JPWO2014065402A1 - Anti-human CD40 monoclonal antibody and use thereof - Google Patents

Abstract

Provision of a monoclonal antibody having a stimulating activity stronger than that of a conventional anti-CD40 monoclonal antibody. An anti-human CD40 monoclonal antibody or an antigen-binding fragment thereof having growth inhibitory activity in the absence of effector cells against human CD40 antigen-expressing cells, comprising a) amino acid residue 21 − of human CD40 amino acid sequence (SEQ ID NO: 1) An epitope comprising 37 or b) an anti-human CD40 monoclonal antibody or antigen-binding fragment thereof that specifically binds to an epitope comprising amino acid residues 38-61 of the human CD40 amino acid sequence (SEQ ID NO: 1).

Description

  The present invention relates to an anti-human CD40 monoclonal antibody useful as an antitumor agent and a pharmaceutical composition containing the same.

CD40 has been identified as an antigen that is expressed on the surface of human B cells (Non-patent Document 1). From the homology of amino acid sequences, the low-affinity NGF receptor, TNF receptor, CD27, OX40, CD30, etc. belong to the TNF receptor family One of the members. CD40 is a type I membrane glycoprotein that can be used in various cell types such as B lymphocytes, dendritic cells, monocyte epithelial cells, fibroblasts, and certain tumor cells such as neoplastic human B cells. Is also known to be expressed. In CD40-deficient mice, thymus-dependent immunoglobulin class switching and germinal center formation are impaired, and an important role of CD40 in cellular and humoral immune responses has been demonstrated (Non-patent Document 2).
The physiological ligand for CD40 is CD40 Ligand (CD154, gp39). CD40L is expressed in activated T lymphocytes and binds to CD40 on the surface of B lymphocytes, which regulates the differentiation and proliferation of B lymphocytes, avoiding spontaneous apoptosis of germinal center B lymphocytes It is responsible for the mechanism (Non-patent Document 3).

  In both T-cell and B-cell malignancies, antitumor effects (proliferation arrest either by apoptosis or not by apoptosis) are often observed when malignant cells are exposed to stimuli that lead to normal lymphocyte activation. Occurs. Growth inhibition induced by this activation has been observed together with signals via either antigen receptors or costimulatory receptors (Non-Patent Documents 4-7). CD40 stimulation with certain stimulating CD40 antibodies or soluble CD40L directly inhibits B cell lymphoma growth (8, 9).

  There is no correlation between the affinity of stimulating anti-CD40 antibodies such as G28-5 (ATCC No. HB-9110) and S2C6 (ATCC No. PTA-110) and the activation of normal B cells by antibodies, but CD40 by antibodies It is known that there is a correlation between ligand inhibition ability and normal B cell activation. These antibodies recognize the N-terminal side from the 61st position of the CD40 amino acid residue (Non-patent Document 10). In addition, HCD122, which is an anti-CD40 antibody that completely inhibits the binding of CD40L, not a stimulating antibody, is known to recognize the 82-86th epitope of CD40 (Patent Document 1).

International Publication No. 2005/044294

Clark et al., PNAS USA 1986; 83: 4494-4498 Kawabe et al., Immunity 1994; 1: 167-168 Kehry, J Immumol 1996; 156: 2345-2348 Ashwell et al., Science 1987; 237: 61-64 Bridges et al., J Immumol 1987; 139: 4242-4549 Page and Defranco, J Immunol 1988; 140: 3717-3726 Beckwith et al., J Natl Cancer Inst 1990; 82: 501-509 Funakoshi et al., Blood 1994; 83: 2787-2794 Francisco et al., Cancer Res 2000; 60: 3225-3231 Hager et al., Scand J Immnol 2003; 57: 517-524

  An object of the present invention is to provide a monoclonal antibody having a stimulating activity stronger than that of a conventional anti-CD40 monoclonal antibody.

  In order to solve the above problems, the present inventor made antibodies against human CD40 and obtained many anti-CD40 monoclonal antibodies. Then, it was found that antibodies that specifically bind to the amino acid residues of the CD40 epitope 21-37 or 38-61 exist in these antibody groups. Furthermore, the present inventors have found that an antibody that binds to the CD40 epitope has a strong growth inhibitory activity against a cancer cell line that expresses the CD40 antigen, thereby completing the present invention.

That is, the present invention provides the following [1] to [16].
[1] An anti-human CD40 monoclonal antibody or an antigen-binding fragment thereof having antiproliferative activity in the absence of effector cells against human CD40 antigen-expressing cells,
a) specifically binds to an epitope comprising amino acid residues 21-37 of the human CD40 amino acid sequence (SEQ ID NO: 1) or b) to an epitope comprising amino acid residues 38-61 of the human CD40 amino acid sequence (SEQ ID NO: 1) An anti-human CD40 monoclonal antibody or an antigen-binding fragment thereof.
[2] The amino acid sequence shown in any one of the following 1) to 16) in the light chain variable region and the heavy chain variable region, or in the amino acid sequence, one or more amino acids are substituted, deleted, added or inserted, The anti-human CD40 monoclonal antibody or the antigen-binding fragment thereof according to [1], which has an amino acid sequence having an identity level of at least 90% or more.
1) SEQ ID NO: 5 and SEQ ID NO: 30,
2) SEQ ID NO: 6 and SEQ ID NO: 31,
3) SEQ ID NO: 9 and SEQ ID NO: 34,
4) SEQ ID NO: 10 and SEQ ID NO: 35,
5) SEQ ID NO: 11 and SEQ ID NO: 36,
6) SEQ ID NO: 14 and SEQ ID NO: 39,
7) SEQ ID NO: 17 and SEQ ID NO: 42,
8) SEQ ID NO: 18 and SEQ ID NO: 43,
9) SEQ ID NO: 19 and SEQ ID NO: 44,
10) SEQ ID NO: 20 and SEQ ID NO: 45,
11) SEQ ID NO: 21 and SEQ ID NO: 46,
12) SEQ ID NO: 22 and SEQ ID NO: 47,
13) SEQ ID NO: 24 and SEQ ID NO: 49,
14) SEQ ID NO: 25 and SEQ ID NO: 50,
15) SEQ ID NO: 26 and SEQ ID NO: 51, and
16) SEQ ID NO: 27 and SEQ ID NO: 52
[3] An amino acid sequence in which each of the light chain variable region and the heavy chain variable region is represented by any of the following, or in the amino acid sequence, one or more amino acids are substituted, deleted, added or inserted, and the level of identity is The anti-human CD40 monoclonal antibody or antigen-binding fragment thereof according to [1] or [2], which has an amino acid sequence of at least 90%.
1) SEQ ID NOs: 9 and 34,
2) SEQ ID NOs: 10 and 35,
3) SEQ ID NOs: 11 and 36,
4) SEQ ID NOs: 14 and 39,
5) SEQ ID NOs: 17 and 42,
6) SEQ ID NOs: 19 and 44,
7) SEQ ID NO: 20 and 45, or 8) SEQ ID NO: 21 and 46
[4] Deposited with NITE under the accession numbers NITE BP-01436, NITE BP-01437, NITE BP-01438, NITE BP-01716, NITE BP-01439, NITE BP-01440, NITE BP-01441, and NITE BP-01442 Hybridoma cell lines PPAT-071-06, PPAT-071-07, PPAT-071-08, PPAT-071-11, PPAT-071-14, PPAT-071-16, PPAT-071-17, and PPAT-071 [18] The anti-human CD40 monoclonal antibody or the antigen-binding fragment thereof according to any one of [1] to [3], which is a monoclonal antibody or an antigen-binding fragment thereof obtained from any one of -18.
[5] The anti-human CD40 monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [4], which is derived from a mouse.
[6] The anti-human CD40 monoclonal antibody or antigen-binding fragment thereof according to [1], which is a humanized antibody.
[7] An antibody humanized using the amino acid sequence containing the complementarity determining region CDR of the anti-human CD40 monoclonal antibody according to any one of [1] to [5] and the amino acid sequence of human immunoglobulin [6] Or an antigen-binding fragment thereof.
[8] An amino acid sequence in which the light chain variable region and the heavy chain variable region are respectively represented by any of the following, or one or more amino acids in the amino acid sequence are substituted, deleted, added or inserted, and the level of identity is The anti-human CD40 monoclonal antibody or antigen-binding fragment thereof according to [6] or [7], which has an amino acid sequence of at least 90% or more.
1) SEQ ID NOs: 58 and 59,
2) SEQ ID NOs: 60 and 61,
3) SEQ ID NOs: 62 and 59,
4) SEQ ID NOs: 63 and 64
5) SEQ ID NOs: 65 and 66,
6) SEQ ID NOs: 60 and 67,
7) SEQ ID NO: 68 and 61, or 8) SEQ ID NO: 69 and 70
[9] The anti-human CD40 monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [8], to which a cytotoxic drug or drug is bound.
[10] The anti-human CD4 monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [8], which is labeled with a radioisotope.
[11] A pharmaceutical composition comprising the anti-human CD40 monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [10].
[12] An antitumor agent for a tumor expressing a human CD40 antigen comprising the anti-human CD40 monoclonal antibody or the antigen-binding fragment thereof according to any one of [1] to [10] as an active ingredient.
[13] A hybridoma that produces the anti-human CD40 monoclonal antibody according to any one of [1] to [5].
[14] The anti-human CD40 monoclonal antibody or the antigen-binding fragment thereof according to any one of [1] to [10] for treating a tumor expressing a human CD40 antigen.
[15] Use of the anti-human CD40 monoclonal antibody or the antigen-binding fragment thereof according to any one of [1] to [10] for the manufacture of a therapeutic agent for a tumor expressing a human CD40 antigen.
[16] A method for treating a tumor expressing human CD40 antigen, comprising administering the anti-human CD40 monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [10] to a patient in need thereof .

  The anti-human CD40 monoclonal antibody of the present invention is an antibody having 21-37- or 36-61-th amino acid residues of human CD40 as an epitope, and strongly suppresses growth against tumor cells expressing CD40 antigen. It has activity and is useful as an anticancer agent.

It is a figure which shows the amino-terminal sequence of human CD40 which concerns on Example 1 and 5 of this invention, and the N terminal side of produced partial length CD40. It is a figure which shows the result of having paralleled the amino acid sequence of human CD40 which concerns on Example 2 and 4 of this invention, and mouse | mouth CD40. It is a figure which shows the result of having paralleled the amino acid sequence of human CD40 which concerns on Example 4 of this invention, and cynomolgus monkey CD40. It is a figure which shows the result of the CD40 epitope analysis in the partial length CD40 expression CHO cell based on Example 5 of this invention. It is a figure which shows the proliferation rate of Ramos cell by each mouse antibody which concerns on Example 6 of this invention. It is a figure which shows the anti-tumor effect in the tumor bearing mouse model of Ramos cell by the mouse antibody which concerns on Example 7 of this invention. FIG. 10 is a graph showing the growth rate of Ramos cells and MOLP-8 cells in the concentration change of the mouse antibody and humanized antibody according to Example 10 of the present invention. It is a figure which shows the anti-tumor effect in the tumor-bearing mouse model of Ramos cell by the humanized antibody which concerns on Example 11 of this invention. It is a figure which shows the sensitivity of Ramos cell, Raji cell, and MOLP-8 cell with respect to DM1SH which concerns on Example 12 of this invention. It is a figure which shows the proliferation rate of Ramos cell, Raji cell, and MOLP-8 cell by DM1 conjugate antibody (PPAT-071-06) based on Example 12 of this invention. It is a figure which shows the proliferation rate of Ramos cell, Raji cell, and MOLP-8 cell by DM1 conjugate antibody (PPAT-071-07) based on Example 12 of this invention. It is a figure which shows the proliferation rate of Ramos cell, Raji cell, and MOLP-8 cell by DM1 conjugate antibody (PPAT-071-18) based on Example 12 of this invention.

Hereinafter, embodiments of the present invention will be described in detail.
1. Definitions The terms used in this specification are defined as follows.

  Two isoforms of human CD40 (encoded by alternative splice transcript variants of this gene) have been identified. The first isoform (also known as “long isoform” or “isoform 1”) is a 277 amino acid precursor polypeptide (SEQ ID NO: 1 (isoform 1 in GenBank accession number NP_001241)). Expressed, which has a signal sequence represented by the first 20 residues. The second isoform (also known as “short isoform” or “isoform 2”) is expressed as a 203 amino acid precursor polypeptide, also represented by the first 20 residues. Have The precursor polypeptides of these two isoforms of human CD40 commonly share its first 165 residues (ie, residues 1-165 of SEQ ID NO: 1). The short isoform precursor polypeptide is encoded by a transcript variant that lacks the coding segment, which results in a frameshift of translation, and the resulting CD40 isoform is contained in the long isoform of CD40 (C-terminus shown in residues 166-277 of SEQ ID NO: 1). In the present invention, “CD40 antigen” or “CD40” encompasses both short and long isoforms of CD40. The anti-CD40 antibodies of the invention bind to an epitope of human CD40 as set forth herein below, which human CD40 is one of either the short or long isoforms of the cell surface antigen. It exists in the same position. “CD40” means “human CD40” unless otherwise noted.

  CD40 antigen is presented on the surface of various cell types. “Characterizing expression” and “expressing” mean that all or a portion of the CD40 antigen is exposed to the outside of the cell. The displayed or expressed CD40 antigen may be fully or partially glycosylated.

  As used herein, “CD40 epitope” means a molecule (eg, a peptide) or a fragment of the molecule capable of immunoreacting with an anti-CD40 antibody, eg, a CD40 antigenic determinant recognized by a monoclonal antibody. Is included. CD40 antigen epitopes may be included in proteins, protein fragments, peptides and the like. An epitope is most commonly a protein, a short oligopeptide, an oligopeptide mimetic (ie, an organic compound that mimics the antibody binding properties of the CD40 antigen), or a combination thereof.

An “antibody” as used herein is a heterotetrameric glycoprotein, typically about 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Is defined as Each light chain is covalently linked to the heavy chain by one disulfide bond to form a heterodimer. A heterotetramer is formed by a disulfide covalent bond between two identical heavy chains of such a heterodimer. The light and heavy chains are linked together by a single disulfide bond, but the number of disulfide bonds between the two heavy chains varies depending on the immunoglobulin isotype. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (V _H ) at the end of the amino acid followed by 3 or 4 constant domains (C _H 1, C _H 2, C _H 3, and C _H 4), and C _H 1 And a hinge region exists between C _H 2. Each light chain has two domains: an amino-terminal variable domain (V _L ) and a carboxy-terminal constant domain (C _L ). V _L domains are associated non-covalently with a V _H domain, whereas the C _L domain usually are covalently linked with C _H 1 domain via a disulfide bond. Certain amino acid residues are thought to form an interface between the light chain variable domain and the heavy chain variable domain (Chothia et al., J MoI Biol 1985; 186: 651-663).

  “Variable” refers to the fact that a particular portion of a variable domain varies widely in sequence between antibodies and is used for the binding and specificity of each particular antibody for that particular antigen. However, the variability is not evenly distributed throughout the variable domain of the antibody. The variability is concentrated in three segments called complementarity determining regions (CDRs) in both the light chain variable domain and the heavy chain variable domain. The more highly conserved portion of the variable domain is called the framework region (FR).

A “complementarity-determining region (CDR)” is a type of sequence within a variable domain that varies greatly between antibodies and is directly involved in the binding and specificity of each specific antibody to its specific antigenic determinant. Regions containing species residues are indicated. Both light and heavy chain variable domains are concentrated in three segments. CDR is Kabat et al, Sequences of Proteins of Immunological Interest, 5 th Ed Public Health Service, National Institutes of Health, 1993:. Bethesda, defined by sequence comparison of MD.. As defined by Kabat, CDR-L1 is around 24-34 residues in the light chain variable domain, CDR-L2 is around 50-56 residues, and CDR-L3 is around 89-97 residues. CDR-H1 is located near 31-35 of the heavy chain variable domain, CDR-H2 is located near 50-65, and CDR-H3 is located near 95-102.

  The three CDRs of each heavy and light chain are separated by a framework region (FR) containing sequences that tend to be less variable. From the amino terminus to the carboxy terminus of the heavy chain variable domain and the light chain variable domain, FR and CDR are arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. FR's rough β-sheet configuration brings the CDRs in each chain close to each other and close to each other. The resulting configuration contributes to the antigen binding site, but not all CDR residues are directly involved in antigen binding.

  FR residues and Ig constant domains are not directly involved in antigen binding, but contribute to antigen binding and / or mediate antibody effector functions. Some FR residues are at least three by directly non-covalently binding to the epitope, interacting with one or more CDR residues, and affecting the boundary between the heavy and light chains The method can have a significant effect on antigen binding. Constant domains are not directly involved in antigen binding, but have a variety of effects, including antibody involvement in antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP). Mediates Ig effector function.

  An “antigen-binding fragment” comprises a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of antigen binding fragments include Fab, Fab ′, F (ab ′) 2 and Fv fragments; bispecific antibodies; linear antibodies (Zapata et. Al., Protein Eng 1995; 8 (10): 1057 -1062); single chain antibody molecules; as well as multispecific antibodies formed from antigen-binding fragments. By papain digestion of the antibody, two identical antigen-binding fragments (called “Fab” fragments) each having a single antigen-binding site, and the remaining “Fc” fragment (this name has the ability to crystallize easily). Is reflected). Pepsin treatment yields an F (ab ′) 2 fragment that has two antigen binding sites and can still crosslink with antigen.

“Fv” is the smallest antigen-binding fragment that contains a complete antigen recognition and binding site. In the two-chain Fv species, this region consists of a dimer of one heavy chain variable domain and one light chain variable domain that are tightly and non-covalently linked. In a single chain Fv species, one heavy chain variable domain and one light chain variable domain associate in a “dimeric” structure whose light and heavy chains are similar to the structure in a two chain Fv species. As such, it can be covalently linked by a flexible peptide linker. In this configuration, the three CDRs of each variable domain interact to define the antigen binding site on the surface of the V _H -V _L dimer. Together, the six CDRs provide antigen binding specificity for the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for the antigen) has the ability to recognize and bind the antigen, but with a lower affinity than the complete binding site.

  The “light chains” of antibodies (immunoglobulins) from any vertebrate species are divided into two distinctly different types (called kappa (κ) and lambda (λ)) based on the amino acid sequences of their constant domains. It is classified.

  Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins are classified into different classes. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG and IgM. It is further divided into subclasses (isotypes) (for example, IgG1, IgG2, IgG3, IgG4, IgA and IgA2). The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Different isotypes have different effector functions. For example, the human IgG1 and IgG3 isotypes mediate antibody-dependent cytotoxicity (ADCC) activity.

  “Cytotoxic agent” refers to a substance that inhibits or suppresses the function of cells and / or causes destruction of cells. The term is intended to include chemotherapeutic agents as well as toxins (enzymatically active toxins of bacterial, fungal, plant or animal origin) and fragments thereof. Such cytotoxic agents can be coupled to antibodies, such as humanized CD40 antibodies, using known standard procedures and used, for example, to treat patients for whom therapy with antibodies is indicated.

A “radioisotope” is a radionuclide such as I ¹³¹ , 1 ¹²⁵ , Y ⁹⁰ and Re ¹⁸⁶ and is coupled to an antibody such as a humanized CD40 antibody using known standard procedures, eg It can be used to treat patients for whom therapy is indicated.

“Tumor expressing CD40” includes acute lymphocytic leukemia, chronic leukemia, lymphoma (eg, Hodgkin's disease or non-Hodgkin's disease), multiple myeloma, carcinoma (eg, colon cancer, pancreatic cancer, breast cancer, ovarian cancer) , Prostate cancer, squamous cell carcinoma, kidney cancer, lung cancer, or esophageal cancer).
“Effector cell” refers to a cell having strong attack power and processing ability in cellular immunity, and examples thereof include killer T cells, helper T cells, suppressor T cells, natural killer cells, and macrophages.

2. The antibody of the present invention or an antigen-binding fragment thereof The antibody of the present invention or an antigen-binding fragment thereof is an anti-human CD40 monoclonal antibody or an antigen-binding fragment thereof having antiproliferative activity in the absence of effector cells against human CD40 antigen-expressing cells. And
a) specifically binds to an epitope comprising amino acid residues 21-37 of the human CD40 amino acid sequence (SEQ ID NO: 1) or b) to an epitope comprising amino acid residues 38-61 of the human CD40 amino acid sequence (SEQ ID NO: 1) Anti-human CD40 monoclonal antibody or antigen-binding fragment thereof.

  The anti-human CD40 monoclonal antibody or antigen-binding fragment thereof of the present invention has a growth inhibitory activity in the absence of effector cells against human CD40 antigen-expressing cells. More specifically, a. Reacts with cells stably expressing human CD40, b. Reacts against a human CD40 expressing cancer cell line, and c. Has the effect of suppressing proliferation of human CD40-expressing cancer cells.

More specifically, the antibody of the present invention reacts with human CD40 stably expressing cells and human CD40 expressing cancer cells in FACS analysis at an antibody concentration of 1.0 μg / mL or less.
In addition, the antibody of the present invention can be obtained by culturing for 3 days in the presence of antibody 3.0 μg / mL against 1 × 10 ⁵ cells / mL of Ramos cells (ATCC No. CRL-1596) which are human CD40-expressing cancer cells. The growth rate of Ramos cells is 70% or less.
Furthermore, the antibody of the present invention has a CD40 amino acid residue after the 21st position in FACS analysis at an antibody concentration of 1.0 μg / mL on cells expressing 6 types of partial length CD40 shortened from the N-terminus of human CD40. Among the antibody groups that have the characteristic of reacting to cells expressing partial length CD40, the antibody group that has the characteristic of not reacting to cells expressing partial length CD40 after the 40th amino acid residue of CD40, or the 62nd and subsequent parts It is a group of antibodies having characteristics that do not react with cells expressing long CD40.

In the antibody of the present invention, preferably, the light chain variable region and the heavy chain variable region are amino acid sequences shown in any one of the following 1) to 16), or one or more amino acids are substituted or deleted in the amino acid sequence: Has an amino acid sequence that is added or inserted and has a level of identity of at least 90% or more.
1) SEQ ID NO: 5 and SEQ ID NO: 30,
2) SEQ ID NO: 6 and SEQ ID NO: 31,
3) SEQ ID NO: 9 and SEQ ID NO: 34,
4) SEQ ID NO: 10 and SEQ ID NO: 35,
5) SEQ ID NO: 11 and SEQ ID NO: 36,
6) SEQ ID NO: 14 and SEQ ID NO: 39,
7) SEQ ID NO: 17 and SEQ ID NO: 42,
8) SEQ ID NO: 18 and SEQ ID NO: 43,
9) SEQ ID NO: 19 and SEQ ID NO: 44,
10) SEQ ID NO: 20 and SEQ ID NO: 45,
11) SEQ ID NO: 21 and SEQ ID NO: 46,
12) SEQ ID NO: 22 and SEQ ID NO: 47,
13) SEQ ID NO: 24 and SEQ ID NO: 49,
14) SEQ ID NO: 25 and SEQ ID NO: 50,
15) SEQ ID NO: 26 and SEQ ID NO: 51, and
16) SEQ ID NO: 27 and SEQ ID NO: 52

In the antibody of the present invention, more preferably, the light chain variable region and the heavy chain variable region are each represented by any of the following amino acid sequences, or one or more amino acids in the amino acid sequence are substituted, deleted, added or inserted: And having an amino acid sequence having a level of identity of at least 90% or more.
1) SEQ ID NOs: 9 and 34,
2) SEQ ID NOs: 10 and 35,
3) SEQ ID NOs: 11 and 36,
4) SEQ ID NOs: 14 and 39,
5) SEQ ID NOs: 17 and 42,
6) SEQ ID NOs: 19 and 44,
7) SEQ ID NO: 20 and 45, or 8) SEQ ID NO: 21 and 46

Among the antibodies of the present invention, the antibody that specifically binds to an epitope including amino acid residues 21-37 of the human CD40 amino acid sequence is preferably an antibody produced by a hybridoma shown in the following 1) to 8).
1) PPAT-071-01
2) PPAT-071-02
3) PPAT-071-18
4) PPAT-071-20
5) PPAT-071-21
6) PPAT-071-22
7) PPAT-071-23
8) PPAT-071-24

Among the antibodies of the present invention, the antibody that specifically reacts with an epitope containing amino acid residues 38-61 of the human CD40 amino acid sequence is preferably an antibody produced by the hybridoma shown in the following 1) to 10). .
1) PPAT-071-03
2) PPAT-071-06
3) PPAT-071-07
4) PPAT-071-08
5) PPAT-071-11
6) PPAT-071-14
7) PPAT-071-15
8) PPAT-071-16
9) PPAT-071-17
10) PPAT-071-19

  Further, among the antibodies of the present invention, accession numbers NITE BP-01436, NITE BP-01437, NITE BP-01438, NITE BP-01716, NITE BP-01439, NITE BP-01440, NITE BP-01441, and NITE BP- Hybridoma cell lines PPAT-071-06, PPAT-071-07, PPAT-071-08, PPAT-071-11, PPAT-071-14, PPAT-071-16, PPAT-071- An antibody produced by any one of 17 and PPAT-071-18 is preferred.

Among the antibodies of the present invention, the antibody produced by the hybridoma is derived from a mouse, but can also be a humanized antibody. The humanized antibody can be obtained, for example, by humanizing using the amino acid sequence containing the complementarity determining region CDR of the mouse-derived anti-human CD40 monoclonal antibody and the amino acid sequence of human immunoglobulin. As such a humanized antibody, each of the light chain variable region and the heavy chain variable region is represented by any of the following amino acid sequences, or one or more amino acids in the amino acid sequence are substituted, deleted, added or inserted: Those having an amino acid sequence with an identity level of at least 90% or more are preferred.
1) SEQ ID NOs: 58 and 59,
2) SEQ ID NOs: 60 and 61,
3) SEQ ID NOs: 62 and 59,
4) SEQ ID NOs: 63 and 64
5) SEQ ID NOs: 65 and 66,
6) SEQ ID NOs: 60 and 67,
7) SEQ ID NO: 68 and 61, or 8) SEQ ID NO: 69 and 70,

  The number of amino acid substitutions, deletions, additions or insertions in the amino acid sequence representing the light chain variable region and heavy chain variable region is preferably 1-10, more preferably 1-8, and 1-6 More preferably. The level of amino acid sequence identity is preferably 91% or more, more preferably 93% or more, and still more preferably 95% or more.

3. Antibody Production Method In the present invention, an antibody that binds to human CD40 can be produced by a method known to those skilled in the art. For example, a monoclonal antibody-producing hybridoma can be basically produced using a known technique as follows. That is, using a CD40 protein or a CD40-expressing cell as a sensitizing antigen, a mammal is immunized according to a normal immunization method. The obtained immune cells can be fused with a known parent cell by an ordinary cell fusion method, and monoclonal antibody-producing cells can be selected by an ordinary screening method.

  Specifically, the monoclonal antibody can be produced as follows. First, a CD40 protein is obtained according to the amino acid sequence of CD40 shown in SEQ ID NO: 1, and this is used as a sensitizing antigen for obtaining an antibody. That is, a gene sequence encoding human CD40 is inserted into a known expression vector system to transform an appropriate host cell, and then stable expression of CD40 on the surface of the host cell is produced, or from the culture supernatant The target CD40 protein is purified by a known method.

  Next, this purified CD40 protein or CD40 stably expressing cells is used as a sensitizing antigen. Alternatively, a partial peptide of CD40 can be used as a sensitizing antigen. In this case, the partial peptide can also be obtained by chemical synthesis according to the amino acid sequence of human CD40.

  The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion. Animals such as mice, rats, hamsters, rabbits, monkeys and the like are used.

  In order to immunize an animal with a sensitizing antigen, a known method is performed. For example, as a general method, a sensitizing antigen is injected into a mammal intraperitoneally or subcutaneously. Specifically, the sensitizing antigen is diluted to an appropriate amount with PBS (Phosphate-Buffered Saline), physiological saline or the like, mixed with an appropriate amount of an ordinary adjuvant, for example, Freund's complete adjuvant, if necessary, and emulsified. Administer to mammals several times every 4-21 days. In addition, an appropriate carrier can be used during immunization with the sensitizing antigen.

  Thus, after immunizing a mammal and confirming that a desired antibody level rises in serum, immune cells are collected from the mammal and subjected to cell fusion. Preferable immune cells include spleen cells.

  Mammalian myeloma cells are used as parent cells to be fused with the immune cells. This myeloma cell is known from various known cell lines such as P3 (P3x63Ag8.653) (Kearney et al., J Immnol 1979; 123: 1548-1550), NS-1 (Kohler. G. and Milstein, C Eur J Immunol 1976; 6: 511-519), SP2 / 0 (Shulman, M. et al., Nature 1978; 276: 269-270) and the like are preferably used.

  The cell fusion between the immune cells and myeloma cells is basically performed by a known method such as the method of Kohler and Milstein, C., Methods Enzymol 1981; 73: 3-46. It can carry out according to etc.

  More specifically, the cell fusion is performed in a normal nutrient culture medium in the presence of a cell fusion promoter, for example. As the fusion accelerator, for example, polyethylene glycol (PEG), Sendai virus (HVJ) or the like is used, and an auxiliary agent such as dimethyl sulfoxide can be added and used to increase the fusion efficiency as desired.

  The use ratio of immune cells and myeloma cells can be arbitrarily set. For example, the number of immune cells is preferably 1 to 10 times that of myeloma cells. As the culture medium used for the cell fusion, for example, RPMI1640 culture medium suitable for growth of the myeloma cell line, MEM culture medium, and other normal culture liquids used for this type of cell culture can be used. Serum supplements such as fetal bovine serum (FBS) can also be used in combination.

  In cell fusion, a predetermined amount of the immune cells and myeloma cells are mixed well in the culture medium, and a PEG solution (for example, an average molecular weight of about 1000 to 6000) preliminarily heated to about 37 ° C. is usually 30-60% ( The target fusion cell (hybridoma) is formed by adding at a concentration of w / v) and mixing. Subsequently, cell fusion agents and the like that are undesirable for the growth of the hybridoma are removed by sequentially adding an appropriate culture medium and centrifuging to remove the supernatant.

  The hybridoma thus obtained is selected by culturing in a normal selective culture solution, for example, a HAT culture solution (a culture solution containing hypoxanthine, aminopterin and thymidine). The culture in the HAT culture solution is continued for a sufficient time (usually several days to several weeks) for cells other than the target hybridoma (non-fusion cells) to die. Subsequently, a normal limiting dilution method is performed, and screening and single cloning of a hybridoma that produces the target antibody are performed.

  In the screening, an antibody that reacts with CD40 can be selected by FACS analysis using CD40 stably expressing cells used as a sensitizing antigen and cancer cells characterizing the expression of CD40. An antibody that reacts with CD40 can also be selected by ELISA using purified CD40 protein. As a preferred embodiment of the antibody of the present invention, a promising method for selecting an antibody to be used for treatment is FACS analysis using cancer cells that characterize the expression of CD40.

  The hybridoma producing the monoclonal antibody thus produced can be subcultured in a normal culture solution, and can be stored for a long time in liquid nitrogen.

  In order to obtain a monoclonal antibody from the hybridoma, the hybridoma is cultured according to a usual method and obtained as a culture supernatant, or the hybridoma is administered to a mammal compatible therewith to proliferate, and as ascites The method of obtaining is adopted. The former method is suitable for obtaining highly pure antibodies, while the latter method is suitable for mass production of antibodies.

4). Cross-reactivity test with antigens derived from animal species other than humans In the present invention, a preferred embodiment includes an antibody that cross-reacts with cynomolgus monkey CD40. Analysis of cross-reactivity of antibodies with antigens of animal species other than humans can be performed by methods known to those skilled in the art, and can be performed by Western blotting or FACS using partial-length antigen-expressing cells. . Specifically, it can be determined by FACS analysis using cells expressing cynomolgus monkey CD40 or mouse CD40.

5. CD40 epitope mapping As a preferred embodiment of the present invention, the following two CD40 epitopes:
1) 21st-37th CD40 amino acid residue 2) An antibody that reacts with 38th-61st CD40 amino acid residue can be mentioned.
Analysis of the epitope recognized by the antibody can be performed by methods known to those skilled in the art, and can be performed by Western blotting or FACS using a partial-length antigen-expressing cell. Specifically, it can be analyzed by FACS using cells expressing six types of partial length CD40 excluding the N-terminal amino acid residue described in the Examples.

  An antibody that recognizes the above region as an epitope can be obtained by methods known to those skilled in the art. For example, a peptide containing the amino acid sequence of the target region is prepared based on the amino acid sequence of human CD40, and the peptide Can be obtained by preparing an antibody using as an immunogen, or by determining the epitope recognized by the obtained antibody and selecting an antibody that recognizes the target epitope after preparing the antibody by a conventional method. Is possible.

6). Proliferation assay As a preferred embodiment of the present invention, an antibody that stimulates cells expressing CD40, that is, an antibody that suppresses proliferation of tumor cells expressing CD40 can be mentioned. The measurement of the proliferation assay can be performed by a method known to those skilled in the art, and can be performed by a method of incorporating H3-thymidine, a color developing method using WST-8 which is a tetrazolium salt, or the like. Specifically, an antibody that acts on CD40 expressed on the surface of a cell such as a B cell or a tumor cell and affects (suppresses or promotes) or does not affect the proliferation of the CD40-expressing cell is selected.

7). Cloning of variable region of anti-human CD40 antibody Regarding the antibody that binds to human CD40 in the present invention, cloning of the variable region can be prepared by methods known to those skilled in the art.

  Specifically, mRNA encoding the variable region of the anti-human CD40 antibody is isolated from the hybridoma producing the anti-human CD40 antibody. Isolation of mRNA is performed by a known method such as guanidine ultracentrifugation (Chirgwin, JM et al., Biochemistry 1979; 18: 5294-5299), AGPC method (Chomczynski, P. et al., Anal Biochem 1987; 162). : 156-159) etc. to prepare total RNA, and mRNA of interest is prepared using mRNA Purification Kit (Pharmacia). Alternatively, mRNA can be directly prepared by using QuickPrep mRNA Purification Kit (Pharmacia).

  The gene encoding the antibody variable region is amplified from the obtained mRNA. Amplification of antibody variable region genes is known to be obtained with SMART RACE cDNA Amplication Kit (Clontech) using oligonucleotides complementary to mouse IgG or mouse κ chain constant region sequences. Alternatively, a cDNA is synthesized from the obtained mRNA using a reverse transcriptase kit (Invitrogen), etc., and the gene encoding the variable region is amplified by PCR using degenerate primer using the prepared cDNA as a template. Can do.

  The desired DNA fragment is purified from the obtained PCR amplification product and ligated with vector DNA. Further, a recombinant vector is prepared from this, introduced into Escherichia coli, etc., and colonies are selected to prepare a desired recombinant vector. Then, the base sequence of the target DNA is confirmed by a known method such as the dideoxynucleotide chain termination method.

  The nucleotide sequence of DNA can identify the amino acid residues and CDRs of the light chain variable region and heavy chain variable region by using publicly available Kabat Database or IMGT (ImMunoGeneTics) Database.

8). Preparation of humanized anti-CD40 antibody As one of the preferred embodiments of the antibody in the present invention, a chimera in which the constant regions of human κ chain and human IgG1 are linked to the variable regions of the light and heavy chains of a mouse antibody that binds to human CD40. Mention may be made of antibodies. Further preferred embodiments include humanized antibodies that bind to human CD40. Humanized antibodies can be produced using known methods.

  A humanized antibody is also called a reshaped human antibody, which is a non-human mammal, for example, a mouse antibody complementarity determining region (CDR) grafted to the CDR of a human antibody, A known genetic recombination technique is also known (WO 96/02576).

  Specifically, for example, when the CDR is derived from a mouse antibody, a DNA sequence designed to link the CDR of the mouse antibody and the framework region (FR) of the human antibody is used as a terminal region of both CDR and FR. Synthesize by PCR using several oligonucleotides prepared to have overlapping parts as primers, or artificially synthesize DNA encoding light chain variable region and heavy chain variable region genes Is also possible.

  As the framework region of the human antibody to be ligated to the CDR, a region in which the complementarity determining region forms a favorable antigen binding site is selected. If necessary, framework region amino acids in the variable region of the antibody may be substituted so that the complementarity determining region of the humanized antibody forms an appropriate antigen binding site (Sato K et al., Cancer Res 1993 ; 53: 851-856).

  The constant region of a humanized antibody is that of a human antibody. For example, Cγ1, Cγ2, Cγ3, Cγ4 can be used for the heavy chain, and Cκ, Cλ can be used for the light chain. In addition, the constant region of a human antibody may be modified to improve the stability of the antibody or its production. Human antibodies used for humanization may be human antibodies of any isotype such as IgG, IgM, IgA, IgE, IgD, but in the present invention, IgG is preferably used, IgG1 or IgG3 is more preferable, and IgG1 is particularly preferable. Is preferred. IgG1 is effective when an antibody is used as an anticancer agent because it has a high cytotoxic activity (Clark MR, Chemical immunology 1997; 65: 88-110).

  In addition, after producing a humanized antibody, amino acids in the variable region or constant region may be substituted with other amino acids.

  Furthermore, the antibody of the present invention may be an antibody with enhanced cytotoxic activity. Examples of antibodies with enhanced cytotoxic activity include antibodies lacking fucose, antibodies in which bisecting N-acetylglucosamine (GlcNAc) is added to the sugar chain, and Fcγ receptor by substituting amino acids in the Fc region. Examples thereof include antibodies whose binding activity with the body has been changed. These antibodies with enhanced cytotoxic activity can be prepared by methods known to those skilled in the art.

  The origin of CDR in the humanized antibody is not particularly limited and may be derived from any animal. For example, sequences of mouse antibody, rat antibody, rabbit antibody, camel antibody and the like can be used, but the CDR sequence of mouse antibody is preferable.

  After obtaining the DNA encoding the variable region of the anti-CD40 humanized antibody of interest, this is incorporated into an expression vector containing DNA encoding the desired human antibody constant region.

  In order to produce the anti-CD40 antibody used in the present invention, an antibody gene is incorporated into an expression vector so as to be expressed under the control of an expression control region such as an enhancer or promoter. Next, host cells are transformed with this expression vector to express the antibody.

  The expression of the antibody gene may be achieved by separately incorporating polynucleotides encoding light or heavy chains into an expression vector and co-transforming host cells, or by using a single polynucleotide encoding light and heavy chains. The host cell may be transformed by incorporating into the expression vector (WO 94/11523).

  In humanization of antibodies, it is usually difficult to perform humanization while maintaining the functional activity of the antibody from which it was derived (for example, it does not affect cell growth). We succeeded in obtaining a humanized antibody having functional activity similar to the mouse antibody. Since humanized antibodies have reduced antigenicity in the human body, they are useful when administered to humans for therapeutic purposes.

9. Anti-CD40 Antibody Conjugated with Cytotoxic Agent Another embodiment of the present invention is a conjugated antibody in which anti-CD40 antibody is bound to various molecules such as cytotoxic drugs and drugs.

  Examples of cytotoxic agents used in the present invention include duocarmycin, duocarmycin analogs and inducers, CC-1065, duocarmycin analogs based on CBI, and duocarmycins based on MCBI. Analogs, duocarmycin analogs based on CCBI, doxorubicin, dolastatin, maytansine, maytansine analogs, DM1, DM2, DM3, DM4, DMI, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP) Monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), methotrexate, metopterin, dichloromethotrexate, 5-fluorouracil, mitomycin C, mitomycin A, carminomycin, aminopterin, vincristine, taxol, taxotere retinoic acid, butyric acid, N8- It can be mentioned cetyl spermidine and camptothecin like, but is not limited thereto.

  The anti-CD40 antibody conjugated with the cytotoxic agent in the present invention can be produced by binding the above-mentioned drug and the anti-CD40 antibody and using a known method. The antibody and the drug may be directly bonded via a linking group or the like possessed by themselves, or may be indirectly bonded via a linker or other substance.

  Examples of the linking group when the drug is directly bonded include a disulfide bond using an SH group and a bond via maleimide. For example, the intramolecular disulfide bond in the Fc region of the antibody and the disulfide bond of the drug are reduced, and both are bonded by a disulfide bond. There is also a method via maleimide. Another method is to introduce cysteine into the antibody by genetic engineering.

  It is also possible to bind the antibody and the drug indirectly via another substance (linker). The linker preferably has one or more functional groups that react with the antibody or drug or both. Examples of functional groups include amino groups, carboxyl groups, mercapto groups, maleimide groups, pyridinyl groups, and the like.

  Examples of linkers include sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC), N-succinimidyl 4- (maleimidomethyl) cyclohexanecarboxylate (SMCC), γ- Maleimidobutyric acid N-succinimidyl ester (GMBS), ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N- (α-maleimidoacetoxy) -succinimide Ester (AMAS), N-succinimidyl 4- (p-maleimidophenyl) -butyrate (SMPB), and N- (p-maleimidophenyl) isocyanate (PMPI), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4 (2-pyridylthio) pentanoate (SPP) and N-succinimidyl (4-iodo-acetyl) aminobenzoic acid ester (SIAB) and the like are exemplified, but not limited thereto. The linker may be, for example, a peptide linker such as valine-citrulline (Val-Cit) or alanine-phenylalanine (Ala-Phe), or the linkers listed above may be used in appropriate combination. good.

  With regard to the method of binding a drug to an antibody, for example, Cancer Research; 68 (22) 9280 (2008), Nature Biotechnology; 26 (8) 925 (2008), Bio Conjugate Chemistry; 19, 1673 (2008), Cancer Research; 68 (15) 6300 (2008) or according to the method described in JP-T-2008-516896.

Ten. Anti-CD40 Antibody Conjugated with Radioisotope Another embodiment of the present invention includes a conjugated antibody in which a radioisotope is labeled with an anti-CD40 antibody. When used as an antitumor agent, cytotoxic radioactive metal elements are preferred. Examples of such cytotoxic radioactive metal elements include yttrium 90 (90Y), rhenium 186 (186Re), rhenium 188 (188Re), copper 67 (67Cu), iron 59 (59Fe), strontium 89 (89Sr), gold 198 (198Au), mercury 203 (203Hg), lead 212 (212Pb), dysprosium 165 (165Dy), ruthenium 103 (103Ru), bismuth 212 (212Bi), bismuth 213 (213Bi), holmium 166 (166Ho), samarium 153 ( 153Sm) and lutetium 177 (177Lu). Among these radioactive metal elements, 90Y, 153Sm, and 177Lu are preferable from the viewpoints of half-life, radiation energy, easy labeling reaction, labeling rate, and complex stability.

  In order to bind these radioactive metals to the anti-CD40 antibody, it is preferable to react the metal chelate reagent with the antibody and react with the radioactive metal element to form a complex. In the conjugate antibody thus obtained, the radioactive metal element is bound to the anti-CD40 antibody via a metal chelating reagent.

  Examples of metal chelating reagents used for such complex formation include (1) 8-hydroxyquinoline, 8-acetoxyquinoline, 8-hydroxyquinaldine, norfloxacin, which is a quinolone compound having a quinoline skeleton, ofloxacin, and spall. Quinoline derivatives such as floxacin; (2) compounds such as chloranilic acid, aluminone, thiourea, tetraphenylarsonium chloride; (3) ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), and similar skeletons Dihydroxyethylglycine, ethylenediaminediacetic acid, ethylenediaminedipropionate hydrochloride, isothiocyanobenzyl EDTA, isothiocyanobenzyl DTPA, methylisothiocyanobenzyl DTPA, cyclohexylisothiocyanobenzyl DTPA, Reimidopropylamidobenzyl EDTA, maleimidodecylamidobenzyl DTPA; (4) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), isothiocyanobenzyl DOTA, etc. Can be mentioned, but is not limited to this.

  Among these metal chelate reagents, isothiocyanobenzyl DOTA, methylisothiocyanobenzyl DTPA, and cyclohexylisothiocyanobenzyl DTPA are easy to introduce metal chelates into antibodies, labeling rate, complex stability, etc. preferable.

  The binding of the radioactive metal element to the anti-CD40 antibody can be performed according to a conventional method. For example, the reaction can be performed by reacting an anti-CD40 antibody with a metal chelate reagent, preparing a labeling precursor in advance, and then reacting with a radioactive metal element.

11. Pharmaceutical Composition In the present invention, examples of the pharmaceutical composition for a tumor expressing CD40 include a composition containing the anti-human CD40 monoclonal antibody or an antibody-binding fragment thereof. Such a pharmaceutical composition preferably includes a pharmaceutically acceptable diluent or carrier in addition to the antibody, and may be a mixture with other antibodies or other agents such as antibiotics. Good. Suitable carriers include, but are not limited to, physiological saline, phosphate buffered saline, phosphate buffered saline glucose solution, and buffered saline. The routes of administration are oral routes and parenteral routes including intravenous, intramuscular, subcutaneous and intraperitoneal injection or delivery.

  In this case, the effective amount administered as a combination of an effective amount of the antibody of the present invention and a suitable diluent and a pharmaceutically usable carrier is 0.1 mg to 100 mg per kg body weight at a time, from 2 days It is administered at 8 week intervals.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

Example 1
Amino acid information of human CD40 With reference to SEQ ID NO: 1 (Genbank accession number NP_001241, FIG. 1), a gene encoding soluble CD40 from the full length to amino acid residues 1-173 was artificially synthesized. Full length CD40 was inserted into the pCXN3 vector. In order to produce soluble CD40-Fc, a gene encoding CD40 at amino acid residues 1-173 was inserted immediately before the Fc gene into a pCXN3 vector in which a gene of Fc region derived from mouse IgG2a was incorporated. Vector inserted with gene encoding full-length CD40 introduced into 293T / 17 (ATCC No. CRL-11268), BA / F3 (DSMZ No. ACC-300), or NIH / 3T3 (ATCC No. CRL-1658) cells Then, a stable expression strain was obtained by culturing in the presence of G418 drug. Expression of the full-length CD40 expression strain was confirmed by FACS analysis using a PE-labeled anti-human CD40 antibody (Santa Cruz). The soluble CD40-Fc vector was transiently introduced into 293T / 17. Using the culture supernatant as a sample, HRP-labeled mouse Fc antibody (Jackson ImmunoResearch) was used to confirm that the soluble CD40-Fc protein was secreted in the supernatant. Thereafter, the protein was purified by passing through a Protein G sepharose column (GE), and the soluble CD40-Fc protein was replaced with PBS. It was confirmed that the soluble CD40-Fc was purified by Western blotting using SDS-PAGE and anti-human CD40 antibody (BD).

Example 2
The immunization to mice and the selection of hybridomas will be described. The homology between human CD40 and mouse CD40 is not high as shown in FIG. 2, and it is considered that antibodies against human CD40 can be obtained by immunizing mice. Immunization was started from 6 to 8 weeks of age using Balb / c mice as immunized animals. The immunization was performed using three immunogens: 1) 293T / 17 or 2) NIH / 3T3, which is a stable expression strain of full-length CD40, and 3) soluble CD40-Fc.

In immunization using cells, the initial immunization was administered with 100 ng / head of pertussis toxin as an adjuvant and 1 × 10 ⁷ cells / head of full-length CD40 expression strain 293T / 17 or NIH / 3T3 intraperitoneally. Two weeks later, the same cells as in the first immunization were immunized with 1 × 10 ⁶ cells / head under conditions without an adjuvant. Thereafter, a total of 4 doses were given at 1 week intervals. Soluble CD40-Fc was given as the first immunization, and CD40-Fc was intraperitoneally administered with 50 μg / head, emulsified with pertussis toxin 100 ng / head and Freud's complete adjuvant. Two weeks later, booster immunization was performed with CD40-Fc 25 μg / head emulsified with Freud's incomplete adjuvant. Thereafter, a total of 4 doses were given at 1 week intervals. Three days before cell fusion, final immunization was performed with full-length CD40-expressing strain BA / F3 at 1.5 × 10 ⁶ cells / head or soluble CD40-Fc 25 μg / head in PBS.

  Cell fusion was performed by extracting spleen cells, mixing mouse myeloma P3-X63Ag8U1, and using PEG1500 (Roche Diagnostics) according to the instruction manual. RPMI1640 medium was added to dilute PEG1500, and PEG1500 was removed by centrifugation. A cell pellet carefully suspended in 10% FBS / RPMI1640 medium was seeded in a 96-well plate at 100 μL / well and cultured overnight. On the next day, RPMI1640 medium (HAT medium) containing 10% FBS / 1 × HAT media supplement / 1 × BM-condimed H1 Hybridoma cloning supplement was added at 100 μL / well. Two to four days after the fusion day, half of the culture medium was replaced with fresh HAT medium, and screening was performed using the culture supernatant after 8 days.

  Screening is performed by FACS analysis using CD9 stable expression cell line and cancer cells IM-9 (ATCC No. CCL-159) and Raji (ATCC No. CCL-86), which characterize the expression of CD40 antigen, and CD40-Fc is fixed. Numerous hybridomas were obtained by phased ELISA. Positive clones were monocloned by the limiting dilution method.

Example 3
The preparation of monoclonal antibodies against human CD40 is described. Mouse ascites was performed to obtain each monoclonal antibody. Monocloned hybridomas were cultured and then collected by centrifugation. The collected hybridoma was washed twice with PBS. Ascites fluid was collected when Balb / c mice that had been adjuvanted with pristane and adjuvanted 1 to 2 weeks before administration of hybridomas were sufficiently ascites collected for 3 to 1.5 months after administration of 1 x 10 ⁷ hybridomas / head. It was collected. The antibody contained in the ascites was precipitated by 50% ammonium sulfate precipitation. The precipitate was dissolved in PBS, and IgG was purified on a Protein G Sepharose column. The antibody was eluted with 0.1M Glycine-HCl pH 2.7 and neutralized with neutralization buffer. Thereafter, the IgG subclass was identified using the Iso Strip mouse monoclonal antibody isotyping kit (Roche Diagnostics) after replacing with PBS. Table 1 shows immunogens and antibody subclasses for the obtained 25 hybridoma clones (PPAT-071-01 to 25).

  In any of the following examples including this example, as well as in the tables or figures shown as test results in the examples, each hybridoma clone producing the human anti-human CD40 monoclonal antibody of the present invention has a symbol. Named. In addition, the term “antibody” after the symbol means an antibody produced by each hybridoma, or a recombinant antibody produced by a host cell carrying an antibody gene isolated from the hybridoma. . In addition, the name of the hybridoma clone may represent the name of an antibody within a range that is clear from the context.

Example 4
Explain the cross-classification between cynomolgus monkey CD40 and mouse CD40 expressing cells. In order to examine cross-reactivity of various anti-CD40 monoclonal antibodies against different types of CD40, cell lines expressing cynomolgus monkey and mouse CD40 were prepared. From the information on the amino acid residues of the cynomolgus monkey CD40 gene (SEQ ID NO: 2, FIG. 3) and mouse CD40 (SEQ ID NO: 3, FIG. 2), DNAs encoding the respective genes were artificially synthesized. These genes inserted into the pCXN3 vector were introduced into CHO cells (ATCC No. CCL-61) and cultured in the presence of G418 drug to obtain a stable expression strain. FACS analysis using 5D12 antibody reported to cross-react with cynomolgus CD40 for cynomolgus monkey CD40-expressing CHO cells and anti-mouse CD40 antibody (eBioscience) for mouse CD40-expressing CHO cells It was confirmed that it was expressed. Each CD40-expressing CHO cell was reacted with an antibody, and the presence or absence of cross-reactivity was examined by FACS analysis.
The results are shown in Table 1. All antibodies did not cross-react with mouse CD40. Except for PPAT-071-12 and PPAT-071-25, it showed a cross-reactivity with cynomolgus monkey CD40.

Example 5
Explain the epitope classification by CD40 epitope-expressing cells. In order to investigate which epitopes on various CD40 monoclonal antibodies recognize various types of CD40, Ellmark et al. (Immunology 2002; 106: 456-463), known from CD40, shortened from the N-terminus of CD40. D1, D1B2, D2, D2B1, D3, D4 (shown in FIG. 3) were artificially synthesized with DNAs encoding genes with the C-terminal addition of VSV-G tags. These genes were inserted into the pCXN3 vector, and the respective vectors were introduced into 293T cells and expressed transiently. The expression of six types of partial length CD40 was confirmed by Western blotting using anti-VSV-G antibody using cell lysate as a sample. Each partial length CD40-expressing 293T cell was reacted with an antibody concentration of 1 μg / mL and subjected to FACS analysis. As shown in FIG. 4, it did not respond to the partial length CD40-expressing cells not containing the CD40 epitope recognized by the antibody. Table 2 shows a summary of the CD40 epitope of each antibody. The antibodies shown in the following 1) to 8) reacted only with D1 (293T expressed from the 21st amino acid of CD40 (mature CD40)).
1) PPAT-071-01
2) PPAT-071-02
3) PPAT-071-18
4) PPAT-071-20
5) PPAT-071-21
6) PPAT-071-22
7) PPAT-071-23
8) PPAT-071-24
The antibodies shown in 1) to 10) below reacted to D1B2 (293T expressed from the 38th amino acid of CD40).
1) PPAT-071-03
2) PPAT-071-06
3) PPAT-071-07
4) PPAT-071-08
5) PPAT-071-11
6) PPAT-071-14
7) PPAT-071-15
8) PPAT-071-16
9) PPAT-071-17
10) PPAT-071-19

  Among the selected hybridomas, PPAT-071-06 (NITE BP-01436), PPAT-071-07 (NITE BP-01437), PPAT-071-08 (NITE BP-01438), PPAT-071-11 (NITE BP -01716), PPAT-071-14 (NITE BP-01439), PPAT-071-16 (NITE BP-01440), PPAT-071-17 (NITE BP-01441) and PPAT-071-18 (NITE BP-01442) ) Was deposited at the National Institute of Technology and Evaluation Patent Microorganisms Deposit Center (2-5-8, Kazusa Kamashichi, Kisarazu, Chiba, Japan) (Deposit Date: NITE BP-01436 to NITE BP-01442 was 2012) October 18, NITE BP-01716 is October 10, 2013).

Example 6
A proliferation assay of mouse antibodies against human CD40 expressing cells (Ramos) is described. Using Ramos cells (ATCC No. CRL-1596), which is a B-cell lymphoma expressing CD40, it was examined whether cell proliferation was affected in the presence of antibodies. Ramos cells were prepared at 2.2 × 10 ⁶ cells / mL in 2% FBS / RPMI1640 medium and seeded on a 24 wells plate at 900 μL (2 × 10 ⁵ cells) / well. To this well, 10 μg / mL of an antibody solution prepared in the same medium was added at 100 μL / well. Thereafter, 200 μL was transferred to a 96-well plate at 100 μL / well (n = 2) for growth assay and cultured at 37 ° C. with 5% CO ₂ . As a control, a well containing only Ramos cells to which 10 μL / well of antibody-free medium was added and a well containing only the medium were applied as controls. Three days later, a 96-well plate was subjected to a proliferation assay using cell counting Kit-8 (Dojindo Laboratories). The growth rate of each antibody-added well was determined by assuming that the culture well of Ramos alone without adding the antibody was 100%. The result is shown in FIG. An antibody with a well (CD95 expression of 2 times or more) showing a growth rate of 70% or less was regarded as an antibody that suppresses proliferation. Many antibodies having CD40 epitopes at amino acid residues 21-37 or 38-61 of CD40 were found to inhibit proliferation. In addition, it has been clarified that many antibodies that suppress the growth of Ramos cells more strongly than the stimulating CD40 antibody G28-5 have been obtained.

Example 7
The antitumor effect in the Ramos transplantation model with anti-human CD40 antibody will be described. Six weeks old CB17 / Icr-scidJc1 mice (Claire Japan) were injected subcutaneously with 5 × 10 ⁶ Ramos cells as tumor cells per mouse. PPAT-071-06 or PPAT-071-18 was intravenously injected on the day the tumor shape reached an average of 230 mm ³ . The dose was 10 mg / kg of anti-CD40 antibody per mouse, and 5 mice each were administered twice a week. The average value of tumor size is shown in FIG. In the group that received 10 mg / kg of anti-CD40 antibody, the size of the tumor in all 5 animals was smaller than that at the first antibody administration, and anti-tumor effects were confirmed in PPAT-071-06 and PPAT-071-18 It was.

Example 8
The cloning of the variable region of the anti-human CD40 antibody will be described. Using total RNA prepared from each hybridoma cultured as a template, cDNA was synthesized using SuperScript III First Strand Synthesis System (Invitrogen) according to the attached instructions. Amplification of the heavy chain variable region was performed by PCR using ExTag polymerase (Takara Bio) as a template using a forward primer containing the degenerate sequence of the nucleic acid encoding the heavy chain signal sequence and a reverse primer specific to the heavy chain constant region. . Amplification of the light chain variable region was performed by PCR using ExTag polymerase as a template using a forward primer containing a degenerate sequence of a nucleic acid encoding a light chain signal sequence and a reverse primer specific to the light chain constant region. The amplified PCR product is purified from a single band of agarose gel electrophoresis using QIAquick Gel Extraction Kit (Qiagen), then cloned into pGEM-T easy vector (Promega), the base sequence is determined, The amino acid residues and CDRs of the chain variable region and light chain variable region were identified. Tables 3 and 4 show the CDR sequences of the light and heavy chains of the antibody having the CD40 epitope at amino acid residues 21-37 or 38-61 of CD40, respectively.

Example 9
Production of anti-CD40 humanized antibody is described. Human antibody sequence data published by IMGT was obtained, and consensus sequences were identified for light chain variable regions and heavy chain variable regions. The amino acid sequence of the resulting light chain variable region is shown in SEQ ID NO: 54, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 55. With reference to the amino acid residues in the framework regions of these human antibody consensus sequences, amino acid residues containing the CDRs of the antibodies shown in 1) to 7) below were transplanted to design humanized antibodies.
1) PPAT-071-06
2) PPAT-071-07
3) PPAT-071-08
4) PPAT-071-11
5) PPAT-071-14
6) PPAT-071-16
7) PPAT-071-17
In addition, a homology search for the PPAT-071-17 antibody was carried out separately from the light chain variable region and heavy chain variable region of PPAT-071-17 from NCBI's Protein BLAST, which was published separately. As a result, it was found that the light chain variable region had high homology with 1UJ3 (SEQ ID NO: 56) and the heavy chain variable region with 3NFP (SEQ ID NO: 57). With reference to amino acid residues in the framework regions of these human antibodies, amino acid residues containing each CDR of PPAT-071-17 were transplanted to design humanized antibodies. Tables 5-1 and 5-2 show the amino acid sequences of the light chain variable region and the heavy chain variable region for each designed humanized antibody.

  The DNA encoding the light chain variable region and heavy chain variable region genes of each humanized antibody designed was artificially synthesized. Expression by inserting the heavy chain variable region gene into the pCXN3 vector with the constant region gene derived from human IgG1, and the light chain variable region gene into the pCXN3 vector with the constant region gene derived from human κ chain The vector was introduced into FreeStyle-293F cells (In vitrogen) to produce each humanized antibody. Each humanized antibody produced was present in the culture supernatant and was confirmed to specifically bind to CD40 on Ramos cells. Humanized antibodies of hsPPAT-071-06, hsPPAT-071-07, hsPPAT-071-14, and hsPPAT-071-18 are further produced, purified on a Protein G column, and further purified on an anion column or gel filtration column And replaced with PBS. As in Examples 4 and 5, each purified humanized antibody was confirmed to have the same characteristics as the original mouse antibody in terms of CD40 epitope and cross-reactivity of heterologous animal CD40.

Example 10
A proliferation assay with humanized antibodies is described. Uses Ramos cells that express CD40 and MOLP-8 (DSMZ No.ACC-569), a multiple myeloma that does not express CD40, to affect cell growth in the presence of mouse and humanized antibodies I investigated. Both Ramos cells and MOLP-8 cells were prepared in 2% FBS / RPMI1640 medium at 3.3 × 10 ⁶ cells / mL, and seeded at 90 μL (3 × 10 ⁵ cells) / well in a 96 well plate. To this well, an antibody solution prepared in the same medium was added at 10 μL / well to a final concentration of 0.01 to 3.0 μg / mL, and cultured at 37 ° C. and 5% CO ₂ . As a control, a well containing only cells to which 10 μL / well of a medium containing no antibody was added and a well containing only the medium were used as controls. After 3 days, proliferation assay was performed using cell counting Kit-8. FIG. 7 summarizes the results showing the growth rate of wells with respect to each concentration of antibody added, with the wells without addition of antibody alone being 100% growth rate. Concentration dependence was confirmed for both the mouse antibody and the humanized antibody, and the growth rate of the humanized antibody was shown to be suppressed although some antibodies showed a slight increase compared to the mouse antibody.

Example 11
The antitumor effect in Ramos transplantation model with anti-CD40 humanized antibody is explained. Six weeks old CB 17 / Icr-scidJc1 mice (CLEA Japan) were injected subcutaneously with 5 × 10 ⁶ Ramos cells as tumor cells per mouse. The day when tumor shapes reached an average of 270 mm ³ , humanized antibodies of hsPPAT-071-06, hsPPAT-071-14, or hsPPAT-071-18 were injected intravenously. The dose was 10 mg / kg of anti-CD40 antibody per mouse, and 5 mice each were administered twice a week. The average value of the size of the tumor shape is shown in FIG. In all 5 animals in the group administered with humanized anti-CD40 antibody 10 mg / kg, the degree of tumor enlargement was delayed compared to the PBS-administered group, confirming the antitumor effect of the humanized antibody.

Example 12
The antitumor effect of an antibody conjugated with a cytotoxic drug will be described. The mouse antibody was chemically cross-linked with a cytotoxic agent to examine whether it had an antitumor effect on the cells. With reference to the crosslinking method of Singh et al. (Methods Mol Biol 2009; 525: 445-467), the anti-CD40 monoclonal antibody crosslinks DM1SH, which is one of the cytotoxic agents maytansinoids, Proliferation assays on Ramos cells in vitro were performed. Sulfo-SMCC (Sulfo-Succinimidyl-4- (N-Maleimidomethyl) Cyclohexane-1-Carboxylate, Pierce) was reacted with 1 mg / mL of antibody at a molar ratio of 30: 1 for 1 hour at room temperature. Then, in order to remove unmodified sulfo-SMCC, it passed through the simple desalting column (Thermo), and the antibody of MCC bridge | crosslinking was obtained. For the MCC-crosslinked antibody, the number of introduced maleimide groups was determined from the absorbance of A280 and 4 PSD. Thereafter, DM1SH was reacted overnight at room temperature at a mole number 1.7 times that of the maleimide group. Free DM1SH was removed as much as possible by passing twice through a simple desalting column. The DM1 concentration and antibody concentration of the final DM1-MCC-antibody were determined by measuring the absorbance of A254 and A223.

In order to examine cytotoxicity against CD40-expressing cells, Ramos cells and Raji cells that characterize the expression of CD40 antigen were selected as target cells, and MOLP-8, which does not show CD40 expression, was selected as a negative control. For cells, prepare 3.3x10 ⁴ cells / mL (Ramos), 5.5x10 ⁴ cells / mL (Raji), 11x10 ⁴ cells / mL (MOLP-8) in 10% FBS / RPMI1640 medium, and add 90μL / well to a 96-well plate. Sowing. DM1-MCC-antibody prepared in the medium was added at 10 μL / well to a final concentration of 0.0001 to 3 μg / mL and cultured for 3 days. Thereafter, a proliferation assay was performed using cell counting Kit-8. Table 6 shows the growth rate of each cell in the well when the DM1-MCC-antibody concentration is 0.1 μg / mL, with the well containing no antibody added as a 100% growth rate. In this culture condition, intact antibodies do not inhibit growth. Moreover, the sensitivity with respect to DM1 of each cell was substantially equivalent (FIG. 9). For PPAT-071-06, PPAT-071-07, and PPAT-071-18, cell proliferation rates are shown in FIGS. 10 to 12 with respect to the added DM1-MCC-antibody concentration. Although the DM1-MCC-antibody solution still contains uncrosslinked DM1SH for growth, the cytotoxic activity of MOLP-8 is observed at a high concentration, but at 0.1 μg / mL, MOLP-8 has a cytotoxic activity. No cytotoxic activity was observed against Ramos and Raji expressing CD40. That is, it was shown that an anti-CD40 antibody conjugated with a cytotoxic drug can specifically kill CD40-expressing cells.

  The antibody of the present invention can be used as an antitumor agent.

Claims (16)

An anti-human CD40 monoclonal antibody or an antigen-binding fragment thereof having antiproliferative activity in the absence of effector cells against human CD40 antigen-expressing cells,
a) specifically binds to an epitope comprising amino acid residues 21-37 of the human CD40 amino acid sequence (SEQ ID NO: 1) or b) to an epitope comprising amino acid residues 38-61 of the human CD40 amino acid sequence (SEQ ID NO: 1) An anti-human CD40 monoclonal antibody or an antigen-binding fragment thereof. The light chain variable region and the heavy chain variable region are the amino acid sequences shown in any one of 1) to 16) below, or one or more amino acids in the amino acid sequence are substituted, deleted, added or inserted, The anti-human CD40 monoclonal antibody or antigen-binding fragment thereof according to claim 1, having an amino acid sequence having a level of at least 90%.
1) SEQ ID NO: 5 and SEQ ID NO: 30,
2) SEQ ID NO: 6 and SEQ ID NO: 31,
3) SEQ ID NO: 9 and SEQ ID NO: 34,
4) SEQ ID NO: 10 and SEQ ID NO: 35,
5) SEQ ID NO: 11 and SEQ ID NO: 36,
6) SEQ ID NO: 14 and SEQ ID NO: 39,
7) SEQ ID NO: 17 and SEQ ID NO: 42,
8) SEQ ID NO: 18 and SEQ ID NO: 43,
9) SEQ ID NO: 19 and SEQ ID NO: 44,
10) SEQ ID NO: 20 and SEQ ID NO: 45,
11) SEQ ID NO: 21 and SEQ ID NO: 46,
12) SEQ ID NO: 22 and SEQ ID NO: 47,
13) SEQ ID NO: 24 and SEQ ID NO: 49,
14) SEQ ID NO: 25 and SEQ ID NO: 50,
15) SEQ ID NO: 26 and SEQ ID NO: 51, and
16) SEQ ID NO: 27 and SEQ ID NO: 52 The light chain variable region and the heavy chain variable region are each amino acid sequences shown below, or one or more amino acids in the amino acid sequence are substituted, deleted, added or inserted, and the level of identity is at least 90% The anti-human CD40 monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, which has the amino acid sequence as described above.
1) SEQ ID NOs: 9 and 34,
2) SEQ ID NOs: 10 and 35,
3) SEQ ID NOs: 11 and 36,
4) SEQ ID NOs: 14 and 39,
5) SEQ ID NOs: 17 and 42,
6) SEQ ID NOs: 19 and 44,
7) SEQ ID NO: 20 and 45, or 8) SEQ ID NO: 21 and 46   Hybridoma cells deposited with NITE under accession numbers NITE BP-01436, NITE BP-01437, NITE BP-01438, NITE BP-01716, NITE BP-01439, NITE BP-01440, NITE BP-01441, and NITE BP-01442 Stocks of PPAT-071-06, PPAT-071-07, PPAT-071-08, PPAT-071-11, PPAT-071-14, PPAT-071-16, PPAT-071-17, and PPAT-071-18 The anti-human CD40 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which is a monoclonal antibody or an antigen-binding fragment thereof obtained from any of the above.   The anti-human CD40 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which is derived from a mouse.   The anti-human CD40 monoclonal antibody or antigen-binding fragment thereof according to claim 1, which is a humanized antibody.   The anti-human CD40 monoclonal antibody according to any one of claims 1 to 5, which is an antibody humanized using the amino acid sequence containing the complementarity determining region CDR of the anti-human CD40 monoclonal antibody and the amino acid sequence of human immunoglobulin. Human CD40 monoclonal antibody or antigen-binding fragment thereof. An amino acid sequence in which the light chain variable region and the heavy chain variable region are each shown in any of the following, or one or more amino acids are substituted, deleted, added or inserted in the amino acid sequence, and the level of identity is at least 90% The anti-human CD40 monoclonal antibody or antigen-binding fragment thereof according to claim 6 or 7 having the amino acid sequence as described above.
1) SEQ ID NOs: 58 and 59,
2) SEQ ID NOs: 60 and 61,
3) SEQ ID NOs: 62 and 59,
4) SEQ ID NOs: 63 and 64
5) SEQ ID NOs: 65 and 66,
6) SEQ ID NOs: 60 and 67,
7) SEQ ID NO: 68 and 61, or 8) SEQ ID NO: 69 and 70   The anti-human CD40 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein a cytotoxic drug or drug is bound thereto.   The anti-human CD4 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, which is labeled with a radioisotope.   A pharmaceutical composition comprising the anti-human CD40 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.   The antitumor agent with respect to the tumor which expresses the human CD40 antigen which contains the anti-human CD40 monoclonal antibody in any one of Claims 1-10, or its antigen binding fragment as an active ingredient.   The hybridoma which produces the anti-human CD40 monoclonal antibody in any one of Claims 1-4.   The anti-human CD40 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, for treating a tumor expressing a human CD40 antigen.   Use of the anti-human CD40 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 for the manufacture of a therapeutic agent for a tumor expressing a human CD40 antigen.   A method for treating a tumor expressing a human CD40 antigen, comprising administering the anti-human CD40 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 to a patient in need thereof.

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