An antibody or a functional fragment thereof, acting agonistically or antagonistically on CD40.

SUMMARY OF THE INVENTION

The functions of DC have been increasingly analyzed recently, so that CD40 has begun to be recognized as a gene important in controlling the functions of DC. Starting from this background, the purpose of the present invention is to provide by employing an evaluation system using DC, an anti-human CD40 antibody or a functional fragment thereof, which is substantially antagonistic also to a human CD40 antigen on the dendritic cell (DC) surface, and an agonistic anti-human CD40 antibody or a functional fragment thereof that is expected to have a therapeutic effect higher than that of the conventional anti-human CD40 antibody.

As a result of intensive studies concerning the preparation of antibodies against human CD40, we have completed the present invention by succeeding in producing a novel agonistic antibody and antagonistic antibody that are thought to have a therapeutic effect against disease higher than that of the conventionally known anti-CD40 antibody. That is, the present invention is as follows.

(1) An antibody against a human CD40, or a functional fragment thereof, having at least one property selected from the following properties (a) to (f) of:

(a) acting on dendritic cells to produce IL-12 in the presence of LPS and IFN.gamma.; (b) having activity to act on dendritic cells causing the cells to mature, which is higher than that of a G28-5 antibody; (c) having activity to promote an established B cell line to express CD95, which is higher than that of the G28-5 antibody; (d) having activity to suppress the proliferation of an established B cell line, which is higher than that of the G28-5 antibody; (e) inducing cell death of an established B cell line; and (f) not inhibiting the binding of CD40 ligands to CD40. (2) The above antibody or the functional fragment thereof of the present invention, wherein the maturation of dendritic cells is performed at a concentration of 20 .mu.g/ml or less. In addition, the antibody or the functional fragment thereof promote the established B cell line to express CD95 at the antibody concentration of 20 .mu.g/ml or less. Examples of the established B cell line include Ramos, HS-Sulton or the like. (3) Furthermore, the above antibody or the functional fragment thereof of the present invention leads to the production of 100 pg/ml or more IL-12 when the antibodies with a concentration of 0.1 .mu.g/ml or more are added to dendritic cells with a concentration of 1.times.10.sup.6 cells/ml, and the production of 1000 pg/ml or more, preferably 10000 pg/ml or more IL-12 when the antibodies with a concentration of 1 .mu.g/ml or more are added. (4) Furthermore, within the antibody concentration range between 0.01 .mu.g/ml and 10 .mu.g/ml, the above antibody or the functional fragment thereof of the present invention, promoting the established B cell line (Ramos cell) to express CD95 with approximately 2 to 3 times or more greater effectiveness than that expressed by a G28-5 antibody as a control. For example, with an antibody concentration of 0.01 .mu.g/ml, the expression is promoted with approximately 2 to 6 times or more greater effectiveness than that expressed by the G28-5 antibody as a control. With an antibody concentration of 0.1 .mu.g/ml, the expression is promoted with approximately 2 to 7 times or more greater effectiveness than that expressed by the G28-5 antibody as a control. With an antibody concentration of 1 .mu.g/ml, the expression is promoted with approximately 2 to 7 times or more greater effectiveness than that expressed by the G28-5 antibody as a control. With an antibody concentration of 10 .mu.g/ml, the expression is promoted with approximately 2 to 6 times or more greater effectiveness than that expressed by the G28-5 antibody as a control. (5) An antibody or a functional fragment thereof, having the amino acid sequences of a heavy chain variable region and a light chain variable region of an antibody that is produced by a hybridoma KM302-1 (Accession No: FERM BP-7578), KM341-1-19 (Accession No: FERM BP-7759), 2105 (Accession No: FERM BP-8024) or F1-102 (Accession No: ATCC PTA-3337).

(6) An antibody or a functional fragment thereof, having amino acid sequences of the mature portions of a heavy chain variable region and a light chain variable region of the antibody produced by a hybridoma F2-103, which are respectively encoded by plasmid DNAs with Accession Nos. ATCC PTA-3302 and ATCC PTA-3303; a heavy chain variable region and a light chain variable region of the antibody produced by a hybridoma F5-77, which are respectively encoded by plasmid DNAs with Accession Nos. ATCC PTA-3304 and ATCC PTA-3305; or a heavy chain variable region and a light chain variable region of the antibody produced by a hybridoma F5-157, which are respectively encoded by plasmid DNAs with Accession Nos. ATCC PTA-3306 and ATCC PTA-3307.

(7) An antibody or a functional fragment thereof, having amino acid sequences of the mature portions of a heavy chain variable region and a light chain variable region of the antibody produced by a hybridoma KM341-1-19, which are respectively represented by SEQ ID NOS: 28 and 30; a heavy chain variable region and a light chain variable region of the antibody produced by a hybridoma 2105, which are respectively represented by SEQ ID NOS: 32 and 34; a heavy chain variable region and a light chain variable region of the antibody produced by a hybridoma 110, which are respectively represented by SEQ ID NOS: 36 and 38; a heavy chain variable region and a light chain variable region of the antibody produced by a hybridoma 115, which are respectively represented by SEQ ID NOS: 40 and 42; a heavy chain variable region and a light chain variable region of the antibody produced by a hybridoma KM643-4-11, which are respectively represented by SEQ ID NOS: 52 and 54; a heavy chain variable region and a light chain variable region of the antibody produced by a hybridoma F2-103, which are respectively represented by SEQ ID NOS: 60 and 62; or a heavy chain variable region and a light chain variable region of the antibody produced by a hybridoma F5-77, which are respectively represented by SEQ ID NOS: 64 and 66. (8) An antibody or a functional fragment thereof, having amino acid sequences of the mature portions of a heavy chain variable region and a light chain variable region that are encoded by nucleic acid sequences isolated from a hybridoma KM341-1-19, which are respectively represented by SEQ ID NOS: 27 and 29; a heavy chain variable region and a light chain variable region that are encoded by nucleic acid sequences isolated from a hybridoma 2105, which are respectively represented by SEQ ID NOS: 31 and 33; a heavy chain variable region and a light chain variable region that are encoded by nucleic acid sequences isolated from a hybridoma 110, which are respectively represented by SEQ ID NOS: 35 and 37; a heavy chain variable region and a light chain variable region that are encoded by nucleic acid sequences isolated from a hybridoma 115, which are respectively represented by SEQ ID NOS: 39 and 41; a heavy chain variable region and a light chain variable region that are encoded by nucleic acid sequences isolated from a hybridoma KM643-4-11, which are respectively represented by SEQ ID NOS: 51 and 53; a heavy chain variable region and a light chain variable region that are encoded by nucleic acid sequences isolated from a hybridoma F2-103, which are respectively represented by SEQ ID NOS: 59 and 61; or a heavy chain variable region and a light chain variable region that are encoded by nucleic acid sequences isolated from a hybridoma F5-77, which are respectively represented by SEQ ID NOS: 63 and 65. (9) An antibody against a human CD40, or a functional fragment thereof, having at least one property selected from the following properties (g) to (j) of: (g) neutralizing the action of ligands on CD40; (h) neutralizing or alleviating one or more effects that ligands, which are for CD40 on an established B cell line, have on CD40-expressing cells, and having agonistic action on CD40 on the above established B cell line weaker than that of 5D12 due to cross-linking by anti-immunoglobulin antibodies; (i) alleviating or neutralizing the action of CD40 ligands on the established B cell line to increase CD95 expression; and (j) having antagonistic action on CD40 expressed on dendritic cells. (10) The antibody or the functional fragment of (9) above can suppress the expression of CD95 in Ramos cells to a level approximately 10% or less than that of a control, when antibodies with a concentration of 0.1 .mu.g/ml are added to the Ramos cells with a concentration of 1.times.10.sup.6 cells/ml supplemented with a saturated amount of CD40L-expressing cells; can suppress the expression of CD95 in Ramos cells to the same level as that of a negative control, when the antibodies with a concentration of 1 .mu.g/ml are added; and can suppress the expression of CD95 in the Ramos cells to the same level as that of the negative control, when the antibodies with a concentration of 10 .mu.g/ml are added. (11) The antibody or the functional fragment thereof of (9) above, wherein the proliferation of tonsillar B cells is suppressed in vitro by approximately 80 to 95% or more, when the antibodies with a concentration between 0.001 .mu.g/ml and 10 .mu.g/ml are added to 1.times.10.sup.5 tonsillar B cells supplemented with soluble CD40L (1 .mu.g/ml). For example, when the antibodies with a concentration between 0.01 .mu.g/ml and 10 .mu.g/ml are added, the proliferation of tonsillar B cells is suppressed by approximately 95% or more. In particular, when the antibodies with a concentration of 0.001 .mu.g/ml are added, the proliferation of tonsillar B cells is suppressed by approximately 80% or more. (12) An antibody or a functional fragment thereof, having amino acid sequences of a heavy chain variable region and a light chain variable region of the antibody produced by a hybridoma KM281-1-10 (Accession No: FERM BP-7579), 4D11 (Accession No: FERM BP-7758) or F4-465 (Accession No. ATCC PTA-3338). Deposited biological material covered by the issued claims in the patent granted from this application will be made available to the public in accordance with 37 C.F.R. .sctn. 1.808.

(13) An antibody or a functional fragment thereof, having amino acid sequences of the mature portions of a heavy chain variable region and a light chain variable region of the antibody produced by a hybridoma KM281-1-10, which are respectively represented by SEQ ID NOS: 44 and 46; a heavy chain variable region and a light chain variable region of the antibody produced by a hybridoma 4D11, which are respectively represented by SEQ ID NOS: 48 and 50; or a heavy chain variable region and a light chain variable region of the antibody produced by a hybridoma F4-465, which are respectively represented by SEQ ID NOS: 56 and 58. (14) An antibody or a functional fragment thereof, having amino acid sequences of the mature portions of a heavy chain variable region and a light chain variable region that are encoded by nucleic acid sequences isolated from a hybridoma KM281-1-10, which are respectively represented by SEQ ID NOS: 43 and 45; a heavy chain variable region and a light chain variable region of an antibody produced by a hybridoma 4D11, which are respectively represented by SEQ ID NOS: 47 and 49; or a heavy chain variable region and a light chain variable region that are encoded by nucleic acid sequences isolated from a hybridoma F4-465, which are respectively represented by SEQ ID NOS: 55 and 57. (15) Examples of the antibody or the functional fragment thereof of (1) to (14) above include human antibodies. (16) A pharmaceutical composition, containing as an active ingredient the antibody or the functional fragment thereof of any one of (1) to (15) above. (17) An immunopotentiating agent, anti-tumor agent or anti-autoimmune disease agent, containing as an active ingredient the antibody or the functional fragment thereof of any one of (1) to (8) above. (18) An immunosuppressive agent, anti-autoimmune disease agent, therapeutic agent against allergies or therapeutic agent against blood coagulation factor VIII-inhibiting syndrome, containing as an active ingredient the antibody or the functional fragment thereof of any one of (9) to (14) above. (19) Here, an epitope of a human CD40 that the monoclonal antibody of the present invention recognizes can be determined by a known method, such as by examining the binding to overlapping synthetic oligopeptides obtained from the primary amino acid sequence of human CD40 (e.g., Ed Harlow and David Lane (eds.), Antibodies: A Laboratory Manual, 1988 Cold Spring Harbor Laboratory Press; U.S. Pat. No. 4,708,871). A peptide library kit with the phage display process (New England BioLabs) can also be used for epitope mapping. The present invention also encompasses an antibody or a functional fragment thereof that recognizes a novel epitope of human CD40 that the antibody or the functional fragment thereof produced by each of the above hybridomas recognizes. (20) The present invention further provides a nucleic acid (RNA or cDNA) encoding at least the variable region of a heavy chain and/or light chain of an antibody isolated from each of the above hybridomas, a vector containing the nucleic acid, and a host cell carrying the nucleic acid.

The present invention will be described in detail. This specification includes part or all of the contents as disclosed in the specification and/or drawings of PCT Application PCT/US01/13672 (filed on Apr. 27, 2001), Japanese Patent Application No. 2001-142482 (filed on May 11, 2001), Japanese Patent Application No. 2001-310535 (filed on Oct. 5, 2001), and U.S. Patent Application U.S. Ser. No. 10/040,244 (filed on Oct. 26, 2001) which are priority documents of the present application.

As described later, we have found that a known monoclonal antibody 5D12 (ATCC No. HB-11339) that is antagonistic to CD40 on B cells is not antagonistic to CD40 on DC. We have further found that many monoclonal antibodies show agonistic activity on their own as a result of cross-linking by anti-immunoglobulin antibodies, even if they are antagonistic antibodies that block the action of CD40L.
 

Claim 1 of 21 Claims

1. An isolated antibody or a functional fragment thereof which binds to human CD40, wherein the antibody or the functional fragment thereof has amino acid sequences of a heavy chain variable region and a light chain variable region of the antibody produced by the hybridoma 4D11 (Accession No: FERM BP-7758).

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