MX2008004225A - Method for production of t cell population - Google Patents

Abstract

Disclosed is a method for production of a T cell population capable of expressing CD45RA and also expressing at least one substance selected from the group consisting of CD62L, CCR7, CD27 and CD28. The method comprises the step of cultivating a cell population containing T cells in the presence of a fibronectin, a fragment thereof or a mixture of the fibronectin and the fragment.

Description

METHOD FOR THE PRODUCTION OF A CELLULAR POPULATION T TECHNICAL FIELD The present invention relates to a method for preparing a population of T cells, which is useful in the medical field.

BACKGROUND OF THE INVENTION A living body is protected from foreign substances mainly by an immune response and an immune system has been established by several cells and the soluble factors produced accordingly. Among these, leukocytes, especially lymphocytes, play a key role. The lymphocytes are classified into two main types, B lymphocyte (which can be referred to hereinafter as B cell) and T lymphocyte (which can be referred to later in this document as a T cell), both of which specifically recognize an antigen and act on the antigen to protect the living body. In the periphery, the T cells consist mainly of CD4 T cells that have the CD marker (Differentiation Grouping) 4 and CD8 T cells that have the CD8 marker. A majority of CD4 T cells is referred to as a helper T cell (referred to subsequently in this document as TH), it involves assistance in the production of antibodies and inductions of various immune responses and differs in Th1 or Th2 which is different in the class of a cytokine produced by antigen stimulation. A majority of CD8 T cells differentiate into cytotoxic T cells (Tc: cytotoxic T lymphocyte, also referred to as a T cell eliminator, which can be referred to herein later as CTL), exhibiting cytotoxic activity by stimulating antigens. For example, in the pathological condition of cancer, immunotherapy has caused an interest in recent years, as a fourth therapy after surgery, chemotherapy and radiotherapy. Since immunotherapy uses immunocompetence possessed inherently by a human, it is said that a physical burden on a patient caused by immunotherapy is slight compared to that caused by other therapies. As immunotherapy, a therapy has been known to include the step of transferring activated cells by lymphokines obtained by the expansion of CTLs or peripheral blood lymphocytes induced ex vivo or the like according to various methods, NKT cells,? DT cells or Similar; a dendritic cell transfer therapy or a peptide vaccine therapy by means of which a induction of specific CTLs for antigens in vivo; Thl cell therapy; immune gene therapy which further includes the step of transducing a gene for which various effects can be expected for the aforementioned ex vivo cells and transferring a transduced cell to the body; and similar. Fibronectin is a gigantic glycoprotein I that has a molecular weight of 250,000, which exists in the blood of an animal, on the surface of a cultured cell or in an extracellular matrix of a tissue and has been known to have several functions. A domain structure of the same is divided into seven portions (Figure 1 and those that follow), where three classes of similar sequences are contained in an amino acid sequence thereof, the repetitions of each of these sequences constitute the sequence complete Three classes of similar sequences are referred to as type I, type II and type III. Among these, type III is constituted by 71 to 96 amino acid residues, where an identity of these amino acid residues is from 17 to 40%. In fibronectin, there are fourteen type III sequences, among which the 8th, 9th or 10th sequence (each one referred to later in this document as III-8, III-9 or III-10) is contained in a domain of cellular link and the 12th, 13th or 14th sequence (each one that is referred subsequently in this document as 111-12, 111-13 or III-14) is contained in a heparin binding domain. further, a binding region to VLA (very late activation antigen) -5 is contained in 111-10 and its core sequence is RGDS. In addition, a region referred to as IIICS exists on the C-terminal side of the heparin binding domain. A region referred to as CS-1 consisting of 25 amino acids and having a binding activity to VLA-4 exists in IIICS (eg, Publications that are not Patent 1 to 3). Among the immunotherapies, in a therapy that includes the step of transferring activated cells with lymphokines obtained by the expansion of CTLs or peripheral blood lymphocytes induced ex vivo or the like according to the actions of IL-2 and an anti-CD3 antibody , with respect to the disadvantages such that both a cytotoxic activity is maintained when the CTLs specific for ex vivo-induced antigens are expanded, that both the lymphocytes can be effectively expanded and the like, the present inventors have already studied the effects of using fibronectin or a fragment thereof (eg, Patent Publications 1 to 3). In recent years, it has been reported that as T lymphocytes used in immunotherapy, in one case where native T cells or central memory T cells are administered in a more undifferentiated state, a much higher therapeutic effect can be expected in administration to a living body, preferably than a case where effector T cells are administered and terminally differentiated (by example, Publications that are not Patents 4 and 5). In addition, also in a cell transfer therapy < dendritic, a therapy of peptide vaccines or the like for which the induction of CTLs specific for antigens is expected in vivo, it is considered that there are few, native T cells, the origins from which CTLs can be induced, for example , in the body of a patient with progressive cancer; therefore, a sufficient effect can not be expected frequently. Publication that is not Patent 1: FIBRONECTIN, ACADEMIC PRESS INC., 1-8, conducted by Deane F. Momer, published in 1988 Publication that is not Patent 2: Kimizuka F. and others eight, J. Biochem. , 1991, 110 (2), 284-291 Publication that is not Patent 3: Hanenberg H. and five others, Human Gene Therapy, 1997, 8 (18), 2193-2206 Publication that is not patent 4: Gattinoni L. and another nine, J. "Clin. Invest., 2005, 115 (6), 1616-1626 Publication that is not Patent 5: Benigni F and ten others, J.

I munol, 2005, 175 (2), 739-748 Patent Publication 1: WO 03/016511 Patent Publication 2: WO 03/080817 Patent Publication 3: WO 2005/019450 DESCRIPTION OF THE INVENTION PROBLEMS TO BE RESOLVED BY THE INVENTION An object of the present invention is to provide a method for preparing a population of T cells, which is effective in administration to a living body.

MEANS FOR RESOLVING THE PROBLEMS A first invention of the present invention relates to a method for preparing a population of cells T, wherein the T cell population expresses CD45RA and expresses at least one selected from the group consisting of CD62L, CCR7, CD27 and CD28; characterized in that the method includes the step of culturing a population of cells containing T cells, in the presence of fibronectin, a fragment thereof or a mixture thereof. In the first invention of the present invention, the total culture days that include the culture step are exemplified by 4 to 14 days. Also, it is exemplified that the culture in the presence of fibronectin, a fragment of the same or a mixture thereof is carried out at least at the beginning of the cultivation and further, it is preferable that the cultivation be carried out for at least one day or more. Further, in the first invention of the present invention, it is exemplified that the culture step in the presence of fibronectin, a fragment thereof or a mixture thereof is carried out in the presence of a CD3 ligand. Also, the CD3 ligand is exemplified by an anti-CD3 antibody. In the first invention of the present invention, the fibronectin fragment is exemplified by a polypeptide (m) that contains at least one of the amino acid sequences shown in SEQ ID Nos: 1 to 8 of the Sequence Listing or a polypeptide ( n) which contains at least one amino acid sequence having a substitution, deletion, insertion or addition of one or the plural number of amino acids in any of the above amino acid sequences, wherein the polypeptide (n) has a function equivalent to that of the previous polypeptide (m). Also, the fibronectin fragment is exemplified by a polypeptide comprising all the amino acid sequences shown in SEQ ID Nos: 1 to 3 and 5 to 8 of the Sequence Listing. In addition, in the first invention of the present invention, the method of preparation which also includes the step of separating expressing cells is also exemplified. at least one selected from the group consisting of CD45RA, CD62L, CCR7, CD27 and CD28. In addition, in the first invention of the present invention, the method of preparation that also includes the step that transduces a foreign gene into the population of cells is also exemplified. In the preparation method, in the transduction of the foreign gene, a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, a lentivirus vector or an ape virus vector can be used. A second invention of the present invention relates to a population of T cells obtained by means of the method of the first invention of the present invention, wherein the population of T cells expresses CD45RA and expresses at least one selected from the group consisting of I CD62L, CCR7, CD27 and CD28. A third invention of the present invention relates to a medicament containing as an effective ingredient the population of T cells obtained by means of the method of the first invention of the present invention, wherein the population of T cells expresses CD45RA and expresses at less . one selected from the group consisting of CD62L, CCR7, CD27 and CD28. A fourth invention of the present invention relates to a method for treating or preventing a disease, which includes the step of administering to a subject an effective amount of the population of T cells obtained by means of the method of the first invention of the present invention, wherein the population of T cells expresses CD45RA and expresses at least one selected from the group consisting of CD62L, CCR7, CD27 and CD28. A fifth invention of the present invention relates to the use of the population of T cells obtained by means of the method of the first invention of the present invention, wherein the population of T cells expresses CD45RA and expresses at least one selected from the group that It consists of CD62L, CCR7, CD27 and CD28 in the manufacture of a medicine. A sixth invention of the present invention relates to a method for preparing a population of T cells, characterized in that the method includes the step of stimulating the population of T cells obtained by means of the method of the first invention of the present invention, wherein the T cell population expresses CD45RA and expresses at least one selected from the group consisting of CD62L, CCR7, CD27 and CD28, with at least one stimulation factor selected from the group consisting of a cell capable of presenting an antigen , a cell that has presented an antigen, an antigen, a ligand of CD3, a ligand of CD28, a cytokine, a chemokine and a cell capable of producing a cytokine. A seventh invention of the present invention relates to a population of T cells obtained by means of the method of the sixth invention of the present invention. An eighth invention of the present invention relates to a medicament containing as an effective ingredient the population of T cells obtained by means of the method of the sixth invention of the present invention. A ninth invention of the present invention relates to a method for treating or preventing a disease, including the step of administering to a subject an effective amount of the population of T cells obtained by means of the method of the sixth invention of the invention. present invention. A tenth invention of the present invention relates to the use of the population of T cells obtained by means of the method of the sixth invention of the present invention in the manufacture of a medicament. An eleventh invention of the present invention relates to a medicament containing: (a) a preparation containing as an effective ingredient the population of T cells obtained by means of the method of the first invention of the present invention, wherein the population of cells T expresses CD45RA and expresses at least one selected from the group consisting of CD62L, CCR7, CD27 and CD28 and (b) a preparation containing as an effective ingredient at least one stimulation factor selected from the group consisting of a cell capable of presenting an antigen, a cell that has presented an antigen, an antigen, a ligand of CD3 , a CD28 ligand, a cytokine, a chemokine and a cell capable of producing a cytokine, wherein the preparations are contained in the medicament as two separate preparations that are administered simultaneously or separately. A twelfth invention of the present invention relates to a method for treating a disease, characterized in that the method includes the following steps (a) and (b) consisting of: • (a) administering to a patient the population of T cells obtained by means of the method of the first invention of the present invention, wherein the population of T cells expresses CD45RA and expresses at least one selected from the group consisting of CD62L, CCR7, CD27 and CD28; and (b) administering to a patient at least one stimulation factor selected from the group consisting of a cell capable of presenting an antigen, a cell that has presented an antigen, an antigen, a ligand of CD3, a ligand of CD28, a cytokine, a chemokine and a cell capable of producing a cytokine.

EFFECTS OF THE INVENTION According to the method of preparation of the present invention, a population of T cells expressing CD45RA and expressing at least one population selected from the group consisting of CD62L, CCR7, CD27 and CD28 is provided. A population of cells obtained by means of the preparation method has a high ratio of T cells which express CD45RA and which express at least one cell selected from the group consisting of CD62L, CCR7, CD27 and CD28, and is extremely useful in a treatment of a disease with cell therapy.

BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] A schematic view showing a fibronectin domain structure. [Figure 2] a graph showing a suppressive action of mouse tumor formation by means of T cell administration.

BEST MODE FOR CARRYING OUT THE INVENTION The present invention has been completed by the findings that, by including the culture step in the presence of fibronectin, a fragment thereof or a mixture of the same (which can be referred to later in this document as the effective ingredient in the present invention), we obtain a population of cells that contains a high ratio of T cells which express CD45RA and which express at least one selected from the group consisting of CD62L, CCR7, CD27 and CD28. Incidentally, the population of T cells expressing CD45RA and expressing at least one selected from the group consisting of CD62L, CCR7, CD27 and CD28 as used herein means a population of T cells that it contains in a ratio high T cells which express CD45RA and which express at least one T cells selected from the group consisting of CD62L, CCR7, CD27 and CD28. Also, a high ratio in this document means that, when the cultivation is carried out under the same conditions except for the presence or absence of the effective ingredient in the present invention, a ratio of the T cells which express CD45RA and which express at least one selected from the group consisting of CD62L, CCR7, CD27 and CD28 in a population of T cells obtained by means of a culture in the presence of the effective ingredient in the present invention is high compared to that in the case of absence of the effective ingredient in the present invention. Is preferable that the T cell population contains the T cells mentioned above in a higher ratio preferably by 5% or more and more preferably by 10% or more, compared to that in the case of the absence of the effective ingredient in the present invention . The ratio of the T cells which express CD45RA and which express at least one selected from the group consisting of CD62L, CCR7, CD27 and CD28, in the resulting cell population, varies depending on various kinds of environmental factors, by example, individual differences and physical conditions of a person who provides cells used in the preparation of T cells such as peripheral blood mononuclear cells (PBMCs). Therefore, it is impossible to unconditionally define the "high relation" mentioned above by a numerical value. Also, the population of T cells obtained by means of the preparation method of the present invention, means a population containing T cells and cells other than T cells, for example, other lymphocytes such as NK cells and a hematocyte component can be contained. different from lymphocytes. The present invention will be explained concretely below. (1) Fibronectin and a Fragment of it Used in the Present Invention. The fibronectin and a fragment thereof described herein may be any of those obtained from nature or those artificially synthesized. The fibronectin and a fragment thereof can be prepared in a substantially pure form from a substance of, natural origin, based on the description, for example, of Ruoslahti E. et al. [J. Biol. Chem., 256 (14), 7277-7281 (1981)]. At this point, the term "substantially pure fibronectin or a fragment of fibronectin" described herein means that fibronectin and a fragment of fibronectin do not contain essentially other proteins that exist together with fibronectin in nature. Each of the fibronectin and a fragment thereof mentioned above can be used alone or in a mixture of plural classes in the present invention. Coincidentally, it is known that there is a large number of fibronectin splice variants. Like the fibronectin used in the present invention, any variant can be used as long as the desired effects of the present invention are exhibited. For example, in the case of plasma-derived fibronectin, it is known that a region referred to as ED-B present upstream of a cell-binding domain and a region referred to as ED-A present between the cell binding domain and the heparin binding domain are deleted. This fibronectin derived from plasma can also be used in the present invention. Useful information on the fibronectin fragments that can be used in the present invention and the preparation of the fragments can be obtained from Kimiduka F. et al [J ". Biochem., 110, 284-291 (1991)], Kornbrihtt AR and collaborators [EMBO J., 4 (7), 1755-1759 (1985)], Sekiguchi K. et al.

[Biochemistry, 25 (17), 4936-4941 (1986)] and the like.

In addition, the nucleic acid sequence encoding fibronectin or the amino acid sequence of fibronectin is disclosed in GenBank Accession No. NM_002026 and NP_002017. In the present invention, the fibronectin fragment is exemplified by, for example, a polypeptide (m) that contains at least one amino acid sequence that contains any of the III-8 regions (amino acid sequence, shown in SEQ. ID NO: 1 of the Sequence Listing), III-9 (amino acid sequence shown in SEQ ID NOs 2 of the Sequence Listing), 111-10 (amino acid sequence shown in SEQ ID NO: 3 of the Sequence Listing) , III-ll (amino acid sequence shown in SEQ ID NO: 4 of the List of Sequences), III-12 (amino acid sequence shown in SEQ ID NO: 5 of the Sequence Listing), 111-13 (amino acid sequence shown in SEQ ID NO: 6 of the Sequence Listing), 111-14 (sequence of amino acids shown in SEQ ID NO: 7 of the Sequence Listing) and CS-1 (amino acid sequence shown in SEQ ID NO: 8 of the Sequence Listing) (see Figure 1) and a polypeptide (n) containing at least one amino acid sequence having a substitution, deletion, insertion or addition of one or a plural number of amino acids in any of the amino acid sequences described above, wherein the polypeptide (n) has a function equivalent to that of the polypeptide mentioned above (m). The length of the fragment is, for example, in terms of the number of amino acids, preferably from 20 to 1000 and more preferably from 100 to 800. At this point, the plural number in this document is a concept that includes the undefined but small number and it is preferably from 2 to 12, more preferably from 2 to 10 and furthermore more preferably from 2 to 8, which is hereinafter referred to the same. In addition, as the fragment, a fragment having a cell adhesion activity and / or a heparin binding activity can preferably be used. The activity of cell adhesion can be evaluated at testing the binding of the fragment (its cell binding domain) used in the present invention to a cell using a known method. For example, the method mentioned above includes a method of Williams D. A. et al. [Nature, 352, 438-441 (1991)]. The method is a method for determining the binding of a cell to an immobilized fragment in a culture dish. In addition, the heparin binding activity can be evaluated by assaying the binding of the fragment (its heparin binding domain) used in the present invention to heparin using a known method. For example, binding of the fragment to heparin can be evaluated in the same way when using heparin, for example, a labeled heparin in place of the cell in the aforementioned method of Williams D. A. et al. In addition, the fibronectin fragment is exemplified by a polypeptide selected from the group consisting of C-274 (amino acid sequence shown in SEQ ID? O: 9 of the Sequence Listing), H-271 (amino acid sequence shown in FIG. SEQ ID? O: 10 of the Sequence Listing), H-296 (amino acid sequence shown in SEQ ID? O: 11 of the Sequence Listing), CH-271 (amino acid sequence shown in SEQ ID? O: 12 of Sequence Listing), CH-296 (amino acid sequence shown in SEQ ID NO: 13 of the Sequence Listing), C-CS1 (amino acid sequence shown in SEQ ID NO: 14 of Sequence Listing) and CH-296Na (amino acid sequence shown in SEQ ID NO: 15 of the Sequence Listing). Each of the above-mentioned fragments CH-271, CH-296, CH-296Na, C-274 and C-CS1 is a polypeptide having a cell binding domain with a binding activity to VLA-5. Also, C-CS1, H-296, CH-296 and CH-296Na are polypeptides having CS-1 with a binding activity to VLA-4. In addition, H-271, H-296, CH-271, CH-296 and CH-296Na are polypeptides that have a heparin binding domain. At this point, CH-296Na is a polypeptide that contains a region of the cell-binding domain to plasma derived fibronectin CS-1. In the present invention, a fragment can also be used in which each of the domains i above is modified. The heparin binding domain of fibronectin consists of three type III sequences (111-12, 111-13 and 111-14). A fragment containing a heparin binding domain having a deletion of one or two of the above type III sequences can also be used in the present invention. For example, the fragments can be exemplified by CHV-89 (amino acid sequence shown in SEQ ID NO: 16 of the Sequence Listing), CHV-90 (amino acid sequence shown in SEQ ID NO: 17 of the Sequence Listing) and CHV-92 (amino acid sequence shown in SEQ ID NO: 18 of the Sequence Listing), which are fragments in which a fibronectin cell binding site (binding domain to VLA-5: Prol239 to Serl515) and one of the two type III sequences are linked or CHV-179 (amino acid sequence shown in SEQ ID NO: 19 of Sequence Listing) and CHV-181 (amino acid sequence shown in SEQ ID NO: 20 of the Sequence List), which are fragments in which the fibronectin cell binding site and two of the type III sequences are link CHV-89, CHV-90 and CHV-92 contain 111-13, 111-14 and 111-12, respectively, and CHV-179 contains 111-13 and 111-14 and CHV-181 contains 111-12 and 111-13 , respectively. In addition, a fragment having an addition of an additional amino acid to each of the fragments mentioned above can be used in the present invention. For example, the fragment can be prepared by the addition of a desired amino acid to each of the fragments mentioned above. For example, H-275-Cys (amino acid sequence shown in SEQ ID NO: 21 of the Sequence Listing) is a fragment having a heparin binding domain of fibronectin and a cysteine residue in a G terminal.

The fragment used in the present invention can be one of those that contain a polypeptide comprising an amino acid sequence having a substitution, deletion, insertion or addition of one or plural number of amino acids in an amino acid sequence of a polypeptide that constitutes the fragment containing at least a part of the amino acid sequence of the naturally occurring fibronectin exemplified above, wherein the polypeptide has a function equivalent to that of the fragment, provided that the desired effects of the present invention are obtained. It is preferable that the substitution or the like of the amino acids be carried out to a degree that can change the physicochemical and similar characteristics of a polypeptide within the range in which the inherent function of the polypeptide can be maintained. For example, it is preferable that substitution or the like of the amino acids be conservative within the range in which the characteristics inherently possessed by the polypeptide are not substantially changed (eg, hydrophobicity, hydrophilicity, electrical charge, pK and the like). For example, it is preferable that the substitution of amino acids be substitutions within each of the groups of: 1. glycine, alanine; 2. valine, isoleucine, leucine; 3. Aspartic acid, glutamic acid, asparagine, glutamine; 4. serine, threonine; 5. lysine, arginine; 6. phenylalanine, tyrosine and that the deletion, addition or insertion of amino acids are the deletion, addition or insertion in amino acids that have characteristics similar to the characteristics of the vicinity of the target site in the polypeptide 'within the range in which they are not changed substantially the characteristics of the vicinity of the target site. At this point, when the fragment used in the present invention has been obtained by means of a genetic engineering technique, in a case where, for example, the polypeptide is prepared using Escherichia coli or the like as host, the methionine in an N-terminus is sometimes suppressed by the effect of methionine peptidase or the like, derived from Escherichia coli, and the polypeptide mentioned above can also be used in the present invention. In other words, a polypeptide having a methionine deletion at an N-terminus of the polypeptides shown in SEQ ID NOs: I 15 and 21 of the Sequence Listing may also be preferably used in the present invention. Substitution or the like of amino acids can be one of those of natural origin that are caused by a difference between species or individuals or it can be one of those artificially induced. The artificial induction can be carried out by means of a known method and is not particularly limited. Artificial induction can be carried out by preparing, for example, a given nucleic acid having a substitution, deletion, addition or insertion of one or a plural number of nucleotides in the nucleic acid encoding the aforementioned region and the given fragment. fibronectin derivative of natural origin, according to a known method, and using the nucleic acid, whereby a polypeptide can be prepared which contains an amino acid sequence having a substitution or the like in the amino acid sequence of the polypeptide constituting the fragments and the like, which have a function equivalent to that of the region mentioned above and the given fragment derived from fibronectin of natural origin. In addition, the phrase "having an equivalent function" refers herein to a portion of the T cells expressing CD45RA and expressing at least one selected from the group consisting of CD62L, CCR7, CD27 and CD28 in a population of T cells obtained when using a polypeptide is higher than that in a population of T cells obtained in the absence of the polypeptide, which is a comparative control. The action mentioned above can be appropriately confirmed according to the method and the like described in Examples 1, 2 and 6 discussed below. In addition, as the fragment comprising a polypeptide having the substitution or the like of the amino acids, a fragment having a cell adhesion activity and / or a heparin binding activity is preferred and a fragment having a heparin domain is also preferred. CS-1 The activity of cell adhesion and heparin binding activity can be evaluated according to the methods mentioned above to determine those activities. As the fragment comprising a polypeptide having the substitution or the like of the amino acids, for example, a fragment having one or more amino acids inserted as a linker, between two different domains can also be used in the present invention. Coincidentally, like fibronectin, similarly to the fragment mentioned above, a polypeptide having an amino acid sequence having a substitution, deletion, insertion or addition of one or plural number of amino acids in an amino acid sequence can be used in the present invention. of the fibronectin polypeptide, wherein the ratio of T cells expressing CD45RA and expressing at least one selected from the group consisting of CD62L, CCR7, CD27 and CD28 in a population of T cells obtained by using the polypeptide is higher than that in a population of T cells obtained in the absence of the polypeptide, which is a comparative control. In addition, as the fibronectin or fragment thereof used in the present invention, as long as the desired effects of the present invention are obtained, a polypeptide having a homology of 50% or more, preferably a polypeptide having a homology of 70% or more, more preferably a polypeptide having a homology of, 90% or more and further more preferably a peptide having a homology of 95% or more, with the amino acid sequence of a polypeptide constituting fibronectin or the fragment thereof, wherein the polypeptide constituting the fibronectin or fragment thereof has a function equivalent to that of fibronectin of natural origin or the fragment containing at least a part of the amino acid sequence of the fibronectin. same as exemplified above. Incidentally, for example, DNASIS Pro Ver.2.6 (manufactured by TAKARA BIO INC. Can be used for the calculation of homology) Incidentally, the fibronectin fragment used much more preferably in the present invention includes a polypeptide which contains amino acid sequence the whole of III-8 (amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing), III-9 (amino acid sequence shown in SEQ ID NO: 2 of the Sequence Listing), 111- 10 (amino acid sequence shown in SEQ ID NO: 3 of the Sequence Listing), III-12 (amino acid sequence shown in SEQ ID NO: 5 of the Sequence Listing), 111-13 (amino acid sequence shown in FIG. SEQ ID NO: 6 of the Sequence Listing), III-14 (amino acid sequence shown in SEQ ID NO: 7 of the Sequence Listing) and CS-1 (amino acid sequence shown in SEQ ID NO: 8 of the List of Sequences), in other words, a polypeptide containing a heparin binding domain, a cell-binding domain and CS-1 and more preferably, the fibronectin fragment includes the CH-296 mentioned above or a polypeptide comprising a sequence of amino acids that has a substitution, deletion, insertion or adding one or the plural number of amino acids in an amino acid sequence of the polypeptide that constitutes the fragment, which has a function equivalent to that of CH-296. Another fibronectin polypeptide preferably used in the present invention includes H-296, CH-271, H-271 and C-CS1, as shown in Example 18 or 'polypeptides comprising an amino acid sequence having a substitution, deletion, insertion or addition of one or the plural number of amino acids in an amino acid sequence of the polypeptide that constitutes the fragment, having a function equivalent to that of those polypeptides. The fibronectin fragment described herein can also be prepared as a fragment of recombinant fibronectin from a genetic recombinant based on, for example, the specification of US Patent Specification No. 5,198,423. For example, each of the fragments mentioned above of H-271 (SEQ ID NO: 10), H-296 (SEQ ID NO: 11), CH-271 (SEQ ID NO: 12) and CH-296 (SEQ ID NO: 13) and a method for obtaining these fragments are described in detail in the specification of this patent. In addition, CH-296Na (SEQ ID NO: 15) and the method of preparation thereof are described in WO 2005/019450. In addition, the fragment C-274 (SEQ ID NO: 9) mentioned above can be obtained according to the method described in the US Patent Specification No. 5,102,988. In addition, the C-CSl fragment (SEQ ID NO: 14) can be obtained according to the method described in the specification of the Patent Gazette Japanese No. 3104178. Each of the aforementioned fragments of CHV-89 (SEQ ID NO: 16), CHV-90 (SEQ ID NO: 17) or CHV-179 (SEQ ID NO: 19) can be obtained according to the method described in the specification of Japanese Patent Gazette No. 2729712. In addition, the CHV-181 fragment (SEQ ID NO: 20) can be obtained according to the method described in WO 97/18318. The CHV-92 fragment (SEQ ID NO: 18) can be obtained by means of a genetic engineering technique using a plasmid constructed in a conventional manner based on the plasmid described in the bibliographies by reference to the specification of the Japanese Patent Gazette No. 2729712 and WO 97/18318. These fragments or fragments which can be derived from these fragments in a conventional manner can be prepared by using microorganisms deposited in the International Patent Organism Depository, National Institute of Advanced Industrial I Science and Technology, Central Tsukuba 6, 1-1, Higashi 1 - i chome, Tsukuba-shi, Ibaraki-ken, Japan (zip code 305-8566) under the following access numbers or can also be prepared by modifying a plasmid carried in each organism according to a known method. FERM BP-2264 (Escherichia coli carrying a plasmid encoding H-271, Deposit Date: January 30, 1989); FERM BP-2800 (Escherichia coli carrying a plasmid encoding CH-296, Deposit Date: May 12, 1989); FERM BP-2799 (Escherichia coli carrying a plasmid that codes CH-271, Deposit date: May 12, 1989); FERM BP-7420 (Escherichia coli carrying a plasmid encoding H-296, Deposit date: May 12, 1989); FERM BP-1915 (Escherichia coli carrying a plasmid encoding C-274, Deposit date: June 17, 1988); FERM BP-5723 (Escherichia coli carrying a plasmid encoding C-CSl, Deposit date: March 5, 1990); FERM BP-10073 (plasmid encoding CH-296Na, Deposit date: July 23, 2004); FERM P-12182 (Escherichia coli carrying a plasmid encoding CHV-89, Deposit date: April 8, 1991); and FERM P-12183 (Escherichia coli carrying a plasmid encoding CHV-179, Deposit date: April 8, 1991). Since fibronectin is a gigantic glycoprotein, it is not necessarily easy in industrial form and in the manufacture of the drug to prepare and use a protein of natural origin. In addition, since fibronectin is a multifunctional protein, some disadvantages caused by a region different from the region that exhibits the effect by the method of the present invention may be considered depending on the conditions of its use. For these reasons, it is preferable that a fragment of fibronectin and more preferably a fragment of recombinant fibronectin obtained as described above, is preferably used herein. invention, from the point of view of availability, ease of handling and security. Furthermore, it is preferable to use the fragment of fibronectin mentioned above, from the point of view of making a high-expansion fold. In addition, the molecular weight of the fibronectin fragment used in the present invention is not particularly limited and is preferably from 1 to 200, kD, more preferably from 5 to 190 kD and even more preferably from 10 to 180 kD. The molecular weight can be determined, for example, by means of SDS-polyacrylamide gel electrophoresis. At this point, in the amino acid sequence of the polypeptide constituting the fibronectin fragment of the present invention, the part of the amino acid sequence different from that of the amino acid sequence of the polypeptide constituting a fibronectin fragment of natural origin is arbitrary and is not particularly limited, as long as the display of the desired effects of the present invention is not inhibited. (2) Method for Preparing the T Cell Population The method for preparing a population of T cells of the present invention will be explained concretely later in this document. The present invention is a method for preparing a population of cells that contains in a high ratio T cells which express CD45RA and which express at least one selected from the group consisting of CD62L, CCR7, CD27 and CD28 and preferably T cells which express CD45RA and which express at least one selected from the group. group consisting of CD62L and CCR7. The method of the present invention is characterized in that the method includes the step of culturing a population of cells containing T cells, in the presence of the aforementioned fibronectin, a fragment 1 thereof or a mixture thereof. CD45RA, CD62L,? CR7, CD27 and CD28 are all markers of lymphocyte cell surface antigens and are known to be expressed in undifferentiated cells such as native T cells. In other words, the T cells which express CD45RA and which express at least one of the group consisting of CD62L, CCR7, CD27 and CD28, contained in a high ratio in a population of cells obtained by means of the preparation method of the present invention can be classified into undifferentiated cells prior to differentiation into memory T cells, ie, natural T cells, in view of a marker phenotype of cell surface antigens. As described in the Publications that are not patents 4 and 5 mentioned previously, it is described that native T cells have a high survival rate, high cell proliferation effect, high accumulation effect for a tumor, high productivity of tumor-specific effector cells, in a living body, when native T cells are they administer to the living body and that the native T cells are useful in the field of cell therapy. In addition, as shown in Example 3 set forth below, the population of T cells obtained by means of the preparation method of the present invention is converted into activated T cells that produce a large amount of IL-2, by stimulating the cell population T for an anti-CD3 antibody or an anti-CD28 antibody. Also, as shown in Example 4 set forth below, since the population of T cells obtained by means of the preparation method of the present invention shows a chemotaxis in response to CCL21 which is a chemokine and the T cell population also has the ability to migrate to the lymph node. In addition, as shown in Examples 5 to 8 discussed below, the stimulation of antigens is applied to the population of T cells, whereby CTLs having specific cytotoxic activities for antigens are induced. In addition, as shown in Example 9 set forth below, since the population of T cells shows a high Viability in the presence of a small amount of IL-2 or the absence of it, compared to a population of T cells prepared in the absence of fibronectin, a fragment thereof or a mixture thereof, it is expected that the population of T cells also show a high viability in a living body. In fact, Example 11 set forth below shows that in a case where the population of T cells obtained by the method of preparation of the present invention is administered to a NOD / scid mouse, as compared to a case that of the cell population T, prepared in the absence of fibronectin, a fragment thereof or a mixture thereof, T cells have a high proportion of graft to the spleen and also have a high percentage of viability. In addition, Example 11 also shows that, since the population of T cells administered also causes a GVHD reaction, cells having high cytotoxic activities are induced. In view of the above, the population of T cells obtained by means of the preparation method of the present invention not only has the phenotype of the cell surface antigen marker, but also a suitable function for use in the field of cell therapy , possessed by native T cells. However, surprisingly, as shown in item (4) of Example 7, item (2) of Example 8, point (4) of Example 15 and point (2) of Example 16, the CTLs induced from a population of T cells obtained by further clamping the population of T cells prepared according to the aforementioned method to the separation step of a population of T cells. cells expressing at least one selected from the group consisting of CD45RA, CD62L, CCR7, CD27 and CD28 mentioned below have high cytotoxic activities and also have high abilities to recognize a specific antigen, compared to those of native T cells obtained from PBMCs and T cells showing the similar phenotype obtained in the absence of the effective ingredient in the present invention. In other words, the population of T cells obtained by means of the separation methods mentioned above has a significantly high cytotoxic activity with differentiation in CTLs, in comparison with those of normal native T cells. In this regard, the T-cell population mentioned above is a population of cells containing novel natural T-cells that have more effective characteristics, which are different from native T cells. The high therapeutic effects of the T-cell population obtained by means of the aforementioned preparation method of the present invention can be similarly expect that both CD8 + and CD4 + cells be displayed in natural T cells. Furthermore, as described in Patent Publication 2 mentioned above, the expansion of T cells is carried out in the presence of fibronectin, a fragment thereof or a mixture thereof, whereby a proliferation rate can be realized very high. Therefore, the population of T cells obtained by means of the method of the present invention is highly suitable for use in the field of cell therapy. The population of cells containing T cells used in the method of preparation of the present invention is exemplified1 by PBMCs, native T cells, memory T cells, hemopoietic stem cells, umbilical cord blood mononuclear cells and the like. In addition, as long as the cells are hematocytes, the cells can be used in the present invention. Any of these cells which are harvested from a living body can be used or cells can be used directly which are obtained via an ex vivo culture, for example, from the population of T cells obtained by means of the method of present invention or those which have been subjected to frozen storage can be used. In addition, for example, a population of cells obtained via of various methods of separating the cells used in the preparation of the aforementioned T cell population obtained from a living body, for example, any population of cells obtained by separating cells such as PBMCs from CD8 + cells or CD4 + cells. Occasionally, in the method for preparing a population of T cells of the present invention, a material can be used which contains the aforementioned cells, e.g., blood such as peripheral blood or umbilical cord blood; one obtained by removing components such as erythrocytes and blood plasma; a bone marrow fluid and the like. It is preferable that the method for preparing a population of T cells of the present invention be carried out on total culture days of preferably four to 14 days. In other words, when the total culture days are from 4 to 14 days, a ratio of the T cells which express CD45RA and which express at least one of the group consisting of CD62L, CCR7, CD27 and CD28 is high. in the resulting T cell population, which makes them highly suitable for use in the field of cell therapy. Occasionally, when the total culture days are less than 4 days, the satisfactory number of cells can not be obtained for use in general immunotherapy. In the present invention, the total cultivation days' are more preferably from 5 to 14 days and even more preferably from 7 to 14 days. In the method for preparing a population of T cells of the present invention, it is preferable that the culture be carried out, in the presence of the effective ingredient in the present invention, particularly preferably, at least in an early stage I of the period. of full culture. It is preferable that the culture in the presence of the effective ingredient in the present invention is carried out more preferably at least at the start of the culture. The culture in the presence of the active ingredient in the present invention can be carried out during the entire period of the culture period or during any part of the period. In other words, the present invention comprises those modalities which include the step mentioned above in a part of the steps for preparing the T cells. It is preferable that the culture in the presence of the effective ingredient in the present invention is carried out during one day or more, more preferably for 3 days or more and even more preferably for 4 days or more, from the start of the culture. In the present invention, the concentration of fibronectin, or a fragment thereof or a mixture thereof during cultivation is not particularly limited and it is, for example, preferably from 0.001 to 500 μg / mL and particularly preferably from 0.01 to 500 μg / mL. In the present invention, it is preferable that the culture in the presence of fibronectin, a fragment thereof or a mixture thereof is carried out in the presence of a CD3 ligand, from the point of view of effectively stimulating a complex of TCR-CD3 with T cells to proliferate the cells. In the present invention, a CD3 ligand is not particularly limited, as long as a substance has a CD3 binding activity and is exemplified by, for example, an anti-CD3 antibody, particularly preferably by an anti-CD4 monoclonal antibody. CD3 and, for example, OKT3. The concentration of a CD3 ligand in the medium is not particularly limited. For example, in a case where the anti-CD3 monoclonal antibody is used, the concentration is, for example, preferably 0.001 to 100 μg / mL and particularly preferably 0.01 to 100 μg / mL. Furthermore, in the present invention, another co-stimulation factor such as a CD28 ligand can be added to introduce the co-stimulation as the occasion demands. For example, an anti-CD28 antibody, CD80, B7-1, B7-2 and the like are exemplified. The means used in the method to prepare a The T cell population of the present invention is not particularly limited and a known medium can be used which is prepared by mixing components necessary to expand T cells. For example, a commercially available medium can be appropriately selected for use. These media may contain cytokines, appropriate proteins and other components in addition to the inherent constituents. Cytokines are exemplified by, for example, IL-2, IL-7, IL-12, IFN-? and the like, preferably a medium containing IL-2 is used. The concentration of IL-2 in the medium is not particularly limited and is, for example, preferably 0.01 to 1 x 105 U / mL and more preferably 0.1 to 1 x 104 U / mL. Also, appropriate proteins are exemplified, for example, by an anti-IL-4 antibody. Also, in addition to the above, a factor of: lymphocyte stimulation such as lectin can be added. The concentration of the component in the medium is not particularly limited, as long as the desired effects can be obtained. In addition, in the culture, serum and plasma can also be added to the medium. The amounts of serum and plasma added to the medium are not particularly limited and are exemplified by from 0% by volume to 20% by volume and the amounts of serum and plasma used can be changed. depending on the stage of, cultivation. For example, serum and plasma can be used by gradually decreasing the concentration of the same. Occasionally, the origin of the serum or plasma may be either autologous (meaning the origin is the same as that of the cultured cell) or non-autologous (meaning that the origin is different from that of the cultured cell). Preferably, autologous serum or plasma can be used, from a safety point of view. [Preparation of a population of T cells of the present invention is usually carried out in a medium containing given components in the presence of the effective ingredient mentioned above in the present invention. The number of cells at the start of the culture used in the present invention is not particularly limited and is exemplified, for example, by preferably from 1 cellulose / mL to 1 × 10 8 cells / mL, more preferably from 1 cell / mL to 5 × 10 7. cells / mL, even more preferably from 1 cell / mL to 2 x 10 7 cells / mL. In addition, culture conditions are not particularly limited and the usual conditions I used for cell culture can be employed. For example, the cells can be cultured under the conditions of 37 ° C in the presence of 5% C02 and, similar. In addition, the medium can be diluted by adding a fresh medium to the solution of cell culture, the medium can be exchanged or the cell culture equipment can be exchanged, at appropriate intervals. The cell culture equipment used in the method for preparing a T-cell population of the present invention is not particularly limited and can be used, for example, a Petri dish, flask, bag, large culture bath, bioreactor and Similar. At this point, as a bag, a C02 gas permeable bag I can be used for cell culture. In addition, in a case where T cells are mass produced industrially, a large culture tank I can be used. In addition, cultivation can be carried out in either an open system or a closed system. Preferably, the culture is carried out in a closed system, from the point of view of the safety of the resulting T cells. Occasionally, the effective ingredient in the present invention, a CD3 ligand, another co-stimulation factor and appropriate proteins, cytokines and other components, contained in the aforementioned medium, can be dissolved to be co-present or the above components can immobilize in an appropriate solid phase, for example, a cell culture equipment of I cells (which includes any of those of a system open and a closed system), such as a Petri dish, a flask or a bag or in a cell culture carrier such as beads, a membrane or a glass slide and can be used. The materials for those solid phases are not particularly limited, as long as the materials can be used for cell culture. When the components are immobilized in, for example, the equipment mentioned above, it is preferable to immobilize a given amount of each component based on the amount of medium that is placed in the equipment so that, with the placement of the medium in the equipment, the medium is contained in a proportion similar to that of a desired concentration in the case where the components dissolve in the medium and are used. The amount of the immobilized components is not particularly limited, as long as the desired effects can be obtained. The carrier mentioned above is used when immersing the carrier in a culture medium in the cell culture equipment during cell culture. When the aforementioned components are immobilized in the carrier mentioned above, it is preferable to immobilize a given amount of each component based on the amount of medium placed in the equipment so that the medium is contained in a proportion similar to that of a desired concentration. with the placement of the carrier in the middle, in the case where the components dissolve in the middle and are used. - The amount of immobilized components is not particularly limited, as long as the desired effects can be obtained. A method for immobilizing the effective ingredient in the present invention, a CD3 ligand and another co-stimulation factor1 in the solid phase is not particularly limited. For example, the substances I mentioned above can be immobilized by bringing them into contact with the solid faise in an appropriate buffer solution. In addition, with respect to immobilization of the fibronectin fragment in the solid phase, immobilization can also be carried out according to the methods described in WO 97/18318 and WO 00/09168. ! Once the various components mentioned above or the effective ingredient in the present invention are immobilized in the solid phase, the population of T cells can be easily separated from the effective ingredient or the like only by separating the population of T cells from the solid phase after that the population of T cells is obtained by means of the method of the present invention, so that the contamination of the population of T cells with the effective ingredient or the like is can prevent Also, the method of preparation of the present invention may additionally include the step of separating a population of T cells expressing at least one selected from the group consisting of CD45RA, CD62L, CCR7, CD27 and CD28, from the population of T cells obtained by means of a culture in the presence of the effective ingredient in the present invention. In other words, the population of T cells obtained by culturing in the presence of the effective ingredient in the present invention as described above contains in a high ratio T cells which express I CD45RA and which express at least one selected from the group consisting of CD62L, CCR7, CD27 and CD28. Therefore, methods for separating cells expressing I at least one surface antigen marker selected from the group consisting of CD45RA, CD62L, CCR7, CD27 and CD28 are additionally carried out and thereby the natural T cells or a population of T cells containing even more natural type T cells can be obtained more effectively. In this case, the T cells to be separated are not particularly limited and are exemplified, for example, by cells expressing CD45RA and are preferably exemplified by cells expressing CD45RA and expressing at least one selected from the group consisting of CD62L, CCR7, CD27 and CD28 and more preferably cells expressing CD45RA and CD62L and cells expressing CD45RA and CCR7. The separation methods are not particularly limited and the separation can be carried out according to a known method, for example, by the use of a cell sorter, magnetic beads, column or the like. For example, when cells expressing CD45RA and CCR7 are separated, separation can be carried out as described in item (3) of Example 3 set forth below. In addition, the T cells prepared by means of the method of the present invention are cloned, whereby the T cells can be maintained as stable T cells. Also, the population of T cells is further cultured according to the method of the present invention or a known method, using the population of T cells obtained by means of the method of the present invention, whereby a population can be obtained recently. of T cells. In addition, the stimulation of antigens or the like is applied according to a known method, for example, in the same manner as Examples 5 to 8 discussed below, using the population of T cells obtained by means of the method of present invention, whereby specific CTLs can be prepared for antigens. The population of T cells obtained by means of the method of the present invention contains in a high ratio T cells which express CD45RA and which express at least one T cell selected from the group consisting of CD62L, CCR7, CD27 and CD28, which are cells T undifferentiated as mentioned above and the T cell population also contains T cells that have cytotoxic activities depending on the class of cells used in the culture. The cytotoxic activity of the T-cell population can also be assessed by means of a test known in vi tro. Since the population of T cells obtained by means of the present invention contains in a high ratio undifferentiated natural T-cells, as mentioned above, the T-cell population obtained by means of the preparation method of the present invention does not necessarily shows a high cytotoxic activity in the previous evaluation system. Diseases for which the T cell population prepared by the method of the present invention is administered are not particularly limited and are exemplified, for example, by cancer, leukemia, malignant tumor, hepatitis and infectious diseases caused by a virus such as influenza or HIV, a bacteria or a fungus, for example tuberculosis, MRSA, VRE and deeply established mycosis. In addition, when a foreign gene is further transduced into it as described below, the effects can also be expected for various genetic diseases. The population of T cells prepared by means of the method of the present invention can also be used for the infusion of donor lymphocytes and the like for the purpose of preventing an infectious disease after bone marrow transplantation or X-ray irradiation, and the remission of recurrent leukemia. In addition, the present invention provides a population of T cells obtained by means of the method for the preparation of a population of T cells of the present invention mentioned above, wherein the population of T cells expresses CD45RA and expresses at least one selected from the group which [consists of CD62L, CCR67, CD27 and CD28. In addition, the present invention provides a medicament (therapeutic agent) that contains the population of T cells as an effective ingredient. The aforementioned therapeutic agent containing the T-cell population is suitably used in immunotherapy. In immunotherapy, T cells suitable for treating a patient are administered to the patient, for example, by means of a method of administration such as intravenous, intra-arterial, subcutaneous or intraperitoneal by injection or drip. The therapeutic agent is very useful for use in the diseases mentioned above and the infusion of donor lymphocytes. The therapeutic agent can be prepared as drops or as an injection, for example, by combining the population of T cells prepared by means of the method of the present invention as an effective ingredient with, for example, a known organic or inorganic carrier which is suitable for parenteral administration, an excipient, a stabilizing agent and the like, according to a method known in the pharmaceutical field. Incidentally, the amount of the T cell population of the present invention contained in the therapeutic agent, the dose of the therapeutic agent and the conditions for the therapeutic agent can be appropriately determined according to the known immunotherapy. For example, the amount of the population of the T cells of the present invention contained in the medicament is not particularly limited and, for example, is exemplified by preferably 1 x 10 to 1 x 10 cells / mL, more preferably 1 x 10 to 1 x 10 cells / mL and even more preferably from 1 x 10 to 1 x 109 cells / mL. Also, the dose of the medicament of the present invention is not particularly limited and is exemplified, for example, preferably from 1 x 106 to 1 x 1012 cells / day, more preferably from 1 x 107 to 5 x 1011 cells / day and even more preferably from 1 x 108 to 2 x 1111 cells / day, per adult per day. In addition, an immunotherapy by the therapeutic agent can be used in combination with a pharmacotherapy or known radiotherapy by means of the administration of a drug or a therapy by means of surgery. The method for preparing a population of T cells of the present invention may further include the step of transducing a foreign gene into T cells. In other words, one embodiment of the present invention provides a method for preparing a population of T cells. which also includes the step of transducing a foreign gene into T cells. At this point, the term "foreign gene" means a gene which is artificially transduced into T cells within which a gene must be transduced and also comprises a gene derived from the same species as that from which the T cells are derived in which a gene must be transduced. By carrying out the method for preparing the T cells of the present invention, the ability for proliferation of cultured T cells is improved. By combining the method for preparing the T cells of the present invention with the step of transducing a gene, expects an increase in the efficiency of gene transduction. A means for transducing a foreign gene is not particularly limited and an appropriate method can be selected from known methods for transducing a gene to be used. The step of transducing a gene can be carried out at any given point during the preparation of a population of T cells. For example, it is preferable to carry out the step simultaneously with or during the course of the aforementioned preparation of the T cell population or after passage, from the I work efficiency point of view. As the method! mentioned above for transducing a gene, any of the methods that use a viral vector and methods without the use of the vector can be employed in the present invention. The details of these methods have already been published in numerous bibliographies. The viral vector mentioned above is not particularly limited and a known viral vector I is used which is ordinarily used in the method for transducing a gene, for example, a retroviral vector, lentiviral vector, adenoviral vector, adeno-associated viral vector , simian viral vector, vaccinia viral vector, sendai viral vector or similar. By way of particularly preferable, as the viral vector is used a retroviral vector, adenoviral vector, viral and adeno-associated vector, lentiviral vector or simian viral vector.

As the viral vector mentioned above, those lacking replication ability are preferable so that the viral vector can not self-replicate in an infected cell. In addition, with transduction of the gene, a substance can also be used which improves the efficiency of gene transduction, such as RetroNectin ™ (registered trademark, I manufactured by TAKARA BIO INC). The retroviral vector and the lentiviral vector are used for the purpose of gene therapy or the like because a foreign gene inserted into the chromosomal DNA can not be stably incorporated into the cell in which the vectors must be transduced. . Since I vectors have a high infection efficiency for cells during division and proliferation, gene transduction is preferably carried out in the preparation step in the present invention. As the method for transducing a gene without using a viral vector, for example, a method using a carrier such as a liposome or ligand-polylysine, calcium phosphate method, electroporation method, method of gun can be used. gene or the like without limit the present invention to them. In this case, a foreign gene incorporated into a plasmid DNA, linear DNA or RNA is transduced. The foreign gene to be transduced in the T cells in the present invention is not particularly limited and any gene which is desirable to be transduced in the cells mentioned above can be selected. As the gene described above, in addition to a gene encoding a protein (e.g., enzymes, cytokines, receptors or the like), for example, a gene encoding an antisense nucleic acid, siRNA (small interfering RNA) or a ribozyme In addition, an appropriate marker gene can be transduced simultaneously which allows the selection of cells in which a gene is transduced. The foreign gene mentioned above can be inserted, for example, into a vector, a plasmid or the like, to be expressed under the control of an appropriate promoter and can be used. In addition, in order to achieve efficient transcription of a gene, other regulatory elements may exist in a vector which cooperate with a promoter or a transcription initiation site, for example, an enhancer sequence or a terminator sequence. In addition, for the purpose of inserting a foreign gene into a chromosome of T cells in which it must be introducing the gene by means of homologous recombination for example, a foreign gene can be ordered between the flanking sequences comprising nucleotide sequences each having homology to the nucleotide sequences located on both sides of the desired target insertion site of the gene in the chromosome The strange gene to be transduced can be one that is of natural origin or can be artificially generated or it can be one - in which the molecules! of DNA having different origins from each other are linked by a known means such as ligation. In addition, the foreign gene may be one that has a sequence in which a mutation is introduced into a sequence of natural origin depending on its purpose, In accordance with the method of the present invention, for example, a gene encoding an associated enzyme with the resistance! A drug used for the treatment of a patient with cancer or the like can be transduced into T cells, providing the T cells with which resistance to a drug. If the T cells described above are used, immunotherapy and drug therapy can be used in combination and, therefore, higher therapeutic effects can be obtained. The drug resistance gene is exemplified, for example, by a gene for multidrug resistance. On the other hand, the reverse of the modality mentioned above, a gene for providing sensitivity to a particular drug can be transduced into T cells, providing the T cells with which i sensitivity to the drug. In this case, the T cells after being transplanted into a living body can be removed when administering the drug. The gene for providing sensitivity to a drug is exemplified, for example, by a thymidine kinase gene. Other genes to be transduced are exemplified by a gene encoding a TCR that recognizes an antigen on the surface of target cells and a gene encoding a chimeric receptor that has an antigen recognition site of an antibody to the surface antigen of target cells and containing an intracellular region of TCR (CD3 or similar). The present invention also provides a method for treating or preventing a disease, including the step of administering to a subject an effective amount of the population of T cells obtained by means of the aforementioned method. The term "subject" as used in this document is not particularly limited and preferably refers to a patient with the disease described above, to which the population of T cells prepared by means of the method of the present invention is administered.

In addition, the term "effective amount" as used herein is an amount of the above-mentioned population of T cells that exhibits a therapeutic or prophylactic effect, in a case where the population of T cells is administered to the subject mentioned above, in comparison with a subject to whom the T-cell population is not administered. The specific effective amount is appropriately established depending on its form of administration, method of administration, purpose of use and age, weight, symptom or the like of a subject and is not constant. The population of T cells can preferably be administered in the same effective amount as the aforementioned medicament. The method of administration is also not limited. For example, the population of T cells can be administered by means of drip, injection or the like, in the same manner as in the case of the aforementioned medicament. In addition, the present invention also provides for the use of the T cell population mentioned above in the manufacture of a medicament. The method for manufacturing the medicament is carried out in the same way as that for the aforementioned medicament. Also, the diseases to which the drug is administered are not particularly limited and are the same as those of the medication mentioned above. In addition, the present invention can prepare a population of activated T cells by stimulating the population of T cells obtained by means of the method of preparation of the present invention mentioned above, wherein the population of T cells expresses CD45RA and expresses at least one selected of the group consisting of CD62L, CCR7, CD27 and CD28, with at least one stimulation factor selected from the group consisting of a cell capable of presenting an antigen, cell that has presented an antigen, antigen, CD3 ligand, CD28 ligand, cytokine, chemokine and cell capable of producing a cytokine. In addition, the present invention provides the population of T cells obtained by means of the aforementioned preparation method. The population of activated T cells targeted as described above can be used as an effective ingredient of a medicament, as with the population of T cells obtained by means of the aforementioned preparation method. At this point, the term "stimulation with a stimulation factor" is not particularly limited, as long as the population of T cells obtained by means of the method of preparation of the present invention mentioned above is activated by a stimulation factor and is exemplified , by example, by carrying out a culture in the coexistence of the population of T cells obtained by means of the method of preparation of the present invention and a stimulation factor. The cell capable of presenting an antigen as used herein is not particularly limited, as long as a cell is generally used as a cell that presents an antigen and is exemplified, for example, by a dendritic cell,? DT cell, monocyte , B cell, T cell, macrophage, fibroblast, Langerhans cell, a population of cells containing at least one cell of the cells mentioned above and which is particularly preferably exemplified by a dendritic cell, dT cell, T cell , B cell, monocyte, macrophage, a population of cells containing at least one cell of the cells mentioned above. Furthermore, the origin of the cell capable of presenting an antigen can be either autologous or non-autologous for a patient to which the cell is administered and is preferably used: an autologous cell. At this point, the cell capable of presenting an antigen as used herein means a cell that is capable of presenting an antigen, but which does not present the antigen. As the cell that has presented an antigen as used in this document, it can be used either cell in which an appropriate antigen has been artificially added to the aforementioned cell capable of presenting the antigen or a cell that already presents an antigen with the harvesting of a living body. The cell that has presented an antigen can also be prepared in the same manner as that described in point (5) of Example 5 set forth below and can be used. At 1 this point, the phrase "cell capable of presenting the antigen" is not included in the meaning of the phrase "cell that has presented an antigen". The antigen as used herein is not particularly limited, so long as the peptide is present in a cell1 that presents an antigen and is recognized by T cells so that the T cells are efficiently activated. For example, the antigen is exemplified by a peptide, glycopeptide, tumor cell extract, product treated with ultrasound of the tumor cell and hydrothermally treated product of tumor cell, virus, bacteria, protein and the like. In addition, the specific examples of a ligand of CD3 and a CD28 ligand are exemplified by a CD3 ligand and a CD28 ligand described above. At this point, the population of T cells which expresses CD45RA and which expresses at least one selected from the group consisting of CD62L, CCR7, CD27 and CD28, obtained by means of the above-mentioned preparation method of the present invention is co-stimulated with an anti-CD3 antibody and an anti-CD28 antibody, as in Example 3 set forth below, whereby a population of activated lymphocytes capable of producing a cytokine. The cytokine as used in this document is not particularly limited, as long as a cytokine can act on! T cells and can activate T cells. For example, la1 cytokine is exemplified by IL-2, IFN- ?, TGF-β, IL-15, IL-7, IFN-a, IL-12, CD40L, IL-27 and the like and is particularly preferably exemplified by IL-2, IFN-? and IL-12, from the point of view of improving a cellular immunity. The chemokine as used herein is not particularly limited, so long as a chemokine acts on T cells and exhibits emigration activity. For example, chemokine is exemplified by RANTES, CCL21, MIP1 a, MIP1 ß, CCL19, CXCL12, IP-10 and MIG. The cell capable of producing a cytokine as used herein is not particularly limited, as long as the cell is capable of producing the aforementioned cytokine. For example, the Thl cell is preferably used, from the viewpoint of improving a cellular immunity.

In addition, the population of T cells obtained by means of the above-mentioned method of preparation of the present invention contains in a high ratio native T-cells that are not differentiated and are not stimulated by an antigen. Therefore, as the stimulation factor used in this document, it is particularly preferable to use a cell capable of presenting the antigen, a cell that has presented an antigen and / or an antigen. For example, as shown in item (5) of Example 5, item (8) of Example 6, item (2) of Example 7, item (1) of Example 8, item (7) of Example 14 and item ( 2) of Example 15 set forth below, stimulation of antigens is provided to the T-cell population obtained by means of the aforementioned preparation method of the present invention, whereby CTLs specific for useful antigens having very high cytotoxic activities can be induced. high and also have high abilities to recognize an antigen. In addition, the culture can be carried out in the presence of the fibronectin mentioned above, a fragment thereof or a mixture thereof and a known component that is used in the culture of T cells, in addition to the aforementioned stimulation factors. . The population of T cells prepared as described above is a very useful population of T cells that have high therapeutic effects. The present invention also provides a method for treating or preventing a disease, including the step of administering to a subject an effective amount of the population of activated T cells obtained by means of the aforementioned method. In addition, the present invention also provides for the use of the population of activated T cells mentioned above in the manufacture of a medicament. A method for manufacturing the medicament is carried out in the same manner as that for the medicament mentioned above. Also, the diseases to which the medicament is administered are not particularly limited and are the same as those of the aforementioned medicament. In addition, the present invention provides a medicament containing: (a) a preparation containing as an effective ingredient the population of T cells obtained by means of the method of preparation of the present invention mentioned above, wherein the population of T cells expresses CD45RA and expresses at least one selected from the group consisting of CD62L, CCR7, CD27 and CD28; and (b) a preparation that contains as an effective ingredient at least one stimulation factor selected from the group consisting of a cell capable of presenting an antigen, cell that has presented an antigen, antigen, CD3 ligand, CD28 ligand, cytokine, chemokine and cell capable of producing a cytokine, wherein the preparations are contained in the medicament as two separate preparations that are administered simultaneously or separately. As mentioned above, the population of T cells obtained by means of the method of the present invention contains in a high ratio natural type T cells. Therefore, the stimulation factors mentioned above are administered simultaneously with or separately from the population of T cells to a patient, whereby the effects of the administered T cell population can be maximally exhibited and therapeutic effects can be exhibited. higher on diseases. At this point, as the stimulation factor, one that can provide the stimulation of antigens is preferable, in other words, one that can be used as a vaccine, for example, at least one selected from the group consisting of a cell capable of present an antigen, cell that has presented an antigen, antigen, CD3 ligand, CD28 ligand, cytokine, chemokine and cell capable of producing a cytokine and a cell capable of presenting an antigen, a cell that has presented an antigen or an antigen is more preferably used in the present invention. A preparation which contains as an effective ingredient the population of T cells obtained by means of the above-mentioned preparation method of the present invention, wherein the T-cell population expresses CD45RA and expresses at a preparation can be prepared and formed in a preparation (a) less one selected from the group consisting of CD62L, CCR7, CD27 and CD28 in the medicament, in the same way as in the case of the medicament containing the T cell population as an effective ingredient as mentioned above. In addition, the content in the medicament, the dose and the manner of administration of the T cell population expressing CD45RA and expressing at least one selected from the group consisting of CD62L, CCR7, CD27 and CD28 are not particularly limited and can they are, for example, the same as those for the medicament containing the population of T cells obtained by means of the method of preparation of the present invention mentioned above. In addition, as (b) a preparation containing as an effective ingredient at least one stimulation factor selected from the group consisting of a cell capable of presenting an antigen, a cell that has presented an antigen, antigen, CD3 ligand, CD28 ligand, cytokine, chemokine and cell capable of producing a cytokine, in the medicament, for example, one formed in a preparation can be used by combining the stimulation factor with an appropriate carrier. Also, as specific examples - of at least one stimulation factor selected from the group consisting of a cell capable of presenting! an antigen, cell that has presented an antigen, antigen, CD3 ligand, CD28 ligand, cytokine, chemokine and cell capable of producing a cytokine, for example, the stimulation factors exemplified above can be used. For example, when a cell capable of presenting an antigen, cell that has presented an antigen, antigen or cell capable of producing a cytokine is used as the stimulation factor, a preparation can be formed by combining the stimulation factor with a pharmaceutical carrier suitable for dripping or injection without particular limitation. In addition, when an antigen peptide I is used, the antigen peptide can be combined with an appropriate adjuvant without particular limitation. Also, when a cytokine or chemokine is used, a preparation can also be formed by incorporating the cytokine or chemokine into a liposome by means of a known method. In addition, as the content of the factor of Stimulation in the preparation can be appropriately selected depending on the kind of stimulation factor used and is not particularly limited. For example, when a cell capable of presenting an antigen, a cell that has presented an antigen or a cell capable of producing a cytokine is used as the stimulation factor, the content is exemplified, for example, by preferably 1 x 103 a 1 x 109 cells / mL, more preferably from 1 x 103 to 1 x 10 8 cells / mL and even more preferably from 1 x 10 4 to 1 x 10 7 cells / mL. In addition, the form of administration of the stimulation factor can be appropriately selected depending on the kind of stimulation factor used and is not particularly limited. For example, the stimulation factor is administered by means of intravenous administration, intra-arterial administration, subcutaneous administration, intraperitoneal administration, oral administration or the like. The dose is not particularly limited, as long as the dose is effective to treat or ameliorate a patient's disease. For example, when a cell capable of presenting an antigen is administered, the dose is exemplified, for example, preferably by 1 x 10 3 to 1 x 10 11 cells / day, more preferably 1 x 10 3 to 1 x 10 10 cells / day and even more preferably from 1 x 104 to 1 x 109 cells / day. As well, When an antigen peptide is administered, the dose is exemplified, for example,! preferably from 0.001 to 100 mg / day, more preferably from 0.003 to 30 mg / day and particularly preferably from 0.01 to 10 mg / day. Incidentally, as a preferable example of administration of preparation (b), for example, it is preferable that a cell capable of presenting an antigen be combined with an antigen to be formed in preparation (d) i and administered. For example, it is preferable that a dendritic cell be combined with an antigen peptide and administered. In this case, the content and dose of the cell capable of presenting an antigen and the antigen in the preparation can be implemented as mentioned above. In addition, the administrations of preparation I (a) and preparation (b) in the medicament are administered simultaneously or separately to a patient. At this point, the term "simultaneously" means that the preparations are administered to a patient as separate preparations simultaneously in terms of time or that preparation (a) and preparation (b) are mixed prior to administration to a patient and administer For example, in a case where the preparation (a) and the preparation (b) | are administered as droplets the present invention comprises a modality such that the Previous preparations: are mixed before administration and administered to a patient. In addition, the term "separately" means that preparation (a) and preparation (b) are administered separately in terms of time. The administration intervals of preparation (a) and preparation (b) are not particularly limited, as long as a stimulation of a stimulation factor contained in preparation (b) is provided to a population of T cells contained in the preparation. preparation (a) in the patient's body. Preferably, it is preferable that preparation (b) be administered after the preparation (a) is administered. Also, the number of times of administration is also not particularly limited and each preparation can be administered once or in divided portions of several times. In addition, the present invention also provides a method for treating a disease, which includes the steps consisting of (a) administering to a patient the population I of T cells obtained by means of the method of preparation of the present invention mentioned above, wherein the T cell population expresses CD45RA and expresses at least one selected from the group consisting of CD62L, CCR7, CD27 and CD28 and (b) administering to a patient at least one stimulation factor selected from the group consisting of a capable cell of presenting an antigen, cell that has presented an antigen, antigen, CD3 ligand, CD28 ligand, cytokine, chemokine and cell capable of producing a cytokine. The treatment method can exhibit very high therapeutic effects, as mentioned above. At this point, as the stimulation factor, one that can provide the stimulation of antigens is preferable, in other words, one that can be used as a vaccine, for example, at least one selected from the group consisting of a capable cell of presenting an antigen, cell that has presented an antigen, antigen, CD3 ligand, CD28 ligand, cytokine, chemokine and cell capable of producing a cytokine and a cell capable of presenting, a cell that has presented an antigen or an antigen they more preferably use in the present invention. In the method of treatment, the step consisting of (a) administering to a patient the population of T cells obtained by means of the method of the present invention is not particularly limited and is carried out, for example, by employing the dose and the administration form similar to those in the method of administration of the aforementioned medicament. In addition, the step consisting in (b) administering to a patient at least one stimulation factor selected from the group consisting of a cell capable of presenting an antigen, a cell that has presented an antigen, antigen, CD3 ligand, CD28 ligand, cytokine, chemokine and a cell capable of producing a cytokine is also not present. Particularly limited and can be carried out, for example, by employing the dose and administration form similar to those for the aforementioned medicament. In addition, the ranges of administrations of step (a) and step of (b) mentioned above can be implemented in the same manner as in the case of the medicament containing the preparation of (a) and the preparation of (b) mentioned previously, in other words, they are not particularly limited as long as a stimulation of the stimulation factor administered by the step of; (b) is added to a population of T cells administered by step (a) in the patient's body and can be carried out simultaneously or separately. Preferably,; it is desirable that the administration step of (b) be carried out after the administration step of (a). Also, the number of times of administration is also not particularly limited and each preparation can be administered once or in divided portions of several times.

EXAMPLES The present invention will be described more specifically below in this document by means of the Examples, without intending to limit the scope of the present invention to them. Example 1: Analysis of CD45RA + CD62L * T cells (1) Isolation and Storage of PBMCs The blood component was collected from a normal human individual donor, obtained with informed consent. The collected blood component was diluted twice with phosphate buffered saline (hereinafter referred to as "PBS"), was placed on a Ficoll ™ plate (manufactured by Amersham Bioscience) and centrifuged at 600 x g for 20 minutes. Peripheral blood mononuclear cells (hereinafter referred to as "PBMCs") in the intermediate layer were collected with a pipette and washed. The Collected PBMCs were suspended in a 90% FBS storage solution (manufactured by Cambrex Corporation) / 10% DMSO (manufactured by SIGMA) or a storage solution of an equivalent volume of CP-1 (manufactured by KYOKUTO PHARMACEUTICAL INDUSTRIAL CO ., LTD.) Containing 8% human serum albumin (manufactured by Baxter Limited, hereinafter referred to as "HSA") and RPMI 1640 (manufactured by SIGMA) and 7i It was stored in liquid nitrogen. During expansion of T cells, these stored PBMCs rapidly melted in a 37 ° C water bath and were washed with RPMI 1640 medium containing 10 μg / mL DNase ™ (manufactured by Calbiochem). After, the number of living cells was calculated by means of the trypan blue staining method. The cells were subjected to each experiment. i (2) Immobilization of Human Anti-CD3 Antibody and Fragment CH-296 A human anti-CD3 antibody and a CH-296 fragment were immobilized in a [culture kit used in the following experiment. Specifically, 1.9 mL each of PBS containing a 'human anti-CD3 antibody (manufactured by JANSSEN PHARMACEUTICAL KK) (final concentration: 5 μg / mL) was added to a 12-well cell culture plate (manufactured by Becton Dickinson ). With the addition,] the CH-296 fragment was added to a group with the addition of CH-296 to have a final concentration of 25 μg / mL. After what; These culture kits were incubated at room temperature for 5 hours, the culture equipment was stored at 4 ° C until use. Immediately before the so, the PBS containing the antibody and CH-296 was removed by aspiration of these culture equipment and then each well was washed. twice with PBS and then once with the RPMI 1640 medium and culture kits were attached to each experiment. (3) Expansion of the Population of T Cells The PBMCs which were prepared in item (1) of Example 1 were suspended in AIM-V (manufactured by Invitrogen) containing 1% human AB serum. (manufactured by Cambrex) (hereinafter referred to simply as "AIM-V at 1%"), to have a density of 1 x 106 cells / mL, to prepare a cell suspension. Then, the 1% AIM-V was added to a plate immobilized with the human anti-CD3 antibody or a plate immobilized with the human anti-CD3 antibody and the CH-296, prepared in point (2) of Example 1, in a volume of 2 -mL / each well, and the above cell suspension was added thereto in a volume of 1 mL / each well. The IL-2 (PROLEUKIN, manufactured by Chiron) was added to the same I to have a final concentration of 1000 U / mL and these plates were attached to the culture at 37 ° C in 5% C02 (zero day of the culture). On or after the fourth day from the start of the culture, a subculture was carried out with a totally new 12.5 cm2 cell culture flask. (manufactured by Becton Dickenson) to which nothing was immobilized using 1% AIM-V and IL-2 was added thereto to have a final concentration of 500 U / mL (volume of culture medium: 6 mL). Specifically, the subculture was carried out at a density of 0.05 x 106 cells / mL on the fourth day from the start of the culture, at a density of 0.1 x 106 cells / mL on the seventh day and at a density of 0.15 x 106 cells / mL in the tenth day On the seventh day, the tenth day and the fourteenth day from the start of the culture, the number of living cells was counted by means of the trypan blue staining method and an expansion fold was calculated by comparing the number of cells counted with the number of cells at the start of the culture. The results are shown in Table 1.

Table 1 1 Expansion fold (times) Day of 10 ° Day of 14 ° Day of Cultivation Cultivation Cultivation Control (Without Immobilization of; x 14 x 257 x 2491 CH-296) CH-296 x 38 x 315 X 4387 As shown in Table 1, in the group in which the immobilized culture equipment was used with CH-296 at an early stage of T-cell expansion, the expansion times of the T-cell population were high in any stage of the culture compared to those of the control group. (4) Analysis of CD45RA + CD62L + T Cells at Every i Day of Culture i The cells prepared on each day of culture at point (3) of Example 1 were washed with PBS and then with I PBS containing 1% bovine serum albumin. % (manufactured by SIGMA, hereinafter referred to as "BSA") (hereinafter referred to as "1% BSA / PBS"). Cells were suspended in 1% BSA / PBS and mouse IgGl labeled with FITC / mouse lgGl labeled with RDl / mouse IgGl labeled with PC5 (manufactured by Beckmann Coulter) was added as a negative control. Similarly, the mouse human anti-CD62L antibody labeled with FITC (manufactured by SIGMA) / mouse human anti-CD45RA antibody labeled with RDl (manufactured by Beckmann Coulter) was added. After I added each of the antibodies, the cells were incubated on ice for 30 minutes. After incubation, the cells were washed with 1% BSA / PBS and then resuspended in PBS. The cells were subjected to flow cytometry (Cytomics FCSOOMR, manufactured by I Beckmann Coulter) and a percentage of CD45RA + CD62L + T cells was calculated. Results are shown in table 2.

Table 2; Percentage of CD45RA + CD62L + T Cells (%) 7th Day of 10 ° Day of 14th Day of Cultivation Cultivation Cultivation Control (Without Immobilization! 29.1 39.8 37.8 of CH-296) CH-296 '48 .9 73.8 51.3 As shown in Table 2, in the group in which immobilized culture equipment was used with the CH-296 fragment, the results of the CD45RA + CD62L + T cell population were obtained higher in the T cells during the culture, in comparison with those of the control group in any of the seventh day, the tenth day and the fourteenth day of the culture. It was clarified from these results that T cells can expand while increasing the percentage of the CD45RA + CD62L + T cell population of the T cell population during expansion from days 7 to 14 by the co-existence of the fragment CH-296 at an early stage of expansion. Example 2: Analysis of CD45RA + CCR7 + T Cells (1) Expansion of the T Cell Population The PBMCs which were prepared in point (1) of Example 1 were suspended in AIM-V containing 3% human AB serum (hereinafter referred to simply as "3% AIM-V") to have a density of 1 x 106 cells / mL, to prepare a cell suspension, then the 3% AIM-V is i added to a plate immobilized with the human anti-CD3 antibody or a plate immobilized with the human anti-CD3 antibody and the CH-296, prepared in point (2) of Example 1, in a volume of 2 mL / well and the previous cell suspension was added thereto in a volume of 1 mL / each well IL-2 was added to the same I to have a final concentration of 1000 U / mL and these plates were attached to the culture at 37 ° C in C02 at 5% (zero day of culture) On the fourth day after the start of the culture, each group was diluted with 1% human AB serum AIM-V to have a density of 0.075 x 106 cells / mL (volume of liquid: 6 mL), the dilution was transferred to a 12.5 cm2 cell culture flask to which nothing was immobilized and IL-2 was added thereto for In a final concentration of 500 U / mL. Serum concentration was determined, assuming that the PBMCs obtained from 30 mL of blood collection were cultured in 10 L of a culture medium. The culture was continued and on the seventh day, each group was transferred to a completely new 25 cm cell culture flask (manufactured by Becton Dickenson), which was not immobilized, using a culture medium (volume of liquid: 12.6 mL), prepared by means of of the dilution with 0.05% human AB serum AIM-V for I have a density of 0.25 x 106 cells / mL. To each group IL-2 was added to have a final concentration of 500 U / mL. On the eleventh day, each group was transferred to an entirely new 25 cm2 cell culture flask to which nothing was immobilized, using a culture medium prepared by means of cloning with the human AB serum.

AIM-V of the same concentration in the serum as of the seventh day to have a density of 1.04 x 106 cells / mL (volume of the liquid: 12.6 mL). IL-2 I was added to each group to have a final concentration of 500 U / mL. On the fourteenth day of the start of the culture, the number of live cells was counted by the means of the trypan blue staining method and an expansion fold was calculated by comparing the number of cells counted with the number of cells. cells at the beginning of the culture. The results are shown in Table 3. ' Table 3 Concentration in Days of Bending of the Serum (%) Cultivation Expansion (times) Control (Without Immobilization 3? 1? 0.05 14 Days x 276 of CH-296) CH-296 3? 1 ? 0.05 14 Days x 688 As shown in Table 3, in the group in which the culture equipment immobilized with CH-296 was used in an early stage of T cell expansion, the times of expansion of the T cells were high compared to those of the control group. At this time, it has been clarified that, the CH-296 fragment is suitably used during the expansion of T cells and in this example, it has been clarified that the CH-296 fragment is also suitably used during a 14-day expansion of T cells (2) Analysis of 'CD45RA + CCR7 + T Cells in Subsets of CD8 + T Cells and CD8 T Cells' after Culturing The cells prepared in step (1) of Example 2 were washed with PBS and then with 1% BSA / PBS Cells were suspended in PBS containing 1% BSA and mouse IgGl labeled with FITC / lgGl from mouse labeled with RDl / mouse IgGl tagged with PC5, + mouse IgGl tagged with ECD was added as a negative control. (manufactured by Beckman Coulter) Similarly, the human anti-CD45RA antibody labeled with RD1 / mouse human anti-CCR7 antibody labeled with FITC (manufactured by R &D Systems) / mouse human anti-CD8 antibody labeled with ECD (ma numbered by Beckmann Coulter). After each of the antibodies, the cells were incubated on ice for 30 minutes. During the course of the incubation, the incubation mixture was shaken gently every ten minutes. After incubation, the cells were washed with 1% BSA / PBS and resuspended in PBS. The cells were subjected to flow cytometry and classified in a CD8 + T cell region and a CD8 T cell region "by the CD8 staining attitude. For each cell population, the percentage of CD45RA + CCR7 + T cells was calculated. The results are shown in Tables 4 and 5.

Table 4 CD8 + T Cells CD45RA + T Cells CCR7 + (%) Control (Non-immobilized CH-296) 11.9 CH-296 37.4 Table 5 CD8 T cells "(CD4 + T cells) CD45RA T cells" CCR7 + (%) Control (Without immobilization of CH-296) 9.7 CH-296 35.0 As shown in Table 4, in the group in which immobilized culture equipment was used with the CH-296 fragment, the results of the highest CD45RA + CCR7 + T cell population in T cells were obtained. during cultivation, compared to those in the control group. This phenomenon was the same even in a case where the CD8 T cells were analyzed "(Table 5), ie a population of cells of which most were CD4 + T cells.It was clarified from these results that the population of cells T can expand while increasing the percentage of the CD45RA + CCR7 + T cell population of T cells by the co-existence of the CH-296 fragment at an early stage of a 14-day expansion .. Example 3: Capacity Analysis of Production of T Cell Cytokine CD45RA + CCR7 + and CD45RA T Cells I CCR7"in a Population of Expanded T Cells Using CH-296 (1) Expansion of the Population of T Cells The PBMCs which were prepared in point (1) of Example 1 were suspended in GT-T503 (manufactured by TAKARA BIO, INC.) Containing 0.5% human AB serum and HSA 0.2% (hereinafter referred to simply as "GT-T503 at 0.5%") to have a density of 0.25 x 106 cells / mL, to prepare a suspension of cells.

Then, the 0.5% GT-T503 was added to a plate 1 immobilized with the human anti-CD3 antibody or a plate immobilized with the human anti-CD3 antibody and the CH-296, prepared in point (2) of Example 1 , in a volume of 0. 5 mL / well and the above cell suspension was added thereto in a volume of 1 mL / well. IL-2 was added thereto to have a final concentration of 1000 U / mL and these plates were subjected to culture at 37 ° C in 5% C02 (zero day of culture). On the fourth day after the start of the culture, the culture medium of each group was diluted approximately 8 times with the 0.5% GT-T503 and 6 mL of the dilution was transferred to a totally new 12.5 cm2 cell culture flask. to which nothing was immobilized and IL-2 was added thereto to have a final concentration of 500 'U / mL. The culture was continued and on the seventh day the cell culture medium of each group was diluted approximately 4.2 times with the GT-T503 at 0. 5% and 12.6 mL of the dilution were transferred to a completely new 25 cm2 cell culture flask to which no immobilization was achieved. IL-2 was added to each group to have a final concentration of 500 U / mL. On the tenth day, the cell culture medium from each group was diluted approximately 2-fold with 0.5% GT-T503 containing 0.2% I HSA and 12.6 mL of the dilution were each transferred to a culture flask. cells of 25 cm2 totally new to which nothing was immobilized. IL-2 was added to each group to have a final concentration of 500 U / mL. On the fourteenth day from the start of culture, the number I of live cells was counted by the staining method with trypan blue and an expansion fold was calculated by comparing the number of cells counted with the number of cells at the start of the culture. The results are shown in Table 6.

Table 6 Expansion Bending (times) Control (Without CH-296 Immobilization) x 172 CH-296 x 581 As shown in Table 6, in the group in which the immobilized culture equipment was used with CH-296 at an early stage of expansion of the T-cell population (hereinafter referred to as "the CH-296 group. "), the fold expansion of the T cell population was high compared to that of the control group. (2) Analysis of CD45RA + CCR7 + T Cells The cells prepared in point (1) of Example 3 were stained with each antibody and analyzed in the same manner as in item (4) of Example 1, with the proviso that combinations of the antibodies were as follows. Specifically, the staining was carried out with mouse IgGl labeled with FITC / mouse lgGl labeled with RDl / mouse IgGl labeled with PC5 and mouse human anti-CD45RA antibody labeled with RDl / mouse human anti-CCR7 antibody labeled with FITC. The stained cells were analyzed with a flow cytometer and a percentage of CD45A + CCR7 + T cells was calculated. i The results are shown in Table 7.! Table 7 CD45RA + T cells CCR7 + (%) Control (Without immobilization of CH-296), 21.2 CH-296 63.7 ! As shown in Table 7, in the CH-296 group, the results of the CD45RA + CCR7 + T cell population were obtained, higher in the T-cell population during culture, compared to those of the control group. . I (3) Isolation of CD45RA + CCR7 + T Cells and CD45RA T Cells "CCR7" After Population Expansion of T Cells The cells prepared at point (1) of Example 3 were stained in the same manner as in FIG. point (2) of Example 2 and then the cells were washed with 1% BSA / PBS and suspended in the GT-T503 medium. The cells were subjected to the classification with Moflo ™ (manufactured by DAKO DENMARK A / S) in CD45RA + CCR7 + T cells and T cells! CD45RA "CCR7" of CD8 + T cells and similarly in T cells CD45RA + CCR7 + and CD45RA T cells "CCR7" of CD8 T cells ", ie a majority of which were CD4 + T cells.

In addition, cells stained before the same classification were subjected to flow cytometry and classified into a region of CD8 + T cells and a region of CD8 T cells according to the staining attitude of CD8. the percentage of CD45RA + CCR7 + T cells for each cell population The results are shown in Tables 8 and 9. i Table 8 CD8 + T cells j CD45RA + T cells CCR7 + (%) Control (Non-immobilized CH-296) 15.9 CH-296 | 52.6 Table 9 CD8 T Cells (CD4 + T Cells) T cells CD45RA + CCR7 + (%) Control (Without immobilization of CH-296) 5.4 CH-296! 34.0 As shown in Table 8, with respect to CD8 + T cells, in the CH-296 group, the results of a higher percentage of the CD45RA + CCR7 + T cell population were obtained in a population of T cells after culture, compared to those in the control group. This phenomenon was the same even in a case where CD8 T cells were analyzed (Table 9), ie a population of cells of which the majority are CD4 + T cells. (4) Immobilization of Human Anti-CD3 Antibody and Human Anti-CD28 Antibody An anti-CD3 antibody Human and a human anti-CD28 antibody were immobilized in a culture kit used in the following experiment, namely, 80 μL of each acipaeta buffer (pH 5.3) containing a human anti-CD3 antibody (2 μg / ml). mL) were added to a 96-well cell culture plate (manufactured by Becton Dickinson), and 80 μL of each acetate buffer containing human anti-CD28 antibody (manufactured by DAKO) was added thereto. DENMARK A / S) (20 μg / mL) The human anti-CD3 antibody was added to have a final concentration of 1 μg / mL and the human anti-CD28 antibody was added to have a final concentration of μg / mL. After these culture kits were incubated at room temperature for 5 hours, the 1 1 culture equipment was stored at 4 ° C until use. Immediately before use, the acetate buffer containing the antibody was removed by aspiration of those culture equipment and then each well was washed twice with PBS and once with the medium GT-T503 and culture equipment. they were subjected to each experiment. (5) Cell Stimulation It has been known that native T cells and central memory T cells produce IL-2 in a large amount upon stimulation with an antigen in the body.

In addition, it has been known that effector memory T cells produce IFN-? or IL-4 in a large amount with stimulation with an antigen. In order to confirm that each i one of the cell fractions obtained in point (3) I of Example 3 maintains these functions, the cytokine production capacity was determined when stimulated with an anti-CD3 antibody and an anti-CD28 antibody. Each of the population of cells obtained in point (3) of Example 3 was collected, the cells were then suspended in 0.5% GT-T503 medium and the number of cells counted. The cells were added to each well of the plate immobilized with the human anti-CD3 antibody and the human anti-CD28 antibody prepared in point (4) of Example 3 to have a density of 2 x 10 5 cells / 0.2 mL and the cells were grown; at 37 ° C in C02 at 5%. After 24 hours, the culture supernatant was collected and used for the experiment for the determination of cytokine according to the ELISA method. (6) Determination of Cytokine Production in Accordance with the ELISA Method As shown in point (3) of Example 3, in group CH-296, the results of a higher percentage of CD45RA + CCR7 + T cells were obtained, compared to those of the control group. In order to confirm that this population of CD45RA + CCR7 + T cells maintains its property with natural T-type cells, the cytokine production capacity of CD45RA + CCR7 + T cells and CD45RA T cells "CCR7" i isolated after the expansion of T cells was evaluated. the population of T cells. The production of IL-2 or IFN-? was determined with an ELISA Development Kit i (manufactured by R &D Systems) and the production of IL-4 was determined with Human Interleukin-4 READY-SET GO! MR (manufactured by eBioscience). The 1 results of each cytokine production in the control group and the CH-206 group are shown in Tables 10 and 11, respectively.

Table 10 Control (Without Immobilization of CH-296) IL-2 (pg / mL) IFN-? (pg / ml_) IL-4 (pg / mL) T cells T cells CD45RA + CCR7 + 286.7 568.0 30.5 CD8 + CD45RA T cells "CCR7" 104.9 1229.0 430.8 T cells T cells CD45RA + CCR7 + 1539.4 107.9 113.9 CD8"T cells CD45RA" CCR7"877.0 1200.0 1004.0 Table 11 Immobilization of CH-296 IL-2 (pg / mL) IFN-? (pg / mL) IL-4 (pg / mL) T Cells CD45RA + T Cells CCR7 + 174.4 251.3 < 0 CD8 + CD45RA T Cells "CCR7" 44.9 526.3 58.1 T Cells CD45RA + CCR7 + T Cells; 1958.2 144.3 20.3 CD8"CD45RA T Cells" CCR7"611.0 587.5 727.3 As shown in Tables 10 and 11, in the control group and the CH-296 group, the production of IL-2 was confirmed. In both groups, the CD8 T cells had a larger amount of cytokine production than that of the CD8 + T cells and the CD45RA + CCR7 + T cells had a larger amount of production than that of the CD45RA T cells "CCR7" It was shown from the above that the population of CD45RA + CCR7 + T cells obtained by the expansion of the T cell population had properties: as natural T cells, on the other hand, IFN-α and IL-4 dominantly produced in the CD45RA T cells "CCR7" in both the control group and the CH-296 group, suggesting that the CD45RA T cell population "CCR7" is a population in which the effector memory function is maintained. : Chemotaxis Analysis of an Expanded T Cell Population Using CH-296 (1) Immobilization of Human Anti-CD3 Antibody and Fragment CH-296 Immobilization was carried out in the same manner as in item (2) of Example 1, except that the PBS containing a human anti-CD3 antibody (final concentration: 5 μg / mL) was added in a volume of 0.45 mL each. (2) Expansion of a Population of T Cells The same procedures were carried out as those of point (1) of Example 3 except that on the fourth day from the start of the culture, the culture medium was diluted approximately 14 times and 10 mL of the dilution was added to a totally new 25 cm2 cell culture flask (manufactred by Corning), that on the eighth day at the beginning of the culture, the culture medium was diluted approximately 2 times and 10 were added. mL of the dilution to a completely new 25 cm2 cell culture flask (manufactured by Corning) and that on the eleventh day from the start of the culture, 10 mL of the GT-T503 medium containing 0.2% HSA was added to each one of the flask. The results of the times of expansion are shown in Table 12. I I Table 12 'Expansion Bending (times) Control (Without CH-296 Freezing) x 414 CH-296: x 646 As shown in Table 12, in the group in which an immobilized culture kit with CH-i 296 was used at an early stage of the expansion of the T-cell population (referred to as "the group" CH-296"), the fold expansion of the T cell population was high compared to that of the control group. (3) Analysis of CD45RA + CCR7 + i T cells For the cells, prepared at point (2) of 1 Example 4, a percentage of CD45RA + I CCR7 + T cells was calculated in the same manner as in point (2) of Example 3 - The results are shown in Table 13.! I T cells CD45RA + CCR7 + (%) Control (Without immobilization of CH-296) '31.5 CH-296 56.8 As shown in Table 13, in the group CH-? 296, the results of a higher CD45RA + CCR7 + T cell population in the cell population were obtained T during cultivation, compared to that of the control group. (4) Analysis of Chemotaxis In group CH-296, the results of a higher percentage of a population of CD45RA + CCR7 + T cells, compared with that of the control group. CCR7-positive cells such as native T cells and central memory T cells show chemotaxis in response to chemokine CCL21. This is an important event for these cells to migrate from the blood vessels to the lymph nodes and it was confirmed whether the cells obtained after the expansion had chemotaxis in response to CCL21. The cells obtained at i (2) of Example 4 were centrifuged, the supernatant was removed and the cells were then suspended in RPMI 1640 medium containing 0.5% BSA (shown hereinafter as a reaction medium). to have a density of 5 x 106 cells / mL. To a lower layer of a Transwell ™ plate (Transwell Polycarbonate, 24-wells, pore sizes 5 μm, manufactured by Corning) was added 600 μL of a reaction medium containing CCL21 (manufactured by R &D Systems) to a final concentration of 1 μg / mL and 100 μL of the prepared cell suspension was added to the upper layer. The number of cells that migrated to the lower layer was determined after culturing at 37 ° C for] 2 hours and the number of i cells remaining in the upper layer. The percentage of cells that induced chemotaxis was calculated from the following formula (1): [Su 1] Percentage (%) of Cells that Induced the Chemotaxis = (Number of cells that migrated to the layer Lower + (Number of Cells That Remained in the Upper Layer + Number of Cells that Migrated to the Layer Bottom)) x 100 The results are shown in Table 14. Table 14 Percentage of Cells that Induced - Chemotaxis (%) Control (Without Immobilization of CH-296) 43.2 CH-296 58.8 As shown in Table 14, the cells that showed chemotaxis in response to CCL21 were confirmed I in both the control group and the CH-296 group and the percentage was higher for the CH-296 group. In other words, it was clarified that the: cells that had the ability to migrate to the nodules; Lymphatics were obtained in a large amount when using: CH-296 with the expansion of the T cell population.

Example 5: Induction of Anti-MART-1 CTLs from an Expanded Cell Population Using CH-296 (1) Immobilization of Human Anti-CD3 Antibody and Fragment CH-296, The same procedures were carried out as in item (1) of Example! 4. (2) Expansion of a T Cell Population The same procedures were carried out as in point (2) of Example 4. The results of the times of expansion are shown in Table 15.

Table 15 Expansion Bending (times) Control (Without Immobilization of CH-296) x 189 CH-296 x 387 As shown in Table 15, in the group in which immobilized culture equipment was used with CH-296 at an early stage of expansion (referred to hereinafter as "the CH-296 group"), the fold of expansion was high compared to that of the control group. (3) Analysis of T cells CD45RA + CCR7 + and CD45RA + CD62L + The cells prepared in point (2) of the Example 5 were stained with each antibody and analyzed in the same manner as in item (4) of Example 1, with the proviso that the combinations of the antibodies were as follows. Specifically, the staining is carried out with mouse IgGl labeled with FITC / mouse lgGl labeled with RDl (both manufactured by DAKO DENMARK A / S), human anti-CD45RA antibody of mouse labeled with RDl / mouse human anti-CCR7 antibody labeled with FITC and mouse anti-human CD45RA antibody labeled with RDl / mouse human anti-CD62L antibody labeled with FITC (hereinafter the human anti-CD62L antibody which is a product manufactured by I eBioscience). These stained cells were analyzed with a flow cytometer and the percentages of CD45RA + CCR7 + T cells and CD45RA + CD62L + T cells were calculated. The results are shown in Table 16.

Table 16 Control (Without Immobilization with CH-296) CH-296 CD45RA + CD62L + T cells (%) 60.2 76.9 T cells CD45RA + CCR7 + (%) 47.1 75.0 As shown in Table 16, in group CH-296, high values were shown in both cell surface markers compared to those in the control group. (4) Storage of Cultured Cells The cells on the fourteenth day of the culture prepared in point (2) of Example 5 were suspended in a storage solution composed of volumes equivalents of 90% FBS DMSO at 10% or CP-1 containing HSA at 8% and RPMI 1640 medium and the suspension was stored in liquid nitrogen. During the induction of CTLs, these stored cultured cells rapidly melted in a 37 ° C water bath and were: washed with RPMI 1640 medium containing 10 μg / mL DNase ™. Then, the number of living cells was calculated by means of the trypan blue-i staining method. The cells were subjected to each experiment. (5) Induction of Specific CTLs for an i Antitumor Associated Antigen (MART-1) 1 Using the cells prepared at point (4) of Example 5, the induction of CTLs I specific for an antitumor associated antigen was carried out. (Melanoma antigen recognized by T cells: MART-1). The induction of specific CTLs for an antitumor associated antigen (MART-1) was carried out by partially modifying a method of Plebanski M. et al. [Eur. J. Immunol. , 25 (6), 1783-1787 (1995)]. 'Specifically, using the PBMCs prepared in item (1) of Example 1 as cells presenting an antigen, - the PBMCs were incubated in a medium I to RPMI 1640 containing 5% human AB serums (hereinafter referred to simply as "5HRPMI") that i contained 40 μg / mL of epitope peptide derived from the MART-1 melanoma antigen (HLA-A2.1 binding peptide derived from the MART-1 melanoma antigen described in the SEQ ID NO: 22 of the List of Sequences) as an antigen peptide and 3 μg / mL of β2 microglobulin (manufactured by Scrips), a mixture of 1.1 mM NEAA, 1 mM sodium pyruvate, 2 mM L-glutamine (all manufactured by Cambrex) and 100 μg / mL of streptomycin sulfate (manufactured by MEIJI SEIKA KAISHA, LTD.) in a 5% C02 incubator at 37 ° C for 2 hours. After the incubation consummation, the mixture was irradiated with X-rays (1.42 C / kg) and the density adjusted with 5HRPMI to 3 μl for 4 × 10 6 cells / mL. On the other hand, the cultured cells prepared at point (4) of Example 5 were suspended in 5HRPMI to have a density of 2 x 10 6 cells / mL and the suspension was added to a 24-well cell culture plate (manufactured by Becton Dickenson) in a volume of 0.5 mL / each well. Each well was added to the antigen-presenting cells prepared by the above method in a volume of 0.5 mL / well and IL-7 (manufactured by R &D Systems) and KLH (manufactured by Calbiochem) were added to have a final concentration of 25 ng / mL and 5 μg / mL, respectively. The plate was cultured in a 5% C02 incubator at 37 ° C. On the first day from the start of the culture, 1 mL of 5HRPMI containing 60 U / mL of IL-2 was added to each well. On the fourth day from the start of the culture, half the volume of the culture supernatant was removed and 1 was added. mL of 5HRPMI containing 60 U / mL of IL-2 to each well. In addition, on the seventh day, the cells presenting an antigen were prepared in the same manner as before and the mixture was then irradiated; with X-rays (1.42 C / kg) and the cells were prepared to have a density of 4 x 106 cells / mL and the cells were added to a totally new 24-well cell culture plate in a volume of 0.5 mL. / each well. Responding cells subjected to a one-week culture (a portion of the cells continued to be cultured for the cytotoxic activity assay) were suspended in 5HRPMI, to have a density of 1.8 to 2.0 x 106 cells / mL and the suspension was added to a similar plate in a volume of 0.5 mL / each well. In addition, IL-7 was added thereto to have a final concentration of 25 ng / mL to stimulate the cells again. On the eighth day from the start of the culture, 1 mL of 5HRPMI containing 60 U / mL of IL-2 was added to each well. On the eleventh day from the start of culture, the cells in each well were suspended and divided into two wells at half volume each and 1 mL of 5HRPMI containing 60 U / mL of IL-2 was added to each well. and the cells continued to be cultivated until the fifteenth day. (6) Determination of Expansion Bending Regarding the cells obtained in point (5) of Example 5, on the seventh day and the fourteenth day from the start of cultivation, the number of live I cells was counted by means of the trypan blue staining method and I an expansion fold was calculated by comparing the number of cells counted with the number of cells at the start of the culture. The results are shown in Table 17.

Table 17 Days of Control Cultivation (Without Immobilization with CH-296 CH-296) Seventh Day x 1.5 x 1.9 Fourteenth Day 'x 6.0 x 8.4 As shown in Table 17, in the population of CTL induced from the CH-296 group, the times of expansion of the CTL population were high compared to those of the control group. In other words, it was clarified that the CTL population could be obtained in a larger amount by carrying out the induction of CTLs from cultured cells in which an immobilized culture kit with CH-296 was used at an early stage of the expansion of the T-cell population. (7) Cytotoxic Activity Assay The cytotoxic activity of CTLs in the Fifteenth day from the start of the culture prepared in the point (5) of Example 5 was evaluated by means of a method to test the cytotoxic activity using calcein-AM [Lichtenfels R: et al., J. "Immunol. Methods 172 ( 2), 227-239 (1994).] Specifically, the T2 cells of a cell line restricted with respect to HLA-A2.1 (ATCC CRL-1992), which were co-cultured overnight with An epitope peptide was cultured in the absence of an epitope peptide, I was suspended in RPMI 1640 medium containing 5% FBS i to have a density of I x 10 6 cells / mL, and then calcein-AM was added. (manufactured by DOJINDO LABORATORIES) to them to have a final concentration of 25 μM and the cells were cultured at 37 ° C for 1 hour.The cells were washed with a medium that did not contain calcein-AM and then the target cells were obtained labeled with calcein, the target cells et Calcein tags were mixed with a 30-fold amount of K562 cells (Human Science Research Resources Bank JCRB0019), to provide cells for testing for cytotoxic activity. At this point, K56¡2 cells were used to exclude non-specific cytotoxic activity by mixed NK cells in responder cells. The CTLs prepared at point (5) of Example 5 were serially diluted with 5HRPMI as effector cells, to have a density of 3 x 105 to 3 x 106 cells / mL. Then, the dilution was previously supplied to each well of a 96-well cell culture plate in a volume of 100 μL / each well and to these plates the cells were added to test the cytotoxic activity in a volume of 100 μL / each well prepared so that the target cells labeled with calcein had a density of 1 x 105 / mL. With the determination, the ratio of the effector cells (E) to the target cells labeled with calcein (T) is expressed as an E / T ratio and the determinations were made in E / T ratios of 30, 10 and 3. The plate containing the above cell suspension was centrifuged at 400 xg for 1 minute and then the cells were incubated in a 5% C02 incubator at 37 ° C for 4 hours. Then, 100 μL of the culture supernatant was collected from each well and the amount of calcein released in the culture supernatant was determined with a fluorescence plate reader (spectrofluorometer) (manufactured by BERTHOLD TECHNOLOGIES) (excited at 485 nm / measured at 538 or 535 nm). The "Specific Cytotoxic Activity (%)" was calculated according to the following formula (2): [Su 2]; Specific Cytotoxic Activity (%) =. { (Measured Value in Each Pocil - Amount Released Minimum) / (Amount * Released Maximum - Amount Released Minimum)} x 100 In the above formula, the minimum released amount is the amount of calcein released in well i that contained only cells to test the cytotoxic activity, which shows the amount of calcein naturally released from target cells labeled with calcein. In addition, the maximum release amount refers to the amount of calcein released when the cells were destabilized - completely by the addition of 0.1% of the Triton X-100MR surfactant (manufactured by Nakalai Tesque Inc.) to the cells to be tested the cytotoxic activity. The results of the cytotoxic activity assay are shown in Table 18.

Table 18 Target Cells EU Cytotoxic Activity (%) Control (Non-immobilization CH-296 of CH-296) T2 30 < 0 < 0 10 < 0 < 0 3 < 0 < 0 T2 + Peptide MART-1 30 62.0 87.2 10 29.1 60.1 3 9.2 26.3 As shown in Table 18, in the CTL population i induced from the CH-296 group, the specific cytotoxic activities of the CTL population were high compared to those of the control group. In other words, it was clarified that cultured cells in which an immobilized culture kit with CH-296 was used at an early stage of expansion of the T cell population could be induced to a CTL population that has an even higher specific cytotoxic activity. Example 6: Allogenic MLR and Induction of CTLs i Anti-MART-1 of Population of Expanded Cells Using I a Bag of Gas Permeable Culture After Stimulation in an Early Stage of CH-296 (1) Immobilization of Anti-CD3 Antibody and Fragment CH-296 A human anti-CD3 antibody and a CH-i fragment 296 were immobilized in a culture equipment used in the following experiment. Specifically, 9 mL each of 1 I PBS containing a human anti-CD3 antibody (final concentration: 5 μg / mL) was added to a 75 cm2 cell culture flask (manufactured by Becton Dickinson). With the addition, * CH-296 was added to a group with the addition of CH-296 to have a final concentration of 25 μg / mL. > '! After these farming equipment is incubated at room temperature for 5 hours, the culture equipment was stored at 4 ° C until use. Immediately before use, the PBS containing the antibody and the CH-296 were removed by aspiration of these culture equipment and then each flask was washed twice with PBS and then once with the RPMI medium and the control equipment. culture were subjected to each experiment. (2) Expansion of Population of T Cells The PBMCs which were prepared in the point (1) of Example 1 were suspended in 0.5% GT-T503 to have a density of 0.5 x 10 6 cells / mL, to prepare a suspension of cells Then, the 0.5% GT-T503 was previously added to a flask immobilized with the human anti-CD3 antibody or a flask immobilized with the human anti-CD3 antibody and the CH-296, prepared in point (1) of Example 6 , in a volume of 21 mL / flask and the previous cell suspension was added thereto in a volume of 9 mL / each flask. IL-2 was added to them to have a final concentration of 1000 U / mL and these flasks were subjected to culture at 37 ° C in 5% C02 (zero day of culture). On the fourth day after the start of the culture, 14 mL of the culture medium of each group and 186 mL of 0.5% GT-T503 were transferred to a gas permeable culture bag (200 cm2 manufactured by TAKARA BIO, INC., code commercial: KB210) to which nothing was immobilized and IL-2 was added further to it to have a final concentration of 500 U / mL. The cultivation continued and on the eighth day, 100 mL of the cell culture medium from each group was removed from the culture bag, leaving 100 mL of the cell culture medium in the bag and the remaining culture medium was diluted 2 times with the 0.5% GT-T503, to constitute a volume of 200 mL of the dilution of cells in the bag. IL-2 was added to each group to have each a final concentration of 500 U / mL. On the eleventh day from the start of the culture, 200 mL of the GT-T503 medium containing 0.2% HSA were added thereto to be diluted 2 times with the cell culture medium of each group. IL-2 was added to each group to have each a final concentration of 500 U / mL. On the fourteenth day from the start of the culture, the number of live cells was counted by the trypan blue staining method and an expansion fold was calculated by comparing the number of cells counted with the number at the start of the culture. The results are shown in Table 19.

Table 19 Expansion fold (times) Control (Without CH-296 Freezing) x 225 CH-296 i x 333 As shown in Table 19, even in a case where the cells were cultured in a gas-permeable culture bag, in the group in which an immobilized culture kit with CH-296 was used in an early stage i of the expansion of the T-cell population (hereinafter referred to as "the CH-296 group"), the fold expansion of the T-cell population was high compared to that of the control group. (3) Analysis of CD45RA + CCR7 + T cells, CD45RA + CD62L + T cells, CD45RA + CD27 + T cells, CD45RA + CD28 + T cells, CD27 + CD28 + T cells, and CD45RA + CDR7 + CD62 + T cells in a T cell population cultured in a culture-permeable culture bag. Gas . The cells prepared in point (2) of Example 6 were stained with each antibody and analyzed in the same manner as in item (4) of Example 1, with the proviso that the combinations of the antibodies were as follows . Specifically, the staining was carried out with mouse IgGl labeled with FITC / mouse lgGl labeled with RDl / mouse IgGl labeled with PC5, human anti-CD45RA antibody of mouse labeled with RD1 / mouse human anti-CCR7 antibody labeled with FITC, mouse human anti-CD45RA antibody labeled with RDl / mouse human anti-CD62L antibody labeled with PC5 (manufactured by Beckmann-Coulter), anti-antibody CD45RA human mouse labeled with RDl / mouse human anti-CD28 antibody labeled with FITC (manufactured by eBioscience), mouse human anti-CD45RA antibody labeled with RDl / mouse human anti-CD27 antibody labeled with PC5 (manufactured by Beckmann- Coulter) and mouse human anti-CD45RA antibody labeled with RDl / mouse human anti-CCR7 antibody labeled with I FITC / anti-CD62L antibody human mouse labeled with PC5. These stained cells were analyzed with a flow cytometer I and the percentages of T cells were calculated.

CD45RA + CCR7 +, CD45RA + CD62L + T cells, CD45RA + CD2D T cells CD45RA + CD28 + T cells, CD27 + CD28 + T cells and CD45RA + CDR7 + CD62L + T cells. The results are shown in Table 20.

Table 20 Control (Non-immobilization of CH-296 CH-296) CD45RA + CCR7 + T cells 24.4% 49.0% CD45RA + CD62L + T cells 40.2% 60.2% CD45RA + CD28 + T cells 36.9% 63.0% CD45RA + CD27 + T cells 46.3% 63.1% CD28 + CD27 + T cells 58.9% 72.1% T Cells CD45RA + CCR7 + CD62L 23.0% 47.3% As shown in Table 20, in the CH-296 group, a high value is shown in any of the cell surface markers, compared to those in the control group. It has been known that CD27 and CD28 have high percentages of expression in undifferentiated cells such as native T cells. Therefore, it was clarified that even when analyzed with these markers, the percentage of the natural T-cell population was increased in the T-cell population by allowing CH-296 to act at an early stage of culture. (4) Allogenic MLR An allogeneic MLR was carried out using the cells prepared in item (2) of Example 6.

Specifically, the PBMCs, prepared in the same manner as in item (1) of Example 1 derived from an allogeneic donor (donor not own: a donor other than the donor I used in item (2) of Example 6), were irradiated with X-rays (0.88 C / kg) and the cells were suspended with 5HRPMI to have a density of 2 x 106 cells / mL (stimulator cells). On the other hand, the cultured cells prepared at point (2) of Example 6 were suspended in 5HRPMI to have a density of 2 x 106 cells / mL (responder cells). Stimulatory cells and prepared responder cells were added to a 24-well cell culture plate in a volume of 0.5 mL / each well. IL-2 was added to each well to have a final concentration of 500 U / mL and then the cells in the plate were cultured in a 5% C02 incubator at 37 ° C. On the third day from the beginning of the crop, 1 mL of 5HRPMI containing 1000 U / mL! of IL-2 was added to each well. On the fifth day from the start of the culture, half the volume of the culture supernatant i was removed and 1 mL of 5HRPMI containing 1000 iU / mL of IL-2 was added to each well. In addition, on the seventh day, the cells in each well were suspended, the suspension was divided into two wells of half I volume each, 1 mL of 5HRPMI containing 1000 U / mL of IL-2 was added to each well and the cultivation continued until the tenth day. (5) Determination of the Expansion Bending Regarding the cells obtained in the point (4) of Example 6, on the seventh day and on the tenth day from the start of the culture, the number of living cells was counted by means of the method of trypan blue staining and an expansion fold was calculated by comparing the number of i cells counted with the number of cells at the start of the culture. The results are shown in Table 21.

Table 21 Days of the Donor of Cells Responding Cells Culture Stimulators Control (Without Immobilization with CH-296 CH-296) Seventh Day A x 1.7 x 2.7 B x 1.6 x 2.7 Tenth Day x 1.8 x 3.0 B x 2.1 x 2.7 As shown in Table 21, in a case where an allogeneic MLR was carried out, using the CH-296 group, the expansion fold after the reaction was high compared to that of the control group. In other words, it was clarified that when an allogeneic MLR was carried out using cultured cells in which an immobilized culture kit with CH-296 was used at an early stage of expansion of the T cell population, the cells they recognized Alloantigens proliferated in an even larger amount. (6) Test of Cytotoxic Activity The cytotoxic activity of the cells on the tenth day from the start of the induction, prepared in point (4) of Example 6, was tested in the same manner as in point (7) of Example 5. At this point, as the target cells with the determination, the PBMCs own or non-own PBMCs which were subjected to blastogenesis (destruction) with phytohemagglutinin (hereinafter referred to simply as "PHA") for 10 days were used as target cells labeled with calcein. During the assay for the cytotoxic activity, a 30-fold amount of K562 cells and target cells labeled with calcein were mixed. The results for the assay of cytotoxic activity I are shown in Table 22.

Table 22 Target Cell Donor E / T Cytotoxic Activity (%) Control Cells (Without Immobilization CH-296 Stimulators of CH-296) A Non-Proprietary Cells 30 50.6 63.8 Destroyed with PHA; 10 29.3 44.1 3 7 16.9 ¡30 < 0 < 0 Own Cells 10 < 0 < 0 Destroyed with PHA 3 < 0 < 0 B Non-Proprietary Cells 30 2 266..66 6 600..11 Destroyed with PHA 10 9.1 38.0 < 0 13.0 Proprietary Cells 30 < 0 < 0 Destroyed with PHA 10 < 0 < 0 3 < 0 < 0 As shown in Table 22, in a case where the allogeneic MLR was carried out using the CH-296 group, the cytotoxic activity specific for alloantigens after the reaction was high, as compared to that of the control group. In other words, in cultured cells in which immobilized culture equipment was used with CH-296 at an early stage of expansion of the T cell population, it was clarified that I could obtain a population of cells that had a even more potent cytotoxic activity for alloantigens through the allogeneic MLR. (7) Storage of Cultured Cells The cells on the fourteenth day of the culture prepared in point (2) of Example 6 were subjected to frozen storage and melted in the same manner as in point (4) of Example 5. cells were attached to each experiment. (8) Induction of CTLs Specific for Antitumor Associated Antigen (MART-1); Using the cells prepared in item (1) of Example 1 and item (7) of Example 6, the induction of specific CTLs for an antitumor associated antigen (MART-1) was carried out in the same manner as in point ( 5) of Example 5 and the culture continued for thirteen days, with the proviso that the modifications were made from the The following way: The characteristics include the cultured cells prepared in the point (7) of Example 6 were suspended in 5HRPMI to have a density of 1 x 106 cells / mL; on the second day from the start of the culture, 1 mL of 5HRPMI containing 60 U / mL of IL-2 was added to each well, on the sixth day from the start of the culture, the responder cells were suspended in 5HRPMI to have a density of 0.3, to 1.3 x 106 cells / mL and the cells that presented an antigen were suspended in them to have a density of 1.6 x 106 cells / mL; and on the seventh day from the start of the culture, 1 mL of 5HRPMI containing 60 U / mL of IL-2 was added to each well and on the tenth day from the start of the culture, the cells in each well were suspended and divided into two wells of half volume each and 1 mL of 5HRPMI containing 60 U / mL of IL-2 was added to each well. (9) Test of Cytotoxic Activity The cytotoxic activity of the CTLs on the thirteenth day from the start of induction, prepared at point (8,) of Example 6 was tested in the same manner as in (7) of Example 5, except that an E / T ratio of 90 was established again. The results of the cytotoxic activity assay are shown in Table 23.

Table 23 E / T Cells Cytotoxic Activity (%) Objective PBMQs Control (Non-immobilization CH-296 of CH-296) T2 90 30.96 N.T. N.T. 30 9.13 23.04 15.78 10 2.42; 9.63 10.56 3 1.94 3.06 3.62 T2 + Peptide 90 59.49 N.T. N.T.

MART-1 30 41.37 25.46 87.53 10 25.29 8.85 77.21 3 7.75 11.26 56.18 NT: not tested As shown in Table 23, in the CTL population induced from the CH-296 group, the specific cytotoxic activity of the CTL population was high, compared to that of the control group and the PBMC group. In other words,; it was clarified that cultured cells in which immobilized culture equipment with CH-296 was used at an early stage of expansion of the T cell population could be induced to a CTL population that has an even higher specific cytotoxic activity .

Example 7: Induction of Anti-MART-1 CTLs from CD45RA * CCR7 + CD8 + T Cells and CD8RAC 'CCR7' CD8 + Cells Expanded Cells (1) Isolation of CD45RA + CCR7 + CD8 + T Cells and CD45RA "CCR7" CD8 + T Cells cells prepared in the point (1) of Example 1 and those prepared in the point (7) of Example I 6 were stained in the same manner as in point (2) of I Example 2. Then, the cells were washed with BSA at 1% / PBS and suspended in an IMDM medium (manufactured by Invitrogen). The cells were subjected to a high-performance cell classifier Moflo and a fraction CD45RA + CCR7 + CD8 + and a fraction CD45RA "CCR7" CD8 + were respectively isolated, obtaining thereby a fraction that was 92 to 99% pure. (2) Induction of Specific CTLs for an Antitumor Associated Antigen (MART-1) Using the cells prepared in item (1) of Example 7, the induction of specific CTLs for an antitumor associated antigen (MART-1) was performed in the same manner as in point (8) of Example 6 and the culture continued for thirteen days, with the proviso that the modifications were made as follows: Characteristics include at the beginning of the culture, the cells at the beginning of the culture and the cells that presented a antigen were added in a volume of 0.25 mL each to a 48-well culture plate (manufactured by Becton Dickenson); and on the second day from the start of the culture, 0.5 mL of 5HRPMI and 'containing 60 U / mL of IL-2 was placed in each well and in. On the fourth day from the start of the culture, half of the volume of the culture supernatant was removed and 0.5 mL of 5HRPMI containing 60 U / mL of IL-2 was added to each well. (3) Determination of the Expansion Bending Regarding the cells obtained in point (2) I of Example 7, on the 13th day from the start of the culture, the number of living cells was counted by means of the blue staining method of trypan and an expansion fold was calculated by comparing the number of cells counted with the number of cells at the start of the culture. The results are shown in Table 24.

Table 24 Crop Start Cells Control Expansion Bending (Without Immobilization of CH-296 CH-296) CD45RA + CDR7 + CD8 + T cells x1.2 x7.8 CD45RA T cells "CCR7" CD8 + x? 8 x1.2 As shown in Table 24, in the population induced CTL using CD45RA + CCR7 + CD8 + T cells isolated from cultured cells in which an immobilized culture kit with CH-296 was used at an early stage of the T-cell population expansion (hereinafter referred to as " group CH-296"), the fold expansion of the CTL population was high, compared to that of the control group. In other words, it was clarified that the CTL population was obtained in a larger quantity at! To carry out the induction of CTLs from the CD45RA + CCR7 + T cells contained in a high percentage in cultured cells in which an immobilized cultivated equipment with CH-296 was used in an early stage of the expansion of the cell population t- (4) Test of Cytotoxic Activity The cytotoxic activity of the CTLs on the thirteenth day from the start of the induction, prepared in point (2) of Example 7 was tested in the same manner as in the point (7) of Example 5. The results for the cytotoxic activity assay are shown in Table 25.

Table 25 Cells E / T Cells - Cytotoxic Activity (%) Start of Objective PBMCs Control (Without CH-296 Immobilization Cultivation of CH-296) T2 T cells 10 8.35 N. T. 18.62 CD45RA + 3 < 0 1.77 9.8 CCR7 + CD8 + T2 + Peptide 10 i 83.59 N. T. 89.49 MART-1 3 i 66.61 20.92 67.07 I T2 T cells 30 '- 17.16 N. T. N. T. I CD45RA "10; 3.19 N. T. N. T. I CCR7"CD8 + 3 | 0.78 10.15 8.96 T2 + Peptide 30; 8.66 N.T. N.T. MART-1 10 < 0 N.T. N.T. 3 < 0 9.06 3.21 N. T. : NOT tested. As shown in Table 25, in the CTL population induced using the CD45RA + CCR7 + CD8 + T cells isolated from the CH-296 group, the specific cytotoxic activity of the CTL population was high, compared to those of the control group and the PBMC group. In addition, the specific cytotoxic activity was high compared to that of the CTL population induced using the CD45RA "CCR7" CD8 + T cells. In other words, it was clarified that the CD45RA + T cells CCR7 + CD8 + which are contained in a high percentage in cultured cells in which an immobilized culture kit with CHt296 was used at an early stage of the expansion of the T cell population could be induced to I a CTL population of the which specific cytotoxic activity was even higher. Example 8: Determination of Ability to Recognize CTL Antigens (1) Induction of Specific CTLs for an Antitumor Associated Antigen (MART-1) i Using cells prepared at point ( 1) of Example 1 and those in point (7) of Example 6, the induction of specific CTLs for an antitumor associated antigen (MART-1) was carried out in the same manner as in item (8) of Example 6 and the cultivation continued for fourteen days. I (2) Determination of the Capacity to Recognize an Antigen The ability to recognize an antigen of the CTLs on the fourteenth day of the start of the induction, prepared in point (1), of Example 8 was determined by partially modifying u? method of Valmori, D. and collaborators [Valmori D. and collaborators, J. Immunol. , 160, 1750-1758 (1998)]. In other words, the T2 cells were suspended in RPMI 1640 medium containing 5% FBS to have a density of 1 x 106 cells / mL, then calcein-AM was added thereto to have a final concentration of 25 μM and the cells were cultured at 37 ° C for 1 hour. The incubated cells were washed with a medium that did not contain calcein-AM and the density was adjusted to 2 x 10 5 cells / mL. Target cells labeled with calcein were supplied in each well of a 96-well cell culture plate in a volume of 50 μL / each well. To each well in which these cells were placed was added 5HRPMI containing an antigen peptide of 0 to 20 μM (epitope peptide derived from the MART-1 melanoma antigen) in a volume of 50 μL / each well. The plate mentioned above was incubated in a 5% C02 incubator at 37 ° C for 1 hour. A suspension of cells containing a 30-fold amount of K562 cells (adjusted to a density of 6 x 10 6 cells / mL) in a volume of 50 μL / each well was added to each well after the incubation. The CTLs prepared in point (1) of Example 8 were adjusted with 5HRPMI to have a density of 6 x 10 6 cells / mL as effector cells and then to each well of the plate the cells were added in a volume of 50 μL / each. well. The plate to! The above cell suspension i was placed in centrifugation at 400 x g for 1 minute and the cells were then incubated in a 5% C02 incubator at 37 ° C for 4 hours. After, 100 μL of the culture supernatant were collected from each well and the amount of calcein released in the culture supernatant was determined with the fluorescence plate reader (spectrofluorometer) (485 nm / 535 nm). The "specific cytotoxic activity (%)" was calculated according to the formula (2) mentioned above. The results of the antigen recognition stability assay are shown in Table 26. The ability to recognize an antigen was expressed as a relative value of the cytotoxic activity at each concentration of aggregated peptide when the cytotoxic activity at an aggregated peptide concentration to the target cells of 10 μM was assumed to be 100.; Table 26 Final Relative Value Concentration of Cytotoxic Activat 10 μM of Peptide Aggregated to the Aggregated Peptide Target Cells (μM) PBMCs Control (Non-immobilized CH-296 of CH-296) 10.0 100 ¡100 100 1.0 124.36: 74.39 91.44 0.1 72.54 0.01 72.77 0.001 29.63: 36.83 74.3 0.0001 18.39 9.8 41.72 0 16.24 5.69 5.16 As shown in Table 26, in the CTL population I induced from the CH-296 group, the target cells were still removed with added antigen peptide of a still lower concentration, as compared to those of the control group and the PBMC group, so! that a capacity to recognize an antigen i specific to the population] of CTL was high. In other words, it was clarified that cultured cells in which a culture kit immobilized with CH-296 was used in an early stage of expansion of the T cell population could be induced to a CTL population having an ability to recognize a specific antigen even more highly.; Example 9: Feasibility Test of a Population 1 I of Expansion Cells of a Population of T Cells 1 Using CH-296 (1) Expansion of a Population of T Cells The procedures were carried out in the same manner as in point (1) of Example 3 except that an acetate buffer (pH 5.3) was used in the immobilization of a human anti-CD3 antibody and a CH-296 fragment and that on the fourth day from the start of the culture, the culture medium was diluted approximately 14 i times and 6.3 ml of the dilution was placed in a totally new 25 cm2 culture flask and on the eighth day from At the start of the culture, the culture medium was diluted approximately 2 times and 6.3 ml of the dilution was transferred to a completely new 25 cm2 culture flask. The results are shown in Table 27.

Table 27 Expansion Bending (times) Control (Without CH-296 Immobilization) x 277 CH-296 x 344 As shown e? Table 27, in the group in which an immobilized culture kit was used with the CH-296 fragment (referred to hereafter as "the CH-296 group") at an early stage of the population expansion of T cells, a high expansion fold was obtained compared to that of the control. (2) Analysis of CD45RA + CCR7 + T cells and CD45RA + CD62L + T cells The cells prepared in point (1) of Example 9 were stained with each antibody and analyzed in the same manner as in point (4) of Example 1, with the condition that the combinations of the antibodies were as follows. Specifically, the staining was carried out with mouse igGl labeled with FITC / mouse lgGl labeled with mouse RDl / IgGl labeled with PC5 and mouse human anti-CCR7 antibody labeled with FITC / mouse human anti-CD45RA antibody labeled with RDl / mouse human CD62L anti-i antibody labeled with PC5. These stained cells were analyzed with a flow cytometer and the percentages' of CD45RA + CCR7 + T cells and CD45RA + CD62L + T cells were calculated. The results are shown in the Tables 28 and 29.

Table 28 T cells CD45RA + CCR7 + (%) Control (Without immobilization of CH-296) 27.2 CH-296 60.5 Table 29 CD45RA + CD62L + T Cells (%) Control (Without CH-296 Immobilization) 71.3 CH-296 84.0 From the results of Tables 28 and 29, in the CH-296 group, the results of the highest CD45RA + CCR7 + T cell population and the highest CD45RA + CD62L + T cell population were obtained, compared to those of the group of control. (3) Cultivation Using Feeder Cells Spot Cells (1) The cells after the expansion obtained in the point (1) of Example 9 were adjusted with 5HRPMI to have a density of 2 x 106 cells / mL and the suspension of! The cells were placed in a 24-well cell culture plate in a volume of 0.5 mL / each well. In addition, the I Own PBMCs prepared in the point (1) of Example 1 were 1 suspended in 5HRPMI, the suspension was then irradiated with X-rays (0.90 C / kg) and, resuspended in 5HRPMI to have a density of 2 x 106 cells / mL (to provide feeder cells). These feeder cells were added to the 24-well cell culture plate I mentioned above in a volume of 0.5 mL / each well and the plate was cultured in a 5% CO 2 incubator at 37 ° C for 7 days. During this period, the culture was carried out without adding cytokines at all IL-2. (4) Analysis of Annexin V + 7AAD + I Cells The cells obtained in point (3) of Example i 9 were harvested and stained according to the protocol of the Annexin V / 7AAD Kit (manufactured by Beckman Coulter) in order to determine the cells that underwent apoptosis. The cells were subjected to flow cytometry and a percentage of Annexin V + 7AAD + cells was determined, ie the cells that underwent the apoptosis for each cell population. The results are shown in Table 30.

Table 30 Annexin cells V + 7AAD + (%) Control (Without immobilization of CH-296) 57.6 J CH-296 44.9 - It was shown from the results of Table 30, in the group in which an immobilized culture equipment with CH-296 was used in an early stage of expansion of the T-cell population, that the population of cells Annexin V + 7AAD + was lower so that the percentage of apoptotic cells was lower, compared to those in the control group. In other words, it was clarified that cells that had high viability can proliferate dominantly, even under the environment of, for example, a low concentration of IL-2 through the use of a CH-296 fragment in the population expansion. of T-cells. Example 10: Studies on the Effects of T-Cell Transfers Using a Singén-Tumor Model in Mouse In order to confirm the effects on T-cell tumors subjected to expansion using CH-296, conducted a test using a syngeneic mouse tumor model. i I (1) Preparation of Mouse CH-296 Mouse CH-296 as shown in SEQ ID I NO: 23 of the Sequence Listing was obtained by designing an I mouse fibronectin fragment based on a sequence of human CH-296 and by constructing a plasmid according to a conventional method, thereby obtaining the mouse CH-296 in a genetic engineering manner using the plasmid. In other words, the HBlOl strain of Escherichia i coli harboring the plasmid was cultured and subjected to induction for expression. Afterwards, the cells were destabilized to prepare crude proteins and the proteins were then subjected to the purification with a cation exchange column, a column of! anion exchange or gel filtration column i to provide a proposed protein. The protein obtained was subjected to bacterial filtration and then it was! stored at -80 ° C until use (2) Preparation of Splenic Lymphocytes from Cancer Carrier Mice I I IMC carcinoma i (hereinafter referred to as "IMC") was implanted in the abdominal cavity of mice CDFi (manufactured by Nippon SLC, Inc.) to generate ascites and ascites were implanted to different mice every 7 days and subcultivated. The ascites on the seventh day from the subculture were harvested,) washed with PBS and then I, 7 suspended in PBS to have a density of 5 x 10 cells / mL. This suspension of cells was implanted subcutaneously to the right side of the abdomen of CDFx mice in a volume of 0.1 mL, to form a solid tumor. After 21 days, the spleen was removed and homogenized in the medium RPMI 1640 using a glass slide. A portion i of the seven mice was collected together in a tube using the RPMI 16,40 medium to form a volume of 45 mL and the mixture was allowed to stand on ice for 5 minutes. The mixture was then transferred to a completely new I tube through a 40 μm Cell Filter (manufactured by Becton Dickenson). After centrifugation, the supernatant was removed and the residue subjected to hemolysis including the process steps consisting of suspending the residue in 2 mL of ACK buffer (NH4C1 0.15 M, 0.01 M KHC03, Na2EDTA 0.01 i mM pH 7.4) add 2 mL of the ACK buffer to the same to suspend the mixture and then add the RPMI 1640 medium to form a volume of a suspension I of 50 mL cells. After centrifugation, the supernatant was removed, the residue was suspended in 10 mL of RPMI 1640 medium and the suspension was transferred to a completely new I tube through the Cell Filter. The middle RPMI 1640 was added for! make up a volume of a cell suspension of 40 mL. Then, the cell suspension was centrifuged, the supernatant was removed and then the residue was suspended in a mixture prepared by mixing equivalent volumes of RPMI 1640 medium and CP-1. I contained HSA at 8% and the suspension was stored in liquid nitrogen until use. , (3) Immobilization of Anti-CD3 Antibody from Mouse and Fragment CH-296 of Mouse i A mouse anti-mouse-CD3 antibody and a mouse CH-296 fragment were immobilized on a culture kit used in the following experiment. Specifically, 400 μL of each acetate buffer (pH 5.3) containing a mouse anti-CD3 antibody (manufactured by R &D Systems) (final concentration: 14 μg / mL) was added to the cell culture plate. 24 wells and culture equipment were incubated overnight at 4 ° C. After the CH-296 prepared in item (1) of Example 10 was added to have a final concentration of 25 μg / mL and the culture kit was incubated at room temperature for an additional 5 hours. Immediately, before use, an acetate buffer containing the antibody and the mouse CH-296 were removed by aspiration of these culture equipment and then each well was washed twice with PBS and then once with the RPMI 1640 medium and culture equipment He stuck to every experiment. (4) Purification of Splenic Lymphocytes with Nylon Fibers - The splenic lymphocytes prepared at point I (2) of Example 10 were purified using nylon fibers in order to increase the purity of the lymphocytes.

A 10 mL syringe (manufactured by TERUMO CORPORATION) was filled with 0.6 g of nylon fibers (manufactured by Wako Puré Chemical Industries, Ltd.), equilibrated with 1 PBS and then sterilized at 121 ° C for 20 minutes. This column was balanced with the RPMI 1640 medium that contained % FBS (manufactured by DAINIPPON PHARMACEUTICAL CO., LTD.) And incubated at 37 ° C in a 5% C02 incubator for 1 hour. The splenic lymphocytes prepared at point (2) of Example 10 were suspended in 2 to 3 mL of the RPMI 1640 medium containing 10% FBS at a density so that it did not exceed 2 x 108 cells, the suspension was applied to the column and then the column was incubated at 37 ° C 1 in a 5% CO 2 incubator for 1 hour. Fifteen milliliters of RPMI 1640 medium containing 10% FBS pre-heated at 37 ° C were added to the column and the eluted cells were harvested. i (5) Expansion of a Population of T Cells from Mouse The lymphocytes prepared in point (4) of Example 10 were suspended in RPMI 1640 medium containing I 10% FBS, a mixture of 0.1 mM NEAA, 1 mM sodium pyruvate and 50 μM 2-mercaptoethanol (manufactured by Nakalai Tesque, Inc.) (hereinafter referred to as "medium mRPMI"), to have a density of 1.5 x 106 cells / mL. Then, the mRPMI medium was previously added to the immobilized plate with the mouse anti-CD3 antibody and the mouse CH-296 prepared in point (3) of Example 10 in an I volume of 0.7 mL / well, the suspension. of previous I cells was added thereto in a volume of 0.5 mL / each well and these plates were cultured at 37 ° C in 5% C0. (zero day of cultivation). On the second day of culture, the cell suspension was diluted using the mRPMI medium to have a density of 1 x 10 5 cells / mL and a complete amount was transferred to a totally new 75 cm 2 cell culture flask which was not nothing was immobilized. At that time, mouse ILr2 (manufactured by R &D Systems) was added to have a final concentration of 100 U / mL or mouse IL-7 (manufactured by E. &D Systems) was added to have a final concentration of 10 ng / mL. On the fifth day of the culture, the subculture was carried out in the same manner as on the second day of cultivation except that the cell suspension was diluted to have a density of 1.5 x 106 cells / mL. On the seventh day of culture, the cells were harvested and clamped to a test with the following syngeneic tumor model. (6) Evaluation of Tumor Rejection Action of the Transfer of a T Cell Population in a Singinal Tumor Model of CDFi-BMI The BMI was implanted subcutaneously to the right side of the abdomen of six week old low CDFi mice anesthesia in the same manner as in item (2) of Example 10. The cells prepared at point (5) of Example 10 were suspended in PBS to have a density of 3 x 10 8 cells / mL and the suspension was administered. of the veins of the tails of the mice in an amount of 0.1 mL (zero day of administration). Mouse IL-2 (3 x 104 U / 0.2 mL) was administered to the abdominal cavity for four consecutive days from day zero administration. As a control, a group was established without the administration of the expanded cells. The size of the tumor was expressed as the tumor area (cm2) by regularly determining the length and width of the tumor up to 21 days after the implantation of BMI. The results are shown in Figure 2. Figure 2 is a graph showing the number of days since the BMI implant and the tumor size, where the solid triangles indicate the control group and the solid circles indicate the group administered with T cells. - As shown in Figure 2, it was discovered that the group administered with the cells that were cultured in the presence of CH-296 (the group administered with T cells) significantly suppresses the formation of a tumor. Example 11: Evaluation of an Expanded Human T Cell Population Using CH-296 by Induction of GVHD in NOD / Scid mice. (1) Expansion of a Cell Population In order to prepare the cells in a large quantity, a culture equipment was changed. Specifically, 21 mL of PBS containing a human anti-CD3 antibody (final concentration: 5 μg / mL) was added to a 175 cm2 culture flask (manufactured by Becton Dickenson). At that time, CH-296 was added to the group with the addition of CH-296, to have a final concentration of 25 μg / mL and the culture equipment was incubated at room temperature for I 5 hours. Immediately before use, the PBS containing the antibody and CH-296i were removed by aspiration of these culture equipment and then each flask was washed twice with PBS and once with the RPMI medium. Fresh PBMCs were prepared by collecting 54 mL of blood from normal human individual donors. The PBMCs were suspended in medium GT-T503 containing 0.5% own plasma and 0.2% HSA (hereinafter referred to as "GT-T503 with 0.5% own plasma"), to have a density of 0.5 x 10G cells / mL, the suspension was added in a volume of 21 mL each to a flask immobilized with an anti-CD3 antibody or a flask immobilized with an anti-CD3 antibody and CH-296, which was previously prepared and IL-2 was added thereto for have a final concentration of 1000 U / mL. These flasks began to be cultivated at 37 ° C in C02 at 5% (zero day of culture). The next day, the GT-T503 with its own 0.5% plasma was added to the flask in a volume of 49 mL each! and IL-2 was added to the culture medium to have a final concentration of 10 μU / mL based on the medium of 1 culture. On the fourth day from the start of the culture, 42 mL of the culture medium of each group and 158 mL of the GT-T503 with its own plasma at 0.5% were transferred to a gas permeable culture bag (600 cm2, manufactured by TAKARA BIO , INC, commercial product code: KB610) to which nothing was immobilized. In addition ^ IL-2 was added to the same i to have a final concentration of 500 U / mL and the culture continued. On the sixth day 'of the start of the culture, the GT-T503 with its own plasma at 0; 5% in a volume of 400 mL and the IL-2 were added to it to obtain a final concentration of IL-2 of 500 U / mL (600 mL of culture medium).

After two days, half of the volume of the culture medium was removed from the 6001 mL of culture medium and the GT-T503 with its own plasma at 0.5% of the same volume and IL-2 (final concentration: 500 U / mL ) were added to it (eighth day of cultivation). On the eleventh day after the start of culture, the GT-T503 medium containing HSA at 0. 2% in a volume of 600 mL and IL-2 were added to have a final concentration of IL-2 of 500 U / mL and the culture continued until the fourteenth day. Cells after culture were suspended in a liquid storage I composed of equivalent volumes of CP-1 i i containing 8% HSA and RPMI 1640 medium and suspension i was stored in liquid nitrogen until use. (2) Clustering J The day before the administration of the cells, the body weights of diejz NOD / scid mice for 8 weeks! of age (produced from CLEA Japan, Inc.) were measured and grouped into 4 groups. The members of the groups in this Example were as follows. Group A: Solvent + Feeder + Human IL-2 + Anti-Asialo Antibody GM1 1 Group B: PBLs + Feeder + Human IL-2 + Anti-Asian Antibody GM1 i Group C: Cells after Expansion (Without Immobilization of CH-296) '+ Feeder + Human IL-2 + i Anti-Asialo GM1' Group D: Cells after Expansion (with Immobilization of CH-296)? + Feeder + Human IL-2 + i Anti-Asialo GM1 i Group A and Group B met n = 2 and Group C and Group D met n = 3. (3) Administration of Anti-Asialo Antibody GM1 It has been known that treatment with anti-asialo antibody GM1 serves to remove NK cells in NOD / scid mice, thereby increasing the grafting of human cells. On the day before administration of the explanted cells, 20 μL of an anti-yeast antibody GM1 (manufactured by Wako Pure Chemicals Industries, Ltd.) was diluted with physiological saline containing 0.4% HSA (hereinafter referred to simply as as "physiological saline solution to which 0.4% HSA was added") and a 0.4 mL portion was administered to the abdominal cavity. (4) Preparation! of Administered Cells and Administration The expanded cells which were stored frozen in the point (1) of Example 11 and the frozen stored PBMCs of the same donor prepared in the same manner as in the dot (1) of Example 1 were rapidly fused in a water bath at 37 ° C. The concentration of peripheral blood lymphocytes (hereinafter referred to as "PBLs") was calculated by assuming that a percentage of the CD3 -positive cells of the PBMCs was 70%. Using a physiological saline solution to which 4% HSA was added as a solvent, a part of PBLs was suspended in the solvent as feeder cells in an amount of 0.5 I x 107 cells / mouse and the PBLs and the cells after the expansion were each suspended in the solvent in an amount of 1.0 x 10 8 cells / mouse so that each has a count of Necessary cells, as cells for administration. All mice were irradiated with X-rays (0.090 C / kg) before administration of the cells and 0.3 mL of the provided cells were administered in the abdominal cavity, starting sequentially from Group A. (5) Administration of IL- 2 Human Human IL-2 used in the expansion of T cells was prepared in a solution with physiological saline to which 0.4% HSA was added to have a concentration of 2 x 104 U / mouse and 0.2 mL of the solution I was administered to the abdominal cavity of the mouse starting from those mice to which the administration of the cells was given at point (4) of Example 11. IL-2 administered once a day for 4 consecutive times from the day of administration of the cells. ! (6) Change with the Time in the Body Weight of Mice After Cell Management! I The body weights were determined in an appropriate interval from the day of administration until the twenty-first day. As a result, in Group B administered with PBLS, a weight loss was observed from the eighth day and a mouse died on the thirteenth day. In the other Groups no mouse died until the twenty-first day and all cases survived. When a percentage of weight loss was observedalthough some fluctuations were found in Group A and Group C during the course of administration, the original body weight was maintained on the twenty-first day and in Group D a slight weight loss trend was observed. In the Xeno-GVHD reaction in which weight loss I is used as an index, it was shown that the reaction was predominantly found in PBLs, showing that cells expanded under the conditions of immobilization with CH-296 were more effective than those under the conditions without immobilization. (7) Grafting of Human T Cells in the Spleen The different Groups of Group B were subjected to the removal of the spleen of the anatomy on the twenty-first day from the beginning of the administration of the cells and a percentage of the CD3-positive cells The spleen in the spleen was analyzed by means of flow cytometry. Since a mouse died on the thirteenth day in Group B, this mouse was subjected to the removal of the spleen immediately after death and the remaining mouse He also underwent an autopsy on the thirteenth day and was subjected to the removal of the spleen. The excised spleen was homogenized with a glass slide and the homogenized spleen was filtered with a nylon mesh and subjected to a centrifuge procedure. The supernatant was removed, the residue was subjected to hemolysis with the ACK buffer and then washed with RPMI 1640 medium to prepare the cells. Staining was carried out using a mouse human anti-CD3 antibody labeled with FITC (manufactured by DAKO DENMARK A / S). As a result, it was shown that the percentage of CD3-positive cells of the splenic cells in Group B analyzed on the thirteenth day was approximately 70% and that in Group D analyzed on the twenty-first day it was approximately 59%. The percentage of positive cells was less than 1% in Group A and several percentages even in Group C i. It could be observed from the results that cells expanded under the conditions of immobilization with CH-296 had a high percentage of graft in the spleen compared to those under conditions without immobilization and increased the percentage for 21 days. Example 12: Lymphocyte expansion with the Addition of IL-2, IL-12, IFN-? o Anti-IL-4 Antibody (1) Immobilization of Human Anti-CD3 Antibody and Fragment CH-296 j I The same procedures were carried out as in item (2) of Example 1, except that an ACD-A solution (pH 5.0) was used in the immobilization of the human anti-CD3 antibody I and the CH-296 fragment and that the amount of the ACD-A solution added to the 12-well cell culture plate was changed to 0.45 mL. i (2) Expansion of a Population of T Cells Using the Medium GT-T503 The PBMCs prepared in the point (1) of Example I 1 were suspended in GT-, 0.5% T503 to have a density of 0.25 x 106 cells / mL and then a 0.5% GT-T503 medium was previously added to the plate immobilized with the human anti-CD3 I antibody or the plate immobilized with the human anti-CD3 antibody and CH-296, prepared at point (1). ) of Example 12 in a volume of 0.5 mL / well. Cell pellets were added in a volume of 1 mL / each well and these plates were cultured at 37 ° C in 5% C02 (zero day of culture). On the fourth day from the start of the culture, the culture medium of each group was diluted approximately 14 times with the medium GT-T503 to the 0. 5% and 6.3 mL of the dilution were transferred to a completely new 25 'cm cell culture flask to which no immobilization was made. The cultivation continued and in the seventh! day, the culture medium of each group was diluted ! approximately 2 times with the 0.5% GT-T503 medium and 6.3 i mL of the dilution were transferred to a totally new 25 cm2 cell culture flask I to which no immobilization was done. On the eleventh day from the start of the culture, the cell culture medium of each group was diluted approximately 2-fold with the GT-T503 medium containing 0.2% HSA and 12.6 mL of the dilution were transferred respectively to a flask of culture of i cells of 25 cm2 totally new to which no immobilized I nothing. At this point, IL-2 was added from the zero day of culture to have a final concentration of 100 U / mL. i In addition, for agents other than IL-2, on the fourth day of culture, (IL-12 (manufactured by R &D; Systems) was added to have a final concentration of 50 IU / mL, IFN-? (manufactured by R &D Systems) was added to have a final concentration of 20 ng / mL or an anti-human IL-4 antibody (manufactured by Becton I Dickenson) was added to have a final concentration of i 2 μg / mL; on the seventh day and the eleventh day of culture, each cytokine and antibody were added to the recently added medium to have the final concentration mentioned above. On the fourteenth day from the start of culture, the number of live cells was counted by means of the trypan blue staining method1 and the fold i of expansion was calculated in a comparison of the number of the cells counted with the number of cells at the beginning of the culture. The results are shown in Table 31. i I I Table 31 i i i Expansion Bending (times) __________ i Control (Without Immobilization of CH-296)] x 388 CH-296! X416 I As shown e? Table 31, in the group in which a culture equipment immobilized with CH-296 was used in an early stage of T cell expansion, the expansion fold of the T cell population was high, compared with that of the control group. (3) Analysis of T Cells CD45RA + CCR7 + and T Cells CD45RA + CD62L + '! I The cells prepared in point (2) of Example 12 were stained with each antibody and analyzed in the same manner as in item (4) of Example 1, with the proviso that the combinations of the antibodies were i in the following manner. Specifically, the staining was carried out with the mouse IgGl labeled with FITC / mouse IgGl labeled with RDl / mouse IgGl labeled with PC5 + mouse IgGl labeled with ECD as a negative control, the human anti-CD45RA antibody against mouse labeled with 1 RDl / mouse human anti-CCR7 antibody labeled with FITC / mouse human anti-CD4 antibody labeled with ECD / mouse human anti-CD8 antibody labeled with PC5 or mouse anti-CD45RA human antibody labeled with RDl / mouse human anti-CD62L antibody labeled with FITC / anti-CD4 antibody Human mouse labeled with ECD / mouse human anti-CD8 antibody labeled with PC5. These stained cells were subjected to flow cytometry and the percentages of CD45RA + CCR7 + T cell and CD45RA + CC62L + T cells were calculated in the entire region of T cells in the CD8 + T cell region or in the CD4 + T cell region. The results are shown in Table 32.

Table 32 CD45RA T Cells + CD45RA + T Cells CCR7 + (%) CD62L + (%) Whole T Cells Control (Without Immobilization 13.8 25.8 of CH-296) j CH-296 40.2 58.4 CD8 + T Control Cells (Without Immobilization i 17.1 33.4 of CH-296) 'CH-296 47.4 69.7 T CD4 + Control Cells (Without Immobilization 7.7 11.6 of CH-296) CH-296 21.8 25.0 As shown e? Table 32, in the group in the which was used a culture kit immobilized with the CH-296 fragment, the results of the highest CD45RA + CCR7 + T cell population and the I population of CD45RA + CD62L + T cells were obtained in the T cells during the I culture, in comparison with those of the control group. i Was clarified from the above in the culture in which i IL-2, IL-12, IFN-α, a human anti-IL-4 antibody were added to a GT-T503 medium that the population of T-cells could be expanded while increasing the percentage of the CD45RA + CCR7 + T cell CD45RA + CD62L + population by allowing the CH-296 fragment to coexist at an early stage of expansion. Example 13: Allogenic MLR of an Expanded Cell Population Using a Gas Permeable Culture Bag After Stimulation in an Early Stage of I CH-296 from Separate PBMCs Recently i (1) Separation of PBMCS from Fresh Blood Fifty milliliters of blood was collected from an individual human normal donor from whom informed consent was obtained and then the PBMCs were separated from the blood collected from ! the same way as at the point (1) of Example 1. The collected PBMCs were subjected to the trypan blue staining and staining methods.

Turk (Turk's stain solution: manufactured by i Nakalai Tesque Inc.), calculating the number of cells and were subjected to each experiment as they were without frozen storage. ! (2) Immobilization of Human Anti-CD3 Antibody and CH-296! The immobilization! it was carried out in the same way as point (1) of 'Example 6. (3) Expansion of a Population of T Cells The PBMCs prepared at point (1) of Example 13 were suspended in GT-T503 with their own plasma at 0.5% to have a density of 0.5 x 106 cells / mL to prepare a suspension of cells. Afterwards, the GT-T503 0. 5% was previously added to a flask immobilized with the human anti-CD3 antibody or a flask immobilized with the human anti-CD3 antibody; and CH-296, prepared in item (2) of Example 13, in a volume of 21 mL / flask and the above cell suspension was added thereto in a volume of 9 mL / flask. The IL-2 was added thereto to have a final concentration of 1000 U / mL and these flasks were subjected to the culture at 37 ° C in 5% C02 (zero day of culture). On the fourth day after the start of the culture, 21 mL of the culture medium of each group and 279 mL of the GT-T503 with own plasma at 0-5% were transferred to a gas-permeable culture bag (300 cm2, manufactured by TAKARA BIO, INC., Commercial code: KB610) to which nothing was immobilized and IL-2 was added in addition to the same to have a final concentration of 500 U / mL. The culture was continued and on the eighth day 150 mL of the cell culture medium of each group was removed from the culture bag, leaving 150 mL of the cell culture medium in the bag and the remaining culture medium was diluted 2 times with the GT-T503 with its own plasma at 0.5%, to form a volume of 300 mL of the cell culture medium of each group; IL-2 was additionally added to each group to have each a final concentration of 500 U / mL. On the eleventh day of initiation of culture, 300 mL of the 0-5% GT-T503 medium containing 0.2% HSA was added thereto to dilute 2 times the cell culture medium of each group. IL-2 was additionally added to each group to each have a final concentration of 500 μU / mL. On the fourteenth day from the start of culture, the number of live cells was counted by the trypan blue staining method and the fold expansion was calculated by comparing the number of counted cells with the number at the start of culture. . The results are shown in Table 33.

Table 33! Expansion Bending (times) Control (Without Immobilization of CH-296) x 885 CH-296 X 1271 As shown in Table 33, still in one case! I where the cells were cultured in a gas permeable culture pocket i, in the group in which an immobilized culture kit with CH-296 was used at an early stage of expansion of the T cell population (referred to herein) successive as "the CH-296 group"), fold expansion of the T cell population was high compared to that of the control group. I (4) Analysis of CD45RA + CCR7 + T cells, CD45RA + CD62L + T cells, CD45RA + CD27 + T cells, CD45RA + T cells CD28 +, CD27 + CD28 + T cells and CD45RA + CDR7 + CD62L + i T cells in a Population of T Cells Cultivated in a Gas Permeable Culture Bag. The cells prepared at point (3) of Example 13 were stained with each antibody and analyzed in the same manner as in item (3) of Example 6. The results are shown in Table 34.

Table 34 Control (Without CH-296 CH-296 Freezing) T Cells CD45RA + CD62L + 77.15% 85.21% T Cells CD45RA + CCR7 + 50.46% 60.17% T Cells CD45RA + CD28 + 65.94% 72.88% T Cells CD45RA + CD27 + 70.93% 85.89% T Cells CD28 + CD27 + 65.54% 76.42% T Cells CD45RA + CCR7 + CD62L + 50.05% 59.32% As shown e? Table 34, in group CH-296, the high values were shown in any of the cellular surface markers, in comparison with those of the control group. (5) Storage of Cultured Cells. Cells on the fourteenth day of the culture prepared at point (3) of Example 13 were subjected to frozen storage and melted in the same manner as in item (4) of Example 5. Cells were attached to each experiment. (6) Allogenic MLR The allogeneic MLR was carried out in the same manner as in item (4) of Example 6 using the cells prepared at item (5) of Example 13, except that the modifications were made to the following way. I Characteristics include stimulator cells and responder cells were prepared to have a cell density of 1 or 2 x 10 6 cells / mL; and on the fifth day, the cells in each well were suspended, the suspension was then divided into two wells of half the volume each, 1 mL of 5HRPMI containing 1000 U / mL of IL-2 was added to each well. and the cultivation continued until the seventh day. i (7) Determination of the Expansion Bending Regarding the cells obtained in point (6) of Example 13, on the seventh day from the start of the culture, the number of live cells was counted by means of the trypan blue staining method and an expansion fold was calculated by comparing the number of cells counted with the number of cells at the beginning of the culture. These results are shown in Table 35.

Table 35 Days of Cultivation Number of Sown Cells Responding Cells at Start of Crop Control (Without Immobilization CH-296 Cells / Well with CH-296) Seventh Day 0.5 x 10"x l .29 X 2.24 1.0 x 10 ° X 2.29 X 3.72 As shown e? Table 35, in a case where the allogeneic MLR was carried out using the CH-I 296 group, the expansion fold after the reaction was high compared to that of the control group. In other words, it was clarified that when an allogeneic MLR was carried out using cultured cells in which an immobilized culture kit with CH-296 was used in an early stage of expansion of a population of T cells, the I cells that recognize an alloantigen are proliferated in an even larger amount. (8) Cytotoxic Activity Test The cytotoxic activity of the cells on the seventh day from the start of the induction, prepared at point (6) of Example 13 was tested in the same manner as in item (6) of Example 6, except that as the target cells in the determination, the PBMCs themselves or the non-own PBMCs which were subjected to blastogenesis (destruction), with PHA for 7 days were used as target cells labeled with calcein.

During the assay for the cytotoxic activity, a 30-fold amount of K562 cells and target cells labeled with calcein were mixed. A ratio of E / T was established from 90 to 3. The results for the assay of the cytotoxic activity I are shown in Table 36.

Table 36 Number of Cells Cells Objective! E / T Cytotoxic activity (%) Sown at the Start Control (Without CH-296 from Cultivation I Cell immobilization / Pocilio CH-296) 0.5 x 106 Non-Proprietary Cells 90 33.18 49.5 Destroyed with 30 17.17 28.31 PHA 10 6.13 10.81 3 3.66 6.12 Proprietary Cells 90 2.01 2.78 Destroyed with 30 < 0 1.67 PHA 10 < 0 < 0 3 4.07 1.1 1.0 x 106 Non-Proprietary Cells 90 69.5 73.42 Destroyed with 30 53.68 66.57 PHA 10 30.29 43.79 3 11.78 18.82 Proprietary Cells 90 3.72 4.21 Destroyed with 30 2.08 3.59 PHA 10 < 0 1.14 3 < 0 < 0 As shown in Table 36, in a case where an allogeneic MLR of the CH-296 group was carried out, the activity after the area of the group of cultivated cells culture immobilizes the expansion that a 1 population of cells that had a cytotoxic activity specific for even stronger alloantigens could be obtained by means of the allogeneic MLR. Example 14: Allogenic MLR and Induction of CTLs i for MART-1 of an Expanded Cell Population i Using a Gas Permeable Culture Bag with the Stimulation in an Early Stage or Subsequent Culture of CH-296 j (1) Immobilization of 'Anti-CD3 Antibody and Fragment CH-296 A human anti-CD3 antibody and a CH-296 fragment were immobilized in a culture kit used in the following experiment. Specifically, 9 mL each of i PBS containing a human anti-CD3 antibody (final concentration: 5 μg / mL) was added to a gas permeable culture bag! (cultivation area: 75 cm2, manufactured by TAKARA 'BIO, INC., commercial code: KB620). With the addition, CH-296 was added to a group with I addition of CH-296, the same volumes of PBS that contained I a human anti-CD3 antibody (final concentration: 5 μg / mL) and CH-296 (final concentration: 25 μg / mL). After these culture kits were incubated at room temperature for 5 hours, the culture equipment was stored at room temperature until use. Immediately before use, the PBS containing the antibody and CH-296 were removed with a syringe from these culture kits and then each bag was washed twice with PBS and once with the RPMI medium and the bags were attached to each experiment. (2) Expansion of a Population of T Cells The same procedures as those in item (3) of Example 13 were carried out except that the bag prepared in item (1) of Example 14, not the culture flask, was used as a culture equipment in the stimulation with CH-296. The results on the fourteenth day after the start of the culture are shown in Table 37.

Table 37 Expansion Bending (times) Control (Without Immobilization of CH-296) x 500 CH-296 x 967 As shown in Table 37, even in one case where the cells were cultured using a gas-permeable culture bag with stimulation at an early stage i or the subsequent culture of CH-296, in the group in which immobilized culture equipment was used with i CH-296 at an early stage of expansion of the T cell population (hereinafter referred to as "the CH-296 group"), the fold expansion of the T cell population was high compared to that of the control group. . i i (3) Analysis of CD45RA + CCR7 + T cells, cells T CD45RA + CD62L +, CD45RA + CD27 + T cells, CD45RA + I cells CD28 +, CD27 + CD28 + T cells and CD45RA + CDR7 + CD62L + I T cells in a Cell Population Is T Grown in a Stock Market Gas Permeable Culture with Early Stage Stimulation or Subsequent Culture of CH-296 i The cells prepared at point (2) of I Example 14 were stained with each antibody and analyzed in the same manner as in point ( 3) of Example 6. The results are shown in Table 38.

Table 38 Control (Non-immobilization CH-296 of CH-296) CD45RA + CD62L + T cells 71.73% 85.03% CD45RA + CCR7 T cells + 47.49% 67.46% CD45RA + CD28 + T cells - 59.81% 79.83% CD45RA + CD27 + T cells 64.69% 85.37% CD28 + CD27 + T cells 59.99% 82.15% T cells CD45RA + CCR7 + CD62L + 46.14% 65.94% I As shown in Table 38, in group CH-296, high values were shown in any of the I cell surface markers, compared to I those in the control group (4) Allogenic MLR1 I The allogeneic MLR was carried out in the same manner as in item (4) of Example 6 using the cells prepared in item (2) of Example 14. (5) Cytotoxic Activity Assay The cytotoxic activity of the cells on the tenth day from the start of the induction, prepared at I point (4) of Example 14, was tested in the same manner as in item (6) of Example 6, except that the 90-day E / T ratio was recently established. The results for the cytotoxic activity assay are shown in Table 39.

Table 39 Cells Objective E / T Cytotoxic activity (%) -Control (Without Immobilization of CH-296 'CH-296) Non-Proprietary Cells 90 65.05 72.73 Destroyed with PHA 30 44.92 61.43 10 21.81 37.2 3 7.23 17.99 Own Cells 90 0.69 0.17 Destroyed with PHA 30 < 0 < 0 10 < 0 < 0 3 < 0 < 0 As shown in Table 39, in a case where the allogeneic MLR of the CH-296 group was carried out, the cytotoxic activities specific for alloantigens I after the reaction were high, compared to those of the control group. In other words, cultured cells in which an immobilized culture kit with CH-296 was used at an early stage of expansion of the T cell population, it was clarified that a population of cells having a even more potent cytotoxic activity for alloantigens via the allogeneic MLR. (6) Storage of Cultured Cells Cells on the fourteenth day of the culture prepared at point (2) of Example 14 were subjected to a frozen storage and were melted in the same manner as in item (4) of Example 5. The cells were attached to each experiment. j (7) Induction of Specific CTLs for a Antitumor Associated Antigen, (MART-1) Using the cells prepared in item (1) of Example 1 and item (6) of Example 14, the induction of CTLs specific for an antitumor associated antigen (MART-1) was in the same way as in the point (5) of Example 5 and the culture was continued for fourteen days, with the proviso that the modifications were made as follows: Characteristics include the responder cells that were suspended in 5HRPMI to have a density of 1 x 106 cells / mL and the suspension was added to a 24-well cell culture plate in a volume of 0.5 mL / each well; on the third day from the start of culture, half the volume of the culture supernatant was removed and 1 mL of 5HRPMI containing 60 U / mL of IL-2 was then added to each well; after one week, the cultured cells were adjusted to a density of 1.5 to 3.0 x 10 6 cells / mL and the cells that presented an antigen were adjusted to a density of 1.0 x 106 cells / mL and each suspension of cells were added in a volume of 0.5 mL / well; and on the tenth day from the start of the culture, half of the I volume of the culture supernatant was removed and 1 mL of I. I 5HRPMI containing 60 U / mL of IL-2 was then added to i each well. i (8) Test of Cytotoxic Activity - The cytotoxic activity of the CTLs on the I fourteenth day from the start of the induction, prepared at point (7), of Example 14 was tested in the same manner as at the point (9) of Example 6. The results of the cytotoxic activity assay are shown in Table 40. - Table 40 Cells Target E / T Cytotoxic Activity (%) PBMÓs Control (Without Immobilization CH-296 of CH-296) T2 90 3.92 < 0 < 0 30 < 0 < 0 < 0 10 < 0 < 0 < 0 3 < 0 < 0 < 0 T2 + Peptide 90 59.86 61.55 65.59 MART-1 30 61.78 56.21 64.66 10 44.21 43.64 51.79 I 3 7.26 20.36 27.06 As shown in Table 40, in the CTL population produced from the CH-296 group, the specific cytotoxic activity of the CTL population was high, compared to that of the control group and the PBMC group. In other words, it was clarified that cultured cells in which a culture kit I immobilized with CH-296 was used at an early stage of expansion of the T cell population could be induced to I a population of CTL that would it had an even higher specific cytotoxic activity. Example 15: Induction of CTLs Anti-MART-1 to Starting from CD45RA T Cells * CCR7 + CD8 + and CD45RA T Cells CCR7"CD8 + Expanded Cell Derivatives Using a Gas Permeable Culture Bag with Stimulation in an Early Stage or Subsequent Cultivation of CH-296 (1) Isolation of CD45RA + CCR7 + CD8 + T cells and CD45RA T cells "CCR7" CD8 + The cells prepared in point (1) of the Example 1 and those prepared in item (6) of Example 14 were isolated in a CD45RA + CCR7 + CD8 + fraction and a CD45RA fraction "CCR7" CD8 + respectively, in the same manner as in item (1) of Example 7, obtaining whereby a fraction that had 93 to 99% purity. (2) Induction of Specific CTLs for an Antitumor Associated Antigen (MART-1) Using the cells prepared in item (1) of Example 15, the induction of CTLs specific for an antitumor associated antigen (MART-1) was carried out in the same manner as in item (7) of Example 14 and I i culture continued for fourteen days, with the proviso that the I modifications were made j as follows: Characteristics include responder cells that were fused in 5HRPMI to have a density of 1 x 10 cells / mL and the suspension was added to a 48-well cell culture plate i in a volume of 0.25 mL / each well; on the first day from the start of culture, 0.5 mL of 5HRPMI containing 60 μU / mL of IL-2 was added to each well; on the third day from the start of the culture, half of the volume of the culture supernatant was removed and 0.5 mL of 5HRPMI containing 60 U / mL of IL-2 was then added to each well; After one week, the cultured cells were adjusted to a density of 0.2 to 1.7 x 10 6 cells / mL and the cells presented with an an antigen was adjusted to a density of 1.0 x 106 cells / mL and each cell suspension was added to a 24-well cell culture plate in a volume of 0.5 mL / well; and on the tenth day from the beginning of the crop, half! The volume of the supernatant of 1 culture was removed and 1 mL of 5HRPMI containing 60 U / mL of IL-2 was then added to each well. (3) Determination of the Expansion Bending As for the cells obtained in point (2) of Example 15, on the fourteenth day from the start of the I culture, the number of living cells was counted by means of the I method of trypan blue staining and a fold expansion by comparing the number of cells counted with the number of cells at the start of the culture. i The results are shown in Table 41.

Table 41 ¡i n '? Cells at the Start of Cultivation 'Control Expansion Bending (Without CH-296 CH-296 Immobilization) CD45RA + CCR7 + CD8 + T cells (%) x 2.36 x 5.38 I CD45RA T cells "CCR7" CD8 + (%) x 3.14 x 4.96 As shown in Table 41, in the I CTL-induced population using CD45RA + CCR7 + CD8 + T cells isolated from cultured cells in which an immobilized I culture kit with CH-296 was used in an early stage of expansion of the T-cell population (hereinafter referred to as "the CH-296 group"), the fold expansion of the CTL population was high, compared to that of the control group. In other words, it was clarified that the CTL population was obtained in a larger amount by carrying out the induction of CTLs of the T cells CD45RA + CCR7 + contained in a high percentage of cultured cells in which a culture kit was used immobilized with CH-296 at an early stage of the expansion of the T cell population. (4) Cytotoxic Activity Assay Cytotoxic activity of the cells on the fourteenth day from the start of the induction, and prepared at point (2) of Example 15 was tested in the same manner as in item (9) of Example 6, except that a ratio of E / T was established from 30 to 3.

The results for the cytotoxic activity assay are shown in Table 42.

Table 42 E / T Cell Cells Cytotoxic Activity (%) Crop Start Target PBMCs Control (Without CH-296 Freeze CH-296) T2 T Cells 30 < 0 N.T. < 0 CD45RA + 10 < 0 < 0 < 0 I CCR7 + CD8 + 3 < 0 < 0 < 0 T2 + Peptide 30 56.77 N.T. 73.96 I MART-1 10 45.7 64.91 73.62 35.36 58.58 58.49 j T T2 30 cells < 0 N.T. < 0 CD45RA "10" < 0 < 0 < 0 CCR7 CD8 * 3 < 0 < 0 < 0 T2 + Peptide 30 9.75 N.T. < 0 I MART-1 10 < 0 1.5 < 0 I 3 < 0 1.5 3.87 N. T. : Not tested. i | As shown e? Table 42, in population I of CTL induced using the CD45RA + CCR7 + CD8 + T cells isolated from the CH-296 group, the specific cytotoxic activities of the CTL population were high, compared to those of the control group and the PBMC group . In addition, the specific cytotoxic activities were high in comparison with those of the CTL population induced using the CD45RA T cells "CCR7" CD8 +. In other words, it was clarified that CD45RA + CDR7 + CD8 I + T cells which are contained in a high percentage in cultured cells in which immobilized culture equipment was used CH-296 at an early stage of expansion of the T cell population could be induced to a CTL population of which the specific cytotoxic activities were even higher. i Example 16: Determination of the Capacity to Recognize an Antigen of the CTLs (1) Induction of CTLs Specific for an Antigen Antitumor Associate (MART-1) I Using the cells prepared in item (1) of Example 1 and item (6) of Example 14, the induction of CTLs specific for an antitumor-associated antigen I (MART-1) was brought to in the same manner as in item (7) of Example 14 and the cultivation continued for fifteen days. J I (2) Determination of the Capacity to Recognize an Antigen The ability to recognize an antigen from CTLs on the fifteenth day from the start of the induction, prepared at point (1) of Example 16 were determined in the same way as in point (2) of i Example 8. The results of the determination of the ability to recognize an antigen are shown in the Table 43. The ability to recognize an antigen was expressed as a relative value of the cytotoxic activity at each concentration of the aggregated peptide when the cytotoxic activity at a concentration of a peptide added to the target cells of 10 μM was assumed to be 100.

Table 43 Final Peptide Concentration Relative Value of Cytotoxic Activity at 10 μM Aggregate to Target Cells Aggregate Peptide i (μM) PBMCs Control (Without CH-296 CH-296 Immobilization) . 0 100 100 100 1.0 101.58 86.33 99.59 0.1 88.24 80.78 89.13 0.01 72.03 64.48 67.28 0.001 25.18 26.72 48.35 0.0001 11.47 2.45 21.59 0.00001 6.32 3.07 15.6 or 8.4 < 0 8.17 As shown e? Table 43, in the CTL population induced from the CH-296 group, still the target cells to which an antigen peptide was added at an even lower concentration were eliminated, compared to those of the control group and the group of PBMC, so that the ability to recognize a specific antigen of the CTL population was high. In other words, it was clarified that cultured cells in which immobilized culture equipment with CH-296 was used at an early stage of expansion of the 1 I population of T cells could be induced to a CTL population that had a capacity to recognize an even higher antigen. Example 17: Capacity Assessment for Produce C-cell Cytokines CD45RA * CCR7 * Derived from Expanded Cells Using a Culture Bag Gas Permeable with Stimulation in an Early Stage and Subsequent Culture of CH-296 (1) Isolation of CD45RA + T Cells CCR7 + and T Cells CD45RA "CCR7" The cells prepared at point (6) of Example 14 were stained and subjected to the classification of! the same way as in point (3) of Example 3. I (2) Immobilization? of the Anti-CD3 Human Antibody and the Anti-CD28 Human Antibody and Cell Stimulation; In order to stimulate the isolated cells, a human anti-CD3 antibody and a human anti-CD28 antibody were immobilized on a culture plate in the same manner as in point (4) of Example 3. Each population of cells obtained in point (1) of Example 17 was suspended in 0.5% GT-T503, cells were added to each well of the plate prepared to have a density of 2 x 10 5 cells / 0.2 mL and if they were cultured at 37 ° C in C02 al %. After 24 hours, the culture supernatant was collected and used for the experiment for the determination of cytokine according to a method of ELISA I (3) Production Determination of IL-2 of Agreement with an ELISA Method In the expansion of lymphocytes for clinical purposes, a gas-permeable culture bag has been used that is capable of culturing the cells in a closed I system. Even when the cells were expanded using the bag in Example 14, in the CH-296 group, the results were obtained for a higher percentage of CD45RA + CCR7 + T cells, e? comparison with those of the control group. In order to confirm that this population of CD45RA + CCR7 + T cells maintained their property as natural type I cells, the ability to produce IL-2 from CD45RA + CCR7 + T cells and CD45RA T cells "CCR7" was evaluated in the same way as at point (6) of Example 3. The results are shown in Table 44.

Table 44 j Immobilization of CH-296) IL-2 (pg / mL) CD8 + T Cells CD45RA + T Cells CCR7 + 347.2 CD45RA Ti Cells "CCR7" < 0 CD8 T cells "T cells CD45RA + CCR7 + 1686.7 T cells, CD45RA" CCR7"215.6! As shown in Table 44, the production of IL-2 in CD45RA + CCR7 + T cells was dominant in both CD8 + T cells and CD8d T cells As already shown in (6) of Example 3, it was also shown in this example that the population of CD45RA + CCR7 + T cells from i-frozen cells that were cultured in a bag using CH-296 had properties that serve as natural T-cells. Example 18: Analysis of CD45RA + CCR7 + T cells in an Expanded T Cell Population Using H-296, CH-271, H-271 or C-CSl | (1) Immobilization of the Human Anti-CD3 Antibody and a Fragment H-296, CH-271, H-271 or C-CSl The same procedures were carried out as in item (1) of Example ^, except that the CH-296 fragment was not used but instead each of the fragments H-296, CH-271, H-271 or C-CSl was added instead of the group with the addition of the fragment to have a final concentration of 25 μg / mL. i (2) Expansion of a Population of T Cells The same procedures as those of point (1) of Example 3 were carried out except that on the fourth day from the start of culture, culture medium i was diluted approximately 14 times and 10 mL of the dilution were added to a 25 cm2 cell culture flask totally new (manufactured by Corning) and that the subculture was not carried out on the seventh day. The culture I continued for 9 days. The results of the expansion times on the eighth day from the start of the culture are shown in Table 45.

Table 45 Expansion (times) Control (Non-immobilization of CH-296) x 207 H-296 x 264 CH-271 x 250 H-271 x 221 C-CS1 x 252 As shown in Table 45, in the group in which an immobilized culture kit with H-I 296, CH-271, H-271 or C-CSl was used at an early stage of the expansion of the T cell population (hereinafter referred to as " the fragment group "), the fold of! The expansion of the T cell population was high compared to that of the control group. (3) Analysis of CD45RA + CCR7 + T cells For the cells prepared in point (2) of Example 18, a percentage of CD45RA + CCR7 + T cells on the ninth day from the start of the culture was calculated in the same manner as in point (2) of Example 3. The Results are shown in Table 46.

Table 46 CD45RA + CCR7 + T Cells (%) Control (Non-immobilized CH-296) I 38.7 H-296 '58.15 CH-271 56.3 i H-271 i 49.75 C-CS1 i 44.79 As shown in Table 46, in the fragment group, the results of the CD45RA + CCR7 + i cell population were obtained highest in the cell population T during cultivation, compared to that of the control group.

INDUSTRIAL APPLICABILITY According to the present invention, a method for producing a population of T cells is provided. The method is suitably used in, for example, immunotherapy, as a population of T cells containing a high percentage of cells. T capable of expressing CD45RA and at least one member selected from the group consisting of CD62L, CCR7, CD27 and CD28. Accordingly, it is expected that the method of the present invention will contribute greatly to the medical fields.

Claims (22)

1. CLAIMS 1. A method for preparing a population of T cells, wherein the T cell population expresses CD45RA t and expresses at least one selected from group I consisting of CD62L, CCR7, CD27 and CD28, characterized in that the method comprises the step that consists in cultivating a population of cells, comprising T cells, in the presence of fibronectin, a fragment thereof or a mixture thereof. j I I 2. Method j according to claim 1, characterized in that the total culture days comprising the culture step are from 4 to 14 days. I 3. The method; according to claim 1 or 2, characterized in that the culture in the presence of fibronectin, a fragment thereof or a mixture thereof is carried out at least at the beginning of the culture. 4. The method according to claim 3, characterized in that the culture in the presence of fibronectin, a fragment thereof or a mixture thereof is carried out for at least a day or more. 5. The method according to any of claims 1 to 4, characterized in that the step of culture in the presence of fibronectin, a fragment thereof or a mixture thereof is carried out in the presence of a CD3 ligand. 6. The method according to claim 5, characterized in that the CD3 ligand is an anti-CD3 antibody. 7. The method according to any of claims 1 to 6, characterized in that the fragment of fibronectin1 is a polypeptide (m) that i comprises at least any of the sequences of! amino acids shown in SEQ ID NOs: 1 to 8 of the sequence listing i or a polypeptide (n) comprising at least one amino acid sequence having a substitution, deletion, insertion or addition of one or plural number of amino acids in any of the amino acid sequences, 'wherein the polypeptide (n) has a function equivalent to that of the polypeptide (m). 8. The method according to claim 7, characterized in that the fibronectin fragment is a polypeptide comprising all the I amino acid sequences shown in SEQ ID NOs: 1 to 3 and 5 to 8 of the sequence listing. 9. The method of compliance with any of! claims 1 to 18, characterized in that in addition it comprises the step consisting in separating a population of I cells expressing at least one selected from the group consisting of CD45RA, CD62L, CCR7, CD27 and CD28. 10. The method according to any of claims 1 to 9, characterized in that it also comprises the step that consists of transducing a foreign gene into the population of cells. 11. The method according to claim 10, characterized in that the foreign gene is transduced using a retrovirus vector, I adenovirus vector, adeno-associated virus vector, lentivirus vector or simian virus vector. 12. A population of T cells obtained by the method according to any of claims 1 to 11, in! where the T-cell population expresses CD45RA and expresses at least one selected from the i group consisting of CD62L) CCR7, CD27 and CD28. 13. A medicament, characterized in that it comprises as an effective ingredient the population of T cells obtained by means of the method according to any of claims 1 to 11, wherein the population of T cells expresses CD45RA and expresses at least one selected from the group which consists of CD62L, CCR7, CD27 and CD28. 14. A method to treat or prevent a characterized in that it comprises the step of administering to a subject an effective amount I of the population of cells, T obtained by means of method I according to any of claims 1 to 11, wherein the population j of cells T expresses CD45RA and i expresses at least one selected from the group consisting of CD62L, CCR7, CD27 and CD28. 15. The use of the population of T cells obtained by means of the method according to any one of claims 1 to 11, wherein the population of TS cells expresses CD45RA and expresses at least one selected from the group consisting of of CD6.2L, CCR7, CD27 and CD28, in the manufacture of a medicament. 16. A method for preparing a population of T cells, characterized in that the method comprises the step of stimulating the population of T cells obtained by means of the method according to any of claims 1 to 11, wherein the population of T cells expresses CD45RA and expresses at least one selected from the group consisting of CD62L, CCR7, CD27 and CD28, with at least one stimulation factor selected from the group. consists of a cell capable of presenting an antigen, cell that has presented an antigen, antigen, CD3 ligand, CD28 ligand, cytokine, chemokine and cell capable of producing a cytokine 17. A population of T cells, characterized in that it is obtained by means of the method according to claim 16. ii 18. A medicament, characterized in that it comprises as an effective ingredient the population of T cells obtained by means of the method according to I claim 16. A method for treating or preventing a disease characterized in that it comprises the step of administering to a subject an effective amount of the population of cells obtained by the method according to the invention. claim 16. 20. The use of the population of T cells obtained by means of the method according to the claim 16 in the manufacture of a medicine. I 21. A medicament, characterized in that it comprises: (a) a preparation comprising as an effective ingredient, the population of T cells obtained by means of the method according to any of claims 1 to 11, where the population of T cells expresses CD45RA and expresses at least one selected from the group consisting of CD62L, CCR7, CD27 and CD28; and (b) a preparation comprising as an effective ingredient at least one stimulation factor selected from the group that consider a cell capable of presenting an antigen, a cell that has presented an antigen, antigen, ligand CD3, CD28 ligand, cytokine, chemokine and cell capable of producing a cytokine, wherein the preparations are comprised in the medicament as two separate preparations and which are administered simultaneously or separately. 22. A method for treating a disease, characterized in that the method comprises the following steps (a) and (b) which consist of: (a) administering a! patient the population of T cells obtained by means of the I method of conformity j with any of claims 1 to 11, where the T-cell population expresses CD45RA and expresses at least one selected from the I group consisting of CD62L, CCR7 , CD27 and CD28; and (b) administering to a patient at least one stimulation factor selected from the group consisting of a cell capable of presenting an antigen, cells that have presented an antigen, antigen, CD3 ligand, CD28 ligand, cytokine. , chemokine and cell capable of producing a cytokine. '