JP2010094123A - Method for producing lymphocyte - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently producing lymphocytes, applicable to adoptive immunotherapy. <P>SOLUTION: The method for producing lymphocytes is to culture lymphocytes or precursor cells of the lymphocytes in a state in contact with (1) a solid phase immobilized with an anti-CD3 antibody by using a solution containing <5 μg/mL of the antibody and/or (2) a solid phase immobilized with a polypeptide having an amino acid sequence derived from fibronectin by using a polypeptide solution containing <25 μg/mL of the polypeptide. The lymphocytes excellent in the rate of engraftment to a living body and maintenance performance in phenotype can be efficiently produced by the method. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

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

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

  T cells are mainly composed of CD4 T cells having a CD (Cluster of Differentiation) 4 marker and CD8 T cells having a CD8 marker in the periphery. Most of the CD4 T cells are called helper T cells, are involved in assisting antibody production and induction of various immune responses, and differentiate into Th1 type or Th2 type different in the type of cytokine produced by antigen stimulation. The majority of CD8 T cells are cytotoxic T cells that exhibit cytotoxic activity upon antigen stimulation [cytotoxic T lymphocytes, also known as killer T cells, hereinafter sometimes referred to as CTL]. Differentiate.

  For example, in the pathology of cancer, immunotherapy has recently attracted attention as the fourth treatment method following surgery, chemotherapy, and radiation therapy. Since immunotherapy uses the immunity inherent in humans, it is said that the physical burden on the patient is lighter than other treatments. Immunotherapy includes the transfer of lymphokine-activated cells, NKT cells, γδ T cells, etc. obtained by culturing in various ways from CTLs or peripheral blood lymphocytes induced outside the body, antigen-specific CTLs in the body Dendritic cell transfer therapy, peptide vaccine therapy, and Th1 cell therapy that are expected to be induced, and immunogene therapy methods in which genes that can be expected to have various effects are introduced outside the body and transferred into the body are known.

  In immunotherapy, in vitro transfer of lymphokine-activated cells obtained by culturing expanded cells by the action of IL (interleukin) -2 and anti-CD3 antibody from CTLs or peripheral blood lymphocytes induced in vitro. The effects of using fibronectin and its fragments on the issues such as how to maintain cytotoxic activity when expanding induced antigen-specific CTLs and how efficiently lymphocytes can be expanded outside of the body are affected. Have already been studied by the present inventors (for example, Patent Documents 1 to 4).

  In recent years, T lymphocytes used for immunotherapy are far more effective when administered to a living body when naïve T cells or central memory T cells are more undifferentiated than effector T cells that have already been terminally differentiated. It has been reported that a high therapeutic effect can be expected (for example, Non-Patent Document 1). Furthermore, even in dendritic cell transfer therapy and peptide vaccine therapy that are expected to induce antigen-specific CTLs in the body, for example, if there are few naive T cells that can be induced by CTLs in the body of a patient with advanced cancer Because of this, it is often impossible to expect a sufficient effect.

International Publication No. 03/016511 Pamphlet International Publication No. 03/080817 Pamphlet International Publication No. 2005/019450 Pamphlet International Publication No. 2007/142300 Pamphlet

J. et al. Clin. Invest. (Journal of Clinical Investment) 2005, 115, 1616-1626

  Regarding the production of lymphokine-activated cells and cytotoxic lymphocytes, the use of fibronectin and its fragments can improve cell proliferation rate and maintain cytotoxic activity. (For example, Patent Documents 1 to 4). However, considering the current situation that adoptive immunotherapy is diversified, further development of cell proliferation methods other than those described in the above literature has been demanded.

  The present inventors have intensively studied various conditions for lymphocyte culture, particularly expansion culture, and have found the conditions for efficient lymphocyte production, thereby completing the present invention. Furthermore, the lymphocytes produced by the method of the present invention were found to retain long telomeres, and the present invention was completed.

That is, the present invention
[1] A method for producing lymphocytes comprising a step of culturing lymphocytes or lymphocyte precursor cells in the presence of an anti-CD3 antibody and a polypeptide having an amino acid sequence derived from fibronectin,
(1) using a solid phase in which the antibody is immobilized using an anti-CD3 antibody solution of less than 5 μg / mL and / or (2) a polypeptide solution having an amino acid sequence derived from fibronectin of less than 25 μg / mL A method for producing lymphocytes, comprising culturing lymphocytes or lymphocyte progenitor cells in contact with a solid phase on which the polypeptide is immobilized;
[2] The method according to [1], wherein the solid phase is a cell culture equipment or a cell culture carrier,
[3] The method according to [1], wherein the polypeptide having an amino acid sequence derived from fibronectin has a binding activity selected from a VLA-4 binding activity, a VLA-5 binding activity, and a heparin binding activity.
[4] The method according to [1], wherein the polypeptide having an amino acid sequence derived from fibronectin has an amino acid sequence derived from fibronectin selected from a CS-1 domain, a cell adhesion domain, and a heparin binding domain.
[5] The method according to [4], wherein the polypeptide having an amino acid sequence derived from fibronectin is a polypeptide having an amino acid sequence selected from SEQ ID NOs: 9 to 34 in the Sequence Listing.
[6] The method according to [1], wherein the lymphocyte is an antigen-specific cytotoxic T lymphocyte or lymphokine-activated cell, and [7] the method according to [1], further including a step of introducing a foreign gene into the lymphocyte. ,
About.
Moreover, it is related with the lymphocyte obtained by the method of [1] description with a long telomere compared with the lymphocyte obtained by single stimulation of the anti-CD3 antibody.

  The present invention provides an efficient method for producing lymphocytes. This method has a high cell proliferation rate, and the lymphocytes obtained by the present invention are preferably used for, for example, adoptive immunotherapy. Therefore, the method of the present invention is expected to greatly contribute to basic research and the medical field.

The present invention relates to an efficient method for producing lymphocytes suitable for medical use.
In the method for producing lymphocytes using an anti-CD3 antibody and a polypeptide having an amino acid sequence derived from fibronectin, the present invention intensively studies and completes the concentration when the active ingredient is immobilized on a solid phase. It has come.

The present invention will be specifically described below.
(1) Polypeptide having an amino acid sequence derived from fibronectin used in the present invention In the present invention, for example, a fibronectin fragment can be used as a polypeptide having an amino acid sequence derived from fibronectin. The fibronectin fragment may be obtained from nature, or may be artificially synthesized or genetically engineered. The nucleic acid sequence encoding fibronectin or the amino acid sequence of fibronectin, which is information for preparing a fragment of fibronectin, is NCBI RefSeq Accession No. NM_002026 and NP_002017.

  In the present invention, fibronectin fragments include, for example, III-8 (SEQ ID NO: 1), III-9 (SEQ ID NO: 2), III-10 (SEQ ID NO: 3), III-11 (SEQ ID NO: 4), III-12 (SEQ ID NO: 5), III-13 (SEQ ID NO: 6), III-14 (SEQ ID NO: 7), and CS-1 (SEQ ID NO: 8) comprising at least one amino acid sequence constituting the region A polypeptide or a polypeptide comprising at least one amino acid sequence in which one or more amino acids are substituted, deleted, inserted or added in any one of the amino acid sequences, and having a function equivalent to that of the polypeptide. The polypeptide which has is illustrated. As a fragment length, 20-1000 are preferable as a number of amino acids, for example, and 100-800 are more preferable. In addition, in this specification, a plurality is a concept including several pieces, 2-12 pieces are preferable, 2-10 pieces are more preferable, 2-8 pieces are still more preferable, and the following is also the same.

  In addition, as the fragment, those having cell adhesion activity and / or heparin binding activity can be preferably used. The cell adhesion activity can be examined by assaying the binding between the fragment (its cell binding domain) used in the present invention and the cell using a known method. For example, such a method includes Williams D.C. A. These methods [Nature, Vol. 352, pp. 438-441 (1991)] are included. This method is a method for measuring the binding of cells to a fragment immobilized on a culture plate. The heparin binding activity can be examined by assaying the binding of the fragment (its heparin binding domain) used in the present invention to heparin using a known method. For example, Williams D. A. In these methods, by using heparin, for example, labeled heparin, instead of cells, the binding between the fragment and heparin can be evaluated in the same manner.

  Further, as fibronectin fragments, C-274 (SEQ ID NO: 9), H-271 (SEQ ID NO: 10), H-296 (SEQ ID NO: 11), CH-271 (SEQ ID NO: 12), CH-296 (SEQ ID NO: 13) And a polypeptide selected from the group consisting of C-CS1 (SEQ ID NO: 14) and CH-296Na (SEQ ID NO: 15).

  Each of the above CH-271, CH-296, CH-296Na, C-274, and C-CS1 fragments is a polypeptide having a cell binding domain having an activity of binding to VLA-5. C-CS1, H-296, CH-296, and CH-296Na are polypeptides having CS-1 having an activity of binding to VLA-4. Furthermore, H-271, H-296, CH-271, CH-296 and CH-296Na are polypeptides having a heparin binding domain. In addition, CH-296Na is a polypeptide containing from cell-binding domain to CS-1 in plasma-derived fibronectin.

  In the present invention, fragments in which each of the above domains is modified can also be used. The heparin binding domain of fibronectin is composed of three type III sequences (III-12, III-13, III-14). Fragments containing heparin binding domains lacking one or two of the type III sequences can also be used in the present invention. For example, CHV-89 (SEQ ID NO: 16), CHV-90 (SEQ ID NO: 17), CHV, which are fragments in which a cell binding site of fibronectin (VLA-5 binding region, Pro1239 to Ser1515) and one type III sequence are combined. Examples include -92 (SEQ ID NO: 18) or CHV-179 (SEQ ID NO: 19) and CHV-181 (SEQ ID NO: 20), which are fragments obtained by binding two type III sequences. CHV-89, CHV-90, and CHV-92 include III-13, III-14, and III-12, respectively, CHV-179 includes III-13 and III-14, and CHV-181 includes III-12. And III-13, respectively.

  A fragment obtained by further adding an amino acid to each of the above fragments can also be used in the present invention. The fragment can be produced, for example, by adding a desired amino acid to each of the above fragments. For example, H-275-Cys (SEQ ID NO: 21) is a fragment having a heparin binding domain of fibronectin and a cysteine residue at the C-terminus.

  In addition, as long as the desired effect of the present invention is obtained, the fragment used in the present invention is a fragment having a function equivalent to that of the fragment containing at least a part of the amino acid sequence of natural fibronectin exemplified above. It may be a recombinant fibronectin fragment consisting of a polypeptide having an amino acid sequence having substitution, deletion, insertion or addition of one or more amino acids in the amino acid sequence of the constituent polypeptide.

  Furthermore, in addition to the recombinant fibronectin fragment, a modified recombinant fibronectin fragment can be used in the present invention. This modified recombinant fibronectin fragment is a polypeptide containing a plurality of amino acid sequences derived from the same fibronectin in the molecule.

  An example of the modified recombinant fibronectin fragment (hereinafter referred to as a modified fragment) is a polypeptide comprising two or more amino acid sequences (m) and one or more amino acid sequences (n) as essential regions. . Here, the amino acid sequence (m) is an amino acid sequence consisting of one or a plurality of amino acid sequences selected from SEQ ID NOs: 5 to 7, which are part of the heparin binding domain of fibronectin. However, the amino acid sequence (m) is an amino acid sequence not including two or more of the same sequences represented by SEQ ID NOs: 5 to 7, and an amino acid composed of a combination of amino acid sequences represented by SEQ ID NOs: 5 to 7 Is an array. For example, the amino acid sequence (m) does not contain two or more amino acid sequences represented by SEQ ID NO: 5, and the same applies to the amino acid sequence represented by SEQ ID NO: 6 or 7. Two or more amino acid sequences (m) present in the polypeptide each represent the same amino acid sequence.

  As the amino acid sequence (m), a polypeptide having the amino acid sequence having heparin binding activity can be preferably used. The heparin binding activity can be measured by the above method.

  The amino acid sequence (m) is preferably contained in the modified fragment, for example, 2 to 5, preferably 2 to 4, more preferably 2 to 3.

  The amino acid sequence (n) is the amino acid sequence represented by SEQ ID NO: 8, which is the CS-1 domain of fibronectin. The amino acid sequence (n) is preferably contained in the modified fragment, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, and preferably in the modified fragment. It is preferable that the same number of amino acid sequences (m) is contained.

  The positional relationship between two or more amino acid sequences (m) and one or more amino acid sequences (n) in the modified fragment is not particularly limited as long as the desired effect of the present invention can be obtained. For example, it is preferable that the amino acid sequence (n) is linked to the C-terminal side with respect to at least one amino acid sequence (m). In the modified fragment, the amino acid sequence (m) is preferably located on the N-terminal side, and the amino acid sequence (n) is preferably located on the C-terminal side. More preferably, in the modified fragment, the amino acid sequence (m) and the amino acid sequence (n) are preferably located alternately.

  The number of amino acids constituting the modified fragment is not particularly limited, but is preferably 100 to 3000 amino acids, more preferably 150 to 2800 amino acids, and still more preferably 200 to 2600 amino acids.

  Furthermore, the modified fragment may contain other amino acid residues as long as the desired effect of the present invention can be obtained. For example, amino acid residues derived from methionine, His tag sequences, linkers, and the like may be included. A polypeptide having an amino acid sequence having one or more amino acid substitutions, deletions, insertions or additions in the amino acid sequence of the modified fragment polypeptide can also be used in the present invention as long as it has a desired effect.

  In the present invention, examples of the modified fragment include a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 22 (H296-H296) and a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 23 (H296-H296-H296-). HT), a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 24 (CH296-CH296-HT), a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 25 (H105-H105-HT), represented by SEQ ID NO: 26 A polypeptide comprising the amino acid sequence (H296-H296-HT), a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 27 (H296-H296-H296), a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 28 (H105-H105), represented by SEQ ID NO: 29 Polypeptide consisting of amino acid sequence (H271-H296), polypeptide consisting of amino acid sequence represented by SEQ ID NO: 30 (H296-H271), polypeptide consisting of amino acid sequence represented by SEQ ID NO: 31 (15aaH105-H105- HT), a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 32 (15aaH105-H105), a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 33 (H105-H105Nc-HT), represented by SEQ ID NO: 34 A polypeptide consisting of an amino acid sequence (H105-H105Nc) is exemplified.

  H296-H296 is a polymorph composed of III-12, III-13, III-14, CS-1, alanine, methionine, III-12, III-13, III-14, CS-1 in this order from the N-terminal side. It is a peptide.

  H296-H296-H296-HT is His tag sequence (MNHKVHHHHHIEGRRH), methionine, III-12, III-13, III-14, CS-1, histidine, methionine, III-12, III-13 in this order from the N-terminal side. , III-14, CS-1, histidine, methionine, III-12, III-13, III-14, and CS-1.

  CH296-CH296-HT has a His tag sequence (MNHKVHHHHHIEGRRH), methionine, III-8, III-9, III-10, aspartic acid, lysine, proline, serine, methionine, III-12, III- in order from the N-terminal side. 13, III-14, CS-1, histidine, methionine, III-8, III-9, III-10, aspartic acid, lysine, proline, serine, methionine, III-12, III-13, III-14, CS A polypeptide composed of -1.

  H105-H105-HT is a polypeptide composed of a His tag sequence (MNHKVHHHHHIIEGRRH), methionine, III-14, CS-1, histidine, methionine, III-14, and CS-1 in this order from the N-terminal side.

  H296-H296-HT has a His tag sequence (MNHKVHHHHHHIEGRH), methionine, III-12, III-13, III-14, CS-1, histidine, methionine, III-12, III-13, III in order from the N-terminal side. -14, a polypeptide composed of CS-1.

H296-H296-H296 is a polypeptide obtained by removing the His tag sequence on the N-terminal side of H296-H296-H296-HT, and is produced by cleaving the His tag sequence from H296-H296-H296-HT by a known method. H105-H105 is a polypeptide obtained by removing the His tag sequence on the N-terminal side of H105-H105-HT, and is produced by cleaving the His tag sequence from H105-H105-HT by a known method. Can do.

  H271-H296 is a polypeptide composed of III-12, III-13, III-14, alanine, methionine, III-12, III-13, III-14, and CS-1 in this order from the N-terminal side.

  H296-H271 is a polypeptide composed of III-12, III-13, III-14, CS-1, alanine, methionine, III-12, III-13, and III-14 in this order from the N-terminal side. .

  15aaH105-H105-HT has a His tag sequence (MNHKVHHHHHIEGRRH), methionine, 15 amino acids on the C-terminal side of III-13, II-14, CS-1, alanine, methionine, alanine, III-14, It is a polypeptide composed of CS-1.

  15aaH105-H105 is a polypeptide obtained by removing the His tag sequence on the N-terminal side of 15aaH105-H105-HT, and can be produced by cleaving the His tag sequence from 15aaH105-H105-HT by a known method.

  H105-H105Nc-HT is a polypeptide composed of a His tag sequence (MNHKVHHHHHIEGRRH), methionine, III-14, CS-1, alanine, methionine, alanine, III-14, CS-1 in this order from the N-terminal side. . The polypeptide differs from the aforementioned H105-H105-HT only in the two amino acids of the linker moiety contained between CS-1 and III-14.

H105-H105Nc is a polypeptide obtained by removing the His tag sequence on the N-terminal side of H105-H105Nc-HT, and can be produced by cleaving the His tag sequence from H105-H105Nc-HT by a known method. The polypeptide differs from the aforementioned H105-H105 only in the two amino acids of the linker moiety contained between CS-1 and III-14.
(2) Method for Producing Lymphocytes Hereinafter, the method for producing lymphocytes of the present invention will be specifically described.

  The method for producing lymphocytes of the present invention is a method comprising culturing lymphocytes or lymphocyte precursor cells in the presence of a polypeptide having an amino acid sequence derived from an anti-CD3 antibody and fibronectin. A polypeptide having an amino acid sequence derived from an anti-CD3 antibody and fibronectin is immobilized on the surface of a solid phase used for culturing lymphocytes or lymphocyte progenitor cells, such as cell culture equipment or a cell culture carrier. used.

  Conventionally, for example, an anti-CD3 antibody has been subjected to immobilization treatment by contacting a solution having a concentration of 5 μg / mL with a solid phase to be immobilized. Even when the concentration of the anti-CD3 antibody solution is less than 5 μg / mL, the present inventors can substantially increase the proliferation rate of lymphocytes when a polypeptide having an amino acid sequence derived from fibronectin is present together. We found that there was no decline. That is, it has been found that the amount of anti-CD3 antibody used can be reduced without reducing its effect.

  The anti-CD3 antibody used in the present invention is not particularly limited, but is preferably an anti-human CD3 monoclonal antibody, more preferably OKT3 [Science, 1979, Vol. 206, Pp. 347-349].

  The immobilization of the anti-CD3 antibody may be carried out by a known method. For example, the same method as the immobilization of the fibronectin fragment described in WO 97/18318 and WO 00/09168 Can be implemented. The concentration of the anti-CD3 antibody in the solution used for immobilization is more than 0 μg / mL and less than 5 μg / mL, preferably 0.001 to 3 μg / mL, more preferably 0.01 to 2 μg / mL, particularly preferably Is immobilized with a CD3 antibody solution having a concentration of 0.1 to 1 μg / mL.

  The solid phase for immobilizing the anti-CD3 antibody can be used for cell culture, and there is no limitation as long as it contacts the above-mentioned cells when lymphocytes or lymphocyte precursor cells are cultured. For example, the anti-CD3 antibody is immobilized on cell culture equipment such as petri dishes, flasks, bags, etc. (including both open and closed systems) or cell culture carriers such as beads, membranes, and glass slides. Can do.

  In the present invention, a polypeptide having an amino acid sequence derived from fibronectin described in (1) above is used together with an anti-CD3 antibody. When the polypeptide is immobilized on a solid phase, a solution having a concentration (for example, 25 μg / mL) conventionally used for immobilization during lymphocyte culture can be used. What is necessary is just to set suitably the density | concentration of the polypeptide solution which has the amino acid sequence derived from fibronectin at the time of fixation so that the proliferation rate of a desired lymphocyte may be obtained. The coexistence of a polypeptide having an amino acid sequence derived from fibronectin suppresses a decrease in the proliferation rate of lymphocytes due to a decrease in the amount of anti-CD3 antibody immobilized. For example, even when the concentration of the anti-CD3 antibody in the solution used for immobilizing the solid phase is 0.01 to 3 μg / mL, the solid phase is 1 μg / mL or more, preferably 5 μg / mL or more. By performing immobilization treatment with a polypeptide solution having an amino acid sequence derived from fibronectin at a concentration, a proliferation rate of lymphocytes comparable to the proliferation rate achieved by the conventional immobilized amount of anti-CD3 antibody can be obtained.

  In another aspect of the present invention, the polypeptide having the amino acid sequence derived from fibronectin described in (1) above is immobilized on a solid phase in a solution containing a lower concentration of the polypeptide than in the prior art. . The concentration of the polypeptide in the solution used for immobilization is greater than 0 μg / mL and less than 25 μg / mL, preferably 0.5-15 μg / mL, particularly preferably 1-10 μg / mL. Is done. In this embodiment, the concentration of the anti-CD3 antibody in the solution used for immobilization of the anti-CD3 antibody is not particularly limited, and may be appropriately set so that a desired lymphocyte proliferation rate is obtained. The same concentration as before (for example, 5 μg / mL) can also be used. The coexistence of the anti-CD3 antibody suppresses a decrease in the proliferation rate of lymphocytes due to a decrease in the amount of immobilized polypeptide having an amino acid sequence derived from fibronectin. For example, even when the concentration of the polypeptide having an amino acid sequence derived from fibronectin in the solution used for immobilization of the solid phase is 1 to 5 μg / mL, 0.05 μg / mL or more for the solid phase, preferably Is a lymphocyte comparable to the growth rate achieved with the amount of immobilized polypeptide having an amino acid sequence derived from conventional fibronectin if the immobilization treatment with an anti-CD3 antibody solution at a concentration of 0.1 μg / mL or more is performed. The growth rate is obtained.

  What is described above is an operation for immobilizing a polypeptide having an amino acid sequence derived from an anti-CD3 antibody and fibronectin. It may be performed stepwise using two types of solutions containing each component individually, or may be performed using one type of solution containing both components.

  As described above, a culture device obtained by an immobilization operation using a solution of a polypeptide having an amino acid sequence derived from an anti-CD3 antibody or fibronectin prepared so as to have a lower concentration than the conventional one is also an aspect of the present invention. .

  The above-mentioned components are immobilized on a culture device with a solution of the polypeptide having an amino acid sequence derived from the anti-CD3 antibody or fibronectin prepared so as to have a lower concentration than the conventional one, and the resulting culture device is used for lymph. By culturing progenitor cells of spheres or lymphocytes, these cells are stimulated, for example, with respect to cell proliferation. In this specification, such a stimulus may be referred to as a “low concentration stimulus”.

  Hereinafter, culturing lymphocytes or lymphocyte progenitor cells in the presence of an anti-CD3 antibody and a polypeptide having an amino acid sequence derived from fibronectin is referred to as the culture step of the present invention. The method for producing lymphocytes of the present invention is carried out by carrying out the culturing process of the present invention during the entire period of culture in lymphocyte production or during an arbitrary partial period. That is, any lymphocyte production process that includes the culture process is included in the present invention. When the culturing step is included in a part of the lymphocyte production process, the culturing step is preferably included in the initial stage of culturing, and more preferably the culturing step is included at the start of culturing. Although the present invention is not particularly limited, the culture step of the present invention is performed for 1 to 7 days, preferably 1 to 5 days from the start of the culture.

  There are no particular limitations on the lymphocytes or their precursor cells used in the present invention. Examples of lymphocytes include CD4 positive cells, CD8 positive cells, naive T cells, memory T cells and the like. As lymphocyte progenitor cells, cells having the ability to differentiate into lymphocytes or cell populations containing such cells can be used in the present invention. Peripheral blood mononuclear cells (PBMC), cord blood mononuclear cells Bone marrow cells, hematopoietic stem cells and the like. The cells used for the culture can be set as appropriate according to the type of lymphocytes to be produced, and the cells can be used either directly or cryopreserved from those collected from the living body. Furthermore, the method of the present invention is also effective for the expansion culture of CTL imparted with antigen-specific cytotoxic activity.

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

  The purpose of the culture process of the present invention is to induce antigen-specific CTL from cells that can become CTL, to maintain antigen-specific CTL, or to expand culture of the above lymphocytes such as antigen-specific CTL and naive T cells. To do. In the method for producing lymphocytes of the present invention, lymphocytes useful for adoptive immunotherapy and the like are produced by culturing lymphocytes by appropriately adjusting the type of cells to be subjected to the method, culture conditions and the like. be able to. In the present specification, the term “lymphocyte” includes a cell group containing lymphocytes, and the term “CTL” includes a cell group containing CTL. The cells used for the culture can be set as appropriate according to the type of lymphocytes to be produced, and the cells can be used either directly or cryopreserved from those collected from the living body.

The cell culture equipment used in the culture process of the present invention is not particularly limited, and for example, a petri dish, a flask, a bag, a large culture tank, a bioreactor and the like can be used. As the bag, for example, can be used CO ₂ gas-permeable cell culture bag. Moreover, when manufacturing a large amount of lymphocytes industrially, a large culture tank can be used. The culture can be carried out in either an open system or a closed system. Preferably, the culture is preferably carried out in a closed system from the viewpoint of the safety of the obtained lymphocytes.

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

Furthermore, it is composed of acidic polysaccharides, acidic oligosaccharides, acidic monosaccharides and salts thereof which are effective in inducing cytotoxic T cells having antigen-specific cytotoxic activity, as described in WO 02/14481. You may culture | cultivate using the compound selected from a group, and the substance selected from the following (A)-(D) of international publication 03/016511 pamphlet with the said component.
(A) a substance having binding activity to CD44 (B) a substance capable of controlling a signal emitted by binding of CD44 ligand to CD44 (C) a substance capable of inhibiting the binding of a growth factor to a growth factor receptor (D) Substance capable of controlling signal emitted by growth factor binding to growth factor receptor The concentration of these components in the medium is not particularly limited as long as a desired effect is obtained. Further, these components may be dissolved in a medium and coexist, or may be used by being immobilized on an appropriate solid phase as described above. In addition, said various substances can be used individually or in mixture of 2 or more types.

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

  In the lymphocyte culture process of the present invention, serum or plasma can be added to the medium. The amount of addition to these media is not particularly limited, but is more than 0 to 20% by volume, especially when using autologous serum or plasma, it is more than 0 to 5% by volume in consideration of the burden on the patient. Is preferred. In the lymphocyte culture process, the serum and plasma concentrations in the medium can be reduced stepwise. For example, when replacing or adding a medium in the lymphocyte culture process, the plasma or plasma used is reduced by adjusting the serum or plasma concentration in the new medium to a low level, and lymphocytes are cultured. be able to. Thus, the ability to reduce the amount of serum or plasma added to the medium is one of the effective effects of the present invention. The origin of serum or plasma may be either self (meaning that the origin is the same as the lymphocyte to be cultured) or non-self (meaning that the origin is different from the lymphocyte to be cultured), Preferably, an autologous material can be used from the viewpoint of safety.

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

(2) -1 Method for Producing Lymphokine Activated Cells Hereinafter, an example of producing lymphokine activated cells by the production method of the present invention (hereinafter sometimes referred to as the method for producing lymphokine activated cells of the present invention) will be described in detail. Describe.

  The method for producing lymphokine-activated cells of the present invention is characterized in that the culture step of the present invention is carried out in the presence of IL-2 using cells capable of becoming lymphokine-activated cells. Here, the culture process of the present invention may be performed during the entire culture period or a part of the culture period, and preferably, the culture process of the present invention is preferably performed at the start of the culture. About the culture | cultivation period of the culture | cultivation process of this invention, for example, 1-10 days, Preferably it is 2-8 days, More preferably, 3-7 days are preferable, and 3 days or more from a viewpoint of implement | achieving a higher expansion culture rate. Is preferred.

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

Further, general conditions for culturing lymphokine-activated cells are known conditions [eg, cell engineering, 1995, 14, 223-227; tissue culture, 1991, 17, 17; The Lancet, 2000, 356, 802-807; see Current Protocols in Immunology, Addendum 17, UNIT 7.7]. The culture conditions are not particularly limited, and the conditions used for normal cell culture can be applied. For example, the culture can be performed under conditions of 37 ° C., 5% CO _{2 and the} like. This culturing is usually carried out for about 2 to 15 days, and as described above, it is preferable to carry out the culturing step of the present invention in the early stage of culturing. Further, a step of diluting the cell culture solution at an appropriate time interval, a step of replacing the medium, or a step of replacing the cell culture equipment may be performed.

The medium to be used is not particularly limited, and a known medium suitable for lymphocyte culture can be used. In addition to the active ingredient of the present invention, an appropriate protein, cytokines, and other components may be included. Preferably, a medium containing IL-2 is used in the present invention. The concentration of IL-2 in the medium is not particularly limited, but is, for example, 0.01 to 1 × 10 ⁵ U / mL, preferably 0.1 to 1 × 10 ⁴ U / mL.

  In one embodiment of the present invention, an anti-CD28 antibody, particularly preferably an anti-human CD28 monoclonal antibody, can coexist in order to give a cell a costimulation. A lymphocyte stimulating factor such as lectin may coexist. Further, as described above, these components can be used by immobilizing them on an appropriate solid phase.

  Cells obtained by the method for producing lymphokine-activated cells of the present invention have a high ratio of CD45RA positive CCR7 positive cells and CD45RA positive CCR7 positive CD62L positive cells as described in Examples 6 and 7. That is, lymphokine activated cells obtained by the method are cells containing a high ratio of naive T cells and are extremely suitable for use in adoptive immunotherapy.

  Furthermore, as described in Example 9, the cells have a strong proliferation / survival action in vivo, and also maintain a high ratio of naive T cells. This indicates that the cells receive antigen presentation from the antigen-presenting cells in vivo and are highly induced by specific CTLs to proliferate and are very useful for adoptive immunotherapy.

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

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

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

  As the gene introduction method, either a method using a viral vector or a method not using the vector can be used in the present invention. A lot of literature has already been published on details of these methods.

  The viral vector is not particularly limited, and is usually a known viral vector used in gene transfer methods, for example, a retrovirus vector, a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, a simian virus vector, a vaccinia virus vector. Alternatively, a Sendai virus vector or the like is used. Particularly preferably, as the virus vector, a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, a lentivirus vector or a simian virus vector is used. As the above-mentioned viral vector, those lacking replication ability are preferable so that they cannot self-replicate in infected cells. In addition, a substance that improves the introduction efficiency at the time of gene introduction may be used. For example, Retronectin (registered trademark, manufactured by Takara Bio Inc.) or other fibronectin fragments can also be used.

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

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

  In the present invention, the foreign gene introduced into the lymphocyte is not particularly limited, and any gene desired to be introduced into the cell can be selected. Examples of such genes include those encoding proteins (eg, enzymes, cytokines, receptors, etc.), as well as those encoding antisense nucleic acids, siRNA (small interfering RNA), and ribozymes. Moreover, you may introduce | transduce simultaneously the suitable marker gene which enables selection of the cell by which the gene was introduce | transduced.

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

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

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

  Other genes to be introduced include a gene encoding a TCR that recognizes the surface antigen of the target cell, an antigen recognition site of an antibody against the surface antigen of the target cell, and an intracellular region of the TCR (such as CD3) Examples include genes encoding chimeric receptors.

(3) Lymphocytes obtained by the production method of the present invention, a medicament containing the lymphocytes, a disease treatment method using the medicament, and the present invention further includes lymphocytes obtained by the production method of the present invention described above. A cell population containing the lymphocyte, a cell subpopulation separated from the progenitor cell population are provided. The present invention also provides a medicament (therapeutic agent) containing the lymphocytes as an active ingredient.

The therapeutic agent containing lymphocytes and cell populations obtained by the production method of the present invention is suitable for use in adoptive immunotherapy. In adoptive immunotherapy, lymphocytes suitable for patient treatment are administered to the patient, for example, via veins. The therapeutic agent is very useful for use in the diseases described below and donor lymphocyte infusion. The therapeutic agent is in accordance with a known method in the pharmaceutical field, for example, using a lymphocyte or a cell population prepared by the method of the present invention as an active ingredient, for example, a known organic or inorganic carrier suitable for parenteral administration, shaping It can be prepared by mixing with agents, stabilizers and the like. In addition, the content of the lymphocytes of the present invention in the therapeutic agent, the dosage of the therapeutic agent, and various conditions relating to the therapeutic agent can be appropriately determined according to known adoptive immunotherapy, although there is no particular limitation, for example, per adult day, Preferably 1 × 10 ⁵ to 1 × 10 ¹² cells / day, more preferably 5 × 10 ^{5 to} 5 × 10 ¹¹ cells / day, and even more preferably 1 × 10 ⁶ to 1 × 10 ¹¹ cells / day Is done. Usually, lymphocytes are administered to veins, arteries, subcutaneous, intraperitoneal, etc. by injection or infusion.

  The administration of the medicament of the present invention is not particularly limited. For example, it can be administered together with a component that can function as a vaccine against a disease to be treated. For example, in the treatment of cancer, tumor antigens, cells capable of presenting antigens, cells presented with antigens, cells derived from tumor tissues that have lost their ability to proliferate due to human manipulation, and extracts from tumor tissues Etc. can also be administered. In the administration of the medicament, lymphocyte stimulating factors such as anti-CD3 antibody, anti-CD28 antibody, cytokine (IL-2, IL-15, IL-7, IL-12, IFN-γ, IFN-α, IFN-β etc.), chemokines and the like can be administered as appropriate. In the present specification, the lymphocyte stimulating factor includes lymphocyte growth factor.

  The disease to which lymphocytes produced by the method of the present invention are administered is not particularly limited. For example, malignant tumor diseases such as cancer and leukemia, and infectious diseases caused by viruses, bacteria and fungi (for example, hepatitis) , Influenza, AIDS, tuberculosis, MRSA infection, VRE infection, deep mycosis). In addition, when a foreign gene is further introduced as described above, an effect is expected for various gene diseases. The lymphocytes produced by the method of the present invention can also be used for bone marrow transplantation, prevention of infection after irradiation, donor lymphocyte infusion for remission of relapsed leukemia, and the like.

  The use of lymphocytes produced by the method of the present invention in the manufacture of a medicament, for example, the use in the manufacture of a medicament for the treatment of cancerous diseases or infectious diseases is also encompassed by the present invention.

  Moreover, the treatment method of the disease using the said pharmaceutical is provided as another aspect of this invention. The method for treating the disease is characterized by using lymphocytes produced by the method for producing lymphocytes described above. Regarding various conditions for administration of the medicaments, disclosure of known adoptive immunotherapy and administration of the aforementioned medicaments is provided. Can be implemented according to

  As described above, lymphokine-activated cells expanded in the presence of a polypeptide having an amino acid sequence derived from an anti-CD3 antibody and fibronectin contain a high proportion of naive T cells. A naive T cell can exhibit a therapeutic effect on a disease for which treatment is desired by receiving an antigen from an antigen presenting cell after transplanted into a living body and differentiating it into a CTL or a helper T cell that recognizes the antigen. It becomes like this.

  As shown in the Examples below, lymphokine-activated cells expanded in the presence of a polypeptide having an amino acid sequence derived from an anti-CD3 antibody and fibronectin are more telomeres than refokine-activated cells expanded by other methods. Is long and can survive for a long time in vivo after infusion. Moreover, the engraftment rate is higher than that of lymphokine-activated cells expanded by other methods. That is, lymphokine-activated cells expanded and cultured in the presence of an anti-CD3 antibody and a polypeptide having an amino acid sequence derived from fibronectin can be stably maintained in vivo and are presented with an antigen at a higher frequency. , Show excellent therapeutic effect. That is, according to the present invention, high in vivo engraftment after administration to a living body expanded in the presence of an immobilized anti-CD3 antibody, a polypeptide having an amino acid sequence derived from immobilized fibronectin and IL-2. This provides lymphokine-activated cells that exhibit a high rate of maintenance of a stable phenotype and are presented with higher frequency of antigen. It is also administered to a living body, including a step of expanding lymphocytes or lymphocyte progenitor cells in the presence of an immobilized anti-CD3 antibody, a polypeptide having an immobilized amino acid sequence derived from fibronectin, and IL-2. The present invention provides a method for producing lymphokine-activated cells that exhibit a high in-vivo engraftment rate and a stable phenotype maintenance rate and present an antigen at a higher frequency.

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

Preparation Example 1 Preparation of a polypeptide (FN fragment) having an amino acid sequence derived from fibronectin C-274 (polypeptide comprising the cell binding domain of fibronectin, SEQ ID NO: 9), H-271 (polypeptide comprising the heparin binding domain of fibronectin) , SEQ ID NO: 10), H-296 (polypeptide consisting of heparin binding domain and CS-1 domain of fibronectin, SEQ ID NO: 11), CH-271 (polypeptide consisting of cell binding domain and heparin binding domain of fibronectin, SEQ ID NO: 12), C-CS1 (polypeptide consisting of a cell binding domain and a CS-1 domain of fibronectin, SEQ ID NO: 14) is described in J. Org. Biochem. (Journal of Biochemistry), 1991, 110, 284-291.

  H296-H296 (SEQ ID NO: 22) and H105-H105 (SEQ ID NO: 28) were prepared based on the description in Patent Document 4.

Example 1 Promotion of Cell Growth in Frozen PBMC-1
(1) Separation and preservation of PBMC After collecting blood components from a healthy person with informed consent, PBMC was collected using Ficoll-paque. The collected PBMC was suspended in RPMI 1640 medium (manufactured by Invitrogen or Wako Pure Chemical Industries), and then CP-1 (manufactured by Kyokuto Pharmaceutical Co., Ltd.) and 25% human serum albumin (Buminate: manufactured by Baxter, hereinafter referred to as HSA) Was added in an equal volume of 17: 8 and stored in liquid nitrogen. At the time of use, these stored PBMCs were rapidly melted in a 37 ° C. water bath, washed with GT-T551 (Takara Bio) containing 10 μg / mL DNase (Calbiochem), and then used for each experiment.

(2) Immobilization of anti-CD3 antibody and CH-296 Anti-CD3 antibody (OKT3, manufactured by Janssen Pharma) and RetroNectin (registered trademark, manufactured by Takara Bio Inc., hereinafter referred to as CH-296) are used for the culture equipment used in the following experiments. ) Was immobilized. That is, an ACD-A solution (manufactured by Terumo) containing only 0.01-5 μg / mL anti-CD3 antibody in a 96-well cell culture plate (Corning), or 0.01-5 μg / mL anti-CD3 antibody and 1 ACD-A solution containing -25 μg / mL CH-296 was added at 80 μL / well. After incubating the culture equipment at room temperature for 5 hours, the ACD-A solution was removed from the culture equipment by suction, and each well was twice treated with Dulbecco's PBS (manufactured by Nissui Pharmaceutical or Invitrogen, hereinafter referred to as DPBS), RPMI1640. The plate was washed once with the medium and used for each experiment.

(3) Cell culture and thymidine incorporation test PBMC 0.3 × 10 ⁵ cells isolated in Example 1- (1) were treated with 0.5% human AB type serum (manufactured by LONZA, hereinafter referred to as serum), final concentration. The anti-CD3 antibody-immobilized plate prepared in Example 1- (2), or suspended in 0.2 mL of GT-T551 containing 200 U / mL IL-2 (formulation name Proleukin, manufactured by Chiron), and anti-CD3 antibody and They were added to CH-296-immobilized plates, and these plates were cultured in a 37 ° C., 5% CO ₂ incubator (culture day 0). On the third day of culture, 0.5% serum-containing GT-T551 containing 0.1 mCi / mL thymidine (Thymidine [methyl-3H]) (manufactured by Perkin Elmer) was added at 10 μL / well at 37 ° C. (5 Incubated for 16 hours in the presence of% CO ₂ . The cultured cells were collected on a FilterMAT (manufactured by SKATORON instruments) using a cell harvester (manufactured by SKATORON instruments), fixed with ethanol (manufactured by SKATORON instruments), washed with water, and dried. The thymidine incorporation activity into the cells was measured by measuring the radioactivity of FilterMAT to which the cells were immobilized using a liquid scintillation counter LS6500 (manufactured by Beckman Coulter) using Aquasol-2 (manufactured by Perkin Elmer) as a scintillation cocktail. evaluated. Two donors were tested, and the radioactivity values (cpm) in each group are shown in Table 1-1 and Table 1-2. In addition, the value in a table | surface shows the average value of N = 4.

  As shown in Table 1-1 and Table 1-2, in both donors, when a plate on which only the anti-CD3 antibody was immobilized was used, thymidine accompanied with a decrease in the concentration of the anti-CD3 antibody upon immobilization. A decrease in uptake activity was observed. However, the thymidine incorporation activity when combined with immobilization with 0.3 μg / mL anti-CD3 antibody solution and immobilization with 5 μg / mL or 25 μg / mL CH-296 solution is 5 μg / mL, which is the conventional protocol. This was equivalent to a combination of an anti-CD3 antibody solution and a 25 μg / mL CH-296 solution. That is, even when the concentration of the anti-CD3 antibody at the time of immobilization was lowered, it was shown that the conventional cell growth promoting effect can be obtained by coexisting CH-296.

Example 2 Promotion of Cell Growth in Frozen PBMC-2
(1) Cell culture and thymidine uptake test Cell culture and thymidine uptake tests were performed in the same manner as in Example 1. However, a 0.1 μg / mL solution was used for immobilization of the anti-CD3 antibody, and H-296, C-274, H-271, CH-271, C-CS1, H296-as FN fragments other than CH-296. H296 and H105-H105 were prepared to the respective concentrations (5-20 μg / mL) in the table, and the anti-CD3 antibody and each FN fragment were immobilized. The resulting radioactivity values for each combination are shown in Table 2. In addition, the value in a table | surface shows the average value of N = 3.

  As shown in Table 2, even when the plate was immobilized with a 0.1 μg / mL anti-CD3 antibody solution, the radioactivity when the FN fragment was further immobilized was that only the anti-CD3 antibody had the above-described radioactivity. It was confirmed that the amount of thymidine incorporation was higher than the radioactivity when immobilized at a concentration. That is, the cell growth promoting effect was shown by the combination of immobilization with a 0.1 μg / mL anti-CD3 antibody solution and immobilization of FN fragments at various concentrations.

Example 3 Promotion of cell proliferation in fresh PBMC (1) Separation of PBMC and inactivated plasma From a healthy human donor with informed consent, 50 mL of blood was collected using heparin sodium as an anticoagulant. After the execution, it was centrifuged to collect a plasma fraction and a cell fraction containing PBMC. The plasma fraction was inactivated at 56 ° C. for 30 minutes, and the supernatant after centrifugation was recovered as inactivated plasma and used for each experiment. As for the cell fraction, PBMCs were collected and washed by Ficoll-paque and used for each experiment.

(2) Cell Culture and Thymidine Uptake Test Cell culture and thymidine uptake test were performed in the same manner as in Example 1. However, the anti-CD3 antibody was immobilized using a 0.01-5 μg / mL anti-CD3 antibody solution, and the CH-296 non-immobilized group and CH-296 were immobilized with a 5 μg / mL solution. And CH-296 immobilization group was prepared. The culture medium used was GT-T551 (hereinafter referred to as 0.6% plasma-containing GT-T551) containing the inactivated plasma to a final concentration of 0.6%.

  The radioactivity values in each group are shown in Table 3. In addition, the value in a table | surface shows the average value of N = 4.

  As shown in Table 3, when stimulation was performed on a plate in which 0.01-1 μg / mL anti-CD3 antibody solution and 5 μg / mL CH-296 solution were combined and immobilized on fresh PBMC The thymidine incorporation activity was confirmed to be comparable when a 5 μg / mL anti-CD3 antibody solution and a 5 μg / mL CH-296 solution were combined. That is, even when immobilized with a low concentration anti-CD3 antibody solution, a cell growth promoting effect was shown by combining with a CH-296 solution.

Example 4 Promotion of Cell Growth in Resting T Cells-1
(1) Preparation of resting T cells PBMC prepared by the same method as in Example 1- (1) was prepared to 1 × 10 ⁶ cells / mL with GT-T551 containing 0.5% serum, and a plate for 48-well cell culture 1 mL was seeded on (Corning). Furthermore, IL-2 was added at a final concentration of 100 U / mL, and Phytoheamagglutinin (manufactured by Sigma) was added to a final concentration of 2 μg / mL, and cultured at 37 ° C. in 5% CO ₂ . On the fourth day of culture, so as to be ^{0.2 × 10 6 cells / mL,} 7 days culture, the 11 day 0.5% serum-containing to a 0.5 × ¹⁰ 6 cells / mL GT-T551 was added. At the time of addition of the medium, IL-2 was added to a final concentration of 50 U / mL each time. Culturing was continued in a T25 cell culture flask (manufactured by Corning), and the cell suspension on day 14 was collected and used as a resting T cell for each experiment.

(2) Cell Culture and Thymidine Uptake Test Cell culture and thymidine uptake tests were performed in the same manner as in Example 1 using the resting T cells prepared in Example 4- (1). However, the anti-CD3 antibody was immobilized with a 0.01-0.3 μg / mL solution, and only the anti-CD3 antibody not using CH-296 was immobilized, and further, 1-10 μg / mL CH-296. A group in which an anti-CD3 antibody immobilized with a solution and CH-296 were immobilized was set. In addition, it tested about the resting T cell derived from two donors. The values of radioactivity under each initial stimulation condition are shown in Table 4-1 (donor A-derived resting T cells) and Table 4-2 (donor B-derived resting T cells). In addition, the value in a table | surface shows the average value of N = 4.

  As shown in Table 4-1, Table 4-2, even when the concentration at the time of immobilization of the anti-CD3 antibody was reduced to 0.01-0.3 μg / mL as an initial stimulus in the resting T cells, When CH-296 was immobilized with a solution of 5 μg / mL or 10 μg / mL in addition to the anti-CD3 antibody, high thymidine incorporation activity was confirmed in any donor. That is, as with fresh PBMCs, it was shown that cell proliferation was promoted in the resting T cells purified to T cells by using a plate fixed by combining a low concentration anti-CD3 antibody solution and a CH-296 solution.

Example 5 Promotion of Cell Growth in Resting T Cells-2
(1) Cell culture and thymidine uptake test Cell culture and thymidine uptake tests were performed in the same manner as in Example 4. However, cell stimulation was performed in the same manner as in Example 2. Table 5 shows the radioactivity values resulting from the initial stimulation conditions. In addition, the value in a table | surface shows the average value of N = 4.

  As shown in Table 5, when immobilized with a 0.1 μg / mL anti-CD3 antibody solution and coexisting with an FN fragment, the radioactivity was higher than when no FN fragment was used, and thymidine incorporation A large amount was confirmed. That is, it was shown that each FN fragment can suppress the decrease in the cell growth promoting effect due to the decrease in the concentration of the anti-CD3 antibody solution at the time of immobilization.

Example 6 Expansion of Lymphocytes by Low-concentration Stimulation Using FN Fragment CH-296 (1) Immobilization of anti-CD3 antibody and CH-296 0.03-10 μg / mL of 12-well plate (Corning) After adding ACD-A solution containing only anti-CD3 antibody or ACD-A solution containing 0.03-10 μg / mL anti-CD3 antibody and 5 μg / mL CH-296 at a rate of 0.45 mL / well, 5% CO in _2, incubated for 5 hours at 37 ° C.. Immediately before use, the ACD-A solution was aspirated and removed, and then each well was washed twice with DPBS and once with RPMI 1640 medium and used for each experiment.

(2) Enlarged culture of lymphocytes 4.77 mL of 0.5% serum-containing GT-T551 was added to the anti-CD3 antibody-immobilized plate prepared in Example 6- (1) or the anti-CD3 antibody and CH-296-immobilized plate. Cell suspension in which the PBMC prepared in Example 1- (1) was suspended in GT-T551 containing 0.5% serum to 1.0 × 10 ⁶ cells / mL. The solution was added at 0.53 mL / well. IL-2 was added to each well to a final concentration of 200 U / mL, and cultured at 37 ° C. in 5% CO ₂ (culture day 0). On the 4th day from the start of the culture, the culture solution of each group was diluted about 8.3 times with 0.5% serum-containing GT-T551, and about 7.8 mL of this diluted solution was prepared in a T25 cell culture flask. Then, IL-2 was added to a final concentration of 200 U / mL. On day 7 and day 10 of culture start, IL-2 was added so that the culture solution of each group was diluted 2-fold with GT-T551 to a final concentration of 200 U / mL, and the resulting culture solution was obtained. 15.6 mL was transferred to a T25 cell culture flask and the culture was continued. On the 7th, 10th, and 13th days after the start of the culture, the number of viable cells was measured, and the expansion culture rate was calculated in comparison with the number of cells at the start of the culture. The results are shown in Table 6-1. Table 6-1 shows the expansion culture rate (times) on each day when the culture start time is 1. In addition, the value in a table | surface shows the average value of N = 2.

  As shown in Table 6-1, by using a low concentration solution and culturing with an anti-CD3 antibody and CH-296-immobilized equipment (referred to as low concentration stimulation conditions), anti-CD3 It was confirmed that expansion of lymphocytes was more strongly promoted compared to stimulation with antibody alone.

(3) Analysis of CD45RA-positive CCR7-positive CD62L-positive cells in lymphocytes after culturing Cells on day 7, 10 and 13 of the culture start prepared in Example 6- (2) were subjected to RD1-labeled mouse anti-human CD45RA. After staining with antibody (manufactured by Beckman Coulter), FITC-labeled mouse anti-human CCR7 antibody (R & D Systems), PC5-labeled mouse anti-human CD62L antibody (manufactured by eBioscience), flow cytometry (Cytics FC500: manufactured by Beckman Coulter) Was analyzed. The results are shown in Table 6-2. Table 6-2 shows the ratio (%) of CD45RA positive CCR7 positive CD62L positive cells. In addition, the value in a table | surface shows the average value of N = 2.

  As shown in Table 6-2, in the group using the culture equipment in which the anti-CD3 antibody and CH-296 were immobilized, the CD45RA positive CCR7 positive CD62L positive in comparison with the control group in which only the anti-CD3 antibody was immobilized. Results with a high proportion of cell populations were obtained. That is, even if the concentration at the time of immobilization of the anti-CD3 antibody is low, by using CH-296 in combination, a CD45RA-positive CCR7-positive CD62L-positive naïve with high differentiation ability into cells exhibiting high cytotoxic activity in vivo. It was revealed that T cells can be proliferated with high efficiency.

Example 7 Expansion of lymphocytes using CH-296, anti-human CD28 antibody, anti-human 4-1BB antibody and CD3 / CD28 beads (1) Anti-human CD3 antibody and CH-296, anti-human CD28 antibody, anti-human 4-1BB antibody immobilization Anti-CD3 antibody (0.3 μg / mL solution) only, anti-CD3 antibody (0.3 μg / mL solution) and CH-296 (5 μg / mL solution), anti-CD3 antibody (0.3 μg / mL solution) ACD-A solution containing anti-CD28 antibody (5 μg / mL solution), anti-CD3 antibody (0.3 μg / mL solution), and anti-4-1BB antibody (5 μg / mL solution), respectively. ² to become so sealed gas permeable culture bags CultiLife (registered trademark) 215 (manufactured by Takara Bio Inc.) by 10.4 mL / bag added, 5% CO ₂ in 3 Incubated at 7 ° C for 5 hours. The bag was subjected to each experiment after removing the ACD-A solution before use and washing with RPMI1640 medium three times.

(2) Enlarged culture of lymphocytes Each PBMC 0.6 × 10 ⁷ cells separated from 4 donors by the same method as in Example 3- (1) was suspended in 120 mL of GT-T551 containing 0.6% plasma, Each of them was added to MultiLife (registered trademark) 215 having different immobilization conditions prepared in Example 7- (1). In the group using CD3 / CD28 beads, CD3 / CD28 beads (Dynabeads CD3 / CD28: manufactured by Invitrogen) washed with 1% serum-containing DPBS were added in an amount of 3 times the number of cells. Next, IL-2 was added to each bag to a final concentration of 200 U / mL, and cultured at 37 ° C. in 5% CO ₂ (culture day 0). On the 4th day from the start of culture, the cells in each CultureLife (registered trademark) 215 were suspended, and 120 mL of this suspension was diluted with 380 mL of 0.6% plasma-containing GT-T551, so that nothing was immobilized. It transferred to the gas-permeable culture bag CultureLife (registered trademark) Eva (manufactured by Takara Bio Inc.). In any group, IL-2 was added to a final concentration of 200 U / mL. On the 7th day from the start of culture, GT-T551 in an amount equal to the cell suspension was added to each bag and diluted 2-fold, and then IL-2 was added so that the final concentration was 200 U / mL. Added. On the 10th day from the start of culture, 500 mL of the cell suspension in each bag was withdrawn, and then an equal amount of GT-T551 was added to the bag, and IL-2 was added so that the final concentration was 200 U / mL. . The number of viable cells was counted on the 10th and 14th days from the start of the culture, and the expansion culture rate was calculated by comparison with the number of cells at the start of the culture. The average value of 4 donors is shown in Table 7-1. Since the CD3 / CD28 bead use group was performed for 3 donors, the average value of 3 donors is shown. In the group using CD3 / CD28 beads, CD3 / CD28 beads were removed from the cell suspension using a magnetic bead separator MPC-1 (manufactured by Invitrogen) at the time of counting the number of living cells. In the table, “+” means “and”, and the same applies to the following tables.

  As shown in Table 7-1, regardless of the number of culture days, by using the culture equipment in which the anti-CD3 antibody and CH-296 are immobilized at the early stage of lymphocyte expansion culture, the expansion is higher than that of other groups. A culture rate was obtained. From this, it was clarified that the combination of anti-CD3 antibody and CH-296 can be suitably used for lymphocyte expansion culture even in a technique using culture equipment in which components are immobilized using a low-concentration solution.

(3) Analysis of CCR7-positive CD45RA-positive T cells Cells on day 10 and day 14 of culture prepared in Example 7- (2) were subjected to CCR7-positive CD45RA-positive T in the same manner as in Example 6- (3). Cell analysis was performed. CD62L is not measured. The average value of 4 donors for the content ratio of each positive cell is shown in Table 7-2. In addition, about the CD3 / CD28 bead use group, it is an average value of 3 donors. Moreover, about 2 donors of the anti-CD3 antibody use group of the culture | cultivation 10th day, since the number of cells required for an analysis was not obtained and it was not implemented, the average value of 2 donors is shown.

  As shown in Table 7-2, regardless of the number of culture days, by using the culture equipment in which the anti-CD3 antibody and CH-296 were immobilized at the initial stage of lymphocyte expansion culture, a higher ratio was obtained compared to the other groups. A CCR7 positive CD45RA positive cell population was obtained. From these examples, it was revealed that naive T cells positive for CCR7 positive CD45RA can be proliferated with high efficiency by applying low concentration stimulation with anti-CD3 antibody and CH-296 at the early stage of lymphocyte expansion culture.

Example 8 Engraftment and naive T cell ratio in NOD / scid mice (1) Preparation of lymphocytes Expanded lymphocytes were cultured in the same manner as in Example 7- (2), and cells on day 14 of culture were used as examples. Cryopreserved by the same method as 1- (1). The concentration at the time of immobilization was 5 μg / mL for anti-CD3 antibody and 25 μg / mL for CH-296. In addition, a group using CD3 / CD28 beads was set.

(2) Mice and group configuration NOD / scid mice (manufactured by CLEA Japan, Inc.) 12 8-week-old females were grouped as shown in Table 8-1 below. In each group, N = 4.

(3) Administration of anti-asialo GM1 antibody It is known that anti-asialo GM1 antibody treatment removes NK cells and enhances human cell survival in NOD / scid mice. On the day before the administration of the cells, 20 μl of anti-asialo GM1 antibody solution (manufactured by Wako Pure Chemical Industries, Ltd.) was diluted with 0.38 mL of 0.4% HSA-containing physiological saline, and the entire amount was administered intraperitoneally to the mice.

(4) Preparation and administration of administration cells Lymphocytes cryopreserved in Example 8- (1) were rapidly thawed in a 37 ° C water bath. Each lymphocyte was suspended in 4% HSA-containing physiological saline so as to be 3.0 × 10 ⁷ cells / 0.3 mL / mouse to prepare cells for administration. Prior to cell administration, all mice were irradiated with X-rays (350R) and prepared cells were administered into 0.3 mL tail vein.

(5) Engraftment of human T cells in peripheral blood On the 7th and 14th days after cell administration, blood was collected from the tail vein of mice and the positive rate of human lymphocytes in the peripheral blood of mice transferred by flow cytometry was determined as% Analysis was carried out as chimerism [human CD3-positive cell content / (human CD3-positive cell content + mouse CD45-positive cell content) × 100%]. For staining, FITC-labeled mouse anti-human CD3 antibody (manufactured by Beckman Coulter) and PE-labeled rat anti-mouse CD45 antibody (manufactured by Beckman Coulter) were used. After the antibody reaction, hemolysis was repeated twice using ACK buffer, and then suspended in DPBS and subjected to flow cytometry. The results are shown in Table 8-2. Table 8-2 shows the positive rate (% chimerism) of human lymphocytes in mouse peripheral blood, and shows the average value of N = 4.

  As shown in Table 8-2, the RN-T group showed higher% chimerism on the 7th and 14th days after the cell administration than the other groups. That is, it was revealed that cells produced using anti-CD3 antibody and CH-296 have higher engraftment ability in vivo in NOD / scid mice.

(6) Analysis of CD45RA-positive CCR7-positive T cells of human T cells in peripheral blood RD1-labeled mouse anti-human CD45RA antibody, FITC-labeled mouse anti-human CCR7 antibody and PC5-labeled mouse anti-antibody in the same manner as in Example 8- (5) Cells were stained with a human CD3 antibody (Beckman Coulter). The content of CD45RA positive CCR7 positive T cells was calculated for the human CD3 positive T cell population. The average of each group is shown in Table 8-3.

  From Table 8-3, about 7th day and 14th day after cell administration, the RN-T group showed a higher value of the CD45RA-positive CCR7-positive T cell content in engrafted human lymphocytes than the other groups. . When stimulated with anti-CD3 antibody and CH-296, CD45RA-positive CCR7-positive naive T cells proliferate efficiently, but the phenotype of NOD / scid mice remains high even in vivo.

Example 9 Study of engraftment and naive T cell ratio of lymphocytes expanded under low concentration stimulation conditions in NOD / scid mice (1) Expansion of lymphocytes Lymphocytes in the same manner as in Example 7- (2) for 10 days Sphere expansion culture was performed. A group in which the concentration at the time of immobilization of anti-CD3 antibody was 5 μg / mL (OKT3-T group), a group in which anti-CD3 antibody was immobilized at 5 μg / mL, and CH-296 was fixed at 25 μg / mL (RN-T group) ) Was added. The group stimulated with CD3 / CD28 beads was not tested.

(2) Mice and group composition 25 NOD / scid mice 9 weeks old females were grouped as shown in Table 9-1 below. Each group had N = 5.

(3) Administration of anti-asialo GM1 antibody An anti-asialo GM1 antibody was administered intraperitoneally on the day before cell administration in the same manner as in Example 8- (3).

(4) Preparation and administration of administration cells Lymphocytes prepared in Example 9- (1) were administered in the same manner as in Example 8- (4). In addition, the cell for administration was suspended in the physiological saline containing 4% HSA so that it might become 5.0 * 10 < ⁷ > cells / 0.3mL / mouse.

(5) Engraftment of human T cells in peripheral blood In the same manner as in Example 8- (5), the% chimerism of human T cells in mouse peripheral blood on day 7 after cell administration was examined.

  The results are shown in Table 9-2. Table 9-2 shows the average value of each group.

  From Table 9-2, the RN (L) -T group, which is a low concentration stimulation condition on the seventh day after cell administration, showed a higher% chimerism than the other groups, similar to the CD28 (L) -T group. That is, it was revealed that cells produced under low-concentration stimulation conditions have a higher engraftment ability at an early stage in the living body of NOD / scid mice.

(6) Analysis of human T cells in peripheral blood CD45RA positive CCR7 positive T cells Staining was carried out in the same manner as in Example 8- (6). The content of CD45RA positive CCR7 positive T cells was calculated for the human CD3 positive T cell population. The average of each group is shown in Table 9-3.

  From Table 9-3, the RN (L) -T group has a higher content of CD45RA-positive CCR7-positive T cells in the transferred human lymphocytes than the other low-stimulation condition groups on the seventh day after cell administration. showed that. Lymphocytes produced using culture equipment that has been immobilized with a low-concentration anti-CD3 antibody solution or CH-296 solution should maintain a high phenotype prior to transplantation even in vivo in NOD / scid mice. Became clear.

Example 10 Examination of engraftment and cell population in NOG mice (1) Expansion of lymphocytes Lymphocyte expansion culture was carried out for 10 days in the same manner as in Example 7- (2). The stimulation conditions were the same as in Example 8- (1).

(2) Mice and group composition NOG mice (NOD / Shi-scid, IL-2γ KO) (manufactured by Central Research Laboratory Animal Research) 12-week-old females with group composition shown in Table 10-1 below did. In each group, N = 4.

(3) Preparation and administration of administered cells The lymphocytes prepared in Example 10- (1) were suspended in a solvent (4% HSA-containing physiological saline) so as to be 5.0 × 10 ⁷ cells / 0.3 mL / mouse. It became cloudy and was administered into the tail vein.

(4) Engraftment of human T cells in peripheral blood In the same manner as in Example 8- (5), human T in mouse peripheral blood on days 8, 14, 21, and 28 after cell administration. The% chimerism of the cells was examined.
The results are shown in Table 10-2. Table 10-2 shows the average value of each group.

  From Table 10-2, the RN-T group showed a higher value than the other groups, particularly at the initial stage until the 21st day after cell administration. That is, it was revealed that cells produced using anti-CD3 antibody and CH-296 have higher engraftment ability at an early stage in the body of NOG mice.

(5) Examination of CD4 and CD8 positive rates of human T cells in mouse peripheral blood In the same manner as in Example 10- (4), humans in peripheral blood on days 14, 21 and 28 after cell administration CD4 and CD8 positive rates in T cells were examined. For staining, a PC5-labeled mouse anti-human CD8 antibody (manufactured by Beckman Coulter, Inc.) was added to the antibody used in Example 8- (5). The content of CD8 positive T cells was calculated for the human CD3 positive T cell population. The content of CD4-positive T cells was determined by subtracting the proportion of CD8-positive T cells from the human CD3-positive T cell population as 100%. The results are shown in Table 10-3.

  From Table 10-3, although the ratio of CD8T cells decreased as the number of days passed after cell administration, the CD8T positive rate was higher in the RN-T group on the 28th day than in the other groups. That is, it has been clarified that cells produced using an anti-CD3 antibody and CH-296 can be engrafted while maintaining a high CD8 T cell ratio in vivo in NOG mice.

(6) Analysis of human T cells in peripheral blood CD45RA positive CCR7 positive T cells Staining was carried out in the same manner as in Example 8- (6). The content of CD45RA positive CCR7 positive T cells was calculated for the human CD3 positive T cell population. The average of each group is shown in Table 10-4.

  From Table 10-4, the RN-T group showed a higher value of the CD45RA-positive CCR7-positive T cell content in engrafted human lymphocytes than the other groups at any stage after cell administration. When produced using an anti-CD3 antibody and CH-296, CD45RA-positive CCR7-positive naive T cells proliferate efficiently, but it was revealed that the phenotype remains high even in vivo in NOG mice.

Example 11 Accumulation of RN-T in lymphoid tissues in NOD / scid mice (1) Preparation of lymphocytes Lymphocyte expansion culture was carried out in the same manner as in Example 7- (2), and the cells on the 14th day were cultured. Cryopreserved in the same manner as in Example 1- (1). For the culture, a bag in which only an anti-CD3 antibody (5 μg / mL solution was used for immobilization) was immobilized, anti-CD3 antibody and CH-296 (5 μg / mL anti-CD3 antibody solution, 25 μg / mL CH-296). A bag in which the solution was used for immobilization) was used. The same CD3 / CD28 bead stimulation group as in Example 7 was set.

(2) Mice and group composition NOD / scid mice (manufactured by CLEA Japan, Inc.) 12 8-week-old females were grouped as shown in Table 11-1. In each group, N = 4.

(3) Administration of anti-asialo GM1 antibody An anti-asialo GM1 antibody was administered intraperitoneally on the day before cell administration in the same manner as in Example 8- (3).

(4) Preparation and administration of administration cells The lymphocytes prepared in Example 11- (1) were administered to NOD / scid mice in the same manner as in Example 8- (4). In addition, the cell for administration was suspended in the physiological saline containing 4% HSA so that it might become 7.0 * 10 < ⁷ > cells / 0.3mL / mouse.

(5) Study on engraftment and absolute number of human T cells in peripheral blood Seven days after cell administration, blood was collected from the tail vein of mice, and the positive rate (% chimerism) and absolute number of transferred human lymphocytes [blood 1 μL Per hCD3-positive cells (cells / μL)] were measured by flow cytometry. That is, an equal amount of blood was added to a mixture of FITC-labeled mouse anti-human CD3 antibody, PE-labeled rat anti-mouse CD45 antibody and Flow-Count (manufactured by Beckman Coulter), which is a suspension of fluorescent particles, and after the reaction, The hemolysis used was applied to flow cytometry. The results are shown in Table 11-2. Table 11-2 shows the positive rate (% chimerism) of human lymphocytes in mouse peripheral blood and the number of hCD3-positive cells (μl / μL) per 1 μL of blood as an average value of N = 4.

  As shown in Table 11-2, the RN-T group on day 7 after cell administration showed a high% chimerism equivalent to that of the other groups, but the number of hCD3-positive cells per 1 μL of blood was higher than that of the other groups. Very high value. That is, it was shown that the cells stimulated with anti-CD3 antibody and CH-296 have high proliferation ability in the body after transplantation.

(6) Analysis of engraftment and accumulation of human T cells in peripheral lymphoid tissue On day 7 after cell administration, the mice were laparotomized under Nembutal anesthesia, and abdominal movements and veins were severed by cutting with scissors. Thereafter, the spleen and right and left inguinal lymph nodes were removed. Inguinal lymph nodes were grouped and used for cell preparation. The removed spleen and lymph nodes were crushed with a glass slide and dispersed, and used as a cell suspension sample for flow cytometry. The spleen was subjected to hemolysis with ACK buffer. For staining, a FITC-labeled mouse anti-human CD3 antibody was used. The results are shown in Table 11-3. Table 11-3 shows the positive rate (%) of human lymphocytes in the spleen and inguinal lymph nodes. The average value of N = 4 is shown for the spleen.

  As shown in Table 11-3, the engraftment of human T cells in mouse peripheral lymphoid tissue on day 7 after cell administration showed high results in the RN-T group for both the spleen and lymph nodes. That is, it was revealed that cells stimulated with anti-CD3 antibody and CH-296 have higher engraftment in peripheral lymphoid tissues in vivo in NOD / scid mice than in the other groups.

  Accumulation of human T cells in the inguinal lymph nodes was analyzed by counting with a Turku stain. The number of human T cells in the inguinal lymph node was determined from the obtained number of cells and the positive rate of human T cells in the lymph node of Table 11-3. The results are shown in Table 11-4.

  As shown in Table 11-4, the results showed that the number of cells in the inguinal lymph node in the RN-T group on day 7 after cell administration was significantly higher than in the other groups. It was also suggested that this increase in the number of cells was due to the accumulation of administered human T cells. That is, it was revealed that cells stimulated with anti-CD3 antibody and CH-296 have a high ability to accumulate in lymph nodes in the body.

(7) Analysis of CD45RA-positive CCR7-positive T cells of human T cells in peripheral lymphoid tissues The cells prepared in Example 11- (6) were subjected to RD1-labeled mouse anti-human in the same manner as in Example 8- (6). Staining was performed with CD45RA antibody, FITC-labeled mouse anti-human CCR7 antibody and PC5-labeled mouse anti-human CD3 antibody. The content of CD45RA positive CCR7 positive T cells was calculated for the human CD3 positive T cell population. The results are shown in Table 11-5. For the spleen, an average value of N = 4 is shown.

  As shown in Table 11-5, the RN-T group in the spleen on day 7 after cell administration was shown to have a higher content of CD45RA-positive CCR7-positive T cells in engrafted human lymphocytes than other groups. However, the content was as high in the lymph nodes. When stimulated with an anti-CD3 antibody and CH-296, naive T cells proliferate efficiently, but human T cells engrafted in the spleen in vivo in NOD / scid mice maintain their high phenotype. It was. Since all human T cells engrafted in lymph nodes have a high content of CD45RA-positive CCR7-positive T cells, it became clear that cells with a high phenotype of naïve T cells tend to accumulate in the lymph nodes.

Example 12 Quantification of telomerase activity in lymphocyte expansion culture, detection of telomere length and analysis of NKG2D positive CD8 positive cells (1) Separation of PBMC and separation of inactivated plasma In the same manner as in Example 3- (1) , PBMC and inactivated plasma were separated and subjected to each experiment.

(2) Immobilization of anti-human CD3 antibody and CH-296 ACD-A solution containing only an anti-human CD3 antibody with a final concentration of 0.3 μg / mL in a cell culture flask (Becton Dickinson) having a culture area of 75 cm ² Alternatively, 9.1 mL of an ACD-A solution containing an anti-human CD3 antibody having a final concentration of 0.3 μg / mL and CH-296 having a final concentration of 5 μg / mL is added, and each is 5 hours in a 37 ° C., 5% CO ₂ incubator. Incubated. Thereafter, the immobilization solution was removed, and the plate was washed twice with PBS and once with RPMI1640 medium immediately before use, and then subjected to each experiment.

(3) Expansion of lymphocytes PBMC 1.05 × 10 ⁷ cells separated in Example 12- (1) were added to 104.5 mL of GT-T551 containing 1% plasma containing IL-2 having a final concentration of 200 U / mL. After suspension, the suspension was added to the anti-CD3 antibody-immobilized flask prepared in Example 12- (2), or the anti-CD3 antibody and CH-296-immobilized flask, and cultured in a 37 ° C., 5% CO ₂ incubator ( Culture day 0). On day 4, the culture solution of each condition was diluted 8.36 times using GT-T551 containing 1% plasma, and 234 mL of this diluted solution was not immobilized at all, so that the bag area would be 300 cm ^2. It was transferred to a sealed gas permeable culture bag, MultiLife (registered trademark) Eva, and IL-2 was further added to a final concentration of 200 U / mL. The culture was continued, and on the seventh day, GT-T551 was added to each bag in an amount equal to the culture solution and diluted two-fold, and then IL-2 was added to a final concentration of 200 U / mL. On the 10th day, 234 mL of the culture solution of each group was extracted, and then IL-2 was added to an equal amount of GT-T551 and a final concentration of 200 U / mL, and the culture was continued until the 15th day. On the 4th, 7th, 10th, and 15th days after the start of culture, the number of viable cells was measured by trypan blue staining, and the expansion culture rate was calculated by comparison with the number of cells at the start of culture. The results are shown in Tables 12-1 and 12-2.

(4) Analysis of CD45RA-positive CCR7-positive cells in lymphocytes after culture Example 6 using the cells on the 4th, 7th, 10th and 15th days of culture prepared in Example 12- (3) -CD45RA positive CCR7 positive cells were analyzed in the same manner as in (3) (CD62L positive cells were not analyzed). The content ratio of each positive cell is shown in Table 12-3 and Table 12-4.

(5) Cell storage The lymphocytes 2 × 10 ⁵ cells on day 4 of culture expanded in Example 12- (3) were collected in a 1.5 mL microtube (manufactured by Treff), 3000 × g, Centrifugation was performed at 4 ° C. for 5 minutes, and the supernatant was removed. 1 mL of PBS was added and centrifuged under the same centrifugation conditions. The supernatant was removed, and the sediment (cells) was stored at −80 ° C. in a deep freezer. At the time of use, these preserved cells were thawed on ice and subjected to a telomerase activity quantitative experiment. In addition, the lymphocytes on the 4th, 7th, 10th, and 15th days after the expansion culture in Example 12- (3) were cryopreserved in the same manner as in Example 1- (1). . At the time of use, these preserved cells are rapidly thawed in a 37 ° C. water bath, washed with RPMI 1640 medium containing 10 μg / mL DNase, and then the number of viable cells is measured by trypan blue staining to detect telomere length. And it used for the analysis experiment of NKG2D positive CD8 positive cell.

(6) Quantification of telomerase activity The sediment (cell) cryopreserved in Example 12- (5) is thawed on ice, and a telomerase activity quantification kit based on the PCR-ELISA method, Telo TAGGG Telomerase PCR ELISA PLUS (Roche Telomerase activity was quantified using a diagnostics company. After the measurement according to the method of the data attached to the kit, the relative telomerase activities (hereinafter referred to as RTA) value was determined from the following formula 1 (Equation 1). The results are shown in Table 12-5.

A _S : Absorbance of sample A _SO : Absorbance of heat-treated sample A _{S, IS} : Absorbance of internal standard A _TS8 : Absorbance of control template A _TS8,0 : Absorbance of lysate A _{TS, IS} : Absorbance of internal standard of control template

  As shown in Table 12-5, in both donors, the flask in which only the anti-CD3 antibody was immobilized was obtained by using the flask in which the anti-CD3 antibody and CH-296 were immobilized in the early stage of lymphocyte expansion culture. Higher RTA values were obtained compared to the use. That is, it was clarified from the Examples that lymphocytes having high telomerase activity can be obtained by applying low concentration stimulation with anti-CD3 antibody and CH-296 in the early stage of lymphocyte expansion culture.

(7) Detection of telomere length Cells prepared by rapidly thawing the lymphocytes cryopreserved in Example 12- (5) in a 37 ° C. water bath and washing with RPMI 1640 medium containing 10 μg / mL DNase Telomere length detection was performed using a telomere length detection kit based on the Fluorescence in situ hybridization method by cytometry, telomere PNA kit / FITC labeling for flow cytometry (manufactured by Dako Japan). After measuring 1301 cells as a comparative control according to the method of the document attached to the kit, a relative telomere length (hereinafter referred to as RTL) value was determined from the following formula 2 (Formula 2). The results are shown in Table 12-6.

PS: FL1 Mean Fluorescence Intensity of sample with probe (average fluorescence intensity, hereinafter referred to as MFI)
P ₀ S: FL1 MFI of the probe-free sample
PC: FL1 MFI of control cells with probe
P ₀ C: FL1 MFI of control cells without probe
IC: DNA index of control cell = 2
IS: DNA index of sample cell = 1

  As shown in Table 12-6, by using a flask in which anti-CD3 antibody and CH-296 were immobilized at the initial stage of lymphocyte expansion culture, compared with the case in which a flask in which only anti-CD3 antibody was immobilized was used. Thus, a high RTL value was obtained. That is, from the examples, it was revealed that lymphocytes retaining longer telomeres can be obtained by applying low concentration stimulation with anti-CD3 antibody and CH-296 at the early stage of lymphocyte expansion culture.

(8) Analysis of NKG2D-positive CD8-positive cells in cultured lymphocytes Lymphocytes on day 10 and day 15 of the cryopreserved culture in Example 12- (5) were rapidly thawed in a 37 ° C. water bath and 10 μg. Cells prepared by washing with RPMI1640 medium containing / mL DNase were treated in the same manner as in Example 6- (3) with FITC-labeled mouse anti-human NKG2D antibody (manufactured by eBioscience) and RD1-labeled mouse anti-human CD8 antibody (Beckman). After staining with Coulter Co.), analysis was performed by flow cytometry. The content ratio of the positive cells is shown in Table 12-7.

  As shown in Table 12-7, in each donor, the flask in which only the anti-CD3 antibody was immobilized was obtained by using the flask in which the anti-CD3 antibody and CH-296 were immobilized at the early stage of lymphocyte expansion culture. A higher NKG2D positive CD8 positive cell ratio was obtained compared to the case of using. NKG2D molecule is an important molecule involved in CD8 positive T cell activation, cytotoxic response and IFN-γ production ability, and it is known that expression is reduced under immunosuppression. It was revealed that NKG2D-positive CD8-positive cells can be obtained with higher efficiency by applying low concentration stimulation with anti-CD3 antibody and CH-296 at the early stage of lymphocyte expansion culture.

Example 13 Examination of Early Activating Antigen in Resting T Cell (1) Preparation of resting T cell A resting T cell was prepared in the same manner as in Example 4. However, on the 0th day of culture, IL-2 was added at a final concentration of 200 U / mL using a cell culture flask (manufactured by Corning) having a culture area of 25 cm ² . 7 days of culture, the 11 day supplemented with 0.5% serum-containing GT-T551 so that 0.75~0.8 × ¹⁰ 6 cells / mL. At this time, IL-2 was added to a final concentration of 100 U / mL. The cell suspension on day 13 was collected and used as a resting T cell for each experiment.

(2) Immobilization of anti-CD3 antibody and CH-296 The anti-CD3 antibody and CH-296 were immobilized on the culture equipment used in the following experiments. That is, ACD-A solution containing only 0.3 μg / mL anti-CD3 antibody or 0.3 μg / mL anti-CD3 antibody and 1.5 μg / mL CH-296 in a 6-well cell culture plate (Corning). The contained ACD-A solution was added at 1 mL / well. After incubating the culture equipment at room temperature for 5 hours, the ACD-A solution was removed from the culture equipment by suction, and each well was washed twice with PBS and once with RPMI 1640 medium, and used for each experiment.

(3) Examination of initial activation antigen of cells Resting T cells 1.5 × 10 ⁶ cells prepared in Example 13- (1) were suspended in 3% of GT-T551 containing 0.5% serum, and Example 13- The plate was added to the anti-CD3 antibody-immobilized plate prepared in (2) or the anti-CD3 antibody and CH-296-immobilized plate, and these plates were cultured in a 37 ° C., 5% CO ₂ incubator. After 16 hours, cells were collected, stained with FITC-labeled mouse anti-human CD69 antibody (manufactured by eBioscience), and analyzed by flow cytometry. After 48 hours, cells were collected, stained with FITC-labeled mouse anti-human CD25 antibody (manufactured by Dako), and analyzed in the same manner. The results are shown in Table 13-1. In addition, the value in a table | surface shows the average value of N = 2.

  As shown in Table 13-1, when the concentration at the time of immobilization of the anti-CD3 antibody was 0.3 μg / mL as an initial stimulus in the resting T cells, CH-296 was added to the anti-CD3 antibody in a 1.5 μg / mL solution. When CD3 was immobilized, an increase in the expression of CD69, which is an early activation antigen of lymphocytes, was confirmed. Similarly, it was confirmed that the expression of CD25 whose expression increases with activation after CD69 is predominantly increased when CH-296 is immobilized on a low concentration anti-CD3 antibody. That is, it was shown that stimulation of cells with a plate immobilized by combining a low-concentration anti-CD3 antibody solution and a CH-296 solution promotes an increase in the expression of the initial activation antigen.

Example 14 Examination of Bcl-xL expression in resting T cells (1) Examination of Bcl-xL expression of cells Anti-CD3 antibody prepared in Example 13- (1) was prepared from the resting T cells prepared in Example 13- (2). They were added to immobilized plates, or anti-CD3 antibody and CH-296 immobilized plates, and these plates were cultured in a 37 ° C., 5% CO ₂ incubator. After 16 hours, the cells were collected, suspended in 1% BSA / PBS, and intracellularly stained with PE-labeled mouse anti-human Bcl-xL antibody (Beckman Coulter) according to the method of IntraPrep Reagent (Beckman Coulter). Was done. The cells were analyzed by flow cytometry. The results are shown in Table 14-1. In addition, the value in a table | surface shows the average value of N = 2.

  As shown in Table 14-1, apoptosis was suppressed when the concentration at the time of immobilization of anti-CD3 antibody was fixed at 0.3 μg / mL and CH-296 was fixed at 1.5 μg / mL as initial stimulation in resting T cells. It was confirmed that the expression intensity of Bcl-xL, which has a function to act, increases.

Example 15 Examination of RN-T cell engraftment over time in NOD / scid mice (1) Preparation of lymphocytes Lymphocyte expansion culture was carried out in the same manner as in Example 7- (2), and cultured on day 14 The cells were cryopreserved in the same manner as in Example 1- (1). The concentration at the time of immobilization of the anti-CD3 antibody and CH-296 was 5 μg / mL for the anti-CD3 antibody and 25 μg / mL for the CH-296.

(2) Mice and group composition Ten 7-week-old female NOD / scid mice were used. On the first day, the third day, the sixth day, the eighth day, and the 14th day after the administration, the final day was two mice, and the engraftment of human T cells in blood and in each tissue was examined.

(3) Administration of anti-asialo GM1 antibody An anti-asialo GM1 antibody was administered intraperitoneally on the day before cell administration in the same manner as in Example 8- (3).

(4) Preparation and administration of administered cells The lymphocytes prepared in Example 15- (1) were administered in the same manner as in Example 8- (4). In addition, the cell for administration was suspended in the physiological saline containing 4% HSA so that it might become 8.0 * 10 < ⁷ > cells / 0.3mL / mouse.

(5) Engraftment of human T cells in blood and tissues On day 1, day 3, day 6, day 8, and day 14 after cell administration, blood was collected or tissues were extracted from mice. The sites examined were the tail vein, inferior vena cava, spleen, inguinal lymph node, bone marrow, and thymus. In the same manner as in Example 8- (5), the% chimerism of human T cells in the tail vein and inferior vena cava was examined. The results are shown in Table 15-1. Further, the positive rate (%) of human lymphocytes in each tissue was examined in the same manner as in Example 11- (6). At this time, spleen and bone marrow were subjected to hemolysis with ACK buffer. The results are shown in Table 15-2.

＊ｎ．ｔ．は組織の摘出ができなかったことを示す。

* N. t. Indicates that the tissue could not be extracted.

  As shown in Table 15-1, RN-T cells showed high% chimerism in the blood from day 3 to day 14 after cell administration. In addition, when time-dependent changes in RN-T cells in the tissues were examined, as shown in Table 15-2, in the spleen, the positive rate (%) of human lymphocytes of 80% or more on the 3rd and 6th days Was confirmed. The inguinal lymph node is a tissue that has not developed in NOD / scid, which is an immunodeficient mouse, but it can be confirmed that it has grown macroscopically after the 6th day, and about 6th and 14th day. A positive rate (%) of human lymphocytes of 45% or more was confirmed. On the 6th day for the thymus and on the 3rd day for the bone marrow, the positive rate (%) of human lymphocytes tended to increase, but on the 14th day, the positive rate decreased to about several percent.

(6) Analysis of CD45RA-positive CCR7-positive T cells of human T cells in blood and tissues In the same manner as in Example 8- (6), the content of CD45RA-positive CCR7-positive T cells was calculated for the human CD3-positive T cell population. . The average result of each group is shown in Table 15-3.

  From Table 15-3, the CD45RA-positive CCR7-positive T cell content (%) showed the same positive rate among the tissues. It was revealed that RN-T cells accumulated in blood and tissues showed a CD45RA-positive CCR7-positive T cell content equal to or higher than that before administration after the sixth day.

Example 16 Accumulation of RN-T in lymphoid tissues in NOD / scid mice (1) Preparation of lymphocytes Lymphocyte expansion culture was performed in the same manner as in Example 7- (2), and cultured on days 10 and 14 The eye cells were cryopreserved in the same manner as in Example 1- (1). The initial stimulation method was the same as in Example 11- (1).

(2) Mice and group composition 24 NOD / scid mice 24 weeks old females were grouped as shown in Table 16-1. In each group, N = 4.

(3) Administration of anti-asialo GM1 antibody An anti-asialo GM1 antibody was administered intraperitoneally on the day before cell administration in the same manner as in Example 8- (3).

(4) Preparation and administration of administration cells The lymphocytes prepared in Example 16- (1) were administered to NOD / scid mice in the same manner as in Example 8- (4). In addition, the cell for administration was suspended in the physiological saline containing 4% HSA so that it might become 7.0 * 10 < ⁷ > cells / 0.3mL / mouse.

(5) Study of engraftment and absolute number of human T cells in peripheral blood In the same manner as in Example 11- (5), tail vein blood was collected from mice on days 5 and 7 after cell administration, and transferred. The percentage of positive human lymphocytes (% chimerism) and the absolute number [number of hCD3-positive cells per 1 μL of blood (cells / μL)] were measured by flow cytometry. The result of the average value of N = 4 is shown in Table 16-2.

  As shown in Table 16-2, the RN-T group on day 5 and day 7 after cell administration showed a high% chimerism equivalent to that of other groups, but on day 5 after cell administration, The number of hCD3-positive cells of was higher than that of the other groups. The% chimerism between 10 and 14 days of culture was similar, but the absolute number of hCD3-positive cells in peripheral blood tended to be higher in cells cultured for 10 days.

(6) Analysis of engraftment and accumulation of human T cells in peripheral lymphoid tissues Analysis of engraftment of human T cells in peripheral lymphoid tissues on day 7 after cell administration in the same manner as in Example 11- (6). did. The average value of N = 4 is shown for the spleen. Each result is shown in Table 16-3.

  As shown in Table 16-3, spleen engraftment of human T cells on day 7 after cell administration showed high results in the RN-T group and the OKT3-T group, and engraftment in lymph nodes was RN. High results were shown in the -T group. In 10 days and 14 days of culture, the 10-day culture group showed slightly higher engraftment. That is, it was revealed that cells stimulated with anti-CD3 antibody and CH-296 have higher engraftment in peripheral lymphoid tissues in the body of NOD / scid mice than in the other groups.

  Next, the accumulation of human T cells in the inguinal lymph nodes was analyzed. The number of human T cells in the inguinal lymph nodes was determined from the number of cells obtained and the positive rate of human T cells in the lymph nodes shown in Table 16-3. The results are shown in Table 16-4.

  As shown in Table 16-4, the number of cells in the inguinal lymph node of the RN-T group on day 7 after cell administration was higher than that of the other groups regardless of the number of culture days. That is, it was revealed that cells stimulated with anti-CD3 antibody and CH-296 have a high ability to accumulate in lymph nodes in the body.

(7) Analysis of CD45RA-positive CCR7-positive T cells of human T cells in peripheral blood and peripheral lymphoid tissues For the cells prepared in Example 16- (6), RD1 was prepared in the same manner as in Example 8- (6). Staining was performed with labeled mouse anti-human CD45RA antibody, FITC-labeled mouse anti-human CCR7 antibody and PC5-labeled mouse anti-human CD3 antibody. The content of CD45RA positive CCR7 positive T cells was calculated for the human CD3 positive T cell population. The results are shown in Table 16-5. For the spleen, an average value of N = 4 is shown.

  As shown in Table 16-5, the content of CD45RA-positive CCR7-positive T cells in human lymphocytes engrafted in peripheral blood, spleen, and inguinal lymph nodes on day 7 after cell administration was higher in the RN-T group. It was shown to be equal to or higher than the group. Naive T cells proliferate efficiently when stimulated with anti-CD3 antibody and CH-296, but human T cells engrafted in peripheral blood and peripheral lymphoid tissues in the body of NOD / scid mice remain high in phenotype It became clear to maintain.

  The present invention provides a method for producing lymphocytes. This method has a high cell growth rate, and the lymphocytes obtained by the present invention are excellent in the engraftment rate in the living body and the ability to maintain the phenotype, and are extremely useful in the medical field including adoptive immunotherapy.

SEQ ID NO: 1; Partial region of fibronectin named III-8.
SEQ ID NO: 2; Partial region of fibronectin named III-9
SEQ ID NO: 3; Partial region of fibronectin named III-10.
SEQ ID NO: 4; Partial region of fibronectin named III-11.
SEQ ID NO: 5; Partial region of fibronectin named III-12.
SEQ ID NO: 6; Partial region of fibronectin named III-13.
SEQ ID NO: 7; Partial region of fibronectin named III-14.
SEQ ID NO: 8; Partial region of fibronectin named CS-1.
SEQ ID NO: 9; Fibronectin fragment named C-274.
SEQ ID NO: 10; Fibronectin fragment named H-271.
SEQ ID NO: 11; Fibronectin fragment named H-296.
SEQ ID NO: 12; Fibronectin fragment named CH-271.
SEQ ID NO: 13; Fibronectin fragment named CH-296.
SEQ ID NO: 14; Fibronectin fragment named C-CS1.
SEQ ID NO: 15; Fibronectin fragment named CH-296Na.
SEQ ID NO: 16; Fibronectin fragment named CHV-89.
SEQ ID NO: 17; Fibronectin fragment named CHV-90.
SEQ ID NO: 18; Fibronectin fragment named CHV-92.
SEQ ID NO: 19; Fibronectin fragment named CHV-179.
SEQ ID NO: 20; Fibronectin fragment named CHV-181.
SEQ ID NO: 21; Fibronectin fragment named H-275-Cys.
SEQ ID NO: 22; Fibronectin fragment named H296-H296.
SEQ ID NO: 23; Fibronectin fragment named H296-H296-H296-HT.
SEQ ID NO: 24; Fibronectin fragment named CH296-CH296-HT.
SEQ ID NO: 25; Fibronectin fragment named H105-H105-HT.
SEQ ID NO: 26; Fibronectin fragment named H296-H296-HT.
SEQ ID NO: 27; Fibronectin fragment named H296-H296-H296.
SEQ ID NO: 28; Fibronectin fragment named H105-H105.
SEQ ID NO: 29; Fibronectin fragment named H271-H296.
SEQ ID NO: 30; Fibronectin fragment named H296-H271.
SEQ ID NO: 31; Fibronectin fragment named 15aaH105-H105-HT.
SEQ ID NO: 32; Fibronectin fragment named 15aaH105-H105.
SEQ ID NO: 33; Fibronectin fragment named H105-H105Nc-HT
SEQ ID NO: 34; Fibronectin fragment named H105-H105Nc.

Claims (7)

A method for producing lymphocytes comprising a step of culturing lymphocytes or lymphocyte progenitor cells in the presence of an anti-CD3 antibody and a polypeptide having an amino acid sequence derived from fibronectin,
(1) using a solid phase in which the antibody is immobilized using an anti-CD3 antibody solution of less than 5 μg / mL and / or (2) a polypeptide solution having an amino acid sequence derived from fibronectin of less than 25 μg / mL A method for producing lymphocytes, comprising culturing lymphocytes or lymphocyte progenitor cells in contact with a solid phase on which the polypeptide is immobilized.   The method according to claim 1, wherein the solid phase is a cell culture equipment or a cell culture support.   The method according to claim 1, wherein the polypeptide having an amino acid sequence derived from fibronectin has a binding activity selected from a VLA-4 binding activity, a VLA-5 binding activity, and a heparin binding activity.   The method according to claim 1, wherein the polypeptide having an amino acid sequence derived from fibronectin has an amino acid sequence derived from fibronectin selected from a CS-1 domain, a cell adhesion domain, and a heparin binding domain.   The method according to claim 4, wherein the polypeptide having an amino acid sequence derived from fibronectin is a polypeptide having an amino acid sequence selected from SEQ ID NOs: 9 to 34 in the sequence listing.   The method according to claim 1, wherein the lymphocyte is an antigen-specific cytotoxic T lymphocyte or a lymphokine activated cell.   The method according to claim 1, further comprising the step of introducing a foreign gene into lymphocytes.