Agonist antibody to human thrombopoietin receptor

1. An agonist antibody to a human thrombopoietin receptor (c-Mpl), wherein the agonist antibody comprises:

(A) antibody constant regions comprising amino acid sequences selected from the following:

(1) amino acid sequences of heavy chain and light chain constant regions of a human antibody, or

(2) an amino acid sequence of a heavy chain constant region with a heavy chain constant region domain substituted between human antibody subclasses, and an amino acid sequence of a light chain constant region of a human antibody,

wherein, the heavy chain constant region of (A)(1) or (A)(2) comprises:

i) an upper hinge portion having the amino acid sequence of SEQ ID NO: 10;

ii) a middle hinge portion having the amino acid sequence selected from the group consisting of:

(a) amino acids 18 to 67 of SEQ ID NO: 101;

(b) amino acids 18 to 37 of SEQ ID NO: 102; and

(c) amino acids 18 to 22 of SEQ ID NO: 103; and

iii) a CH1 Region having the amino acid sequence of amino acids 1 to 5 of SEQ ID NO: 99; and

iv) a CH2 region having the amino acid sequence of (a) amino acids 18 to 25 of SEQ ID NO: 100, or (b) amino acids 18 to 25 of SEQ ID NO: 99, or

the heavy chain constant region of (A)(1) or (A)(2) comprises:

i) an upper hinge portion having the amino acid sequence of SEQ ID NO: 11;

ii) a middle hinge portion having the amino acid sequence of amino acids 18 to 22 of SEQ ID NO: 104; and

iii) a CH1 Region having the amino acid sequence of amino acids 1 to 5 of SEQ ID NO: 99; and

iv) a CH2 region having the amino acid sequence of (a) amino acids 18 to 25 of SEQ ID NO: 100, or (b) amino acids 18 to 25 of SEQ ID NO: 99; and

(B) antibody variable regions capable of binding to and activating a human thrombopoietin receptor;

wherein the agonist antibody has the following properties:

(a) that the antibody induces colony formation at a concentration of 10,000 ng/ml or lower as determined by the CFU-MK colony formation assay using human umbilical cord blood derived CD34+ cells; and

(b) that the antibody has a maximal activity at least 50% higher than that of PEG-rHuMGDF, which comprises the amino acid sequence as shown in SEQ ID NO: 1 having the following structure and has been pegylated at the N terminus, and a 50% effective concentration (EC50) of 100 nM or less in the cell proliferation assay using UT7/TPO cells:

(SEQ ID NO: 1)

PEG-NH-SPAPPACDLRVLSKLLRDSHVLHSRLSQCPEVHPLPTPVLLPA

VDFSLGEWKTQMEETKAQDILGAVTLLLEGVMAARGQLGPTCLSSLLGQL

SGQVRLLLGALQSLLGTQLPPQGRTTAHKDPNAIFLSFQHLLRGKVRFLM

LVGGSTLCVRRAPPTTAVPS-COOH.

2. The antibody according to claim 1 having the properties (a) and (b):

(a) that the antibody induces colony formation at a concentration of 1,000 ng/ml or lower as determined by the CFU-MK colony formation assay using human umbilical cord blood derived CD34+ cells; and

(b) that the antibody has a maximal activity at least 70% higher than that of PEG-rHuMGDF and an EC 50 of 10 nM or less in the cell proliferation assay using UT7/TPO cell.

3. The antibody according to claim 1 having the properties (a) and (b):

(a) that the antibody induces colony formation at a concentration of 100 ng/ml or lower as determined by the CFU-MK colony formation assay using human umbilical-cord-blood-derived CD34+ cells; and

(b) that the antibody has a maximal activity at least 90% higher than that of PEG-rHuMGDF and an EC 50 of 1 nM or less in the cell proliferation assay using UT7/TPO cell.

4. The antibody according to claim 1 , wherein the heavy chain and light chain variable regions are selected from the group consisting of following (1) to (8):

(1) a heavy chain variable region comprising the amino acid sequence as shown in SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence as shown in SEQ ID NO: 3;

(2) a heavy chain variable region comprising the amino acid sequence as shown in SEQ ID NO: 4 and a light chain variable region comprising the amino acid sequence as shown in SEQ ID NO: 5;

(3) a heavy chain variable region comprising the amino acid sequence as shown in SEQ ID NO: 6 and a light chain variable region comprising the amino acid sequence as shown in SEQ ID NO: 7;

(4) a heavy chain variable region comprising the amino acid sequence as shown in SEQ ID NO: 8 and a light chain variable region comprising the amino acid sequence as shown in SEQ ID NO: 9;

(5) a heavy chain variable region comprising the amino acid sequence as shown in SEQ ID NO: 2, and a light chain variable region comprising an amino acid sequence comprising a deletion(s), substitution(s), addition(s), or insertion(s) of one or several amino acid residues of the framework region in the amino acid sequence as shown in SEQ ID NO: 3;

(6) a heavy chain variable region comprising the amino acid sequence as shown in SEQ ID NO: 4, and a light chain variable region comprising an amino acid sequence comprising a deletion(s), substitution(s), addition(s), or insertion(s) of one or several amino acid residues of the framework region in the amino acid sequence as shown in SEQ ID NO: 5;

(7) a heavy chain variable region comprising the amino acid sequence as shown in SEQ ID NO: 6, and a light chain variable region comprising an amino acid sequence comprising a deletion(s), substitution(s), addition(s), or insertion(s) of one or several amino acid residues of the framework region in the amino acid sequence as shown in SEQ ID NO: 7; and

(8) a heavy chain variable region comprising the amino acid sequence as shown in SEQ ID NO: 8, and a light chain variable region comprising an amino acid sequence comprising a deletion(s), substitution(s), addition(s), or insertion(s) of one or several amino acid residues of the framework region in the amino acid sequence as shown in SEQ ID NO: 9.

5. The antibody according to claim 1 , wherein the agonist antibody to human c-Mpl is a human antibody.

6. The antibody according to claim 5 , wherein the antibody is selected from the group consisting of the following antibodies (1) to (8):

(1) an antibody comprising a heavy chain comprising the amino acid sequence as shown in SEQ ID NO: 2 and a light chain comprising the amino acid sequence as shown in SEQ ID NO: 3;

(2) an antibody comprising a heavy chain comprising the amino acid sequence as shown in SEQ ID NO: 4 and a light chain comprising the amino acid sequence as shown in SEQ ID NO: 5;

(3) an antibody comprising a heavy chain comprising the amino acid sequence as shown in SEQ ID NO: 6 and a light chain comprising the amino acid sequence as shown in SEQ ID NO: 7;

(4) an antibody comprising a heavy chain comprising the amino acid sequence as shown in SEQ ID NO: 8 and a light chain comprising the amino acid sequence as shown in SEQ ID NO: 9;

(5) an antibody comprising a heavy chain comprising the amino acid sequence as shown in SEQ ID NO: 2, and a light chain comprising an amino acid sequence comprising a deletion(s), substitution(s), addition(s), or insertion(s) of one or several amino acid residues of the framework region in the amino acid sequence as shown in SEQ ID NO: 3;

(6) an antibody having a heavy chain comprising the amino acid sequence as shown in SEQ ID NO: 4, and a light chain comprising an amino acid sequence comprising a deletion(s), substitution(s), addition(s), or insertion(s) of one or several amino acid residues of the framework region in the amino acid sequence as shown in SEQ ID NO: 5;

(7) an antibody having a heavy chain comprising the amino acid sequence as shown in SEQ ID NO: 6, and a light chain comprising an amino acid sequence comprising a deletion(s), substitution(s), addition(s), or insertion(s) of one or several amino acid residues of the framework region in the amino acid sequence as shown in SEQ ID NO: 7; and

(8) an antibody having a heavy chain comprising the amino acid sequence as shown in SEQ ID NO: 8, and a light chain comprising an amino acid sequence comprising a deletion(s), substitution(s), addition(s), or insertion(s) of one or several amino acid residues of the framework region in the amino acid sequence as shown in SEQ ID NO: 9.

7. A pharmaceutical composition comprising, as an active ingredient, an antibody according to claim 1 .

8. A pharmaceutical composition for increasing platelets comprising, as an active ingredient, an antibody according to claim 1 .

9. The pharmaceutical composition for increasing platelets according to claim 8 , which is used for promoting platelet recovery when bone marrow transplantation or umbilical cord blood transplantation is carried out.

10. A therapeutic composition for thrombocytopenia comprising, as an active ingredient, an antibody according to claim 1 .

11. The therapeutic composition for thrombocytopenia according to claim 10 , wherein the thrombocytopenia is a disease selected from the group consisting of the following diseases (1) to (6):

(1) idiopathic thrombocytopenic purpura (ITP);

(2) thrombocytopenia caused after cancer chemotherapy;

(3) aplastic anemia;

(4) osteomyelodysplasia syndrome (MDS);

(5) thrombocytopenia attributable to hepatic diseases; and

(6) thrombocytopenia caused after bone marrow transplantation or umbilical cord blood transplantation.

12. A pharmaceutical composition for increasing blood cells used for promoting blood cell recovery after hematopoietic stem cell transplantation, comprising, as an active ingredient, the antibody according to claim 1 .