JP2006265155A - Cancer immunotherapy - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a therapeutic method for cancers expressing higher therapeutic effects than those of conventional methods. <P>SOLUTION: The therapeutic agent for cancers is prepared by combining a TNF-associated apoptosis-inducing factor with lymphocyte activating factor. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to cancer immunotherapy by a combination therapy of cell death induction therapy and lymphocyte activation therapy.

  At present, treatment for cancer is carried out with three pillars: surgical therapy, chemotherapy with anticancer agents, and radiation therapy, but in recent years immunotherapy has been established as a fourth option.

  As an immunological approach to cancer, adoptive immunotherapy for transferring LAK (lymphokine-activated killer) and TIL (tumor-infiltrating lymphocytes) has been performed for a long time. It was difficult to selectively kill, and clear clinical results and effectiveness were not obtained.

  However, with the advancement of molecular biological technology, tumor antigens have been identified, and cancer antigen-specific cytotoxic T cells (CTLs) induction using dendritic cells using or presenting them have been identified. Tumor-specific treatment has been started.

  On the other hand, anti-HER2 / neu antibody and anti-CD20 antibody, which are tumor-specific antibodies, have been prepared, and these antibodies are good at directly inducing cell death against tumors and thereby inducing cytotoxic T cells (CTL). Clinical results have been obtained, the importance of direct cell death induction on cancer cells has been demonstrated, and immunotherapy for cancer has again attracted attention (zum Buschenfelde CM, et al. Cancer Res 62 (2 ), 2244-2247, 2002: Non-patent document 1; Selenko N, et al., Leukemia 15 (10), 1619-1626, 2001: Non-patent document 2).

  Also, research on dendritic cells as antigen-presenting cells necessary for inducing an antigen-specific immune response, research on functional molecules involved in T cell-antigen-presenting cell interactions such as costimulatory molecules, and tumor rejection Research on identification of antigenic peptides has been conducted energetically, and the necessary conditions for applying tumor antigen-specific cytotoxic T cells (CTLs) to clinical treatment have been clarified by the results of basic research from various fields. Yes.

  However, (i) cytotoxic T cells (CTLs) due to cancer antigen expression mutations resulting from genetic instability of tumor cells and acquisition of resistance to cell death induction by anti-HER2 / neu antibody or anti-CD20 antibody ) And tumor growth and metastasis escaped from antibody therapy, and (ii) a vast number of facilities, equipment, technology, human resources, etc. necessary for induction of cytotoxic T cells (CTL), and immunotherapy is widely clinical There are many obstacles to applying to.

In addition, Takeda et al. (Takeda K. et al., J Exp Med 199 (4) 437-448, 2004: Non-Patent Document 3) describes TRAIL (Tumor necrosis factor-induced apoptosis-inducing ligand) cell death (hereinafter referred to as “cell death”). It has been reported that an agonistic antibody (MD5-1) against DR5, which is a receptor that induces apoptosis, can be induced and administered to induce tumor rejection.
zum Buschenfelde CM, et al. , Cancer Res 62 (2), 2244-2247, 2002 Selenko N, et al. Leukemia 15 (10), 1619-1626, 2001 Takeda K, et al. , J Exp Med 199 (4) 437-448, 2004.

  As described above, various researches have been made on cancer treatment methods, but no treatment methods that still have a sufficient therapeutic effect have been established. Therefore, establishment of a therapeutic method that exhibits a higher therapeutic effect is desired.

  The present invention has been made to solve the above problems, and provides the following cancer therapeutic agent and cancer treatment kit.

(1) A cancer therapeutic agent comprising a combination of a TNF-related cell death inducing factor and a lymphocyte activator.
(2) The cancer therapeutic agent according to (1), wherein the TNF-related cell death inducing factor is an agonistic antibody against TRAIL receptor.
(3) The cancer therapeutic agent according to (2), wherein the agonistic antibody to the TRAIL receptor is an anti-DR5 antibody.
(4) The cancer therapeutic agent according to (1), wherein the lymphocyte activator is an agonistic antibody against a costimulatory molecule.
The cancer therapeutic agent as described in.
(5) The cancer therapeutic agent according to (4), wherein the agonistic antibody against the costimulatory molecule is an agonistic antibody against a CD28 family member or an agonistic antibody against a TNFR family member having no death receptor.
(6) The cancer therapeutic agent according to (5), wherein the agonistic antibody against the CD28 family member is an anti-CD28 antibody, an anti-ICOS antibody, or a combination thereof.
(7) The cancer therapeutic agent according to (5), wherein the agonistic antibody against the TNFR family member having no death receptor is an anti-CD40 antibody, an anti-4-1BB antibody, or a combination thereof.
(8) The cancer therapeutic agent according to (1), wherein the TNF-related cell death inducing factor is an anti-DR5 antibody, and the lymphocyte activating factor is an anti-CD40 antibody and an anti-4-1BB antibody.
(9) The cancer therapeutic agent according to any one of (1) to (8), further comprising an antagonistic antibody against a receptor that transmits an inhibitory signal.
(10) The cancer therapeutic agent according to (9), wherein the antagonistic antibody to the receptor that transmits the inhibitory signal is an anti-CTLA-4 antibody.

(11) A cancer therapeutic agent for use in combination with a lymphocyte activator, comprising a TNF-related cell death inducing factor.
(12) The cancer therapeutic agent according to (11), wherein the TNF-related cell death inducing factor is an agonistic antibody against TRAIL receptor.
(13) The cancer therapeutic agent according to (12), wherein the agonistic antibody to the TRAIL receptor is an anti-DR5 antibody.
(14) The cancer therapeutic agent according to (11), wherein the lymphocyte activator contains an agonistic antibody against a costimulatory molecule.
(15) The cancer therapeutic agent according to (14), wherein the agonistic antibody against the costimulatory molecule is an agonistic antibody against a CD28 family member or an agonistic antibody against a TNFR family member having no death receptor.
(16) The cancer therapeutic agent according to (15), wherein the agonistic antibody against the CD28 family member is an anti-CD28 antibody, an anti-ICOS antibody, or a combination thereof.
(17) The cancer therapeutic agent according to (15), wherein the agonistic antibody against a TNFR family member having no death receptor is an anti-CD40 antibody, an anti-4-1BB antibody, or a combination thereof.
(18) The cancer therapeutic agent according to any one of (11) to (17), further comprising an antagonistic antibody against a receptor that transmits an inhibitory signal.
(19) The cancer therapeutic agent according to (18), wherein the antagonistic antibody to the receptor that transmits the inhibitory signal is an anti-CTLA-4 antibody.

(20) A cancer therapeutic agent for use in combination with a cell death inducer, comprising a lymphocyte activator.
(21) The cancer therapeutic agent according to (20), wherein the lymphocyte activator is an agonistic antibody against a costimulatory molecule.
(22) The cancer therapeutic agent according to (21), wherein the agonistic antibody against the costimulatory molecule is an agonistic antibody against a CD28 family member or an agonistic antibody against a TNFR family member having no death receptor.
(23) The cancer therapeutic agent according to (22), wherein the agonistic antibody against the CD28 family member is an anti-CD28 antibody, an anti-ICOS antibody, or a combination thereof.
(24) The cancer therapeutic agent according to (22), wherein the agonistic antibody against the TNFR family member having no death receptor is an anti-CD40 antibody, an anti-4-1BB antibody, or a combination thereof.
(25) The cancer therapeutic agent according to (20), wherein the cell death inducer contains a TNF-related cell death inducer.
(26) The therapeutic agent for cancer according to (25), wherein the TNF-related cell death inducing factor is an agonistic antibody against TRAIL receptor.
(27) The cancer therapeutic agent according to (26), wherein the agonistic antibody to the TRAIL receptor is an anti-DR5 antibody.
(28) The cancer therapeutic agent according to any one of (20) to (27), further comprising an antagonistic antibody against a receptor that transmits an inhibitory signal.
(29) The cancer therapeutic agent according to (28), wherein the antagonistic antibody to the receptor that transmits the inhibitory signal is an anti-CTLA-4 antibody.

(30) The cancer therapeutic agent according to any one of (1) to (29), which is used in combination with a chemotherapeutic agent.
(31) The cancer therapeutic agent according to any one of (1) to (29), which is used in combination with radiation therapy.

(32) A cancer treatment kit by a combination therapy of cell death induction therapy and lymphocyte activation therapy,
A drug containing a TNF-related cell death inducing factor;
A cancer treatment kit comprising a drug containing a lymphocyte activator.
(33) The cancer treatment kit according to (32), wherein the TNF-related cell death inducing factor is an agonistic antibody against a TRAIL receptor, and the lymphocyte activator is an agonistic antibody against a costimulatory molecule.
(34) The cancer treatment kit according to (33), wherein the agonistic antibody to the TRAIL receptor is an anti-DR5 antibody, and the agonistic antibody to the costimulatory molecule is an anti-CD40 antibody and an anti-4-1BB antibody.
(35) The cancer treatment kit according to any of (32) to (34), further comprising an antagonistic antibody against a receptor that transmits an inhibitory signal.
(36) The cancer treatment kit according to (35), wherein the antagonistic antibody to the receptor that transmits the inhibitory signal is an anti-CTLA-4 antibody.

  ADVANTAGE OF THE INVENTION According to this invention, the treatment method of the cancer which has a higher therapeutic effect is provided. According to the present invention, tumor rejection can be induced even at a stage where tumor growth has progressed. The present invention provides a new cancer therapy that can eradicate cancer at a high rate.

  The present inventors examined TNF-related cell death inducers such as anti-DR5 antibodies and lymphocyte activators such as anti-CD40 antibodies and anti-4-1BB antibodies that are agonistic antibodies to costimulatory molecules. The present invention has been completed by finding that the combined administration of can induce cancer antigen-specific cytotoxic T cells (CTLs) more efficiently, and an unexpectedly high therapeutic effect can be obtained. That is, the cancer treatment method of the present invention is characterized in that cell death induction therapy using a TNF-related cell death inducer is combined with lymphocyte activation therapy using a lymphocyte activation factor.

(TNF-related cell death inducing factor)
In the present specification, “TNF-related cell death inducing factor” specifically binds to a receptor of a cell death inducing cytokine belonging to the tumor necrosis factor (TNF) family, and cell death of tumor cells (or A molecule capable of inducing apoptosis).

  TNF-α, lymphotoxin-α (or TNF-β), Fas ligand (hereinafter abbreviated as FasL), TRAIL (or Apo-2 ligand), Apo-3 ligand (hereinafter abbreviated as Apo3L), etc. As receptors for cell death-inducing cytokines (death factors) belonging to the TNF family, TNF receptor type I (hereinafter abbreviated as TNFR-I), Fas, DR (death receptor) 3, DR4, DR5, etc. are known, respectively. Yes.

  These death factor receptors belong to the TNF receptor family and are structurally similar to each other. In the extracellular region, the cysteine residue-rich domain repeat structure and in the intracellular region induce apoptosis called death domain. It has an essential structure. In addition, a death factor called DR6 has recently been cloned (Pan GH et al., FEBS Letters 431 (3): 351-356 (1998)) and induces apoptosis and controls Th balance involved in the development of allergy in the immune system. It has been shown to be important for the activation of B cells involved in immune diseases and the control of proliferation (Zhao H et al., Journal of Experimental Medicine 194 (10): 1441-8 (2001); Kazuhiko Oya 3 others, MBL Research Reagent Magazine, Vol. 2, No. 1, pages 1-9, August 2002. Information related to the DR6 sequence can be found in public databases such as GenBank Accession No. AF068868, AF322069, etc. Registered Obtained as an array information.

  TNF-α and lymphotoxin-α bind to TNFR-1 (Goodwin et al., Mol. Cell. Biol. 11 (6), 3020-3026 (1991)). Information relating to the sequence of TNFR-1 can be found in, for example, GenBank Accession No. Obtained as sequence information registered in a public database by M59378 or the like.

  Fas (also called CD95 or Apo-I) is a receptor belonging to the TNF receptor superfamily, to which a Fas ligand (hereinafter abbreviated as FasL) binds. Fas is involved in the induction of apoptosis via FasL (eg, Behrmann et al., European Journal of Immunology 24 (12): 3057-62 (1994)).

  TRAIL (also known as Apo2L) binds to DR4 and DR5. TRAIL can induce rapid caspase-dependent apoptosis in transformed cells, but rarely in normal cells. DR4 and DR5 transmit TRAIL's apoptosis-inducing signal downstream. These ligands and receptors are widely distributed in various tissues, and specific cancer cells and cultured cell lines are also sensitive to TRAIL (eg, Burns and El-Deary, Journal of Biological Chemistry 276 (41): 37879-86 (2001); Wiley et al., Immunity 3 (6): 673-682 (1995); Piti et al, J. Biol. Chem. 271 (22) 12687-12690 (1996)). Information relating to the sequences of DR4 and DR5 can be found in, for example, GenBank Accession No. BD056810 and the like, and GenBank Accession No. It is obtained as sequence information registered in a public database in AB054004, AF012535, AF016268, and the like.

  Apo3L binds to DR3. DR3, discovered as an Apo3L death receptor, is structurally and functionally similar to TNFR-I and, like TNFR-I, is a nuclear transcription factor κB (hereinafter abbreviated as NFκB). It can transmit both activation and apoptosis induction signals. TNFR-I is expressed in various tissues and cells, whereas DR3 is restricted to spleen, thymus, peripheral blood cells and activated T cells. Conversely, the expression of Apo3L, which is a ligand for DR3, is recognized in many tissues and cells. For DR3, the following documents are referred to. Chinnaiyan et al Science 274 (5289): 990-992 (1996); Wang et al Molecular Cellular Biology 21 (10): 3451-61 (2001). Information related to the sequence of DR3 is, for example, GenBank Accession No. It is obtained as sequence information registered in a public database in AB051850, AB051851, AF026607, AF0266071, U72763, and the like.

  The “TNF-related cell death inducing factor” used in the present invention is a molecule that can specifically bind to a receptor for a cell death-inducing cytokine belonging to the TNF family as described above, and induce apoptosis of tumor cells. Include agonistic antibodies against TNF receptor type I (TNFR-I), Fas, DR3, DR4, DR5, DR6, etc. (in this specification, anti-TNFR-I antibody, anti-Fas antibody, anti-DR3 antibody, anti-DR3 antibody, respectively). DR4 antibodies, anti-DR5 antibodies, anti-DR6 antibodies, etc.) may be used. In particular, agonistic antibodies (anti-DR4 antibody and anti-DR5 antibody) against TRAIL receptors DR4 and DR5 can be used more preferably, and anti-DR5 antibodies are most preferably used.

  Antibodies against human death receptors are preferred, but not limited thereto, against death receptors derived from various species including other mammals such as mice, rats, primates, horses, pigs, rabbits, poultry, etc. Antibodies can also be used in the present invention.

  Anti-DR5 antibody binds to Fc receptors on cells belonging to the innate immune system, induces apoptosis in tumor cells, and at the same time activates antigen-presenting cells to which antibodies are bound via activated Fc receptors. Tumor-specific CTLs that promote cell presentation and have cytotoxic mechanisms other than TRAIL induced thereby exert antitumor effects even on TRAIL resistant mutants.

  In the present invention, a lymphocyte activator is further administered to activate the immune mechanism, and finally the tumor including the TRAIL resistant mutant is eradicated. The therapeutic method of the present invention is very promising as a new anti-tumor antibody therapy that simultaneously activates innate immunity and acquired immunity.

  Each of the above antibodies is preferably a monoclonal antibody, which is commercially available from suppliers well known to those skilled in the art (eg, BD. Pharmingen, eBioscience, etc.) or made using well-known techniques such as hybridoma methods. Can do.

  In the present specification, the “cell death induction therapy” refers to a method for treating cancer through administration of the TNF-related cell death inducing factor to a subject. “Treatment” or “therapy” also refers to therapeutic therapy, prophylactic therapy, and protective therapy.

  In the present specification, the “cell death inducing agent” refers to an agent that induces apoptosis of cancer cells, and is typically an agent containing a TNF-related cell death inducing factor.

  As used herein, a “subject” is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, apes, humans, domestic animals, sport animals and pets. In the present specification, the “patient” is assumed to be a human.

(Lymphocyte activator)
In the present specification, the term “lymphocyte activator” refers to a molecule that can specifically bind to a costimulatory molecule and activate or enhance a cancer antigen-specific cytotoxic T cell.

  In the present specification, the term “costimulatory molecule” refers to a T binding to a peptide when acting together with a peptide / MHC complex bound by a T cell receptor (TCR) on the surface of the T cell. It is intended to encompass any single signal molecule (co-signal molecule) or a combination of multiple co-signal molecules that induce co-stimulatory effects to achieve cell activation. Thus, the term complexed with other costimulatory molecules (one or more) on an antigen presenting matrix, such as an antigen presenting cell (APC), fragments thereof (alone or other molecules (one or more)). Or as part of a fusion protein) that activates T cells by binding to the cognate ligand together with the peptide / MHC complex when the TCR on the T cell surface specifically binds the peptide. Include.

  In general, it is known that two independent signals are required for full activation of T cells. One is a signal transmitted by recognizing a peptide in which a TCR binds to a major histocompatibility complex (MHC) on an antigen-presenting cell. The other is an auxiliary signal transmitted by a co-stimulatory molecule, and this auxiliary signal induces sufficient activation of T cells, maintenance of cell proliferation, avoidance of cell death, differentiation of effector T cells and memory T cells. Is important to.

  To date, many molecules that induce this auxiliary signal have been found. For example, CD28 molecules on T cells can interact with the B7 molecule group (B7-1, B7-2) on APC. It plays a crucial role in T cell proliferation, such as transmitting activation signals to cells and inducing the production of many cytokines. For example, ICOS is known as another auxiliary signal molecule belonging to the CD28 family. Furthermore, CTLA-4 is known as a receptor that binds to the B7 molecule group as in CD28 and, in contrast to CD28, suppresses T cell activity, and also belongs to the CD28 family that transmits inhibitory signals. PD-1 is also known as a receptor.

In addition to the CD28 family, OX40, 4-1BB, CD27, CD40, CD30, HVEM, etc. belonging to the TNF receptor (hereinafter abbreviated as TNFR) family are known as molecules that transmit auxiliary signals (for example, Croft M , Nat Rev Immunol. 3: 609-620, 2003, Smith et al, Cell 76 (6): 959-962 (1994)). These molecules are members of the TNFR family that do not have a death domain in the intracellular region, and bind to TRAF (TNF receptor-associated factor) via a TRAF binding motif. TRAF is an adapter molecule for transmitting a signal to a cell when a ligand is bound to TNFR, and is involved in activation of c-Jun NH ₂ terminal protein kinase (hereinafter abbreviated as JNK) and NFκB. .

  CD28 belongs to the immunoglobulin superfamily. CD28 is a molecule that is constitutively expressed on T cells. When CD28 stimulates naive T cells that have recognized an antigen by TCR, CD28 transmits a strong auxiliary signal to T cells. As a result, IL-2 production and IL-2 receptor expression are induced, and cell cycle rotation begins. In addition, CD28 signal suppresses apoptosis of T cells by inducing the expression of Bcl-XL (Boussiotis et al., Immunol Rev 153, 5, 1996; Lenschow et al., Annu Rev immunol 14, 233, 1996). . Information relating to the sequence of CD28 is, for example, GenBank Accession No. It is obtained as sequence information registered in a public database by AF411057, BC064058 or the like.

  CTLA-4 is a similar molecule to CD28, has a stronger binding force, and has been reported as a receptor that antagonizes CD28 and suppresses activation signals from the B7 family. Is known to make CD28-mediated T cell activation more efficient (Chen, Nat. Rev. Immunol. 4 (5) 336-347 (2004), Egen et al. Nat. Immunol 3 (7) 611-618 (2002)). Information related to the CTLA-4 sequence is, for example, GenBank Accession No. It is obtained as sequence information registered in a public database such as NM005214, L15006, X05719.

  PD-1 belongs to the CD28 family and is known as a receptor that mediates inhibitory signals from PDL-1 and B7-DC, and information related to this sequence can be found in, for example, GenBank Accession No. It is obtained as sequence information registered in public databases such as L27440 and E06067 (Okaki et al., Curr. Opin. Immunol. 14 (6) 779-782 (2002).

  Unlike CD28, ICOS is hardly expressed in naive T cells, and its expression increases with activation. ICOS is considered not to bind to B7-1 and B7-2 but to B7h. ICOS has been shown to transmit auxiliary signals to T cells, similar to CD28. Cross-linking ICOS with anti-ICOS antibody or B7h-Ig in the presence of anti-CD3 antibody induces an enhanced proliferative response compared to anti-CD3 antibody alone. ICOS hardly induces IL-2 production, and IL-4, IL-5, IL-10, IFN-IFN-γ, and TNF-α production are enhanced. It is thought that it mainly plays a role in the expression of effector functions (Sharp et al., Rev Immunol 2,116, 2002). Information related to the ICOS sequence is, for example, GenBank Accession No. It is obtained as sequence information registered in a public database by AJ277832, AF411058, AF411059, AL645744, etc.

  OX40 (also referred to as CD134) is not expressed on naive T cells, but is expressed on activated T cells. Its expression is induced by TCR stimulation and further enhanced by CD28 stimulation. The expression of OX40L is not observed in resting blood cells, but is expressed in activated B cells, activated dendritic cells, and vascular endothelial cells (Watts and DeBeneette, Curr Immuno Immunol 11, 286, 1999). OX40 transmits a signal downstream by binding a TRAF family molecule to an intracellular region. OX40 binds to TRAF2, TRAF3, TRAF5 and induces activation of JNK and NFκB (Arch and Thompson, Mol Cell Biol 18, 558, 1998; Kawamata et al., J Biol Chem 2 73, 5808, 1998). . The structure of human OX40 cDNA is described in Latza U et al. , European Journal of Immunology 24: 677-683, 1994. Information relating to the sequence of OX40 is, for example, GenBank Accession No. It is obtained as sequence information registered in a public database by AJ277151, X85214, or the like.

4-1BB (also referred to as CD137) is expressed in activated T cells and NK cells, and is said to prolong the activation and survival of T cells (particularly CD8 ⁺ T cells). (Linda D, et al., J Immunol 168: 3755-3762, 2002; Melero I, et al., Nat Med. 3: 682-685, 1997). ). The ligand 4-1BBL is expressed in activated B cells, mature dendritic cells, activated macrophages, etc. (Watts and DeBeneette, supra). 4-1BB is a few auxiliary signal molecules that enhance IL-2 production, similar to CD28. 4-1BB binds to intracellular regions with TRAF1, TRAF2, and TRAF3, and induces activation of JNK and NFκB (Arch and Thompson, supra; Sauli et al., J Exp Med 187, 1849, 1998). Information related to the sequence of 4-1BB is, for example, GenBank Accession No. It is obtained as sequence information registered in a public database by BD250417, U03397, U02567, J04492, and the like.

  CD27 is constitutively expressed in naive T cells, and its expression level is greatly increased by activation. Expression is also observed in activated B cells. The ligand CD70 is transiently expressed on activated T and B cells. In addition, an auxiliary signal via CD27 induces activation of cytotoxic T cells (Lens et al., Semin Immunol 10, 491, 1998). TRAF2, TRAF3, and TRAF5 bind to the intracellular region, and as a result, activation of JNK and NFκB is induced (Akiba et al., J Biol Chem 273, 133 53, 1998; Yamamoto et al., J Immunol). 161, 4753, 1998). Information relating to the sequence of CD27 is, for example, GenBank Accession No. It is obtained as information registered in a public database by M63928, L24493, L24494, etc.

  CD40 is mainly expressed in antigen-presenting cells such as B cells, dendritic cells, macrophages, and the production of IL-12 is induced by a signal via CD40, and the subsequent enhancement of IFN-γ production and suppression of IL-10 production are induced. It induces a cellular immune response predominantly Th1 and, as a result, assists the induction of cancer antigen-specific cytotoxic T cells (CTLs) and leads to tumor rejection (French RR, et al., Nat Med. 5: 548-553, 1999). TRAF5 and TRAF6 are known as signal transduction factors downstream of CD40 (see, for example, Junichiro Inoue, “Mechanism of cytokine signal transduction by TRAF6” Molecular Medicine Vol. 39 Immunization 2003 (2002)). Information relating to the sequence of CD40 is, for example, GenBank Accession No. It is obtained as sequence information registered in a public database by AH001877, M83312, etc.

  CD30 (also referred to as CD153) is known as a marker protein for Hodgkin lymphoma and is mainly expressed in activated T cells, B cells, and Reed Sternberg cells and binds to CD153 (CD30 ligand). In vitro studies have shown that CD30 mediates various activations, differentiation or apoptosis. CD30 has been shown to bind to TRAF2 and TRAF5 and to activate NF-κB and JNK / p38 MAPK (Aizawa et al., J Biol Chem. 272 (4): 2042-5; 1997). . Information related to the sequence of CD30 is, for example, GenBank Accession No. AJ272029, AJ289159, AJ308521, AJ308522, AJ409011, AJ409012, etc. are obtained as sequence information registered in a public database.

  HVEM has been shown to bind to two cellular ligands, secreted LT-α (TNF-β) and LIGHT (Mauri et al., Immunity 8 (1): 21-30; 1998). In addition, the intracellular region of HVEM binds to TRAF-1, TRAF-2, TRAF-3, and TRAF-5, indicating that HVEM is linked to a signal transduction pathway that activates the immune response. (Marsters et al., Journal of Biological Chemistry 272 (22): 14029-14232; 1997). This interaction activates NFκB and AP-1. Information relating to the sequence of HVEM is, for example, GenBank Accession No. Obtained as sequence information registered in a public database by AY466111, BD091696, etc.

  The “lymphocyte activator” used in the present invention is typically a single substance that specifically binds to a co-stimulatory molecule as described above and activates a cancer antigen-specific cytotoxic T cell. A molecule or a combination of molecules. Preferably, the “lymphocyte activator” used in the present invention is an agonistic antibody against an auxiliary signal molecule belonging to the CD28 family or TNFR family (eg, anti-CD28 antibody, anti-ICOS antibody, anti-OX40 antibody, anti-4- 1BB antibody, anti-CD27 antibody, anti-CD40 antibody, anti-CD30 antibody, anti-HVEM antibody, etc.) or antagonistic antibodies against molecules belonging to the CD28 family that suppress auxiliary signals (eg anti-CTLA-4 antibody, anti-PD-1 antibody, etc.) In particular, anti-4-1BB antibody or antibody CD40 antibody is used as an agonistic antibody against an auxiliary signal molecule, and anti-CTLA-4 antibody is used as an antagonistic antibody against a molecule belonging to the CD28 family that suppresses an auxiliary signal. Good Properly, the combination of a combination or anti-4-1BB antibody and anti-CD40 antibody and anti-CTLA-4 antibody and anti-4-1BB antibody and anti-CD40 antibody is most preferred.

  In addition, antibodies to human costimulatory molecules are preferred, but not limited thereto, and assistance derived from various species including other mammals such as mice, rats, primates, horses, pigs, rabbits, poultry, etc. Antibodies to stimulatory factors can also be used in the present invention.

  Each of the above antibodies is preferably a monoclonal antibody, which is commercially available from suppliers well known to those skilled in the art (eg BD Phramingen, eBioscience, etc.) or methods well known to those skilled in the art (eg hybridoma methods) Can be made.

  In the present specification, “lymphocyte activation therapy” refers to a method for treating cancer by administering the lymphocyte activator to a subject. The “lymphocyte activator” refers to a drug containing a lymphocyte activator.

  As used herein, the terms “cancer”, “cancerous” or “malignant” are used to refer to a physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinomas including adenocarcinomas, lymphomas, blastomas, melanomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodkin and non-Hodkin lymphoma, lymphoma, pancreatic cancer, glioblastoma, cervical cancer, Liver cancer such as ovarian cancer, liver cancer and hepatoma, bladder cancer, breast cancer, colon cancer, glioblastoma, endometrial cancer, salivary gland cancer, renal cell carcinoma such as renal cell carcinoma and Wilm tumor, basal cell carcinoma species, It includes melanoma, prostate cancer, spawning cancer, thyroid cancer, testicular cancer, esophageal cancer and various types of head and neck cancer.

  In the present specification, the term “chemotherapeutic agent” is used in the meaning commonly used in the art, and is a compound useful for the treatment of cancer, which inhibits or suppresses cell function and / or disrupts cells. Refers to what causes. Examples of chemotherapeutic agents include adriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosine arabinoside ("Ara-C"), cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids such as paclitaxel (taxol, Bristol) -Myers Squibb Oncology, Princeton, NJ) and doxetaxel (Taxotere, Rhone-Poulenc Rorer, Antony, Rance), Toxotere, methotrexate, cisplatin, melphalan, phosphatine, melphalan, CPT-1 C, mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide Daunomycin, carminomycin, aminopterin, dactinomycin, mitomycin, esperamicins (see U.S. Pat. No. 4,675,187), Melphalan and other related nitrogen mustards. This definition also includes hormonal agents that act to modulate or inhibit hormonal effects on tumors, such as tamoxifen and onapristone.

As used herein, the term “radiotherapy” is used in the sense commonly used in the art and uses radioisotopes (eg, I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , and Re ¹⁸⁶ ) to determine cell function. Refers to a therapy that treats cancer by inhibiting or suppressing and / or causing cell destruction.

(antibody)
Antibodies that can be used in the present invention are produced by polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies or chimeric antibodies, single chain antibodies, Fab fragments, F (ab ′) fragments, Fab expression libraries Fragments, anti-idiotype (anti-Id) antibodies, and epitope binding fragments of any of the above. Preferably, monoclonal antibodies are used in the present invention.

  As used herein, "agonistic antibody" to a molecule means an antibody that specifically binds to the molecule and activates or increases its ability to induce a given biological activity. For example, an agonistic antibody to DR5 is an antibody to DR5 that, when bound to DR5, activates or increases the ability to induce caspase-dependent apoptosis. Here, the ability to induce caspase-dependent apoptosis corresponds to the original biological activity of DR5 that is exerted when the receptor-ligand pair TRAIL-DR5 is formed. In contrast, an antibody that reduces the ability of the molecule to induce a given biological activity is called an “antagonistic antibody”. The agonistic or antagonistic antibodies to specific molecules used in the present invention are preferably monoclonal antibodies, which are obtained commercially from suppliers well known to those skilled in the art (eg BD Phramingen, eBioscience, etc.). Or can be made and identified by techniques well known in the art.

  In the present specification, the “antibody” refers to an immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule, that is, a molecule containing an antigen-binding site that immunospecifically binds to an antigen. The immunoglobulin molecules of the invention can be any type of immunoglobulin molecule (eg, IgG, IgE, IgM, IgD, IgA, and IgY), any class (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). Or it may be a subclass of an immunoglobulin molecule.

Most preferably, the antibody is a human antigen binding antibody fragment, which includes Fab, Fab ′ and F (ab ′) ₂ , Fd, single chain Fvs (scFv), single chain antibody, disulfide bond Fvs (sdFv) As well as fragments containing either the _VL or _VH domains. Antigen-binding antibody fragments, including single chain antibodies, can contain variable regions alone or in combination with all or part of those listed below: hinge region, CH1 domain, CH2 domain and CH3 domain. In addition, antigen-binding fragments that also include any combination of variable region and hinge region, CH1 domain, CH2 domain, and CH3 domain may also be used in the present invention. The antibodies used in the present invention can be from any animal origin including birds and mammals. Preferably, the antibody is derived from human, murine, donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.

  The antibodies used in the present invention can be monospecific, bispecific, trispecific or more multispecific antibodies. Multispecific antibodies can be specific for different epitopes of the polypeptide of interest, or are specific for both the polypeptide of interest and a heterologous epitope (eg, a heterologous polypeptide or solid support material). It can be. For example, PCT Publication WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt et al. Immunol. 147: 60-69 (1991); U.S. Pat. Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920, 5, 601,819; Kostelny et al., J. MoI. Immunol. 148: 1547-1553 (1992).

  The antibodies used in the present invention may be those commercially available from suppliers well known to those skilled in the art, but can also be produced by any suitable method known in the art. For example, polyclonal antibodies against an antigen of interest can be produced by various procedures well known in the art. For example, the polypeptide of interest can be administered to various host animals (including but not limited to rabbits, mice, rats, etc.) to induce the production of serum containing polyclonal antibodies specific for the antigen. . Various adjuvants can be used to increase the immunological response, depending on the host species, and include Freund (complete and incomplete), mineral gels such as aluminum hydroxide, lysolecithin, etc. These include surfactants, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and corynebacterium parvum. It is not limited. Such adjuvants are also well known in the art.

  Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display techniques, or combinations thereof. For example, monoclonal antibodies can be produced using hybridoma technology known in the art, including, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd Edition, 1988). Reference is made to the method described by Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY 1981). As used herein, the term “monoclonal antibody” is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the antibody from which it is generated. Thus, the term “monoclonal antibody” is not limited to antibodies produced through hybridoma technology. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridomas and recombination and phage display techniques.

  Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. Briefly, mice can be immunized with a polypeptide of interest or cells that express such a peptide. Once an immune response is detected (eg, an antibody specific for the antigen is detected in the mouse serum), the mouse spleen is collected and the splenocytes isolated. The splenocytes are then fused to any suitable myeloma cells (eg, cells from cell line SP20 available from ATCC) by well-known techniques. Hybridomas are selected and cloned by limiting dilution. The hybridoma clones are then assayed by methods known in the art for cells secreting antibodies capable of binding the polypeptide of interest. Ascites fluid, which generally contains high levels of antibodies, can be produced by immunizing mice with positive hybridoma clones.

  Therefore, the antibody used in the present invention can be produced by a method comprising culturing a monoclonal antibody as well as a hybridoma cell that secretes the antibody used in the present invention. Preferably, the hybridoma is a hybridoma clone that fuses myeloma cells and splenocytes isolated from a mouse immunized with the antigen of the present invention, and then secretes an antibody capable of binding the polypeptide of interest. Produced by screening for hybridomas arising from the fusion.

Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, the Fab and F (ab ′) ₂ fragments of the present invention can be obtained using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F (ab ′) ₂ fragments). Can be produced by proteolytic cleavage of immunoglobulin molecules. The F (ab ′) ₂ fragment contains the variable region, the light chain constant region and the CH1 domain of the heavy chain.

  Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art, including the phage display methods described above using antibody libraries derived from human immunoglobulin sequences (US Pat. Nos. 4,444,887 and 4). PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741).

(Formulation)
The TNF-related cell death inducing factor and lymphocyte activating factor used in the present invention are preferably used in a carrier. Suitable carriers and their formulations are described in Remington's Pharmaceutical Sciences, 16th ed., Edited by Oslo et al. , 1980, Mack Publishing Co. It is described in. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to make the formulation isotonic. Examples of the carrier include physiological saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to 8, more preferably about 7.4 to 7.8. Further, the carrier includes a sustained-release preparation such as a semi-permeable matrix of a solid hydrophobic polymer containing an antibody, and this matrix is in the form of a molded product such as a film, liposome or microparticle. Yes. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of antibody being administered. The carrier may be lyophilized or in the form of an aqueous solution.

  Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and buffers such as phosphate, citrate and other organic acids; anticorrosives including ascorbic acid and methionine. Preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3 -Pentanol; and m-cresol); low molecular weight (less than 10 residues) polypeptide; protein such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer such as polyvinylpyrrolidone; glycine, glutamine, a Amino acids such as paragin, arginine, histidine or lysine; monosaccharides such as glucose, mannose or dextrin, disaccharides or other carbohydrates, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol, salt formation such as sodium Counterions; metal complexes (eg, Zn-protein complexes); and / or nonionic surfactants such as TWEEN®, PLURONICS ™ or polyethylene glycol (PEG).

  A formulation may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. The TNF-related cell death inducing factor or lymphocyte activating factor used in the present invention is, for example, a microcapsule prepared by coacervation or interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsule and poly- (, respectively. Methyl methacrylate) microcapsules can be encapsulated in colloidal drug delivery systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. et al. Ed .; 1980.

The formulation used for in vivo administration must be sterile. This is easily accomplished by filtration through a sterile filter membrane. Sustained release preparations may be prepared. Suitable examples of sustained release preparations include solid hydrophobic polymer semipermeable matrices containing antibodies, which are in the form of shaped articles such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (eg, poly (2-hydroxyethyl-methacrylate), or poly (vinyl alcohol), polylactide (US Pat. No. 3,773,919), L-glutamic acid and γ-ethyl-L- Glutamate copolymers, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT ^™ (injectable microspheres consisting of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-3-hydroxybutyric acid While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for over 100 days, certain hydrogels release proteins for a shorter time.

(Form of treatment)
The therapeutic agent for cancer of the present invention is administered intravenously as a bolus or continuously infused over a period of time, intramuscularly, intraperitoneally, intracerebral spinal cord, subcutaneously, intraarticularly, intracerebral fluid, or arachnoid space according to a known method. It is administered to a mammal, preferably a human, by the route of internal, oral, topical, intralesional or inhalation (into an aerosolized nasal cavity, into the lung) and by sustained or sustained release methods. In some cases, administration can also be performed by minipump inflow using various commercially available devices.

  Effective doses and schedules for administration of the cancer therapeutics of the invention can be determined empirically, and making such determinations is within the skill in the art. Effective dosages or amounts of drugs used alone are currently considered to range from about 1 μg / kg to about 100 mg / kg body weight or more per day for each active ingredient. Interspecies scaling of doses is described, for example, by Mordenti et al., Pharmaceut. Res. 8: 1351; 1991, and can be performed by methods known in the art.

  In the combination therapy of the cell death induction therapy and lymphocyte activation therapy of the present invention, the TNF-related cell death inducer and lymphocyte activation factor used in the present invention are applied to the subject at the same time or at intervals. Can be administered. For example, a TNF-related cell death inducing factor is first administered to the subject followed by a lymphocyte activator. In another preferred embodiment, a TNF-related cell death inducing factor and a lymphocyte activator are first administered in combination, and in the subsequent administration schedule, the dose of only the TNF-related cell death inducing factor is decreased over time. Alternatively, administration of only the TNF-related cell death inducing factor may be discontinued.

  When in vivo administration of the cancer therapeutic agent of the present invention is used, the normal dosage is from about 10 ng / kg to 100 mg / kg of each active ingredient per day for the body weight of the mammal, preferably depending on the route of administration, About 1 μg / kg / day to 10 mg / kg / day. Specific dosage and delivery method guidelines are given in the literature; see, eg, US Pat. Nos. 4,657,760, 5,206,344, or 5,225,212. It is expected that different formulations will be effective for different therapeutic compounds and different diseases, for example, administration targeting one organ or tissue will require delivery in a different manner than other organs or tissues Is done. One skilled in the art understands that the dose of drug that must be administered may vary depending on, for example, the mammal receiving the drug, the route of administration, and other drugs or treatments being administered to the mammal. It will be possible.

  Depending on the cell type and / or severity of the disease, for example, one or more separate doses or continuous infusions, each active ingredient is about 1 μg / kg to 15 mg / kg (eg 0.1-20 mg / kg). kg) antibody is the first candidate dose to the patient. Depending on the factors discussed above, typical daily dosages range from about 1 μg / kg to 100 mg / kg or more. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs.

  In some cases, prior to administration of a cancer therapeutic of the present invention, a mammal or patient is tested to determine the level or activity of a TNF-related cell death inducer and / or lymphocyte activator used in the present invention. be able to. Such tests may be performed by serum samples or peripheral blood leukocyte ELISA or FACS.

  It is contemplated that additional therapies may be used in the method. One or more other therapies are known in the art, including but not limited to, radiation therapy, cytokines, growth inhibitors, chemotherapeutic agents, cytotoxic agents, tyrosine kinase inhibitors Administration of rasfarnesyltransferase inhibitors, angiogenesis inhibitors, and cyclin dependent kinase inhibitors. In addition, therapies are based on therapeutic antibodies that target tumor antigens such as Rituxan® or Herceptin® as well as anti-angiogenic antibodies such as anti-VEGF.

  Preparation methods and dosage schedules for chemotherapeutic agents are either used according to manufacturer's instructions or determined empirically by skilled practitioners. Preparation methods and dosage schedules for such chemotherapy are also described in Chemotherapy Service, M. et al. C. Edited in Perry, Williams &; Wilkins, Baltimore, MD; 1992. The chemotherapeutic agent may also be given, for example, prior to, or after administration of the antagonist. The antagonist may also be combined with an anti-estrogenic compound such as tamoxifen or an anti-progesterone such as onapristone (see EP 616812) at a dose known for such molecules.

(kit)
In another embodiment of the present invention, a kit is provided that contains substances useful for the treatment of the aforementioned diseases. The kit typically comprises a container and a label. Suitable containers include, but are not limited to, for example, bottles, vials, syringes, test tubes, and the like. The container can be formed of various materials such as glass or plastic. The container contains a composition effective for the treatment of a medical condition and has a sterile entry / exit port (eg, the container is a bag or vial for intravenous solution with a stopper pierceable by a hypodermic needle. Good). The active agents in the composition are TNF-related cell death inducers and lymphocyte activators. The label on or associated with the container indicates that the composition is used for the treatment of the selected medical condition. The article of manufacture further comprises a second container containing a pharmaceutically acceptable buffer, such as phosphate buffered saline, lingual solution and glucose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts including instructions for use.

  EXAMPLES Hereinafter, although this invention is demonstrated more concretely using an Example, the scope of the present invention is not limited by these Examples.

Example 1
Materials and Methods Experimental Animals 6-8 week old BALB / c mice and CB17-scid (SCID) mice were purchased from Nippon Charles River Co., Ltd. All mice were bred in a SPF (specific pathogen-free) facility, and experiments were conducted according to the guidelines for animal experiments at Juntendo University School of Medicine.

2. Experimental tumor BALB / c mouse-derived TRAIL-sensitive breast cancer 4T1 (Pulski BA, et al: Reduction of Established Spontaneous Mamma in the United States of America and the United States of America)
cell-based tumor vaccines. Cancer Res 58 (7): 1486-1493 (1998)), TRAIL-sensitive renal cancer R331-mock, and R331-FLIP (TRAIL resistance) introduced with a FLIP gene that becomes caspase-dependent apoptosis resistant (Seki N., et al. Cancer Res.63: 207-213 (2003)) was used. The tumor cells were cultured and passaged in RPMI 1640 medium containing 10% FBS in the presence of 37 ° C. and 5% CO ₂ and used for the experiment.

3. Antibodies Agonic anti-mouse DR5 monoclonal antibody (MD5-1) (Takeda K, et al., J Exp Med 199 (4) 437-448 (2004)), agonistic anti-mouse CD40 monoclonal antibody (FGK45) (Rolink A. , Et al. Immunity 5: 319-330 (1996)), agonistic anti-mouse 4-1BB monoclonal antibody (3H3) (Shuford WW, et al. J. Exp. Med. 186: 47-55 (1997). )), An antagonistic anti-mouse CTLA-4 monoclonal antibody (UC10-4F10) (Walunas TL, et al, Immunity 1: 405-413 (1994)). In addition, anti-hamster control Ig (UC8-1B9) and anti-rat IgG _2a (R35-95; BD Pharmingen) were used as control antibodies. MD5-1, FGK45, 3H3, UC8-1B9 were prepared by the hybridoma method in our laboratory (for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd edition, 1988). Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY 1981)), used in experiments.

4). Mouse subcutaneous tumor model BALB / c mice were inoculated with 4T1 tumor cells subcutaneously on the back. 100 μg of each antibody was started from the time when the size of the tumor mass reached 3 × 3 mm ² −8 × 8 mm ^2, and intraperitoneally administered four times every 3 days. The size of the tumor mass was measured with a caliper every 1-2 days, calculated as major axis × minor axis (mm ² ), and expressed as the mean ± SD (mm ² ) of 5-10 animals in each group.

5. Evaluation of Tumor Specific Rejection Naive BALB / c mice and mice rejected by treatment were again inoculated with 4T1 tumor subcutaneously in the back and their growth was compared. In addition, 5 × 10 ⁷ splenic T cells collected from naive BALB / c mice and mice that rejected 4T1 tumors by treatment were inoculated into 2 × 10 ⁵ 4T1 tumor cells subcutaneously and then to naive SCID mice on the second day. Transfer and tumor growth were compared.

Further, R331-mock or R331-FLIP was inoculated into BALB / c mice, and then the above-mentioned antibody was administered when the tumor mass reached 3 × 3 mm ^2, and the growth of the tumor was compared. R331-mock and R331-FLIP were simultaneously inoculated subcutaneously on the right and left dorsal regions, respectively, and when the antibody reached 3 × 3 mm ² , the above-mentioned antibody was administered, and tumor growth was compared.

(Example 2)
Therapeutic effect of Mix Therapy When the size of the tumor mass reached 3 × 3 mm ^2, it was divided into a single administration group, a two antibody combination administration group, a three antibody combination administration group (Mix therapy) group, and a control antibody administration group. The transition of the tumor mass size was compared. FIG. 1 shows the results. In addition, the downward arrow in the figure indicates the time point when the antibody was administered (the same applies to other figures described below). Although some tumor rejection was induced in the group containing anti-4-1BB antibody (2 out of 10 cases alone or in combination with anti-CD40 antibody, 4 out of 10 cases in combination with anti-DR5 antibody), 3 A synergistic effect was observed by the combined administration of the antibody (Mix therapy), and a significantly high induction of tumor rejection was observed with a tumor rejection rate of 80% (8 of 10 cases) (FIG. 1 and Table 1).

(Example 3)
Therapeutic effect due to difference in size of tumor mass in Mix Therapy Next, the therapeutic effect in a more advanced state was examined. The treatment was started when the size of the tumor mass reached 3 × 3 mm ² , 5 × 5 mm ² , and 8 × 8 mm ² , respectively. Similarly 5 80% even rejection rates in × 5 mm ² and high therapeutic effect as 3 × 3 mm ² was obtained. Although tumor rejection could not be induced even at 8 × 8 mm ² , significant tumor shrinking effect and growth inhibitory effect were observed as compared with the control antibody administration group (FIG. 2 and Table 1). This result indicates that Mix therapy is very useful in clinical applications because it is often found in some advanced state when treating human cancer in the clinic.

Example 4
Therapeutic effect of single administration of anti-DR5 antibody or anti-DR5 antibody alone is administered only once, and other drugs (anti-CD40 antibody and anti-4-1BB antibody) are administered 4 times, and its anti-tumor effect ((Shown as “Mix (1-4-4)” in FIG. 3)). As shown in FIG. 3, even when the number of administrations of anti-DR5 antibody was only once, the same result as in Example 3 in which anti-DR5 antibody was administered four times in the same manner as other drugs (rejection rate) 80%) was obtained. From this, it was shown that although apoptosis induction for tumors is important, provision of tumor antigens for induction of apoptosis is sufficient at the initial stage of treatment (induction by single administration). Although there are still many unclear points, reports on the toxicity of recombinant TRAIL are scattered (Jo M et al., Nat. Med. 6: 564-567 (2000); Qin J et al., Nat Med. 7: 385-386 (2001)). However, no obvious toxicity was observed in this experiment, and it was considered possible to reduce the toxicity of TRAIL by reducing the number of administrations.

Induction of tumor-specific cytotoxic T cells Furthermore, 60 days after rejection of the primary inoculated tumor, the same tumor was re-inoculated, and complete rejection was induced (FIG. 4A). When 5 × 10 ⁷ spleen T cells collected from the tumor-rejected mice were introduced into SCID mice 2 days after 4T1 tumor inoculation, a significant tumor growth inhibitory effect was observed (FIG. 4B). In addition, in vitro experiments have confirmed the presence of CTL. From these results, it was suggested that the induction of CTL that can be caused by the administration of anti-DR5 antibody alone was efficiently enhanced by the combined use of agonistic antibodies to costimulatory molecules and led to tumor rejection, as reported previously. .

(Example 5)
Effect of induction of tumor-specific cytotoxic T cells against TRAIL-sensitive and non-sensitive strains A tumor that became TRAIL-resistant by introducing FLIP resistant to caspase-dependent apoptosis into R331, a TRAIL-sensitive renal cancer (R331-FLIP) ) And mock tumor (R331-mock) maintaining TRAIL sensitivity, the effect of Mix therapy was examined. R331-FLIP also showed some tumor growth inhibitory effect, which seems to be the effect of antibody administration against costimulatory molecules, but no rejection was induced, whereas R331-mock had 80% rejection. (FIG. 5A. Next, R331-mock and FLIP were simultaneously inoculated subcutaneously on the right and left dorsal regions, respectively, to examine the effects of Mix therapy. Not only R331-mock but also R331-FLIP rejected almost simultaneously ( 80% rejection) was induced, suggesting that tumor rejection of mutants via FasL and perforin other than TRAIL by CTL was performed very rapidly (FIG. 5B). The emergence of TRAIL-insensitive tumors in therapeutic strategies targeting TRAIL receptors Although tumor enlargement due to escape becomes a problem (Qin J et. Nat Med. 7: 385-386 (2001)), the results obtained this time indicate that the mix therapy is based on some sensitive tumors, It was shown that it is possible to eradicate the whole tumor by capturing immunologically specifically.

(Example 6)
Therapeutic effect of anti-CTLA-4 monoclonal antibody administration in combination with Mix Therapy 4T1 tumor 2 × 10 ⁵ cells were inoculated subcutaneously into mice, and when the tumor size reached 5 × 5 mm ² , the mixture was treated with the mix therapy group, mix therapy + anti-CTLA -4 antibody administration group and control antibody administration group, and tumor growth of each mouse was compared (n = 10).

  As shown in FIG. 6, in the control group (see FIG. 6A), no therapeutic effect on the tumor was observed, but in the Mix therapy group, there was a significant tumor growth inhibitory effect (rejected in 9 out of 13 cases). Observed (see FIG. 6B). Further, it should be noted that, when Mix therapy and anti-CTLA-4 antibody administration are used in combination, a better tumor growth inhibitory effect is seen compared to the case of Mix therapy alone, and in all cases (13 of 13 cases) Tumor rejection was observed (see FIG. 6C). Thus, the therapeutic effect of Mix therapy is further enhanced by using a factor that suppresses the action effect of CTLA-4 (anti-CTLA-4 antagonistic antibody (blocking antibody)) to suppress the activity of T cells. It was shown that

(Discussion)
As specifically shown in the above examples, according to the cancer treatment method of the present invention, the entire tumor cells can be immunologically used as a step to induce cell death in TRAIL-sensitive cells without identifying a tumor-specific antigen. It is possible to induce many types of CTL specific to a comprehensive cancer antigen and thereby escape from immunological treatment of cancer cells by loss of TRAIL sensitivity and mutation of cancer antigen Can reduce the possibility of causing Since there are some reports that TRAIL receptor expression is relatively frequent in cancer in humans, the cancer therapy according to the present invention can be implemented as a first-line therapy for more patients. . In addition, even when metastasis is taken into account, it is difficult to produce an effect in detail due to the problem of drug delivery or the like, and recurrence due to a temporary effect is a problem, but the cancer treatment method according to the present invention According to the above, it is considered that the effect of secondary CTL induction reaches a long-term and more detailed effect, and the cancer can be driven out from the living body. Furthermore, in the cancer treatment method according to the present invention, since cell death may be induced once in the tumor cells, the tumor cell death can be efficiently induced, but the immune function is also suppressed. Combination with chemotherapy or radiation therapy with anticancer agents may also be possible. Thus, the treatment method according to the present invention as specifically shown in the Examples is a simple and more versatile cancer treatment method, and further provides an efficient economic effect.

  The present invention can induce tumor-specific CTL more efficiently and strongly, and can exhibit a high therapeutic effect that tumor rejection can be induced even at a stage where tumor growth has progressed. It is useful as a new cancer therapy that can eradicate cancer.

10 is a graph showing the effect of Mix Therapy in Example 2. When 4T1 tumor 2 × 10 ⁵ cells were inoculated subcutaneously and the tumor mass reached 3 × 3 mm ² , anti-DR5 antibody, anti-CD40 antibody and anti-4-1BB antibody were administered alone, and two antibodies were administered The mice were divided into 3 antibody administration (Mix therapy) group and control antibody administration group, and the tumor growth of each mouse was compared (n = 10). 10 is a graph showing an effect of a difference in tumor mass size in Mix therapy in Example 3. When BALB / c mice were inoculated subcutaneously with 2 × 10 ⁵ cells of 4T1 tumor and the tumor masses reached 3 × 3 (■), 5 × 5 (●), and 8 × 8 (▲) mm ² , respectively. Then, mix therapy was performed 4 times in total every 3 days. Each group was expressed as the mean ± SD (mm ² ) of 5-10 animals. 6 is a graph showing the therapeutic effect of a single administration of anti-DR5 antibody in Example 4. When the size of the tumor mass reached 3 × 3 mm ² , the anti-DR5 antibody was only administered for the first time, and the anti-CD40 antibody and the anti-4-1BB antibody were intraperitoneally administered four times every three days (■). As a control, naive BALB / c mice were simultaneously inoculated with 4T1 tumor (□). Each group was expressed as the mean ± SD (mm ² ) of 5-10 animals. 4 is a graph showing the induction of tumor-specific cytotoxic T cells in Example 4. (A) BALB / c mice that rejected 4T1 by Mix therapy were re-inoculated with 2 × 10 ⁵ 4T1 tumors and growth was observed (■). As a control, naive BALB / c mice were simultaneously inoculated with 4T1 tumor (□). Each group was expressed as the mean ± SD (mm ² ) of 5-10 animals. (B) Naive SCID mice were inoculated subcutaneously with 2 × 10 ⁵ 4T1 tumors. Two days after tumor inoculation, 5 × 10 ⁷ splenic T cells isolated from naive BALB / c mice (◯) and BALB / c mice (●) that rejected 4T1 were transferred from the tail vein, and tumor growth was observed. . As a control (□), naïve SCID mice were simultaneously inoculated with 4T1 tumor. Each group was expressed as the mean ± SD (mm ² ) of 5-10 animals. It is a graph which shows the effect by the induction | guidance | derivation of the tumor specific cytotoxic T cell with respect to the TRAIL sensitive strain | stump | stock and non-sensitive strain | stump | stock in Example 5. (A) BALB / c mice were inoculated with R331-mock (◯: control group, ●: Mix therapy group) or R331-FLIP (□: control group, ■: Mix therapy group), and then the size of the tumor mass was When reaching 3 × 3 mm ² , Mix therapy or control antibody was administered, and tumor growth was observed. Each group was expressed as the mean ± SD (mm ² ) of 5-10 animals. (B) R331-mock (circle) and R331-FLIP (square) are simultaneously inoculated subcutaneously on the right and left dorsal backs, respectively, and when reaching 3 × 3 mm ² , Mix therapy (filled in black) or control antibody (outlined) And tumor growth was observed. Each group was expressed as the mean ± SD (mm ² ) of 5-10 animals. 10 is a graph showing the effect of anti-CTLA antagonistic antibody administration on Mix therapy in Example 6. From the left, the results are shown for (A) a control antibody administration group, (B) a mix therapy group, and (C) a mix therapy + anti-CTLA-4 antibody administration group. 4T1 tumor 2 × 10 ⁵ cells were inoculated subcutaneously and treatment was initiated when the tumor mass size reached 5 × 5 mm ² to compare the tumor growth of individual mice.

Claims (36)

  A cancer therapeutic agent comprising a combination of a TNF-related cell death inducer and a lymphocyte activator.   The cancer therapeutic agent according to claim 1, wherein the TNF-related cell death-inducing factor is an agonistic antibody against a TRAIL receptor.   The cancer therapeutic agent according to claim 2, wherein the agonistic antibody to the TRAIL receptor is an anti-DR5 antibody.   The cancer therapeutic agent according to claim 1, wherein the lymphocyte activator is an agonistic antibody against a costimulatory molecule.   The cancer therapeutic agent according to claim 4, wherein the agonistic antibody against the co-stimulatory molecule is an agonistic antibody against a CD28 family member or an agonistic antibody against a TNFR family member having no death receptor.   The cancer therapeutic agent according to claim 5, wherein the agonistic antibody to the CD28 family member is an anti-CD28 antibody, an anti-ICOS antibody, or a combination thereof.   The cancer therapeutic agent according to claim 5, wherein the agonistic antibody against the TNFR family member not having a death receptor is an anti-CD40 antibody, an anti-4-1BB antibody, or a combination thereof.   The cancer therapeutic agent according to claim 1, wherein the TNF-related cell death inducing factor is an anti-DR5 antibody, and the lymphocyte activating factor is an anti-CD40 antibody and an anti-4-1BB antibody.   Furthermore, the cancer therapeutic agent in any one of Claims 1-8 containing the antagonistic antibody with respect to the receptor which transmits an inhibitory signal.   The cancer therapeutic agent according to claim 9, wherein the antagonistic antibody to the receptor that transmits the inhibitory signal is an anti-CTLA-4 antibody.   A cancer therapeutic agent for use in combination with a lymphocyte activator, comprising a TNF-related cell death inducing factor.   The cancer therapeutic agent according to claim 11, wherein the TNF-related cell death inducing factor is an agonistic antibody against a TRAIL receptor.   The cancer therapeutic agent according to claim 12, wherein the agonistic antibody against the TRAIL receptor is an anti-DR5 antibody.   The cancer therapeutic agent according to claim 11, wherein the lymphocyte activator contains an agonistic antibody against a costimulatory molecule.   The cancer therapeutic agent according to claim 14, wherein the agonistic antibody to the co-stimulatory molecule is an agonistic antibody to a CD28 family member or an agonistic antibody to a TNFR family member having no death receptor.   The cancer therapeutic agent according to claim 15, wherein the agonistic antibody to the CD28 family member is an anti-CD28 antibody, an anti-ICOS antibody, or a combination thereof.   The cancer therapeutic agent according to claim 15, wherein the agonistic antibody to the TNFR family member having no death receptor is an anti-CD40 antibody, an anti-4-1BB antibody, or a combination thereof.   Furthermore, the cancer therapeutic agent in any one of Claims 11-17 containing the antagonistic antibody with respect to the receptor which transmits an inhibitory signal.   The cancer therapeutic agent according to claim 18, wherein the antagonistic antibody to the receptor that transmits the inhibitory signal is an anti-CTLA-4 antibody.   A cancer therapeutic agent for use in combination with a cell death inducer, comprising a lymphocyte activator.   21. The cancer therapeutic agent according to claim 20, wherein the lymphocyte activator is an agonistic antibody against a costimulatory molecule.   The cancer therapeutic agent according to claim 21, wherein the agonistic antibody against the co-stimulatory molecule is an agonistic antibody against a CD28 family member or an agonistic antibody against a TNFR family member having no death receptor.   The cancer therapeutic agent according to claim 22, wherein the agonistic antibody against the CD28 family member is an anti-CD28 antibody, an anti-ICOS antibody, or a combination thereof.   23. The cancer therapeutic agent according to claim 22, wherein the agonistic antibody against a TNFR family member having no death receptor is an anti-CD40 antibody, an anti-4-1BB antibody, or a combination thereof.   The cancer therapeutic agent according to claim 20, wherein the cell death inducer contains a TNF-related cell death inducer.   26. The cancer therapeutic agent according to claim 25, wherein the TNF-related cell death inducing factor is an agonistic antibody against TRAIL receptor.   27. The cancer therapeutic agent according to claim 26, wherein the agonistic antibody against the TRAIL receptor is an anti-DR5 antibody.   Furthermore, the cancer therapeutic agent in any one of Claims 20-27 containing the antagonistic antibody with respect to the receptor which transmits an inhibitory signal.   The cancer therapeutic agent according to claim 28, wherein the antagonistic antibody to the receptor that transmits the inhibitory signal is an anti-CTLA-4 antibody.   The cancer therapeutic agent according to any one of claims 1 to 29, which is used in combination with a chemotherapeutic agent.   The cancer therapeutic agent according to any one of claims 1 to 29, which is used in combination with radiation therapy. A kit for cancer treatment by a combination therapy of cell death induction therapy and lymphocyte activation therapy,
A drug containing a TNF-related cell death inducing factor;
A cancer treatment kit comprising a drug containing a lymphocyte activator.   The cancer treatment kit according to claim 32, wherein the TNF-related cell death inducing factor is an agonistic antibody against a TRAIL receptor, and the lymphocyte activator is an agonistic antibody against a costimulatory molecule.   34. The cancer treatment kit according to claim 33, wherein the agonistic antibody to TRAIL receptor is an anti-DR5 antibody, and the agonistic antibody to the costimulatory molecule is an anti-CD40 antibody and an anti-4-1BB antibody.   The cancer treatment kit according to any one of claims 32 to 34, further comprising an antagonistic antibody against a receptor that transmits an inhibitory signal.   36. The cancer treatment kit according to claim 35, wherein the antagonistic antibody against the receptor that transmits the inhibitory signal is an anti-CTLA-4 antibody.

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