Anti-TRAIL-R antibody

Abstract

Anti-TRAIL-R1 and R2 antibodies or functional fragments thereof, having at least one property selected from the following (a) to (c) of: (a) having activity to induce apoptosis in carcinoma cells expressing TRAIL-R1 and / or TRAIL-R2; (b) not having effect on normal human cells expressing TRAIL-R1 and / or TRAIL-R2; and (c) not inducing human hepatocyte toxicity, and an anti-TRAIL-R1 and R2 antibodies or functional fragments thereof, having the following properties: having activity to induce apoptosis in carcinoma cells independently of exogenous factors and as a monomer of an antibody.

Description

[0001] This a Continuation-In-Part Application of PCT / JP02 / 04816 filed May 17, 2002, and is incorporated herein in its entirety by reference.FIELD OF THE INVENTION [0002] The present invention relates to an anti-TRAIL receptor (TRAIL-R) antibody recognizing a TRAIL receptor 1 (TRAIL-R1) or a TRAIL receptor 2 (TRAIL-R2), which are cell membrane molecules involved in apoptosis. [0003] Furthermore, the present invention relates to a prophylactic or therapeutic agent, which contains anti-TRAIL-R antibody as an active ingredient and is used against diseases caused by cells expressing TRAIL-R, and in particular relates to a therapeutic agent used against malignant tumors. [0004] Furthermore, the present invention relates to a prophylactic or therapeutic agent, which contains anti-TRAIL-R antibody having the activity to induce apoptosis as a monomer independently of exogenous factors as an active ingredient and is used against diseases caused by cells expressing TRAIL-R, and in particular ...

AI Technical Summary

Benefits of technology

[0034] A first purpose of the present invention is to provide a novel antibody or a molecule analogous thereto, which is capable of binding to human TRAIL-R1 and / or human TRAIL-R2 and induces apoptosis specifically in carcinoma cells, without inducing damage to normal human hepatocytes to which a recombinant human TRAIL protein can cause damages. The novel antibody or a molecule analogous thereto also includes an antibody or a molecule analogous thereto which is capable of inducing apoptosis independently of exogenous factors and as a monomer in carcinoma cells. A second purpose of the present invention is to provide a prophylactic or therapeutic agent comprising the above antibody or a molecule analogous thereto as an active ingredient against various malignant tumors including solid tumors that are currently difficult to treat.

[0070] Surprisingly, the novel human anti-TRAIL-R2 monoclonal antibody of the present invention has been revealed to have no side effect of inducing cytotoxicity against not only cells derived from a normal human tissue, but also normal hepatocytes for which cytotoxicity by the recombinant human TRAIL protein is a concern. We have obtained a novel anti-TRAIL-R2 monoclonal antibody. That is, we have completed the present invention by succeeding for the first time in the world in producing a novel monoclonal antibody provided with possible advantages of improved safety and therapeutic effects. The monoclonal antibody is preferably a human antibody. Its antigenicity, which is always a problem in the case of a mouse-derived antibody, has already been avoided.

[0094] Immunization can be performed once, or repeatedly (multiple times) at appropriate intervals (intervals of preferably 3 days to 1 week or intervals of 2 weeks). Subsequently, the antibody titer against the antigen in the serum of the immunized animal is measured, and the animals showing sufficiently increased antibody titers are used as a source of antibody-producing cells, so that the effect of the following steps can be enhanced. Generally, antibody-producing cells derived from animals 3 to 5 days after the final immunization are preferably used for the following cell fusion step.

[0104] Hybridomas that have been shown to produce specific antibodies by measuring antibody titer in a manner similar to that described in (2) are transferred to another plate and then subjected to cloning. Examples of the cloning method include the limiting dilution method wherein dilution is performed to cause each well of a plate to contain one hybridoma, followed by culturing; the soft agar method, wherein culturing is performed in a soft agar medium and then colonies are collected; a method wherein each cell is picked with a micromanipulator and then the cell is cultured; and the sorter clone method, wherein one cell is separated with a cell sorter. The limiting dilution method is convenient, and is often used.

[0108] Mass culture is performed by the roll-streak system using a large culture bottle, or by the spinner culture method. The supernatant in the mass culture is purified using a method known by a person skilled in the art such as gel filtration, so that anti-TRAIL-R1 and R2 monoclonal antibodies which are contained in the prophylactic or the therapeutic agent of the present invention as an active ingredient can be obtained. Furthermore, proliferation of the hybridoma intraperitoneally in, for example, mice of the same line (e.g., BALB / c) or Nu / Nu mice, rats, guinea pigs, hamsters or rabbits makes it possible to obtain ascites containing a large amount of anti-TRAIL-R1 and R2 monoclonal antibodies which are contained in the prophylactic or the therapeutic agent of the present invention as an active ingredient. As a convenient purification method, for example, a commercially available monoclonal antibody purification kit (e.g., MAbTrap GII kit; Amersham Pharmacia Biotech) can also be used.

[0135] The antibody of the present invention includes antibodies having a property of showing an LD50 value for normal cells (human hepatocytes) of, for example, 0.01 μg / ml (10 ng / ml) or more, or preferably 0.1 μg / ml or more when the LD50 value is measured under the above conditions. It can be said that the higher the LD50 value against normal cells, the higher the safety. Thus, antibodies having LD50 values of 2 to 100 μg / ml are further preferred. The antibody of the present invention includes antibodies having a property of showing the LD50 value for carcinoma cells of, for example, 100 μg / ml or less, or preferably 0.7 μg / ml or less when the LD50 value is measured under the above conditions. It can be said that lower the LD50 value against carcinoma cells, the stronger the activity to kill tumor cells. Thus, antibodies having LD50 values of 0.02 to 0.11 μg / ml are further preferred. In particular, the E-11-13 antibody, the L-30-10 antibody and the KMTR1 antibody of the present invention have properties of showing LD50 values for human hepatocytes of 2 to 100 μg / ml or more (for example, 2 to 24 μg / ml, preferably, 100 μg / ml) and LD50 values for carcinoma cells of 0.02 to 0.11 μg / ml. That is, these antibodies have both safety for normal cells and an apoptosis-inducing effect on tumor cells. Furthermore surprisingly, the antibody of the present invention significantly suppressed tumor cell proliferation in a tumor-bearing mouse model.

Problems solved by technology

It is thought that when cells which should originally be eliminated by apoptosis are not removed, this may cause cancer, lupus, herpes virus infection, hepatitis and other problems.

However, such selectivity has not yet been supported theoretically since TRAIL receptors are also expressed in normal cells.

Because agonistic anti-Fas antibodies, which induce apoptosis in hepatocytes, induce fulminant hepatitis in a very short time and thus cause death in mice and chimpanzees, cell death induction by TRAIL on hepatocytes has attracted attention as a particularly significant issue.

For example, in the case of a different antigen such as TRAIL, it has not been demonstrated whether or not agonistic antibodies having no toxicity can be obtained.

Furthermore, no research has been done based on the idea of whether hepatotoxicity can be avoided by adding TRAIL-R1 / R2 selectivity to agonistic antibodies.

A drug whose action mechanism is apoptosis induction by the recombinant human TRAIL may cause damages to normal tissues, particularly the liver and the brain, even if it is able to remove carcinoma cells.

In addition, if antibodies are those against TRAIL receptors, antibodies that may be obtained can avoid causing damage to the liver, which is unable to avoid with the recombinant human TRAIL, and have equivalent apoptosis-inducing activity against carcinoma cells.

Since a polymer antibody induces the formation of aggregates and may possibly cause adverse side effects, a polymer antibody is generally removed as much as possible.

Thus, in case where a polymer alone possesses the activity, the activity is lost by the increase of the purity of a monomer antibody.

Under these circumstances, an antibody which needs exogenous factors to exert its activity cannot always take anti-tumor effects.

However, since these antibodies have problems that include a shortening of a half life in blood, antigenicity and the like, and the usefulness as a pharmaceutical antibody may be lost, there are many problems to be solved.

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