0

5

10

15

20

25

30

35

40

Pe

g-r

hM

GD

F (

5.9

)

Pe

g-r

hM

GD

F (

0.5

9)

Pe

g-r

hM

GD

F (

0.0

6)

MA

01

G4

34

4U

(6

.7)

MA

01

G4

34

4U

(0

.67

)

MA

01

(6

.7)

MA

01

(0

.67

)

DW

MK

co

lon

ies

MA01G4344U Peg-rhMGDF

a b

Supplementary Figure 2. Stimulation of CFU-MK colonies from human cord blood CD34+ cells by purified antibodies.

(a) The result of CFU-MK colony assay with human cord blood–derived CD34+ cells. Agonist activities of purified MA01 and MA01G4344U for primary cells were tested. MA01 and MA01G4344U stimulated human cord blood–derived CD34 cells to make CFU-MK colonies without any other supportive cytokines. The graph shows CFU-MK colony counts per slide (mean +/- SD). The concentrationsof added agents (nM) are indicated. (b) Morphology of CFU-MK colonies obtained from human cord blood CD34+ cells treated by MA01G4344U (6.7 nM) and Peg-rhMGDF (5.9 nM). The boxed areas in the upper images (10x objective lens) are shown at higher magnification (20x) in the lower images. Scale bar = 150 mm.

Mouse Mpl promoter

5.5 kb

Human Mpl CDS

2 kb

Human Mpl UTR

1.6 kb

a

+ - + - + - + -

Human Mpl

Mouse Mpl

Tg Tg Wt Wtb

Supplementary Figure 3: Human Mpl-Tg mouse construct and expression of human Mpl.

(a) Construction of the transgene. The human Mpl coding sequence (CDS) was connected to a murine Mpl promoter. (b) Expression of human Mpl mRNA in bone marrow cells (RT-PCR). +; RT(+), –; RT(–).

0.0

0.5

1.0

1.5

2.0

0 5 10 15 20 25 30

Days after administration

Ser

um C

onc.

(m

g/m

L)

MA01G4344U

MA01G4PE

Peg-rhMGDF

Supplementary Figure 4: Serum concentration of the antibodies and Peg-rhMGDF in human Mpl-Tg mice.

The graph shows mean serum concentration (mg/mL) of the agonist antibodies and Peg-rhMGDF after single intraperitoneal administration (3 mg/mouse). Blood sampling was performed at 2, 8, 24 and 48 h after Peg-rhMGDF administration or 1, 7, 14, 21 and 28 days after antibody administration. Each time point contains data from three animals.

Antibody

PBS

CD41

Cel

l cou

nt

11.7 ± 1.6 %

10 0

10 1 10 2

10 3

200

400

600

800

1000

10 0

10 1 10 2

10 3

26.5 ± 1.1 %

200

400

600

800

1000

Cel

l cou

nt

Supplementary Figure 5: Proportion of CD41+ cells in bone marrow of Tg mice treated with MA01G4344U

Bone marrow mononuclear cells were analyzed by flow cytometry two weeks after antibody (MA01G4344U, 3 mg/mouse) or PBS treatment. The histogram of representative data is shown. Blue line indicates the isotype control. The mean +/- SD of percentage of CD41+ cells is indicated. MA01G4344U increased the proportion of CD41+ cells in bone marrow of Tg mice.

IgG1 VDKRV EPKSCDKTHT CPPCP APELLGGP

IgG2 VDKTV ERK CCVECPPCP APPVAGP

IgG3 VDKRV ELKTPLGDTTHT CPRCP(EPKSCDTPPPCPRCP)x3 APELLGGP

IgG4 VDKRV ESKYGPP CPSCP APEFLGGP

IgG4PE VDKRV ESKYGPP CPPCP APEFEGGP

IgG3344 VDKRV ELKTPLGDTTHT CPRCP(EPKSCDTPPPCPRCP)x3 APELLGGP

IgG4344 VDKRV ELKTPLGDTTHT CPRCP(EPKSCDTPPPCPRCP)x3 APELLGGP

IgG4344U VDKRV CPPCP APEFEGGPESKTPLGDTTHT

CH1 Upper hinge Middle hinge CH2

Supplementary Table 1: Comparison of the hinge sequences of natural human IgGs and hybrid antibodies

Human IgG3 has a long middle hinge. G3344 and G4344 have the entire hinge region of IgG3. G4344U has a part of the upper hinge region of IgG3 and the other parts of IgG4.

1

Supplementary Methods

Generation of monoclonal antibodies The coding sequence of human Mpl (Genbank: NM_005373) was amplified by PCR using KOD-Plus DNA polymerase (Toyobo, Tokyo, Japan). The amplified DNA was constructed in a pEF6 expression vector (Invitrogen, Carlsbad, CA, USA) in the EcoRI - XbaI site (pEF6/hMpl). The construct was stably transfected to L929 (mouse fibroblast) and FM3A (mouse mammary tumor) cells using Lipofectamine (Invitrogen) reagent, to make the human Mpl-expressing cell lines L929-hMpl and FM3A-hMpl. Human antibody–producing mice (KM-mice TM, Kirin, http://www.tcmouse.com/) were immunized by L929-hMpl or FM3A-hMpl cells (2 x 106 – 5 x

106 cells/mouse/wk, iv) or recombinant soluble portion of human Mpl (5 μg/mouse/wk, sc) with or without Freund complete or incomplete or Ribi adjuvant. Serum samples were collected every week or every two weeks, and antibody titers were examined by enzyme-linked immunosolvent assay (ELISA) or flowcytometry analysis. After the detection of anti-human Mpl antibodies, splenocytes were isolated from immunized mice and fused with mouse SP2/O myeloma cells. Hybridomas expressing anti-human Mpl antibodies were screened with the supernatants by ELISA or flowcytometry. Agonist antibodies were screened by a UT-7/TPO cell proliferation assay (kindly provided by Dr. Norio Komatsu, University of Yamanashi, Japan) as described below. Human gamma and human kappa were confirmed by flowcytometory analysis. Limiting dilutions were performed to clone the hybridoma cells. After the second limiting dilution, antibody genes were isolated from the hybridoma clones.

Recombinant antibodies Complementary DNA (cDNA) coding variable regions of both heavy and light chains were amplified from total RNAs of hybridomas using rapid amplification of the 5’ cDNA ends (5’ RACE) methods and then cloned into a pCR4 cloning vector (Invitrogen). They were into the antibody expression vector N5KG1 (Biogen IDEC, Cambridge, MA, USA). The constructs were then transfected into Hek293 or CHO cells, and antibodies were purified from the supernatants with protein A-conjugated sepharose colomns (GE Healthcare, Malmo, Sweden). Furthermore, we made antibody expression vectors encoding domain subclass-converted derivatives, IgG4PE, IgG3344, IgG4344 and IgG4344U. IgG4PE is a human IgG4 variant with Ser228Pro and Leu235Glu mutations to reduce the monomer fraction of natural IgG4 and to reduce antibody-dependent cellular cytotoxicity (ADCC). IgG3344 is a hybrid antibody of IgG3 and IgG4PE, which have the CH1-hinge region of IgG3 and the CH2 -CH3 regions of IgG4PE. IgG4344 is also a hybrid whose CH1, CH2 and CH3 are IgG4PE and the hinge region is IgG3. IgG4344U is a variant of IgG4344, which has the upper hinge of IgG3 and the other parts of

2

IgG4PE. The upper hinge is a part of the hinge region at the N-terminus side of the first inter-heavy chain disulfide bond (residues between 216 and 226 in Kabat numbering). We made the MA01 derivatives MA01G4PE, MA01G4344 and MA01G4344U, and the MA02 derivatives MA02G3344 and MA02G4344U.

Cell proliferation assay UT-7/TPO cells were usually cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) (Invitrogen) containing 10% FBS and 5 ng/ml Peg-rhMGDF. For the proliferation assay, cells were deprived of Peg-rhMGDF for at least 6 hours. Then, cells (3x104 cells/well) and samples were distributed into each well of a 96-well plate. After a 48-hour incubation, cell numbers were measured using water-soluble tetrazolium salt-8 (WST-8 reagent, Dojindo, Kumamoto Japan). WST-8 is reduced by dehydrogenase activity of a viable cell to produce a yellow color called formazan dye.

Flow cytometry analysis FM3A-hMpl cells were used for the flowcytometry analysis. Cells (4x105 cells/reaction) were suspended in the staining medium (2% FBS, 0.1% NaN3, 1 mM EDTA in PBS). Samples were added to the suspension and placed on ice for 30 minutes. After washing with the staining medium, secondary antibody, RPE-conjugated goat anti-human IgG Fab’2 (Southern biotech, Birmingham) was added. To confirm human kappa, anti-human kappa secondary antibody was used. Then samples were incubated for 30 minutes on ice again. The analysis was performed with a FACSCalibur flowcytometry system (Becton Dickinson, Franklin Lakes, NJ, USA).

Colony assay To assess the agonist activities for human primary cells, a colony assay was performed with purified mAbs. A MegacultTM-C system (StemCell Technologies, Vancouver, BC, Canada) was used. CD34+ cells were purified from human cord blood (kindly donated by healthy volunteers after

informed consent was obtained) by using anti-CD34 antibody-coated magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany). Five thousand cells per slide were cultured in collagen-based semi solid media without serum for 7 to 10 days. After dehydration and fixation, immunostaining with anti-human CD41 antibody was performed for the detection of megakaryocyte colonies. The number of colonies was counted under light microscopy (Model: BX50F4, Olympus, Tokyo, Japan). All the experiments using human subjects were conducted according to the protocols approved by the institutional research ethics committee.

3

Signaling analysis Phosphorylation of signaling proteins was analyzed by immunoblotting with phosphoprotein-specific antibodies. The antibodies used are as follows: anti-phospho-Jak2 (Tyr1007/1008) (Upstate, #07-606), anti-Jak2 (Upstate, #06-255), anti-phospho-STAT5 (Tyr694) (Cell Signaling Technology, #9351), anti-STAT5 (Cell Signaling Technology, #9352), anti-phospho-p44/42 Map Kinase (Thr202/Tyr204) (Cell Signaling Technology, #9101), anti-p44/42 Map Kinase (Cell Signaling Technology, #9102), anti-phospho-Akt (Ser473) (Cell Signaling Technology, #9101S) and anti-Akt (Cell Signaling Technology, #9102). The procedure was as follows. UT-7/TPO cells were deprived of TPO for 6 hrs. Then, cells were distributed to each well of a 6-well plate at 2 x 106 cells/well. Agonist antibodies or TPO was added to each well, and the plate was incubated. Cells were harvested after 5-min or 30-min stimulation, and whole-cell extracts were prepared by using PhosphoSafeTM Extraction Buffer (EMD Biosciences, San Diego, CA, USA). Samples were resolved by SDS-PAGE and electroblotted onto polyvinylidene fluoride (PVDF) membranes. The membranes were probed with the antibodies above. The ECL system (GE Healthcare) was used for detection.

Platelet aggregation Citrated human platelet-rich plasma (PRP) was collected from healthy volunteers who gave

informed consent. Up to 10 μg/mL of antibodies were added to the PRP. Platelet aggregation was evaluated by turbidity measurement using the NBS Hematracer 801 system (Niko Bioscience, Tokyo, Japan). All the experiments using human subjects were conducted according to the protocols approved by the institutional research ethics committee.

Transgenic mice A 5.5-kb sequence containing the murine Mpl promoter region was amplified by PCR and cloned into the pBluescript plasmid vector (Primers: Forward 5’-TAGTCCCAGCATTTATGAAACAGAAGCAAGG-3’, Reverse 5’-GCCGGCACTGTGTGCCTGCC-3’). Then, the human Mpl coding sequence (2 kb) and the 3’-untranslated region (3’UTR) (1.6 kb) were amplified and cloned into the plasmid downstream of the murine promoter. Polyadenylation sequences were included in the 3’UTR. The total length of the construct was 9.1 kb. The construct DNA was isolated from the vector sequence and injected into fertilized C57BL/6 one-cell embryos. The embryos were transplanted into foster mothers. Three weeks after birth, transgenic mice were identified by genomic PCR with specific primers for the human Mpl 3’UTR sequence: Forward 5’-CTGCCACCATGCCTAATTTT-3’, Reverse 5’-ACATGGCAAAACCCTGTCTC-3’. Expression of human Mpl was examined by reverse transcription (RT) PCR. Total RNAs were

4

extracted from whole bone marrow cells using ISOGEN (Nippon Gene, Tokyo, Japan). cDNAs were prepared with oligo-dT primers and Superscript III reverse transcriptase (Invitrogen). Human Mpl was amplified by PCR using the above primer set. To evaluate in vivo efficacy, the

Tg mice were treated with MA01G4344U (3 or 10 μg/mouse) or MA01G4PE (3 μg/mouse) or Peg-rhMGDF (3 μg/mouse). Wildtype/control littermates were treated with 10 μg/mouse of MA01G4344U. Each group contained six animals. All treatments were single intraperitoneal injections (at Day 0). Blood samples were collected from the retro-orbital plexus under anesthesia, and platelet counts were measured using the KX-21 Hematology Analyzer system (Sysmex, Kobe, Japan). The effects of antibody on progenitor cells were examined by CFU-MK assay with bone marrow mononuclear cells from Tg mice. Whole bone marrow cells were flushed out from the femurs two weeks after antibody administration. Then mononuclear cells (MNC) were separated by the ficoll density gradient method. Colony assay was performed by a MegacultTM-C system as described above. The incubation period was 7 days. Detection was by staining megakaryocytes for acetylcholinesterase activity. All the animal experiments were conducted according to the protocols approved by the institutional animal ethics committee.

Pharmacokinetic analysis Blood sampling was performed at 2, 8, 24 and 48 h after Peg-rhMGDF administration or 1, 7, 14, 21 and 28 days after antibody administration. Serum antibody concentrations were determined by sandwich ELISA with two anti-human gamma chain antibodies. Serum concentrations of Peg-rhMGDF were determined using Human Thrombopoietin Quantikine ELISA Kit (R&D systems, Minneapolis, USA). The data sets were analyzed using pharmacokinetic software (non-compartment model).

Flowcytometric analysis of mouse bone marrow cells Two weeks after antibody treatment, bone marrow MNC were prepared as described above. Rat anti-mouse CD41-PE conjugated (BD pharmingen, #558040) was used to detect megakaryocytic cells. 30,000 cells in each sample were analyzed. The analysis was performed by a FACSCalibur flowcytometry system.

Statistical Analysis Student’s t-test was used to determine statistical difference in the results. A p value of less than 0.05 was considered to be statistically significant.