ES2359854T3 - ANTI-CD20 MONOCLONAL ANTIBODY. - Google Patents

Abstract

A murine anti-CD20 monoclonal antibody that has cell growth inhibitory activities that include apoptosis against cells expressing the human CD20 antigen in the culture of cells expressing the CD20 antigen without effector cells, where the amino acid sequences of the variable region of chain H and the variable region of chain L are SEQ ID NOS: 1 and 7.

Description

Technical Field

The present invention relates to a monoclonal antibody directed to the human CD20 antigen. The present invention is further related to a chimeric anti-CD20 monoclonal antibody and a humanized anti-CD20 monoclonal antibody produced by gene recombination, as well as a therapeutic agent for a B cell mediated tumor or an immune disease containing any of these antibodies as an active ingredient.

10 Background Technique

Monoclonal antibodies are known that recognize the CD20, B1, 2B8 antigen (the name of the chimeric antibody is rituximab), 1F5, 2H7 and so on. Above all, rituximab, a chimeric anti-CD20 monoclonal antibody developed by IDEC Pharmaceuticals Corporation, U.S., has established itself as a standard therapeutic agent for low-grade non-Hodgkin lymphoma (NHL), and is found to have an effect

Therapeutic in many B-cell mediated immune diseases. For example, it is said to be effective for, in addition to malignant tumors such as chronic lymphatic leukemia, autoimmune diseases in which a pathogenic autoantibody appears to be involved such as autoimmune and purple hemolytic anemia. idiopathic thrombocytopenic, and inflammatory diseases such as chronic rheumatoid arthritis and multiple sclerosis (Non-patent documents 14 to 17).

20 CD20 is a molecule present on the cell surface of B lymphoma and its expression is seen in normal B cells in peripheral blood, spleen, tonsil and bone marrow and so on, as well as B cells in most malignant tumors. This molecule, comprising 297 amino acid residues, penetrates a cell membrane four times, and has the C-terminal and the N-terminal inside the cell, and has only the extracellularly exposed loop with a sugar-free chain consisting of 43 amino acid residues between the third and fourth domains of

25 transmembrane (Non-patent documents 1 and 9). It is considered that the CD20 molecule usually exists as a tetramer, and additionally forms a heterocomplex with other minor components (Non-patent document 18). Because the CD20 protein is not secreted from the cell or divided, and additionally, it is taken strongly within the cell by binding the antibody, a cytotoxic mechanism based on an antibody directed against a target cell can be expected to work effectively. (Non-patent documents 1 to 3).

30 Despite its small molecular size, CD20 shows epitope diversity partially due to the effect of its expression form as a complex outside the cell, and the binding of antibodies to its various different mediated biological responses. For example, activities such as regulation by decreasing B cell receptors, increasing expressions of MHC class II antigens and adhesion molecules, activating Ca2 + released in the presence of hyper-crosslinking, inhibiting antigen 1 associated with the function of

Non-homotypic adhesion-dependent lymphocyte, induction of apoptosis and the opposite activity, promotion of cell growth, vary significantly (Non-patent documents 4 to 13). Typical examples of the anti-CD20 antibody, rituximab, B1, 1F5 and 2H7, also have different biological characteristics and functions, and a reference to a "monoclonal antibody that binds to CD20" does not specify only its biological properties.

The molecule that constitutes the extracellular domain of CD20 is insoluble. Although the CD20 molecule derived from a

Cellular lysate or as a recombinant gene protein can be solubilized by using a strong surfactant or alkali, it is difficult to maintain the natural three-dimensional structure under such a treatment condition. Therefore, a CD20 positive B cell is used as an immunogen to obtain antibodies. However, its immunostimulatory property is weak, and it is not easy to obtain clones of cells that produce the mature antibody.

As of 2005, rituximab, a chimeric mouse / human antibody, is only the anti-CD20 monoclonal antibody

45 approved as a therapeutic agent. Because chimeric molecules with heterologous molecules have antigenicity, they are not generally preferred as therapeutic agents. However, antiCD20 antibodies have a property of objectifying and eliminating all B cells that include normal cells, and therefore they are said to have substantially no antigenicity. However, examples have been reported in which a neutralizing antibody is induced during the treatment period, although they count only several percentages, and

50 would more easily be induced depending on the dose and dosage period. Therefore, the development of a humanized antibody having a sequence more closed to that of the human or a human antibody is desired. Another disadvantage of the chimeric antibodies is the short life of the blood, and the useful life β is only 3 or 4 days. The effective rate of rituximab alone against recurrence of low-grade NHL is much less than 50% in a clinical study with the United States, which indicates that 50% or more of patients do not respond or respond poorly to rituximab. The response rate in patients with moderate NHL malignancy is even lower, being only approximately 30% (Non-patent document 14). Therefore, it is necessary to investigate the factors and background of the different responses in patients, and at the same time the development of an antibody that has a superior effect is desired.

5 Non-patent document 1: Leukocyte Fact Book 2nd Edition, Academic Press Non-patent document 2: Stashenko P et al., J. Immunol., 1980, 125: 1678-85 Non-patent document 3: Anderson KC et al., Blood, 1984, 63: 1424-33 Non-patent document 4: Shan D et al., Blood, 1998, 91: 1644-52 Non-patent document 5: Flieger D et al., Cell Immunol., 2000, 204: 55-63

10 Non-patent document 6: Mathas S et al., Cancer Res., 2000, 60: 7170-6 Non-patent document 7: Cardarelli PM et al., Cancer Immunol. Immunother., 2002, 51: 15-24 Non-patent document 8: Pedersen IM et al., Blood, 2002, 99: 1314-9 Non-patent document 9: Deans JP et al., Immunol., 2002, 107: 176- 82 Non-patent document 10: Golay JT et al., J. Immunol., 1992, 149: 300-8

15 Non-patent document 11: Bourger I et al., Eur. J. Immunol., 1993, 23: 768-71 Non-patent document 12: White MW et al., J. Immunol., 1991, 146: 846-53 Document Patent No. 13: Shan D et al., Cancer Immunol. Immnother., 2000, 48: 673-83 Non-patent document 14: Coiffier B et al., Blood, 1998, 92: 1927-32 Non-patent document 15: Edward JC et al., Rheumatology (Oxford), 2001, 40: 205-11

20 Non-patent document 16: Zaja F et al., Heamatologica, 2002, 87: 189-95 Non-patent document 17: Perrotta S et al., Br. J. Haematol., 2002, 116: 465-7 Non-patent document 18 : Polyak MJ et al., Blood, 2002, 99: 3256-62 WO 2005/000901 describes monoclonal antibodies that bind to CD20 and pharmaceutical compositions that

They contain them.

25 Davies A.J. et al (Q. J. Nucl. Med. Mol. Imaging (2004) Vol 48 (4) 305-316) describes the administration of iodine [131I] tositumomab that targets the CD20 antigen. WO 03/068821 describes human, chimeric and humanized anti-CD20 antibodies and fusion proteins of the

CD20 antibody that bind to CD20. Uchida et al (J. Exp. Med. (2004) Vol 199, pages 1659-1669 discusses the mechanisms involved in the

30 anti-CD20 antibody immunotherapy. Description of the Invention An object of the present invention is to provide a monoclonal antibody that has biological functions.

superior to those of conventional anti-CD20 monoclonal antibody therapeutic agents. The inventors of the present invention obtain murine anti-CD20 monoclonal antibodies that specifically bind to human CD20 antigen by using two or more B cell strains positive for CD20 antigen, mammalian cells made biotechnologically to express human CD20 antigen in their cell membranes, and the human CD20 protein fused with the glutathione S-transferase (GST) protein in a combination

arbitrary as an immunogen. Some of them have direct cell growth inhibitory activities that include apoptosis in a cell culture that expresses CD20 in vitro without effector cells. Additionally, regardless of the presence or absence of cell growth inhibitory activities such as apoptosis, these antibodies, which include other selected murine anti-CD20 monoclonal antibodies, are imparted

5 with antibody-dependent cell complement or cell-mediated cytotoxicity by chimerization. By humanizing the amino acid sequences of the antibodies determined by having the most desirable biological activities among them, the anti-CD20 monoclonal antibodies that can be used as a therapeutic agent are prepared. Thus, the present invention has been carried out.

The present invention provides the following.

10 (1) A murine anti-CD20 monoclonal antibody that has cell growth inhibitory activities that include apoptosis against cells expressing the human CD20 antigen in the culture of cells expressing the CD20 antigen without effector cells, where the amino acid sequences of the variable region of chain H and the variable region of chain L are SEQ ID Nos: 1 and 7.

(2) A hybridoma that produces the anti-CD20 monoclonal antibody according to (1).

15 (3) A chimeric anti-CD20 monoclonal antibody, where the amino acid sequence of the variable region of the anti-CD20 monoclonal antibody is fused according to (1) and the amino acid sequence of the human immunoglobulin constant region.

(4) A humanized anti-CD20 monoclonal antibody using the region's amino acid sequence

Complementarity determinant (CDR) of the variable region of the anti-CD20 monoclonal antibody according to (1) and a human immunoglobulin amino acid sequence.

(5)

The anti-CD20 monoclonal antibody according to any one of (3) to (4), which has cytotoxicity against cells expressing the CD20 antigen in the presence of a human complement.

(6)

A mammalian cell incorporated with a nucleotide sequence encoding the amino acid sequence of the anti-CD20 monoclonal antibody according to any one of (3) to (5).

25 (7) The mammalian cell according to (6), which is a Chinese hamster ovary (CHO) cell.

(8) A diagnostic agent comprising the anti-CD20 monoclonal antibody according to any one of (1)

or (3) to (5) as an active ingredient.

(9) A therapeutic agent comprising the anti-CD20 monoclonal antibody according to any one of (3) to (5) as an active ingredient.

The amino acid residues in the amino acid sequences of the monoclonal antibodies defined above can be replaced with other amino acid residues as much as the secondary structures and their biological properties are not significantly altered, and such monoclonal antibodies from which the sequences of the amino acids are changed. Amino acid, as mentioned above, falls within the scope of the present invention.

Brief Description of the Drawings

35 [Fig. 1] Structure of a vector for the expression of a recombinant antibody, pNOW-Ab.

[Fig. 2] Structure of a vector for the expression of a protein, pNOW-Ag.

[Fig. 3] Sequences of the primers for the cloning of the human CD20 gene.

[Fig. 4] Amino acid sequences of the H-chain and L-chain variable regions of murine anti-CD20 monoclonal antibodies.

40 [Fig. 5] Results of apoptosis test using murine anti-CD20 monoclonal antibodies.

[Fig. 6] Results of the cell growth inhibition test using murine anti-CD20 monoclonal antibodies.

[Fig. 7] Complement-dependent cytotoxicity test results using chimeric anti-CD20 monoclonal antibodies.

[Fig. 8A] Amino acid sequences of the H-chain and L-chain variable regions of humanized anti-CD20 monoclonal antibodies and the nucleotide sequences corresponding thereto.

[Fig. 8B] Amino acid sequences of the H-chain and L-chain variable regions of humanized anti-CD20 monoclonal antibodies and the nucleotide sequences corresponding thereto.

5 [Fig. 9] Results of the cell growth inhibition test using humanized anti-CD20 monoclonal antibodies.

Best Mode to Carry Out the Invention

In the present invention, the term "antibody" is used in a meaning that encompasses the antibody in the general meaning, the H chain and the L chain that constitute them, and their fragments.

The present invention relates to an anti-CD20 monoclonal antibody that binds to the human CD20 antigen in a cell membrane and has desirable biological activities to induce a therapeutic effect.

The antibody according to a first embodiment of the present invention is a monoclonal antibody that specifically binds to the human CD20 antigen in a cell membrane and has cell growth inhibitory activities that include apoptosis against cells expressing the human CD20 antigen in the culture of 15 cells expressing the CD20 antigen without the help of effector cells. This is originally a murine anti-CD20 monoclonal antibody, and additionally includes an anti-CD20 monoclonal antibody obtained by chimerizing or humanizing that antibody. These antibodies have direct cell growth inhibitory activities that include apoptosis against cells expressing human CD20 antigen in vitro culture of cells expressing CD20 antigen without the help of effector cells. These anti monoclonal antibodies

20 humanized or chimerized CD20s have a cell-mediated cytotoxicity dependent on antibody and / or complement.

The binding property for a CD20 antigen on a cell membrane can be examined by ELISA for cells, in which cells expressing CD20 such as SB cells and Raji cells adhere to a plate and react with a monoclonal antibody that It will be tested. However, because the levels of CD20 antigen expression of these cells are insufficient, the reactivity is not high. Therefore, in the present invention, an ELISA method is performed for cells, in which the CHO cells in which the CD20 is expressed in a large amount by gene recombination (CD20 / CHO cells) adhere to a plaque and reacted with a monoclonal antibody to be tested. In a preliminary test of the present invention, it is confirmed that the ELISA for cells using CD20 / CHO cells shows a pattern similar to that observed

30 in ELISA for cells using SB cells or Raji cells in a reactivity test of a monoclonal antibody, and shows high sensitivity (see example, Checking ELISA detection method for CD20 / CHO cells, Table 1).

Direct cell growth inhibitory activities can be determined in an in vitro culture of cells expressing human CD20 antigen without effector cells by a usual method (Miyamoto T et al., Avian 35 Dis., Vol. 46 (1), 10-16). Additionally, the capacity that induces apoptosis can be determined by a test using flow cytometry (annexin V staining / propidium iodide (PI)).

Examples of the antibody according to the first embodiment include mouse anti-CD20 monoclonal antibodies having a combination of the amino acid sequences of SEQ ID NOS: 1 and 7, for the H chain variable region and the variable chain region L, as well as the anti-CD20 monoclonal antibodies obtained by chimerizing or humanizing those antibodies. These antibodies exhibit direct cell growth inhibitory activities that include apoptosis against cells expressing human CD20 antigen in vitro culture of cells expressing CD20 antigen without the help of effector cells. These antibodies also have antibody-dependent and / or complement-dependent cell-mediated cytotoxicity. The present invention also includes a hybridoma that produces a murine antibody, and a mammalian cell (CHO cell in the examples)

Incorporated with a nucleotide sequence corresponding to any one of the amino acid sequences of the chimeric or humanized antibodies.

Chimerization is carried out by fusing the amino acid sequence of the murine H-chain variable region of a murine monoclonal antibody and the H-chain constant region amino acid sequence of human immunoglobulin, and the amino acid sequence of the variable region of L chain and the 50 amino acid sequence of the human immunoglobulin L chain constant region (Ishida T et al., Nippon Rinsho, Vol. 60, No 3, 439-444). Humanized antibodies are designed using an amino acid sequence of the CDR variable region of a murine monoclonal antibody and a human immunoglobulin amino acid sequence. Preferred humanized anti-CD20 monoclonal antibodies as therapeutic agents are selected at

compare the characteristics of several designated antibodies (Padlan EA, Mol. Immunol., Vol. 28, No 4/5, 489-498; Wu TT and Kabat EA, Mol. Immunol., Vol. 29, No 9, 1141-1146 ; Padlan EA et al., FASEB J., Vol. 9, 133-139).

Humanized or chimerized anti-CD20 monoclonal antibodies have additional complement dependent cytotoxicity (CDC), and antibody dependent cell mediated cytotoxicity (ADCC) in the presence

5 of effector cells. For the test methods for these CDC and ADCC, the commonly used methods are referred to as (Manches O et al., Blood, 2003, 101 (3), 949-54; Idusogie EE et al., J. Immunol., 2000 , 164, 41784184).

the chimeric anti-CD20 monoclonal antibodies and the humanized anti-CD20 monoclonal antibodies described as the antibodies of the first embodiment can be expected to exhibit a superior effect as a therapeutic agent for B cell mediated malignant tumors and immunological diseases in which the cells are involved B or the antibodies produced by B cells, and an object of the present invention is to use them in the development of a therapeutic agent containing a humanized or chimeric anti-CD20 monoclonal antibody, desirably a humanized anti-CD20 monoclonal antibody, as an active ingredient . Examples of target diseases include non-Hodgkin lymphoma, Hodgkin lymphoma, chronic lymphatic leukemia, leukemia

Acute lymphatic, chronic rheumatoid arthritis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, systemic lupus erythematosus, anti-phospholipid antibody syndrome, Sjogren's syndrome, Crohn's disease, scleroderma, multiple sclerosis, type I diabetes, and so established.

The murine monoclonal antibody of the present invention can be prepared by the following method.

As an immunogen for sensitization, the SB cell or the Raji cell can be used as a cell strain that expresses the human CD20 antigen, and the CHO cell made to express the human CD20 antigen in combination. Additionally, a human CD20 protein fused with GST (GST-CD20) can be used as a complementary sensitizing antigen.

A hybridoma that produces a monoclonal antibody can be prepared by a series of procedures that include (1) immunization of an animal to be immunized (mouse), (2) lymphocyte preparation of the immunized animal, 25 (3) preparation of progenitor cells, (4) cell fusion of lymphocytes and progenitor cells, (5) detection and

(6) cloning (Biochemistry Experiment Method: Monoclonal Antibody, written by Ailsa M. Campbell, translated by Toshiaki Osawa, Tokyo Kagaku Dozin Co., Ltd., 1989). An anti-CD20 monoclonal antibody that specifically binds to the CD20 antigen on a cell surface can be cloned by reacting a monoclonal antibody to be tested with an ELISA system for cells in which CD20 / CHO cells are immobilized in a license plate. Commercially available expression vectors can also be used. However, because the CD20 antigen needs to be expressed in the CHO cell at a high density, a high mammalian cell expression vector, pNOW (Japanese Patent No. 3582965) can be used. A selection criterion of the monoclonal antibody is the display of reactivity comparable to or greater than a positive control.

A chimeric or humanized antibody can be prepared according to a usual gene recombination method. For example, pNOW-Ab, which contains 2 sets of multicloning sites positioned in tandem to produce the antibody, and is previously incorporated with the genes encoding the human H chain and the constant L chain region, can be used as a vector of expression (Fig. 1).

[Example 1]

Preparation, chimerization and humanization of the monoclonal antibodies directed to the CD20 antigen as well as the test for characteristics of the antibodies obtained will be explained below with reference to the examples.

(1) Preparation of immunogen to sensitize mice

The human CD20 gene is obtained from a cDNA collection by using a 5 'primer of SEQ ID NO: 13 and

A 3 'primer of SEQ ID NO: 14, which are specific to the gene encoding the total human CD20 molecule (Multiple Choice cDNA human spleen, Origene Technologies, Inc., 6 Taft Court, Suite 100, Rockville, MD 20850). Specifically, the primers shown in Figure 3 are used. The CD20 gene is incorporated into pNOW-Ag (Fig. 2) as a high-expression vector for mammalian cells, and is transfected into CHO cells as the host cells. Recombinant CHO cells (CD20 / CHO cells) that express CD20 molecules at a high level on their cell surfaces are established by FACS analysis. Cells that show 5 or more times of higher fluorescence intensity compared to the SB cell in stained with FITC labeled antiCD20 monoclonal antibodies are defined as those that show high expression. GST-CD20, the complementary immunogen, is prepared by fusing GST into the N-terminal of 43 amino acid residues of the CD20 extracellular domain by using the vector pGEX-4T2 (G et al. AM, Electrophoresis, Vol. 20 (2): 344 -348).

(2)

Immunogen Preparation

SB cells or Raji cells are grown in 10% RPMI 1640 medium added to FCS. CD20 / CHO cells are grown in CHO-S-SFM II medium (GIBCO, Cat. No. 12052-098) added to 800 µg / ml of G418. These cultures are centrifuged (1100 rpm, 5 minutes), then cells with Dulbecco PBS (-) are added and suspended, and the suspension is centrifuged again. This washing procedure is repeated once again, and a suspension prepared by adding physiological saline to the cells (cell count: 1 to 3 x 107 / ml) is used for immunization. The pGEX-4T2 incorporated with GSTCD20 is introduced into competent E. coli cells. The competent cells are lysed after culture, and GST-CD20 is crudely purified from the lysed cells, and then solubilized by the addition of 0.1 N sodium hydroxide.

(3)

Immunization and cell fusion

As the animals to be immunized, female mice 7 to 11 weeks old are used. SB cells, Raji cells or CD20 / CHO cells are repeatedly administered two or three times at intervals of several number of days, then a different cell antigen (SB cells, Raji cells or CD20 / CHO cells) is selected and used. for final immunization. The count of administered cells is 1 to 3 x 107 per mouse despite the cell type. Additionally, complementary immunization is performed by using GST-CD20 for a part of the mouse. Three days after the final immunization, cells are removed from the mice, and suspended in the RPMI medium, and a fusion reaction with mouse myeloma (NS-1) is carried out in the presence of PEG-1500 ( Oi, VT et. Herzenberg, 1980, in: Selected Methods in Cellular Immunology; Mishell B et al. (Freeman and Co., San Francisco, CA) p.351).

(4)

Establishment of the CD20 / CHO cell ELISA detection method

Several murine and 2B8 anti-CD20 monoclonal antibodies are reacted by using 96-well plates to which SB cells, Raji cells, CD20 / CHO cells and CHO CD20 progenitor cell line adhere. It is confirmed that in these cell ELISA tests, similar trends are observed for antibody concentrations, and it is found that relative comparisons between antibodies and with a control are also possible. Due to the high density of the surface cell antigens adhered to the plate in the ELISA for CD20 / CHO cells, an absorbance is observed at a level that sufficiently allows detection even with a relatively low concentration of the test antibody sample , and it is found to be a sensitive measurement system. Specific measurement results are shown in Table 1.

Table 1 Comparison of ELISA for SB cells, ELISA for Raji cells and ELISA for CD20 / CHO cells

Antibody

ELISA for Raji cells (A492) Antibody concentration (ng / ml) ELISA for SB cells (A492) Antibody concentration (ng / ml)

1000 320 100 32 10 3

1000 320 100 32 10 3

1K1228

1,683 1,170 0.678 0.326 0.148 Nt 1,265 0.931 0.452 0.192 0.082 Nt

1K1257

1,775 1,263 0.782 0.445 0.177 0.096 1,509 1,143 0.570 0.276 0.119 0.055

1K1402

1,228 0.525 0.214 0.102 0.061 0.049 0.763 0.400 0. 152 0.095 0.066 0.057

1K1422

0.748 0.277 0.121 0.057 0.046 0.049 0.403 0.147 0.071 0.039 0.036 0.051

1K1428

1,196 0.514 0.222 0.104 0.067 0.057 1,111 0.509 0.252 0.108 0.058 0.061

1K1936A

0.887 0.376 0.191 0.105 0.058 0.058 0.959 0.445 0.264 0.120 0.067 0.065

2B8

0.329 0.121 0.055 0.038 0.034 0.046 0.337 0.091 0.045 0.038 0.037 0.053

NC

0.035 0.028

Antibody

ELISA for Raji cells (A492) Antibody concentration (ng / ml) ELISA for SB cells (A492) Antibody concentration (ng / ml)

1000 320 100 32 10 3

1000 320 100 32 10 3

1K1228

3,056 2,704 2,091 1,275 0.572 0.265 0.081 0.040 0.038 0.034 0.038 Nt

1K1257

3,184 2,576 1,924 1,223 0.604 0.271 0.095 0.053 0.038 0.039 0.037 0.029

1K1402

1,877 1,574 1,486 0.824 0.389 0.174 0.385 0.170 0.085 0.063 0.070 0.065

1K1422

2,230 1,327 0.842 0.238 0.105 0.068 0.164 0.068 0.046 0.047 0.035 0.067

1K1428

2,544 2,448 2,190 0.967 0.463 0.283 0.564 0.213 0.091 0.056 0.050 0.060

1K1436A

2,432 2,369 2,278 1,101 0.559 0.286 0.375 0.153 0.072 0.043 0.043 0.060

2B8

2,293 1,664 1,090 0.561 0.174 0.089 0.056 0.040 0.045 0.035 0. 044 0.060

NC

0.070 0.029

(5) ELISA detection for cells

ELISA is performed for cells by using a 96-well plate to which the CD20 / CHO cells or the CHO cells (CD20 progenitor cell line) adhere, and wells are selected in which antibodies specifically reactive to CD20 are produced. 2B8 is used as a positive control, and a mouse 5 monoclonal antibody directed to the human CD3 antigen (BD PharMingen) is used as a negative control. Specifically, CD20 / CHO cells or CHO (progenitor cell line) cells adhered to a 96-well plate coated with poly-Lysine (Asahi Techno Glass Corporation, Cat. No. 11-023-018) are used for ELISA ce cell. A blocking solution (0.2% gelatin and 0.5% BSA solution in PBS) in a volume of 150 µl is added to each well and allowed to stand at 37 ° C for 1 hour. Then, the plate is washed 5 times with 150 mM NaCl and 0.05% Tween 20 aqueous solution, and 100 µl of each sample (diluted culture supernatant solution) is added to each well to develop the primary reaction at 37 ° C for 1 hour. After washing, 100 µl of a diluted solution of an antibody labeled [rabbit anti-mouse IgG (H + L) anti-mouse antibody (Jackson Lab., Code No. 315-035-003) or rabbit IgG antibody (Fc) HRP-labeled anti-mouse (Jackson Lab., Code No. 315-035-008)] is added to each well to develop the secondary reaction at 37 ° C for 1 hour. For the preparation of the mez clas de

For the primary and secondary reactions, a solution is used as the blocking solution. After washing, 100 µl of a color development solution (OPD) is added to each well, 30 minutes later, 50 µl of 4 N H2SO4 is added to terminate the reaction, and the absorbance at 492 nm is measured (A492 ). Then, wells that show reactivity comparable to or significantly greater than that of 2B8 are selected.

(6)

Cloning

Cloning is carried out by the limited solution method. The cells are seeded in a 96-well plate and cultured, then ELISA is performed for cells for CD20 cells / CHO cells for the culture supernatant of a well containing 1 colony to select a clone that produces a specific antibody.

(7)

Preparation of purified antibody

The clone that produces a specific antibody is grown in 10% RPMI 1640 medium added to FCS. When the cell density becomes approximately 5 x 105 / ml, the medium is replaced with a serum-free medium, ASF104N (Ajinomoto Co. Inc.), and cultures are continued. Then, 2 to 4 days later, the culture medium is centrifuged, and the culture supernatant is collected and subjected to purification using a G protein column. The eluted monoclonal antibody solution is dialyzed against 150 mM NaCl. The solution is subjected to filtration sterilization using a filter that has a pore size of 0.2 µm and is used as a test antibody (CD20 anti-human mouse monoclonal antibody).

Monoclonal antibody clones that show binding affinity comparable to those of positive control are selected by ELISA for CD20 / CHO cells. The gene sequences of the variable regions of these antibodies are determined, and their amino acid sequences are determined as a result. The sequences

35 of the H-chain variable region and the L-chain variable region of typical antibodies are shown in SEQ ID NOS: 1 and 7, SEQ ID NOS: 2 and 8, SEQ ID NOS: 3 and 9, SEQ ID NOS : 4 and 10, SEQ ID US: 5 and 11, SEQ ID US: 6 and 12, SEQ ID US: 15 and 17, and SEQ ID US: 16 and 18 (Figure 4). Additionally, the biological characteristics of these clones are investigated.

Biological characteristic test (1): Apoptosis induction test

Apoptosis that induces the ability of test compounds is determined by flow cytometry (annexin V / PI staining). 2B8 is used as a positive control, and the mouse monoclonal antibody directed to human CD3 (BD PharMingen) is used as a negative control. The procedures are as follows.

The MEBCYTO Apoptosis Equipment (MBL, Cat. No. 4700, Lot. 20) is used.

Raji cells are centrifuged, and then suspended in a fresh RPMI 1640 medium (Sigma, Cat. No. R8758, Lot

45 44K2416) containing 10% FCS (inactivated) (ICN, Cat. No. 2916754, Lot 8005C), and 1 ml of the suspension at a density of 5 x 105 cells / ml is added to each well of a plate 12 wells Twenty wells are used for each antibody, and each antibody is added in a final concentration of 2 µg / ml or 4 µg / ml (3 wells x 2 different concentrations x 2 time points, 12 wells in total).

One day and two days after the start of the culture, the culture medium containing approximately 2 x 105 cells is collected, and centrifuged, and then the cells are washed once with PBS. Subsequently, 85 µl of a cell binding buffer is added to suspend the cells in the buffer. Additionally, 10 µl of annexin V-FITC and 5 µl of PI is added to the suspension, mixed sufficiently, and allowed to react at room temperature for 15 minutes with light protection. Measurement is performed using flow cytometry (FACS Calibur, Becton Dickinson), and the results are analyzed using CellQuest (Becton Dickinson).

Measurement results of 8 types of typical murine anti-CD20 monoclonal antibodies, positive control (2B8), and negative control (anti-CD3 antibody) are shown in Figure 5. In general, apoptosis that induces capacity. 2B8 is said to be very high. Still compared to this, the clone of which the amino acid sequences of the H-chain variable region and the L-chain variable region are those of SEQ ID NOS: 2 and 8 (1K1791) that shows an activity that induces markedly greater apoptosis . Cell death is also clearly seen due to apoptosis with the clones of which the amino acid sequence of the H chain variable region and the L chain variable region are those of SEQ ID NOS: 1 and 7 (1K1422) and SEQ ID NOS: 15 and 17

10 (1K0924).

Biological characteristic test (2): cell growth inhibition test

A suspension of 5 x 104 cells / ml of Raji cells is prepared with 10% RPMI 1640 medium added by FCS, and added to a 96-well plate in a volume of 100 µl / well, and culture is performed. After 24 hours, 50 µl / well of each antibody solution is added at an antibody concentration of 0.01 µg / ml, 0.1 µg / ml or 11-15 µg / ml, and culture is continued. Seventy-two hours after the antibody addition, 10 µl / well of a color development solution, Cell Counting Equipment 8 (Dojindo Laboratories, Cat. No. 343-07623, Lot SG076) is added, the cells are cultured for an additional 4 hours, and then the absorbance at 492 nm is measured. Live cell counts of the 8 typical murine anti-CD20 monoclonal antibodies and the positive control (2B8) are shown in Figure 6 as percentages based on that of the negative control (100%). You can estimate the

20 cell growth inhibitory effect at the base of the reduced live cell count index compared to that of the negative control. Clear inhibition of cell growth is observed with 1K0924, 1K1422, 1K1791 and 2B8 as the positive control, and the inhibition is particularly marked with 1K1791. This trend is consistent with the results of the apoptosis induction test.

Preparation of chimeric antibodies

The genes encoding the H-chain and L-chain variable regions of each murine antibody are incorporated into pNOW-Ab, a high-expression vector for the CHO cell that already contains the genes encoding the constant regions of the h-chain and of L () chain of human immunoglobulin as a cassette. Each expression vector is transfected into CHO cells, and clones that show high productivity for each antibody are selected.

30 Test for CD20 antigen binding property of chimeric antibodies

The 8 prepared types of chimeric anti-CD20 monoclonal antibodies are examined for reactivity to human CD20 antigen by ELISA for CD20 / CHO cells. Rituximab (c2B8) is used as a positive control. The test results are shown in Table 2. The values measured in the ELISA for cells (A492) reflect the intensity of the binding property. These antibodies show affinity substantially comparable to or greater

35 than that of the control except that c1K0924 and c1K1422 tend to show slightly lower affinity than that of the control.

Table 2 ELISA test for CD20 / CHO cells of anti-CD20 chimeric antibodies

Antibody

100 32 ELISA for CD20 / CHO cells (A492) Antibody concentration (ng / ml) 10 3 1 0

c1K0924

1,423 0.724 0.391 0.186 0.094 0.032

c1K1228

2,226 1,580 0.701 0.289 0.120 0.032

c1K1402

2,449 1,621 0.737 0.349 0.116 0.032

c1K1422

1,919 0.912 0.357 0.151 0.077

c1K1712

2,292 1,683 0.793 0.359 0.145

c1K1736

2,428 1,548 0.748 0.320 0.122

c1K1782

2,101 1,017 0.505 0.169 0.074

c1K1791

2,231 1,458 0.745 0.276 0.108

c2B8

2,147 1,143 0.536 0.226 0.088

CDC chimeric antibody test

The 8 prepared types of chimeric anti-CD20 monoclonal antibodies are examined for CDC activity. Rituximab (c2B8) is used as a positive control. RC-K8 (obtained from Kochi Medical School) is used as the 5 target cells. As a medium for use, HBV (basal medium: RPMI-1640, additives: 0.1% BSA, 20 mM HEPES (pH 7.2), 2 mM glutamine, 100 units / ml penicillin G, 100 µg / ml streptomycin ) are prepared and used. The target cells are washed with RHB and resuspended at 106 cells / ml. In a volume of 50 µl of each of the solutions of the chimeric test antibodies and rituximab having different concentrations, 50 µl of 4 times diluted solution of a commercially available human complement (Quidel, 10 San Diego, CA, Cat. A113 ), and 50 µl of a cell suspension containing 106 cells / ml are added to each well of a 96-well flat bottom culture plate (black). The antibody concentration in the 150 µl / well mixture is set at 0.1, 1 and 10 µg / ml. To promote complement-mediated cell lysis, the mixture is incubated under the conditions of 37 ° C and 5% CO 2 for 2 hours. To the mixture is added 50 µl of alamar blue (undiluted prescription from AccuMed International, Biosource, Cat. DAL1100), and the reaction is allowed to add

15 overnight under the same conditions. The plate is left at room temperature for 10 minutes to cool, and the fluorescence at 590 nm is measured for emission with excitation at 530 nm when using a fluorescence microplate reader. The results are represented in terms of fluorescence intensity (RFU). The CDC activity index is calculated according to the following equation:

% CDC activity = 100 x {RFU (antibody not added) - RFU (antibody added)} / {RFU (antibody not added 20) - RFU (Triton X - 100 added)}

The results are shown in Figure 7. Except for c1K1712, the antibodies show CDC activities substantially comparable to or greater than those of C2B2 as the positive control.

Preparation of humanized antibodies

Humanized antibodies are designed based on the CDR variable region of the anti-CD20 monoclonal antibody of

25 murine 1K1791. By developing structural analysis based on the amino acid sequences and additionally changing the designed method, 4 types of humanized sequences are prepared for each of the H and L chains (Padlan EA, Mol. Immunol., Vol. 28, No 4/5, 489-498; Wu TT and Kabat EA, Mol. Immunol., Vol. 29, No 9, 1141-1146; Padlan EA et al., FASEB J., Vol. 9, 133-139). Antibodies are prepared with all possible combinations of the four types for each of the H chain and the L chain. These sequences are shown.

30 amino acid in SEQ ID US: 19 and 23, SEQ ID US: 19 and 24, SEQ ID US: 19 and 25, SEQ ID US: 19 and 26, SEQ ID US: 20 and 23, SEQ ID US: 20 and 24, SEQ ID US: 20 and 25, SEQ ID US: 20 and 26, SEQ ID US: 21 and 23, SEQ ID US: 21 and 24, SEQ ID US: 21 and 25, SEQ ID US: 21 and 26, SEQ ID US: 22 and 23, SEQ ID US: 22 and 24, SEQ ID US: 22 and 25, and SEQ ID US: 22 and 26 (Figures 8A and 8B).

The amino acid sequences of these 4 types of H-chain variable regions and 4 types of variable regions of

The L-chain is converted into the DNA sequences (nucleotide) with codons most frequently used in the human gene sequences, and some of these nucleotides properly considered in CHO host cells are changed without changing the original amino acid residues (Kim CH et al., Gene, 1997, 15; 199 (1-2): 293301). Specifically, it is used as the nucleotide sequences that correspond to the amino acid sequences are those of SEQ ID NO: 27 that correspond to SEQ ID NO: 19, SEQ ID NO: 28 which

40 correspond to SEQ ID NO: 20, SEQ ID NO: 29 that correspond to SEQ ID NO: 21, SEQ ID NO: 30 that correspond to SEQ ID NO: 22, SEQ ID NO: 31 that correspond to SEQ ID NO: 23, SEQ ID NO: 32 that correspond to SEQ ID NO: 24, SEQ ID NO: 33 that correspond to SEQ ID NO: 25, and SEQ ID NO: 34 that correspond to SEQ ID NO: 26 (Fig. 8A and Fig. 8B). In these nucleotide sequences, nucleotides can be replaced with other nucleotides both when the corresponding amino acid sequences are not altered.

45 The 8 total types of nucleotide sequences of the H-chain variable regions (SEQ ID NOS: 27 to 30) and the L-chain variable regions (SEQ ID NOS: 31 to 34) are synthesized (Takara Shuzo Co., Ltd.) and incorporated into pNOW-Ab, an expression vector for mammalian cells that contain a multicloning site. Expression vectors incorporated with each of these humanized antibody genes are transfected into CHO cells, and clones that show high productivity for each antibody are selected.

Biological characteristic and cell growth inhibition tests for humanized antibodies

A suspension containing 5 x 104 / ml of Raji cells is prepared with 10% RPMI 1640 medium added to

5 FCS, and added to a 96-well plate in a volume of 100 µl / well, and culture is performed. After 24 hours, 50 µl / well of each antibody solution is added at an antibody concentration of 0.5 µg / ml, and culture is continued. Seventy-two hours after the addition of the antibody, 10 µl / well of a color development solution, Cell Counting Equipment 8 (Dojindo Laboratories, Cat. No. 343-07623, Lot SG076) is added, the culture is performed for an additional 4 hours, and then the absorbance at 492 nm is measured. The live cell count of

10 15 types (27 clones) of 16 types of humanized antibodies derived from 1K1791 and c2B8 (another name of rituximab) as the positive control are shown in Figure 9 as indices based on those of the negative control (100%). The cell growth inhibitory effect can be estimated on the basis of the reduced live cell count index compared to that of the negative control, and the growth inhibitory effect is observed for all clones in this test.

15 Monoclonal antibody names and sequence numbers described in this specification and the accompanying drawings are summarized as follows.

Table 3

Murine antibody name

H-string variable region Variable region of string L Chimeric Antibody Name Humanized variable region (H chain / L chain)

1K1422

SEQ ID NO: 1 SEQ ID NO: 7 c1K1422

1K1791

SEQ ID NO: 2 SEQ ID NO: 8 c1K1791 SEQ ID US: 19 and 23

SEQ ID US: 19 and 24

SEQ ID US: 19 and 25

SEQ ID US: 19 and 26

SEQ ID US: 20 and 23

SEQ ID US: 20 and 24

SEQ ID US: 20 and 25

SEQ ID US: 20 and 26

SEQ ID US: 21 and 23

SEQ ID US: 21 and 24

SEQ ID US: 21 and 25

SEQ ID US: 21 and 26

SEQ ID US: 22 and 23

SEQ ID US: 22 and 24

SEQ ID US: 22 and 25

SEQ ID US: 22 and 26

1K1712

SEQ ID NO: 3 SEQ ID NO: 9 c1K1712

1K1402

SEQ ID NO: 4 SEQ ID NO: 10 c1K1402

1K1736

SEQ ID NO: 5 SEQ ID NO: 11 c1K1736

1K1782

SEQ ID NO: 6 SEQ ID NO: 12 c1K1782

1K0924

SEQ ID NO: 15 SEQ ID NO: 17 c1K0924

1K1228

SEQ ID NO: 16 SEQ ID NO: 18 c1K1228

The hybridomas that produce these monoclonal antibodies are named on the basis of the names of the antibodies produced, and are intentionally deposited with the National Institute of Advanced Industrial Science and Technology, Depositary of the International Patent Agency (Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba

5 shi, Ibaraki-ken, 305-8566, Japan) on March 28, 2006 under the provisions of the Budapest Treaty, and assigned access numbers of FERM BP-10587 (1K1422), FERM BP-10591 (1K1791), FERM BP -10588 (1K1712), FERM BP10586 (1K1402), FERM BP-10589 (1K1736), FERM BP-10590 (1K1782), FERM BP-10584 (1K0924), and FERM BP10585 (1K1228).

Industrial Applicability

The present invention provides a monoclonal antibody that has biological activities for use as a therapeutic agent.

Sequence Listing

<110> BioMedics Inc.

<120> Anti-CD20 monoclonal antibody 15 <130> C6084-PCT

<150> JP 2005-103093

<151> 2005-03-31

<150> JP 2005-378466

<151> 2005-12-28 20 <160> 34

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<400> 14

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Claims (6)

1. A murine anti-CD20 monoclonal antibody that has cell growth inhibitory activities that include apoptosis against cells expressing the human CD20 antigen in the culture of cells expressing the CD20 antigen without effector cells, wherein the amino acid sequences of the variable region of string H and the 5 variable region of string L are SEQ ID NOS: 1 and 7.

2.

A hybridoma that produces the anti-CD20 monoclonal antibody according to claim 1.

3.

A chimeric anti-CD20 monoclonal antibody, wherein the amino acid sequence of the variable region of the anti-CD20 monoclonal antibody according to claim 1 and the amino acid sequence of the human immunoglobulin constant region are fused.

4. A humanized anti-CD20 monoclonal antibody using the amino acid sequence of the CDR variable region of the anti-CD20 monoclonal antibody according to claim 1 and a human immunoglobulin amino acid sequence.

5. The anti-CD20 monoclonal antibody according to any one of claims 3 to 4, which has cytotoxicity against cells expressing the CD20 antigen in the presence of a human complement.

An isolated mammalian cell incorporated with a nucleotide sequence encoding the amino acid sequence of the anti-CD20 monoclonal antibody according to any one of claims 3 to 5.

7.

The mammalian cell according to claim 6, which is a CHO cell.

8.

A diagnostic agent comprising the anti-CD20 monoclonal antibody according to any one of claims 1, or 3 to 5, as an active ingredient.

A therapeutic agent comprising the anti-CD20 monoclonal antibody according to any one of claims 3 to 5 as an active ingredient.