ⁷ cells / mL, and then CP-1 (manufactured by Kyokuto Pharmaceutical) and 25% human serum albumin (buminate). : Baxter) was added and suspended in an equal volume of 17: 8 and stored in liquid nitrogen. For expanded lymphocyte culture, these preserved PBMCs were rapidly thawed in a 37 ° C. water bath, washed with a test medium containing 10 μg / mL DNase (manufactured by Calbiochem), and the number of viable cells was determined by trypan blue staining. Calculated and used for each experiment.

(2) Immobilization of anti-human CD3 antibody and CH-296 The culture equipment used in the following experiments has an anti-human CD3 antibody (OKT3, manufactured by Janssen Pharma) and the amino acid sequence represented by SEQ ID NO: 13 in the sequence listing. An FN fragment [trade name: Retronectin (registered trademark), manufactured by Takara Bio Inc., hereinafter referred to as CH-296] was immobilized. That is, in a 24-well cell culture plate (Corning), an ACD-A solution containing anti-human CD3 antibody having a final concentration of 5 μg / mL and CH-296 having a final concentration of 25 μg / mL or only CH-296 having a final concentration of 25 μg / mL. After adding the ACD-A solution containing 0.45 mL / well, the culture equipment was incubated at room temperature for 5 hours and then stored at 4 ° C. until use. Immediately before use, the ACD-A solution containing the antibody and CH-296 was aspirated and removed from the culture equipment, and each well was washed twice with PBS and once with RPMI 1640 and used for each experiment.

(3) Preparation of CD8-Removed Cells 1 × 10 of PBMC prepared in Example 1- (1) using PBS containing 1% human AB serum (Cambrex) (hereinafter referred to as 1% HS / PBS) After suspension to ⁷ cells / mL, CD8 positive selection beads (Dynabeads M-450 CD8: manufactured by Invitrogen) washed with 1% HS / PBS are 2 × 10 ⁷ beads per PBMC 1 × 10 ⁷ cells. And gently agitated at 4 ° C. for 1 hour using a rotator, and then allowed to stand on a magnetic separator MPC-1 (manufactured by Dynal) for 2 to 3 minutes to recover non-bead-bound cells (hereinafter referred to as “beads”). Described as CD8-depleted cells). The collected CD8-removed cells are centrifuged at 500 × g for 5 minutes, and then GT-T503 (produced by Takara Bio Inc., hereinafter referred to as 2% GT-T503) containing 2% human AB serum and 0.2% human serum albumin or 2 × 10 ⁶ cells / mL using 5% human AB serum, 2 mM L-Glutamine (Cambrex), X-VIVO15 containing 20 mM Hepes (Cambrex, hereinafter referred to as 5% X-VIVO15) It was suspended in.

(4) Expansion culture of cells prepared in Example 1- (3) 2% on the anti-human CD3 antibody and CH-296 immobilized plate or CH-296 immobilized plate prepared in Example 1- (2) After adding GT-T503 or 5% X-VIVO15 at 0.5 mL / well, the cell suspension prepared in Example 1- (3) was added at 0.5 mL / well. In a well in which only CH-296 is immobilized, cells containing anti-CD3 antibody / anti-CD28 antibody beads washed with 1% HS / PBS (Dynabeads CD3 / CD28 T cell Expander: manufactured by Invitrogen: hereinafter referred to as T cell Expander) Three times the amount was added. Subsequently, IL-2 (Proleukin: manufactured by Chiron) was added to a final concentration of 200 U / mL for the 2% GT-T503 culture group and a final concentration of 100 U / mL for the 5% X-VIVO15 culture group. These plates were cultured at 37 ° C. in a 5% CO ₂ incubator (culture day 0). On the 3rd day from the start of the culture, the 2% GT-T503 culture group was diluted about 6 times with 2% GT-T503, and 4 mL of the diluted solution was not immobilized on a 12-well cell culture plate (Corning). In the 5% X-VIVO culture group, 0.8 mL of the culture solution was transferred to a 12-well cell culture plate on which nothing was immobilized. On the 6th day of culture, the 2% GT-T503 culture group was diluted about 2-fold, and then transferred to a standing T25 cell culture flask (Corning Inc.) in which 6 mL of the diluted solution was not immobilized. About the% X-VIVO15 culture group, after diluting so that it might become 0.5 * 10 < ⁶ > cells / mL, 1.5 mL of dilution liquid was moved to the 12-well cell culture plate which has not fix | immobilized anything. On the 10th day of the culture, the 2% GT-T503 culture group was diluted about 2-fold, then transferred to a standing T25 cell culture flask in which 10 mL of the diluted solution was not immobilized, and cultured at 5% X-VIVO15. About the group, after diluting so that it might be set to 0.5 * 10 < ⁶ > cells / mL, it moved to what set up the T25 cell culture flask which did not fix | immobilize 3 mL of diluents. On any culture day, IL-2 was added so that the final concentration was 200 U / mL in the 2% GT-T503 culture group and the final concentration was 100 U / mL in the 5% X-VIVO15 culture group. Table 1 shows the expansion rate on day 6 after the start of the culture. The expansion culture rate was calculated by measuring the number of viable cells by trypan blue staining and comparing it with the number of cells at the start of culture. In the table, “+” means “and”, and the same applies to the following tables.

  As shown in Table 1, the group in which the anti-CD3 antibody and CH-296 were immobilized in the early stage of CD8-removed cell expansion culture obtained a higher expansion culture rate than the group stimulated with T cell Expander and CH-296. It was. That is, it has been clarified that the combination of anti-CD3 antibody and CH-296 is suitably used during the CD8-removed cell culture in the initial stimulation.

(5) Analysis of CD45RA-positive CCR7-positive T cells The cells on day 12 of culture obtained in Example 1- (4) were PBS containing 1% bovine serum albumin (BSA) (manufactured by Sigma) (hereinafter 1). % BSA / PBS). Cells were suspended in 1% BSA / PBS, and FITC-labeled mouse IgG ₁ / RD1-labeled mouse IgG ₁ / PC5-labeled mouse IgG ₁ (manufactured by Beckman Coulter, Inc.) was added as a negative control. Similarly, RD1-labeled mouse anti-human CD45RA antibody (manufactured by Beckman Coulter) / FITC-labeled mouse anti-human CCR7 antibody (manufactured by R & D Systems) was added to the expression detection cells, followed by incubation on ice for 30 minutes. After incubation, the cells were washed with PBS containing 0.1% BSA and resuspended in PBS. The cells were subjected to flow cytometry (Cytomics FC500: manufactured by Beckman Coulter, Inc.), and the content of CD45RA positive CCR7 positive T cells was calculated for each cell population. The results are shown in Table 2.

  As shown in Table 2, the group in which the anti-CD3 antibody and CH-296 were immobilized in the early stage of CD8-removed cell expansion culture was compared with the group stimulated with T cell Expander and CH-296, and the CD45RA positive CCR7 positive T The cell content was high. CD45RA-positive CCR7-positive T cells are a phenotype found in naive T cells, and an effect can be expected when lymphocytes after expanded culture are returned to the body. Therefore, it was revealed that the combination of anti-CD3 antibody and CH-296 can proliferate naive T-like cells with high efficiency.

(6) Analysis of IL-4 negative IFN-γ positive T cells The cells on day 12 of culture obtained in Example 1- (4) were obtained from 10% fetal bovine serum (MP Biomedicals). ) were suspended RPMI1640 (to be less than 10FRPMI as described) to 2 × ¹⁰ 6 cells / mL containing, in advance 10FRPMI to 0.4 mL / well each added with 48 well cell culture plate (Corning) 0. 2 mL / well was infused. PMA (Phorobol 12-myristate 13-acetate: manufactured by BIOMOL) at a final concentration of 50 ng / mL, Ionomycin (manufactured by Sigma) at a final concentration of 1 μg / mL, and Brefeldin A (manufactured by Sigma) at a final concentration of 10 μg / mL. After adding to each well, it was cultured for 4 hours in a 37 ° C., 5% CO ₂ incubator. Cells after completion of the culture were collected and washed with PBS, 1% BSA / PBS. The cells were suspended in 1% BSA / PBS, ECD-labeled mouse IgG ₁ or ECD-labeled mouse anti-human CD4 antibody (both manufactured by Beckman Coulter) was added, and the mixture was incubated on ice for 30 minutes, and then IntraPrep Regent (Beckman Coulter, Inc.). ), Intracellular staining was performed with FITC-labeled mouse anti-human IL-4 antibody (manufactured by eBioscience) and RD1-labeled mouse anti-human IFN-γ antibody (manufactured by Beckman Coulter). As negative controls, FITC-labeled mouse IgG ₁ (Beckman Coulter) and PE-labeled mouse IgG ₁ (Dako) were used. The cells were subjected to flow cytometry, and the content of IL-4 negative IFN-γ positive T cells was measured for each cell population. The results are shown in Table 3. In all conditions, it was confirmed that the content of CD4 positive T cells was 85% or more.

  Table 3 shows that cells stimulated with anti-CD3 antibody and CH-296 had a higher content of IL-4 negative IFN-γ positive than those stimulated with T cell Expander and CH-296. . Th1-type activated CD4-positive T cells are known to produce IFN-γ, and Th1-type CD4-positive T cells useful for cellular immunity by initial stimulation with anti-CD3 antibody and CH-296 The content was shown to be high.

Example 2 Expansion of CD4-positive T cells using CH-296-2
(1) Expansion culture of CD8-removed cells CD8-removed cells were prepared from the PBMCs separated and stored by the method described in Example 1- (1) by the method described in Example 1- (3). Using the plate fixed by the method (2), expansion culture was performed in the same manner as in Example 1- (4). However, a 12-well cell culture plate is used as the culture equipment, and at the start of culture, cell suspension is added 1 mL / well at a time to 2% GT-T503 or 5% X-VIVO15 at 3 mL / well. did. Further, Dynabeads CD3 / CD28 (manufactured by Invitrogen: hereinafter referred to as CD3 / CD28 beads) was used as the anti-CD3 antibody / anti-CD28 antibody bead. On the 3rd day from the start of the culture, the 2% GT-T503 culture group was diluted about 3 times with 2% GT-T503, and 6 mL of the diluted solution was not immobilized on any T25 cell culture flask (manufactured by Corning) The 5% X-VIVO culture group was transferred to a standing T25 cell culture flask in which no culture solution (3.4 mL) was immobilized. On the 6th day of culture, the 2% GT-T503 culture group was diluted about 2.2 times, and then transferred to a standing T25 cell culture flask in which 10.5 mL of the diluted solution was not immobilized. For the X-VIVO15 culture group, 1.6 mL of the culture solution was diluted with 5% X-VIVO15 to 0.5 × 10 ⁶ cells / mL and then transferred to a T25 cell culture flask. On the 10th day of culture, 9.5 mL of 2% GT-T503 medium was added for the 2% GT-T503 culture group, and the conditions for the CD3 antibody and CH-296 were about 5% X-VIVO15 culture group. The condition with 8.5 mL, CD3 / CD28 beads and CH-296 added about 11.5 mL of medium. Table 4 shows the expansion rate on day 6 after the start of the culture.

  As shown in Table 4, the group in which the anti-CD3 antibody and CH-296 were immobilized in the early stage of CD8-removed cell expansion culture obtained a higher expansion culture ratio than the stimulation group of CD3 / CD28 beads and CH-296. It was. That is, it has been clarified that the combination of the anti-CD3 antibody and CH-296 is suitably used for the expansion culture of CD8-removed cells in the initial stimulation.

(2) Analysis of IL-4 negative IFN-γ positive T cells The cells on day 12 of culture obtained in Example 2- (1) were IL-4 negative by the method described in Example 1- (6). IFN-γ positive T cells were measured. The results are shown in Table 5. In all conditions, it was confirmed that the content of CD4 positive T cells was 85% or more.

  As shown in Table 5, cells primed with anti-CD3 antibody and CH-296 show IL-4 negative IFN-γ positive T even when compared to cells stimulated with different CD3 / CD28 bead products. Results with high cell content were obtained, indicating that Th1-type CD4-positive T cells proliferate preferentially.

Example 3 Expansion of CD4-positive T cells using CH-296-3
(1) Preparation of CD3 / CD28 bead selection cells CD8-removed cells were prepared in the same manner as in Example 1- (3) using PBMC separated and stored by the method described in Example 1- (1). However, the cell concentration after preparation was 4 × 10 ⁶ cells / mL. The required amount of CD8-removed cells is taken into a 1.5 mL screw-type tube (manufactured by Assist), centrifuged at 500 × g for 5 minutes, the supernatant is removed, and 5 × 10 ⁷ cells / mL using 1% HS / PBS It prepared so that it might become. Three times the amount of CD3 / CD28 beads washed with 1% HS / PBS was added to the number of cells, and gently stirred at 4 ° C. for 30 minutes using a rotator. After completion of the reaction, an appropriate amount of 1% HS / PBS was added and gently stirred, and then allowed to stand in MPC for 2 to 3 minutes to remove non-bead-bound cells. The bead-bound cells were first suspended in 5% X-VIVO15 so that the number of cells collected in a 1.5 mL screw-type tube was 4 × 10 ⁶ cells / mL. Hereinafter, this cell is referred to as a T cell selection cell.

(2) Expansion culture of CD4 positive T cells (i) Preparation of group without T cell selection Anti-CD3 antibody and CH-296 or CH-296 alone were immobilized in the same manner as in Example 1- (2) 12 The CD8-removed cells prepared in Example 3- (1) were added to the well cell culture plate at 0.5 mL / well. Of these, to wells in which only CH-296 was immobilized, CD3 / CD28 beads washed with 1% HS / PBS were added in 3 times the number of cells, and each was made up to 4 mL using 5% X-VIVO15. Cells prepared and treated under these conditions were taken as the group without T cell selection.
(Ii) Preparation of group with T cell selection The T cell selection cell prepared in Example 3- (1) was added to a well in which only CH-296 was immobilized in the same manner as in Example 1- (2). Wells were added and adjusted to 4 mL with 5% X-VIVO15. Cells treated under these conditions were designated as T cell selection group.
(Iii) above (i), the plates of the (ii), 37 ° C., and culturing was started at 5% CO ₂ incubator (culture day 0). On day 3 of culture start, the T cell selection group was diluted approximately 6.3 times with 5% X-VIVO15, and a T25 cell culture flask in which 20 mL of the diluted solution was not fixed was set up. For the group with T cell selection, dilute 3.2 mL of the cell culture solution to 0.05 × 10 ⁶ cells / mL, and set up a T25 cell culture flask in which no dilution was immobilized. Moved. On the 6th day of culture, 20 mL of diluted solution diluted with 2-fold using 5% X-VIVO15 was added to the group without T cell selection, and 20 mL of diluted solution was diluted about 4.7 times with the group with T cell selection. Each was transferred to a T25 cell culture flask in which nothing was immobilized, and the culture was continued for 10 days. In addition, all IL-2 was added so that it might become final concentration of 100 U / mL. Table 6 shows the expansion culture rate on the 6th day after the start of the culture.

  As shown in Table 6, the initial stimulation with anti-CD3 antibody and CH-296 alone showed the highest expansion culture rate. That is, it became clear that the combination of anti-CD3 antibody and CH-296 is suitable.

(3) Analysis of IL-4 negative IFN-γ positive T cells The cells on day 10 of culture obtained in Example 3- (2) were IL-4 negative by the method described in Example 1- (6). The content of IFN-γ positive T cells was measured. The results are shown in Table 7. In all conditions, it was confirmed that the content of CD4 positive T cells was 85% or more.

  As shown in Table 7, cells initially stimulated with anti-CD3 antibody and CH-296 had a content of IL-4 negative IFN-γ positive T cells compared to cells stimulated with CD3 / CD28 beads. Was expensive. That is, it was revealed that Th1 type CD4 positive T cells proliferate preferentially in the combination of anti-CD3 antibody and CH-296 in the initial stimulation.

Example 4 Influence of the ratio of CD4 positive T cells and CD8 positive T cells at the start of culture in expansion culture of CD8 positive CD45RA positive CCR7 positive T cells (1) Immobilization of anti-human CD3 antibody and CH-296 Example 1 It carried out like (2). However, the culture equipment used for immobilization was a 12-well cell culture plate, and a group for immobilizing only anti-CD3 antibody was also set.

(2) Preparation of CD8-positive T cells CD8-positive T cells are separated from PBMCs prepared in Example 1- (1) by the same method as in Example 1- (3). Was removed to obtain bead-bound cells. The collected bead-bound cells are suspended to 1 × 10 ⁸ cells / mL using 1% HS / PBS, and then detacher beads (DETACHABEAD CD4 / CD8: manufactured by Invitrogen) are used as cells 1 × 10 ⁷ cells. On the other hand, 10 μL was added and gently agitated at room temperature for 1 hour using a rotator, and then allowed to stand in MPC for 2 to 3 minutes to recover non-bead-bound cells (hereinafter referred to as CD8-positive T cells). The collected CD8 positive T cells are centrifuged at 500 × g for 5 minutes, and then GT-503 (hereinafter referred to as 0.5% GT-T503) containing 0.5% human AB serum and 0.2% human serum albumin is used. And suspended at 0.25 × 10 ⁶ cells / mL.

(3) Preparation of CD4-positive T cells After suspending the PBMC prepared in Example 1- (1) to 1 × 10 ⁷ cells / mL using 1% HS / PBS, 1% HS / PBS in washed CD4 positive selection beads (CD4 positive Isolation Kit: manufactured by Invitrogen, hereinafter, the reagents used in this step using the Kit supplied) added at 1 × 10 ⁷ beads per PBMC1 × ¹⁰ 7 cells Then, after gently stirring at 4 ° C. for 1 hour using a rotator, the beads were left undissolved in MPC for 2 to 3 minutes to remove bead-unbound cells, and bead-bound cells were recovered (recovered in a bead-bound state). Thereafter, in the same manner as in Example 4- (2), non-bead-bound cells were collected using detacher beads (hereinafter referred to as CD4-positive T cells), centrifuged at 500 × g for 5 minutes, 0.5 It was suspended using% GT-T503 so that it might become 0.25 * 10 < ⁶ > cells / mL.

(4) Preparation of monosite The PBMC prepared in Example 1- (1) was suspended in 1% HS / PBS to 1 × 10 ⁸ cells / mL, and then per 1 × 10 ⁷ cells of PBMC. 20 μL of Blocking Reagent (Monocyte Negative Isolation Kit: manufactured by Invitrogen) . Thereafter, the supernatant was removed by centrifugation 5 min at 500 × g, consisting of monocyte negative selection beads washed PBMC were suspended in 1% HS / PBS and ^{PBMC 1 × 10 7 cells 1 ×} 10 7 beads per Then, the mixture was gently stirred at 4 ° C. for 15 minutes using a rotator, and then allowed to stand in MPC for 2 to 3 minutes to recover non-bead-bound cells (hereinafter referred to as monosite). The collected monosite was centrifuged at 500 × g for 5 minutes, and then suspended with 0.5% GT-T503 so as to be 1 × 10 ⁵ cells / mL.

(5) Expansion culture of cells prepared in Example 4- (2), (3), and (4) Immobilization of anti-CD3 antibody alone or anti-CD3 antibody and CH-296 in Example 4- (1) After adding 0.5% GT-T503 at 0.25 mL / well to the prepared plate, 0.25 mL / well of the monosite suspension prepared in Example 4- (4) was added. As a ratio of CD4 positive T cell: CD8 positive T cell, 5 conditions of CD4 positive T cell: CD8 positive T cell = 0: 1, 0.5: 1, 1: 1, 2: 1, 4: 1 are set. Example 4-, so as to meet the ratio of the individual conditions, and so that the total number of cells in each well is 0.25 × 10 ⁶ cells (excluding the cells prepared in Example 4- (4)). The CD8 positive T cell suspension and the CD4 positive T cell suspension prepared in (2) and (3) were added. Thereafter, IL-2 was added to each well to a final concentration of 200 U / mL, and these plates were cultured in a 37 ° C., 5% CO ₂ incubator (culture day 0). On the 4th day from the start of the culture, a T-25 cell culture flask (manufactured by Corning) was prepared by diluting about 14 times with 0.5% GT-T503 and fixing 10 mL of the diluted solution. Moved. On the 7th day of culture, it was diluted about 2-fold with 0.5% GT-T503 and transferred to a standing T-25 cell culture flask in which 10 mL of the diluted solution was not immobilized. On the 11th day of culture, it was diluted about 2-fold using GT-T503 containing 0.2% human serum albumin (hereinafter referred to as 0% GT-T503) to a total volume of 20 mL. The 14th day of culture was regarded as the experiment end date. On any culture day, IL-2 was added to a final concentration of 200 U / mL.

(6) Analysis of CD8 + CD45RA + CCR7 + T cells PBMC prepared on the 0th day of culture and each cell on the 14th day of culture obtained in Example 4- (5) are the same as in Example 1- (5). The antibody staining was performed by the method described above, and the content of CD8-positive CD45RA-positive CCR7-positive T cells was calculated. Here, the negative control of Example 1- (5) was used as the negative control, and the RD1-labeled mouse anti-human CD45RA antibody / FITC-labeled mouse anti-human CCR7 antibody and PC5-labeled mouse anti-human were similarly used for the expression detection cells. CD8 antibody (manufactured by Beckman Coulter) was added. Table 8 shows the expansion rate of CD8-positive CD45RA-positive CCR7-positive T cells on the 14th day of culture. In addition, the CD8 positive CD45RA positive CCR7 positive T cell expansion culture rate in the table is the number of CD8 positive CD45RA positive CCR7 positive T cells contained in the cell population on the 14th day of culture and the CD8 contained in the cell population at the start of the culture. It calculated by comparing with the number of positive CD45RA positive CCR7 positive T cells. The number of CD8-positive CD45RA-positive CCR7-positive T cells was obtained by multiplying the total number of cells obtained by the ratio of CD8-positive CD45RA-positive CCR7-positive T cells.

  As shown in Table 8, as the ratio of CD4 positive T cells to CD8 positive T cells in the cell population at the start of culture increases, the expansion culture rate of CD8 positive CD45RA positive CCR7 positive T cells increases. It was shown that the effect was more remarkable when expansion culture was performed using CH-296 in addition to the anti-CD3 antibody.

Example 5 Suppressive effect of IL-5 and IL-9 production of T cells obtained by combined stimulation of anti-human CD3 antibody and CH-296 (1) Anti-human CD3 antibody, anti-human CD28 antibody, and CH-296 Immobilization Immobilization of anti-human CD3 antibody alone or a plate combining anti-human CD3 antibody and CH-296 was carried out in the same manner as in Example 4- (1). Further, immobilization combining an anti-human CD3 antibody and an anti-human CD28 antibody was prepared in an ACD-A solution to a final concentration of 5 μg / mL, and was performed in the same manner as in Example 4- (1).

(2) Preparation of CD4-positive T cells CD4-positive T cells were prepared in the same manner as in Example 4- (3).

(3) Expansion culture of CD4-positive T cells After adding 0.5% GT-T503 at 0.5 mL / well to each of the immobilized plates prepared in Example 5- (1), Example 5- (2 The CD4 positive T cell suspension prepared in step 1) was added at 1.0 mL / well. Thereafter, IL-2 was added to each well to a final concentration of 500 U / mL, and these plates were cultured in a 37 ° C., 5% CO ₂ incubator (culture day 0). On the 4th day from the start of culture, the mixture was diluted about 14 times with 0.5% GT-T503, and 2.0 mL of the diluted solution was transferred to a 12-well cell culture plate on which nothing was fixed. On the 7th day of culture, it was diluted about 2-fold with 0.5% GT-T503, and 2.0 mL of the diluted solution was transferred to a 12-well cell culture plate on which nothing was immobilized. On the 11th day of culture, the solution was diluted approximately 2-fold with 0% GT-T503 to a total volume of 4.0 mL, and the culture was continued until the 14th day of culture. On any passage day, IL-2 was added to a final concentration of 500 U / mL. Table 9 shows the expansion rate on the 14th day after the start of the culture.

  As shown in Table 9, it was found that the group in which the anti-CD3 antibody and CH-296 were combined obtained a high expansion culture rate as compared with the control group. Furthermore, a higher expansion culture rate was obtained when the anti-CD3 antibody and CH-296 were combined than when the anti-CD3 antibody and anti-CD28 antibody were combined.

(4) Detection of IL-5 in the culture supernatant IL-5 in the culture supernatants on the second, fourth, seventh, and eleventh days in the expansion culture in Example 5- (3) The concentration was measured using a human IL-5 ELISA KIT (manufactured by PIERCE). For measurement, the instructions attached to the kit were followed. However, the culture supernatant used was collected prior to the subculture operation except for the second day of culture, and was frozen and stored at −80 ° C. until the measurement date before use at room temperature.

(5) Detection of IL-9 in culture supernatant 0.5 mg / mL Purified anti-human IL-9 (manufactured by BioLegend, MH9A4) (hereinafter referred to as Capture antibody) was added to 20 mM glycine, 30 mM NaCl, pH 9. The resulting solution was diluted to 2.5 μg / mL with the solution of No. 2 (hereinafter referred to as “Coating buffer”), added to an ELASA plate (manufactured by Nunc) at 100 μL / well, and stored at 4 ° C. protected from light. The plate preserved the next day was taken out, PBS containing 0.05% Tween 20 (manufactured by Sigma) (hereinafter referred to as “Wash buffer”) was added at 200 μL / well, and the supernatant was removed with an aspirator. This operation was repeated three times to wash the inside of the well. Next, PBS containing 1% BSA (hereinafter referred to as “Blocking Buffer”) was added at 300 μL / well, and left in a 37 ° C. CO ₂ incubator for 2 hours. Thereafter, 200 μL / well of wash buffer was added, and the supernatant was removed with an aspirator. This operation was repeated three times to wash the inside of the well. Next, in Example 5- (3), the culture supernatant was collected on the second day, the fourth day, the seventh day, and the eleventh day during the expansion culture. And frozen at -80 ° C. until the measurement date, thawed at room temperature and added to 100 μL / well, and standard IL-9 (10-fold serial dilution from 500 pg / mL to 5 pg / mL) Were added at 100 μL / well and allowed to stand at room temperature for 2 hours. Wash buffer was added at 200 μL / well, and the supernatant was removed with an aspirator. This operation was repeated three times to thoroughly wash the inside of the well. Biotin anti-human IL-9 (MH9A3) (hereinafter referred to as detection antibody) diluted 10-fold with PBS and diluted 2000-fold with Blocking buffer was added at 100 μL / well, and ₂ in a CO ₂ incubator. Left for hours. Wash buffer was added at 200 μL / well, and the supernatant was removed with an aspirator. After repeating this operation three times, SA-HRP (Hts, HE101) diluted 10-fold with PBS and diluted 500-fold with Blocking buffer was added at 100 μL / well and allowed to stand at room temperature for 30 minutes. After that, Wash buffer was added at 200 μL / well, and the supernatant was removed with an aspirator. This operation was repeated three times. ABTS Peroxidase Substrate (manufactured by KPL, 50-66-01) (hereinafter referred to as “substrate”) was added at 100 μL / well, and light-shielded at room temperature for 10 minutes (shortened as needed while watching the state). 50 μL / well of 150 mM oxalic acid (hereinafter referred to as “stop solution”) was added, and measurement was performed with a plate reader (wavelength 405 nm).

  Tables 10 and 11 show the concentrations of IL-5 and IL-9 (pg / mL) contained in the supernatant cultured under each stimulation condition for each number of culture days. In this table, the cytokine production ratio under each stimulation condition when the concentration of each cytokine under the stimulation condition combining the anti-CD3 antibody and CH-296 is 1 is shown. N. in the table. T.A. Means Not Test.

  As shown in Table 10 and Table 11, the concentrations of IL-5 and IL-9 contained in the culture supernatant throughout the culture period are different under the stimulation conditions in which anti-CD3 antibody and CH-296 are combined. It was found to be lower than the stimulation conditions with only the antibody and the stimulation conditions in which the anti-CD3 antibody and the anti-CD28 antibody were combined. From the above, stimulation with a combination of anti-CD3 antibody and CH-296 gives a higher expansion rate than stimulation with a combination of anti-CD3 antibody and anti-CD28 antibody, and IL-5 and IL-5 during T cell proliferation -9 production was shown to be low.

Example 6 Preparation of H204 (1) Construction of H204 expression vector III-13 (amino acid sequence represented by SEQ ID NO: 6 in the sequence listing), III-14 (amino acid sequence represented by SEQ ID NO: 7 in the sequence listing) and In order to obtain a modified FN fragment (H204) in which CS-1 (amino acid sequence represented by SEQ ID NO: 8 in the sequence listing) was linked in order from the N-terminus, an expression vector was constructed as follows.

(I) Construction of CH-296 expression vector CH-296 (polypeptide consisting of FN cell-binding domain, heparin-binding domain and CS-1 domain) expression vector (pCH102) was transformed into Escherichia coli HB101 / pCH102 (FERM BP-2800 This was prepared based on the description in US Pat. No. 5,198,423.

  From the base sequence of CH-296 described in SEQ ID NO: 24 of the Sequence Listing (see International Publication No. 03/080817 pamphlet), synthetic primer CH296-NdeF and synthetic primer H296 having the base sequences described in SEQ ID NO: 25 and 26 of the Sequence Listing -HindR was synthesized with a DNA synthesizer and purified by a conventional method. The synthetic primer CH-296-NdeF has a recognition sequence for the restriction enzyme NdeI at base numbers 9 to 14, and a base sequence corresponding to amino acid numbers 1 to 6 of the amino acid sequence of CH-296 at base numbers 15 to 32. Synthetic DNA. The synthetic primer H296-HindR has a recognition sequence of restriction enzyme HindIII as base numbers 11 to 16, and a base sequence corresponding to amino acid numbers 570 to 574 of the amino acid sequence of CH-296 (SEQ ID NO: 13) as base number 20 -34 is a synthetic DNA.

  PCR was performed using the synthetic primers. PCR reaction conditions are shown below. Specifically, about 0.1 μg of pCH102 as template DNA, 5 μL of 10 × Ex Taq Buffer (manufactured by Takara Bio Inc.), 5 μL of dNTP mixed solution (manufactured by Takara Bio Inc.), 10 pmol synthetic primer CH296-NdeF, 10 pmol synthetic primer H296 -HindR, 0.5 U TaKaRa Ex Taq (manufactured by Takara Bio Inc.) was added, and sterilized water was added to make the total volume 50 μL. The reaction solution was set in TaKaRa PCR Thermal Cycler SP (manufactured by Takara Bio Inc.), and 30 cycles of reaction were performed with 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 3 minutes. After completion of the reaction, 5 μL of the reaction solution was subjected to 1.0% agarose gel electrophoresis, and the target DNA fragment of about 1.7 kbp was confirmed. The remaining PCR reaction solution was electrophoresed, the fragment was recovered and purified, and ethanol precipitation was performed. The recovered DNA after ethanol precipitation was suspended in 10 μL of sterilized water and double-digested with restriction enzyme NdeI (Takara Bio) and restriction enzyme HindIII (Takara Bio). The NdeI and HindIII digests were extracted and purified by 1.0% agarose gel electrophoresis to obtain NdeI-HindIII digested DNA fragments.

  Next, a pCold14ND vector described later was digested with NdeI and HindIII, mixed with the NdeI-HindIII digested DNA fragment, and ligated using a DNA ligation kit (manufactured by Takara Bio Inc.). Thereafter, E. coli JM109 was transformed with 10 μL of the ligation reaction solution, and the transformant was grown on an LB medium (containing 50 μg / mL of ampicillin) containing agar at a concentration of 1.5% (w / v). The recombinant plasmid into which the DNA fragment encoding the target protein was inserted was confirmed by sequencing and designated pCold14NDCH-296.

(Ii) Construction of H204 expression vector Synthetic primers NVS-F-Nco and pC-TER having the base sequences described in SEQ ID NOs: 27 and 28 in the sequence listing from the base sequence of CH-296 described in SEQ ID NO: 24 in the sequence listing It was synthesized with a DNA synthesizer and purified by a conventional method. The synthetic primer NVS-F-Nco has the recognition sequence of the restriction enzyme NcoI as base numbers 10 to 15, and the base sequence corresponding to amino acid numbers 371 to 377 of the amino acid sequence of CH-296 (SEQ ID NO: 13) as base numbers 18-38 synthetic DNA. The synthetic primer pC-TER is a synthetic DNA having a sequence that anneals to the 3 ′ TER portion of the pCold14 vector described in SEQ ID NO: 28 in the Sequence Listing.

  The pCold04NC2 vector was prepared according to the method described in Examples 1 to 6 of WO99 / 27117. This pCold04NC2 vector is referred to herein as the pCold14 vector. Moreover, a vector in which the NcoI site of the pCold14 vector was converted to an NdeI site by a conventional method was prepared. This vector is referred to herein as a pCold14ND vector.

  PCR was performed using the synthetic primers. PCR reaction conditions are shown below. That is, about 0.1 μg of pCold14NDCH-296 constructed in Example 6- (1)-(i) as template DNA, 10 μL of 10 × Pyrobest Buffer II (manufactured by Takara Bio Inc.), 8 μL of dNTP mixed solution, 20 pmol of synthetic primer NVS-F-Nco, 20 pmol of synthetic primer pC-TER, 2.5 U of Pyrobest DNA Polymerase (manufactured by Takara Bio Inc.) were added, and sterilized water was added to make a total volume of 100 μL. The reaction solution was set in TaKaRa PCR Thermal Cycler SP, and 30 cycles of reaction were performed with 95 ° C. for 30 seconds and 68 ° C. for 2 minutes as one cycle. After completion of the reaction, 5 μL of the reaction solution was subjected to 1.0% agarose gel electrophoresis, and the target DNA fragment of about 0.6 kbp was confirmed. The remaining PCR reaction solution was ethanol precipitated, the recovered DNA was suspended in sterilized water, and double digested with restriction enzyme NcoI (Takara Bio) and restriction enzyme XbaI (Takara Bio). The NcoI and XbaI digests were extracted and purified by 1.0% agarose gel electrophoresis to obtain NcoI-XbaI digested DNA fragments.

  Next, a pCold14 vector digested with NcoI and XbaI was prepared, mixed with the NcoI-XbaI digested DNA fragment, and ligated using a DNA ligation kit. Thereafter, E. coli JM109 was transformed with 10 μL of the ligation reaction solution, and the transformant was grown on an LB medium (containing 50 μg / mL of ampicillin) containing agar at a concentration of 1.5% (w / v).

  A plasmid into which a DNA fragment encoding a protein in which III-13, III-14 and CS-1 were sequentially linked was confirmed by sequencing, and this recombinant plasmid was designated as pCold14-H204. This pCold14-H204 is a plasmid containing a base sequence encoding the amino acid sequence of amino acid numbers 371 to 574 of CH-296. The protein is named H204, its amino acid sequence is shown in Sequence Listing 29, and its base sequence is shown in Sequence Number 30 in the Sequence Listing.

(2) Expression and purification of H204 Escherichia coli BL21 was transformed with pCold14-H204 prepared in (1) above, and the transformant was transformed into an LB medium containing agar at a concentration of 1.5% (w / v) ( (Including 50 μg / mL ampicillin). The grown colonies were inoculated into 30 mL of LB liquid medium (containing 50 μg / mL of ampicillin) and cultured at 37 ° C. overnight. The entire amount was inoculated into 3 L of the same LB medium and cultured at 37 ° C. until the logarithmic growth phase. In this culture, a 5 L mini jar fermenter (manufactured by Biott) was used under the conditions of 120 r / min and Air = 1.0 L / min. After the culture, the mixture was cooled to 15 ° C., IPTG was added to a final concentration of 1.0 mM, and cultured at 15 ° C. for 24 hours to induce expression. Thereafter, the cells were collected by centrifugation and resuspended in about 40 mL of a cell disruption solution [50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 50 mM NaCl]. The cells were disrupted by ultrasonic disruption, and separated into a supernatant extract and a precipitate by centrifugation (11,000 r / min, 20 minutes). The obtained supernatant extract was dialyzed against 2 L of buffer A [50 mM Tris-HCl (pH 7.5), 50 mM NaCl], and purified by ion exchange chromatography using about 40 mL of the extract as follows. . That is, a column (Φ4 cm × 20 cm) in which 100 mL of SP-Sepharose (manufactured by Amersham Pharmacia) in a resin volume was saturated with buffer A was prepared, and a sample after dialysis was applied thereto. Thereafter, the target protein was eluted with 250 mL of buffer A and 250 mL of buffer B [50 mM Tris-HCl (pH 7.5), 1 M NaCl] with a concentration gradient of 50 mM to 1 M sodium chloride. Fractions containing 5 mL each were fractionated, and about 100 mL of a fraction containing a large amount of the target protein having a molecular weight of about 23 kDa was collected by 10% SDS-PAGE, and dialyzed against 2 L of buffer A. Next, a column (Φ3 cm × 16 cm) in which 50 mL of Q-Sepharose (manufactured by Amersham Pharmacia) in a resin volume was saturated with buffer A was prepared, and a sample after dialysis was applied thereto. Then, the target protein was eluted with a concentration gradient of 50 mM to 1 M sodium chloride with 250 mL of buffer A and 250 mL of buffer B. Fractions of 5 mL each were confirmed and confirmed by 10% SDS-PAGE. About 100 mL of a fraction containing a large amount of target protein having a molecular weight of about 23 kDa was recovered, and 2 L of buffer D [50 mM sodium carbonate buffer (pH 9.5)] Dialysis was performed. Thereafter, the solution was concentrated to 10 mL with Centricon-10 (manufactured by Millipore), and further confirmed with 10% SDS-PAGE. Then, when the protein concentration was measured using a MicroBCA kit (Pierce), it was 2.2 mg / mL (calculated from the molecular weight of about 96 μM).

Example 7 Construction of H296-CS1 (1) Construction of H296-CS1 Expression Vector In order to obtain a modified FN fragment (H296-CS1) to which the represented amino acid sequence was linked, an expression vector was constructed as follows.

(I) Construction of H-296 expression vector An expression vector for H-296, which is a polypeptide consisting of amino acid numbers 278 to 574 of the amino acid sequence of CH-296 (described in SEQ ID NO: 13 in the sequence listing), was prepared as follows. It was constructed.

  First, a synthetic primer H296-NcoF having a base sequence shown in SEQ ID NO: 31 in the sequence listing was synthesized from a base sequence of CH-296 shown in SEQ ID NO: 24 in the sequence listing using a DNA synthesizer and purified by a conventional method. In the synthetic primer H296-NcoF, the recognition sequence of the restriction enzyme NcoI is represented by nucleotide numbers 11 to 16, and the nucleotide sequence corresponding to amino acid numbers 278 to 283 of the amino acid sequence of CH-296 (SEQ ID NO: 13) is represented by nucleotide numbers 13 to 30 synthetic DNA.

  PCR was performed using the synthetic primer H296-NcoF and the synthetic primer H296-HindR. PCR reaction conditions are shown below. That is, about 0.1 μg of pCH102, 5 μL of 10 × Ex Taq Buffer, 5 μL of dNTP mixed solution, 10 pmol of synthetic primer H296-NcoF, 10 pmol of synthetic primer H296-HindR, 0.5 U of TaKaRa Ex Taq were added as template DNA. Then, sterilized water was added to make the total volume 50 μL. The reaction solution was set in TaKaRa PCR Thermal Cycler SP, and 30 cycles of reaction were performed with 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 3 minutes as one cycle.

  After completion of the reaction, 5 μL of the reaction solution was subjected to 1.0% agarose gel electrophoresis, and the target DNA fragment of about 0.9 kbp was confirmed. The remaining PCR reaction solution was electrophoresed, the fragment was recovered and purified, and ethanol precipitation was performed. The recovered DNA after ethanol precipitation was suspended in 10 μL of sterilized water and double-digested with restriction enzyme NcoI and restriction enzyme HindIII. The NcoI and HindIII digests were extracted and purified by 1.0% agarose gel electrophoresis to obtain NcoI-HindIII digested DNA fragments.

  Next, a pCold14 vector digested with NcoI and HindIII was prepared, mixed with the NcoI-HindIII digested DNA fragment, and ligated using a DNA ligation kit. Thereafter, E. coli JM109 was transformed with 20 μL of the ligation reaction solution, and the transformant was grown on an LB medium (containing 50 μg / mL of ampicillin) containing agar at a concentration of 1.5% (w / v).

  The plasmid in which the target DNA fragment was inserted was confirmed by sequencing, and this recombinant plasmid was designated as pCold14-H296. This pCold14-H296 is a plasmid containing a base sequence encoding the amino acid sequence of amino acid numbers 278 to 574 of CH-296.

(Ii) Construction of H296-CS1 expression vector Synthetic primer NC2-5′UTR having the base sequence described in SEQ ID NO: 32 in the sequence listing from the base sequence of CH-296 described in SEQ ID NO: 24 in the sequence listing, described in SEQ ID NO: 33 Synthetic primer CS1-no. st-CS1-R, a synthetic primer CS1-no. St-CS1-F and synthetic primer FN-5630F having the base sequence described in SEQ ID NO: 35 were synthesized by a DNA synthesizer and purified by a conventional method. The synthetic primer NC2-5′UTR is a synthetic DNA that anneals to the 5′UTR portion of the pCold14 vector. In addition, synthetic primer CS1-no. In st-CS1-R, the base sequence corresponding to amino acid numbers 554 to 550 of the amino acid sequence of CH-296 (SEQ ID NO: 13) is changed to base numbers 1 to 15, and the amino acid sequence of CH-296 (SEQ ID NO: 13) is further changed. A synthetic DNA having base sequences corresponding to amino acid numbers 574 to 569 at base numbers 16 to 33. In addition, synthetic primer CS1-no. In st-CS1-F, the nucleotide sequence corresponding to amino acid numbers 570 to 574 of the amino acid sequence of CH-296 (SEQ ID NO: 13) is changed to base numbers 1 to 15, and further the amino acid sequence of CH-296 (SEQ ID NO: 13). It is a synthetic DNA having base sequences corresponding to amino acid numbers 550 to 555 at base numbers 16 to 33. Synthetic primer FN-5630F is a synthetic DNA having base numbers 2 to 28 corresponding to amino acid numbers 426 to 434 of the amino acid sequence of CH-296 (SEQ ID NO: 13).

  PCR was performed using the synthetic primers. PCR reaction conditions are shown below. That is, about 0.1 μg of pCold14NDCH-296 constructed in Example 6- (1)-(i) as template DNA, 10 μL of 10 × Pyrobest Buffer II, 8 μL of dNTP mixed solution, 20 pmol of synthetic primer NC2-5′UTR, 20 pmol of synthetic primer CS1-no. st-CS1-R and 2.5 U Pyrobest DNA Polymerase were added, and sterilized water was added to make the total volume 100 μL. The reaction solution was set in TaKaRa PCR Thermal Cycler SP, and the reaction was carried out for 30 cycles of 98 ° C. for 1 minute, 58 ° C. for 1 minute, 72 ° C. for 4 minutes and 72 ° C. for 10 minutes. After completion of the reaction, 5 μL of the reaction solution was subjected to 1.0% agarose gel electrophoresis, and the target DNA fragment of about 1.8 kbp was confirmed. The remaining PCR reaction solution was extracted and purified by 1.0% agarose gel electrophoresis, and the recovered DNA was suspended in 10 μL of sterile water. This DNA fragment was defined as 1L.

  Furthermore, PCR was performed using the synthetic primer. PCR reaction conditions are shown below. That is, about 0.1 μg of pCold14NDCH-296 constructed in Example 6- (1)-(i) as template DNA, 10 μL of 10 × Pyrobest Buffer II, 8 μL of dNTP mixed solution, 20 pmol of synthetic primer CS1-no. St-CS1-F, 20 pmol synthetic primer pC-TER, 2.5 U Pyrobest DNA Polymerase were added, and sterilized water was added to make a total volume of 100 μL. The reaction solution was set in TaKaRa PCR Thermal Cycler SP, and the reaction was carried out for 30 cycles of 98 ° C. for 1 minute, 58 ° C. for 1 minute, 72 ° C. for 4 minutes and 72 ° C. for 10 minutes. After completion of the reaction, 5 μL of the reaction solution was subjected to 1.0% agarose gel electrophoresis, and the target DNA fragment of about 0.3 kbp was confirmed. The remaining PCR reaction solution was extracted and purified by 1.0% agarose gel electrophoresis, and the recovered DNA was suspended in 10 μL of sterile water. This DNA fragment was 3 L.

  Next, PCR was carried out using 1L and 3L as template DNA. PCR reaction conditions are shown below. Template DNA 1L and 3L diluted 100-fold with sterilized water 1 μL, 10 μL 10 × Pyrobest Buffer II, 8 μL dNTP mixture, 20 pmol synthetic primer FN-5630F, 20 pmol synthetic primer pC-TER, 2.5 U Of Pyrobest DNA Polymerase was added, and sterilized water was added to make a total volume of 100 μL. The reaction solution was set in TaKaRa PCR Thermal Cycler SP, and the reaction was performed at 30 ° C. for 1 minute at 96 ° C. for 1 minute, 58 ° C. for 1 minute, and 72 ° C. for 2 minutes for 5 minutes at 72 ° C. After completion of the reaction, 5 μL of the reaction solution was subjected to 1.0% agarose gel electrophoresis, and the target DNA fragment of about 0.7 kbp was confirmed. The remaining PCR reaction solution was recovered by 1.0% agarose gel electrophoresis, the DNA was suspended in sterilized water, and double digested with restriction enzyme SacI (Takara Bio Inc.) and restriction enzyme XbaI. The digested SacI and XbaI was extracted and purified by 1.0% agarose gel electrophoresis to obtain a SacI-XbaI digested DNA fragment of about 0.5 kbp.

  Next, pCold14-H296 constructed in Example 7- (1)-(i) was prepared by digesting with SacI and XbaI, mixed with the SacI-XbaI digested DNA fragment, and ligated using a DNA ligation kit. . Thereafter, E. coli JM109 was transformed with 10 μL of the ligation reaction solution, and the transformant was grown on an LB medium (containing 50 μg / mL of ampicillin) containing agar at a concentration of 1.5% (w / v). The recombinant plasmid in which the DNA fragment encoding the protein in which CS-1 was linked to the target H-296 was confirmed by sequencing, and this recombinant plasmid was designated as pCold14-H296-CS1.

  This pCold14-H296-CS1 includes a base sequence in which a base sequence encoding the amino acid sequence of amino acids 550-574 of CH-296 is linked to a base sequence encoding the amino acid sequence of amino acids 279-574 of CH-296. It is a plasmid. The protein is named H296-CS1, its amino acid sequence is shown in SEQ ID NO: 36 in the sequence listing, and its base sequence is shown in SEQ ID NO: 37 in the sequence listing.

(2) Expression and purification of H296-CS1 E. coli BL21 was transformed with pCold14-H296-CS1 prepared in (1) above, and expression and purification of H296-CS1 were carried out in the same manner as in Example 6- (2). went. Note that the amounts of the culture solution, cell disruption solution, resin volume, buffer A, buffer B, buffer D, fraction solution, and concentrate were appropriately changed as necessary. When the obtained concentrated liquid was confirmed by 10% SDS-PAGE, the target protein having a molecular weight of about 35 kDa was detected in almost a single band, which was designated as H296-CS1. Thereafter, when the protein concentration was measured using a MicroBCA kit, it was 2.1 mg / mL (calculated from the molecular weight, about 60 μM).

Example 8 Preparation of H296-CS2 (1) Construction of H296-CS2 expression vector To obtain a modified FN fragment (H296-CS2) in which another CS-1 was linked to the C-terminus of H296-CS1, Thus, an expression vector was constructed.

(I) Construction of H296-CS2 expression vector Synthetic primer FN-5630F and synthetic primer CS1-no. PCR was performed using st-CS1-R. PCR reaction conditions are shown below. That is, about 2 ng of pCold14-H296-CS1 constructed in Example 7- (1)-(ii) as template DNA, 10 μL of 10 × Pyrobest Buffer II, 8 μL of dNTP mixed solution, 20 pmol of synthetic primer FN-5630F, 20 pmol of Synthetic primer CS1-no. st-CS1-R and 2.5 U Pyrobest DNA Polymerase were added, and sterilized water was added to make the total volume 100 μL. The reaction solution was set in TaKaRa PCR Thermal Cycler SP, and 30 cycles of reaction were performed, with 1 cycle of 96 ° C for 1 minute and 68 ° C for 2 minutes. After completion of the reaction, 5 μL of the reaction solution was subjected to 1.0% agarose gel electrophoresis, and the target DNA fragment of about 0.5 kbp was confirmed. The remaining PCR reaction solution was extracted and purified by 3.0% agarose gel electrophoresis, and the recovered DNA was suspended in 10 μL of sterile water. This DNA fragment was designated as H296-CS1 DNA fragment.

  Furthermore, PCR was performed using the above DNA fragment. PCR reaction conditions are shown below. That is, the DNA fragment 3L prepared in Example 7- (1)-(ii) as a template DNA was diluted 100 times, and the DNA fragment H296-CS1 DNA fragment 0.5 μL, 2 μL of 10 × Pyrobest BufferII, 1. 6 μL of dNTP mixed solution and 2.5 U of Pyrobest DNA Polymerase were added, and sterilized water was added to make a total volume of 20 μL. The reaction solution was set in TaKaRa PCR Thermal Cycler SP, and the reaction was carried out for 3 cycles with 1 cycle of 96 ° C. for 1 minute and 68 ° C. for 2 minutes. After the reaction, 20 pmol synthetic primer FN-5630F, 20 pmol synthetic primer pC-TER, 3 μL 10 × Pyrobest Buffer II, 2.4 μL dNTP mixed solution, 2.5 U Pyrobest DNA Polymerase were added, and sterilized water was added. The total volume was 50 μL. The reaction solution was set in TaKaRa PCR Thermal Cycler SP, and 30 cycles of reaction were carried out with 96 ° C. for 1 minute and 68 ° C. for 2 minutes as one cycle. 5 μL of the reaction solution was subjected to 1.0% agarose gel electrophoresis, and the target DNA fragment of about 0.8 kbp was confirmed. The remaining PCR reaction solution was extracted and purified by 1.0% agarose gel electrophoresis, collected, and double-digested with restriction enzyme SacI and restriction enzyme XbaI. The digested SacI and XbaI were extracted and purified by 1.0% agarose gel electrophoresis to obtain an approximately 0.6 kbp SacI-XbaI digested DNA fragment.

  Next, pCold14-H296 constructed in Example 7- (1)-(i) was prepared by digesting with SacI and XbaI, mixed with the SacI-XbaI digested DNA fragment, and ligated using a DNA ligation kit. . Thereafter, E. coli JM109 was transformed with 10 μL of the ligation reaction solution, and the transformant was grown on an LB medium (containing 50 μg / mL of ampicillin) containing agar at a concentration of 1.5% (w / v). A recombinant plasmid in which a DNA fragment encoding another protein in which CS-1 is linked to the target H296-CS1 was inserted was confirmed by sequencing, and this recombinant plasmid was designated as pCold14-H296-CS2. .

  This pCold14-H296-CS2 has a base sequence encoding the amino acid sequence of amino acid numbers 279-574 of CH-296 and two base sequences encoding the amino acid sequence of amino acid numbers 550-574 of CH-296 linked to each other. A plasmid containing the sequence. The protein is named H296-CS2, its amino acid sequence is shown in SEQ ID NO: 38 of the sequence listing, and its base sequence is shown in SEQ ID NO: 39 of the sequence listing.

(2) Expression and purification of H296-CS2 Escherichia coli BL21 was transformed with pCold14-H296-CS2 prepared in (1) above, and expression and purification of H296-CS2 were performed in the same manner as in Example 6- (2). went. Note that the amounts of the culture solution, cell disruption solution, resin volume, buffer A, buffer B, buffer D, fraction solution, and concentrate were appropriately changed as necessary. When the obtained concentrated liquid was confirmed by 10% SDS-PAGE, the target protein having a molecular weight of about 38 kDa was detected in almost a single band, which was designated as H296-CS2. Thereafter, the protein concentration was measured using a MicroBCA kit and found to be 0.9 mg / mL (approximately 24 μM calculated from the molecular weight).

Example 9 Preparation of H114-HT and H114 (1) Construction of H114-HT expression vector His tag sequence, III-13 (amino acid sequence represented by SEQ ID NO: 6 in the sequence listing) and CS-1 (from the N-terminus) In order to obtain a modified FN fragment (H114-HT) in which the amino acid sequence represented by SEQ ID No. 8 in the sequence listing was linked, an expression vector was constructed as follows.

(I) Construction of H114-HT expression vector Synthetic primer 13-Nde-F having the base sequence described in SEQ ID NO: 40 in the sequence listing, based on the base sequence of CH-296 described in SEQ ID NO: 24 in the sequence listing, described in SEQ ID NO: 41 A synthetic primer 13-CS1-R having the nucleotide sequence of SEQ ID NO: 42, a synthetic primer 13-CS1-F having the nucleotide sequence of SEQ ID NO: 42, and a synthetic primer CS1-R-Xba having the nucleotide sequence of SEQ ID NO: 43 And purified by a conventional method. The synthetic primer 13-Nde-F has a recognition sequence for the restriction enzyme NdeI as base numbers 6 to 11, and a base sequence corresponding to amino acid numbers 371 to 377 in the amino acid sequence of CH-296 (SEQ ID NO: 13). Synthetic DNA having 12-32. In addition, the synthetic primer 13-CS1-R has the nucleotide sequence corresponding to amino acid numbers 555 to 550 of the amino acid sequence of CH-296 (SEQ ID NO: 13) as base numbers 1 to 18, and further the amino acid sequence of CH-296 (sequence). No. 13) is a synthetic DNA having base sequences 19 to 36 corresponding to amino acid numbers 459 to 454. In addition, the synthetic primer 13-CS1-F has a nucleotide sequence corresponding to amino acid numbers 454 to 459 of the amino acid sequence of CH-296 (SEQ ID NO: 13) as base numbers 2 to 19, and further an amino acid sequence of CH-296 (sequence). No. 13) is a synthetic DNA having a base sequence corresponding to amino acid numbers 550 to 555 at base numbers 20 to 37. In addition, the synthetic primer CS1-R-Xba has a base sequence corresponding to amino acid numbers 574-569 of the amino acid sequence of CH-296 (SEQ ID NO: 13) based on the recognition sequence of the restriction enzyme XbaI. Synthetic DNA having numbers 19 to 36.

  PCR was performed using the synthetic primers. PCR reaction conditions are shown below. That is, about 0.1 μg of pCold14-H204 constructed in Example 6- (1)-(ii) as template DNA, 20 μL of 5 × PrimeStar Buffer (manufactured by Takara Bio Inc.), 8 μL of dNTP mixed solution, 20 pmol of synthetic primer 13-Nde-F, 20 pmol synthetic primer 13-CS1-R, 2.5 U PrimeSTAR HS DNA Polymerase (manufactured by Takara Bio Inc.) were added, and sterilized water was added to make a total volume of 100 μL. The reaction solution was set in TaKaRa PCR Thermal Cycler PERSONAL (manufactured by Takara Bio Inc.), and 30 cycles of reaction were performed in NORM mode with 98 ° C. for 10 seconds and 60 ° C. for 45 seconds as one cycle. After completion of the reaction, 5 μL of the reaction solution was subjected to 3.0% agarose gel electrophoresis, and the target DNA fragment of about 0.28 kbp was confirmed. This was designated H114-1.

  Furthermore, PCR was performed using the synthetic primer. PCR reaction conditions are shown below. That is, about 0.1 μg of pCold14-H204 constructed in Example 6- (1)-(ii) as template DNA, 20 μL of 5 × PrimeSTAR Buffer, 8 μL of dNTP mixed solution, 20 pmol of synthetic primer 13-CS1-F, 20 pmol of synthetic primer CS1-R-Xba and 2.5 U of PrimeSTAR HS DNA Polymerase were added, and sterilized water was added to make a total volume of 100 μL. The reaction solution was set in TaKaRa PCR Thermal Cycler PERSONAL, and the reaction was performed in NORM mode at 96 ° C. for 30 seconds, 58 ° C. for 1 minute, and 72 ° C. for 1 minute for 30 cycles and 72 ° C. for 3 minutes. After completion of the reaction, 5 μL of the reaction solution was subjected to 3.0% agarose gel electrophoresis, and the target DNA fragment of about 0.09 kbp was confirmed. This was designated H114-2.

  Next, PCR was performed using H114-1 and H114-2 as template DNA. PCR reaction conditions are shown below. As template DNA, H114-1 and H114-2 were 0.5 μL, 20 μL of 5 × PrimeSTAR Buffer, 8 μL of dNTP mixed solution, 20 pmol of synthetic primer 13-Nde-F, 20 pmol of synthetic primer CS1-R-Xba, 2 .5 U PrimeSTAR HS DNA Polymerase was added, and sterilized water was added to make up a total volume of 100 μL. The reaction solution was set in TaKaRa PCR Thermal Cycler PERSONAL, and 30 cycles of reaction were performed in NORM mode with 98 ° C. for 10 seconds, 58 ° C. for 15 seconds, and 72 ° C. for 30 seconds. After completion of the reaction, 5 μL of the reaction solution was subjected to 3.0% agarose gel electrophoresis, and the target DNA fragment of about 0.36 kbp was confirmed. The remaining PCR reaction solution was subjected to ethanol precipitation, and the recovered DNA was suspended in sterilized water and double-digested with restriction enzyme NdeI and restriction enzyme XbaI. The NdeI and XbaI digests were extracted and purified by 3.0% agarose gel electrophoresis to obtain NdeI-XbaI digested DNA fragments.

  Next, a pColdI vector (Takara Bio Inc.) digested with NdeI and XbaI was prepared, mixed with the NdeI-XbaI digested DNA fragment, and ligated using a DNA ligation kit. Thereafter, E. coli JM109 was transformed with 10 μL of the ligation reaction solution, and the transformant was grown on an LB medium (containing 50 μg / mL of ampicillin) containing agar at a concentration of 1.5% (w / v). The recombinant plasmid inserted with the DNA fragment encoding the protein of the target III-13 and CS-1 linked was confirmed by sequencing, and this recombinant plasmid was designated as pColdI-H114-HT.

  This pColdI-H114-HT has a base sequence encoding the amino acid sequence of amino acid numbers 371-459 of CH-296 and a base sequence encoding the amino acid sequence of amino acid numbers 550-574 of CH-296 linked. It is a plasmid containing a DNA sequence encoding a polypeptide having a His tag sequence and methionine at the end. The protein is named H114-HT, its amino acid sequence is shown in SEQ ID NO: 44 of the sequence listing, and its base sequence is shown in SEQ ID NO: 45 of the sequence listing.

(2) Expression and purification of H114-HT The pColdI-H114-HT prepared in (1) above was used to transform E. coli BL21, and the transformant was transformed into 1.5% (w / v) agar. Growth was carried out on LB medium containing 50 μg / mL ampicillin. The grown colonies were inoculated into 500 mL of LB liquid medium (containing 50 μg / mL of ampicillin) and cultured at 37 ° C. until OD = 0.4 to 0.6 (about 5 to 8 hours). Then, after cooling to 15 ° C., IPTG was added to a final concentration of 1.0 mM, and cultured at 15 ° C. for 24 hours to induce expression. Thereafter, the cells were collected by centrifugation, about 2.5 g of a part thereof was suspended in 12.5 mL of Equilibration / Wash Buffer [50 mM Tris-HCl (pH 8.0), 300 mM NaCl], and then ultrasonically disrupted. The cells were disrupted by the above, and the supernatant extract was obtained by centrifugation (11,000 r / min 20 minutes). Next, 4 mL of TALON His-tag purified resin (manufactured by Clontech) was saturated with Equilibration / Wash Buffer, and the supernatant extract was applied. After stirring at 4 ° C. for 1 hour, the resin was washed twice with 50 ml of Equilibration / Wash Buffer, and the target protein bound to the resin was washed with 20 mL of Elution Buffer [50 mM Tris-HCl (pH 8.0), 300 mM NaCl, Elution with 0.1% CHAPS, 150 mM Imidazole]. The obtained eluate was concentrated to 10 ml with VIVASPIN (manufactured by Sartorius) and analyzed with 2100 Bioanalyzer (manufactured by Agilent Technologies). As a result, the target protein having a molecular weight of about 15 kDa was detected as an almost single band. 0.5 mg / mL (approximately 33 μM calculated from molecular weight).

(3) Purification of H114 Next, cleavage of the His tag sequence for 5 mg of the H114-HT purified protein purified in (2) above was performed overnight at 4 ° C. using Factor Xa Cleavage Capture Kit (manufactured by Merck). After that, the target protein without His tag was purified. The cleavage of the His tag and purification of the target protein were performed according to the operation manual attached to the Kit. The solution after cleavage was diluted 100 times or more with Stock Buffer [25 mM sodium citrate buffer (pH 6.0)] and concentrated to 1 mL using VIVASPIN. When the obtained concentrated solution was analyzed by 2100 Bioanalyzer, the target protein having a molecular weight of about 13 kDa was detected as an almost single band, and the protein concentration was 0.86 mg / mL (calculated from the molecular weight, about 66 μM ). The amino acid sequence of H114 is shown in SEQ ID NO: 46 in the sequence listing, and the base sequence predicted to encode the protein is shown in SEQ ID NO: 47 in the sequence listing.

Example 10 Preparation of H117-HT and H117 (1) Construction of H117-HT expression vector In order from the N terminus, His tag sequence, III-12 (amino acid sequence represented by SEQ ID NO: 5 in the sequence listing) and CS-1 ( In order to obtain a modified FN fragment (H117-HT) linked with the amino acid sequence represented by SEQ ID NO: 8 in the sequence listing, an expression vector was constructed as follows.

(I) Construction of H117-HT expression vector From the base sequence of CH-296 described in SEQ ID NO: 24 in the sequence listing, synthetic primer H296-NdeF having the base sequence described in SEQ ID NO: 48 in the sequence listing, the base described in SEQ ID NO: 49 A synthetic primer 12-CS1-R having a sequence and a synthetic primer 12-CS1-F having a base sequence described in SEQ ID NO: 50 were synthesized by a DNA synthesizer and purified by a conventional method. The synthetic primer H296-NdeF is a synthetic DNA having a recognition sequence for the restriction enzyme NdeI at base numbers 9 to 14, and a base sequence corresponding to amino acid numbers 279 to 285 of the amino acid sequence of CH-296 at base numbers 15 to 35. It is. In addition, the synthetic primer 12-CS1-R has a nucleotide sequence corresponding to amino acid numbers 555 to 550 of the amino acid sequence of CH-296 (SEQ ID NO: 13) as base numbers 1 to 18, and further an amino acid sequence of CH-296 (sequence). No. 13) is a synthetic DNA having nucleotide sequences 19 to 33 corresponding to amino acid numbers 370 to 366. Further, the synthetic primer 12-CS1-F has a nucleotide sequence corresponding to amino acid numbers 366 to 370 of the amino acid sequence of CH-296 (SEQ ID NO: 13) as base numbers 1 to 15, and further an amino acid sequence of CH-296 (sequence). No. 13) is a synthetic DNA having a base sequence corresponding to amino acid numbers 550 to 555 at base numbers 16 to 33.

  PCR was performed using the synthetic primers. PCR reaction conditions are shown below. That is, about 0.1 μg of pCold14-H296 constructed in Example 7- (1)-(i) as a template DNA, 20 μL of 5 × PrimeSTAR Buffer, 8 μL of dNTP mixed solution, 20 pmol of synthetic primer H296-NdeF, 20 pmol of Synthetic primer 12-CS1-R, 2.5 U PrimeSTAR HS DNA Polymerase were added, and sterilized water was added to make the total volume 100 μL. The reaction solution was set in TaKaRa PCR Thermal Cycler PERSONAL, and 30 cycles of reaction were performed in NORM mode with 98 ° C. for 10 seconds and 68 ° C. for 30 seconds as one cycle. After completion of the reaction, 5 μL of the reaction solution was subjected to 3.0% agarose gel electrophoresis, and the target DNA fragment of about 0.3 kbp was confirmed. This was designated as H117-1.

  Furthermore, PCR was performed using the synthetic primer and the synthetic primer CS1-R-Xba. PCR reaction conditions are shown below. That is, about 0.1 μg of pCold14-H296 constructed in Example 7- (1)-(i) as template DNA, 20 μL of 5 × PrimeSTAR Buffer, 8 μL of dNTP mixed solution, 20 pmol of synthetic primer 12-CS1-F, 20 pmol of synthetic primer CS1-R-Xba and 2.5 U of PrimeSTAR HS DNA Polymerase were added, and sterilized water was added to make a total volume of 100 μL. The reaction solution was set in TaKaRa PCR Thermal Cycler PERSONAL, and 30 cycles of reaction were performed in NORM mode with 98 ° C. for 10 seconds, 58 ° C. for 15 seconds, and 72 ° C. for 30 seconds. After completion of the reaction, 5 μL of the reaction solution was subjected to 3.0% agarose gel electrophoresis, and the target DNA fragment of about 0.09 kbp was confirmed. This was designated as H117-2.

  Next, PCR was carried out using H117-1 and H117-2 as template DNA. PCR reaction conditions are shown below. As template DNA, H117-1 and H117-2 were 0.5 μL, 20 μL of 5 × PrimeSTAR Buffer, 8 μL of dNTP mixed solution, 20 pmol of synthetic primer H296-NdeF, 20 pmol of synthetic primer CS1-R-Xba, 2.5 U, respectively. PrimeSTAR HS DNA Polymerase was added, and sterilized water was added to make a total volume of 100 μL. The reaction solution was set in TaKaRa PCR Thermal Cycler PERSONAL, and 30 cycles of reaction were performed in NORM mode with 98 ° C. for 10 seconds, 58 ° C. for 15 seconds, and 72 ° C. for 30 seconds. After completion of the reaction, 5 μL of the reaction solution was subjected to 1.0% agarose gel electrophoresis, and the target DNA fragment of about 0.4 kbp was confirmed. The remaining PCR reaction solution was subjected to ethanol precipitation, and the recovered DNA was suspended in sterilized water and double-digested with restriction enzyme NdeI and restriction enzyme XbaI. The NdeI and XbaI digests were extracted and purified by 1.0% agarose gel electrophoresis to obtain NdeI-XbaI digested DNA fragments.

  Next, a pColdI vector digested with NdeI and XbaI was prepared, mixed with the NdeI-XbaI digested DNA fragment, and ligated using a DNA ligation kit. Thereafter, E. coli JM109 was transformed with 10 μL of the ligation reaction solution, and the transformant was grown on an LB medium (containing 50 μg / mL of ampicillin) containing agar at a concentration of 1.5% (w / v). A recombinant plasmid inserted with a DNA fragment encoding the protein of the target III-12 and CS-1 linked was confirmed by sequencing, and this recombinant plasmid was designated as pColdI-H117-HT.

  In this pColdI-H117-HT, the base sequence encoding the amino acid sequence of amino acid numbers 279-370 of CH-296 and the base sequence encoding the amino acid sequence of amino acid numbers 550-574 of CH-296 are linked, and the N It is a plasmid containing a DNA sequence encoding a polypeptide having a His tag sequence and methionine at the end. The protein is named H117-HT, its amino acid sequence is shown in SEQ ID NO: 51 of the sequence listing, and its base sequence is shown in SEQ ID NO: 52 of the sequence listing.

(2) Expression and purification of H117-HT E. coli BL21 was transformed with the pColdI-H117-HT prepared in (1) above, and expression and purification of H117-HT was performed in the same manner as in Example 9- (2). went. The culture solution, the amount of cells used, the resin volume, the Equilibration / Wash Buffer, the Elution Buffer, and the concentration of the concentrate were appropriately changed as necessary. When the obtained concentrated solution was analyzed by 2100 Bioanalyzer, the target protein having a molecular weight of about 15 kDa was detected as an almost single band, and the protein concentration was 1.2 mg / mL (calculated from the molecular weight, about 80 μM). .

(3) Purification of H117 Next, the His tag sequence for 5 mg of the H117-HT purified protein purified in (2) above was cleaved overnight at 4 ° C. using Factor Xa Cleavage Capture Kit. The target protein without tag was purified. The cleavage of the His tag and purification of the target protein were performed according to the operation manual attached to the Kit. The solution after cleavage was diluted 100 times or more with Stock Buffer, and concentrated to 2 mL using VIVASPIN. When this concentrated solution was analyzed by 2100 Bioanalyzer, the target protein having a molecular weight of about 13 kDa was detected as an almost single band, and the protein concentration was 0.36 mg / mL (calculated from the molecular weight of about 28 μM). The amino acid sequence of H117 is shown in SEQ ID NO: 53 in the sequence listing, and the base sequence predicted to encode the protein is shown in SEQ ID NO: 54 in the sequence listing.

Example 11 Preparation of H206-HT and H206 (1) Construction of H206-HT expression vector His tag sequence, III-12 (amino acid sequence represented by SEQ ID NO: 5 in the sequence listing), III-13 (from the N-terminus) In order to obtain a modified FN fragment (H206-HT) in which CS-1 (amino acid sequence represented by SEQ ID NO: 8 in the sequence listing) and CS-1 (amino acid sequence represented by SEQ ID NO: 8 in the sequence listing) are sequentially linked, Thus, an expression vector was constructed.

(I) Construction of pCold14-H271 vector Synthetic primers 12-Nco-F2 and H271-BamR having the base sequences described in SEQ ID NOs: 55 and 56 in the sequence listing from the base sequence of CH-296 described in SEQ ID NO: 24 in the sequence listing Was synthesized by a DNA synthesizer and purified by a conventional method. The synthetic primer 12-Nco-F2 has a recognition sequence for the restriction enzyme NcoI as base numbers 7 to 12, and a base sequence corresponding to amino acid numbers 279 to 285 of the amino acid sequence of CH-296 (SEQ ID NO: 13 in the sequence listing) Is a synthetic DNA having base numbers 12 to 32. Further, the synthetic primer H271-BamR has a recognition sequence for the restriction enzyme BamHI in base numbers 8 to 13, and further a base sequence corresponding to amino acid numbers 549 to 544 in the amino acid sequence of CH-296 (SEQ ID NO: 13 in the sequence listing). It is a synthetic DNA having base numbers 19 to 36.

  PCR was performed using the synthetic primers. PCR reaction conditions are shown below. That is, about 0.1 μg of pCold14-H296 constructed in Example 7- (1)-(i) as template DNA, 10 μL of 10 × Pyrobest Buffer II, 8 μL of dNTP mixed solution, 20 pmol of synthetic primer 12-Nco-F2, 20 pmol of synthetic primer H271-BamR and 2.5 U of Pyrobest DNA Polymerase were added, and sterilized water was added to make a total volume of 100 μL. The reaction solution was set in TaKaRa PCR Thermal Cycler SP, and 30 cycles of reaction were performed with 98 ° C. for 10 seconds and 68 ° C. for 1 minute as one cycle. After completion of the reaction, 5 μL of the reaction solution was subjected to 1.0% agarose gel electrophoresis, and the target DNA fragment of about 0.8 kbp was confirmed. The remaining PCR reaction solution was subjected to ethanol precipitation, the recovered DNA was suspended in sterilized water, and double digested with restriction enzyme NcoI and restriction enzyme BamHI (manufactured by Takara Bio Inc.). The NcoI and BamHI digests were extracted and purified by 1.0% agarose gel electrophoresis to obtain an NcoI-BamHI digested DNA fragment.

  Next, a pCold14 vector digested with NcoI and BamHI was prepared, mixed with the NcoI-BamHI digested DNA fragment, and ligated using a DNA ligation kit. Thereafter, E. coli JM109 was transformed with 10 μL of the ligation reaction solution, and the transformant was grown on an LB medium (containing 50 μg / mL of ampicillin) containing agar at a concentration of 1.5% (w / v). The recombinant plasmid into which the target DNA fragment was inserted was designated as pCold14-H271.

(Ii) Construction of pCold14-H105 vector A synthetic primer AID-F-Nco having a base sequence described in SEQ ID NO: 57 in the sequence listing was synthesized from a base sequence of CH-296 described in SEQ ID NO: 24 in the sequence listing using a DNA synthesizer. And purified by a conventional method. The synthetic primer AID-F-Nco has a recognition sequence of the restriction enzyme NcoI as base numbers 10 to 15, and a base sequence corresponding to amino acid numbers 460 to 466 of the amino acid sequence of CH-296 (SEQ ID NO: 13) as base numbers. 18-38 synthetic DNA.

  PCR was performed using the synthetic primer and the synthetic primer CS1-R-Xba. PCR reaction conditions are shown below. That is, about 0.1 μg of pCold14-H296 constructed in Example 7- (1)-(i) as template DNA, 10 μL of 10 × Pyrobest Buffer II, 8 μL of dNTP mixed solution, 20 pmol of synthetic primer AID-F-Nco, 20 pmol of synthetic primer CS1-R-Xba and 2.5 U of Pyrobest DNA Polymerase were added, and sterilized water was added to make a total volume of 100 μL. The reaction solution was set in TaKaRa PCR Thermal Cycler SP, and 30 cycles of reaction were performed with 98 ° C. for 10 seconds and 68 ° C. for 1 minute as one cycle. After completion of the reaction, 5 μL of the reaction solution was subjected to 1.0% agarose gel electrophoresis, and the target DNA fragment of about 0.35 kbp was confirmed. The remaining PCR reaction solution was subjected to ethanol precipitation, and the recovered DNA was suspended in sterilized water and double-digested with restriction enzyme NcoI and restriction enzyme XbaI. The NcoI and XbaI digests were extracted and purified by 1.0% agarose gel electrophoresis to obtain NcoI-XbaI digested DNA fragments.

  Next, a pCold14 vector digested with NcoI and XbaI was prepared, mixed with the NcoI-XbaI digested DNA fragment, and ligated using a DNA ligation kit. Thereafter, E. coli JM109 was transformed with 10 μL of the ligation reaction solution, and the transformant was grown on an LB medium (containing 50 μg / mL of ampicillin) containing agar at a concentration of 1.5% (w / v). The recombinant plasmid into which the target DNA fragment was inserted was designated as pCold14-H105.

(Iii) Construction of H206-HT expression vector PCR was performed using the above recombinant plasmid. PCR reaction conditions are shown below. That is, about 0.1 μg of pCold14-H271 constructed in Example 11- (1)-(i) as a template DNA, 20 μL of 5 × PrimeSTAR Buffer, 8 μL of dNTP mixed solution, 20 pmol of synthetic primer H296-NdeF, 20 pmol of Synthetic primer 13-CS1-R, 2.5 U PrimeSTAR HS DNA Polymerase were added, and sterilized water was added to make up a total volume of 100 μL. The reaction solution was set in TaKaRa PCR Thermal Cycler PERSONAL, and 30 cycles of reaction were performed in NORM mode with 98 ° C. for 10 seconds and 60 ° C. for 1 minute as one cycle. After completion of the reaction, 5 μL of the reaction solution was subjected to 1.0% agarose gel electrophoresis, and the target DNA fragment of about 0.6 kbp was confirmed. This was designated as H206-1.

  Furthermore, PCR was performed. PCR reaction conditions are shown below. That is, about 0.1 μg of pCold14-H105 constructed in Example 11- (1)-(ii) as template DNA, 20 μL of 5 × PrimeSTAR Buffer, 8 μL of dNTP mixed solution, 20 pmol of synthetic primer 13-CS1-F, 20 pmol of synthetic primer CS1-R-Xba and 2.5 U of PrimeSTAR HS DNA Polymerase were added, and sterilized water was added to make a total volume of 100 μL. The reaction solution was set in TaKaRa PCR Thermal Cycler PERSONAL, and the reaction was performed in NORM mode at 96 ° C. for 30 seconds, 58 ° C. for 1 minute, and 72 ° C. for 1 minute for 30 cycles and 72 ° C. for 3 minutes. After completion of the reaction, 5 μL of the reaction solution was subjected to 2.0% agarose gel electrophoresis, and the target DNA fragment of about 0.08 kbp was confirmed. This was designated H206-2.

  Next, PCR was performed using H206-1 and H206-2 as template DNA. PCR reaction conditions are shown below. As template DNA, H206-1 and H206-2 were each 0.5 μL, 20 μL of 5 × PrimeStar Buffer, 8 μL of dNTP mixed solution, 20 pmol of synthetic primer H296-NdeF, 20 pmol of synthetic primer CS1-R-Xba, 2.5U PrimeSTAR HS DNA Polymerase was added, and sterilized water was added to make a total volume of 100 μL. The reaction solution was set in TaKaRa PCR Thermal Cycler PERSONAL, and 30 cycles of reaction were performed in NORM mode with 96 ° C. for 15 seconds, 58 ° C. for 15 seconds, and 72 ° C. for 90 seconds. After completion of the reaction, 5 μL of the reaction solution was subjected to 1.0% agarose gel electrophoresis, and the target DNA fragment of about 0.63 kbp was confirmed. The remaining PCR reaction solution was subjected to ethanol precipitation, and the recovered DNA was suspended in sterilized water and double-digested with restriction enzyme NdeI and restriction enzyme XbaI. The NdeI and XbaI digests were extracted and purified by 1.0% agarose gel electrophoresis to obtain NdeI-XbaI digested DNA fragments.

  Next, a pColdI vector digested with NdeI and XbaI was prepared, mixed with the NdeI-XbaI digested DNA fragment, and ligated using a DNA ligation kit. Thereafter, E. coli JM109 was transformed with 10 μL of the ligation reaction solution, and the transformant was grown on an LB medium (containing 50 μg / mL of ampicillin) containing agar at a concentration of 1.5% (w / v). The recombinant plasmid inserted with the DNA fragment encoding the protein to which the desired III-12, III-13 and CS-1 were sequentially linked was confirmed by sequencing, and this recombinant plasmid was identified as pColdI-H206-HT. did.

  This pColdI-H206-HT has a base sequence encoding the amino acid sequence of amino acid numbers 279-459 of CH-296 and a base sequence encoding the amino acid sequence of amino acid numbers 550-574 of CH-296 linked to each other. It is a plasmid containing a DNA sequence encoding a polypeptide having a His tag sequence and methionine at the end. The protein is named H206-HT, its amino acid sequence is shown in SEQ ID NO: 58, and its base sequence is shown in SEQ ID NO: 59.

(2) Expression and purification of H206-HT Escherichia coli BL21 was transformed with pColdI-H206-HT prepared in (1) above, and expression and purification of H206-HT were conducted in the same manner as in Example 9- (2). went. The culture solution, the amount of cells used, the resin volume, the Equilibration / Wash Buffer, the Elution Buffer, and the concentration of the concentrate were appropriately changed as necessary. When the obtained concentrated solution was analyzed by 2100 Bioanalyzer, the target protein having a molecular weight of about 25 kDa was detected as a substantially single band, and the protein concentration was 1.5 mg / mL (calculated from the molecular weight, about 60 μM). .

(3) Purification of H206 Next, the His tag sequence for 5 mg of the H206-HT purified protein purified in (2) above was cleaved overnight at 4 ° C. using the Factor Xa Cleavage Capture Kit, and then His was used. The target protein without tag was purified. The cleavage of the His tag and purification of the target protein were performed according to the operation manual attached to the Kit. The cut solution was diluted 100 times or more with Stock Buffer and concentrated to 1 mL using VIVASPIN. When this concentrated solution was analyzed with 2100 Bioanalyzer, the target protein having a molecular weight of about 23 kDa was detected as a substantially single band, and the protein concentration was 0.72 mg / mL (calculated from the molecular weight of about 31 μM). The amino acid sequence of H206 is shown in SEQ ID NO: 60 in the sequence listing, and the base sequence predicted to encode the protein is shown in SEQ ID NO: 61 in the sequence listing.

Example 12 Preparation of H296R-HT and H296R (1) Construction of H296R-HT expression vector In order from the N terminus, His tag sequence, CS-1 (amino acid sequence represented by SEQ ID NO: 8 in the sequence listing), III-12 (Amino acid sequence represented by SEQ ID NO: 5 in the sequence listing), III-13 (amino acid sequence represented by SEQ ID NO: 6 in the sequence listing) and III-14 (amino acid sequence represented by SEQ ID NO: 7 in the sequence listing) In order to obtain a modified FN fragment (H296R-HT) linked to, an expression vector was constructed as follows.

(I) Construction of H296R-HT expression vector From the base sequence of CH-296 described in SEQ ID NO: 24 in the sequence listing, synthetic primer CS1-Nde-F having the base sequence described in SEQ ID NO: 62 in the sequence listing and SEQ ID NO: 63 described DNA synthesis of synthetic primer CS1-12-R having the nucleotide sequence of SEQ ID NO: 64, synthetic primer CS1-12-F having the nucleotide sequence of SEQ ID NO: 64, and synthetic primer H271-R-Xba having the nucleotide sequence of SEQ ID NO: 65 And purified by a conventional method. The synthetic primer CS1-Nde-F has a recognition sequence of the restriction enzyme NdeI as base numbers 6 to 11, and a base sequence corresponding to amino acid numbers 550 to 557 of the amino acid sequence of CH-296 (SEQ ID NO: 13) as base numbers. Synthetic DNA having 12-35. Further, the synthetic primer CS1-12-R was prepared by changing the nucleotide sequence corresponding to amino acid numbers 283 to 279 of the amino acid sequence of CH-296 (SEQ ID NO: 13) to base numbers 3 to 17, and further the amino acid sequence of CH-296 (sequence). No. 13) is a synthetic DNA having a base sequence corresponding to amino acid numbers 574 to 570 at base numbers 18 to 32. In addition, the synthetic primer CS1-12-F includes a base sequence corresponding to amino acid numbers 570 to 574 of the amino acid sequence of CH-296 (SEQ ID NO: 13) to base numbers 2 to 16, and an amino acid sequence of CH-296 (sequence). No. 13) is a synthetic DNA having nucleotide sequences 17 to 31 corresponding to amino acid numbers 279 to 283. The synthetic primer H271-R-Xba has a base sequence corresponding to amino acid numbers 549 to 544 of the amino acid sequence of CH-296 (SEQ ID NO: 13) and the recognition sequence of restriction enzyme XbaI to base numbers 10 to 15 Synthetic DNA having numbers 19 to 36.

  PCR was performed using the synthetic primers. PCR reaction conditions are shown below. That is, about 0.1 μg of pCold14-H105 constructed in Example 11- (1)-(ii) as template DNA, 20 μL of 5 × PrimeSTAR Buffer, 8 μL of dNTP mixed solution, 20 pmol of synthetic primer CS1-Nde-F, 20 pmol of synthetic primer CS1-12-R and 2.5 U of PrimeSTAR HS DNA Polymerase were added, and sterilized water was added to make the total volume 100 μL. The reaction solution was set in TaKaRa PCR Thermal Cycler PERSONAL, and 30 cycles of reaction were performed in NORM mode with 96 ° C. for 10 seconds, 58 ° C. for 15 seconds, and 72 ° C. for 1 minute. After completion of the reaction, 5 μL of the reaction solution was subjected to 3.0% agarose gel electrophoresis, and the target DNA fragment of about 0.1 kbp was confirmed. This was designated H296R-1.

  Furthermore, PCR was performed. PCR reaction conditions are shown below. That is, about 0.1 μg of pCold14-H271 constructed in Example 11- (1)-(i) as template DNA, 20 μL of 5 × PrimeSTAR Buffer, 8 μL of dNTP mixed solution, 20 pmol of synthetic primer CS1-12-F, 20 pmol of synthetic primer H271-R-Xba and 2.5 U of PrimeSTAR HS DNA Polymerase were added, and sterilized water was added to make a total volume of 100 μL. The reaction solution was set in TaKaRa PCR Thermal Cycler PERSONAL, and 30 cycles of reaction were performed in NORM mode with 96 ° C. for 10 seconds, 58 ° C. for 15 seconds, and 72 ° C. for 1 minute. After completion of the reaction, 5 μL of the reaction solution was subjected to 1.0% agarose gel electrophoresis, and the target DNA fragment of about 0.8 kbp was confirmed. This was designated H296R-2.

  Next, PCR was performed using the above H296R-1 and H296R-2 as template DNA. PCR reaction conditions are shown below. As template DNA, H296R-1 and H296R-2 were each 0.5 μL, 20 μL of 5 × PrimeStar Buffer, 8 μL of dNTP mixed solution, 20 pmol of synthetic primer CS1-Nde-F, 20 pmol of synthetic primer H271-R-Xba, 2 .5 U PrimeSTAR HS DNA Polymerase was added, and sterilized water was added to make up a total volume of 100 μL. The reaction solution was set in TaKaRa PCR Thermal Cycler PERSONAL, and 30 cycles of reaction were performed in NORM mode with 96 ° C. for 10 seconds, 58 ° C. for 15 seconds, and 72 ° C. for 1 minute. After completion of the reaction, 5 μL of the reaction solution was subjected to 1.0% agarose gel electrophoresis, and the target DNA fragment of about 0.9 kbp was confirmed. The remaining PCR reaction solution was subjected to ethanol precipitation, and the recovered DNA was suspended in sterilized water and double-digested with restriction enzyme NdeI and restriction enzyme XbaI. The NdeI and XbaI digests were extracted and purified by 1.0% agarose gel electrophoresis to obtain NdeI-XbaI digested DNA fragments.

  Next, a pColdI vector digested with NdeI and XbaI was prepared, mixed with the NdeI-XbaI digested DNA fragment, and ligated using a DNA ligation kit. Thereafter, E. coli JM109 was transformed with 10 μL of the ligation reaction solution, and the transformant was grown on an LB medium (containing 50 μg / mL of ampicillin) containing agar at a concentration of 1.5% (w / v). The desired recombinant plasmid inserted with a DNA fragment encoding a protein linked with CS-1, III-12, III-13 and III-14 in this order from the N-terminal was confirmed by sequencing, and this recombination was confirmed. The plasmid was designated as pColdI-H296R-HT.

  This pCold I-H296R-HT has a base sequence encoding the amino acid sequence of amino acids 550-574 of CH-296 and a base sequence encoding the amino acid sequence of amino acids 279-549 of CH-296 linked to each other. It is a plasmid containing a DNA sequence encoding a polypeptide having a His tag sequence and methionine at the end. The protein is named H296R-HT, its amino acid sequence is shown in SEQ ID NO: 66 in the sequence listing, and its base sequence is shown in SEQ ID NO: 67 in the sequence listing.

(2) Expression and purification of H296R-HT E. coli BL21 was transformed with pColdI-H296R-HT prepared in (1) above, and expression and purification of H296R-HT was performed in the same manner as in Example 9- (2). went. The culture solution, the amount of cells used, the resin volume, the Equilibration / Wash Buffer, the Elution Buffer, and the concentration of the concentrate were appropriately changed as necessary. When the obtained concentrated solution was analyzed with 2100 Bioanalyzer, the target protein having a molecular weight of about 34 kDa was detected as a nearly single band, and the protein concentration was 0.71 mg / mL (calculated from the molecular weight of about 21 μM). .

(3) Purification of H296R Next, the His tag sequence for 5 mg of the H296R-HT purified protein purified in (2) above was cleaved overnight at 4 ° C. using Factor Xa Cleavage Capture Kit. The target protein without tag was purified. The cleavage of the His tag and purification of the target protein were performed according to the operation manual attached to the Kit. The solution after cleavage was diluted 100 times or more with Stock Buffer and concentrated to 1.5 mL using VIVASPIN. When this concentrated solution was analyzed by 2100 Bioanalyzer, the target protein having a molecular weight of about 32 kDa was detected as an almost single band, and the protein concentration was 0.55 mg / mL (calculated from the molecular weight of about 17 μM). The amino acid sequence of H296R is shown in SEQ ID NO: 68 in the sequence listing, and the base sequence predicted to encode the protein is shown in SEQ ID NO: 69 in the sequence listing.

Example 13 Preparation of H105-HT and H105 (1) Construction of H105-HT expression vector In order from the N terminus, His tag sequence, III-14 (amino acid sequence represented by SEQ ID NO: 7 in the sequence listing) and CS-1 ( In order to obtain a modified FN fragment (H105-HT) in which the amino acid sequence represented by SEQ ID NO: 8 in the sequence listing was linked, an expression vector was constructed as follows.

(I) Construction of H105-HT expression vector A synthetic primer H105-NdeF having a base sequence described in SEQ ID NO: 70 in the sequence listing was synthesized with a DNA synthesizer from the base sequence of CH-296 described in SEQ ID NO: 24 in the sequence listing. The product was purified by a conventional method. The synthetic primer H105-NdeF is a synthetic DNA having the recognition sequence of the restriction enzyme NdeI at base numbers 9 to 14, and further the base sequence corresponding to amino acid numbers 460 to 466 of the amino acid sequence of CH-296 at base numbers 15 to 35 It is.

  PCR was performed using the synthetic primer and synthetic primer H296-HindR. PCR reaction conditions are shown below. Specifically, about 0.1 μg of pCH102, 5 μL of 10 × Ex Taq Buffer, 5 μL of dNTP mixed solution, 10 pmol of synthetic primer H105-NdeF, 10 pmol of synthetic primer H296-HindR, 0.5 U of TaKaRa Ex Taq were added as template DNA. Then, sterilized water was added to make the total volume 50 μL. The reaction solution was set in TaKaRa PCR Thermal Cycler SP, and 30 cycles of reaction were performed with 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 3 minutes as one cycle. After completion of the reaction, 5 μL of the reaction solution was subjected to 1.0% agarose gel electrophoresis, and the target DNA fragment of about 0.35 kbp was confirmed. The remaining PCR reaction solution was electrophoresed, the fragment was recovered and purified, and ethanol precipitation was performed. The recovered DNA after ethanol precipitation was suspended in 10 μL of sterilized water and double-digested with restriction enzyme NdeI and restriction enzyme HindIII. The NdeI and HindIII digests were extracted and purified by 1.0% agarose gel electrophoresis to obtain NdeI-HindIII digested DNA fragments.

  Next, a pColdI vector digested with NdeI and HindIII was prepared, mixed with the NdeI-HindIII digested DNA fragment, and ligated using a DNA ligation kit. Thereafter, E. coli JM109 was transformed with 20 μL of the ligation reaction solution, and the transformant was grown on an LB medium (containing 50 μg / mL of ampicillin) containing agar at a concentration of 1.5% (w / v). The recombinant plasmid into which the target DNA fragment was inserted was confirmed by sequencing, and this recombinant plasmid was designated as pColdI-H105-HT. This pColdI-H105-HT is a plasmid containing a DNA sequence encoding a polypeptide having a His tag sequence and a methionine at the N-terminal of the base sequence encoding the amino acid sequence of amino acid numbers 460 to 574 of CH-296. The protein is named H105-HT, its amino acid sequence is shown in SEQ ID NO: 71 of the sequence listing, and its base sequence is shown in SEQ ID NO: 72 of the sequence listing.

(2) Expression and purification of H105-HT E. coli BL21 was transformed with pColdI-H105-HT prepared in (1) above, and expression and purification of H105-HT was performed in the same manner as in Example 9- (2). went. The culture fluid, the amount of cells used, the resin volume, the Equilibration / Wash Buffer, and the Elution Buffer were appropriately changed as necessary. The obtained eluate was diluted 100 times or more with Stock Buffer and concentrated to 1 mL using VIVASPIN. When this concentrated solution was analyzed by 2100 Bioanalyzer, the target protein having a molecular weight of about 15 kDa was detected as an almost single band, and the protein concentration was 0.23 mg / mL (calculated from the molecular weight of about 15 μM).

(3) Purification of H105 Next, the His tag sequence for 1 mg of the H105-HT purified protein purified in (2) above was cleaved overnight at 4 ° C. using the Factor Xa Cleavage Capture Kit, and then His was used. The target protein without tag was purified. The cleavage of the His tag and purification of the target protein were performed according to the operation manual attached to the Kit. The solution after cleavage was diluted 100 times or more with Stock Buffer and concentrated to 0.3 mL using VIVASPIN. When this concentrated solution was analyzed by 2100 Bioanalyzer, the target protein having a molecular weight of about 13 kDa was detected as a substantially single band, and the protein concentration was 0.4 mg / mL (calculated from the molecular weight of about 31 μM). The amino acid sequence of H105 is shown in SEQ ID NO: 73 in the sequence listing, and the nucleotide sequence predicted to encode the protein is shown in SEQ ID NO: 74 in the sequence listing.

Example 14 Construction of CH105-HT (1) Construction of CH105-HT expression vector His tag sequence, FN C-domain (region from III-8 to III-10), amino acid sequence “EIDKPSMA” from the N-terminus, In order to obtain a modified FN fragment (CH105-HT) linked with III-14 and CS-1, an expression vector was constructed as follows.

(I) Construction of CH105-HT expression vector pCold14-H105 constructed in Example 11- (1)-(ii) was digested with NcoI and XbaI and digested with NcoI and XbaI by 1.0% agarose gel electrophoresis. Was extracted and purified to obtain an NcoI-XbaI digested DNA fragment of about 0.35 kbp, which was designated as an H105 DNA fragment.

  EXAMPLE 6 pCold14NDCH-296 constructed in (1)-(i) was digested with NdeI and NcoI, and the NdeI and NcoI digests were extracted and purified by 1.0% agarose gel electrophoresis, and about 0.8 kbp An NdeI-NcoI digested DNA fragment was obtained and used as a C domain DNA fragment.

  Next, a pColdI vector digested with NdeI and XbaI was prepared, mixed with the H105 DNA fragment and C domain DNA fragment, and ligated using a DNA ligation kit. Thereafter, E. coli JM109 was transformed with 10 μL of the ligation reaction solution, and the transformant was grown on an LB medium (containing 50 μg / mL of ampicillin) containing agar at a concentration of 1.5% (w / v). A recombinant plasmid in which a DNA fragment encoding a protein having H105 linked to the target C domain was inserted was confirmed by sequencing, and this recombinant plasmid was designated as pColdI-CH105-HT.

  In this pColdI-CH105-HT, the base sequence encoding the amino acid sequence of amino acid numbers 1-278 of CH-296 and the base sequence encoding the amino acid sequence of amino acid numbers 460-574 of CH-296 sandwich the “A”. And a DNA sequence encoding a polypeptide having a His tag sequence and methionine at its N-terminus. The protein is named CH105-HT, its amino acid sequence is shown in SEQ ID NO: 75 of the sequence listing, and its base sequence is shown in SEQ ID NO: 76 of the sequence listing.

(2) Expression and purification of CH105-HT E. coli BL21 was transformed with pColdI-CH105-HT prepared in (1) above, and expression and purification of CH105-HT was conducted in the same manner as in Example 9- (2). went. The culture fluid, the amount of cells used, the resin volume, the Equilibration / Wash Buffer, and the Elution Buffer were appropriately changed as necessary. The obtained eluate was diluted 100 times or more with Stock Buffer and concentrated to 1 mL using VIVASPIN. When this concentrated solution was analyzed by 2100 Bioanalyzer, the target protein having a molecular weight of about 45 kDa was detected in almost a single band, and the protein concentration was 5.9 mg / mL (calculated from the molecular weight of about 131 μM).

Example 15 Construction of CH-296mut2 (1) Construction of CH296mut2 expression vector To obtain a modified FN fragment (CH296mut2) obtained by converting amino acids corresponding to amino acid numbers 543 to 548 of the amino acid sequence of CH-296 (SEQ ID NO: 13). An expression vector was constructed as follows.

(I) Construction of pColdICH-296-HT expression vector PCR was performed using synthetic primer CH296-NdeF and synthetic primer H296-HindR. PCR reaction conditions are shown below. That is, about 0.1 μg of pCH102, 5 μL of 10 × Ex Taq Buffer, 5 μL of dNTP mixed solution, 10 pmol of synthetic primer CH296-NdeF, 10 pmol of synthetic primer H296-HindR, 0.5 U of TaKaRa Ex Taq were added as template DNA. Then, sterilized water was added to make the total volume 50 μL. The reaction solution was set in TaKaRa PCR Thermal Cycler SP, and 30 cycles of reaction were performed with 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 3 minutes as one cycle. After completion of the reaction, 5 μL of the reaction solution was subjected to 1.0% agarose gel electrophoresis, and the target DNA fragment of about 1.7 kbp was confirmed. The remaining PCR reaction solution was electrophoresed, the fragment was recovered and purified, and ethanol precipitation was performed. The recovered DNA after ethanol precipitation was suspended in 10 μL of sterilized water and double-digested with restriction enzyme NdeI and restriction enzyme HindIII. The NdeI and HindIII digests were extracted and purified by 1.0% agarose gel electrophoresis to obtain NdeI-HindIII digested DNA fragments.

  Next, a pColdI vector digested with NdeI and HindIII was prepared, mixed with the NdeI-HindIII digested DNA fragment, and ligated using a DNA ligation kit. Thereafter, E. coli JM109 was transformed with 20 μL of the ligation reaction solution, and the transformant was grown on an LB medium (containing 50 μg / mL of ampicillin) containing agar at a concentration of 1.5% (w / v). The recombinant plasmid into which the target DNA fragment was inserted was designated as pColdICH-296-HT.

(Ii) Construction of pColdICH-296mut2-HT vector Synthetic primers HBD2mutF and HBD2mutR having the base sequences described in SEQ ID NOs: 77 and 78 in the sequence listing were synthesized by a DNA synthesizer and purified by a conventional method. The synthetic primers HBD2mutF and HBD2mutR are designed to convert a base sequence corresponding to an amino acid sequence corresponding to 543 to 548 among base sequences corresponding to amino acid numbers 537 to 554 of the amino acid sequence of CH-296 (SEQ ID NO: 13). Synthetic DNA.

  PCR was performed using the synthetic primers. PCR reaction conditions are shown below. That is, pColdI-CH296-HT 100 pg constructed in Example 15- (1)-(i) as template DNA, 25 μL of 2 × PrimeStar Max Premix (manufactured by Takara Bio Inc.), 10 pmol synthetic primer HBD2mutF, 10 pmol synthetic primer HBD2mutR was added, and sterilized water was added to make the total volume 50 μL. The reaction solution was set in TaKaRa PCR Thermal Cycler PERSONAL, and 30 cycles of reaction were performed in NORM mode with 98 ° C. for 10 seconds, 55 ° C. for 15 seconds, and 72 ° C. for 30 seconds. After completion of the reaction, 2.5 μL of the reaction solution was subjected to 1.0% agarose gel electrophoresis, and the target DNA fragment of about 6.2 kbp was confirmed. E. coli JM109 was transformed with 2 μL of the remaining PCR reaction solution, and the transformant was grown on LB medium (containing 50 μg / mL of ampicillin) containing 1.5% (w / v) concentration of agar. The recombinant plasmid into which the target DNA fragment was inserted was designated as pColdICH-296mut2-HT.

  This pColdICH-296mut2-HT was digested with NdeI and XbaI, and the NdeI and XbaI digests were extracted and purified by 1.0% agarose gel electrophoresis to obtain an NdeI-XbaI digested DNA fragment of about 1.7 kbp.

  Next, a pCold14ND vector digested with NdeI and XbaI was prepared, mixed with the NdeI-XbaI digested DNA fragment, and ligated using a DNA ligation kit. Thereafter, E. coli JM109 was transformed with 10 μL of the ligation reaction solution, and the transformant was grown on an LB medium (containing 50 μg / mL of ampicillin) containing agar at a concentration of 1.5% (w / v). The recombinant plasmid into which the target DNA fragment was inserted was confirmed by sequencing, and this recombinant plasmid was designated as pCold14ND-CH296mut2.

  This pCold14NDCH-296mut2 is a plasmid containing a base sequence encoding an amino acid sequence obtained by converting the amino acid sequence of amino acid numbers 543 to 548 contained in III-14 of CH-296 from “LIGRKK” to “VLAKRR”. The protein is named CH296mut2, its amino acid sequence is shown in SEQ ID NO: 79 in the sequence listing, and its base sequence is shown in SEQ ID NO: 80 in the sequence listing.

(2) Expression and purification of CH296mut2 Escherichia coli BL21 was transformed with pCold14NDCH-296mut2 prepared in (1) above, and expression of CH296mut2 was induced in the same manner as in Example 9- (2). Thereafter, the cells were collected by centrifugation, and about 2 g of a part thereof was resuspended in 10 mL of a cell disruption solution. The cells were disrupted by ultrasonic disruption, and separated into a supernatant extract and a precipitate by centrifugation (11,000 r / min, 20 minutes). About 20 mL of 50 mM Tris-HCl (pH 7.5) was added to the obtained supernatant extract to adjust the salt concentration. After saturation of a 5 ml HiTrap SP column with buffer A using a chromatography system, about 30 mL of the extract was applied, and 25 mL of buffer C [50 mM Tris-HCl (pH 7.5), 300 mM NaCl] was applied. The target protein was eluted. 25 mL of the eluate was diluted 100 times or more with Stock Buffer and concentrated to 5 mL using VIVASPIN. When this concentrated solution was analyzed by 2100 Bioanalyzer, the target protein having a molecular weight of about 63 kDa was detected in almost a single band, and the protein concentration was 2.2 mg / mL (calculated from the molecular weight of about 35 μM).

Example 16 Construction of CH296mut3 (1) Construction of CH296mut3 expression vector To obtain a modified FN fragment (CH296mut3) obtained by converting amino acids corresponding to amino acid numbers 569 to 572 of the amino acid sequence of CH-296 (SEQ ID NO: 13), An expression vector was constructed as described above.

(I) Construction of pColdICH-296mut3-HT vector From the base sequence of CH-296 described in SEQ ID NO: 24 in the sequence listing, synthetic primers HBD3mutF and HBD3mutR having the base sequences described in SEQ ID NO: 81 and 82 in the sequence listing were used as DNA synthesizers. And purified by a conventional method. The synthetic primers HBD3mutF and HBD3mutR are designed to convert a base sequence corresponding to the amino acid sequence corresponding to 569 to 572 out of the base sequence corresponding to amino acid numbers 563 to 574 of the amino acid sequence of CH-296 (SEQ ID NO: 13). Synthetic DNA.

  PCR was performed using the synthetic primers. PCR reaction conditions are shown below. Specifically, 100 pg of pColdI-CH296-HT constructed in Example 15- (1)-(i) as template DNA, 25 μL of 2 × PrimeStar Max Premix, 10 pmol of synthetic primer HBD3mutF, 10 pmol of synthetic primer HBD3mutR, and sterile water were added. In addition, the total volume was 50 μL. The reaction solution was set in TaKaRa PCR Thermal Cycler PERSONAL, and 30 cycles of reaction were performed in NORM mode with 98 ° C. for 10 seconds, 55 ° C. for 15 seconds, and 72 ° C. for 30 seconds. After completion of the reaction, 2.5 μL of the reaction solution was subjected to 1.0% agarose gel electrophoresis, and the target DNA fragment of about 6.2 kbp was confirmed. E. coli JM109 was transformed with 2 μL of the remaining PCR reaction solution, and the transformant was grown on LB medium (containing 50 μg / mL of ampicillin) containing 1.5% (w / v) concentration of agar. The recombinant plasmid into which the target DNA fragment was inserted was designated as pColdICH-296mut3-HT.

  This pColdICH-296mut3-HT was digested with NdeI and XbaI, and the NdeI and XbaI digests were extracted and purified by 1.0% agarose gel electrophoresis to obtain an NdeI-XbaI digested DNA fragment of about 1.7 kbp.

  Next, a pCold14ND vector digested with NdeI and XbaI was prepared, mixed with the NdeI-XbaI digested DNA fragment, and ligated using a DNA ligation kit. Thereafter, E. coli JM109 was transformed with 10 μL of the ligation reaction solution, and the transformant was grown on an LB medium (containing 50 μg / mL of ampicillin) containing agar at a concentration of 1.5% (w / v). The recombinant plasmid into which the target DNA fragment was inserted was confirmed by sequencing, and this recombinant plasmid was designated as pCold14ND-CH296mut3.

  This pCold14NDCH-296mut3 is a plasmid containing a base sequence encoding an amino acid sequence in which the amino acid sequence of amino acid numbers 569 to 572 contained in CS-1 of CH-296 is converted from “LDVP” to “LELP”. This region is known as a VLA4 binding domain. The protein is named CH296mut3, its amino acid sequence is shown in SEQ ID NO: 83 of the sequence listing, and its base sequence is shown in SEQ ID NO: 84 of the sequence listing.

(2) Expression and purification of CH296mut3 Escherichia coli BL21 was transformed with pCold14NDCH-296mut3 prepared in (1) above, and CH296mut3 was expressed and purified in the same manner as in Example 15- (2). In addition, the liquid volume of the culture solution, the cell disruption solution, 50 mM Tris-HCl (pH 7.5), buffer A, buffer C, Stock Buffer, and the concentrate was appropriately changed as necessary. When the obtained concentrated solution was analyzed by 2100 Bioanalyzer, the target protein having a molecular weight of about 63 kDa was detected as an almost single band, and the protein concentration was 2.5 mg / mL (calculated from the molecular weight of about 40 μM). .

Example 17 Construction of CH-296mut4 (1) Construction of CH296mut4 Expression Vector Modified FN fragment in which amino acids corresponding to amino acid numbers 543 to 548 and amino acid numbers 569 to 572 of the amino acid sequence of CH-296 (SEQ ID NO: 13) were converted ( In order to obtain CH296mut4), an expression vector was constructed as follows.

(I) Construction of pColdICH-296mut4-HT vector PCR was performed using synthetic primer HBD3mutF and synthetic primer HBD3mutR. PCR reaction conditions are shown below. That is, pColdICH-296mut2-HT 100 pg constructed in Example 15- (1)-(iii) as template DNA, 25 μL of 2 × PrimeStar Max Premix, 10 pmol of synthetic primer HBD3mutF, 10 pmol of synthetic primer HBD3mutR, and sterilized water were added. In addition, the total volume was 50 μL. The reaction solution was set in TaKaRa PCR Thermal Cycler SP, and 30 cycles of reaction were performed in NORM mode with 98 ° C. for 10 seconds, 55 ° C. for 15 seconds, and 72 ° C. for 30 seconds. After completion of the reaction, 5 μL of the reaction solution was subjected to 1.0% agarose gel electrophoresis, and the target DNA fragment of about 6.2 kbp was confirmed. E. coli JM109 was transformed with 2 μL of the remaining PCR reaction solution, and the transformant was grown on LB medium (containing 50 μg / mL of ampicillin) containing 1.5% (w / v) concentration of agar. The recombinant plasmid into which the target DNA fragment was inserted was designated as pColdICH-296mut4-HT.

  This pColdICH-296mut4-HT was digested with NdeI and XbaI, and the NdeI and XbaI digests were extracted and purified by 1.0% agarose gel electrophoresis to obtain an NdeI-XbaI digested DNA fragment of about 1.7 kbp.

  Next, a pCold14ND vector digested with NdeI and XbaI was prepared, mixed with the NdeI-XbaI digested DNA fragment, and ligated using a DNA ligation kit. Thereafter, E. coli JM109 was transformed with 10 μL of the ligation reaction solution, and the transformant was grown on an LB medium (containing 50 μg / mL of ampicillin) containing agar at a concentration of 1.5% (w / v). The recombinant plasmid in which the target DNA fragment was inserted was confirmed by sequencing, and this recombinant plasmid was designated as pCold14ND-CH296mut4.

  In this pCold14NDCH-296mut4, the amino acid sequence of amino acid numbers 543 to 548 included in III-14 of CH-296 is changed from “LIGRKK” to “VLAKRR”, and further amino acid numbers 569 to 572 included in CS-1 of CH-296. Is a plasmid comprising a base sequence encoding an amino acid sequence converted from “LDVP” to “LELP”. The protein is named CH296mut4, its amino acid sequence is shown in SEQ ID NO: 85, and its base sequence is shown in SEQ ID NO: 86 in the sequence listing.

(2) Expression and purification of CH296mut4 Escherichia coli BL21 was transformed with the pCold14NDCH-296mut4 prepared in (1) above, and CH296mut4 was expressed and purified in the same manner as in Example 15- (2). In addition, the liquid volume of the culture solution, the cell disruption solution, 50 mM Tris-HCl (pH 7.5), buffer A, buffer C, Stock Buffer, and the concentrate was appropriately changed as necessary. When the obtained concentrated solution was analyzed by 2100 Bioanalyzer, the target protein having a molecular weight of about 63 kDa was detected as an almost single band, and the protein concentration was 1.6 mg / mL (calculated from the molecular weight of about 25 μM). .

Example 18 Expansion of lymphocytes using FN fragments (H204, H296-CS1, H296-CS2, H206, H114, H117, H296R, H105-HT, H105, CH105-HT) (1) Anti-human CD3 antibody and Immobilization of FN fragment Anti-human CD3 antibody and FN fragment (H296-CS1, H296-CS2, H206, H114, H117, H296R, CH105-HT) were used in the culture equipment used in the following experiments as Example 4- (1). Immobilization was performed in the same manner. However, at the time of FN fragment immobilization, the FN fragment was added to ACD-A solution (pH 5.0) or PBS so as to have a final concentration of 25 to 100 μg / mL. The concentration of the FN fragment was adjusted in order to keep the amount of each FN fragment immobilized on the culture equipment constant. In addition, FN fragments (H204, H105-HT, H105) were directly added to the culture solution at a final concentration of 0.33 to 0.73 μg / mL at the start of culture without being immobilized on culture equipment. . The temperature at the time of immobilization was room temperature or 37 ° C. (in the presence of 5% CO ₂ ). In this example, H204 prepared in Example 6, H296-CS1 prepared in Example 7, H296-CS2 prepared in Example 8, H206 prepared in Example 11, and H206 prepared in Example 9 were prepared as FN fragments. H114, H117 prepared in Example 10, H296R prepared in Example 12, H105-HT prepared in Example 13, H105 prepared in Example 13, and CH105-HT prepared in Example 14 were used.

(2) Expansion of lymphocytes After suspending PBMC prepared in Example 1- (1) in 0.5% GT-T503 so as to be 0.5 × 10 ⁶ cells / mL, Example 18- ( 0.5% GT-T503 was added at 1.0 mL / well to the anti-human CD3 antibody-immobilized plate prepared in 1) or the anti-human CD3 antibody and FN fragment-immobilized plate, and the cell suspension was Added 5 mL / well. Subsequently, IL-2 was added to a final concentration of 1000 U / mL, and these plates were cultured at 37 ° C. in 5% CO ₂ (culture day 0). On the 4th day from the start of culture, the culture solution of each group was diluted 14.3 times with 0.5% GT-T503, and a 25 cm ² cell culture flask in which 10 mL of the diluted solution was not immobilized was set up. IL-2 was added to a final concentration of 500 U / mL. The culture was continued, and on the 8th day, the culture solution of each group was diluted 2-fold with 0.5% GT-T503, and a new 25 cm ² cell culture flask in which 10 mL of the diluted solution was not fixed was set up. IL-2 was added to a final concentration of 500 U / mL. On the 11th day of culture, the solution was diluted about 2-fold with 0% GT-T503 to make a total volume of 20 mL. On the 14th day after the start of the culture, the number of viable cells was measured by trypan blue staining, and the expansion culture rate was calculated in comparison with the number of cells at the start of the culture. Based on the calculated expansion culture rate, the cell proliferation ratio was calculated when the expansion culture rate was 1 when stimulated with anti-CD3 antibody alone. The results are shown in Table 12.

  As shown in Table 12, FN fragments (H204, H296-CS1, H296-CS2, H206, H114, H117, H296R, H105-HT, H105, CH105-HT) were immobilized in the early stage of lymphocyte expansion culture. In the group in which the equipment was used or added directly to the medium, a higher cell growth rate was obtained compared to the control group. That is, the FN fragment (H204, H296-CS1, H296-CS2, H206, H114, H117, H296R, H105-HT, H105, CH105-HT) is clearly used in lymphocyte expansion culture. It was.

Example 19 Analysis of CD45RA-positive CCR7-positive cells, CD45RA-positive CD62L-positive cells, CD45RA-positive CCR7-positive CD62L-positive cells of the lymphocytes obtained in Example 18 On the 14th day from the start of culture prepared in Example 18- (2) The cells were analyzed for CD45RA positive CCR7 positive cells, CD45RA positive CD62L positive cells, CD45RA positive CCR7 positive CD62L positive cells in the same manner as in Example 1- (5), and the content ratio was measured. However, in addition to the RD1-labeled mouse anti-human CD45RA antibody / FITC-labeled mouse anti-human CCR7 antibody, a PC5-labeled mouse anti-human CD62L antibody (manufactured by eBioscience) was used as the staining antibody. Based on the content ratio of each positive cell, the positive cell ratio when each content ratio when stimulated with only the anti-CD3 antibody was set to 1 was calculated. The results are shown in Table 13.

  As shown in Table 13, in the group using culture equipment in which the FN fragment (H296-CS1, H296-CS2, H206, H114, H117, H296R, CH105-HT) was immobilized, compared to the control group A result with a high ratio of the CD45RA positive CCR7 positive cell population, the CD45RA positive CD62L positive cell population, and the CD45RA positive CCR7 positive CD62L positive cell population was obtained. By using FN fragments (H296-CS1, H296-CS2, H206, H114, H117, H296R, CH105-HT), naïve T-like cells with high differentiation potential into cells exhibiting high cytotoxic activity in vivo can be obtained. It became clear that it can proliferate efficiently.

Example 20 Expansion of Lymphocytes Using CH-296 Mutants (CH296mut2, CH296mut3, CH296mut4) (1) Immobilization of anti-human CD3 antibody, CH-296 and three types of CH-296 mutants Anti-human CD3 antibody, CH-296, and three types of CH-296 mutants (CH296mut2, CH296mut3, CH296mut4) were immobilized on the culture equipment used in the same manner as in Example 4- (1). However, when each FN fragment was immobilized, the FN fragment was added to ACD-A solution (pH 5.0) or PBS so that the final concentration was 4 to 50 μg / mL. The concentration of the FN fragment was adjusted in order to keep the amount of each FN fragment immobilized on the culture equipment constant. In this example, CH-296, CH296mut2 prepared in Example 15, CH296mut3 prepared in Example 16, and CH296mut4 prepared in Example 17 were used as FN fragments. Note that CH296mut2 was introduced into the heparin-binding domain of CH-296, CH296mut3 was introduced into the VLA4-binding domain of CH-296, and CH296mut4 was introduced into the CH-296 heparin-binding domain and VLA4-binding domain through amino acid substitution. It is a mutant. In addition, a group in which only the anti-CD3 antibody was immobilized was also set.

(2) Enlarged culture of lymphocytes Expanded culture of lymphocytes was carried out in the same manner as in Example 18- (2). On the 14th day after the start of the culture, the number of viable cells was measured by trypan blue staining, and the expansion culture rate was calculated in comparison with the number of cells at the start of the culture. Based on the calculated expansion culture rate, the cell proliferation ratio was calculated when the expansion culture rate was 1 when stimulated with anti-CD3 antibody alone. The results are shown in Table 14.

  As shown in Table 14, when CH-296 or a mutant thereof was used, the cell proliferation ratio was higher than when only the anti-CD3 antibody was used. In addition, the group using the culture equipment in which the CH-296 mutant was immobilized in the early stage of lymphocyte expansion culture had a lower cell proliferation ratio than the group using the culture equipment in which CH-296 was immobilized. The suppression effect was the strongest with CH296mut4. That is, it was revealed that the heparin binding domain and the VLA4 binding domain are important for the cell proliferation action of CH-296 during lymphocyte expansion culture.

Example 21 Analysis of CD45RA-positive CCR7-positive cells, CD45RA-positive CD62L-positive cells, and CD45RA-positive CCR7-positive CD62L-positive cells of the lymphocytes obtained in Example 20 On the 14th day from the start of the culture prepared in Example 20- (2) The cells were analyzed for CD45RA-positive CCR7-positive cells, CD45RA-positive CD62L-positive cells, and CD45RA-positive CCR7-positive CD62L-positive cells by the same method as in Example 19, and the content ratio was measured. Based on the content ratio of each positive cell, the positive cell ratio when the content ratio upon stimulation with only the anti-CD3 antibody was set to 1 was calculated. The results are shown in Table 15.

  As shown in Table 15, when CH-296 or a variant thereof was used, the CD45RA positive CCR7 positive cell ratio, the CD45RA positive CD62L positive cell ratio, and the CD45RA positive were compared with the case where only the anti-CD3 antibody was used. The ratio of CCR7 positive CD62L positive cells was high. In addition, the group using the culture equipment in which the CH-296 mutant is immobilized is compared with the group using the culture equipment in which the CH-296 is immobilized, the CD45RA positive CCR7 positive cell population, the CD45RA positive CD62L positive cell population. As a result, a low ratio of the CD45RA positive CCR7 positive CD62L positive cell population was obtained. The suppression effect was the strongest with CH296mut4. That is, it was revealed that the VLA-4 binding domain and the heparin binding domain are important for the naive T-like cell proliferation action of CH-296 during lymphocyte expansion culture.

Example 22 Expansion of lymphocytes using inhibitory peptides (VLA4-Inhibitor and HBD2) (1) Immobilization of anti-human CD3 antibody and CH-296 Anti-human CD3 antibody and CH- 296 was immobilized in the same manner as in Example 1- (2). However, 0.24 mL / well of immobilization solution was added and immobilized at room temperature or 37 ° C. (in the presence of 5% CO ₂ ) for 5 hours.

(2) Reaction with peptide Prior to initial stimulation, the peptide used in this example (a peptide that specifically binds to a functional site of VLA-4 binding domain or heparin binding domain on the cell surface) is reacted with cells. Thus, in order to verify whether or not the target effect is inhibited, the following peptide reaction was performed.

PBMC prepared by the same method as in Example 1- (1) was suspended using GT-T503 so that the concentration was 0.5 × 10 ⁶ cells / mL. Thereafter, VLA4-Inhibitor (amino acid sequence: Phenylac-Leu-Asp-Phe-Pro-NH ₂ , manufactured by BACHEM) or FN fragment (FN amino acid sequence from 1954 to 1959: amino acid number 84 of amino acid sequence of SEQ ID NO: 7) -89: GT-T503 containing H-Leu-Ile-Gly-Arg-Lys-Lys-OH (manufactured by BACHEM, hereinafter referred to as HBD2) is added, and PBMC is finally 0.15 × 10 ⁶ cells / mL (This solution is hereinafter referred to as a cell suspension). At that time, the final concentration of each peptide in the cell suspension was adjusted to 100 and 50 μg / mL. To the control group, the same amount of DMSO (Hybrimax: manufactured by Sigma) as each peptide was added. The reaction with the peptide was carried out by allowing it to stand at room temperature for 30 minutes.

(3) Expanded culture of lymphocytes The cell suspension subjected to peptide reaction in Example 22- (2) was added to a final concentration of 0.5% human AB serum, 0.2% human serum albumin, 1000 U / mL IL- The required amount was added so as to be 2. The cell suspension was seeded at 0.12 × 10 ⁶ cells / well on the anti-human CD3 antibody and CH-296 immobilized plate prepared in Example 22- (1), and these plates were incubated at 37 ° C. The cells were cultured in a 5% CO ₂ incubator (culture day 0). Tables 16 and 17 show the cell growth ratios on the 4th day after the start of the culture. The cell growth ratio was defined as the cell growth ratio when the number of viable cells was measured by trypan blue staining and the control group with DMSO added was compared with each cell number being 1.

  As shown in Tables 16 and 17, the cell proliferation ratio was decreased by reacting with VLA4-Inhibitor or HBD2 before the start of lymphocyte expansion culture as compared with the control group. That is, it was revealed that the VLA-4 binding domain and the heparin binding domain are important for the cell proliferation effect of CH-296 during lymphocyte expansion culture.

Example 23 Expansion of Lymphocytes Using FN Fragment (C274) (1) Immobilization of anti-human CD3 antibody and FN fragment The FN fragment (C274) having the sequence was immobilized in the same manner as in Example 4- (1).

(2) Enlarged culture of lymphocytes Using the anti-human CD3 antibody-immobilized plate prepared in Example 23- (1), or the anti-human CD3 antibody and C274-immobilized plate, the same as in Example 18- (2) The lymphocytes were expanded by this method. The results are shown in Table 18.

  As shown in Table 18, in the group using the culture equipment in which the FN fragment (C274) was immobilized at the initial stage of lymphocyte expansion culture, a higher expansion culture rate was obtained compared to the control group. That is, it was revealed that the FN fragment (C274) is suitably used during lymphocyte expansion culture.

(3) Analysis of CD45RA-positive CCR7-positive T cells and CD45RA-positive CCR7-positive CD62L-positive T cells The cells on day 14 after the start of culture prepared in Example 23- (2) were CD45RA-positive CCR7-positive in the same manner as in Example 21. T cells and CD45RA positive CCR7 positive CD62L positive T cells were analyzed and the content ratio was measured. The results are shown in Table 19.

  As shown in Table 19, the ratio of the CD45RA-positive CCR7-positive cell population and the CD45RA-positive CCR7-positive CD62L-positive cell population in the group using the culture device in which the FN fragment (C274) was immobilized was compared with the control group. High results were obtained. It was revealed that naive T-like cells can be proliferated with high efficiency by using the FN fragment (C274).

Example 24 Analysis of Th1-type cell ratio in lymphocyte expansion culture using CH-296 (1) Immobilization of anti-human CD3 antibody and CH-296 fragment Anti-human CD3 antibody and CH were used as culture equipment used in the following experiments. -296 was immobilized in the same manner as in Example 4- (1).

(2) Enlarged culture of lymphocytes Expanded culture of lymphocytes was carried out in the same manner as in Example 18- (2). However, IL-2 is added so that the final concentration is 500 U / mL at the start of culture (culture 0 day), and each group becomes 0.015 × 10 ⁶ cells / mL on the 4th day of culture start. Diluted with 0.5% GT-T503 (volume 10 mL), transferred to a 25 cm ² cell culture flask in which nothing was immobilized, and added IL-2 to a final concentration of 500 U / mL . Cells on day 7 after the start of culture were collected, the number of viable cells was counted by trypan blue staining, and the expansion culture rate was calculated by comparison with the number of cells at the start of culture. The results are shown in Table 20.

  As shown in Table 20, in the group using the culture equipment in which CH-296 was immobilized at the initial stage of lymphocyte expansion culture, the result of the cultivated lymphocyte expansion culture rate being higher than that of the control group was obtained. It was.

(3) Analysis of CD45RA-positive CCR7-positive T cells The cells on day 7 of culture obtained in Example 24- (2) were analyzed for CD45RA-positive CCR7-positive T cells in the same manner as in Example 1- (5). The content ratio was measured. However, 1% BSA / PBS was used for cell washing after the antibody reaction. The results are shown in Table 21.

  As shown in Table 21, in the group using the culture container in which CH-296 was immobilized, the CD45RA-positive CCR7-positive cell content ratio in the lymphocytes on the seventh day of culture was higher than that in the control group. was gotten.

(4) Analysis of IL-4 producing cells The cells on day 7 of culture obtained in Example 24- (2) were suspended in 10 FRPM so as to be 4 × 10 ⁶ cells / mL, and 10 FRPMI was then added to 0 in advance. 0.1 mL / well was poured into a 48-well cell culture plate added with 3 mL each. PMA was added to each well to a final concentration of 50 ng / mL, Ionomycin to a final concentration of 1 μg / mL, and Brefeldin A to a final concentration of 10 μg / mL, followed by culturing in a 37 ° C., 5% CO ₂ incubator for 4 hours. Cells after completion of the culture were collected and washed with PBS, 1% BSA / PBS. The cells were suspended in 1% BSA / PBS, ECD-labeled mouse IgG ₁ or ECD-labeled mouse anti-human CD4 antibody (Beckman Coulter) was added, and incubated on ice for 30 minutes, followed by FITC labeling according to the method of IntraPrep Reagent. Intracellular staining was performed with mouse anti-human IL-4 antibody and RD1-labeled mouse anti-human IFN-γ antibody. As negative controls, FITC-labeled mouse IgG ₁ (manufactured by Beckman Coulter) and PE-labeled mouse IgG ₁ (manufactured by Dako or Beckman Coulter) were used. The cells were subjected to flow cytometry, and the ratio of IL-4 production positive cells to the whole measured cells was analyzed. The results are shown in Table 22. The results of analysis of IL-4 negative IFN-γ positive T cells are shown in Table 23, and the results of analysis of CD4 positive IL-4 negative IFN-γ positive T cells are shown in Table 24.

  As shown in Table 22, in the group using the culture vessel in which CH-296 was immobilized, the positive rate of IL-4 producing cells in the whole lymphocytes in culture was lower than that in the control group. Obtained. From the examples, it was revealed that the content ratio of IL-4 producing cells, ie, Th2 type cells, can be suppressed by allowing CH-296 to act at the early stage of expansion of lymphocytes. As shown in Tables 23 and 24, in the group using the culture vessel in which CH-296 was immobilized, the ratio of IL-4 negative IFN-γ positive cells in culture was higher than that in the control group. Results were obtained. From this example, it was revealed that the content ratio of IL-4 negative IFN-γ positive cells, ie Th1 type cells, can be increased by allowing CH-296 to act at the early stage of expansion of lymphocytes. From the above results, it was shown that the combination of anti-CD3 antibody and CH-296 can efficiently culture Th1-type T cells useful for cellular immunity.

Example 25 Expansion of Lymphocytes Using FN Fragment (CH-296, H296-H296) and Anti-4-1BB Antibody-1
(1) Immobilization of anti-human CD3 antibody and FN fragment (CH-296, H296-H296) and anti-4-1BB antibody Anti-CD3 antibody and FN fragment (CH-296, H296-) were used in the culture equipment used in the following experiments. H296) and an anti-4-1BB antibody (manufactured by R & D Systems), which is a costimulatory factor, was immobilized in the same manner as in Example 4- (1). However, H296-H296 and anti-4-1BB antibody were immobilized to a final concentration of 5 μg / mL.

(2) Enlarged culture of lymphocytes PBMC 0.53 × 10 ⁶ cells prepared in Example 1- (1) containing 5.3% human AB-type serum-containing KBM551 (hereinafter referred to as 0.6% KBM551) 5.3 mL And added to the plate prepared in Example 25- (1), IL-2 was added to a final concentration of 200 U / mL, and the cells were cultured at 37 ° C. in 5% CO ₂ (culture day 0). Eye). On day 4 of culture, the cells in each well were suspended, a portion of which was diluted 8.3 times with 0.6% KBM551, and 7.8 mL of the diluted solution was not fixed to 25 cm ² cells. The culture flask was transferred to a standing one, and IL-2 was added to a final concentration of 200 U / mL. The culture was continued, and on day 7, an equal amount of plasma-free KBM551 was added to the culture solution of each group and diluted 2-fold, and IL-2 was added so that the final concentration was 200 U / mL. On the 10th day after the start of culture, the cell culture solution of each group was diluted 2-fold with plasma-free KBM551, and 15.6 mL of the diluted solution was added to a new 25 cm ² cell culture flask on which nothing was immobilized. Each moved. IL-2 was added to a final concentration of 200 U / mL in each group. On the 10th and 14th day after the start of the culture, the number of viable cells was measured by trypan blue staining method, and the expansion culture rate was calculated in comparison with the number of cells at the start of the culture. The results are shown in Table 25.

  As shown in Table 25, the anti-CD3 antibody alone or the anti-CD3 antibody and the anti-4-1BB antibody were immobilized by using a culture device in which CH-296 or H296-H296 was immobilized at the early stage of lymphocyte expansion culture. Compared with the group using the cultured equipment, a high expansion rate of lymphocytes was obtained regardless of the culture period. From this, it was revealed that CH-296 or H296-H296 is preferably used during lymphocyte expansion culture.

(3) Analysis of CCR7-positive CD45RA-positive T cells and CCR7-positive CD62L-positive CD45RA-positive T cells The cells on day 10 or day 14 of culture prepared in Example 25- (2) were subjected to CCR7 in the same manner as in Example 19. Analysis of positive CD45RA positive T cells and CCR7 positive CD62L positive CD45RA positive T cells was performed. Table 26 shows the results of the 10th day of culture, and Table 27 shows the results of the 14th day of culture, in which the content ratio of each positive cell was calculated.

  As shown in Table 26 and Table 27, by using culture equipment in which CH-296 or H296-H296 is immobilized at the initial stage of lymphocyte expansion culture, only anti-CD3 antibody or anti-CD3 antibody and anti-4-1BB are used. Compared with the group using the culture apparatus on which the antibody was immobilized, a CCR7-positive CD45RA-positive T cell population and a CCR7-positive CD62L-positive CD45RA-positive T cell population were obtained at a high ratio regardless of the culture period. From this example, it was revealed that naive T-like cells can be proliferated with high efficiency by using CH-296 or H296-H296 at the early stage of lymphocyte expansion culture.

Example 26 Expanded culture of lymphocytes using CH-296, anti-human CD28 antibody, anti-human 4-1BB antibody and CD3 / CD28 beads (1) Separation of PBMC and inactivated plasma Obtaining informed consent From a healthy human donor, blood was collected for 50 mL using heparin sodium as an anticoagulant, and then centrifuged at 700 × g for 20 minutes. The plasma fraction, which is the supernatant after centrifugation, and the cell fraction containing PBMC were collected. The plasma fraction was inactivated at 56 ° C. for 30 minutes, and then centrifuged at 900 × g for 30 minutes. The supernatant after centrifugation was recovered as inactivated plasma and used for each experiment. The cell fraction containing PBMC was diluted with DPBS, overlaid on Ficoll-paque, and centrifuged at 700 × g for 20 minutes. The PBMC in the intermediate layer was collected and washed with a pipette, and the number of viable cells was calculated using a Turku staining solution (manufactured by Nacalai Tesque) and used for each experiment.

(2) Immobilization of anti-human CD3 antibody and CH-296, anti-human CD28 antibody, anti-human 4-1BB antibody The anti-CD3 antibody, CH-296, anti-CD28 antibody, anti-4- 1BB antibody was immobilized. That is, only anti-CD3 antibody (final concentration 5 μg / mL), anti-CD3 antibody (final concentration 5 μg / mL) and CH-296 (final concentration 25 μg / mL), anti-CD3 antibody (final concentration 5 μg / mL) and anti-CD28 antibody (Final concentration 5 μg / mL), ACD-A solution containing anti-CD3 antibody (final concentration 5 μg / mL) and anti-4-1BB antibody (final concentration 5 μg / mL) sealed with a bag area of 86 cm ² 10.4 mL / bag was added to a permeable culture bag, MultiLife 215 (manufactured by Takara Bio Inc.), and incubated at 37 ° C. in 5% CO ₂ for 5 hours. In addition, the bag was washed three times with RPMI 1640 before use and then subjected to each experiment.

(3) Enlarged culture of lymphocytes PBMC 0.6 × 10 ⁷ cells separated in Example 26- (1) 0.6% inactivated plasma-containing KBM551 (manufactured by Takara Bio Inc., hereinafter containing 0.6% plasma) (Described as KBM551) suspended in 120 mL and added to each of the MultiLife 215 having different immobilization conditions prepared in Example 26- (2). Regarding CD3 / CD28 bead conditions, CD3 / CD28 beads (Dynabeads CD3 / CD28) washed with 1% HS / PBS were added in an amount of 3 times the number of cells. IL-2 was added to a final concentration of 200 U / mL and cultured at 37 ° C. in 5% CO ₂ (culture day 0). On the fourth day of culture, cells in each CultureLife 215 were suspended, and 120 mL of this suspension was diluted with 380 mL of 0.6% plasma-containing KBM551, and nothing was immobilized on the gas-permeable culture bag MultiLifeEva ( Transferred to Takara Bio). At this time, for the CD3 / CD28 bead stimulation group, the CD3 / CD28 beads were removed using the magnetic bead separator MPC-1 after cell suspension. In any group, IL-2 was added to a final concentration of 200 U / mL, and the cells were cultured at 37 ° C. in 5% CO ₂ . On the 7th day from the start of the culture, each culture life Eva was added with 0% plasma-containing KBM551 (hereinafter referred to as plasma-free KBM551) in the same amount as the cell suspension and diluted twice. IL-2 was added so that it might become 200 U / mL. On the 10th day from the start of the culture, the number of viable cells was counted using the trypan blue staining method, and the expansion culture rate was calculated by comparison with the number of cells at the start of the culture. The results are shown in Table 28.

  As shown in Table 28, by using a culture device in which anti-CD3 antibody and CH-296 were immobilized at the initial stage of lymphocyte expansion culture, only anti-CD3 antibody, anti-CD3 antibody and anti-CD28 antibody, anti-CD3 antibody and A high expansion culture rate was obtained as compared with the group using the culture equipment on which the anti-4-1BB antibody was immobilized or the CD3 / CD28 beads. From this, it became clear that CH-296 is suitably used during lymphocyte expansion culture.

(4) Analysis of CCR7-positive CD45RA-positive T cells The cells on day 10 of culture prepared in Example 26- (3) were analyzed for CCR7-positive CD45RA-positive T cells in the same manner as in Example 1- (5). It was. The results of calculating the content ratio of each positive cell are shown in Table 29.

  As shown in Table 29, by using a culture device in which anti-CD3 antibody and CH-296 were immobilized at the early stage of lymphocyte expansion culture, only anti-CD3 antibody, anti-CD3 antibody and anti-CD28 antibody, anti-CD3 antibody and A CCR7-positive CD45RA-positive T cell population was obtained at a higher ratio as compared with the group using the culture equipment on which the anti-4-1BB antibody was immobilized or the CD3 / CD28 beads. From this example, it was revealed that naive T-like cells can be proliferated with high efficiency by using CH-296 in the early stage of lymphocyte expansion culture.

Example 27 Allogeneic mixed lymphocyte reaction (MLR) using cultured lymphocytes
(1) Cryopreservation and thawing of cultured cells Stock 10 day cultured cells prepared in Example 26- (3) were suspended in RPMI 1640, and CP-1 and 25% HSA were mixed at a ratio of 17: 8. An equal amount of was added, suspended, and stored in liquid nitrogen. At the time of use, these preserved cultured cells were rapidly thawed in a 37 ° C. water bath, washed with RPMI 1640 containing 10 μg / mL DNase, and subjected to each experiment after calculating the number of viable cells by trypan blue staining.

(2) Allogeneic MLR
Allogeneic MLR was performed using the cells prepared in Example 27- (1). The cultured cells thawed in Example 27- (1) were treated with 5% human AB serum, 2 mM L-glutamine, 1 mM Sodium pyruvate, 1 × NEAA Mixture (all manufactured by Cambrex), 100 μg / mL streptomycin sulfate (manufactured by Meiji Seika Co., Ltd.). ) Containing RPMI1640 (hereinafter referred to as 5HRPMI) to 2 × 10 ⁶ cells / mL (hereinafter referred to as responder cells). On the other hand, PBMC derived from Allogeneic donor (non-self donor: donor different from the donor described in Example 26- (1)) prepared by the same method as in Example 1- (1) was used as an X-ray irradiation apparatus (manufactured by HITEX). 260 type) was irradiated with X-rays (0.88 C / kg), washed with 5HRPMI, and adjusted to 2 × 10 ⁶ cells / mL (hereinafter referred to as Stimulator cells). To each 24-well cell culture plate, 0.5 mL / well of each cell suspension prepared such that the ratio of responder cells: stimulator cells was 1: 1 was added. Next, IL-2 was added to each well to a final concentration of 500 U / mL and cultured at 37 ° C. in 5% CO ₂ (culture day 0). On the second day of culture, 1 mL of 5HRPMI was added to each well, and IL-2 was added to a final concentration of 500 U / mL (final volume: 2 mL / well). After suspending the cells in each well on the 4th day from the start of the culture, a half amount was divided into 2 wells, and IL-2 was added to all wells to 1 mL of 5HRPMI and a final concentration of 500 U / mL (final volume: 2 mL / well). The culture was continued until day 7 of the culture, the number of viable cells was measured by trypan blue staining method, and the expansion culture rate was calculated in comparison with the number of cells at the start of the culture. The results are shown in Table 30.

  As shown in Table 30, lymphocytes using an anti-CD3 antibody and CH-296-immobilized culture equipment at the initial stage of lymphocyte expansion culture were anti-CD3 antibody only, anti-CD3 antibody and anti-CD28 antibody, and anti-CD3 antibody. It was revealed that non-self antigen-recognizing cells proliferate at a higher expansion rate in Allogeneic MLR as compared to the group using culture equipment immobilized with anti-4-1BB antibody or CD3 / CD28 beads. From this, it became clear that CH-296 is suitably used during lymphocyte expansion culture.

(3) Preparation of Phytohemaglutinin (PHA) blast cells A PBMC derived from Allogeneic donor prepared by the same method as in Example 1- (1) was prepared to 1 × 10 ⁶ cells / mL with 5HRPMI, and a plate for 6-well cell culture 3 mL each was seeded on (Corning). PHA (manufactured by Sigma) was added to a final concentration of 2 μg / mL so that IL-2 had a final concentration of 100 U / mL, and cultured at 37 ° C. in 5% CO ₂ . On the fourth day of culture, 12 mL of 5HRPMI was added to the culture solution, and IL-2 was added to a final concentration of 100 U / mL, and then the cell suspension was distributed to a 6-well cell culture plate at 5 mL / well. The culture was continued until 7 days after the start of the culture.

(4) Measurement of non-self-specific cytotoxic activity The non-self-specific cytotoxic activity of the cells 7 days after the start of culture prepared in Example 27- (2) was measured using Calcein-AM. Act [Lichtenfels R. (Lichtenfelds R. et al.), J. et al. Immunol. Methods, Vol. 172, No. 2, pp. 227-239 (1994)]. The PHA blast cells blasted in Example 27- (3) were suspended in RPMI 1640 containing 5% FBS so as to be 1 × 10 ⁶ cells / mL, and then calcein− was prepared to a final concentration of 25 μM. AM (manufactured by Dojindo Laboratories) was added and cultured at 37 ° C. for 2 hours. The cells were washed with a medium not containing Calcein-AM, and then used as Calcein-labeled target cells. Seventy-seven days after the start of culture prepared in Example 27- (2), effector cells were used as effector cells, and after serial dilution with 5HRPMI to 3 × 10 ⁶ cells / mL, a 96-well cell culture plate (Becton Dickinson) Alternatively, 100 μL / well was dispensed into each well. Calcein-labeled target cells were mixed with 30 times the amount of K562 cells, adjusted so that the calcein-labeled target cells were 1 × 10 ⁵ / mL, and 100 μL / well was added. The K562 cells were used to eliminate nonspecific cytotoxic activity by NK cells mixed in responder cells. At this time, the ratio of the effector cells (E) to the Calcein-labeled target cells (T) was shown as an E / T ratio, and the measurement was performed at an E / T ratio of 30. The plate containing the cell suspension was centrifuged at 210 × g for 1 minute, and then incubated at 37 ° C. for 4 hours in the presence of 5% CO ₂ . After 4 hours, 100 μL of culture supernatant was collected from each well, and the amount of calcein released into the culture supernatant was measured with a fluorescent plate reader [Berthold technologies (excitation 485 nm / measurement 538 nm)]. “Cytotoxic activity (%)” was calculated according to the following formula 1.
Formula 1: Cytotoxic activity (%) =
{(Measured value of each well-minimum release amount) / (maximum release amount-minimum release amount)} × 100

  In the above formula, the minimum release amount is the Calcein release amount of a well containing only Calcein labeled target cells, and indicates the Calcein spontaneous release amount from Calcein labeled target cells. The maximum release amount indicates the release amount of Calcein when the cell is completely destroyed by adding 0.1% surfactant Triton X-100 (manufactured by Nacalai Tesque) to Calcein-labeled target cells. The measurement results are shown in Table 31.

  As shown in Table 31, lymphocytes using culture equipment in which anti-CD3 antibody and CH-296 were immobilized at the early stage of lymphocyte expansion culture were anti-CD3 antibody only, anti-CD3 antibody and anti-CD28 antibody, and anti-CD3 antibody. And the non-self antigen-specific cytotoxic activity was high compared with the group which used the culture equipment or CD3 / CD28 bead which fix | immobilized anti-4-1BB antibody. From this, it became clear that CH-296 is suitably used during lymphocyte expansion culture.

Example 28 Expansion of CD4-positive T cells using CH-296-4
(1) Immobilization of anti-human CD3 antibody and H296-H296 Anti-human CD3 antibody and FN fragment (CH-296, H296-H296) were used in the same manner as in Example 4- (1) for the culture equipment used in the following experiments. Immobilized by the method. However, conditions for immobilizing only CH-296 were also set, and the FN fragment (H296-H296) was immobilized to a final concentration of 3 μg / mL.

(2) Preparation of CD8-removed cells and preparation of CD3 / CD28 bead selection cells (CD3 / CD28 bead-binding cells) Example 1 using PBMC separated and stored by the method described in Example 1- (1) -CD8-removed cells were prepared by the same method as (3), and T cell selection cells (CD3 / CD28 bead-binding cells) using CD3 / CD28 beads were prepared by the same method as in Example 3- (1). The prepared cells were suspended in 2% GT-T503 and 5% X-VIVO15 so that the cell concentration was 1.0 × 10 ⁶ cells / mL.

(3) Expansion culture of CD4-positive T cells 2 mL of 2% GT-T503 and 5% X-VIVO15 were added to the plate prepared in Example 28- (1), and anti-CD3 antibody and FN fragment ( In the plate immobilized with CH-296 or H296-H296), the CD8-removed cells prepared in Example 28- (2) were used, and in the plate on which only CH-296 was immobilized, Example 28- (2). 2 mL each of the CD3 / CD28 bead-bound cells prepared in (cells stimulated with CD3 / CD28 beads) was added, followed by culturing at 37 ° C. in 5% CO ₂ (culture day 0). On the third day after the start of the culture, 2.5 mL of the culture solution is transferred to a T25 cell culture flask on which nothing is fixed, and is further allowed to stand until the last day of culture (hereinafter referred to as static culture). And conditions for culturing while shaking using LABO SHAKER (manufactured by RIO CRAFFT) (hereinafter referred to as shaking culture). On the 3rd, 5th, 7th, and 10th days after the start of culture, the cells are diluted with each medium so that the cell concentration becomes 0.5 × 10 ⁶ cells / mL, and the total culture volume is 12. From the next culture day, when 5 mL was reached, 8.75 mL of the culture supernatant was replaced with a new medium, and the culture was continued until day 12 of the culture start. In any culture day, IL-2 was added so that the 2% GT-T503 culture group had a final concentration of 200 U / mL, and the 5% X-VIVO15 culture group had a final concentration of 100 U / mL. Table 32 shows the expansion culture rate on the 5th day from the start of culture under shaking conditions. In addition, Table 33 shows the expansion culture rate on the 5th and 12th days after the start of the culture under the stationary conditions.

  As shown in Tables 32 and 33, in the group stimulated with anti-CD3 antibody and CH-296 or H296-H296 regardless of the culture conditions, medium conditions, and culture days, stimulation was performed with CD3 / CD28 beads and CH-296. A higher expansion rate than that of the group obtained was obtained. That is, it was shown that the combination of anti-CD3 antibody and CH-296 or H296-H296 can be suitably used in expansion culture of CD4 positive cells.

(4) Analysis of CD45RA-positive CCR7-positive T cells The cells on day 12 of culture obtained under shaking culture conditions in Example 28- (3) were treated with CD45RA-positive cells in the same manner as in Example 1- (5). The content of CCR7 positive T cells was calculated. The results are shown in Table 34.

  As shown in Table 34, the group stimulated with anti-CD3 antibody and CH-296 or H296-H296 in either medium compared to the group stimulated with CD3 / CD28 beads and CH-296. The content of positive CCR7-positive T cells was high. That is, it became clear that the combination of anti-CD3 antibody and CH-296 or H296-H296 can proliferate naive T-like cells with high efficiency.

(5) Analysis of IL-4 negative IFN-γ positive T cells and IL-4 positive IFN-γ negative T cells Example 24- About the cells on the 12th day of culture obtained in Example 28- (3) The content ratio of IL-4 negative IFN-γ positive T cells and IL-4 positive IFN-γ negative T cells was measured by the same method as in (4). The results of the IL-4 negative IFN-γ positive T cell ratio are shown in Table 35, and the results of the measurement of the content ratio of IL-4 positive IFN-γ negative T cells are shown in Table 36. Note that analysis was performed on CD4 positive cells under all conditions.

  As shown in Table 35 and Table 36, the group stimulated with anti-CD3 antibody and CH-296 or H296-H296 was stimulated with CD3 / CD28 beads and CH-296 regardless of the culture conditions and medium conditions. Compared with the group, the ratio of IL-4 negative IFN-γ positive T cells was high, and the ratio of IL-4 positive IFN-γ negative T cells was low. It is known that Th1-type activated CD4-positive T cells produce IFN-γ, and Th2-type activated CD4-positive T cells produce IL-4. That is, in the stimulation during lymphocyte culture, the combination of anti-CD3 antibody and CH-296 or H296-H296 proliferates predominantly Th1-type CD4-positive T cells, suppresses Th2-type CD4-positive cell proliferation, and It was revealed that a cell population suitable for cell immunization can be produced.

(6) Analysis of CCR4-positive T cells The cells on day 12 of culture obtained in Example 28- (3) were treated with PE-labeled anti-human CCR4 antibody (R & D Systems) in the same manner as in Example 1- (5). CCR4-positive T cells were measured using FITC-labeled anti-human CD4 antibody. Here, FITC-labeled mouse IgG1 and RD1-labeled mouse IgG1 (manufactured by Beckman Coulter, Inc.) were used as negative controls. The content ratio of CD4-positive CCR4-positive T cells was calculated for each cell population. The results are shown in Table 37.

  As shown in Table 37, the group initially stimulated with anti-CD3 antibody and CH-296 or H296-H296 in any culture condition and medium condition is the group stimulated with CD3 / CD28 beads and CH-296. In comparison, it was shown that the content ratio of CD4-positive CCR4-positive T cells was low. CCR4 is known to be selectively expressed on Th2-type CD4-positive T cells. That is, it became clear that the combination of anti-CD3 antibody and CH-296 or H296-H296 suppressed the proliferation of Th2-type CD4 positive cells in stimulation during lymphocyte culture.

(7) Analysis of regulatory T cells (CD4-positive CD25-positive Foxp3-positive T cells) Cells on day 12 of culture obtained in Example 28- (3) were washed with PBS, 1% BSA / PBS. The cells were suspended in 1% BSA / PBS, and FITC-labeled mouse anti-human CD25 antibody (manufactured by Dako Cytomation) and ECD-labeled mouse anti-human CD4 antibody were added, followed by incubation at room temperature in the dark for 15 minutes. Here, FITC-labeled mouse IgG1 and ECD-labeled mouse IgG1 were used as negative controls. After washing with PBS, FIXATION / PERMEABILIZATION WORKING SOLN. (Manufactured by eBioscience) was added and reacted at 4 ° C. in the dark for 30 minutes. Pereabilization buffer (manufactured by eBioscience) was added, and the supernatant was removed by centrifugation at 4 ° C. and 300 g for 5 minutes. The Pereabilization buffer was added again, PE-labeled mouse anti-human Foxp3 antibody (manufactured by eBioscience) was added, and the mixture was incubated at 4 ° C. in the dark for 30 minutes. At this time, PE-labeled mouse IgG1 was added as a negative control. After washing with a pereabilization buffer, the supernatant was removed, and the suspension was again suspended in the pereabilization buffer. The cells were subjected to flow cytometry, and the content ratio of CD4-positive CD25-positive Foxp3-positive T cells was calculated for each cell population. The results are shown in Table 38.

  As shown in Table 38, the group stimulated with anti-CD3 antibody and CH-296 or H296-H296 in any culture condition compared to the group stimulated with CD3 / CD28 beads and CH-296, It was revealed that the content of CD4-positive CD25-positive Foxp3-positive T cells was low. CD4-positive CD25-positive Foxp3-positive T cells are known as regulatory T cells and are known to have a function of suppressing various immune response reactions. That is, the combination of an anti-CD3 antibody and CH-296 or H296-H296 suppresses the proliferation of CD4 positive regulatory T cells and has a low content ratio of CD4 positive regulatory T cell cells that have an adverse effect in cellular immunotherapy according to the present invention. It was revealed that a cell population suitable for the therapy can be produced.

Example 29 Expansion culture of CD4-positive T cells using CH-296-5
(1) Expansion culture of CD4-positive T cells 2% GT-T503 or 5% X-VIVO15 is added to a plate fixed in the same manner as in Example 28- (1), and anti-CD3 antibody and CH-296 are added. CD8-removed cells prepared by the same method as in Example 28- (2) were prepared on the immobilized plate, and prepared by the same method as Example 28- (2) on the plate on which only CH-296 was immobilized. CD3 / CD28 bead-binding cells are added, and the initial seeding cell number so that the final total number of cells per well is 1 × 10 ⁶ cells or 2 × 10 ⁶ cells (both are 4 mL in total volume) for each stimulation condition. Conditions were set and these plates were cultured at 37 ° C. in 5% CO ₂ (culture day 0). On the third day from the start of culture, 2 mL of the culture solution was transferred to a T25 cell culture flask on which nothing was fixed, and the culture was continued under shaking culture conditions. However, at the time of shaking culture, shaking was performed using Mild Mixer (manufactured by TAITEC). Thereafter, on the 6th and 7th days after the start of the culture, the cells were diluted with each medium so that the cell concentration became 0.5 × 10 ⁶ cells / mL, and the next culture day when the total culture volume became 10 mL. 7 ml of culture supernatant was replaced with fresh medium. The culture was continued until the 10th day after the start of the culture. In any culture day, IL-2 was added so that the 2% GT-T503 culture group had a final concentration of 200 U / mL, and the 5% X-VIVO15 culture group had a final concentration of 100 U / mL. Table 39 shows the expansion rate on the sixth day after the start of the culture.

  As shown in Table 39, in any medium condition and initial seeding cell number, the group stimulated with anti-CD3 antibody and CH-296 was expanded larger than the group stimulated with CD3 / CD28 beads and CH-296. The rate was obtained. That is, it was shown that the combination of an anti-CD3 antibody and CH-296 is preferably used during lymphocyte expansion culture.

(2) Analysis of CD45RA-positive CD62L-positive T cells The cells on day 10 of culture obtained in Example 29- (1) were analyzed for the content ratio of CD45RA-positive CD62L-positive T cells in the same manner as in Example 19. . Table 40 shows the results of measuring the content ratio of CD45RA-positive CD62L-positive T cells.

  As shown in Table 40, the group stimulated with anti-CD3 antibody and CH-296 at any initial seeding cell number was CD45RA positive compared to the group stimulated with CD3 / CD28 beads and CH-296. The CD62L positive T cell content ratio showed a high value. That is, it became clear that the combination of anti-CD3 antibody and CH-296 can proliferate naive T-like cells with high efficiency.

(3) Analysis of IL-4 negative IFN-γ positive T cells and IL-4 positive IFN-γ negative T cells Example 24- About the cells on the 10th day of culture obtained in Example 29- (1) The content ratio of IL-4 negative IFN-γ positive T cells and IL-4 positive IFN-γ negative T cells was measured by the method described in (4). The results for IL-4 negative IFN-γ positive T cells are shown in Table 41, and the results for IL-4 positive IFN-γ negative T cells are shown in Table 42. Note that analysis was performed on CD4 positive cells under all conditions.

  As shown in Table 41, Table 42, the group stimulated with anti-CD3 antibody and CH-296 compared to the group stimulated with CD3 / CD28 beads and CH-296, IL-4 negative IFN-γ. The content ratio of positive T cells was high, and the content ratio of IL-4 positive IFN-γ negative T cells was low. That is, it is clear that Th1-type CD4-positive T cells proliferate preferentially and suppress the growth of Th2-type CD4-positive cells by stimulation with a combination of anti-CD3 antibody and CH-296 during lymphocyte expansion culture. It became.

(4) Analysis of CCR4-positive T cells The cells on day 10 of culture obtained in Example 29- (1) were used in the same manner as in Example 28- (6), and the content ratio of CD4-positive CCR4-positive T cells. Was calculated. The results are shown in Table 43.

  As shown in Table 43, cells stimulated with anti-CD3 antibody and CH-296 were compared to cells stimulated with CD3 / CD28 beads and CH-296 at any medium condition and initial seeded cell number. The content ratio of CD4-positive CCR4-positive T cells was low. That is, it was revealed that the proliferation of Th2-type CD4-positive cells was suppressed by combining anti-CD3 antibody and CH-296 during lymphocyte expansion culture.

Example 30 Expansion of CD4-positive T cells using CH-296-6
(1) Expansion culture of CD4-positive T cells Expansion of CD4-positive T cells was performed in the same manner as in Example 29- (1). However, 2% GT-T503 was used as the medium, and the initial stimulation period with CH-296 was 4 days. Further, on the 6th, 7th and 9th days from the start of culture, the medium was changed and the cells were diluted (hereinafter referred to as culture method A). On the third day of culture, transfer 2 mL of the culture solution to a T25 cell culture flask on which nothing is immobilized, add each new medium to make the total culture solution volume 10 mL, and then start culture 7 days. For each eye, a group of 10 mL of a new medium was added and the total culture volume was 20 mL, and the culture was continued under stationary culture conditions (hereinafter referred to as culture method B). IL-2 was added to a final concentration of 200 U / mL on any culture day, and the culture was continued until the 10th day. Table 44 shows the expansion culture rate for the culture method A on the 6th day from the start of culture, and Table 45 shows the expansion culture rate for the culture method B on the 7th day of the culture start.

  As shown in Table 44 and Table 45, in any initial cell number condition and culture method, the group initially stimulated with anti-CD3 antibody and CH-296 is the group stimulated with CD3 / CD28 beads and CH-296. A higher expansion rate was obtained. That is, it was shown that it is preferable to stimulate with a combination of anti-CD3 antibody and CH-296 during lymphocyte expansion culture.

(2) Analysis of CD45RA-positive CCR7-positive T cells and CD45RA-positive CD62L-positive T cells The cells on day 10 of culture obtained in Example 30- (1) were treated in the same manner as in Example 19 with the same method as in Example 19. The content ratio of cells and CD45RA positive CD62L positive T cells was calculated. The results are shown in Tables 46 and 47.

  As shown in Tables 46 and 47, the groups stimulated with anti-CD3 antibody and CH-296 in any initial seeding cell number and culture method were the groups stimulated with CD3 / CD28 beads and CH-296. In comparison, the CD45RA positive CCR7 positive T cell content ratio and the CD45RA positive CD62L positive T cell content ratio showed high values. That is, it became clear that the combination of anti-CD3 antibody and CH-296 can proliferate naive T-like cells with high efficiency.

  According to the present invention, it is possible to produce a cell population in which the cell proliferation rate, the ratio of Th1-type CD4-positive T cells, Th2-type CD4-positive T cells, CD4-positive naive T-like cells, and CD4-positive regulatory T cells are arbitrarily changed. it can. The cell population obtained by the production method is extremely useful for the treatment of diseases caused by cell therapy.

SEQ ID NO: 1; Partial region of fibronectin named III-8.
SEQ ID NO: 2; Partial region of fibronectin named III-9.
SEQ ID NO: 3; Partial region of fibronectin named III-10.
SEQ ID NO: 4; Partial region of fibronectin named III-11.
SEQ ID NO: 5; Partial region of fibronectin named III-12.
SEQ ID NO: 6; Partial region of fibronectin named III-13.
SEQ ID NO: 7; Partial region of fibronectin named III-14.
SEQ ID NO: 8; Partial region of fibronectin named CS-1.
SEQ ID NO: 9; Fibronectin fragment named C-274.
SEQ ID NO: 10; Fibronectin fragment named H-271.
SEQ ID NO: 11; Fibronectin fragment named H-296.
SEQ ID NO: 12; Fibronectin fragment named CH-271.
SEQ ID NO: 13; Fibronectin fragment named CH-296.
SEQ ID NO: 14; Fibronectin fragment named C-CS1.
SEQ ID NO: 15; Fibronectin fragment named CH-296Na.
SEQ ID NO: 16; Fibronectin fragment named CHV-89.
SEQ ID NO: 17; Fibronectin fragment named CHV-90.
SEQ ID NO: 18; Fibronectin fragment named CHV-92.
SEQ ID NO: 19; Fibronectin fragment named CHV-179.
SEQ ID NO: 20; Fibronectin fragment named CHV-181.
SEQ ID NO: 21; Fibronectin fragment named H-275-Cys.
SEQ ID NO: 22; Fibronectin fragment named H296-H296.
SEQ ID NO: 23; Fibronectin fragment named H105-H105.
SEQ ID NO: 24; Polynucleotide coding Fibronectin fragment named CH-296.
SEQ ID NO: 25; Primer CH-296-NdeF.
SEQ ID NO: 26; Primer H296-HindR.
SEQ ID NO: 27; Primer NVS-F-Nco.
SEQ ID NO: 28; Primer pC-TER.
SEQ ID NO: 29; Fibronectin fragment named H204.
SEQ ID NO: 30; Polynucleotide coding Fibronectin fragment named H204.
SEQ ID NO: 31; Primer H296-NcoF.
SEQ ID NO: 32; Primer NC2-5 'UTR.
SEQ ID NO: 33; Primer CS1-no.st-CS1-R.
SEQ ID NO: 34; Primer CS1-no.st-CS1-F.
SEQ ID NO: 35; Primer FN-5630F.
SEQ ID NO: 36; Fibronectin fragment named H296-CS1.
SEQ ID NO: 37; Polynucleotide coding Fibronectin fragment named H296-CS1.
SEQ ID NO: 38; Fibronectin fragment named H296-CS2.
SEQ ID NO: 39; Polynucleotide coding Fibronectin fragment named H296-CS2.
SEQ ID NO: 40; Primer 13-Nde-F.
SEQ ID NO: 41; Primer 13-CS1-R.
SEQ ID NO: 42; Primer 13-CS1-F.
SEQ ID NO: 43; Primer CS1-R-Xba.
SEQ ID NO: 44; Fibronectin fragment named H114-HT.
SEQ ID NO: 45; Polynucleotide coding Fibronectin fragment named H114-HT.
SEQ ID NO: 46; Fibronectin fragment named H114.
SEQ ID NO: 47; Polynucleotide coding Fibronectin fragment named H114.
SEQ ID NO: 48; Primer H296-NdeF.
SEQ ID NO: 49; Primer 12-CS1-R.
SEQ ID NO: 50; Primer 12-CS1-F.
SEQ ID NO: 51; Fibronectin fragment named H117-HT.
SEQ ID NO: 52; Polynucleotide coding Fibronectin fragment named H117-HT.
SEQ ID NO: 53; Fibronectin fragment named H117.
SEQ ID NO: 54; Polynucleotide coding Fibronectin fragment named H117.
SEQ ID NO: 55; Primer 12-Nco-F2.
SEQ ID NO: 56; Primer H271-BamR.
SEQ ID NO: 57; Primer AID-F-Nco.
SEQ ID NO: 58; Fibronectin fragment named H206-HT.
SEQ ID NO: 59; Polynucleotide coding Fibronectin fragment named H206-HT.
SEQ ID NO: 60; Fibronectin fragment named H206.
SEQ ID NO: 61; Polynucleotide coding Fibronectin fragment named H206.
SEQ ID NO: 62; Primer CS1-Nde-F.
SEQ ID NO: 63; Primer CS1-12-R.
SEQ ID NO: 64; Primer CS1-12-F.
SEQ ID NO: 65; Primer H271-R-Xba.
SEQ ID NO: 66; Fibronectin fragment named H296R-HT.
SEQ ID NO: 67; Polynucleotide coding Fibronectin fragment named H296R-HT.
SEQ ID NO: 68; Fibronectin fragment named H296R.
SEQ ID NO: 69; Polynucleotide coding Fibronectin fragment named H296R.
SEQ ID NO: 70; Primer H105-NdeF.
SEQ ID NO: 71; Fibronectin fragment named H105-HT.
SEQ ID NO: 72; Polynucleotide coding Fibronectin fragment named H105-HT.
SEQ ID NO: 73; Fibronectin fragment named H105.
SEQ ID NO: 74; Polynucleotide coding Fibronectin fragment named H105.
SEQ ID NO: 75; Fibronectin fragment named CH105-HT.
SEQ ID NO: 76; Polynucleotide coding Fibronectin fragment named CH105-HT.
SEQ ID NO: 77; Primer HBD2mutF.
SEQ ID NO: 78; Primer HBD2mutR.
SEQ ID NO: 79; Fibronectin fragment namedCH-296mut2.
SEQ ID NO: 80; Polynucleotide coding Fibronectin fragment namedCH-296mut2.
SEQ ID NO: 81; Primer HBD3mutF.
SEQ ID NO: 82; Primer HBD3mutR.
SEQ ID NO: 83; Fibronectin fragment named CH-296mut3.
SEQ ID NO: 84; Polynucleotide coding Fibronectin fragment namedCH-296mut3.
SEQ ID NO: 85; Fibronectin fragment named CH-296mut4.
SEQ ID NO: 86; Polynucleotide coding Fibronectin fragment named CH-296mut4.

Claims (10)

A method for producing a cell population, comprising the following steps.
Step (1): removing a CD8-positive T cell from a cell population containing T cells, or collecting a CD4-positive T cell to prepare a cell population to be cultured, and step (2): the step (1) (B) culturing the cell population obtained in the following (A) and (B): (A) at least one selected from the group consisting of fibronectin, a fibronectin fragment and a mixture of fibronectin fragments CD3 ligand The method according to claim 1, comprising collecting or removing a cell subpopulation prior to performing step (1) or step (2). The method according to claim 1 or 2, further comprising the step of further culturing the cell population obtained in step (2) in the absence of (A) and / or (B) according to claim 1. Furthermore, the method of any one of Claims 1-3 including the process of introduce | transducing a gene into the cell population obtained at the process (1) or the process (2). The manufacturing method of any one of Claims 1-4 whose cell population obtained is at least 1 selected from the group which consists of the following.
Cell population (a): Cell population containing Th1-type CD4 positive T cells Cell population (b): Cell population containing CD4 positive CD45RA positive CCR7 positive T cells (c): CD4 positive CD45RA positive CD62L positive T cells Cell populations containing A cell population obtained by the production method according to claim 1. The cell population according to claim 6, wherein the cell population is at least one selected from the group consisting of:
Cell population (1): Cell population with high CD4 positive naive T cell content cell population (2): Cell population with high Th1 type CD4 positive T cell content cell population (3): Th2 type CD4 positive T cell content rate Low cell population Cell population (4): Cell population with low CD4 positive regulatory T cell content A medicament comprising the cell population according to claim 6 or 7. Use of a cell population according to claim 6 or 7 for the manufacture of a medicament. A method for treating or preventing a disease, comprising a step of administering an effective amount of the cell population according to claim 6 or 7 to a subject.