JP4741906B2 - Method for producing lymphocytes - Google Patents

Description

  The present invention relates to a method for obtaining lymphocytes 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 sometimes referred to as B cells) and T lymphocytes (hereinafter sometimes referred to as T cells). It recognizes specifically and acts on this to protect the living body.

T cells, CD has a (Cluster of Differentiation) 4 marker, primarily helper T cells involved in the induction of antibody production aid and various immune responses (hereinafter referred to as _{T H),} having a CD8 marker mainly cytotoxic T cells that exhibit cytotoxic activity _{[T C;} are cytotoxic T lymphocytes (cytotoxic T lymphocyte), also known as killer T cells. Hereinafter, it may be described as CTL). CTL, which plays the most important role in recognizing, destroying and removing tumor cells and virus-infected cells, does not produce antibodies that react specifically to antigens like B cells, but targets Major histocompatibility complex (MHC: sometimes called human leukocyte antigen (HLA) in humans) present on the surface of the cell membrane directly recognizes and acts on antigens (antigen peptides) derived from target cells associated with class I molecules To do. At this time, the T cell receptor on the surface of the CTL membrane (hereinafter referred to as TCR) specifically recognizes the above-mentioned antigen peptide and MHC class I molecule, and the antigen peptide is self-derived or non-self-derived To determine if it is. And the target cell judged to be non-self-derived is specifically destroyed and removed by CTL.

  In recent years, treatment methods with heavy physical burdens on patients such as drug treatment methods and radiotherapy methods have been reviewed, and interest in immunotherapy methods with light physical burdens on patients has increased. Adopted to expand lymphocytes and transfer them to patients after inducing CTLs that specifically react to target antigens from human lymphocytes ex vivo or without induction The effectiveness of immunotherapy is drawing attention. For example, it has been suggested that adoptive immunotherapy is an effective treatment for viral infections and tumors in animal models (eg, Non-Patent Documents 1 and 2). In this treatment method, it is important to maintain or increase the number of cells while maintaining or enhancing the antigen-specific injury activity of CTL.

  In adoptive immunotherapy as described above, in order to obtain a therapeutic effect, it is necessary to administer cytotoxic lymphocytes having a certain number of cells or more. That is, obtaining the number of cells ex vivo in a short time is the biggest problem.

  In order to maintain and enhance the antigen-specific injury activity of CTL, a general method is to repeat stimulation using a target antigen when inducing a response specific to the antigen for CTL. However, this method usually reduces the number of finally obtained CTLs and does not provide a sufficient number of cells.

  Next, regarding the preparation of antigen-specific CTL, using autologous CMV-infected fibroblasts and IL-2 (for example, Non-Patent Document 6), or anti-CD3 monoclonal antibody (anti-CD3 mAb) and IL-2, A method for isolating and mass-culturing CMV-specific CTL clones (for example, Non-Patent Document 7) has been reported.

Further, Patent Document 1 discloses a REM method (rapid expansion method). This REM method is a method of propagating in a short period of time the initial population of T cells comprising an antigen-specific CTL and _{T H} (Expand). That is, individual T cell clones can be proliferated to provide a large amount of T cells, and anti-CD3 antibody, IL-2, and PBMC (peripheral blood mononuclear cell, peripheral blood mononuclear cells that have been made non-proliferative by irradiation ) And Epstein-Barr virus (hereinafter abbreviated as EBV) infected cells, the number of antigen-specific CTLs is increased.

  Patent Document 2 discloses a modified REM method, which uses a non-dividing mammalian cell line that expresses a T cell stimulating component distinct from PBMC as feeder cells, and uses PBMC. This is a method of reducing the amount.

  Examples of lymphocytes effective for treatment of diseases other than CTL include, for example, lymphokine-activated cells (for example, Non-Patent Document 3), tumor-infiltrating lymphocytes induced using high concentrations of interleukin-2 (IL-2). (TIL) (for example, Non-Patent Documents 4 and 5) are known.

  Lymphokine activated cells are functional cells having cytotoxic activity obtained by adding IL-2 to peripheral blood (peripheral blood leukocytes), umbilical cord blood, tissue fluid, etc. containing lymphocytes and culturing in vitro for several days. It is a group. In the step of culturing lymphokine-activated cells, the addition of anti-CD3 antibody further accelerates the growth of lymphokine-activated cells. The lymphokine-activated cells thus obtained have a non-specific cytotoxic activity against various cancer cells and other targets.

  Fibronectin is a huge glycoprotein with a molecular weight of 250,000 present in the blood of animals, on the surface of cultured cells, and in the extracellular matrix of tissues, and is known to have a variety of functions. The domain structure is divided into seven parts (refer to FIG. 1 below), and the amino acid sequence includes three types of similar sequences. Yes. Three types of similar sequences are called type I, type II, and type III. Of these, type III is composed of 71 to 96 amino acid residues, and the concordance rate of these amino acid residues. Is 17-40%. There are 14 type III sequences in fibronectin, of which 8th, 9th and 10th (hereinafter referred to as III-8, III-9 and III-10, respectively) are cell binding domains, The 12th, 13th, and 14th (hereinafter referred to as III-12, III-13, and III-14, respectively) are contained in the heparin-binding domain. In addition, III-10 includes a VLA (very late activation antigen) -5 binding region, and the core sequence is RGDS. A region called IIICS exists on the C-terminal side of the heparin-binding domain. IIICS has a region called CS-1 that has binding activity to VLA-4 consisting of 25 amino acids (for example, Non-Patent Documents 8 to 10).

  In the production of lymphokine-activated cells and cytotoxic lymphocytes, the use of fibronectin and its fragments to improve the cell proliferation rate and maintain the cytotoxic activity has already been studied by the present inventors. (For example, Patent Documents 3, 4 and 5). However, when considering application to adoptive immunotherapy, the method described in the above literature is never satisfactory, and there is a need for an expansion culture method that does not use feeder cells from the viewpoint of further improvement of cell proliferation rate and safety. It was.

Greenberg, P.M. D. Written and published in 1992, Advances in Immunology Reuser P. Three others, Blood, 1991, Vol. 78, no. 5, P 1373 to 1380 Ridell S. A. Four others, J. Immunol. 1991, Vol. 146, no. 8, P2795- 2804 Greenberg P.M. D. Another one, J. Immunol. Methods, 1990, Vol. 128, no. 2, P189-201 Rosenberg S. A. Et al. Engl. J. et al. Med. 1987, Vol. 316, no. 15, P889-897 Rosenberg S. A. Et al. Engl. J. et al. Med. 1988, Vol. 319, no. 25, P1676 ~ 1680 Ho M.H. Nine others, Blood, 1993, Vol. 81, no. 8, P2093-2101 Deane F.D. By Momer, published in 1988, FIBRONECTIN, ACADEMIC PRESS INC. , P1-8 Kimizuka F.M. Eight others, J. Biochem. 1991, Vol. 110, no. 2, p284-291 Hanenberg H.M. 5 others, Human Gene Therapy, 1997, Vol. 8, no. 18, p2193-2206 International Publication No. 96/06929 Pamphlet WO 97/32970 pamphlet International Publication No. 03/016511 Pamphlet International Publication No. 03/080817 Pamphlet International Publication No. 2005/019450 Pamphlet

  An object of the present invention is to provide an efficient method for producing lymphocytes suitable for medical use.

The first invention of the present invention relates to a method for producing lymphocytes, comprising a culturing step in the presence of the following polypeptide (X) and / or polypeptide (Y).
Polypeptide (X): containing two or more polypeptides (m) comprising at least one amino acid sequence selected from SEQ ID NO: 1 to 3 in the sequence listing, and SEQ ID NO: 4 in the sequence listing Including one or more polypeptides (n) consisting of the amino acid sequence represented,
However, polypeptide (X) includes any amino acid sequence selected from SEQ ID NOs: 1 to 3 in the sequence listing,
Polypeptide (Y): at least one amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence of the polypeptide (X), and the substitution, deletion, insertion or addition Is a polypeptide (Y) contained in the polypeptide (m) and / or the polypeptide (n), and has a function equivalent to that of the polypeptide (X).
In the first invention of the present invention, examples of the polypeptide (m) include polypeptides comprising the amino acid sequences represented by SEQ ID NOs: 1, 2 and 3 in the sequence listing. Examples of the polypeptide (X) include those comprising two polypeptides (m). Examples of the polypeptide (X) include those comprising two polypeptides (n). Moreover, as polypeptide (X), the polypeptide which has an amino acid sequence shown to sequence number 5 of a sequence table is illustrated. In the first invention of the present invention, the produced lymphocytes are exemplified by cytotoxic T lymphocytes or lymphokine activated cells.
The present invention also provides the production method of the first invention of the present invention further comprising a step of introducing a foreign gene into lymphocytes. In the method, examples of the foreign gene include retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, lentivirus vectors, and simian virus vectors.

  The second invention of the present invention relates to lymphocytes obtained by the production method of the first invention of the present invention.

  The third invention of the present invention relates to a pharmaceutical comprising, as an active ingredient, lymphocytes obtained by the production method of the first invention of the present invention.

  The fourth invention of the present invention is the amino acid sequence (j) set forth in SEQ ID NO: 5 in the sequence listing, or an amino acid sequence in which one or more amino acids are deleted, inserted, added or substituted in the amino acid sequence (j) ( The polypeptide having k), wherein the polypeptide having the amino acid sequence (k) has a function equivalent to that of the polypeptide having the amino acid sequence (j).

The fifth invention of the present invention relates to a nucleic acid selected from the following.
A polypeptide having the amino acid sequence (j) set forth in SEQ ID NO: 5 in the sequence listing or the amino acid sequence (k) in which one or more amino acids are deleted, inserted, added or substituted in the amino acid sequence (j). A nucleic acid (r) encoding a polypeptide wherein the polypeptide having the amino acid sequence (k) has a function equivalent to that of the polypeptide having the amino acid sequence (j),
A nucleic acid (s) comprising the base sequence set forth in SEQ ID NO: 6 in the Sequence Listing,
A nucleic acid (t) that hybridizes under stringent conditions with a nucleic acid (s) comprising the base sequence set forth in SEQ ID NO: 6 in the sequence listing, wherein the polypeptide encoded by the nucleic acid (t) is A nucleic acid (t) having a function equivalent to the polypeptide having the amino acid sequence (j) described in SEQ ID NO: 5.

  The present invention provides a method for producing lymphocytes. This method has a high cell proliferation rate, and the lymphocytes obtained according to the present invention are suitably used, for example, for adoptive immunotherapy. Therefore, the method of the present invention is expected to make a great contribution to the medical field. The present invention also provides a novel modified recombinant fibronectin fragment.

  In the present invention, lymphocytes are cultured in the presence of a modified recombinant fibronectin fragment having a portion of the fibronectin heparin-binding domain overlapping to obtain lymphocytes having a high expansion rate and high cytotoxic activity. And the present invention has been completed.

  Hereinafter, the present invention will be specifically described.

(1) Polypeptide (X) or polypeptide (Y) used in the present invention
Polypeptide (X) described in the present specification includes two or more polypeptides (m), one or more polypeptides (n) as essential regions, and any one selected from SEQ ID NOs: 1 to 3 It is a polypeptide containing such an amino acid sequence redundantly.

  The polypeptide (m) is a polypeptide comprising at least one amino acid sequence selected from SEQ ID NOs: 1 to 3 that is part of the heparin-binding domain of fibronectin in the sequence. In the sequence listing, SEQ ID NOs: 1 to 3 show partial amino acid sequences of the heparin-binding domain of fibronectin and are amino acid sequences of III-12, III-13, and III-14, respectively (see FIG. 1).

  As said polypeptide (m), what has heparin binding activity can be used conveniently. The heparin binding activity can be examined by assaying the binding between the polypeptide (m) and heparin using a known method. For example, Williams D.C. A. Et al. [Williams D. et al. A. , Et al. , Nature, Vol. 352, pp. 438-441 (1991)], by using heparin instead of cells, for example, labeled heparin and the polypeptide (m). Assessment of binding to heparin can be performed.

  Particularly preferably, the polypeptide (m) includes all of the amino acid sequences represented by SEQ ID NOs: 1 to 3 in the sequence listing, that is, polypeptides including all of III-12, III-13, and III-14. Illustrated.

  The polypeptide (m) is a polypeptide comprising at least one amino acid sequence selected from SEQ ID NOs: 1 to 3 in the sequence listing in the sequence, and is desired as the polypeptide (X) of the present invention. As long as the above-described effects can be obtained, the above-described amino acid sequence may be directly linked, or another amino acid sequence may be included between two amino acid sequences. For example, a plurality of amino acids may be included as a linker. good.

  The polypeptide (m) is contained in two or more in the polypeptide (X), and each of these two or more polypeptides (m) has a different amino acid sequence as long as the above requirements are satisfied. Or may be the same. The polypeptide (m) is preferably contained in the polypeptide (X), for example, 2 to 5, preferably 2 to 4, particularly preferably 2 to 3.

  Polypeptide (n) is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing, which is the CS-1 domain of fibronectin. The polypeptide (n) is preferably contained in the polypeptide (X), for example, 1 to 5, preferably 1 to 4, particularly preferably 1 to 3.

  As described above, the polypeptide (X) contains two or more polypeptides (m), one or more polypeptides (n), and the polypeptide (X) contains SEQ ID NOs: 1 to 3 in the Sequence Listing. Is a polypeptide that is required to include any one of the amino acid sequences selected from the above, and includes other amino acid residues as long as the desired effect of the present invention can be obtained. Also good. For example, an amino acid residue derived from a linker or the like may be contained.

  The positional relationship between two or more polypeptides (m) and one or more polypeptides (n) in the polypeptide (X) is not particularly limited as long as the desired effect of the present invention can be obtained. However, for example, those in which the polypeptide (n) is linked to the C-terminal side with respect to at least one polypeptide (m) are preferred. In addition, it is preferable that the polypeptide (m) is located on the N-terminal side in the polypeptide (X) and the polypeptide (n) is located on the C-terminal side. Particularly preferably, it is preferable that the polypeptide (m) and the polypeptide (n) are alternately located in the polypeptide (X).

In the present invention, as the polypeptide (X), for example, a polypeptide (H296-H296) having an amino acid sequence represented by SEQ ID NO: 5 in the sequence listing is preferably used. For H296-H296, use the plasmid deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, Aichi 1-chome, 1-chome, Tsukuba-shi, Ibaraki, 305-8566, Japan. Can be manufactured;
FERM P-20602 (plasmid containing nucleic acid encoding H296-H296; date of deposit July 22, 2005).
H296-H296 is composed of methionine, III-12, III-13, III-14, CS-1, alanine, methionine, III-12, III-13, III-14, CS-1 in this order from the N-terminal side. Polypeptide. H296-H296 is a novel polypeptide produced for the first time in the present invention. H296-H296 will be described in detail in “(4) H296-H296” described later.

  Polypeptides (X) and (Y) used in the present invention are CH-, which is a known fibronectin fragment in the lymphocyte production method of the present invention described later, as described in Examples 9 to 14 described later. Even when compared with H.296 (polypeptide consisting of fibronectin cell-binding domain, heparin-binding domain, CS-1 domain) and H-296 (polypeptide consisting of fibronectin heparin-binding domain and CS-1 domain) It is a very useful polypeptide because it has a high proliferation rate and can maintain a high cytotoxic activity when used in the production of the CTLs of the present invention. Furthermore, by using polypeptide (X) or (Y) for the expansion culture of CTL as shown also in (2) -1-3 mentioned later, high cell proliferation rate and cytotoxic activity are obtained without using feeder cells. There is a very useful advantage that maintenance can be realized. The number of amino acids constituting these polypeptides (X) or (Y) is not particularly limited, but is preferably 100 to 3000 amino acids, more preferably 150 to 2800 amino acids, and even more preferably 200 to 2600 amino acids. It is.

  As useful information regarding the preparation of polypeptide (X), information regarding fibronectin can be found in Kimika F. [Kimiduka 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. In addition, the nucleic acid sequence encoding fibronectin or the amino acid sequence of fibronectin is described in Genbank Accession No. NM_002026 and NP_002017.

  The polypeptide (Y) described in the present specification includes at least one amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in the amino acid sequence of the aforementioned polypeptide (X), The substitution, deletion, insertion or addition is a polypeptide (Y) contained in the polypeptide (m) and / or the polypeptide (n), and has a function equivalent to that of the polypeptide (X) It is a polypeptide. As the substitution, deletion, insertion or addition, for example, one in which at least one of substitution, deletion, insertion or addition of 1 to 20 amino acids has occurred, more preferably substitution of 1 to 5 amino acids , Deletions, insertions or additions are exemplified.

  The amino acid substitution and the like are preferably such that the physicochemical properties of the polypeptide (X) can be changed within a range in which the original function of the polypeptide (X) can be maintained. For example, amino acid substitution and the like are preferably conservative within a range that does not substantially change the properties (eg, hydrophobicity, hydrophilicity, charge, pK, etc.) of the original polypeptide (X). 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, amino acid deletion, addition, and insertion are properties of amino acids around the target site in the polypeptide (X) having properties similar to those around the target site Deletion, addition, and insertion within a range that does not substantially change is preferable.

  Amino acid substitution or the like may be naturally occurring due to species differences or individual differences, or may be artificially induced. Artificial induction may be performed by a known method, and is not particularly limited. For example, by a known method, one or more bases are substituted, deleted, or deleted in the nucleic acid encoding the polypeptide (X) described above. A predetermined nucleic acid added or inserted can be produced, and a polypeptide (Y) containing an amino acid sequence having a substitution in the amino acid sequence of the polypeptide (X) can be produced.

  In addition, in the present specification, “having an equivalent function” means that when a lymphocyte described below is produced using a polypeptide (X) as a comparative control, an equivalent cell proliferation rate is obtained, Or it means that equivalent cytotoxic activity is maintained. That is, the function can be appropriately confirmed by carrying out in accordance with the method for producing lymphocytes described later, particularly the method described in Examples 2 to 21 described later. Moreover, as polypeptide (Y), what has heparin binding activity is suitable. The heparin binding activity can be evaluated according to the activity measuring method. The polypeptide (X) and the polypeptide (Y) can be used alone or in a mixture of a plurality of types for use in the lymphocyte production method described below.

(2) Method for Producing Lymphocytes Hereinafter, the method for producing lymphocytes of the present invention will be specifically described. The method of the present invention includes a culture step in the presence of the aforementioned polypeptide (X) and / or polypeptide (Y) (hereinafter sometimes referred to as the culture step of the present invention). This is a method for producing lymphocytes. In the present specification, polypeptide (X) and / or polypeptide (Y) may be referred to as an active ingredient of the present invention.

  The method for producing lymphocytes of the present invention is carried out by carrying out the culturing process of the present invention during the entire period of culture in lymphocyte production or during an arbitrary partial period. That is, any lymphocyte production process that includes the culture process is included in the present invention.

  The lymphocytes obtained by the production method of the present invention are not particularly limited. For example, cytotoxic T lymphocytes (CTL), lymphokine activated cells, tumor infiltrating lymphocytes (TIL), NK cells, naive cells Memory cells, cell populations containing at least one of these cells, and the like. Of these, the present invention is particularly suitable for the production of CTL and lymphokine activated cells. These production methods will be specifically described in (2) -1 and (2) -2 described later. In this specification, the lymphokine-activated cell is a functional cell population obtained by adding IL-2 to peripheral blood (peripheral blood leukocytes), umbilical cord blood, tissue fluid or the like containing lymphocytes and culturing for several days. Indicates. Such a cell population may be generally referred to as lymphokine-activated killer cells (LAK cells), but the cell population includes cells that are not cytotoxic (for example, naive cells). Therefore, in the present specification, the cell population is referred to as lymphokine activated cells.

  In addition, when evaluating the cytotoxic activity of lymphocytes obtained by the production method of the present invention, it can be evaluated by a known method, for example, cytotoxicity of cytotoxic lymphocytes to target cells labeled with a radioactive substance, a fluorescent substance, etc. The activity can be evaluated by measuring radioactivity and fluorescence intensity derived from target cells destroyed by cytotoxic lymphocytes. It can also be detected by measuring the amount of cytokines such as GM-CSF and IFN-γ that are specifically released from cytotoxic lymphocytes and target cells. In addition, it can be directly confirmed by using an antigen peptide-MHC complex labeled with a fluorescent dye or the like. In this case, for example, cytotoxic lymphocytes are contacted with a first fluorescent marker coupled with a cytotoxic lymphocyte-specific antibody and then contacted with an antigen peptide-MHC complex coupled with a second fluorescent marker. The cytotoxic activity of cytotoxic lymphocytes can be assessed by analyzing the presence of double-labeled cells with a flow cytometer

  The culture process of the present invention is intended to induce CTLs from cells that can become CTLs, maintain CTLs, or expand CTLs and other lymphocytes as described above. In the method for producing lymphocytes of the present invention, lymphocytes useful for adoptive immunotherapy and the like are produced by culturing lymphocytes by appropriately adjusting the type of cells to be subjected to the method, culture conditions and the like. be able to. In this specification, lymphocyte means a cell group containing lymphocytes, and CTL means a cell group containing CTL. In addition, although the cell used for culture | cultivation can be suitably set according to the kind of lymphocyte to manufacture, the said cell can use either what was extract | collected from the biological body as it is, or cryopreserved.

The cell culture equipment used in the culture process 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, for example, can be used CO ₂ gas-permeable cell culture bag. Moreover, when manufacturing a large amount of lymphocytes industrially, a large culture tank can be used. The culture can be carried out in either an open system or a closed system. Preferably, the culture is preferably carried out in a closed system from the viewpoint of the safety of the obtained lymphocytes.

  Ingredients used in the medium including the active ingredient of the present invention (for example, anti-CD3 antibody, anti-CD28 antibody, interleukin-2 and the like described later) are dissolved in the medium and coexist, and an appropriate solid phase, for example Cell culture equipment such as petri dishes, flasks, bags, etc. (including both open and closed systems), or cell culture carriers such as beads, membranes, glass slides, etc. . The material of the solid phase is not particularly limited as long as it can be used for cell culture. For example, when the component is immobilized on the device, the device is prepared so that when the medium is placed in the device, the ratio is the same as the desired concentration when the component is dissolved in the medium. It is preferable to fix a certain amount of each component with respect to the amount of medium to be added, but the amount of the component to be fixed is not particularly limited as long as a desired effect is obtained. The carrier is used by immersing it in a culture solution in a cell culture equipment during cell culture. When immobilizing the component on the carrier, when the carrier is placed in the medium, the amount of medium to be placed in the equipment so that the ratio is the same as the desired concentration when the component is dissolved in the medium. It is preferable to fix a certain amount of each component to the above, but the amount of the component to be immobilized is not particularly limited as long as a desired effect is obtained.

The culture conditions may be those known in the art for lymphocytes, and the conditions used for normal cell culture can be applied. For example, it is possible to 37 ° C., and cultured under conditions, such as 5% CO _2. In addition, a fresh medium can be added to the cell culture medium for dilution at an appropriate time interval, the medium can be replaced with a fresh one, or the cell culture equipment can be replaced. The culture medium used and other components used at the same time can be appropriately set depending on the type of lymphocyte to be produced, as will be described later.

  For example, immobilization of polypeptide (X) and / or polypeptide (Y) is similar to immobilization of fibronectin fragments described in WO 97/18318 and WO 00/09168. It can be implemented by a method. If the above-mentioned various components and the active ingredient of the present invention are immobilized on a solid phase, the lymphocytes can be obtained only by separating the lymphocytes from the solid phase after obtaining the lymphocytes by the method of the present invention. And the active ingredient of the present invention can be easily separated, and mixing of the active ingredient into the lymphocytes can be prevented.

Furthermore, it comprises acidic polysaccharides, acidic oligosaccharides, acidic monosaccharides and salts thereof, which are effective for inducing cytotoxic T cells having antigen-specific cytotoxic activity, as described in WO 02/14481. You may culture | cultivate using the compound selected from a group, and the substance selected from the following (A)-(D) of international publication 03/016511 pamphlet with the said component.
(A) a substance having binding activity to CD44 (B) a substance capable of controlling a signal emitted by binding of CD44 ligand to CD44 (C) a substance capable of inhibiting the binding of a growth factor to a growth factor receptor (D) Substances that can control signals emitted by growth factors binding to growth factor receptors

Examples of the substance having CD44 binding activity include CD44 ligand and / or anti-CD44 antibody. Examples of the substance capable of controlling the signal emitted by the binding of CD44 ligand to CD44 include various phosphorylase and dephosphorase inhibitors or activators. Substances that can inhibit the binding of growth factors to growth factor receptors include, for example, growth factor binding activity that inhibits growth factors from binding to growth factor receptors by forming complexes with growth factors. Examples thereof include a substance or a substance that has a binding activity to a growth factor receptor and inhibits the growth factor from binding to the growth factor receptor. Furthermore, examples of the substance capable of controlling a signal emitted when a growth factor binds to a growth factor receptor include various phosphorylase and dephosphorase inhibitors or activators. The concentration of these components in the medium is not particularly limited as long as a desired effect is obtained. Further, these components may be dissolved in a medium and coexist, or may be used by being immobilized on an appropriate solid phase as described above.
In addition, said various substances can be used individually or in mixture of 2 or more types.

  In the present invention, the presence of the component means that the active component is present in a state where it can exert its function when lymphocytes are cultured, and the presence state is not particularly limited. For example, when the active ingredient is dissolved in the medium to be used, the content of the active ingredient of the present invention in the culture medium is not particularly limited as long as the desired effect is obtained. It is 0001-10000 microgram / mL, More preferably, it is 0.001-10000 microgram / mL, More preferably, it is 0.005-5000 microgram / mL, Especially preferably, it is 0.01-1000 microgram / mL.

  As a medium used in the lymphocyte culture process of the present invention, a known medium used for cell culture may be used, and various cytokines and the like may be added according to the type of lymphocyte to be produced as described later. It ’s fine.

  In the lymphocyte culture process of the present invention, serum or plasma can be added to the medium. The amount added to these media is not particularly limited, but it is 0 to 20% by volume, particularly when using autologous serum or plasma, it is 0 to 5% by volume in consideration of the burden on the patient. preferable. The origin of serum or plasma may be either self (meaning that the origin is the same as the lymphocyte to be cultured) or non-self (meaning that the origin is different from the lymphocyte to be cultured), Preferably, an autologous material can be used from the viewpoint of safety.

For example, in the method of the present invention, when lymphocyte expansion culture is performed, the number of cells at the start of the culture used in the present invention is not particularly limited, but for example, 1 cell / mL to 1 × 10 ⁸ cells / mL. And preferably 1 cell / mL to 5 × 10 ⁷ cells / mL, more preferably 1 cell / mL to 2 × 10 ⁷ cells / mL.

(2) -1 Method for Producing CTL Hereinafter, an example of producing CTL by the production method of the present invention (hereinafter sometimes referred to as the method for producing CTL of the present invention) will be described in detail.

  When CTL is produced by the production method of the present invention, culture for inducing cells having the potential to become CTL into CTL (the method for inducing CTL of the present invention), culture for maintaining CTL (of the present invention) The culturing step of the present invention can be carried out in CTL maintenance method) or culture for expanding CTL (CTL expansion method of the present invention).

(2) -1-1 Method for Inducing CTL of the Present Invention The lymphocyte culture process of the present invention for the induction of CTL will be described. Induction of CTL is performed by culturing cells capable of becoming CTL together with appropriate antigen-presenting cells in order to confer CTL with the ability to recognize a desired antigen.

  Examples of cells having the ability to become CTL include peripheral blood mononuclear cells (PBMC), naive cells, memory cells, umbilical cord blood mononuclear cells, hematopoietic stem cells, or lymphokine-activated cells. Any of these cells collected from a living body, or those obtained through in vitro culture can be used as they are or after cryopreservation. The antigen-presenting cell is not particularly limited as long as it has the ability to present an antigen to be recognized to T cells. For example, monocyte, B cell, T cell, macrophage, dendritic cell, fibroblast, etc. The desired antigen can be presented, and if necessary, the proliferation can be deactivated by X-ray irradiation or the like and used in the present invention. In addition, stimulation with antigen-presenting cells can be performed in several steps during the CTL culture period.

In the induction of CTL, the medium may preferably contain appropriate proteins, cytokines and other components in addition to the active ingredient of the present invention. Preferably, a medium containing interleukin-2 (hereinafter sometimes referred to as IL-2) is used in the present invention. The concentration of IL-2 in the medium is not particularly limited. For example, it is preferably 0.01 to 1 × 10 ⁵ U / mL, more preferably 0.1 to 1 × 10 ⁴ U / mL. is there.

In addition, general conditions for induction of CTL may be in accordance with known conditions (Bednarek MA et al., J. Immunol., Vol. 147, No. 12, P4047-4053, 1991). The culture conditions are not particularly limited, and the conditions used for normal cell culture can be used. For example, the culture can be performed under conditions of 37 ° C., 5% CO _{2 and the} like. This culture is usually carried out for about 2 to 15 days. Further, a step of diluting the cell culture solution at an appropriate time interval, a step of replacing the medium, or a step of replacing the cell culture equipment may be performed.

  Thus, by carrying out the culture process of the present invention to induce CTL, the cell growth rate after CTL induction is high, and furthermore, when the induced CTL is subjected to expansion culture, a very high cell growth rate is realized. can do. Furthermore, the CTL induced in this way has a feature that even if it is maintained or expanded for a long period of time, it maintains a high cytotoxic activity as observed immediately after induction. This maintenance culture and expansion culture can be preferably carried out by the CTL maintenance culture method and expansion culture method described later, but other known methods, that is, the aforementioned polypeptide (X) and / or polypeptide ( Even if CTL maintenance culture or expansion culture is carried out in the absence of Y), the obtained cells maintain high cytotoxic activity as described above, and a high cell proliferation rate is observed. These effects are significantly higher than those obtained by inducing CTL in the presence of H-296 (see Examples 9 to 14 described later), which is a known fragment of fibronectin, for example. . Furthermore, when expansion culture is performed using CTL obtained by the CTL induction method of the present invention, a high cell proliferation rate can be realized without using feeder cells. In addition, stable CTLs can be maintained, for example, by cloning induced CTLs.

(2) -1-2 Method for Maintaining CTLs of the Present Invention The method for maintaining CTLs of the present invention is a method for maintaining CTLs while maintaining their cytotoxic activity. This method is characterized in that the above-described culture step of the present invention is carried out during the maintenance culture of CTL, whereby the cytotoxic activity of CTL can be maintained.

  The CTL to which the method can be applied is not particularly limited, and is obtained by a CTL obtained by a known method, a CTL obtained by the CTL induction method of the present invention described above, or a CTL expansion culture method described later. Used to maintain CTL. Here, CTL means a cell population containing CTL.

  The medium to be used is not particularly limited, and a known medium can be used, and it contains appropriate proteins, cytokines (particularly IL-2), and other components as in the above-described induction of CTL. Also good.

The general conditions for maintenance culture of CTL may be according to known methods (for example, Carter J. et al., Immunology, Vol. 57, No. 1, P123-129, 1996). The culture conditions are not particularly limited, and the conditions used for normal cell culture can be used. For example, the culture can be performed under conditions of 37 ° C., 5% CO _{2 and the} like. There is no particular limitation on the number of culture days. Further, a step of diluting the cell culture solution at an appropriate time interval, a step of replacing the medium, or a step of replacing the cell culture equipment may be performed.

(2) -1-3 CTL expansion culture method of the present invention The CTL expansion culture method of the present invention is a method of rapidly expanding the number of cells while maintaining the cytotoxic activity of CTL. The method is characterized in that the above-described culture step of the present invention is carried out during CTL expansion culture.

  The CTL to which the method can be applied is not particularly limited. For example, CTL obtained by a known method, CTL obtained by the CTL induction method of the present invention described above, or maintenance culture by the CTL maintenance method described above. CTL is used.

  The medium to be used is not particularly limited, and a known medium can be used, and it may contain appropriate proteins, cytokines, and other components as in the above-described induction of CTL.

  The general conditions for CTL expansion culture may be according to known methods (for example, Carter J. et al., Immunology, Vol. 57, No. 1, P123-129, 1996).

  In the CTL expansion culture method of the present invention, it is preferable to co-culture with an anti-CD3 antibody, particularly preferably an anti-human CD3 monoclonal antibody, in addition to the active ingredient, from the viewpoint of realizing a high expansion culture rate. The concentration of the anti-CD3 antibody in the medium is not particularly limited. For example, 0.001 to 100 μg / mL, particularly 0.01 to 100 μg / mL is preferable. Further, it can be co-cultured with an anti-CD28 antibody, particularly preferably an anti-human CD28 monoclonal antibody, as a costimulation. It is also possible to co-culture lymphocyte stimulating factors such as lectins.

Moreover, in the expansion culture method of CTL of this invention, it can also co-culture with a suitable feeder cell as needed. In the CTL expansion culture method, the feeder cells are not particularly limited as long as the feeder cells stimulate CTL in cooperation with the anti-CD3 antibody and activate the T cell receptor or each stimulating receptor. In the present invention, the feeder cells are not particularly limited. For example, autologous or non-autologous PBMC and EBV-B cells are used. From the viewpoint of safety, cells other than EBV-B may be used. preferable. Usually, feeder cells are used after depriving of proliferation ability by irradiation with X-rays or the like or treatment with a drug such as mitomycin. In addition, what is necessary is just to determine the content in the culture medium of a feeder cell according to a well-known method, and there is no limitation in particular, For example, 1-1 * 10 < ⁷ > cells / ml is suitable.

  However, it is preferable not to use feeder cells in adoptive immunotherapy from the viewpoint of patient burden and safety. One feature of the CTL expansion culture method of the present invention is that a high expansion culture rate is achieved even when feeder cells are not used. In other words, the present invention provides a method for expanding CTL without using feeder cells. When CTL is produced without using feeder cells, the CTL used in the expansion culture method of the present invention is obtained by the above-described CTL induction method of the present invention from the viewpoint of realizing a high expansion culture rate. It is desirable to use CTL.

(2) -2 Method for Producing Lymphokine Activated Cells Hereinafter, an example of producing lymphokine activated cells by the production method of the present invention (hereinafter sometimes referred to as the method for producing lymphokine activated cells of the present invention) will be described in detail. Describe.

  The method for producing lymphokine-activated cells of the present invention is carried out by subjecting cells having the ability to become lymphokine-activated cells in the presence of IL-2 to the culture of the present invention.

  The cells having the ability to become lymphokine-activated cells are not particularly limited, and include, for example, peripheral blood mononuclear cells (PBMC), NK cells, cord blood mononuclear cells, hematopoietic stem cells, and these cells. Examples include blood components, and any blood cell can be used. In addition, materials containing the cells, for example, blood such as peripheral blood and umbilical cord blood, blood from which components such as red blood cells and plasma are removed, bone marrow fluid, and the like can be used.

Moreover, the general conditions for culturing lymphokine activated cells are known conditions [for example, cell engineering, Vol. 14, no. 2, p223-227, (1995); cell culture, 17, (6), p192-195, (1991); THE LANCET, Vol. 356, p802-807, (2000); see Current Protocols in Immunology, supplement 17, UNIT 7.7]. The culture conditions are not particularly limited, and the conditions used for normal cell culture can be applied. For example, the culture can be performed under conditions of 37 ° C., 5% CO _{2 and the} like. This culture is usually carried out for about 2 to 15 days. Further, a step of diluting the cell culture solution at an appropriate time interval, a step of replacing the medium, or a step of replacing the cell culture equipment may be performed.

  The medium to be used is not particularly limited, and a known medium can be used, and it may contain appropriate proteins, cytokines, and other components as in the above-described induction of CTL.

  In the method for producing lymphokine-activated cells of the present invention, it is preferable to co-culture with an anti-CD3 antibody, particularly preferably an anti-human CD3 monoclonal antibody, in addition to the active ingredient, from the viewpoint of realizing a high expansion culture rate. The concentration of the anti-CD3 antibody in the medium is not particularly limited. For example, 0.001 to 100 μg / mL, particularly 0.01 to 100 μg / mL is preferable. Further, it can be co-cultured with an anti-CD28 antibody, particularly preferably an anti-human CD28 monoclonal antibody, as a costimulation. It can also be co-cultured with lymphocyte stimulating factors such as lectins.

(2) -3 Method for Producing Lymphocytes of the Present Invention Comprising a Foreign Gene Introducing Step The present invention also provides a method for producing lymphocytes further comprising the step of introducing a foreign gene in the above-described method for producing lymphocytes. provide. That is, this invention provides the manufacturing method of the lymphocyte which further includes the process of introduce | transducing a foreign gene into a lymphocyte as the one aspect | mode. The term “foreign” refers to being out of the lymphocytes targeted for gene transfer.

  By performing the method for producing lymphocytes of the present invention, particularly the method for expanding lymphocytes, the proliferation ability of the cultured lymphocytes is enhanced. Therefore, an increase in gene introduction efficiency is expected by combining the method for producing lymphocytes of the present invention with a gene introduction step.

  The means for introducing the foreign gene is not particularly limited, and an appropriate one can be selected and used by a known gene introduction method. The gene transfer step can be performed at any time during the production of lymphocytes. For example, it is preferable to carry out at the time of cell proliferation in the method for expanding CTL of the present invention or the method for producing lymphokine-activated cells.

  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.

  The viral vector is not particularly limited, and is usually a known viral vector used in gene transfer methods, for example, a retrovirus vector, a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, a simian virus vector, a vaccinia virus vector. Alternatively, a Sendai virus vector or the like is used. Particularly preferably, as the virus vector, a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, a lentivirus vector or a simian virus vector is used. As the above-mentioned viral vector, those lacking replication ability are preferable so that they cannot self-replicate in infected cells.

  Retroviral vectors and lentiviral vectors can stably incorporate foreign genes inserted into the chromosomal DNA of cells into which the vectors are introduced, and are used for gene therapy and other purposes. Since the vector has high infection efficiency with respect to dividing and proliferating cells, it is suitable for gene transfer in the lymphocyte production process, for example, the expansion culture process in the present invention.

  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 or linear DNA is introduced.

  In the present invention, the foreign gene introduced into the lymphocyte is not particularly limited, and any gene desired to be introduced into the cell can be selected. Examples of such a gene include those encoding an antisense nucleic acid, siRNA (small interfering RNA), and a ribozyme in addition to those encoding proteins (eg, enzymes, cytokines, receptors, etc.). Moreover, you may introduce | transduce simultaneously the suitable marker gene which enables selection of the cell by which gene introduction was carried out.

  The foreign gene can be used by inserting it into a vector, a plasmid or the like so that it can be expressed under the control of an appropriate promoter, for example. 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 the chromosome of a lymphocyte to be introduced by homologous recombination, for example, bases having homology to the base sequences on both sides of the desired target insertion site of the gene in the chromosome A foreign gene may be placed between flanking sequences consisting of sequences. 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 depending on the purpose.

  According to the method of the present invention, for example, a gene encoding an enzyme related to resistance to a drug used for treatment of a patient such as cancer can be introduced into lymphocytes to impart drug resistance to the lymphocytes. . By using such lymphocytes, it is possible to combine adoptive immunotherapy and drug therapy, and thus it is possible to obtain a higher therapeutic effect. An example of the drug resistance gene is a multidrug resistance gene.

  On the other hand, conversely to the above-described embodiment, a gene that imparts sensitivity to a specific drug can be introduced into lymphocytes to impart sensitivity to the drug. In such a case, it becomes possible to remove lymphocytes after transplantation into a living body by administration of the drug. An example of a gene that confers sensitivity to a drug is a thymidine kinase gene.

(3) Lymphocytes obtained by the production method of the present invention, a drug containing the lymphocytes, a lymphocyte culture medium containing the active ingredient of the present invention. The present invention is obtained by the production method of the present invention described above. Provide lymphocytes. The present invention also provides a medicament (therapeutic agent) containing the lymphocyte as an active ingredient. In particular, the therapeutic agent containing the lymphocyte is suitable for use in adoptive immunotherapy. In adoptive immunotherapy, lymphocytes suitable for treating a patient are administered to the patient, for example, by intravenous administration. The therapeutic agent is very useful for use in the aforementioned diseases and donor lymphocyte infusion. The therapeutic agent is in accordance with a known method in the pharmaceutical field, for example, using the lymphocyte prepared by the method of the present invention as an active ingredient, for example, a known organic or inorganic carrier suitable for parenteral administration, an excipient, It can be prepared by mixing with a stabilizer or the like. The content of the lymphocytes of the present invention in the therapeutic agent, the dose of the therapeutic agent, and various conditions relating to the therapeutic agent can be determined as appropriate according to known adoptive immunotherapy.

  The disease to which lymphocytes produced by the method of the present invention are administered is not particularly limited, and examples thereof include infectious diseases caused by cancer, malignant tumors, hepatitis, viruses such as influenza, bacteria, and fungi. Illustrated. In addition, when a foreign gene is further introduced as described above, an effect is expected for various gene diseases. The lymphocytes produced by the method of the present invention can also be used for bone marrow transplantation and donor lymphocyte infusion for the purpose of preventing infection after irradiation.

  As another aspect of the present invention, a medium containing the active ingredient of the present invention is provided. The medium further comprises other arbitrary components, for example, medium components used for known cell culture, proteins, cytokines (preferably IL-2), and other desired components. The content of the active ingredient of the present invention in the medium is not particularly limited as long as the desired effect of the present invention is obtained. For example, the active ingredient in the medium used in the method of the present invention, etc. According to the content of, it can be appropriately determined if desired. As one aspect of the culture medium of the present invention, a medium containing a cell culture carrier on which the active ingredient of the present invention is immobilized, and a cell culture device on which the active ingredient of the present invention is immobilized are provided. A medium is included.

(4) H296-H296
In the present invention, the amino acid sequence (j) (H296-H296) described in SEQ ID NO: 5 in the Sequence Listing, or an amino acid sequence in which one or more amino acids are deleted, inserted, added or substituted in the amino acid sequence (j) A polypeptide having (k), wherein the polypeptide having amino acid sequence (k) has a function equivalent to that of the polypeptide having amino acid sequence (j), and encodes the same Nucleic acid (r) is also provided. Examples of the nucleic acid include a nucleic acid (s) composed of the base sequence described in SEQ ID NO: 6 in the Sequence Listing (nucleic acid encoding H296-H296), and a nucleic acid (s) composed of the base sequence described in SEQ ID NO: 6 in the Sequence Listing. A nucleic acid (t) that hybridizes under stringent conditions, wherein the polypeptide encoded by the nucleic acid (t) is equivalent to the polypeptide having the amino acid sequence (j) described in SEQ ID NO: 5 of the Sequence Listing A nucleic acid (t) having a function is exemplified. In the present specification, the novel polypeptide may be referred to as the polypeptide of the present invention, and the nucleic acid encoding it may be referred to as the nucleic acid of the present invention.

  Hereinafter, the polypeptide of the present invention, the nucleic acid of the present invention, and the method for producing the polypeptide will be described. As long as the polypeptide of the present invention has a desired function in the production of lymphocytes as described above, a sequence in which one or more substitutions, deletions, insertions or additions in the amino acid sequence have occurred. Are also included in the polypeptide of the present invention. As the polypeptide of the present invention other than H296-H296, any one or more of substitution, deletion, insertion or addition of 1 to 20 amino acids preferably occurs in the amino acid sequence shown in SEQ ID NO: 5 of the Sequence Listing. More preferably 1 to 10 amino acid substitutions, deletions, insertions or additions, more preferably 1 to 5 amino acid substitutions, deletions, insertions or additions. Examples in which any one or more of the additions are generated are exemplified. 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. In detail, the method for producing the polypeptide can be carried out in the same manner as described in the above-mentioned “(1) Polypeptide (X) or polypeptide (Y) used in the present invention”.

  The nucleic acid represented by SEQ ID NO: 6 in the sequence listing encoding the polypeptide of the present invention encodes a gene encoding natural fibronectin or a recombinant fibronectin fragment such as CH-296 and H-296 described above. Using a gene, a nucleic acid encoding each domain constituting the polypeptide of the present invention can be obtained, and these can be obtained by connecting them by a known method. For example, as described in Example 1 described later, a nucleic acid encoding a heparin-binding domain and a CS-1 domain can be acquired from a nucleic acid encoding CH-296, and these can be acquired by overlapping them together. .

  In addition, the nucleic acid of the present invention includes those in which one or more substitutions, deletions, insertions or additions of one or more of the nucleotide sequences of the nucleic acid represented by SEQ ID NO: 6 in the Sequence Listing have occurred. . For example, one in which at least one of substitution, deletion, insertion or addition of 1 to 60 bases has occurred from the base sequence described in SEQ ID NO: 6 in the sequence listing, more preferably substitution or deletion of 1 to 30 bases Examples include one in which any one or more of insertion or addition has occurred, and more preferably one in which any one or more of substitution, deletion, insertion or addition of 1 to 15 bases has occurred. 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.

Furthermore, it hybridizes with a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 6 under stringent conditions and encodes 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. Such nucleic acids are also included in the nucleic acids of the present invention. The above “stringent conditions” are not particularly limited, and the temperature and salt concentration at the time of hybridization, preferably further at the time of washing, are selected according to the DNA to be hybridized to the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 6. Although be set by appropriately determining, 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 Conditions described in documents such as Harbor Laboratory 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 cleaning is performed under conditions of 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 (a lower percentage of formamide results in reduced stringency), salt concentration, or temperature. For example, lower stringency conditions are: 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.

  On the other hand, a polypeptide having the amino acid sequence represented by SEQ ID NO: 5 in the sequence listing can be obtained by genetic engineering using the nucleic acid thus obtained. 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. As described above, the plasmid containing the nucleic acid encoding H296-H296 has been deposited as FERM P-20602.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further more concretely, this invention is not limited to these description at all.

Preparation Example 1 Preparation of H-296 H-296 (polypeptide consisting of fibronectin heparin-binding domain and CS-1 domain) used Escherichia coli HB101 / pHD102 (FERM BP-7420), which was used in US Pat. No. 5,198. , 423.

Preparation Example 2 Preparation of CH-296 CH-296 (polypeptide consisting of fibronectin cell-binding domain, heparin-binding domain and CS-1 domain) was Escherichia coli HB101 / pCH102 (FERM BP-2800), which was used as a US patent. It was prepared based on the description in No. 5,198,423.

Example 1 Production of H296-H296 Among the operations described herein, basic operations such as plasmid preparation and restriction enzyme digestion were published in 2001 by Cold Spring Harbor Laboratory, T.W. Edited by T. Maniatis et al., Molecular Cloning: A Laboratory Manual Third Edition (Molecular Cloning: A Laboratory Manual 3rd ed.).

(1) Construction of expression vector (i) Construction of H-296 expression vector A poly consisting of amino acids 279 to 575 (base numbers 835 to 1725) from the N-terminal side of the amino acid sequence of CH-296 described in SEQ ID NO: 7 in the sequence listing In order to express a mutant protein (H296-H296) in which the peptide is H-296 and two H-296s are linked, an expression vector was constructed as follows. See FIG.

  First, synthetic primers H296-NcoF and H296 having the base sequences described in SEQ ID NOs: 9 and 10 from the base sequence of CH-296 described in SEQ ID NO: 8 of the Sequence Listing (see International Publication No. 03/080817 pamphlet). -HindR was synthesized with 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 279 to 284 of the amino acid sequence of CH-296 (SEQ ID NO: 7) is represented by nucleotide numbers 13 to 30 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 571 to 575 of the amino acid sequence of CH-296 (SEQ ID NO: 7) 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 of synthetic primer H296-NcoF, 10 pmol of 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 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 is suspended in 10 μl of sterilized water, double digested with restriction enzyme NcoI (Takara Bio) and restriction enzyme HindIII (Takara Bio), and the NcoI is subjected to 1.0% agarose electrophoresis. -The HindIII digest was extracted and purified to obtain an NcoI-HindIII digested DNA fragment.

  Next, a pCold04NC2 vector was prepared according to the method described in Examples 1 to 6 of International Publication No. 99/27117 pamphlet (hereinafter, this pCold04NC2 vector will be referred to as a pCold14 vector).

  Next, the pCold14 vector is cleaved with the same restriction enzyme used when preparing the NcoI-HindIII digested DNA fragment, and the end is dephosphorylated, and mixed with the NcoI-HindIII digested DNA fragment. The DNA ligation kit (manufactured by Takara Bio Inc.) was used for ligation. Thereafter, E. coli JM109 was transformed with 20 μl of the ligation 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 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 279 to 575 of CH-296.

(Ii) Construction of H296-H296 expression vector Next, a synthetic primer H296-NcoR having a base sequence described in SEQ ID NO: 11 in the sequence listing is used as a DNA synthesizer from the base sequence disclosed in International Publication No. 03/080817 pamphlet. And purified by a conventional method. In the synthetic primer H296-NcoR, the recognition sequence of the restriction enzyme NcoI is represented by nucleotide numbers 10 to 15, and the nucleotide sequence corresponding to amino acid numbers 575 to 570 of the amino acid sequence of CH-296 (SEQ ID NO: 7) is represented by nucleotide numbers 17 to 34 is a synthetic DNA. PCR was performed using the synthetic primer and a primer (NC2-5′UTR) that annealed to the 5′UTR portion of the NC2 vector described in SEQ ID NO: 12 in the Sequence Listing. PCR reaction conditions are shown below.

  That is, about 0.1 μg of pCold14-H296 as template DNA, 10 μl of 10 × pyrobest buffer (manufactured by Takara Bio Inc.), 8 μl of dNTP mixed solution (manufactured by Takara Bio Inc.), 20 pmol NC2-5′UTR, 20 pmol synthetic primer H296-NcoR, 5 U pyrobest DNA polymerase (manufactured by Takara Bio Inc.) was added, and sterilized water was added to make the total volume 100 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 96 ° C. for 1 minute and 68 ° C. for 4 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 recovered and purified using a Bio rad column, and ethanol precipitation was performed. The recovered DNA after ethanol precipitation was suspended in 39 μl of sterilized water, digested with restriction enzyme NcoI (Takara Bio Inc.) in a reaction solution made up to a total volume of 50 μl, and the NcoI-NcoI digest was extracted by 1.0% agarose electrophoresis. Purification gave an NcoI-NcoI digested DNA fragment.

  Next, pCold14-H296 prepared in (i) is digested with the restriction enzyme NcoI, and the end is dephosphorylated, mixed with the NcoI-NcoI digested DNA fragment, and the DNA ligation kit (Takara Bio Inc.). The product was connected using Thereafter, E. coli JM109 was transformed with 20 μl of the ligation 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 plasmid in which the target DNA fragment was inserted was confirmed by sequencing, and this recombinant plasmid was designated as pCold14-H296-H296. The plasmid was named and displayed as plasmid pCold14-H296-H296, and from July 22, 2005, the National Institute of Advanced Industrial Science and Technology (AIST) (Postal code 305-8656)) as FERM P-20602. This pCold14-H296-H296 is a plasmid containing a base sequence encoding the amino acid sequence of amino acid numbers 279 to 575 of CH-296 in a form in which two amino acid “A” are sandwiched between them. The amino acid sequence of the protein is shown in SEQ ID NO: 5 in the sequence listing, and the base sequence is shown in SEQ ID NO: 6 in the sequence listing.

(2) Expression and purification Escherichia coli BL21 was transformed with pCold14-H296-H296 prepared in (1) above, and the transformant was transformed into LB medium containing agar at 1.5% (w / v) concentration ( (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-liter mini jar fermenter (manufactured by Biott) was used under the conditions of 120 rpm 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 rpm, 20 minutes). This was dialyzed with 2 l of buffer A [50 mM Tris-HCl (pH 7.5), 50 mM NaCl], and about 40 ml thereof was further purified by ion exchange chromatography as follows.

  That is, a column (Φ4 cm × 20 cm) in which SP-Sepharose (manufactured by Amersham Pharmacia) with a resin volume of 100 ml was saturated with buffer A [50 mM Tris-HCl (pH 7.5), 50 mM NaCl] was prepared. A sample after dialysis was applied. 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 each containing 5 ml were fractionated, and about 100 ml of a fraction containing a large amount of the target protein having a molecular weight of about 64.6 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 (made 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 from 50 mM to 1 M sodium chloride with 250 ml of buffer A and 250 ml of buffer B. When the fraction containing only the target protein was examined by 10% SDS-PAGE, it was contained in the non-adsorbed fraction. About 100 ml of this fraction was recovered and 2 l of buffer D [50 mM sodium carbonate buffer, pH 9.5]. The dialysis was performed.

  Thereafter, the solution was concentrated to about 5 times with Centricon-10 (Millipore) and further confirmed with 10% SDS-PAGE. As a result, the target protein having a molecular weight of about 64.6 kDa was detected in a single band. Was designated as H296-H296. Thereafter, when the protein concentration was measured using a MicroBCA kit (Pierce), it was 2.16 mg / ml (calculated from the molecular weight of about 33.4 mM).

Example 2 Induction of specific cytotoxic activity-retaining CTL using H296-H296 (1) Isolation and storage of PBMC After collecting components from a human healthy donor possessing HLA-A2.1, the collected blood was subjected to phosphate buffered physiology. The solution was diluted 2-fold with saline (PBS), overlaid on Ficoll-paque (manufactured by Amersham Biosciences), and centrifuged at 600 × g 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) at 5 × 10 ⁷ cells / ml, and then CP-1 (manufactured by Kyokuto Pharmaceutical Co., Ltd.) and 25% human serum albumin (buminate: An equivalent amount of a stock solution mixed with Baxter (Baxter) at a ratio of 17: 8 was added, suspended, and stored in liquid nitrogen. At the time of CTL induction, these stored PBMCs were rapidly melted in a 37 ° C. water bath, washed with RPMI 1640 medium containing 10 μg / ml DNase (manufactured by Calbiochem), and the number of viable cells was calculated by trypan blue staining. It used for each experiment.

(2) Immobilization of H296-H296 fragment H296-H296 was immobilized on the culture equipment (container) used in the following experiments. That is, 1 ml of PBS containing H296-H296 (final concentration 25 μg / ml) was added to a 24-well cell culture plate (Becton, Dickinson and Company) and incubated overnight at 4 ° C. The plate was washed twice with PBS before use and then washed once with RPMI 1640 medium.

(3) Induction of anti-influenza virus memory CTL Anti-influenza virus memory CTL was induced by the method of Bednarek et al. [Bednarek M. et al. A. et al. J. et al. Immunology. 147, 4047-4053 (1991)] was partially modified. Specifically, RPMI1640 medium containing 5% human AB serum, 1 mM sodium pyruvate, 2 mM L-glutamine, 1 × NEAA Mixture (all manufactured by Cambrex), 1% streptomycin-penicillin (manufactured by Nacalai Tesque) ( The PBMC prepared in Example 2- (1) is suspended in 4 HR cells (hereinafter abbreviated as 5HRPMI) to 1 × 10 ⁶ cells / ml, and then 1 ml is added to the 24-well cell culture plate prepared in Example 2- (2). / Well and incubated in a 5% CO ₂ wet incubator for 1.5 hours at 37 ° C. to separate plastic adherent monocytes (control plate without H296-H296 immobilization treatment) use). Thereafter, non-adherent cells were collected using RPMI 1640 medium and stored on ice as responder cells. The isolated monocytes contained 5 μg / ml of influenza virus protein-derived epitope peptide (matrix protein-derived HLA-A2.1 binding peptide described in SEQ ID NO: 13 in the Sequence Listing) and 1 μg / ml of β2 microbe as antigenic peptides. 0.5 ml each of 5HRPMI containing globulin [manufactured by Scripts] was added, incubated at room temperature for 2 hours, and then irradiated with X-rays (5500R) to obtain antigen-presenting cells. The peptide solution is aspirated and removed from each well, and the wells are washed with RPMI 1640 medium, and the responder cells stored on ice are suspended in 5 HRMI to 1 × 10 ⁶ cells / ml and presented with 1 ml / well of antigen. After addition on the cells, the plates were incubated at 37 ° C. in 5% CO ₂ . On the first day after the start of the culture, 1 ml of 5HRPMI containing 60 U / ml of IL-2 (Proleukin: manufactured by CHIRON) was added to each well. On the fifth day, half of the culture supernatant was removed, and then the same IL -2 containing medium was added by 1 ml. On the seventh day, antigen-presenting cells were prepared in the same manner as described above, and then responder cells cultured for one week were collected and centrifuged at 440 × g for 10 minutes. After centrifugation, the responder cells were suspended in 1 ml of 5HRPMI, added 1 ml / well on the prepared antigen-presenting cells, and restimulated. At this time, a plate in which H296-H296 was immobilized was used (a non-immobilized plate was used as a control). On the first day after restimulation, 1 ml of 5HRPMI containing 60 U / ml IL-2 was added to each well. On the fourth day, half of the culture supernatant was removed, and then 1 ml of the same medium as before removal was added. The culture was further continued for 3 days to induce CTL. Table 1 shows the cell growth rate after 14 days of culture. The cell growth rate was determined as the growth rate by the ratio of the number of cells at the end of induction to the number of responder cells at the start of CTL induction.

  As a result, the group in which H296-H296 was immobilized showed a higher growth rate than the control group in which H296-H296 was not immobilized. That is, it was revealed that the use of H296-H296 during CTL induction increases the cell proliferation rate.

Example 3 Expansion culture using CTL feeder cells of Example 2 (1) Immobilization of H296-H296 fragment H296-H296 was immobilized on culture equipment (container) used in the following experiment. That is, PBS containing H296-H296 (final concentration 25 μg / ml) was added to each 12.5 cm ² flask (Becton, Dickinson and Company) in an amount of 2.7 ml and incubated overnight at 4 ° C. The flask was washed twice with PBS before use and then once with RPMI 1640 medium.

(2) Expansion culture using feeder cells of CTL of Example 2 CTL induced in the presence of H296-H296 prepared in Example 2- (3) (control was CTL induced in the absence of H296-H296) It adjusted to 3 * 10 < ⁴ > cells / ml. On the other hand, HLA-A2.1 non-retained allogeneic PBMC collected by the same method as in Example 2- (1) was mixed with 2 donors, then irradiated with X-rays (3300R), washed with a medium, and then 4 × 10 ⁶ cells / Adjusted to ml (referred to as feeder cells). These 3 × 10 ⁴ cells CTL and 4 × 10 ⁶ cells feeder cells were mixed with 5 ml of 10% Hyclone FBS (HyClone), 1 mM sodium pyruvate, 2 mM L-glutamine, 1 × NEAA Mixture, 1% streptomycin-penicillin. In an RPMI 1640 medium (hereinafter abbreviated as 10 Hyclone RPMI), further added with an anti-CD3 antibody (manufactured by Janssen Kyowa Co., Ltd.) having a final concentration of 50 ng / ml, placed in a 12.5 cm ² flask, and in a 37 ° C. wet CO ₂ incubator. Cultured for 14 days. At this time, the H296-H296 immobilized flask of Example 3- (1) was used (a non-immobilized flask was used as a control). During this period, no peptide stimulation was applied, and 5 ml of 10 Hyclone RPMI and IL-2 at a final concentration of 120 U / ml were added on the first day of the culture, and the culture supernatant was added every 2-3 days after the start of the culture. After half removal, 5 ml of 10 Hyclone RPMI containing 60 U / ml IL-2 was added to each flask and expanded for 14 days. The results are shown in Table 2. The growth rate at the time of expansion culture is the ratio of the number of cells at the end of expansion culture to the number of cells at the start of expansion culture as the growth rate. Obtained as a ratio of numbers.

  As a result, the CTL of the group in which H296-H296 was immobilized during CTL induction and expansion culture showed a higher growth rate than the control group in which H296-H296 was not immobilized. That is, it was revealed that the use of H296-H296 during CTL induction and expansion culture increases the cell proliferation rate.

Example 4 Measurement of Cytotoxic Activity of CTL of Example 3 The cytotoxic activity of CTL prepared in Example 3 on the 14th day after the start of expansion culture was determined by measuring the cytotoxic activity using Calcein-AM [Lichtenfels R. (Lichtenfelds R. et al.), J. et al. Immunol. Methods, Vol. 172, No. 2, pp. 227-239 (1994)]. HLA-A2.1-bearing EBV transform B cells (cell name 221A2.1) cultured in the presence or absence of epitope peptide overnight are treated with 5% FBS (fetal bovine serum) to 1 × 10 ⁶ cells / ml. , Calcein-AM (manufactured by Dojindo Laboratories) was added to a final concentration of 25 μM, followed by incubation at 37 ° C. for 1 hour. The cells were washed with a medium not containing Calcein-AM, and then used as Calcein-labeled target cells. Calcein-labeled target cells were mixed with 30 times the amount of K562 cells (ATCC CCL-243) to prepare cells for measuring cytotoxic activity. Note that K562 cells were used to eliminate non-specific damaging activity by NK cells mixed in responder cells.

The anti-influenza virus memory CTL prepared in Example 3 was used as effector cells, and after serial dilution with 5HRMI to 1 × 10 ^{5 to} 3 × 10 ⁶ cells / ml, a 96-well cell culture plate (manufactured by Becton, Dickinson and Company) 100 μl / well was dispensed in each well, and 100 μl / well of cells for measuring cytotoxic activity, adjusted to a Calcein-labeled target cell concentration of 1 × 10 ⁵ / ml, was added thereto. At this time, the ratio of the effector cells (E) to the Calcein-labeled target cells (T) was shown as the E / T ratio, and the E / T ratios 30, 10, 3, 1 were measured. The plate containing the cell suspension was centrifuged at 400 × g for 1 minute and then incubated for 4 hours in a 37 ° C. wet CO ₂ incubator. After 4 hours, 100 μl of the culture supernatant was collected from each well, and the amount of calcein released into the culture supernatant was measured with a fluorescent plate reader (manufactured by Berthold technologies) (excitation 485 nm / measurement 538 nm). “Specific cytotoxic activity (%)” was calculated according to the following formula 1.

Formula 1: Specific 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 the well containing only the cells for measuring cytotoxic activity, and indicates the Calcein spontaneous release amount from the Calcein-labeled target cells. Further, 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 the cell for measuring cytotoxic activity. Further, how much the cytotoxic activity before expansion culture was maintained at an E / T ratio of 3 was calculated according to the following equation 2 as “maintained cytotoxic activity (%)”.

Formula 2: Cytotoxic activity maintenance (%) =
[Cytotoxic activity after expansion culture (%) / Cytotoxic activity before expansion culture (%)] × 100

Table 3 shows the measurement results.

  The cytotoxic activity against Calcein-labeled target cells cultured in the absence of peptide was almost 0%, confirming that there was no nonspecific activity. In the cytotoxic activity against Calcein-labeled target cells cultured in the presence of the peptide, the group in which the H296-H296 fragment was immobilized at the time of CTL induction and expanded culture had specific and high cytotoxic activity even after 14 days of expanded culture. Was holding. On the other hand, in the control group in which H296-H296 was not immobilized both during CTL induction and during expansion culture, the activity was clearly reduced. That is, it has been clarified that by using H296-H296 during CTL induction and expansion culture, expansion of CTL can be performed while maintaining a specific and high cytotoxic activity for a long time.

Example 5 Measurement of TCR Vβ17-positive cell content ratio in CTL cell population of Example 3 The peptide derived from HLA A2.1-binding influenza virus protein used for CTL induction was recognized by TCR Vβ17-positive T cells. Lehner et al. [Lehner P. et al. J. et al. et al. , J .; Exp. Med. 181: 79-91 (1995)]. Therefore, by measuring the positive cell content ratio of TCR Vβ17, it can be an index of anti-influenza virus memory CTL.
CTL of 2 × 10 ⁵ cells prepared in Example 3 was washed with PBS, suspended in 15 μl of PBS containing 1% bovine serum albumin (BSA) (manufactured by Sigma), and FITC-labeled mouse IgG1 or FITC-labeled mouse Anti-human TCR Vβ17 antibody (both manufactured by BECKMAN COULTER) was added, followed by incubation on ice for 30 minutes. After incubation, the cells were washed twice with PBS containing 0.1% BSA and suspended in PBS containing 1% BSA. The cells were subjected to flow cytometry using Cytomics FC500 (BECKMAN COULTER) to measure the content of TCR Vβ17 positive cells. Table 4 shows the measurement results.

  CTL immobilized with H296-H296 at the time of CTL induction and expansion culture showed a clearly higher positive cell content than the control group that was not immobilized. The CTL after induction, that is, the content of TCR Vβ17 positive cells before expansion culture, was 78.0% for the group to which H296-H296 was immobilized, and 73.0% for the control group that was not immobilized. . That is, it was confirmed that by using H296-H296 at the time of CTL induction and expansion culture, expansion culture was possible while maintaining a high content of anti-influenza virus memory CTL.

Example 6 Expansion culture without using CTL feeder cells of Example 2 (1) Immobilization of anti-CD3 antibody and H296-H296 fragment Culture equipment (container) used in the following CTL expansion culture experiments without using feeder cells H296-H296 was immobilized on the plate. That is, 160 μl of PBS containing an anti-CD3 antibody (final concentration 5 μg / ml) and H296-H296 (final concentration 25 μg / ml) was added to a 96-well cell culture plate and incubated overnight at 4 ° C. (as a control, anti-CD3 Immobilize antibody only). The plate was washed twice with PBS before use and then washed once with RPMI 1640.

(2) Expanded culture of CTL without using feeder cells CTL induced in the presence of H296-H296 prepared in Example 2- (3) (control was CTL induced in the absence of H296-H296) was washed with 5HRPMI. Thereafter, 1 × 10 ⁵ cells were suspended in 300 μl of 5HRPMI, and culture was started in a 37 ° C. wet CO ₂ incubator using the 96-well culture plate prepared in Example 6- (1). On the first day of culture, IL-2 was added to a final concentration of 120 U / ml, and after 5 days from the start of culture, the cells were passaged to a non-immobilized plate every 2-3 days. IL-2 at a concentration of 100 U / ml was added. During this period, no stimulation by the peptide was applied, and expansion culture was performed for 13 days. Table 5 shows the cell proliferation rate.

  The group in which H296-H296 was immobilized at the time of induction and expansion culture showed a high growth rate compared to the non-immobilized control group. That is, by using H296-H296 at the time of induction and expansion culture, expansion culture of CTL became possible without using feeder cells.

Example 7 Measurement of Cytotoxic Activity of CTL of Example 6 CTL specific cytotoxic activity of the CTL obtained in Example 6- (2) was measured in the same manner as in Example 4. The results are shown in Table 6.

  The cytotoxic activity against Calcein-labeled target cells cultured in the absence of peptide was almost 0%, confirming that there was no nonspecific activity. Regarding the cytotoxic activity against Calcein-labeled target cells cultured in the presence of peptides, the CTL of the group to which H296-H296 was added at the time of induction and expansion was more specific after 13 days of expansion than the control group. And retained high cytotoxic activity. That is, it is clear that by using H296-H296 at the time of CTL induction and expansion culture, expansion culture can be performed by a method that does not use feeder cells while maintaining a specific and high cytotoxic activity for a long period of time. Became.

Example 8 Measurement of TCR Vβ17 positive cell content ratio in CTL cell population of Example 6 2 × 10 ⁵ cells CTL prepared in Example 6- (2) were TCR Vβ17 positive in the same manner as in Example 5. The cell content ratio was measured. The control group using CTL expanded in the absence of H296-H296 was not tested because the number of cells obtained after expanded culture was small. The results are shown in Table 7.

  In the group where H296-H296 was used for induction and expansion, high positive cell content was shown. The CTL after induction, that is, the content of TCR Vβ17 positive cells before expansion culture, was 78.0% for the group in which H296-H296 was immobilized. That is, by using H296-H296 at the time of induction and expansion culture, it was confirmed that expansion culture was possible while maintaining a high content ratio of anti-influenza virus memory CTL in a system not using feeder cells.

Example 9 Induction of CTL retaining specific cytotoxic activity using H296-H296 and H-296 (1) Immobilization of H296-H296 and H-296 fragments H296 was used in culture equipment (containers) used in the following experiments. -H296 and H-296 were immobilized. Specifically, 1 ml each of PBS containing H296-H296 and H-296 (final concentration 25 μg / ml) was added to a 24-well cell culture plate and incubated overnight at 4 ° C. The plate was washed twice with PBS before use and then washed once with RPMI 1640 medium.

(2) Induction of anti-influenza virus memory CTL Induction of anti-influenza virus memory CTL in the same manner as in Example 2- (3) using PBMC separated and stored by the method described in Example 2- (1) Went. The culture plate used at this time was the same as that prepared in Example 9- (1) (a plate on which H296-H296 and H-296 were not immobilized was used as a control). Table 8 shows the cell proliferation rate on day 14 after the start of induction.

  As a result, the group in which H296-H296 was immobilized showed the highest growth rate, and then the group in which H-296 was immobilized, and the control group that was not immobilized had a low growth rate compared to these. That is, it became clear that the use of H296-H296 or H-296 during CTL induction increases the cell growth rate, and the use of H296-H296 has a higher effect.

Example 10 Measurement of Cytotoxic Activity of CTL of Example 9 CTL specific cytotoxic activity of the CTL obtained in Example 9 was measured in the same manner as in Example 4. The results are shown in Table 9.

  The cytotoxic activity against Calcein-labeled target cells cultured in the absence of peptide was almost 0%, confirming that there was no nonspecific activity. In the cytotoxic activity against Calcein-labeled target cells cultured in the presence of the peptide, the CTL of the group in which H296-H296 or H-296 was immobilized at the time of induction retained a specific and high cytotoxic activity compared to the CTL of the control group Was. That is, by using H296-H296 or H-296 at the time of CTL induction, it became possible to induce CTL having high cytotoxic activity.

Example 11 Measurement of content ratio of TCR Vβ17 positive cells in CTL cell population of Example 9 The CTL of 2 × 10 ⁵ cells prepared in Example 9 was measured for the content ratio of TCR Vβ17 positive cells in the same manner as in Example 5. Measurements were made. The results are shown in Table 10. In addition, the Vβ17 positive cell content rate of the responder cells before CTL induction was 2.5%.

  The group using H296-H296 or H-296 at the time of induction showed a higher positive cell content ratio than the control group not used. That is, it became clear that anti-influenza virus memory CTL can be efficiently induced by using H296-H296 or H-296 during induction.

Example 12 Expansion culture without using CTL feeder cells of Example 9 (1) Immobilization of anti-CD3 antibody and H296-H296 or H-296 Culture equipment used in CTL expansion culture experiments without using the following feeder cells H296-H296 or H-296 was immobilized on (container). Specifically, 160 μl of PBS containing anti-CD3 antibody (final concentration 5 μg / ml) and H296-H296 or H-296 (final concentration 25 μg / ml) was added to a 96-well cell culture plate and incubated overnight at 4 ° C. (control) Only immobilize anti-CD3 antibody). The plate was washed twice with PBS before use and then washed once with RPMI 1640.

(2) Expanded culture of CTL without using feeder cells After washing CTL induced in the presence of H296-H296 or H-296 prepared in Example 9 (control was induced in the absence) with 5HRPMI, 1 × 10 ⁵ cells were suspended in 300 μl of 5HRPMI, and culture was started in a 37 ° C. wet CO ₂ incubator using the 96-well culture plate prepared in Example 12. IL-2 was added to a final concentration of 500 U / ml on the first day of culture, and after 5 days, the cells were passaged to a plate that had not been immobilized using 5HRPMI, and a final concentration of 500 U / ml was obtained. -2 was added. That is, the cells were passaged at 1 × 10 ⁵ cells / ml on the 5th day after the start of culture, 1.5 × 10 ⁵ cells / ml on the 8th day, and 3.2 × 10 ⁵ cells / ml on the 12th day. Went. During this period, no stimulation by the peptide was applied, and expansion culture was performed for 15 days. Table 11 shows the cell proliferation rate.

  The group in which H296-H296 was immobilized at the time of induction and expansion culture showed the highest growth rate, and then the group to which H-296 was immobilized at the time of induction and expansion culture, the control group that was not immobilized had the lowest growth rate. showed that. That is, by using H296-H296 or H-296 during induction and expansion culture, CTL can be expanded without using feeder cells, and the effect is higher when H296-H296 is used. It was done.

Example 13 Measurement of Cytotoxic Activity of CTL of Example 12 Specific cytotoxic activity of CTL was measured for the CTL obtained in Example 12- (2) in the same manner as in Example 4. The results are shown in Table 12.

  The cytotoxic activity against Calcein-labeled target cells cultured in the absence of peptide was almost 0%, confirming that there was no nonspecific activity. Regarding the cytotoxic activity against Calcein-labeled target cells cultured in the presence of peptides, the CTLs of the group to which H296-H296 was added during induction and expansion maintained specific and high cytotoxic activity even after 15 days of expansion. Was. The CTL of the group to which H-296 was added during induction and expansion culture was lower than that of the group to which H296-H296 was added, but retained specific cytotoxic activity. That is, by using H296-H296 or H-296 at the time of CTL induction and expansion culture, expansion culture can be performed by a method that does not use feeder cells while maintaining specific cytotoxic activity for a long period of time. As a result, H296-H296 was more effective.

Example 14 Measurement of content ratio of TCR Vβ17 positive cells in CTL cell population of Example 12 CTL of 2 × 10 ⁵ cells prepared in Example 12- (2) was TCR Vβ17 positive in the same manner as in Example 5. The cell content ratio was measured. The results are shown in Table 13.

  The group using H296-H296 during induction and expansion showed the highest positive cell content ratio, followed by H-296 using the induction and expansion cultures, and the non-immobilized control group being the lowest positive The cell content was shown. The CTL after induction, that is, the content of TCR Vβ17 positive cells before expansion culture, was 50.6% for the control group, 73.5% for the group in which H296-H296 was immobilized, and H-296 was immobilized. It was 73.2% for the selected group. That is, by using H296-H296 at the time of induction and expansion culture, it was confirmed that expansion culture was possible while maintaining a high content ratio of anti-influenza virus memory CTL in a system not using feeder cells.

Example 15 Expansion culture without using CTL feeder cells of Example 9 assuming 30 mL blood collection (1) Immobilization of anti-CD3 antibody and H296-H296 or CH-296 fragment In a CTL expansion culture experiment without using the following feeders H296-H296 or CH-296 was immobilized on the culture equipment (container) to be used. That is, 160 μl of PBS containing anti-CD3 antibody (final concentration 5 μg / ml) and H296-H296 or CH-296 (final concentration 25 μg / ml) was added to a 96-well cell culture plate and incubated at 4 ° C. overnight (control). Only immobilize anti-CD3 antibody). The plate was washed twice with PBS before use and then once with RPMI medium.

(2) Expansion of CTL without using feeder cells CTL induced with H296-H296 in Example 9 was treated with 3% human AB serum, 1 mM sodium pyruvate, 2 mM L-glutamine, 1 × NEAA Mixture, 1% After washing with RPMI 1640 medium containing streptomycin-penicillin (hereinafter abbreviated as 3HRPMI), 1 × 10 ⁵ cells were suspended in 300 μl of 3HRPMI and placed in the 96-well culture plate prepared in Example 15- (1) at 37 ° C. The culture was started in a wet CO ₂ incubator. At this time, in the culture scale using PBMC 3 × 10 ⁷ cells expected to be obtained from 30 mL blood collection, the maximum amount of human AB serum used from induction to expansion culture is 15 mL, and the maximum RPMI medium usage is 10 L. Restricted expansion culture was performed. On the first day of culture, IL-2 having a final concentration of 500 U / ml was added. On the fifth day, 1 × 10 ⁵ cells / ml was applied to a plate not immobilized using 1% human AB serum RPMI medium. Passaging was performed, and IL-2 having a final concentration of 500 U / ml was added. On the 8th day after the start of the culture, the cells were subcultured at 1.5 × 10 ⁵ cells / ml on a plate not immobilized using 0.2% human AB type serum RPMI medium, to a final concentration of 500 U / ml. IL-2 was added, and only IL-2 having a final concentration of 500 U / ml was added on the 12th day. During this period, the cells were expanded for 15 days without any stimulation by the peptide. Table 14 shows the cell proliferation rate.

  The CTL of the group in which H296-H296 or CH-296 was immobilized during expansion culture showed a higher growth rate than the group in which H296-H296 or CH-296 was not immobilized during expansion culture. That is, with respect to CTL obtained using H296-H296 at the time of induction, a high cell proliferation effect was observed by using H296-H296 or CH-296 during expansion culture.

Example 16 Induction of CTL having specific cytotoxic activity using H296-H296 (autologous plasma)
Anti-influenza virus memory CTL was induced in the same manner as in Example 2- (3) using PBMC separated and stored by the method described in Example 2- (1). At that time, RPMI medium (hereinafter abbreviated as 5 Plasma RPMI) containing 5% autologous plasma, 1 mM sodium pyruvate, 2 mM L-glutamine, 1 × NEAA Mixture, 1% streptomycin-penicillin was used. Table 15 shows the cell proliferation rate on the 14th day after the initiation of induction thus prepared.

  In the group in which H296-H296 was immobilized at the time of induction, a higher growth rate was obtained than in the control group in which H296-H296 was not immobilized. That is, it has been clarified that the cell growth rate is increased by using H296-H296 at the time of CTL induction in a medium containing autologous plasma.

Example 17 Measurement of the content ratio of TCR Vβ17 positive cells in the CTL cell population of Example 16 The CTL of 2 × 10 ⁵ cells prepared in Example 16 was measured for the content ratio of TCR Vβ17 positive cells in the same manner as in Example 5. Measurements were made. The results are shown in Table 16. In addition, the Vβ17 positive cell content rate of the responder cells before CTL induction was 2.5%.

  The group in which H296-H296 was immobilized at the time of induction showed a higher positive cell content ratio than the control group in which H296-H296 was not immobilized. That is, it became clear that anti-influenza virus memory CTL can be efficiently induced by using H296-H296 during induction.

Example 18 Expansion using feeder cells of CTL of Example 16 Example using CTL induced in the presence of H296-H296 prepared in Example 16 (control was CTL induced in the absence of H296-H296) CTL expansion culture was performed in the same manner as in 3. Under the present circumstances, it culture | cultivated using 5PlasmaRPMI culture medium and expanded culture for 14 days. Table 17 shows the cell proliferation rate after 14 days.

  The group in which H296-H296 was immobilized at the time of induction and expansion culture showed a higher growth rate than the group to which H296-H296 was not immobilized. That is, it has been clarified that the use of H296-H296 during induction or expansion culture increases the cell proliferation rate.

Example 19 Measurement of CTL cytotoxic activity of Example 18 CTL specific cytotoxic activity of the CTL obtained in Example 18 was measured in the same manner as in Example 4. The results are shown in Table 18.

  The cytotoxic activity against Calcein-labeled target cells cultured in the absence of peptide was almost 0%, confirming that there was no nonspecific activity. Regarding the cytotoxic activity against Calcein-labeled target cells cultured in the presence of peptides, the group in which H296-H296 was immobilized during induction and expansion culture retained specific and high cytotoxic activity even after 14 days of expansion culture. It was. On the other hand, the activity of the control group in which H296-H296 was not immobilized during induction and expansion was clearly reduced. In other words, by using H296-H296 at the time of CTL induction and expansion culture in the expansion culture using autologous plasma, it is possible to expand the CTL while maintaining a specific and high cytotoxic activity for a long period of time. It became clear that

Example 20 Measurement of TCR Vβ17 positive cell content ratio in the CTL cell population of Example 18 The CTL of 2 × 10 ⁵ cells prepared in Example 18 was measured for the TCR Vβ17 positive cell content ratio in the same manner as in Example 5. Measurements were made. The results are shown in Table 19.

  The group in which H296-H296 was immobilized at the time of induction and expansion culture clearly showed a higher positive cell content ratio than the non-immobilized control group. In addition, as shown in Table 16, the content of TCR Vβ17 positive cells for the CTL after induction, that is, before expansion culture, was 80.1% for the group in which H296-H296 was immobilized, and the group that was not immobilized Was 72.1%. In other words, it is clear that in the expansion culture using autologous plasma, by using H296-H296 at the time of CTL induction and expansion culture, it is possible to perform the expansion culture while maintaining a high content ratio of the anti-influenza virus memory CTL. became.

Example 21 Expanded culture of lymphocytes (lymphokine activated cells) using H296-H296 (1) Separation and storage of PBMC After collecting components from a healthy human donor with informed consent, blood was collected. Was diluted 2-fold with phosphate buffered saline (PBS), overlaid on Ficoll-paque, and centrifuged at 500 × g for 20 minutes. Middle layer peripheral blood mononuclear cells (PBMC) were collected with a pipette and washed. The collected PBMC was suspended in a storage solution composed of 90% FBS / 10% DMSO (manufactured by SIGMA) and stored in liquid nitrogen. At the time of lymphocyte expansion culture, these preserved PBMCs were rapidly thawed in a 37 ° C. water bath, washed with RPMI 1640 medium containing 10 μg / mL DNase, and subjected to each experiment after calculating the number of viable cells by trypan blue staining.

(2) Immobilization of anti-human CD3 antibody and H296-H296 fragment The anti-human CD3 antibody and H296-H296 were immobilized on the culture equipment used in the following experiments. Specifically, 1.9 mL of PBS containing anti-human CD3 antibody (final concentration 5 μg / mL) was added to each 12-well cell culture plate (Becton Dickinson). At this time, H296-H296 prepared in Example 1 was added to the H296-H296 addition group so as to have a final concentration (25 μg / mL).
These culture devices were incubated at room temperature for 5 hours and then stored at 4 ° C. until use. Immediately before use, PBS containing the antibody and H296-H296 was aspirated and removed from these culture equipment, and each well was washed twice with PBS and once with RPMI medium for each experiment.

(3) Enlarged culture of lymphocytes Example 21- (1) so that AIM-V containing 3% humanAB serum (Invitrogen, hereinafter abbreviated as 3% AIM-V) is 1 × 10 ⁶ cells / mL. After suspending the PBMC prepared in step 3, 3% AIM-V was added at 2 mL / well to the anti-human CD3 antibody-immobilized plate prepared in Example 21- (2) or the anti-human CD3 antibody and H296-H296-immobilized plate. The cell solution was added in an amount of 1 mL / well. 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, each group was diluted with AIM-V containing 1% humanAB serum so that the concentration was 0.075 × 10 ⁶ cells / mL (liquid volume 6 mL), and no culture solution was immobilized. It was transferred to a 12.5 cm ² cell culture flask, and IL-2 was added to a final concentration of 500 U / mL. The serum concentration was determined on the assumption that PBMC obtained from 30 mL blood collection was cultured in 10 L of medium. In the control group (H296-H296 non-immobilized), AIM-V containing 0.1% humanAB serum was added so that each group had 0.25 × 10 ⁶ cells / mL on the seventh day. And a new 25 cm ² cell culture flask in which no culture medium (volume 12.6 mL) diluted with AIM-V containing 0.09% humanAB serum was immobilized in the H296-H296 immobilized group. I moved it to a stand. In either case, IL-2 was added so as to have a final concentration of 500 U / mL. On the 10th day after the start of the culture, the cell solution was diluted to 0.413 × 10 ⁶ / mL using AIM-V containing human AB serum having the same serum concentration as that on the 7th day (fluid volume 12.6 mL). ), Each transferred to a standing 25 cm ² cell culture flask with nothing immobilized. IL-2 was added to a final concentration of 500 U / mL in each group. On the 15th 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 by comparison with the number of cells at the start of the culture. The results are shown in Table 20.

  As shown in Table 20, in the group using the culture equipment in which H296-H296 was immobilized at the initial stage of lymphocyte expansion culture, the expansion culture rate of lymphocytes was higher than that in the control group. From this, it was revealed that H296-H296 is preferably used during lymphocyte expansion culture.

  The present invention provides a method for producing lymphocytes. This method has a high cell proliferation rate, and the lymphocytes obtained according to the present invention are suitably used, for example, for adoptive immunotherapy. Therefore, the method of the present invention is expected to make a great contribution to the medical field.

It is a schematic diagram which shows the domain structure of fibronectin. It is a figure which shows the preparation methods of H296-H296.

SEQ ID NO: 1; Partial region of fibronectin named III-12.
SEQ ID NO: 2; Partial region of fibronectin named III-13.
SEQ ID NO: 3; Partial region of fibronectin named III-14.
SEQ ID NO: 4; Partial region of fibronectin named CS-1.
SEQ ID NO: 5; Fibronectin fragment named H296-H296.
SEQ ID NO: 6; Polynucleotide coding Fibronectin fragment named H296-H296.
SEQ ID NO: 7; Fibronectin fragment named CH-296.
SEQ ID NO: 8; Polynucleotide coding Fibronectin fragment named CH-296.
SEQ ID NO: 9; Primer H296-NcoF.
SEQ ID NO: 10; Primer H296-HindR.
SEQ ID NO: 11; Primer H296-NcoR.
SEQ ID NO: 12; Primer NC2-5 'UTR.
SEQ ID NO: 13; Designed peptide based on matrix-protein derived from influenza virus.

Claims (6)

Producing how lymphocytes, characterized in that it comprises the step of culturing in the presence of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 5. The production method according to claim 1, wherein the lymphocyte is a cytotoxic T lymphocyte or a lymphokine activated cell. The production method according to claim 1 or 2 , further comprising a step of introducing a foreign gene into lymphocytes. The production method according to claim 3 , wherein the foreign gene is introduced using a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, a lentivirus vector or a simian virus vector. Polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 5. A nucleic acid selected from:
Nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 6 nucleic acid, or the Sequence Listing encoding the polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 5.

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