JP2005124568A - Method for activating cell, method for producing the cell using the same, and pharmaceutical composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for activating a cell, capable of simply obtaining a cytotoxic T cell having nonspecific cytotoxic activity on various tumor cells, virally infected cells, etc., in a short period, to provide a method for producing the cell using the same, and to provide a pharmaceutical composition. <P>SOLUTION: This pharmaceutical composition has a CD3 agonist which gives stimulation (main stimulation) to the cytotoxic T cell or a precursor of the cytotoxic T cell (hereinafter, referred to as the precursor) through a CD3 molecule of the cytotoxic T cell or the precursor, and further has a CD52 agonist which gives stimulation (auxiliary stimulation) to the cytotoxic T cell or the precursor through a CD52 molecule of the cytotoxic T cell or the precursor. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to cytotoxic T cells or precursor cells thereof for the purpose of inducing differentiation, promoting proliferation and enhancing functions of cytotoxic T cells (CD8 killer T cells, CTL) that attack tumor cells, virus-infected cells, and the like. The present invention relates to a cell activation method for activating immune cells, a cell production method using the same, and a pharmaceutical composition.

JP 2003-102471 A

  Examples of so-called cancer immunotherapy that treats cancer using the immune function of the living body include, for example, a vaccine using a tumor-specific antigen, a BCG that enhances immunity, or a monoclonal antibody that specifically attacks cancer cells. Examples thereof include a method of using the preparation, and so-called adoptive immunotherapy in which immunocompetent cells taken out of the living body are appropriately treated to have an anticancer function and the immunocompetent cells having the anticancer function are returned to the living body. As adoptive immunotherapy, for example, monocytes (mononuclear cells) are removed from the peripheral blood of the living body, the extracted monocytes are differentiated to proliferate dendritic cells, and the proliferated dendritic cells are treated with tumor antigens, Examples include so-called dendritic cell therapy in which dendritic cells treated with tumor antigens are returned to the living body. In such dendritic cell therapy, it is believed that dendritic cells induce cytotoxic T cells that specifically attack tumor cells in vivo. In addition, for example, natural killer cells (hereinafter referred to as NK cells) or natural killer T cells (hereinafter referred to as NKT cells) having non-specific anticancer activity are taken out of the living body and activated to be activated. A method of returning to the living body after NK cells or NKT cells proliferate is also considered as one of adoptive immunotherapy.

  As a pioneering therapy of adoptive immunotherapy in which cells are activated outside the body and returned to the body, for example, so-called LAK (lymphokine-activated killer) therapy can be mentioned (Lotze M et al., Cancer Res. 41: 4420, 1981). LAK therapy has been available since the 1980s when recombinant human interleukin 2 (hereinafter referred to as IL-2), which is a lymphocyte growth factor, became available, and peripheral blood lymph collected from cancer patients. LAK cells having antitumor activity, which is one of NK cells obtained by culturing spheres in the presence of high concentration of IL-2, were transferred into the body of a cancer patient, and simultaneously with this transfer, a high dose of IL-2 was introduced. The therapy to be administered. Although such LAK therapy has been reported to have an effect on 11 of 25 cases in malignant melanoma and renal cancer, no effect has been observed in the follow-up test after that report. The therapeutic effect of LAK therapy alone is difficult to recognize due to poor medical evidence.

  Research on immunocompetent cells that have infiltrated tumor tissues even after LAK cells have become available, and as a result, cells that recognize tumor cell antigens and show specific cytotoxic activity against the tumor cells Was found to be cytotoxic T cells, lymphocytes (TIL) infiltrating the tumor tissue were collected instead of peripheral blood, and the collected TILs were proliferated using IL-2 in vivo. So-called TIL therapy was attempted to revert to. There are reports that TIL therapy is effective for malignant melanoma, locally recurrent breast cancer, cancerous pleural effusion, cancerous ascites, hepatocellular carcinoma, colon cancer, non-Hodgkin lymphoma, etc., but the collection of TIL is complicated. In addition, since TIL requires a long-term culture for proliferation, there is a risk that TIL accumulation in the tumor and antitumor activity may be reduced, and cells that suppress the antitumor effect may appear. There is also.

  In order to induce cytotoxic T cells having strong activity against tumor cells, T cells as precursor cells of cytotoxic T cells and antigen-presenting cells such as dendritic cells are cocultured. It is said that it is necessary to stimulate T cells. That is, in order to induce cytotoxic T cells, a T cell receptor (TCR) of a T cell having a CD8 molecule recognizes an antigen peptide bound to an HLA class 1 molecule of an antigen presenting cell. In addition to the main stimulation, it is necessary to give the CD8 T cell a costimulation through the CD28 molecule of the T cell and the like. For this reason, research on specific antigens expressed only in tumor cells and their epitopes has been advanced, and new tumor antigens are being discovered one after another.

  A method for inducing differentiation of dendritic cells treated with tumor antigens or tumor antigen-specific cytotoxic T cells in vitro in large quantities and returning them to the living body using the new tumor antigens discovered by the above-described research as clues Has been studied. As such a method, for example, a tumor-specific cytotoxic T cell or a cytotoxic T cell clone is induced and proliferated by culturing dendritic cells incorporating a tumor antigen together with autologous peripheral blood lymphocytes. There is a method (Dudley ME, et al., J. Immunother. 25: 243-51, 2002, Yee C, et al., Pro. Natl. Acad. Sci. USA., 99: 16168-73, 2002).

  In Japan, in addition to the methods described above, self-activating lymphocyte transfer therapy for cancer has been performed as a highly advanced medical treatment at some universities and private medical facilities. As such self-activating lymphocyte transfer therapy, for example, peripheral blood lymphocytes are stimulated with anti-CD3 antibody, cultured for a long period of time with IL-2, proliferated, and the peripheral blood lymphocytes thus proliferated are in the body of a cancer patient. The so-called CD3-LAK therapy that returns to the above is mentioned as a relatively simple therapy, and the cells that are activated by the CD3-LAK therapy and have antitumor activity are mainly CD8 T cells, and the activity used in the LAK therapy Not NK cells (Ochoa AC et al., J. Immunol. 138: 2728, 1987). CD3-LAK therapy is currently used for various cancers, and it has been reported that a recurrence-inhibiting effect after hepatocellular carcinoma healing is particularly recognized (Takayama T et al., Lancet 356). : 802, 2000).

  However, in the CD3-LAK therapy as described above, the anti-tumor activity of activated T cells returned to the living body is low (Geller RL et al., J. Immunol. 146: 3280, 1991), and this CD3-LAK The response rate (completion response + partial response) in which cancer was actually reduced by the therapy is around 10% (Goto S et al., Anticancer Res. 22: 2461, 2002).

  The reason why the CD3-LAK therapy has a low response rate to cancer is that, firstly, the proliferated cytotoxic T cells have low perforin expression, and secondly, chemokine receptors for migration to lymph nodes, It is possible that CCR7 disappears from cytotoxic T cells.

  Regarding the first reason for the low response rate of CD3-LAK therapy to cancer, in order to express a large amount of perforin having a function of piercing the cell membrane of a target tumor cell, a cytotoxic T cell or a precursor cell thereof must be an antigen. It is considered necessary to receive two signals from a specific T cell receptor (TCR) and a costimulatory molecule (usually a CD28 molecule), and a cytotoxic T cell or its progenitor cell can When only a signal is received, it is considered that perforin in the proliferated cytotoxic T cells does not increase sufficiently. When such perforin is low, it is considered that cytotoxic T cells cannot significantly attack cancer cells because the function of piercing the surface tissue of cancer cells is significantly reduced.

  Regarding the second reason why CD3-LAK therapy has a low response rate to cancer, cytotoxic T cells proliferated by CD3-LAK therapy are required to migrate to peripheral lymph nodes. Since chemokine receptors, particularly CCR7, do not express so much, cytotoxic T cells administered to the body cannot reach peripheral lymph nodes, and thus cannot exert an effect on cancer that has metastasized to peripheral lymph nodes. It is possible. In addition, it is considered to be extremely important to prevent cancer metastasis to peripheral lymph nodes that cause tumor immune reaction.

  In addition, when the immunotherapy as described above is used as a practical treatment, there are several problems as follows. As such a problem, it takes a complicated operation and a lot of time to induce tumor-specific cytotoxic T cells and to proliferate the cytotoxic T cells to such an extent that a therapeutic effect can be obtained. It is difficult to easily proliferate cytotoxic T cells in a short period of time, and since there are still few known tumor antigens, it is difficult to make tumor-specific cytotoxic T cells compatible with a wide variety of cancers. In addition, it is difficult to deal with tumor lymphocytes (TIL) removed from living bodies to all patients, and lymphocytes activated by stimulation with anti-CD3 antibody alone have low perforin expression and CCR7 Since it has also disappeared, it is difficult to increase the response rate for cancer.

  On the other hand, the present inventor has shown that regulatory T cells derived from CD4 positive T cells having an inhibitory action on other T cells are induced by the action of anti-4C8 antibody (Masuyama, J. et. al., J. Immunol. 169: 3710, 2002), and the induction of such regulatory T cells is also shown in Patent Document 1. The present inventor has also discovered that anti-CD52 antibody (anti-4C8 antibody) has transvascular endothelial cell migration inhibitory activity (Masyama, J. et al., J. Exp. Med. 189: 979, 1999; WO 99/12972). However, the anti-C4C8 antibody or the like gives a costimulation to a cytotoxic T cell or its progenitor cell via a CD52 molecule, and a tumor cell or a virus-infected cell based on the cytotoxic T cell or progenitor cell to which the costimulation is given No research has been done to kill them.

  The present invention has been made in view of the above-mentioned points, and an object of the present invention is to short-term cytotoxic T cells having nonspecific cytotoxic activity against various tumor cells, virus-infected cells and the like. It is an object of the present invention to provide a cell activation method, a cell production method using the same, and a pharmaceutical composition that can be easily obtained.

  Another object of the present invention is to provide cytotoxic T cells having non-specific cytotoxic activity against various tumor cells, virus-infected cells and the like, and pharmaceutical compositions containing the same. .

  In the cell activation method according to the first aspect of the present invention, a cytotoxic T cell or a progenitor cell thereof is stimulated by a CD3 agonist and a CD52 agonist.

  According to the cell activation method of the first aspect, since it has the above-described configuration, the cytotoxicity T cell or its progenitor cell is mainly stimulated by the CD3 agonist and the CD52 agonist via the CD3 molecule and the CD52 molecule. And by giving a costimulation, it is possible to cause a strong activation of the cytotoxic T cell or its progenitor cell, and to induce strong differentiation in the thus activated cytotoxic T cell or its progenitor cell. Therefore, it is possible to easily increase the number of cytotoxic T cells that express chemokine receptors, particularly CCR7, and can migrate to the lymph nodes in a short period of time. Thus, cytotoxic T cells having nonspecific cytotoxic activity against various tumor cells, virus-infected cells and the like can be obtained. Moreover, the cytotoxic T cell obtained by using the cell activation method of the first aspect can more accurately separate normal cells from tumor cells than other immune cells. In addition, the cytotoxic T cells obtained based on the cell activation method of the first aspect are stimulated again by the cell activation method, so that they have a large amount of perforin and CCR7, Cytotoxic T cells exhibiting strong and non-specific cytotoxic activity against tumor cells, virus-infected cells and the like can be obtained. The cell activation method according to the first aspect capable of exhibiting such an effect is directed to treatment of diseases / pathological conditions such as malignant tumors and viral infections directly (administration of an agonist to a living body) or indirectly (cytotoxicity). It can be used for the production of T cells). In the cell activation method according to the first aspect capable of exhibiting such an effect, the present inventor has intensively studied the efficacy of CD52 agonists such as anti-4C8 antibody on cytotoxic T cells or progenitor cells thereof, and CD3 agonists and CD52 agonists. When a cytotoxic T cell or a progenitor cell thereof is stimulated (co-stimulated) via CD3 molecule and CD52 molecule to be activated by activation, the cytotoxic T cell or the progenitor cell obtained by the activation is activated. However, it can be constructed for the first time based on the result of newly finding that chemokine receptors, particularly CCR7 are highly expressed.

  In the cell activation method of the second aspect of the present invention, in the cell activation method of the first aspect, the CD52 agonist has an anti-4C8 antibody or a fragment thereof.

  In the cell activation method of the third aspect of the present invention, in the cell activation method of the first or second aspect, the CD52 agonist has campus-1H or a fragment thereof.

  According to the cell activation method of the second or third aspect, since it has the above-described configuration, cytotoxic T cells or progenitor cells thereof by anti-4C8 antibody or fragment thereof or campus-1H or fragment thereof By giving a costimulatory effect, it is possible to express a chemokine receptor, particularly CCR7, in many cytotoxic T cells, and in particular to a cytotoxic T cell or a progenitor cell thereof by an anti-4C8 antibody or a fragment thereof. By giving the chemokine receptor, in particular, CCR7 can be expressed in more cytotoxic T cells.

  In the cell activation method according to the fourth aspect of the present invention, in the cell activation method according to any one of the first to third aspects, the CD52 agonist is one of CD52 molecules of cytotoxic T cells or progenitor cells thereof. It has an antibody or fragment thereof that binds to a site and another antibody or fragment thereof that binds to another site of the CD52 molecule of the cytotoxic T cell or its progenitor cell.

  In the cell activation method of the fifth aspect of the present invention, in the cell activation method of the fourth aspect, the antibody or fragment thereof that binds to one site of the CD52 molecule of a cytotoxic T cell or a progenitor cell thereof, The other antibody or fragment thereof that consists of an anti-4C8 antibody or fragment thereof and binds to other sites of the CD52 molecule of the cytotoxic T cell or its precursor cell consists of campus-1H or a fragment thereof.

  According to the cell activation method of the fourth or fifth aspect, since it has the above-described configuration, for example, an anti-4C8 antibody or a fragment thereof is bound to one site of the CD52 molecule, and in addition, the campus By binding -1H or a fragment thereof to other sites of the CD52 molecule, cytotoxic T cells or progenitor cells thereof can be suitably activated.

  In the cell activation method according to the sixth aspect of the present invention, in the cell activation method according to any one of the first to fifth aspects, the CD3 agonist binds to a CD3 molecule of a cytotoxic T cell or a progenitor cell thereof. It has an anti-CD3 antibody or fragment thereof.

  In the cell activation method according to the seventh aspect of the present invention, in the cell activation method according to any one of the first to sixth aspects, at least one of the CD3 agonist and the CD52 agonist is at least one of a humanized antibody and a human antibody. have.

  In the cell activation method of the eighth aspect of the present invention, in the cell activation method of the seventh aspect, the humanized antibody is mouse humanized antibody or rat humanized antibody campus-1H.

  The cell activation method according to the ninth aspect of the present invention is the cell activation method according to any one of the first to eighth aspects, wherein the cytotoxic T cell or its cell is in vitro or in vivo by a CD3 agonist and a CD52 agonist. Stimulates progenitor cells.

  A cell activation method according to a tenth aspect of the present invention is the cell activation method according to any one of the first to ninth aspects, wherein the peripheral blood, lymph node, thymus, bone marrow, tumor, pleural effusion is obtained using a CD3 agonist and a CD52 agonist. Stimulates cytotoxic T cells or their progenitor cells that can be present in ascites or umbilical cord blood.

  The cell production method of the present invention produces cytotoxic T cells using the cell activation method according to any one of the first to tenth aspects. Moreover, the cytotoxic T cell of the present invention can be obtained by the cell activation method according to any one of the first to tenth aspects. Furthermore, the present invention also provides a pharmaceutical composition containing cytotoxic T cells obtained by the cell activation method of any one of the first to tenth aspects, which is different from the pharmaceutical composition of each aspect described below. It can be configured as an aspect.

  The pharmaceutical composition of the first aspect of the present invention has a CD3 agonist and a CD52 agonist that give stimulation to cytotoxic T cells or their precursor cells.

  According to the pharmaceutical composition of the first aspect, since it has the above-described configuration, the CD3 agonist and the CD52 agonist can mainly stimulate the cytotoxic T cell or its precursor cell via the CD3 molecule and the CD52 molecule. By giving a costimulation, it is possible to cause strong activation of the cytotoxic T cell or its progenitor cells, and to induce strong differentiation in the thus activated cytotoxic T cell or its progenitor cells. Proliferation can be promoted and, therefore, cytotoxic T cells that express chemokine receptors, particularly CCR7, and can migrate to the lymph nodes can be easily increased in a short period of time. Thus, cytotoxic T cells having nonspecific cytotoxic activity against various tumor cells, virus-infected cells and the like can be obtained. Moreover, the cytotoxic T cell obtained by using the pharmaceutical composition of the first aspect can more accurately separate normal cells from tumor cells than other immune cells. In addition, the cytotoxic T cells obtained using the pharmaceutical composition of the first aspect are stimulated again with the pharmaceutical composition, so that they have a large amount of perforin and CCR7, and various tumor cells. In addition, cytotoxic T cells exhibiting strong and non-specific cytotoxic activity against virus-infected cells and the like can be obtained. The pharmaceutical composition of the first aspect capable of exhibiting such an effect is directly (for example, administration of an agonist to a living body) or indirectly (cytotoxic T) for the treatment of diseases / pathological conditions such as malignant tumors and viral infections. For example, cell production). The pharmaceutical composition of the first aspect capable of exhibiting such an effect has been studied by the present inventor as to the efficacy of CD52 agonists such as anti-4C8 antibody on cytotoxic T cells or their progenitor cells, and the use of CD3 agonists and CD52 agonists. When a cytotoxic T cell or a progenitor cell thereof is stimulated (co-stimulated) via CD3 molecule and CD52 molecule and activated, the cytotoxic T cell or the progenitor cell obtained by the activation is activated. The chemokine receptor, particularly CCR7, can be constructed for the first time based on the result of newly finding that it is highly expressed.

  In the pharmaceutical composition of the second aspect of the present invention, in the pharmaceutical composition of the first aspect, the CD52 agonist has an anti-4C8 antibody or a fragment thereof.

  In the pharmaceutical composition of the third aspect of the present invention, in the pharmaceutical composition of the first or second aspect, the CD52 agonist has campus-1H or a fragment thereof.

  According to the pharmaceutical composition of the second or third aspect, since it has the above-described configuration, it can be treated with cytotoxic T cells or progenitor cells thereof by anti-4C8 antibody or a fragment thereof or campus-1H or a fragment thereof. By applying costimulation, a number of cytotoxic T cells can express chemokine receptors, particularly CCR7, and in particular, the cytotoxic T cells or their progenitor cells are costimulated by anti-4C8 antibodies or fragments thereof. As a result, more cytotoxic T cells can express chemokine receptors, particularly CCR7.

  In the pharmaceutical composition of the fourth aspect of the present invention, in the pharmaceutical composition of any one of the first to third aspects, the CD52 agonist is present at one site of the CD52 molecule of the cytotoxic T cell or its precursor cell. It has an antibody or fragment thereof that binds and another antibody or fragment thereof that binds to other sites on the CD52 molecule of the cytotoxic T cell or its progenitor cells.

  In the pharmaceutical composition of the fifth aspect of the present invention, in the pharmaceutical composition of the fourth aspect, the antibody or fragment thereof that binds to one site of the CD52 molecule of cytotoxic T cells or progenitor cells thereof is anti-4C8. Another antibody or fragment thereof consisting of an antibody or fragment thereof that binds to other sites of the CD52 molecule of the cytotoxic T cell or its progenitor cells consists of campus-1H or a fragment thereof.

  According to the pharmaceutical composition of the fourth or fifth aspect, since it has the above-described configuration, for example, an anti-4C8 antibody or a fragment thereof is bound to one site of the CD52 molecule, By binding 1H or a fragment thereof to other sites of the CD52 molecule, cytotoxic T cells or progenitor cells thereof can be suitably activated.

  In the pharmaceutical composition of the sixth aspect of the present invention, in the pharmaceutical composition of any one of the first to fifth aspects, the CD3 agonist is an anti-CD3 that binds to a CD3 molecule of a cytotoxic T cell or a precursor cell thereof. Has an antibody or fragment thereof.

  In the pharmaceutical composition of the seventh aspect of the present invention, in the pharmaceutical composition of any one of the first to sixth aspects, at least one of the CD3 agonist and the CD52 agonist has at least one of a humanized antibody and a human antibody. doing.

  In the pharmaceutical composition according to the eighth aspect of the present invention, in the pharmaceutical composition according to the seventh aspect, the humanized antibody is mouse humanized antibody or rat humanized antibody campus-1H.

  The pharmaceutical composition of the ninth aspect of the present invention has a CD52 agonist that stimulates cytotoxic T cells or progenitor cells thereof.

  According to the pharmaceutical composition of the ninth aspect, since it has the above-described configuration, cytotoxicity is caused by applying costimulation to a cytotoxic T cell or its progenitor cell via a CD52 molecule by a CD52 agonist. Cytotoxicity when sex T cells or their progenitor cells are given a major stimulus via CD3 molecules based on the binding of MHC-1 molecules to the cytotoxic T cells or their T cell receptors, etc. T cells or their progenitor cells can be strongly activated, and thus activated cytotoxic T cells or their progenitor cells can be strongly induced to promote proliferation. In addition, cytotoxic T cells that express chemokine receptors, particularly CCR7, and can migrate to lymph nodes can be easily increased in a short period of time. It can be obtained cytotoxic T cells with non-specific cytotoxic activity against cells. The pharmaceutical composition of the ninth aspect capable of exhibiting such an effect is obtained by the inventor's earnest study on the efficacy of CD52 agonists such as anti-4C8 antibody on cytotoxic T cells or their progenitor cells as described above. It can be configured for the first time based on the results obtained.

  In the pharmaceutical composition of the tenth aspect of the present invention, in the pharmaceutical composition of the ninth aspect, the CD52 agonist has an anti-4C8 antibody or a fragment thereof.

  In the pharmaceutical composition of the eleventh aspect of the present invention, in the pharmaceutical composition of the ninth or tenth aspect, the CD52 agonist has campus-1H or a fragment thereof.

  According to the present invention, a cell activation method capable of easily obtaining a cytotoxic T cell having non-specific cytotoxic activity against various tumor cells, virus-infected cells and the like in a short period of time and the use thereof are used. Cell production methods as well as pharmaceutical compositions may be provided. Moreover, according to this invention, the cytotoxic T cell which has non-specific cytotoxic activity with respect to various tumor cells, virus infection cells, etc., and a pharmaceutical composition containing this can be provided.

  Next, examples of embodiments of the present invention will be described in more detail. The present invention is not limited to these examples.

  The pharmaceutical composition of this example stimulates cytotoxic T cells or progenitor cells via CD3 molecules of cytotoxic T cells or progenitor cells of cytotoxic T cells (hereinafter referred to as progenitor cells) (primary stimulation). And a CD52 agonist that gives stimulation (costimulation) to the cytotoxic T cell or progenitor cell via the CD52 molecule of the cytotoxic T cell or progenitor cell.

In this example, the cytotoxic T cell or progenitor cell to which stimulation (costimulation) by CD3 agonist and CD52 agonist is given has at least CD8 molecule, CD3 molecule and CD52 molecule. Cytotoxic T cells or progenitor cells consist of mononuclear cells that can be present in peripheral blood, lymph nodes, thymus, bone marrow, tumor, pleural effusion, ascites or umbilical cord blood, preferably peripheral blood units that can be present in peripheral blood. It consists of a nuclear sphere. The progenitor cells are embodied by, for example, CD8 T cells (including CD8 ⁺ CD28 ⁻ T cells and the like) before activation.

  The CD3 agonist in this example mainly acts on a CD3 molecule expressed on the surface of a cytotoxic T cell or progenitor cell, and transmits a signal into the cytotoxic T cell or progenitor cell via this CD3 molecule. Made of substances that cause irritation. The CD52 agonist in this example acts on a CD52 molecule expressed on the cell surface of a cytotoxic T cell or progenitor cell, and transmits a signal into the cytotoxic T cell or progenitor cell via this CD52 molecule. Consists of substances that cause costimulation.

  In this example, the CD3 agonist contains an anti-CD3 antibody as a main active ingredient. The anti-CD3 antibody is recognized by the cytotoxic T cell or progenitor cell by binding to the CD3 molecule of the cytotoxic T cell or progenitor cell. Here, instead of or in addition to the anti-CD3 antibody, the CD3 agonist may contain a recognized anti-CD3 antibody fragment that binds to and recognizes the CD3 molecule in the same manner as the anti-CD3 antibody. The fragment preferably consists of a Fab fragment having an antigen recognition site.

  The anti-CD3 antibody may be configured as a humanized antibody (including mouse humanized antibody, rat humanized antibody, etc.) or a human antibody.

  Anti-CD3 antibodies that are agonistic to the CD3 molecule are embodied by, for example, OKT3 (ATCC CRL-8001), UCHT1 (BD Pharmingen), HIT3a (BD Pharmingen).

  In addition to the anti-CD3 antibody described above, the CD3 agonist may contain any molecule that can be a natural or synthetic ligand for the CD3 molecule, for example, formation of a complex consisting of a T cell antigen receptor and a CD3 molecule. And various substances that can lead to signal transduction to cytotoxic T cells or progenitor cells via CD3 molecules, in particular antibodies having an agonistic action or fragments thereof, such antibodies or fragments thereof can be , OT145 (Posnett et al., 1986, Proc. Natl. Acad. Sci. USA, 83: 7888-92), an antibody against the human T cell antigen receptor Vbeta6.7. In addition to the above-mentioned anti-CD3 antibody, the CD3 agonist recognizes T cell antigen receptors such as HLA molecules as soluble MHC-1 molecules, tetramer molecules having HLA molecules and antigen peptides, and has an agonistic action. It may contain substances that can result.

  In this example, the CD52 agonist contains an anti-4C8 antibody as one of the anti-CD52 antibodies as a main active ingredient. In this example, the anti-4C8 antibody is recognized by the cytotoxic T cell or progenitor cell by binding to the CD52 molecule of the cytotoxic T cell or progenitor cell. Here, the CD52 agonist may contain a fragment of an anti-4C8 antibody that binds to and is recognized in the same manner as the anti-4C8 antibody, instead of or in addition to the anti-4C8 antibody. It preferably consists of a Fab fragment having an antigen recognition site. In addition, the production of cytokines such as interferon γ (IFN-γ) and IL-2 having an antitumor effect can be strongly induced in cytotoxic T cells or progenitor cells to which costimulation is given by an anti-4C8 antibody or a fragment thereof. .

  The class of anti-4C8 antibodies is IgG3. The anti-4C8 antibody may be configured as a humanized antibody (including mouse humanized antibody, rat humanized antibody, etc.) or a human antibody.

  A humanized antibody can be obtained, for example, by transplanting the variable region of an anti-CD52 antibody produced by hybridoma JM-1 into a human antibody framework.

  The human antibody is, for example, a mouse that retains a non-rearranged human antibody gene and produces a human antibody specific for the antigen by auditing the antigen (eg, Tomizuka et al., 2000, Proc. Natl. Acad. Sci). USA., 97: 722).

  A CD52 agonist contains, instead of or in addition to an anti-4C8 antibody, a natural or synthetic ligand that binds to the CD52 molecule, in other words, any molecule that induces a signal through the CD52 molecule or an antibody against the CD52 molecule as an antigen. The molecule or antibody may recognize any part of the CD52 molecule and can transmit signals to cytotoxic T cells or progenitor cells via the CD52 molecule. Anything is acceptable. The CD52 agonist may contain, for example, campus-1H (Campath-1H) or a fragment thereof binding to the CD52 molecule as an active ingredient instead of or in addition to the anti-4C8 antibody. It preferably consists of a Fab fragment having an antigen recognition site.

  Campus-1H in this example means rat humanized antibody campus-1H (rat humanized antibody Campath-1H) that binds to the CD52 molecule. Campus-1H is known, for example, as a therapeutic agent for patients with B cell lymphoma and as an antibody that removes peripheral blood lymphocytes. In this example, costimulation is performed on cytotoxic T cells or progenitor cells. Used as one of the antibodies to give.

  The CD52 agonist can be obtained by contacting a cell expressing a CD52 molecule with a candidate compound and performing screening using the interaction or the interaction with CD52 for detecting a CD52 stimulation response as an index.

  The CD52 agonist may contain any molecule that can be a natural or synthetic ligand for the CD52 molecule in addition to the anti-4C8 antibody described above. Anti-4C8 antibody and a molecule that can serve as a ligand immunize human T cells according to a report by Masuyama et al. (Masyuyama, J. et al., 1999, J. Exp. Med. 189: 979-989; WO99 / 12972). It is possible to obtain by selecting from the monoclonal antibody obtained from the isolated animal using as an index the suppression of in vitro extravasation of T cells.

  An anti-CD52 antibody comprising an anti-4C8 antibody or an antibody that binds to a CD52 molecule other than the anti-4C8 antibody is obtained by adding human T cells to human umbilical vein vascular endothelial cells (HUVEC) cultured in a monolayer on a collagen gel for a certain period of time. Later, it is possible to obtain T cells that have migrated under HUVEC by using them as antigens.

  A suitable antibody contained in a CD52 agonist can be easily obtained by combining the step of selecting an antibody that recognizes the same epitope as the anti-4C8 antibody and the step of evaluating the ability to induce differentiation and proliferation of cytotoxic T cells. can get.

  When activating progenitor cells using the pharmaceutical composition of the present example, the progenitor cells are mainly stimulated by the CD3 molecule via the CD3 molecule of the progenitor cells, and the progenitor cells are stimulated by the CD52 agonist together with the main stimulation by the CD3 agonist. The progenitor cells are costimulated via the CD52 molecule. The primary stimulation of cytotoxic T cells or progenitor cells by CD3 agonists is made through the main stimulus transmission pathways involved in the differentiation of cytotoxic T cells or progenitor cells. Primary stimulation with CD3 agonists elicits a response that promotes the differentiation of cytotoxic T cells or progenitor cells. Costimulation of cytotoxic T cells or progenitor cells by a CD52 agonist is performed through a stimulus transmission pathway other than the main stimulus transmission pathway. CD52 agonists co-stimulate cytotoxic T cells or progenitor cells at the same time as, or before or after, the main stimulation by the CD3 agonist. Progenitor cells to which main stimulation and costimulation are given by the CD3 agonist and CD52 agonist are induced based on these stimulations to differentiate and proliferate to become cytotoxic T cells having cytotoxic activity. Such cytotoxic T cells, as shown in the respective examples described later, are highly expressed by chemokine receptors, particularly CCR7, and therefore migrate to the organs and lymph nodes subordinate to the organs. It has a high chemotaxis to such an extent that it is capable of killing tumor cells and virus-infected cells.

  In addition, when the cytotoxic T cells comprising naïve T cells or memory T cells are activated using the pharmaceutical composition of this example, as in the case of activating progenitor cells, the cytotoxic T cells are mainly treated with cytotoxic T cells. Gives stimuli and accessory stimuli. Cytotoxic T cells to which major and minor stimuli are given proliferate by inducing differentiation based on these stimuli. The cells activated and proliferated using the pharmaceutical composition of this example are cytotoxic T cells proliferated upon stimulation by a CD52 agonist having a CD3 agonist and an anti-4C8 antibody or an anti-4C8 antibody fragment. In some cases, cytotoxic T cells having large amounts of perforin and CCR7 can be obtained. Cytotoxic T cells with high amounts of perforin and CCR7 may exhibit higher chemotaxis to CCR7 ligand and higher killing ability against tumor cells, virus infected cells, and the like.

  The pharmaceutical composition of this example may be used by being contained in a drug consisting of a buffer solution, capsule, granule, powder, syrup, or the like that is orally administered to a living body, and used for subcutaneous injection, intramuscular injection, intravenous injection It may be used by being contained in a medicine composed of an injection, infusion, suppository, etc. administered parenterally into a living body by injection or the like, and a pharmaceutical composition administered in vivo may be lyophilized. It may be used by being contained in a drug together with various additives such as carriers acceptable for therapeutic effects, pH buffering agents, stabilizers, and excipients. Here, the medicament containing the pharmaceutical composition preferably further contains a physiologically acceptable diluent or carrier, such a physiological saline, phosphate buffered saline, and the like. , Phosphate buffered saline glucose solution, buffered saline, or a combination of these as appropriate. In addition, the pharmaceutical composition includes cytotoxic cells such as cytotoxic T cells and progenitor cells collected from patients or non-patients, particularly T cells and peripheral blood, lymphocytes, lymph node cells, thymocytes containing these cells. May be used ex vivo for umbilical cord blood, tumor tissue, etc. In this case, cytotoxic T cells activated in vitro, differentiated, proliferated, or function-enhanced may be used in-house or elsewhere. It is moved into the living body of the house.

  The administration method and dose of the pharmaceutical composition are appropriately determined in the context of preclinical studies and clinical trial processes. When a pharmaceutical composition is orally administered, the dose is usually about 0.01 mg to 1000 mg per day for an adult, and such oral administration is performed once or several times. In addition, when the pharmaceutical composition is administered parenterally, the dose is usually about 0.01 mg to 1000 mg per dose for an adult. In addition, the dosage of the pharmaceutical composition by oral administration or parenteral administration is determined according to age, body weight, and disease symptoms.

  Further, the pharmaceutical composition may be used in a so-called ex vivo method that can be said to reproduce an experimental system developed in a basic research field almost as it is in a therapeutic field. Since the ex vivo method involves activating cytotoxic T cells or progenitor cells in vitro, administration of the pharmaceutical composition into the living body may be caused by in vivo absorption, metabolism, or interference by unknown factors. In view of the possibility that the expected therapeutic effect may not be achieved, it is considered to be a method with a relatively low risk in practical use.

  The hybridoma producing the monoclonal antibody is an anti-4C8 antibody produced by Hybridoma JM-1 (deposited with the accession number FERMBP-7757 dated September 26, 2001 at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary). Can be easily combined by using as an indicator that T cell staining by the test antibody is suppressed, in other words, recognizing the same epitope as the anti-CD52 antibody produced by hybridoma JM-1. It is possible to obtain.

  Cytotoxic T cells activated by the pharmaceutical composition of this example proliferate more easily in the presence of IL-2. Cytotoxic T cells proliferated in the presence of IL-2 induce more perforin by being re-stimulated with anti-4C8 antibody, and the killing ability against tumor cells, virus-infected cells, etc. is further enhanced. It is preferable that the second costimulation with the anti-4C8 antibody or fragment thereof against the cytotoxic T cell is given together with the main stimulation with the CD3 agonist.

  According to the pharmaceutical composition as described above, since it has a CD3 agonist and a CD52 agonist, the cytotoxicity T cell or its progenitor cell is mainly stimulated by the CD3 agonist and the CD52 agonist via the CD3 molecule and the CD52 molecule. And by giving a costimulation, it is possible to cause a strong activation of the cytotoxic T cell or its progenitor cell, and to induce strong differentiation in the thus activated cytotoxic T cell or its progenitor cell. Therefore, it is possible to easily increase the number of cytotoxic T cells that express chemokine receptors, particularly CCR7, and can migrate to the lymph nodes in a short period of time. Thus, cytotoxic T cells having nonspecific cytotoxic activity against various tumor cells, virus-infected cells and the like can be obtained. Further, the cytotoxic T cells obtained by using the pharmaceutical composition of this example can more accurately separate normal cells from tumor cells than other immune cells. In addition, the cytotoxic T cells obtained using the pharmaceutical composition of this example are stimulated again with the pharmaceutical composition, so that they have a large amount of perforin and CCR7, and various tumor cells, viruses Cytotoxic T cells exhibiting strong and nonspecific cytotoxic activity against infected cells and the like can be obtained. Further, when the CD52 agonist has an anti-4C8 antibody or a fragment thereof, or campus-1H or a fragment thereof, cytotoxic T cells or progenitor cells thereof by the anti-4C8 antibody or a fragment thereof or campus-1H or a fragment thereof. By giving a costimulatory effect, it is possible to express a chemokine receptor, particularly CCR7, in many cytotoxic T cells. By giving the chemokine receptor, in particular, CCR7 can be expressed in more cytotoxic T cells. Furthermore, when the CD52 agonist has an anti-4C8 antibody or fragment thereof and campus-1H or a fragment thereof, for example, the anti-4C8 antibody or fragment thereof is bound to one site of the CD52 molecule, and the campus-1H Alternatively, a cytotoxic T cell or a progenitor cell thereof can be preferably activated by binding a fragment thereof to another site of the CD52 molecule. The pharmaceutical composition of this example can be used directly (for example, administration of an agonist to a living body) or indirectly (for example, production of cytotoxic T cells) for the treatment of diseases / pathological conditions such as malignant tumors and viral infections. . The pharmaceutical composition of this example capable of exhibiting such an effect has been studied by the present inventor with regard to the efficacy of CD52 agonists such as anti-4C8 antibody on cytotoxic T cells or progenitor cells thereof. When the cytotoxic T cell or its progenitor cell is stimulated (co-stimulated) via CD52 molecule and activated, the cytotoxic T cell or its progenitor cell obtained by the activation becomes a chemokine It can be constructed for the first time based on the result of newly finding that a receptor, particularly CCR7 is highly expressed.

  The other pharmaceutical composition of this example contains a CD52 agonist having an anti-4C8 antibody or a fragment thereof as a main active ingredient. According to such a pharmaceutical composition, for example, by giving a costimulation to a cytotoxic T cell or a precursor cell thereof via a CD52 molecule by a CD52 agonist, the cytotoxic T cell or a precursor cell thereof is cytotoxic. Strong activation of cytotoxic T cells or their progenitor cells when given a major stimulus via CD3 molecule based on the binding of MHC-1 molecules to the T cell receptor of T cells or their progenitor cells Proliferation can be promoted by causing strong differentiation induction of the thus activated cytotoxic T cells or their progenitor cells, and high expression of chemokine receptors, particularly CCR7. Cytotoxic T cells that can migrate to lymph nodes can be easily increased in a short period of time, and thus non-specific cells for various tumor cells, virus-infected cells, etc. It can be obtained cytotoxic T cells with harm activity.

  In Example 1, a test is performed on the expression of CCR7 and perforin in cytotoxic T cells induced by costimulation with a CD52 agonist.

First, mononuclear cells were separated from peripheral blood of a healthy person by density gradient centrifugation using Ficoll-Paque Plus (Amersham Pharmacia), the separated mononuclear cells were passed through a nylon wool column, and a flow-through fraction was collected. T cells were obtained. Next, CD8 T cells having CD8 molecules were isolated from T cells by negative selection using MACS CD8 ⁺ T cell Isolation Kit (Milteny Biotec). In addition, immobilization of anti-CD3 antibody and anti-4C8 antibody or campus-1H (Alemtumumab) on a plate was carried out by diluting each well of a 48-well plate (Costar) with phosphate buffer (PBS) to 100 ng / ml. 330 μl of CD3 antibody (ORTHOCLONE OKT3, Ortho Biotec) was added and treated at 4 ° C. for 24 hours. After treatment, anti-CD3 antibody was removed from each well and adjusted to 5 μg / ml with PBS or Campus-1H (campus-1H ( (CAMPATH, Berlex) was added in an amount of 330 μl, and the mixture was allowed to stand at 4 ° C. for 24 hours.

Next, the isolated CD8 T cells were suspended in RPMI 1640 medium (10% FCS + 15 mM HEPES + 1% penicillin + 1% streptomycin) at a concentration of 1 × 10 ⁶ / ml and treated with anti-CD3 antibody and anti-4C8 antibody or campus-1H. 0.5 to 1 × 10 ⁶ were added to each hole of the plate. After culturing at 37 ° C. for 3 to 4 days using a CO ₂ incubator, adjust to 1 × 10 ⁶ / ml, transfer to another culture vessel, add IL-2 (20 U / ml) and further culture Continued. The culture medium was changed by half every other day, and IL-2 (20 U / ml) was added every time the medium was changed. After 14 days from the start of culture, T cells were collected and washed. 99% or more of the collected T cells were CD8 positive and TCRαβ positive.

The CD8 T cells were given a first costimulation and then the CD8 T cells were given a second costimulation via the CD52 molecule. First, 2 ml of anti-CD3 antibody diluted to 100 ng / ml with PBS was treated for 24 hours, and after treatment, 2 ml of anti-CD52 antibody (anti-4C8 antibody (5 μg / ml) or campus-1H (50 μg / ml)) was treated for 24 hours. Then, 1 × 10 ^{7 of the} collected T cells were added to a 6-well plate on which anti-CD3 antibody and anti-4C8 antibody were immobilized, and cultured in a CO ₂ incubator. After 3 to 4 days from the start of the culture, CD8 T cells were collected, and such CD8 T cells were subjected to experiments.

CD3-LAK cells (CD3-LAK) to be compared were obtained by the following general method. In each well of a 48-well plate solid-phased with an anti-CD3 antibody (5 μg / ml), 1 × 10 ⁶ T cells, which are a nylon wool column passage fraction of peripheral blood mononuclear cells, were seeded. IL-2 (100 U / ml) was added after 24 hours and 72 hours after sowing, and cultured for a total of 5 days. Next, the cells were transferred and cultured for about 10 days while IL-2 (20 U / ml) was added every other day for growth.

After 3 × 10 ⁵ CD8 T cells (hereinafter referred to as CD52-CTL) treated with costimulation of CD52 molecule were suspended in 50 μl of the culture solution, 3 μl of FITC-labeled anti-CCR7 antibody (Dako) was added, and the mixture was on ice. And left in the dark for 30 minutes. After standing, it was washed twice with PBS supplemented with 0.1% bovine serum albumin, fixed with 1% paraformaldehyde, and the expression of cell surface CCR7 (positive) of living cells was examined with FACScan. On the other hand, after 3 × 10 ⁵ CD3T-LAK cells were suspended in 50 μl of the culture solution, 3 μl of FITC-labeled anti-CCR7 antibody (Dako) was added and allowed to stand on ice and in the dark for 30 minutes. After standing, it was washed twice with PBS supplemented with 0.1% bovine serum albumin, fixed with 1% paraformaldehyde, and the expression of cell surface CCR7 of living cells was examined with FACScan. Perforin in each cell was stained with FITC-labeled anti-perforin antibody (Ancell) by treating the cells with Cytofix / Cytoperm Kit (Pharmingen).

As a result of the test as described above, as shown in FIG. 1, about 18% of CCR7 of the pre-CD8 T cell is positive, whereas about 5% of CCR7 of the CD3-LAK cell is on campus 1-H. Approximately 70% of CCR7 of CD52-CTL stimulated by and about 80% of CCR7 of CD52-CTL stimulated by anti-4C8 antibody were positive. The CCR7 ⁺ expression (%) of CD52-CTL co-stimulated with anti-4C8 antibody or campus 1-H greatly exceeded the CCR7 ⁺ expression (%) of CD8 T cells before stimulation (pre-CD8 T cells). This suggests that CCR7 ⁺ is expressed in the majority of CD52-CTLs. CD3-LAK cells CCR7 ⁺ expression (%), since the front is below CCR7 ⁺ expression CD8T cells (%), that they've CCR7 ⁺ disappeared from CD3-LAK cells has been suggested . In addition, the average fluorescence intensity of perforin in cells of CD52-CTL is significantly increased in CD52-CTL after the second stimulation than in CD52-CTL before the second stimulation, as shown in FIG. This suggests that the intracellular perforin of CD52-CTL increases markedly after the second stimulation.

  In Example 2, a test on chemotaxis (chemotaxis) reaction to CCR7 ligand was performed, and it was examined whether or not chemotactic activity was enhanced by the reaction of C52 highly expressed CD52-CTL to CCR7 ligand. To do.

The chemotaxis assay was performed using Transwell (5 μm pore, polycarbonate, Costar) for 24-well plates. The CCR7 ligand chemokine CCL21 (R & D) was added to Transwell's lower chamber at concentrations of 10 ng / ml, 100 ng / ml and 1000 ng / ml, respectively. As a negative control, CCL20 (R & D), which is a ligand of CCR6 that is not expressed in CD52-CTL and CD3-LAK, was used. In the upper chamber, 5 × 10 ⁵ CD52-CTL or CD3-LAK were added and cultured at 37 ° C. After 90 minutes from the start of the culture, the cells that migrated to the lower chamber were counted. The chemotaxis activity (%) was determined using the following formula (1).

(Equation 1)

Chemotaxis activity = number of cells moving in the lower chamber / number of cells added in the upper chamber (1)

  As a result of the test as described above, as shown in FIG. 3 (b), the chemotaxic activity (%) of CCL21 against CCR7 of CD52-CTL markedly increased from a chemokine concentration of 100 ng / ml to 1000 ng / ml. On the other hand, the increase in the chemotaxis activity (%) of CCL20 relative to CCR6 of CD52-CTL was not particularly observed. In addition, as shown in FIG. 3 (a), the chemotaxic activity (%) of CCL21 with respect to CCR7 for CD3-LAK with respect to the chemokine concentration is higher than that of CCL21 with respect to CCR7 of CD52-CTL. There was no reaction. Therefore, it can be seen that CD52-CTL migrates actively due to the chemotactic response of CCR7 and CCL21, and when such CD52-CTL is administered in vivo, the majority of the CD52-CTL is lymph node It is suggested that can be reached.

  In Example 3, a test on the cytotoxic activity of CD52-CTL against cancer cells and normal cells is performed.

For analysis of cytotoxic activity, a standard 4 hour ⁵¹ Cr release assay was used. The target cells were labeled with ⁵¹ Cr100 μCi and then washed, and 1 × 10 ⁴ washed target cells were placed in each well of a 96-well round bottom plate (ICN), and an E / T ratio (effector cell / CD52-CTL was added at the target cell ratio). Cultivation was carried out at 37 ° C. for 4 hours in a CO ₂ incubator, and ⁵¹ Cr radioactivity released in 100 μl of the supernatant after the cultivation was measured with a gamma counter. Cytotoxic activity (%) was determined using the following formula (2).

(Equation 2)

Cytotoxic activity = (test release-spontaneous release) / (maximum release-spontaneous release) x 100 (2)

The spontaneous release of ⁵¹ Cr in the target cells was always less than 20% of the maximum release.

  As target tumor cells, KatoIII (gastric cancer), TE11 (esophageal cancer) and Daudi (Burkitt lymphoma) were used, and human peripheral blood mononuclear cells stimulated with Con-A (10 μg / ml) as a normal cell control for 3 days were used. Using.

  As a result of the test as described above, as shown in FIG. 4, CD52-CTL has a cytotoxic activity against KatoIII and TE11 depending on the CD52-CTL / target cell ratio (E / T ratio) (E / T ratio). %). CD52-CTL also suggested cytotoxic activity (%) against Daudi independent of the CD52-CTL / target cell ratio. CD52-CTL showed no cytotoxic activity (%) against ConA-Blasts (Con-A) as normal cells.

  In Example 4, a test on the cytotoxic activity of CD52-CTL cultured for 24 hours against tumor cells is performed.

Tumor cell lines include KatoIII (stomach cancer), TE11 (esophageal cancer), TE2 (esophageal cancer), WiDr-TC (colon cancer), SK-MEL-28 (malignant melanoma), LCSC # 1 (lung adenocarcinoma) LU65 (small cell lung cancer) was used. Target tumor cells 1 × 10 ⁵ / ml (SK-MEL-28 only 0.5 × 10 ⁵ / ml) are seeded in 100 μl in each well of a 96-well flat bottom plate, and cultured at 37 ° C. for 3 hours, 5% CO ₂ Cultivated until the tumor cells were well attached to the 96-well flat bottom plate. To this, 100 μl of CD52-CTL was added at a predetermined E / T ratio, and cultured at 37 ° C. for 24 hours in a 5% CO ₂ incubator. After culturing, the formazan conversion ability reflecting the metabolic activity of living cells was examined, and the cytotoxic activity of CD52-CTL was calculated. Such cytotoxic activity (%) was measured by adding 10% of WST-1 (Roche Diagnostics) to the culture solution for 2 hours, measuring with an ELISA plate reader at two wavelengths of A492nm / A620nm, It calculated | required using Numerical formula (3) of these. TS means only target cells, BG0 means background of culture medium only, E means target cells and CD52-CTL (effector cells), BGEC means background of effector cells only To do.

(Equation 3)

Cytotoxic activity = [{(TS-BG0)-(E-BGEC)} / (TS-BG0)] × 100 (3)

  As a result of conducting the test as described above, as shown in FIG. 5, the cytotoxic activity (%) depending on the E / T ratio of CD52-CTL induced by costimulation via CD52 molecule by anti-4C8 antibody is as follows: It is strong against each of the six types of tumor cells and shows a non-specific tendency towards these tumor cells.

  In Example 5, a test on the enhancing action of IL-2 on CD52-CTL is performed.

  The cytotoxic activity of CD52-CTL against SK-MEL-28 was measured after culturing at an E / T ratio of 2.5, 5, 10 and 20 for 24 hours, respectively. As a result of the measurement, as shown in FIG. 6, it was shown that the cytotoxic activity (%) depending on the E / T ratio is increased by adding IL-2 (100 U / ml).

  Example 6 compares the cytotoxic activity (%) of CD52-CTL given costimulation with anti-4C8 antibody and the cytotoxic activity (%) of CD52-CTL given costimulation with campus-1H Perform the test.

  The cytotoxic activity against tumor cells of CD52-CTL given costimulation by anti-4C8 antibody and the cytotoxic activity against tumor cells of CD52-CTL given costimulation by Campus-1H are 5, 10, 20, Each was measured at an E / T ratio of 40. As a result of the measurement, as shown in FIG. 7, the cytotoxic activity (%) depending on the E / T ratio of CD52-CTL subjected to the costimulation by the anti-4C8 antibody was reduced by the campus-1H. It was suggested that it is higher than the cytotoxic activity (%) depending on the E / T ratio of a given CD52-CTL. In addition, the cytotoxic activity (%) depending on the E / T ratio of CD52-CTL given costimulation by campus-1H is slightly inferior to CD52-CTL given costimulation by anti-4C8 antibody. However, it is considered that a sufficient antitumor effect can be obtained even if CD52-CTL given a costimulation by such campus-1H is used for treatment of tumor cells and the like.

  In Example 7, cytotoxicity of CD3-LAK cells and CD52-CTL cells against monolayer cultured LCSC # 1 (obtained from Tohoku University Medical Research Center for Aging Medicine, including lung adenocarcinoma and other cancer cells) Test for activity comparison.

  LCSC # 1 was cultured in RPMI 1640 supplemented with 10% FCS until it became a single layer in a 24-well plate (Corning). In the same manner as in Example 1, T cells were obtained by separating peripheral blood mononuclear cells (PBMC) from healthy individuals and further passing through a nylon wool column. The T cell antibody stimulation obtained in this way has a range of solid-phase antibody concentrations effective for T cell stimulation (anti-CD3 antibody 100 ng / ml-5 μg / ml, anti-4C8 antibody and campus-). 1H 1-50 μg / ml), an antibody amount different from that in Example 1 was used.

  CD3-LAK cells were induced by stimulation for 3 days using a 48-well plate on which an anti-CD3 antibody (100 ng / ml) was immobilized, and then the addition of IL-2 (100 U / ml) was repeated every 3 days. The culture was grown for 2 weeks while replacing the large plate.

The CD52-CTL cells that were stimulated once were subjected to the first CD52 costimulation on T cells for 3 days using a plate on which an anti-CD3 antibody (100 ng / ml) and an anti-4C8 antibody (5 μg / ml) were immobilized. Afterwards, IL-2 (100 U / ml) was added every 3 days and cultured for 2 weeks while replacing large plates. As described above, the CD52-CTL cells that were stimulated twice were given a second CD52 costimulation for 3 days to some T cells that were given the first CD52 costimulation as described above. Obtained by culturing. In such a culture method, T cells proliferated 100 to 1000 times. Further, in such a culture method, CD8 ⁺ T cells accounted for about 80%.

1 × ¹⁰ 6 / ml in cell number CD3-LAK cells, 1 × ¹⁰ 6 / ml in cell number once stimulated CD52-CTL cells and 1 × ¹⁰ 6 / ml in cell number of 2 stimulations CD52- Each 10% FCS-added RPMI 1640 in which each of the CTL cells floated was added in 2 ml to each well of a 24-well plate in which LCSC # 1 was monolayered, and co-cultured in the presence of IL-2 (100 U / ml). . On the seventh day of co-culture, LCSC # 1 was separated by trypsin treatment, and directly microscopically stained with trypan blue staining to count the number of living cells of the LCSC # 1. The experiment was performed in triplicate.

  In the test in Example 7, the results as shown in FIG. 8 were obtained. According to such a result, the cytotoxic activity against LCSC # 1 by CD3-LAK cells was 8%, and the cytotoxic activity against LCSC # 1 by CD52-CTL cells stimulated once was 38%, which was stimulated twice. The cytotoxic activity against LCSC # 1 by CD52-CTL cells was 80%.

  According to the results of the test in Example 7, it was found that the cytotoxic activity of CD52-CTL cells after single stimulation was significantly increased compared with the cytotoxic activity of CD3-LAK cells, It is recognized that the cytotoxic activity of the CD52-CTL cells stimulated twice is further significantly increased as compared with the cytotoxic activity of CD3-LAK cells. Moreover, according to the result of the test in Example 7, it is recognized that the CD52-CTL cells have a stronger killing effect on the cancer cells LCSC # 1 than the CD3-LAK cells.

  In Example 8, a test on the life-prolonging effect of CD52-CTL cells on the lethal effect of intraperitoneal administration of LCSC # 1 to SCID mice is performed.

  Here, whether or not administration of CD52-CTL cells showed a higher life-spanning effect than CD3-LAK cells was examined using cancer-bearing animals in which LCSC # 1 was actually transferred into the abdominal cavity.

  LCSC # 1 was cultured and monolayered in the same manner as in Example 7 and briefly treated with trypsin. After separation by trypsin treatment, washed and suspended in PBS was used. The T cell culture medium used was AIM-V (Invitrogen) supplemented with 5% autologous plasma in consideration of actual clinical use.

T cells were obtained from healthy individuals in the same manner as in Example 7. CD3-LAK cells were induced by stimulation for 3 days using a 48-well plate on which an anti-CD3 antibody (100 ng / ml) was immobilized, and then the addition of IL-2 (100 U / ml) was repeated every 3 days. The one grown in culture for 2 weeks while using a large plate was used. The CD52-CTL cells were subjected to the first stimulation for 3 days using a plate on which T cells were immobilized on campus-1H (20 μg / ml) instead of anti-CD3 antibody (100 ng / ml) and anti-4C8 antibody. Then, IL-2 (100 U / ml) was added every 3 days, and the cells were grown for 2 weeks in place of a large plate. After the growth, the second stimulation was performed for 3 days, and the last 24 hours were IL-2. What was performed in the presence of (100 U / ml) was used. The CD3-LAK cells and CD52-CTL cells thus obtained were each administered 4 × 10 ⁷ into the mouse abdominal cavity as described below.

  The day before the administration of LCSC # 1, in order to suppress the activity of NK cells with nonspecific antitumor activity, anti-Asialo GM1 antibody (Wako Pure Chemical Industries, 100 μg / mouse) was used in SCID mice (7 weeks old, female, obtained from CLEA Japan). ) Was administered intraperitoneally. Three SCID mice were divided into three groups (Group A, Group B and Group C).

Group A consisted of 3 SCID mice that were intraperitoneally transferred only with LCSC # 1 (1 × 10 ⁷ mice / mouse). In group B, LCSC # 1 (1 × 10 ⁷ cells / mouse) was transferred into the peritoneal cavity, and CD3-LAK cells (4 × 10 ⁷ cells / mouse) were injected into the peritoneal cavity on the third and fifth days after the transfer. It consisted of 3 SCID mice administered inside. In Group C, LCSC # 1 (1 × 10 ⁷ cells / mouse) was transferred into the peritoneal cavity, and CD52-CTL (4 × 10 ⁷ cells / mouse) was injected intraperitoneally on the third and fifth days after the transfer. Consists of 3 SCID mice administered to In addition, human IL-2 (5000 U) was injected into the lumbar subcutaneous skin of each of the SCID mice of Group B and Group C simultaneously with the intraperitoneal administration of CD3-LAK cells and CD52-CTL cells. As a control, the same amount of human IL-2 (5000 U) was also injected into Group A.

  In the test in Example 8, the results as shown in FIG. 9 were obtained. According to such results, the SCID mice of Group A, which were administered only LCSC # 1 alone, showed cancerous peritonitis in which cancer cells were seeded in multiple organs in the abdominal cavity, and the last one died on the 40th day. In addition, the average survival time of the SCID mice in Group A was 31.3 days. Moreover, according to such a result, all the SCID mice of the B group and the C group were eventually dead, the average survival time of the SCID mice of the B group was 38.3 days, and the SCID mice of the C group The average survival time was 47.6 days.

  According to the results of the test in Example 8, it can be seen that the average survival days of the SCID mice of Group B and Group C were respectively extended as compared to the average survival days of the SCID mice of Group A. Moreover, according to such a result, the group C (p = 0.025, Logrank test) administered with CD52-CTL cells has a statistically significant life-prolonging effect as compared with the group A. The B group does not have a statistically significant life-prolonging effect compared to the A group, and the C group has a statistically significant life-prolonging effect compared to the B group. (P = 0.025, Logrank test).

It is explanatory drawing regarding the test result in Example 1. FIG. It is explanatory drawing regarding the test result in Example 1. FIG. (A) And (b) is explanatory drawing regarding the test result in Example 2. FIG. It is explanatory drawing regarding the test result in Example 3. FIG. It is explanatory drawing regarding the test result in Example 4. FIG. It is explanatory drawing regarding the test result in Example 5. FIG. It is explanatory drawing regarding the test result in Example 6. FIG. It is explanatory drawing regarding the test result in Example 7. FIG. It is explanatory drawing regarding the test result in Example 8. FIG.

Claims (24)

  A cell activation method in which a cytotoxic T cell or a progenitor cell thereof is stimulated by a CD3 agonist and a CD52 agonist.   The cell activation method according to claim 1, wherein the CD52 agonist has an anti-4C8 antibody or a fragment thereof.   The cell activation method according to claim 1 or 2, wherein the CD52 agonist has campus-1H or a fragment thereof.   A CD52 agonist is an antibody or fragment thereof that binds to one site of the CD52 molecule of a cytotoxic T cell or its progenitor cell, and another that binds to another site of the CD52 molecule of the cytotoxic T cell or its progenitor cell. The cell activation method according to any one of claims 1 to 3, which comprises the antibody or fragment thereof.   The antibody or fragment thereof that binds to one site of the cytotoxic T cell or its progenitor cell CD52 molecule comprises an anti-4C8 antibody or fragment thereof, and the cytotoxic T cell or its progenitor cell CD52 molecule other 5. The cell activation method according to claim 4, wherein the other antibody or fragment thereof that binds to the site comprises campus-1H or a fragment thereof.   6. The cell activation method according to any one of claims 1 to 5, wherein the CD3 agonist has an anti-CD3 antibody or a fragment thereof that binds to a CD3 molecule of a cytotoxic T cell or a progenitor cell thereof.   The cell activation method according to any one of claims 1 to 6, wherein at least one of the CD3 agonist and the CD52 agonist has at least one of a humanized antibody and a human antibody.   The cell activation method according to claim 7, wherein the humanized antibody is mouse humanized antibody or rat humanized antibody campus-1H.   The cell activation method according to any one of claims 1 to 8, wherein the cytotoxic T cell or a progenitor cell thereof is stimulated by a CD3 agonist and a CD52 agonist in vitro or in vivo.   10. The cytotoxic T cell or its progenitor cell that can be present in peripheral blood, lymph node, thymus, bone marrow, tumor, pleural effusion, ascites or umbilical cord blood is stimulated by a CD3 agonist and a CD52 agonist. The cell activation method according to Item.   The cell manufacturing method which manufactures a cytotoxic T cell using the cell activation method as described in any one of Claims 1-10.   A cytotoxic T cell obtained by the cell activation method according to any one of claims 1 to 10.   A pharmaceutical composition comprising the cytotoxic T cell according to claim 12.   A pharmaceutical composition comprising a CD3 agonist and a CD52 agonist for stimulating cytotoxic T cells or progenitor cells thereof.   The pharmaceutical composition according to claim 14, wherein the CD52 agonist has an anti-4C8 antibody or a fragment thereof.   The pharmaceutical composition according to claim 14 or 15, wherein the CD52 agonist has campus-1H or a fragment thereof.   A CD52 agonist is an antibody or fragment thereof that binds to one site of the CD52 molecule of a cytotoxic T cell or its progenitor cell, and another that binds to another site of the CD52 molecule of the cytotoxic T cell or its progenitor cell. The pharmaceutical composition according to any one of claims 14 to 16, which comprises an antibody or a fragment thereof.   The antibody or fragment thereof that binds to one site of the cytotoxic T cell or its progenitor cell CD52 molecule comprises an anti-4C8 antibody or fragment thereof, and the cytotoxic T cell or its progenitor cell CD52 molecule other 18. The pharmaceutical composition according to claim 17, wherein the other antibody or fragment thereof that binds to the site comprises campus-1H or a fragment thereof.   The pharmaceutical composition according to any one of claims 14 to 18, wherein the CD3 agonist has an anti-CD3 antibody or a fragment thereof that binds to a CD3 molecule of a cytotoxic T cell or a progenitor cell thereof.   The pharmaceutical composition according to any one of claims 14 to 19, wherein at least one of the CD3 agonist and the CD52 agonist has at least one of a humanized antibody and a human antibody.   21. The pharmaceutical composition according to claim 20, wherein the humanized antibody is mouse humanized antibody or rat humanized antibody campus-1H.   A pharmaceutical composition comprising a CD52 agonist that stimulates cytotoxic T cells or progenitor cells thereof.   The pharmaceutical composition according to claim 22, wherein the CD52 agonist has an anti-4C8 antibody or a fragment thereof.   24. The pharmaceutical composition according to claim 22 or 23, wherein the CD52 agonist has campus-1H or a fragment thereof.

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