WO2005012363A1 - Targeting inflammation inducer - Google Patents

Abstract

It is intended to provide a conjugate, a fused protein or a complex each having a tumor-specific antibody as a targeting member and an Fas ligand, its activity region or its derivative as an effector member, a tumor site-specific inflammation inducer containing the same, a remedy for tumor and so on. Thus, it is possible to provide an inflammation inducer which has a high specificity to a target site (in particular, a tumor site) with little systemic side effect and/or a high efficacy, an antitumor agent having a tumor site-specific effect of inducing inflammation and so on.

Description

 Description Targeted Inflammation Inducer Technical Field

 The present invention relates to a conjugate specific to a target site, particularly a tumor site, an inflammation-inducing agent, or an antitumor agent having a tumor site-specific inflammation-inducing effect. Background art

 Fas ligand (FasL) induces cell apoptosis by binding to its receptor, Fas! [It is a type-membrane protein and exhibits activity as a trimer or higher multimer. A portion of F as L undergoes degradation by the processing enzyme, meta-oral protease, and is converted to a soluble form.However, this soluble form of F as L has less or no apoptosis-inducing activity compared to membrane form F as L. are doing. However, a fusion protein of FasL with an aggregate or a multimerizing peptide obtained by cross-linking soluble FsL with an antibody, such as a fusion protein of FAG with FLAG tag or isoleucine zipper (as used herein) And FIZ-FasL; W001 / 90382: Patent Document 1) has a strong apoptosis-inducing activity like membrane-bound FasL, but at the same time has systemic side effects, especially Hepatotoxicity also develops. For this reason, there is a limit in systemically administering soluble aggregates of FasL (WO 97/02290: Patent Document 2) and multimers to expect an antitumor effect.

The Fa s -Fa s L system plays an important role in immune system self-tolerance and homeostasis in both humans and mice. Fa s L also functions as a cytotoxic molecule for cytotoxic T cells (CTLs). For example, the eyes and testes are known to be tissues that are immune to destructive attacks on the immune system, or immu neprivile ge dorg an. It is said that F as L is expressed in these tissues and suppresses inflammation by inducing apoptosis in inflammatory infiltrating cells. In addition, it has been pointed out that some cancer cells express FasL and may use it for immune evasion. On the other hand, ectopic expression of Fa s L in cancer cells and transplanted tissues may lead to neutrophil infiltration. It has been reported that it causes refusal and accelerates rejection. As a mechanism, it has been pointed out that FasL induces apoptosis in inflammatory cells such as neutrophils, and at the same time, induces the processing and release of IL-11i3 to enhance inflammation.

 In recent years, antibody drugs for diseases such as cancer have been developed and used clinically. These are thought to have high specificity for cancer cells, but are not always highly effective. Generally, it is necessary to administer high doses, and there are concerns about side effects. In addition, the effectiveness of the antibody also depends on the expression level of the antigen on the cell, so that the efficacy rate and the cure rate are limited.

Disclosure of the invention

 An object of the present invention is to provide an inflammation-inducing agent having high specificity at a target site, particularly a tumor site, low systemic side effects, and high Z or efficacy, or an anti-tumor agent having a tumor site-specific inflammation-inducing effect To provide an agent. Further, the present invention provides a method for inducing inflammation by specifically accumulating neutrophils and the like at a target site, and treating a tumor by accumulating neutrophils and the like specifically at a target site to induce inflammation. Methods and kits for those methods are provided.

 The present invention has been made to achieve the above object, and typical embodiments thereof will be described below.

 (1) a target cell, tissue or site, for example, a tumor-specific antigen, specifically an antibody to carcinoembryonic antigen (CEA) or an antigen-binding fragment thereof or a derivative thereof as a targeting moiety; Or a conjugate, a fusion protein or a complex containing the active region or a derivative thereof as an effector portion.

(2) a target cell, tissue or site, for example, a tumor-specific antigen, specifically an antibody to carcinoembryonic antigen (CEA) or an antigen-binding fragment thereof or a derivative thereof as a targeting moiety; Or a tumor site-specific inflammation-inducing agent comprising a conjugate, a fusion protein or a complex containing the active region or a derivative thereof as an effector moiety.

(3) a target cell, tissue or site, for example, a tumor-specific antigen, specifically an antibody to carcinoembryonic antigen (CEA) or an antigen-binding fragment thereof or a derivative thereof as a targeting moiety, Or their active regions or their An agent for treating a tumor, comprising a conjugate containing a derivative as an effector moiety, a fusion protein or a complex.

 (4) Targeting section containing an antibody against a target cell, tissue or site, for example, a tumor-specific antigen, specifically, carcinoembryonic antigen (CEA) or an antigen-binding fragment thereof or a derivative thereof (targeting Administering an effector moiety (effect agent or effector substance) containing a Fas ligand or its active region or a derivative thereof simultaneously or at appropriate time intervals. A method for inducing inflammation by accumulating neutrophils in a target site-specific manner.

 (5) Targeting section containing an antibody against a target cell, tissue or site, for example, a tumor-specific antigen, specifically, carcinoembryonic antigen (CEA) or an antigen-binding fragment thereof or a derivative thereof (targeting Agent or targeting substance) and an effector portion (effector substance or effector substance) containing Fas ligand or its active region or a derivative thereof simultaneously or at an appropriate time interval. A method for treating tumors by accumulating neutrophils in a target site-specific manner and inducing inflammation.

 (6) Targeting section containing an antibody against a target cell, tissue or site, for example, a tumor-specific antigen, specifically, carcinoembryonic antigen (CEA) or an antigen-binding fragment thereof or a derivative thereof (targeting A kit comprising an agent or targeting substance) and an effector moiety (effector agent or effector substance) containing Fas ligand or an active region thereof or a derivative thereof. Brief Description of Drawings

 FIG. 1 is a graph showing the results of measuring the number of neutrophils infiltrating into the peritoneal cavity after administering various conjugates and the like to SCID mice into which HT-29 cells were transplanted intraperitoneally. The first to third columns from the left show the results for the group not transplanted with HT-29 cells, and the fourth to tenth columns from the left show the results for the group transplanted with HT-29 cells. I have.

FIG. 2 shows the results of measuring the number of neutrophils infiltrating into the peritoneal cavity after administration of various conjugates and the like to SCID mice in which LS-180 cells were transplanted intraperitoneally. Column 1, 2, 3, and 4 are described in the saline-administered (non-transplanted) group, 3-180 transplanted (non-administered) group, shFasL (Pichia) -administered (non-transplanted) group, and Example 5, respectively. Shown are the results of the CM 001—F 9 18 (B 3) -shF as L (Pichia) complex-administered (LS-180 transplantation) group of the present invention.

 FIG. 3 is a diagram showing the results of measuring the number of neutrophils infiltrating into the peritoneal cavity after administration of various conjugates and the like to SCID mice in which LS-180 cells were transplanted intraperitoneally. Columns 1, 2, 3, 4, 5, 6, 7, and 8 are the saline-administered (non-implanted) group, LS-180 transplanted (non-administered) group, and CM001 described in Example 3 (2), respectively. -F as L (A I'd) administration (non-transplant) group, CM00 l—F as L (A 1 d) administration (LS—180 transplant) group, CM00 1— described in Example 3 (1) F as L (EDC) -administered (non-implanted) group, CM00 1—F as L (EDC) -administered (LS-180 transplant) group, anti-CEA antibody (CM00 1) -administered (non-implanted) group and anti-CEA The results of the group administered with the antibody (CM001) (transplanted with LS-180) are shown.

 FIG. 4 shows the results of measuring the number of surviving LS-180 cells in the peritoneal cavity after administration of various conjugates and the like to SCID mice in which LS-180 cells were implanted intraperitoneally. Columns 1, 2, and 3 are the LS-180 transplanted (non-administered) group, the CM001-FasL (Aid) -administered (LS-180 transplanted) group described in Example 3 (2), and Example 3 respectively. The result of the CM001-FasL (EDC) administration group (LS-180 transplant) described in (1) is shown. BEST MODE FOR CARRYING OUT THE INVENTION

 The present invention relates to a conjugate comprising a targeted portion and an effector portion, which can be specifically delivered to a target site, a target cell or tissue, in particular, a tumor site, cell or tissue, It is an inflammation-inducing agent containing a fusion protein or a complex, or an antitumor agent having a tumor site-specific inflammation-inducing effect.

 When the conjugate, fusion protein or complex is generalized, it can be represented by the following formula [I].

TLE [I] In the above formula [I], T is a targeting part, L is a linker part and may not be present, and E is an effector part (function or effect (inflammation induction, neutrophil attraction) Expression part). The bond between T, L and Ε includes a covalent bond or a non-covalent bond. In some cases, the L part may be composed of two or more parts. (In this specification, when the L part is composed of η parts, each part is described as Ll, L2, Ln Sometimes) . In addition, two or more types of T and Z or two or more types of E may be bonded to the L part. T, L and Ε are preferably derived from the animal species to be used or a molecule compatible therewith. If human, it is a molecular species having low immunogenicity in a human, more preferably a human. There are several possible combinations and types of bonds, examples of which are summarized in Table 1. Table 1 Combination of each part Generalized example Specific example Example pattern

 Τ-Ε Anti-tumor antibody-FasL fusion anti-CEA human antibody-human Fa9

 Synthetic protein s L Extracellular region fusion protein Antitumor antibody-FasL compound Anti-CEA antibody—FasL compound 1 Chemical conjugate

TLE antitumor antibody Rinka Bae flop anti-CEA antibody - (G 1 y ₄ S e

Tide—FasL fusion protein r) ₃ —Human FasL extracellular region

 Area

 Anti-tumor antibody-Insoluble carrier-Anti-CEA antibody S e ρ h a r 4

 Chemical combination of F a S L o s e— F I Z— Fa sL

Anti-tumor antibody anti-CEA antibody— (CH ₂ ) ₆ — 3 (1) Chemical conjugate of F as L FI ZF as L 3 (2)

T-L?腫 瘍 瘤 Face Lin, anti-CEA antibody—anti-human F a s 5

 Body Chemical Conjugate L Antibody

 Anti-tumor antibody-dextran Anti-CEA antibody-dextran 6-anti-FasL antibody chemistry Mono-human anti-FasL antibody

 Union

 T-L · ί ί ί 饥 饥 in F inFas Li Antibody against CEA antibody Fas5

 Complexes of chemotherapeutic conjugates and FasL antibodies and shFasL (pi-L complex Qia)

Anti-tumor antibody-Dextran Anti-CEA antibody-Dextran 6-Anti-FasL antibody chemical binding-Anti-human FasL antibody and F-conjugated and FasL complex IZ-FasL complex Anti-tumor antibody and anti-Ig antibody Anti-CE A human antibody and anti-human

 -F a s L complex I Antibody-sh F a s L (pi

 Kia) complex

 T-L 1 · anti-tumor antibody-avidin and anti-CEA antibody-avidin and 7

 L 2-E Complex of biotinylated Fas L Biotinylated F I Z—Fas L

 Complex of body

 Anti-tumor antibody monopeptide T a anti-CE A antibody—His Tag · ί / ιΤ a g 饥 -body —Fasg · anti-His T a gi body— s

 L complex hF a s L (Pichia) complex Anti-tumor antibody mono- and anti-sugar chains Anti-CE A antibody-cyaryl S S 执 —Fas L EX · C A 54/61 antibody—

 s hF a s L (Pichia)

 T · L · E Piotinylated anti-tumor antibody, a Piotinylated anti-CE A antibody, a

 Vidin and biotinylated F a Vidin and biotinylated sh F

 Complex of sL Complex of sL (Pichia)

L · E Insoluble carrier-bound anti-Fas L protein A beads · Anti-? a 8

 Antibody · FasL complex sL antibody · FIZ—FasL In Table 1 above, “one” indicates a covalent bond, and “·” indicates a non-covalent bond. Also, in the generalized examples of Table 1, antibodies include fragments, parts or derivatives of antibodies having antigenic activity, and FassL includes its active region or derivative.

The “targeting part” is a substance (target-specific binding substance) having an activity of specifically binding to a target site, a cell or a target tissue, or a target molecule present therein, or derived from such a target-specific binding substance. And has a function of specifically binding the conjugate of the present invention to a target site, cell or target tissue. Examples of the target-specific binding substance include an antibody against a cell surface antigen, a ligand for a cell surface receptor, a receptor for a cell surface ligand, a lectin-like substance for a cell surface sugar chain, and the like, and preferably an antibody. The antibody may be a fragment, a part or a derivative of the antibody as long as it has the activity, for example, the ability to bind to a target cell or the like. For example, Fab (fr agmentof antigenbinding), Fab, (Fab) ₂ , Fab—Fc ', single-chain antibody (scFv), diabody, disulfide-stabilized antibody (dsFv), CDR-containing peptides, etc. Antibodies whose ADCC activity is enhanced by modifying the sugar chain are also useful as the targeting moiety. There is no particular limitation, and the antibody may be classified into any of them, for example, an antibody whose isotype is IgG. Alternatively, it is an antibody of subclass IgG4 in terms of lack of complement fixation, and IgM in terms of high ADCC activity. When used for humans, human antibodies, humanized antibodies or antibodies derived from human genes are preferred. These can be produced according to a known method. Specific examples include antibodies against so-called tumor markers, such as the anti-CEA antibody used in the examples, and anti-tumor antibody drugs that are commercially available or under development. When these antibody drugs are used as the targeting moiety of the conjugate or the like of the present invention, it is expected that the action of the antibody drug and the effect of the effector moiety work synergistically. Preferred examples of antibody drugs include Herceptin Trast uz um ab), R it ux an (R ituximab), My 1 otarg (G e mt uz umab), C amp ath (A l emt uz umab), Z enepax (D ac 1 iz um ab), Remi cade (Av akine), Syn agis (P a 1 ibit umab) and the like, and more preferably Herceptin (Tr astuz umab, R it ux an (R ituximab) It is.

 Target sites, cells or tissues include tumor cells, infected cells such as viruses, pathogens (such as bacteria, fungi or protozoa), autoantibody-producing cells, self-recognizing lymphocytes such as self-reactive CTLs, and synovial cells. This includes cells or tissues that are harmful or unnecessary for living organisms, such as cells transferred from outside by cell therapy or cells into which genes have been introduced by gene therapy, such as tumors, Includes cancer and ascites cancer and their metastatic lesions and disseminated tissues.

The target molecule is preferably a molecule which specifically exists in the target cell or tissue or a molecule whose expression tissue is limited and whose action on the tissue is therapeutically acceptable, and more preferably a molecule which is present only in the target cell or tissue. preferable. As used herein, “specifically present in a target cell or tissue” means that its abundance is qualitatively or quantitatively higher than that of cells other than the target. For example, tumor markers such as CEA, CA 54/601, CA125 and CA19-9, lymphocyte differentiation antigens such as CD20 and CD33, and also involved in the growth, function or survival of target cells, especially tumor cells Molecules (such as HER2) are also useful as targets. Other target molecules include IL-2 receptor, IL-6 receptor, TNF-Q !, Fa s, Fa s L, etc. Can be. When the target molecule is Fa s L (when the target cells are Fa s L-expressing tumor or autoreactive CTL expressing Fas L), the F918-7-3 antibody ( W097 / 02290) or an antibody that recognizes the N-terminal region of the FasL extracellular region, such as a humanized antibody thereof, and an N-terminal deletion of the FasL extracellular region (such as nd 32) may be used in combination.

A part of the linker is a part that connects the T part and the E part, and various molecular species can be applied. In some cases, a part of the linker has a function as a tag, a spacer, or a carrier. For example, a peptide linker such as 1 (G 1 y ₄ Ser) ₃ —, a MAP linker, a His—Tag, a spacer molecule of an appropriate length, for example, an alkylene such as 1 (CH ₂ ) ₆ — Group, avidin and Z or biotin, antibodies or antigen-binding fragments or derivatives thereof, polymers such as ribosomes and dextran having a functional group introduced as necessary, polymers such as polyvinyl alcohol having an appropriate molecular weight, polylysine and Sugar chains and the like.

In some cases, the L portion may be composed of two or more moieties, including, for example, a T—L 1 covalent conjugate and an L 2—E covalent conjugate (see Table 1). In this case, at least one linker is part of an animal species to be used, for example, a molecular species having low immunogenicity in humans if human, preferably derived from humans, and usually has a concentration in blood or tissue. Is low, or has a limited expression in tissues, and its effects on the tissue are therapeutically acceptable, especially in the relatively early stages of ontogeny, for example, in the fetal period, but not or almost completely in adults. Components that are not expressed are preferred. Examples thereof include CEA, sialyl S SEA-1 antigen, sialyl S SEX, monophytoprotein, etc., which are fetal cancer antigens, and antigenic fragments thereof. Sialyl S SEA-1 antigen and Sialyl S SEX are also carbohydrate antigens. In addition, the ligand is not limited to proteinaceous molecules, but includes low-molecular-weight ligands such as avidin and biotin, and sugar chains and lectin-like substances. By using these components, side effects in the living body can be reduced, and the targeting portion and a part of the effector can be efficiently bonded. Furthermore, by increasing the amount of L1-part binding to one molecule of the T-part, the amount of L2-E part binding to the target can be increased. Such methods of administration are useful for enhancing efficacy and reducing Z or side effects. That is, Even when the number of target molecules on the target cell to which the targeting moiety binds is small and a significant effect cannot be obtained with ordinary antibody drugs, the amount of binding of the functional moiety to the target can be increased. Also, the L part may be composed of two or more types, and multiple effects are expected by simultaneously using different functions of the E part of the L2-E part that specifically binds to each L part. You can do it. Specifically, by using a combination of DNA having F as L in the E portion and TLR (T oil Lake Receptor) agonist, for example, DNA having an unmethylated CpG sequence which is TLR 9 agonist. The anti-tumor effect by inflammation induction and the induction of anti-tumor immunity by TLR stimulation can be expected.

An “effector moiety” is a moiety that expresses some biological activity at a target site bound via a targeting moiety. Preferably, it induces or promotes infiltration, migration, or accumulation of inflammatory cells, such as neutrophils, lymphocytes, macrophages, monocytes, eosinophils and basophils, especially neutrophils, at the target site It has an activity, an activity of inducing or inducing inflammation by accumulating neutrophils at a target site, or an activity of removing target cells and the like. Specific examples thereof include chemokines such as Fas L and TRAI L (Tumor necrosis factor (TNF) — related ap tosis—indueing 1 igand and IL-8, and active regions or derivatives thereof, and are preferred. Is F as L or an active region or derivative thereof. Chemokines include GCP-2, GROa, GROi3, GROa, NAP-2, ENA-78, RANTES, MI-la, MCP-1, MCP-2, MCP-3, MCP-4MP IF-1 and Lkn-1 etc. Examples of the active region or derivative of FasL include FsL extracellular region and its N-terminal amino acid deletion. (Described as nd 5, nd 12, nd 20, nd 32, nd41, nd 42 depending on the number of deleted residues) (WO 95/13293 and WO 97/02290), production of recombinant Pichia yeast Soluble human Fas L (WO 97/02290, sometimes referred to as sh Fas L (Pichia) in this specification), membrane-type Fa s L was cleaved by a processing enzyme And soluble FasL fragments (Tan aka et al., Nature Media, 2: 317-322, 1996 and WO 97/02290) and FIZ-FasL. Here, the bond between T, L and 含 む includes a covalent bond or a non-covalent bond. In the case of a covalent bond, the bond can be made chemically or genetically. The details of the coupling method are described in the examples, but those methods and methods similar thereto are exemplified. In the case of chemical conjugates, they can be linked using known chemical conjugation techniques. Generally, this linkage will be through an amine or sulfhydryl group. The connection may be a severable connection or a non-severable connection.

 Specific examples of non-cleavable linkers include carbodiimides (EDO-based, sulfhydryl-maleimide-based, and periodate-based). Reacts to activate the carboxyl group, which binds to the amino group of the second protein, resulting in a non-cleavable amide bond between the two proteins.

 In the sulfhydryl monomaleimide system, a sulfhydryl group is introduced into the amine group of one of the above proteins using a compound such as a trout reagent. Another protein is reacted with an NH S ester (such as gamma-maleimidobutyric acid NH S ester (GMBS)) to create a maleimide derivative that is reactive toward sulfhydryl groups. The two modified proteins are then reacted to form a non-cleavable covalent bond.

 Periodate coupling requires the presence of an oligosaccharide group on the part to be linked, ie, either the T, L or ま た は part. When these groups are on the protein to be delivered (for example, horseradish peroxidase (HRΡ)), the activity capable of reacting with the amino group on the carrier is on the protein to be delivered. Forming sex aldehydes. Also, an active aldehyde group can be formed from carbohydrate groups present on the antibody molecule. These groups can then be reacted with an amino group on the protein to be delivered to form a stable conjugate. Alternatively, a stable conjugate can be obtained by reacting an antibody oxidized with periodate with a hydrazine derivative of the protein to be delivered.

In addition, the sulfhydryl (SH) method can be used. Disulphide (S-S) bond to mercaptoethanol, dithiothreitol, mercaptoethanol A free SH group can be created by reduction with luminin (MEA). In the case of IgG, however, the MEA reduces the S—S bond at the hinge part and releases the free SH group via the released SH group. To pull it.

 If the bond between any of T, L and Ε is non-covalent, do not form a complex beforehand and administer them at the same time or at appropriate time intervals (ie two or more steps). Stepwise administration method) is also possible. Such an administration method is useful for enhancing the effect and reducing the adverse effects or side effects, particularly hepatotoxicity. That is, when the number of target molecules on the target cell to which the targeting moiety binds is small and no significant effect is obtained, and Ζ or when the number of binding of the effector moiety per targeting moiety or target molecule is increased. Useful for In addition, by selecting a molecular species whose activity is reconstituted by accumulating on target cells in a liquid phase, that is, in blood, the activity is low, so that not only effects but also side effects can be reduced. For example, when the number of target molecules such as antigens presented on a tumor is small, the number of F aS L conjugates bound to target cells is also limited, and sufficient effects may not be obtained. The use of conjugates with an increased number of effectors per targeting site to increase activity may also increase toxicity. In order to solve these problems, an anti-tumor antibody modified with a molecule having a binding region for a specific ligand or the like, for example, an avidin-labeled anti-CEA antibody as shown in Example 7 is first administered to the antibody on the tumor surface. After binding, Piotin-labeled FasL is administered at appropriate time intervals. The previously administered avidin-labeled antibody binds to the tumor surface, and specifically binds and accumulates the biotin-labeled FasL, which increases the activity only at the tumor site, resulting in low toxicity and high antitumor activity Is shown. In this case, by using a multivalent binding region on the antibody, for example, avidin multimer, the amount of FasL bound can be increased even when the number of molecules displayed on the tumor surface is limited. . Systemic toxicity can be avoided by using a weakly active F a s L alone.

The dosing interval is the concentration of the previously administered drug in the blood and Z or target tissue, metabolic properties such as half-life or elimination curves, systemic effector moiety alone or in complex with the targeting moiety and / or linker moiety. It can be appropriately determined in consideration of sexual side effects and the like. Preferably, select a time when the blood concentration of the previously administered drug is sufficiently low and the target tissue concentration is maintained at a level where the drug can exert its efficacy. Use such an administration method In this case, antibody fragments (such as Fab or scFv) are preferable to intact antibodies (such as IgG) from the viewpoints of metabolic rate and permeability to target tissues.

 The conjugate and the inflammation-inducing agent of the present invention are specific to a target site, specifically, a tumor tissue and the like, and have reduced side effects, particularly hepatotoxicity. Pathogens (bacteria, fungi, protozoa, etc.), autoantibody-producing cells, self-recognizing lymphocytes such as self-reactive CTLs, synovial cells, cells transferred from outside by cell therapy, etc., or genes introduced by gene therapy It can be used for the purpose of preventing and / or treating cells or tissues harmful or unnecessary to living organisms such as cells, in particular, tumors, cancers and ascites cancers, and metastases and disseminated tissues thereof. To experimentally confirm these pharmacological effects, for example, antitumor effects, transplant human tumor cells expressing the target molecule into immunodeficient mice such as nude mice or Scid mice (subcutaneous, intradermal, intraperitoneal) Later or in the renal capsule) at the appropriate time, and then administer the drug once or multiple times, and confirm by determining the number of tumor cells, the weight or size of the formed tumor, or the survival time . In addition, transplantation of mouse tumor cells expressing the target molecule into mice with normal immunity such as B a1 b / c mice and C57 / BL6 mice

(Subcutaneous, intradermal, intraperitoneal, or under the renal capsule, etc.), then administer the drug at an appropriate time, and determine the number of tumor cells, the weight or size of the formed tumor, or the survival time. The pharmacological effect can be confirmed. In this case, the same tumor is transplanted again into a mouse whose tumor has remitted by administration of the conjugate or the like of the present invention, and the number of tumor cells, the weight or size of the formed tumor, or the survival period is determined. It can be confirmed that antitumor immunity is induced by the conjugate of the present invention. Furthermore, the same cells as the transplanted tumor cells are previously killed by irradiation or the like, mixed with the conjugate of the present invention, and administered subcutaneously to the tumor-engrafted mouse. It can be confirmed that anti-tumor immunity is induced by using the weight or size of the tumor mass or the survival time as an index. According to this method, a therapeutic effect can be obtained by combining the conjugate of the present invention with a tumor removed by surgery or the like or a cell used for transfer in cell therapy, even if the conjugate of the present invention does not directly reach the tumor site. It is confirmed that. The inflammation-inducing agent and the like of the present invention are usually formulated together with pharmaceutically acceptable additives and the like. These additives include excipients, bulking agents, binders, Agents, disintegrants, lubricants, surfactants, dispersants, buffers, preservatives, solubilizers, preservatives, flavoring agents, soothing agents, stabilizers, isotonic agents, etc. You. The administration route can be oral or parenteral, but since it is a peptide, it is preferably porous or continuous IV, intramuscular, subcutaneous, transmucosal (nasal or rectal) or targeted It is administered parenterally, such as by local injection into tissues. The dose of the inflammation-inducing agent and the like of the present invention is appropriately determined in consideration of the disease, disease state, patient condition, and the like.For example, it is preferably 0.001 to 1000 mg / body per 1 kg of body weight at a time. Can be selected from the range of 0.01 to 100 mg / body, and is administered once or multiple times a day.

 The present invention also relates to a targeting moiety comprising an antibody against a target cell, tissue or site, for example, a tumor-specific antigen, specifically carcinoembryonic antigen (CEA) or an antigen-binding fragment or derivative thereof. And a kit comprising an effector portion (effector agent or effector substance) containing a Fas ligand or an active region or derivative thereof. The components of the kit of the present invention are the above-mentioned conjugates or the like or at least one or more of the respective components, an inflammation-inducing agent, or an antitumor agent.

 In the kit of the present invention, when there is a non-covalent bond in the bond between the T portion, the L portion and the E portion (for example, T—L • Ε, T′L-E, T-L described in Table 1) 1 · L 2 -T or T · L · Ε) is preferably at least one of covalently bonded moieties (eg, Τ—L, L_E, T-L1, L2—T) , More preferably all. In a preferred embodiment, each part, for example, in the case of <1 L>, <1 L and <1> is administered in a dosage form (for injection, purified water for injection or physiological saline to which an appropriate stabilizer is added if necessary). It can be contained in a separate container in the form of a solution previously dissolved in a solution such as saline) or in a storage form (in the case of an injection, a lyophilized formulation). Example

Hereinafter, the present invention will be described more specifically with reference to examples, but these are shown as examples, and the present invention is not limited thereto. Abbreviations used in the following description are based on common abbreviations in the relevant field. The Example 1: Preparation of anti-CEA antibody-FasL chemical conjugate (direct)

 Mouse anti-CEA monoclonal antibody (CM001) (Hatada et al., New Medicine 38, 401-404, 1983, and Tsutsumi et al., Am. J. Clin. Pat. 82, 535-542, 1984) 2.5 mg was diluted with 0.1 M phosphate buffer (pH 7.2) containing 0.15 M NaCl to 5 mg / ml. Subsequently, 5 mg of sodium metaperiodate (Nacalai) was added and oxidized at 25 ° C for 30 minutes to aldehyde-convert the sugar chains of the antibody. Subsequently, 0.2 M carbonate buffer (pH 9.5) was added to activate the aldehyde group, and immediately replaced with 0.1 M phosphate buffer (pH 8.0) containing 0.8 M NaCl. Equimolar amounts of FIZ-FasL were added and reacted overnight at room temperature. The next day, add sodium borohydride dissolved at 4 mg / ml, leave at 4 ° C for 2 hours, replace with physiological saline, and chemically bind anti-CEA antibody-FasL with no linker The body was prepared. Example 2: Preparation of anti-CEA antibody-FIZ-FasL chemical conjugate using heterobifunctional crosslinker

 (1) F I Z-F a s L and s hF a s L (Pichia)

 FIZ-F. As L was used in accordance with the example of WO 01/90382, and shFasL (Pichia) and a mouse anti-FasL monoclonal antibody (F918—7—3) were used in the example of WO 97/02290. Prepared according to

(2) Dissolve 5 mg of mouse anti-CEA monoclonal antibody (CM00 1) in 0.1 M phosphate buffer (pH 6.0) containing 5 mM EDTA, and adjust the final concentration to 50 mM with 2-mercaptoet hylam. ine—HCl was added and reacted at 37 ° C. for 90 minutes. After cooling on ice, the mixture was replaced with a 2 OmM phosphate buffer containing 0.15 M NaCl and ImM EDTA to prepare Fab-Fc '. Next, FIZ-FasL lmg was replaced with 0.1 M phosphate buffer (PH 7.2) containing 0.8 M NaC1 to prepare 2 mg / ml. There 0.25mg of Su lfo— MB S (PI ERCE) was added and reacted at 37 for 30 minutes to bind the linker. Excess linker was removed with a NAP column (Amersham Bioscience) and mixed with FabFc 'in a 1: 1 (molar ratio). The reaction was allowed to proceed overnight at 4 ° C, and replaced with physiological saline to prepare an anti-CEA-FIZFasL chemical conjugate.

(3) 5 mg of mouse anti-CEA monoclonal antibody (CM001) was dissolved in 0.1 M Na acetate buffer (pH 4.2) to adjust to 5 mg / ml. Pepsin (Sigma) adjusted to 5 mg / nil was added as an antibody: pepsin (weight ratio: 33: 1) and allowed to stand at 37 ° C. for 16 hours. After the pH was raised to 8 with 0.5 N NaOH to stop the reaction, the mixture was dialyzed against 5 mM Tris-HCl (pH 8). Next, gradient elution of Tris-HCl (pH 8) of 5 to 50 OmM was performed using a DEAE-ceululose column (Whatman) to obtain Fb,) 2. After substituting with 0.1 M phosphate buffer (pH 6.0) containing ImM EDTA, add 2-mercaptoetylamine-HC1 to a final concentration of 50 mM, and in 37 Reacted for 90 minutes. After cooling on ice, Fab 'was prepared by substituting with 2 OmM phosphate buffer containing 0.15 M NaCl and ImM EDTA. Next, FIZ-FasL and Fab, activated in (2), are mixed at 1: 1 (molar ratio), reacted at 4 ° C overnight, replaced with physiological saline, and anti-CE A-FIZFas The L chemical conjugate was prepared. Example 3: Preparation of anti-CEA antibody-FasL chemical conjugate using diaminolinker

M

 (1) CM001—F a s L (EDO

Dissolve 0.25 mg of FI ZF as L in 0.1 M MES buffer (pH 5.5) containing 0.8 M NaCl, and then add 10-fold molar amount of diaminohexane (Wako Pure Chemical) or p-Fenium Rangeiam (Wako Pure Chemical) was added. Then, 6.251 was added with 0.1 mg / ml of water-soluble rupoimide (EDC, Dojindo Chemical) dissolved in 0.1 M MES buffer (pH 5.5), and reacted at room temperature for 2 hours. The reaction solution was replaced with a phosphate buffer (pH 8.0) containing 0.8 M NaCl using a NAP column, and the aldehyde-modified CM001 antibody prepared according to Example 1 was used at a ratio of 1: 1 (molar). (Ratio). The reaction solution obtained was replaced with physiological saline, and FIZ—Fas L—

(CH ₂ ) ₆ _CM001 antibody chemical conjugate (CM001—Fas L (sometimes referred to as EDO) was prepared. (2) CM001 ~ F as L (Aid)

Activated by adding 50 1 of 0.2M carbonate buffer (pH 9.5) to 2.5 mg of the aldehyde-modified CM001 antibody prepared according to Example 1, and 20-fold molar diaminohexane (Wako Pure Chemical Industries) or p-Phenylenediamine (Wako Pure Chemical Industries) was added and reacted at 25 for 2 hours. Next, 4 mgZm 1 of sodium borohydride was added, and the mixture was reacted at 4 for 2 hours. After replacing the buffer with 0.1 M MES (pH 5.5), FIZ-FasL dissolved in 0.1 M ^^ 3 buffer (<<: 5.5) containing 0.8 M NaCl and 1: 1 ( (Molar ratio), and added 0.1 mg Zm 1 of a water-soluble compound, lupoimide (EDC, Dojindo Chemical) 6.251, and reacted at room temperature for 2 hours. The obtained reaction solution was replaced with physiological saline to prepare a CM001 antibody— (CH ₂ ) ₆ —FIZ—FasL chemical conjugate (sometimes described as CM001—FasL (Aid)). . Example 4: Preparation of anti-CEA antibody—Sepharo se—FIZ—Fas L conjugate (CM001—Sepha rose—FIZ—Fas L)

 (1) Preparation of ligand solution

FIZ—FasL (2.83 mg / mL) 850 / zL was applied to a PD10 column (Amersham am Biosci ence), and 1 MNaCl 1 aqueous solution (pH 4.5) was used as a developing solution. Fractionation was performed and fraction 4 was used as a FIZ-FasL ligand solution. On the other hand, after diluting 76 ml of anti-CEA antibody (35.5 5 mg / mL) with 850 L of 11 ^ NaCl aqueous solution (pH 4.5), the solvent was replaced with a PD 10 column, and fraction 4 of the anti-CEA antibody Used as a standard solution. The absorbance at 280 nm of each ligand solution and each sample applied to the PD10 column was measured to determine the protein concentration of each ligand solution. As a result, the protein concentration of both ligand solutions was 2 mgZmL. (2) Coupling reaction

 10 mL of EAH—Sepharose4B (AmershamBioscinecce) was washed with 2000 mL of 1 M NaCl aqueous solution (pH 4.5). Thereafter, EAH-Sepharose4B was suspended in the same solution, and each 0.1 mL of the gel was dispensed into five tubes. Next, the FIZ-FasL ligand solution is diluted with a 1 M NaC1 aqueous solution (pH 4.5) to obtain final concentrations of 0, 0.1, 0.25, 0.4 and 0.5 mgZmL. And added 1.4 mL to each tube. Furthermore, 1-Ethyl—3— (3-dimime thyl aminop ropyl) —carb odi imide HC 1 (dojinkagaku) was added to a final concentration of 10 mgZmL. Coupling was performed for 3 hours while inverting and stirring. After the completion of the coupling reaction, each gel suspension was centrifuged at 1 000 g for 2 minutes to recover the supernatant. Next, the anti-CEA antibody ligand solution was diluted with a 1 M aqueous solution of NaCl (pH 4.5) to obtain final concentrations of 1, 0.4, 0.25, 0.1, and 0 mgZmL. — 1.4 mL was added to the coupled gel suspension with FasL. Furthermore, 1-E t hy 1- 3— (3-d ime t hy l am i nopro py l) carbodiimide HC 1 (Dojin Chemical Lot GP 169) was adjusted to a final concentration of 10 mg / mL. The mixture was added, and coupling was performed at 4 ° C., under agitation and overturning. After the completion of the coupling reaction, each gel suspension is packed in a column, and the anti-CEA antibody ligand solution is recovered. After washing with 50 mL of physiological saline (Otsuka Pharmaceutical Co., Ltd.), the anti-CEA antibody—Sepharos e_F IZ—Fas L conjugate. The EAH-Sepharose4B carrier obtained by performing the above operation without adding the FlZ-FassL ligand solution and the anti-CEA antibody ligand solution was used as a control carrier. Example 5: Preparation of anti-CEA antibody-anti-FasL antibody chemical conjugate using homobivalent NHS ester cross-linking agent (CM00 1-F 918 (B 3))

1 mg of mouse anti-CEA monoclonal antibody (CM001) and 2 mg of mouse anti-FasL monoclonal antibody (F91 8—7—3, WO 97/02290) It was dissolved in a 0.1 M phosphate buffer (pH 7.2) containing 15 M NaCl. Subsequently, a homobivalent NHS ester cross-linking agent BS ³ [Bis (sulfos uccinimidy 1) ssuberate] (PI ERCE) was added thereto in an amount equivalent to 10-fold the molar amount of the antibody, and reacted at room temperature for 30 minutes. 'After completion of the reaction, add 2M Tris' HCl (pH 7.4) to a final concentration of 5 OmM, and buffer the resulting anti-CEA antibody-anti-Fas L antibody conjugate buffer with a NAP column. (Amersham Biosciences) and replaced with saline. The protein concentration was calculated from the absorbance at 280 nm using the extinction coefficient (1 mgZml). The conjugates of this Example and Example 6 were used after reacting with FEZFasL for several tens minutes to form a complex in Examples 13 and 14 described later. The conjugates of Examples 5 to 7 were allowed to react in advance with FEZFasL or the like for a certain period of time to form a complex with FIA-FasL or the like, or simultaneously or separately without forming a complex beforehand. It can also be administered to form a complex at the target site. Example 6: Preparation of an anti-CEA antibody-dextran-anti-human Fas L antibody chemical conjugate (CM001-F918 (Dex))

1.5 mg of mouse anti-CEA monoclonal antibody (CM001) lmg and mouse anti-FasL monoclonal antibody (F918-7-3) 0.15M NaCl in 0.1 M phosphate buffer (pH 7.2 ) Dissolved in lml. Subsequently, both antibodies were mixed with dextran (deXtran) according to the manual of Aldehyde activateddextran coupling kit (PI ERCE) dissolved in 1 ml, and allowed to react at room temperature overnight. Then, VI VAS PIN The solution was concentrated using (VI VA SCIENCE), and the solvent of the obtained anti-CEA antibody-anti-FasL antibody chemical conjugate was replaced with physiological saline using a NAP column (Amersham Bioscience). The protein concentration was calculated from the absorbance at 280 nm using the extinction coefficient (lmg / ml). Example 7: Preparation of anti-CEA antibody 'FasL complex using avidin-biotin binding— (CM001 · FasL_ (Bio)) 2.5 mg of mouse anti-CEA monoclonal antibody (CMO 01) was diluted with 0.1 M phosphate buffer (pH 7.2) containing 0.15 M NaCl to be 5 mg / ml. Subsequently, 5 mg of sodium metaperiodate (Nacalai) was added and oxidized at 25 ° C for 30 minutes to aldehyde convert the sugar chain of the antibody. Subsequently, 0.2 M carbonate buffer (pH 9.5) was added to activate the aldehyde group, and immediately avidin (PI ERCE) dissolved in 0.83 mg of 0.01 M carbonate buffer (pH 9.5) was added. And reacted at 25 for 2 hours. Next, sodium borohydride dissolved in 4 mgZml was added, the mixture was allowed to stand at 4 ° C for 2 hours, and the buffer was replaced with 0.1 M phosphate buffer (ρΗ7.2) containing 0.15 M NaC1 . Next, a biotinylated Fas L was prepared. That is, 0.25 mg of FIZ_FasL was dissolved in 0.8 M NaCl-containing phosphate buffer (PH 8.0), and then 28 g of D—Biotinyl—ε—dissolved in DMF. Aminocaproic acid N-hydroxy succin imi de ester (Roche) was added and reacted at room temperature for 30 minutes. After completion of the reaction, unbound biotin was removed using a NAP column (Amersham Bioscience) to prepare purified biotinylated FasL. Next, in order to prepare an anti-CEA antibody-FasL complex using an avidin-biotin bond, the avidinated CM001 antibody and the biotinylated FasL were mixed at a ratio of 1: 4 (molar ratio), and the mixture was stirred at room temperature. After the reaction for 30 minutes, the buffer was replaced with physiological saline to prepare an avidinated anti-CEA antibody and a POTOTINIDORI FIZ-FasL complex. Example 8: Preparation of FasL complex using protein A-conjugated beads

As a complex having no target cell binding region, a FasL conjugate using Protein A beads (PRO SEP—rA High Capacity, Millipore) was prepared. 150 g of F918-7_3 antibody and 50 g of FIZ-FasL were mixed and incubated at room temperature for 30 minutes to form a complex of antibody and FasL. This and 100? 1 ^ 03 £? — Eight High Capacity beads are suspended in 1 mL of physiological saline, incubated at room temperature for 30 minutes, and then RPMI 1640 medium containing 10% fetal serum (Sigma). After washing three times, protein A beads FI ZF as L complex was prepared. As a control, beads to which only FIZ-FasL was not added were also prepared. Example 9: Preparation of anti-CE A antibody-Fa s L fusion protein

 F as L and the target site binding domain are encoded on one gene and prepared as one polypeptide. Specifically, a primer is designed and synthesized using the nucleotide sequence of a known anti-CE A human antibody (for example, GenB Ank accession numbers AB 107216 and AB 107217), and the primer is designed at the 3 ′ end of the nucleotide sequence of the heavy chain. The 5'-end of the gene encoding the extracellular domain of FasL or FIZFasL, which is a highly active form of FasL, is linked so as to encode a continuous peptide. An expression plasmid is prepared by incorporating this gene into a PEF-BOS (described in JP-A-2-242687) plasmid. A light chain expression plasmid is similarly constructed. The two expression plasmids are simultaneously introduced into COS-1 cells, and the protein is expressed and secreted in the supernatant. That is, 1 g of the plasmid is dissolved in 2 1 OmM Tris-HC1 (pH 7.4) / ImM ethylenediaminetetraacetic acid solution. 0.7 mL of D-MEM (Nissui Pharmaceutical Co., Ltd.) containing 0.2 mg / mL DEAE-dextran and 5 OmM Tris-HC1 (pH 7.4) was added to each of these, Make a DNA-DEAE dextran mixture. A DNA-DEAE dextran mixture is added dropwise to COS-1 cells cultured in monolayer until semi-confluent in a 6-well plate, and cultured at 37 ° C in a C02 incubator. After 4 hours, remove the DNA-DEAE dextran mixture, replace with D-MEM containing 10% FBS (Gibco), and incubate for another 96 hours. The culture supernatant of the COS-1 cells into which the plasmid has been introduced is collected and used in the following Examples. (2) Purification of fusion protein

Using the C〇S-1 cell culture supernatant containing the anti-CEA antibody-FasL fusion prepared by the method of (1), the anti-Fas ligand antibody F919-9-118 (International Publication No. WO097 / 02290) is purified as follows by affinity chromatography using Sepharose 4B carrier immobilized. That is, and The COS-1 cell culture supernatant containing the anti-CEA antibody and Fas L is passed through a 0.45 m pore size filter (Millipack 60: Millipore), and the filtrate is recovered. I do. Purified on a F919-9-118-Sepharose 4 BFF column (φ103.2 cmX6.2 cm) pre-equilibrated with PBS (phosphate— bufferedsa 1 ine) in a cold place At a flow rate of 1 OmLZ min. After applying the starting materials, flow PBS at 15.3 mL / min through the column (Wash 1), and then wash the column with 1 mo 1 / L NaC 1 / PBS under the same conditions (Wash 2). . Subsequently, 5 Ommo 1 / Lg lycine-NaOH (pH 11) is passed through the column at 1 OmL / min to perform the elution operation. Immediately add 10 mL of lmol / LTris-HC1 (pH 8) per 4 OmL of each eluted fraction and store in a cool place. Example 10: Analysis of conjugate etc.

 (1) SDS—PAGE

 SDS-PAGE was performed on the prepared Fa s L conjugate under non-reducing conditions, and silver staining was performed. The band of FasL that did not bind became thinner, and the band of the anti-CEA antibody moved to the polymer side due to the binding of FasL.

(2) Enzyme immunoassay (EIA)

The following EIA method was used to confirm the presence of the CEA binding site and the FasL binding site or FasL molecule in the same molecule in order to confirm the properties of the prepared FasL-conjugate and the like. The conjugate of the anti-CEA antibody and the anti-FasL antibody F918-17-3 in Examples 5 and 6 was detected by the following method. First, 2.58 // 1111 ^ of Oriental yeast was dissolved in PBS (pH 7.2) and added to a 96-well plate at 501 ^/ we11. After incubating for 1 hour at 37, the plate was washed 5 times with ion-exchanged water, and then 100 zl / well 1 of PBS (pH 7.2) containing 2% stabi1 iGuard (SuRModics) was added. Blocked. Next, add 25 1 each of the conjugate solution described in Examples 5 and 6 and PBS (pH 7.2) containing 10 ig / mL FIZ-FasL to the plate, and incubate at 37 ° C for 1 hour. did. After washing three times with 0.9% NaC1 containing 0.05% Tween 20, containing 0.5% g / m1 peroxidase-labeled anti-Fas containing 10% rat serum and 1% mouse serum. L antibody F919-9-18 (described in International Publication No. WO 97/02290) / PBS (pH 7.2) was added at 50 1 / we 11 and incubated at 37 ° C for 1 hour. After washing Likewise, the addition of TMB (B i oFX), the reaction with 0. 5M H ₂ S_〇 ₄ stops, the measured absorbance at 450 nm with a plate absorption meter (E- Max), a concentration-dependent Absorbance increased, and the formation of a complex was confirmed.

The conjugate of the anti-CEA antibody of Examples 1, 2, 3, and 7 and FasL was detected by the following method. Anti-mouse Igs antibody (DAKO) was dissolved in PBS (pH 7.2) at 10 g / ml and immobilized on a 96-well plate at 37 ° C for 1 hour. After washing five times with ion-exchanged water, 1001 Zwe 11 containing PBS (pH 7.2) containing 2% stabii iGuard was added for blocking. Next, 50 lZwe 11 of the conjugate solution described in Examples 1, 2, 3 and 7 was added to the plate, and reacted at 37 ° C for 1 hour. After washing 3 times with 0.9% NaC 1 containing 0.05% Tween 20, 0.5 gZm1 of peroxidase-labeled anti-FasL antibody F 9 19-containing 10% rat serum and 1% mouse serum 9/18 / PBS (pH 7.2) was added at 50 1 / we 11 and incubated at 37 ° C for 1 hour. After washing Likewise, the addition of TMB (B i oFX), where the reaction with 0. 5M H ₂ S_〇 ₄ was stopped and measured 4 50 nm of absorbance at plate Photometer (E- Max), a concentration-dependent Absorbance gradually increased, and formation of a complex was confirmed. No increase in absorbance was observed with the anti-CEA antibody used alone as the control.

(3) Flow cytometry (FACS)

FI ZF as L—Anti-CEA antibody-Sepharose 4B conjugate is bound to a fluorescently labeled antibody human FasL antibody and anti-mouse IgG antibody and analyzed by Cellcytometer FC500 (Beckman Coulter). . Sepharose 4B alone does not have fluorescence, but Sepharose 4B to which FasL and anti-CEA antibody are bound has two types of fluorescence detected on the same particle. It is confirmed that it is labeled with. Example 11: Invitro apoptosis-inducing action

Apoptosis-inducing activities of various Fas ligands were examined by luciferase assay shown below. First, pAc t—Luc, which incorporates a luciferase gene into an expression vector, pAc t—C (Hisashi H., et. A1, 1990, Cell, 63, p 303-312) using the actin promoter. The plasmid was introduced into Jurkat cells, a human T cell-derived cell line, and JA11 cells, which express constitutively expressing luciferase, were established. The JA11 cells were suspended in RPMI 1640 medium (Sigma) containing 10% FBS at a concentration of 1.25 × 10 ⁶ cells / mL, and the suspension was placed in a 96-well plate with 80 LZwe 1 1 (1 × 10 ⁵ cells Zwell). Next, the Fas ligand extracellular domain was diluted to a concentration to be assayed in RPMI 1640 medium containing 10% FBS. Further, in order to polymerize FasL, anti-FasL antibody F918-7-3 was added in an amount of 3 times and 10 times the amount of FsL, whereby the FasLs were cross-linked to each other and added. After about 20 hours cultured in C0 ₂ incubator of the above F a solution containing the as L was added to 20 L / we 1 1 that it crowded the previous cell planted example so Ueru 37 ° C, the apoptosis-inducing activity It was measured. 10 luciferase activity measurement reagents (Picker Gene LT 1.5, Toyo Ink Co., Ltd.)! ^ / 61 1 was added, left at room temperature for 30 minutes, and then measured for luminescence. The apoptosis-inducing activity was determined by subtracting the value of the gel in which no cells were implanted as a background, and setting the value of the gel similarly measured in a medium containing no Fas ligand protein as 100%.

Cell viability (%) = (luminance of assay cells—luminance of cells without cells) X 100 (luminance of cells without FsL conjugate / absorbance of cells without cells and absorbance of cells without cells) FI of positive target ZF as L showed strong apoptosis-inducing activity on JAl1 cells. However, shFasL (Pichia) showed almost no apoptosis-inducing activity by itself, but shFasL (Pichia) also showed no apoptosis-inducing activity. Cross-linking with the anti-FasL antibody F918-73 provided a scaffold, and polymerization enhanced the apoptosis-inducing activity. Example 12: In Vivo neutrophil migration activity

 (Intraperitoneal transplant)

 A mixture of various Fas ligand conjugates prepared in Examples 3, 4, 5, 6 and 7 and HT-29 (low-expressing strain) or LS-180 cells (high-expressing strain), which are CEA-expressing tumor cells, Alternatively, SCID mice (6 weeks old, 6 weeks old, or the like) were administered at a dose of 500 L / mouse of a saline suspension of the Fas ligand conjugate alone or the CEA-expressing tumor cells alone in Examples 3, 4, 5, 6, and 7. Was injected into the abdominal cavity of Japanese charles liver. Eighteen hours after the transplantation, the intraperitoneal exudate cells were obtained by washing the intraperitoneal cavity with PBS supplemented with 0.1% BSA.

(Analysis of infiltrating cells in the abdominal cavity)

The intraperitoneal exudate cells were washed with PBS containing 0.1% BSA, centrifuged (1500 rpm, 5 minutes), suspended in 1 mL of PBS containing 0.1% BSA, and counted. In addition, the number of neutrophils in the exudate cells was counted as Ly-6G (Gr-1) -positive cells using a cell cytometer try. That is, ⁵ × 10 ⁵ intraperitoneal infiltrating cells were suspended in 50 L of PBS supplemented with 0.1% BSA, and 1 g of FITC-labeled anti-mouse Ly-6G (Gr-1) antibody (Japan (Kutton Dickin Son) The reaction was carried out on ice for 60 minutes. 0.1 Wash twice with 2 mL of PBS containing 1% BSA. • Centrifuge (1,500 rpm, 5 minutes, 4 ° C), suspend in 1 mL of washing buffer, and use Cell Cylometer FC 500 The analysis was performed using (Beckman's Coulter) (Fig .:! ~ 3). Compared with saline-treated animals, the HT-29 or LS-180 transplanted CEA-expressing tumors showed a slight infiltration of neutrophils into the peritoneal cavity. In addition, administration of FasL or the FasL conjugate or the complex alone showed little infiltration of neutrophils intraperitoneally. On the other hand, various FasL conjugates were administered together with this tumor. The animals had marked neutrophil infiltration into the peritoneal cavity. It was shown that the FasL conjugate accumulated on the tumor surface induced neutrophil migration. In addition, even when FasL was bound to the mouse Tin A beads instead of the tumor cells, neutrophil migration could be significantly induced as compared to the beads alone. Example 13: In Vivo antitumor activity

 (1) Investigation of the inhibitory effect of transplanted cells on survival

The tumor cell engraftment inhibitory effect was evaluated by implanting tumor cells that had been fluorescently labeled in advance. LS—180 cells, 6 10 ⁶ ? 83, Suspend in 10 mL and treat for 7 minutes in the presence of 0.1 mo1 / L CFSE (Dojindo). After that, the staining was stopped by adding 1 OmL of FBS, washed 3 times with 2 mL of PBS containing 0.1% BSA, and centrifuged (1,500 rpm, 5 minutes) to obtain CFSE-stained tumor cells. Vesicles were obtained. When this reagent is taken into cells, it is cleaved by intracellular esterase and emits fluorescence similar to that of FITC. At the same time, the amino groups meet and covalently bind to intracellular proteins, thereby fluorescently labeling the cells. According to the method of Example 12 (intraperitoneal transplantation), 1.5 × 10 ^{6 of the} labeled cells were transplanted alone or simultaneously with the Fas ligand conjugate into the peritoneal cavity of SCID mice (Nippon Charls River). Eighteen hours after the transplantation, the exuded cells in the abdominal cavity were collected, and the number of surviving labeled tumor cells was counted using a cell cytometer. That is, peritoneal exudate cells were collected according to the method of Example 12, and the total number of peritoneal exudate cells was counted. Further, ⁵ × 10 ⁵ recovered cells were suspended in 0.1 mL of PBS containing 0.1% of BSA, and the resulting cells were suspended in Plopi idium Iodide (hereinafter abbreviated as PI, Institute of Medical Biology). Treated on ice for 30 minutes in the presence. Dead cells were fluorescently labeled with PI. After washing the cells twice with 2 mL of PBS containing 0.1% BSA, analyze the abundance ratio of CFSE-positive PI-negative cells and CFSE-labeled cells with a flow cytometer Cytomics FC 500 (Beckman Cole Yuichi) did. The results were multiplied by the total peritoneal exudate cell count to determine the number of viable tumor cells (Figure 4).

In the group to which only LS180, a tumor cell labeled with CFSE, was transplanted, 6.2 × 10 ⁵ live tumor cells were observed, but the Fas L-binding substance LS180 + CM001 / Fa The groups treated with sL (Aid) or LS180 + CM001 / FasL (EDC) showed a significant decrease in the number of viable tumor cells. This revealed that the administration of the Fas ligand conjugate exerts an antitumor effect. (2) Study of side effects ''

 Before collecting the infiltrating cells in the abdominal cavity in Example 13 (1), blood was collected from the mouse orbit and serum was separated by centrifugation at 5,000 rpm for 10 minutes at room temperature. The concentration of GPT in the serum was examined using Drychem 5000 (Fuji Photo Film). No increase in serum GPT was observed upon administration of any of the Fas L conjugates or complexes. Although F as L is feared for systemic toxicity, particularly hepatotoxicity, it has been found that the F as L conjugate or complex of the present invention exhibits an antitumor effect without causing systemic toxicity. . Example 14: Study on effect of prolonging survival time of tumor-transplanted mice

 Tumor cells (HT-29 and LS-180) having target antigens such as CEA are transplanted (subcutaneously or intraperitoneally) into SC ID mice. Immediately after or several days after transplantation, the conjugate used in Examples 12 and 13 is administered once or multiple times, and the size of the tumor mass, the number of tumor cells, or the viability is determined over time. In the mouse to which the conjugate or the like of the present invention has been administered, the tumor size or the number of engrafted tumor cells decreases, and the survival time of the individual increases. Example 15: Two-stage administration method

Tumors are implanted into mice in the same manner as in Examples 13 and 14. Tumor-transplanted mice were treated with CM001—F918 (B3) and shFasL (Pichia) of Example 5, CM001—F918 (Dex) and shFa sL (Pichia) or fruit of Example 6. Two-stage administration was carried out using three combinations of avidinated CM 001 and biotinylated FIZFasL of Example 7, and evaluation was performed in the same manner as in Examples 13 and 14. CM001-F918 (B3) of Example 5 of targeting part, CM001—F918 (D eX) of Example 6 and avidinated CM001 of Example 7 and s as effector part The administration intervals of hFasL (Pichia) and the biotinylated FasL of Example 7 should be 1 hour, 6 hours, 24 hours or 7 days. In the mouse to which the conjugate or the like of the present invention has been administered, the tumor size or the number of engrafted tumor cells decreases, and the survival period of the individual increases. The incidence of side effects such as hepatotoxicity is low. Industrial applicability

 The conjugate or the like of the present invention accumulates at the target site depending on the target specificity of the targeting portion, and the activity of the effector portion is expressed or reconstituted in a target site-specific manner. Therefore, a safe drug with high efficacy and reduced systemic side effects is provided.

Claims

The scope of the claims 1. A conjugate, fusion protein or complex containing a tumor-specific antibody as a targeting moiety and a Fas ligand or an active region or derivative thereof as an effector moiety.  2. A tumor site-specific inflammation-inducing agent comprising a tumor-specific antibody as a targeting moiety and a conjugate, fusion protein or complex containing a Fas ligand or an active region or derivative thereof as an effector moiety.  3. A tumor therapeutic agent comprising a tumor-specific antibody as a targeting moiety and a conjugate, fusion protein or complex containing a Fas ligand or an active region or derivative thereof as an effector moiety.