doi:10.1182/blood-2007-04-083998Prepublished online July 9, 2007;   

 Lacy, David A Egan, John E Harlan, Richard R Lesniewski and Edward B ReillyZhihong Liu, Vincent S Stoll, Peter J DeVries, Clarissa G Jakob, Nancy Xie, Robert L Simmer, Susan E binding siteA potent erythropoietin mimic human antibody interacts through a novel

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A potent erythropoietin mimic human antibody

interacts through a novel binding site

Running titile: EPOR AB ACTIVATES THROUGH A NOVEL BINDING SITE

Zhihong Liu1, Vincent S. Stoll1, Peter J. DeVries1, Clarissa G. Jakob1, Nancy Xie1,

Robert L. Simmer1, Susan E. Lacy2, David A. Egan1, John E. Harlan1, Richard R.

Lesniewski1 and Edward B. Reilly1

1From the Global Pharmaceutical Research and Development, Abbott Laboratories,

Abbott Park, IL 60064, USA; 2Global Pharmaceutical Research and Development,

Abbott Bioresearch Center, Worcester, MA 01605, USA.

Corresponding author: Edward B. Reilly, Abbott Laboratories, R4CD, AP31-4, 200

Abbott Park Rd. Abbott Park, IL 60064-6199 (Tel.: 1-847-9370815, Fax: 1-847-9381336,

Email:ed.reilly@abbott.com

Blood First Edition Paper, prepublished online July 9, 2007; DOI 10.1182/blood-2007-04-083998

Copyright © 2007 American Society of Hematology

For personal use only. by guest on June 2, 2013. bloodjournal.hematologylibrary.orgFrom

Abstract

Recombinant human erythropoietin (rHu-EPO) is used to treat anemia by activating the

erythropoietin receptor (EPOR) in erythroid progenitor cells leading to proliferation and

differentiation into mature red blood cells. To allow less frequent dosing, a

hyperglycosylated version of EPO has been developed with a longer half-life. In

principle, an agonistic antibody targeting EPOR would offer an even longer half-life,

support robust monthly dosing, and, unlike EPO products, reduce the risk of pure red cell

aplasia. The efficiency of signaling and corresponding potency of previously reported

antibody mimics are generally suboptimal compared to EPO and not suitable for clinical

use. Here we describe a potent, fully human, agonistic antibody (ABT007) targeting

EPOR that supports potent, more sustained, and less pulsatile elevation of hematocrit in a

human EPOR expressing-transgenic mouse model compared to standard doses of

rHu-EPO while requiring less frequent dosing. Resolution of the crystal structure of the

EPOR extracellular domain (ECD) complexed to the ABT007 Fab fragment, determined

at 3.2 ÅÅ, identifies a binding site that is consistent with a novel mechanism of receptor

activation based on a unique, antibody-imposed, conformational change. These results

demonstrate that a symmetric molecule can serve as a potent activator of the EPOR.

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Introduction

EPO, a naturally occurring hematopoietic growth factor produced by the kidney, is the

primary regulator of erythropoiesis1. Recombinant human EPO has important clinical

uses in patients with anemia associated with renal disease and cancer. Analogs of rHu-

EPO with extended serum half-lives have been developed and shown to provide a clinical

advantage by allowing maintenance of stable hemoglobin levels with less frequent

dosing2,3. A full length human agonistic antibody targeting the EPOR would offer an

longer serum half-life and may support even less frequent dosing regimens that could

better match with many chemotherapy regimens and may provide better convenience for

both predialysis and peritoneal dialysis patients who need to attend the clinic only

infrequently. In addition, an antibody EPO mimic is unlikely to induce pure red cell

aplasia, a condition associated with some forms of rHu-EPO due to the formation of rHu-

EPO-induced neutralizing antibodies4.

Mouse monoclonal antibodies (mAbs) raised to the soluble ECD of the human EPOR

have been described that mimic EPO activation by inducing ligand-dependent cell

proliferation and differentiation5,6. These mAbs, however, activate the EPOR less

efficiently than the natural hormone does and consequently are less potent agonists and

unsuitable for clinical use. Crystal structure of the EPO-(EPOR)2 complex reveals that

EPO binds two distinct sites of the two cell surface EPORs and that asymmetric

molecules may therefore be required for optimal signaling7.

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This report describes a fully human agonistic antibody, ABT-007, that effectively

stimulates both proliferation and erythroid differentiation. Since ABT007 exhibits a high

degree of selectivity and does not recognize rodent EPOR, mice expressing the human

EPOR transgene were used to establish its in vivo efficacy. Surprisingly, the activation

signal achieved with the symmetric molecule, ABT007 is sufficient to support potent, and

more sustained erythropoiesis in animal models compared to standard doses of rHu-EPO.

We examined the crystal structure of human monomeric EPOR ECD complexed with a

single antibody fragment of ABT007 (Fab-EPOR) to better understand the molecular

basis for the erythropoietic potency of ABT007. Resolution of the resulting crystal

structure identified a unique EPOR non-linear epitope distinct from the EPO binding site

resulting in a receptor conformation that supports activation. In vivo properties of

ABT007 may be further enhanced by extended serum half-life of the human antibody and

its fast off rate from the receptor which permits continuous stimulation.

Materials and Methods

Mice and cell lines. Mouse EPOR / /human EPOR transgenic mice were obtained

from Dr. Constance Noguchi of the NIH. F36E cell line

+

8 was purchased from Cell Bank

(RIKEN bioResourse Center, Ibaraki, Japan). Fresh human bone marrow cells were

obtained from Cambrex (New Jersey). All animal studies were conducted in accordance

with the guidelines established by the Abbott Laboratories Institutional Animal Care and

Use Committee.

Generation of ABT007. XenoMouse® mice (XenoMouse XG2, Amgen Fremont Inc.,

Fremont, CA) were immunized with soluble EPOR9 coupled to a universal T-cell

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epitope10. The specific titers obtained from Xenomouse® animals were determined by

enzyme-linked immunosorbent assay (ELISA) with immobilized biotinylated EPOR on

streptavidin plates (Sigma-Aldrich, St. Louis, MO). B cells from the harvested animals

were cultured and those secreting EPOR specific antibodies were isolated using the

XenoMax approach as described in Babcook et al.,TM 11. EPOR specific wells were

identified by ELISA. Supernatants from several thousand wells were tested. Those wells

testing positive for binding were screened in cell proliferation assays using EPO

dependent human cell lines (see below). Single plasma cells producing HuMabs of the

desired specificity were isolated by a plaque forming assay, mRNA was extracted and

reverse transcriptase PCR was conducted to generate cDNA. Recombinant antibody,

harvested as cell culture supernatant from transfected cells, was purified over protein A

Sepharose columns. ABT007 was one of the several recombinant antibodies identified

based on its ability to stimulate the proliferation of EPO responsive cells. It was

engineered for improved potency using yeast display technology12.

In vitro assays. F36E cells were maintained in RPMI 1640 media with 10% FBS and

5 U/ml of rHu-EPO (Epogen®, Amgen). Prior to assays, cells were cultured overnight at

a density of 4.0 to 5.0 x 105 cells/ml in growth medium without EPO. Cells were

recovered, washed and resuspended at a density of 1.0 x 106 cells/ml in assay medium

(RPMI 1640 + 10% FBS) and 50 µl of cells added to wells of a 96-well microtiter plate.

50 µl each of ABT007, isotype control antibody, or EPO standards in assay medium were

added to wells and the plates were incubated in a humidified incubator at 37°C with a 5%

CO2 atmosphere. After 72 hours, 20 µl of Promega Cell Titer 96 Aqueous® MTS reagent

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were added to all wells. Plates were incubated at 37°C with a 5% CO2 atmosphere for 4h

and the optical density at 490 nm was determined using a microplate reader. To measure

formation of erythroid colonies, fresh human bone marrow cells or bone marrow

harvested from mouse EPOR / /human EPOR transgenic mice+ were resuspended at

106

cells/ml in IMDM-2% FBS. Cells (0.3 ml) were added to 12 ml tubes containing 2.6

ml Methocult (Stem Cell Technologies), 66 µl stem cell growth factor (Sigma, 1 μg/ml),

and EPO, NESP (Amgen), ABT007, or isotype control at the concentrations shown. 50

ng/ml of EPO, 50 ng/ml of NESP and 1 μg/ml of ABT007 correspond to 1.66 nM, 1.35

nM, and 6.6 nM, respectively. After mixing, 1.1 ml of the Methocult suspension was

added to a 35-mm non-tissue culture treated sterile petri dish and incubated at 37oC, 5%

CO2 for 2 weeks.

Transgenic mouse model for erythropoiesis. Male mouse EPOR / /human EPOR+

transgenic mice were dosed subcutaneously (4-6 mice per treatment group) with NESP

(novel erythropoiesis stimulating protein; Amgen) at 3, 12 and 20 μg/kg on days 0 and 14

or ABT007 on day 0 only, at 0.2, 0.8 and 1.6 mg/kg. A human IgG2 isotype control

antibody was dosed at 1.6 mg/kg on day 0 only. 25 μl of blood were collected weekly via

orbital bleed from each animal and hematocrit was measured using a HESKA® Vet ABC-

Diff hematology analyzer.

Protein preparation and crystallization. A soluble form of mature EPOR ECD,

representing residues 1 to 225, was expressed in E. coli and refolded and purified as

described9. In order to facilitate the generation of Fab fragments, ABT007, was re-

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engineered as an IgG1 human antibody and subjected to papain cleavage13. Samples for

crystallization contained 1:1 complexes of EPOR ECD and ABT007 Fab fragments at a

concentration of 14 mg/ml in 20 mM HEPES, 150 mM NaCl, 1 mM NaN3 at pH 7.5.

Crystallization was carried out using the hanging drop vapor diffusion method at 17oC

combining 2 μl protein with 2 μl of reservoir solution consisting of 15% PMME5000 and

600 mM Li2SO4. Protein crystals grew to approximately 0.8 x 0.1 x 0.1 mm in two weeks

time. The cryopreservative was made using 80% reservoir solution and 20% glycerol.

Crystals were flash frozen in liquid nitrogen for data collection after quick passage

through the cryopreservative. Diffraction data were collected at the Industrial

Macromolecular Crystallography Association beamline ID-17 at Argonne National

Laboratory and processed to 3.2 ÅÅ resolution using HKL200014. The crystals are space

group P212121 and unit cell parameters a = 117.95, b = 156.17, c = 164.20 with three

Fab's bound to three EPOR's in the asymmetric unit based on Matthews parameter

calculations.

Structure determination and refinement. The structure was solved using a

combination of Phaser15 and Molrep16 for molecular replacement. The search model used

in Phaser for the Fab fragment was 1JPT17, and an ensemble of EPOR structures 1CN4,

1EBA18, 1EBP19 and 1EER were used to search for EPOR portions. This procedure

identified two Fab/EPOR complexes in the asymmetric unit. One of these Fab/EPOR

complexes was then used as a search model in Molrep to identify the third Fab/EPOR

complex in the asymmetric unit with the first two complexes from Phaser held fixed. The

resulting structure shows well-determined electron density for three copies of EPOR, two

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well-defined copies of the ABT007 Fab, while the third copy has well-defined density of

the light and heavy chains in the complementarity determining regions. The conserved

domains of the light and heavy chains of the third copy are solvent exposed and not well

ordered. Refinement was initiated with multiple rounds of visual inspection and manual

fitting in Quanta (Accelrys Software, Inc., San Diego, CA) and refinement using

CNX20, 21 followed by a final refinement using refmac22 to refine the structure to 3.2 ÅÅ

resolution with an Rwork = 23% and Rfree = 32%.

Results

ABT007 stimulates erythropoiesis

BIAcore analysis confirmed that ABT007 binds to EPOR with a Kd value of 30 nM and a

fast off rate of 4.8X10-3 s-1 (S.E.L. and E.B.R. unpublished data). ABT007 stimulated the

proliferation of the F36E EPO-dependent cell line8 with maximal proliferative activity

similar to that observed with EPO (Fig. 1A). The maximal response occurred at

concentrations approximately ten-fold higher than EPO on a molar basis. Increasing

concentrations of both EPO and ABT007 resulted in a bell shaped activation curve, most

likely explained by involvement of EPOR-ligand/antibody interactions in nonproductive

1:1, not 2:1, complexes5,6. Similar results were observed with UT-7/EPO an EPO

dependant human megakaryoblastic leukemia cell line23. Since growth and

differentiation of erythroid progenitor cells depend on EPO, ABT007 was tested for its

ability to support the formation of erythroid colonies from human bone marrow

containing CD34+ progenitor cells. The addition of ABT007 to hematopoietic progenitor

cells induced the formation of erythroid colonies, which were readily identified by the

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hemoglobinization of cells in the colony (Fig. 1B, D and E). The maximum colony

number observed with ABT007 was somewhat reduced compared to the maximum

colony number observed with EPO and required a 5-40-fold greater concentration on a

molar equivalent basis.

ABT007 does not recognize rodent EPOR (E.B.R unpublished data) and so transgenic

mice generated by rescuing genetic knockout mice lacking the murine EPOR gene with

the human EPOR transgene24,25 were used to establish in vivo efficacy of ABT007. These

mice exhibit the transgene in hematopoietic tissues at levels comparable to endogenous

murine EPOR, and the reticulocyte and hematocrit responses to dosing with EPO

observed in these animals are similar to those seen with inbred strains of mice. ABT007

supported the formation of erythroid colonies from transgenic mouse-derived

hematopoietic precursors comparable to that seen with human progenitor cells, indicating

that ABT007 does recognize the human transgene receptor (Fig. 2).

ABT007 dosed every 4 weeks sustains hematocrit increases in animals

The ability of ABT007 to elevate hematocrit in a dose- and time- responsive manner was

compared to that of NESP, a long acting, hyperglycosylated analog of rHu-EPO and

currently in clinical use for anemia treatment as Aranesp®. Successful antibody therapy

generally requires higher dose requirements than other protein therapeutics. A single

administration of ABT007, at concentrations >0.2 mg/kg, results in a dose-dependent rise

in hematocrit that is sustained for at least four weeks (Fig. 3). The hematocrit achieved

with a single dose of ABT007 is at least equivalent to that observed with a clinically

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relevant dose (3 μg/kg) of NESP26 administered every two weeks. ABT007 dosing results

in more stable erythropoiesis with less fluctuation than that observed following NESP

dosing (Fig. 3). Additionally, increases in hematocrit were similar following either

subcutaneous or intravenous administration of ABT007 (unpublished data).

ABT007 Fab interacts with novel EPOR binding site

Protein fragment complementation assays and crystallographic studies indicate that

EPOR exists as a preformed dimer19,27. EPO binding to the receptor, through

nonequivalent high and low affinity binding sites28, triggers the switch between a self-

associated, inactive conformation and an active, ligand-bound conformation. A full-

length IgG type antibody raised to EPOR ECD, by virtue of its bivalency, may also

induce the interaction of two receptors and stabilize the active conformation. The in vivo

potency of ABT007 may be at least partially attributed to the enhanced serum half-life of

the fully human antibody. In fact, it has been demonstrated that NESP, despite its lower

affinity for EPOR, has greater in vivo activity than that of EPO due to a longer serum

half-life29. We postulated, however, that the binding conformation imposed by ABT007

might also contribute to the activation of the receptor and subsequent enhanced

erythropoiesis. In fact, ABT007 binds to EPOR under non-denaturing conditions, but not

under denaturing conditions, suggesting that the epitope recognized by ABT007 is a

nonlinear, conformational epitope. In order to map the EPOR binding site and elucidate

the molecular basis of this interaction, a soluble form of rHu-EPOR ECD9 was generated,

and the crystal structure of human monomeric EPOR ECD complexed with a single Fab

fragment of ABT007 (Fab-EPOR) was determined at 3.2 ÅÅ resolution by molecular

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replacement. The crystal structure of Fab-EPOR (Fig. 4) confirmed that ABT007 binds

EPOR through a non-linear, conformationally defined epitope that includes residues from

EPOR region E25-V112 (Table 1). The primary hydrophobic interactions are extended

cation/π stacking interactions involving residues Y33 from antibody heavy chain, R99

from EPOR, H110 from EPOR and H91 from antibody light chain. In addition to the

extended stacking interactions, there is a series of hydrogen bonds between light chain

N31 to EPOR-R111, light chain E32 to EPOR-R111, light chain R30 to both EPOR-E25

and the main chain carbonyl of EPOR-L26 and heavy chain L100 main chain to EPOR-

E97 that further stabilize the complex. There are additional van der Waals interactions

between other residues of both the light and heavy chains and residues V112, P107 and

H110 of EPOR that complete the interactions between the Fab and EPOR. Finally, W64

of EPOR is an additional contact residue that interacts with heavy chain Y33 and may

also interact with EPOR R99 side chain thereby stabilizing the EPOR conformation.

Comparison of the Fab-EPOR complex with the previously determined crystal structure

of EPO complexed to EPOR shows there is no overlap of contact residues (Fig. 4). F93

and F205 of EPOR, which are the basis of essential hydrophobic interactions with

EPO7,27, do not participate in the interaction with Fab. Coordinates of the x-ray structure

of the ABT007 Fab/EPOR complex have been deposited in the RCSB Protein Data Bank

under accession number 2JIX.

It has been demonstrated that the activating efficiency of EPOR is dependent on the

orientation imposed by bound ligands, and that asymmetric molecules may be required

for optimal EPOR activation7, ,19 27. The fact that ABT007 is a symmetric molecule that

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binds to a distinct Fab-EPOR binding site suggests that ABT007 may induce a unique

conformation on the receptor and provide a basis for a novel mechanism of activation. To

explore this possibility further, as shown in Fig. 5, we superimposed two Fab-EPOR

complexes (to mimic the bivalency of ABT007) onto the conformation of two adjacent

receptors induced by EPO binding. Although it is conceivable that 1:1 monomeric

antibody-receptor complexes could support this model, this is unlikely since the bell

shaped activation curve observed for ABT007 (Fig. 1) is consistent with non-productive

binding at high antibody concentrations where 1:1 complexes are likely to predominate.

Additionally, in this model (Fig. 5) the distance between the carboxyl termini of the two

antigen-specific Fabs is ≥ 113 ÅÅ and cannot be accommodated by a single antibody

molecule. A more attractive model of activation is based on a conformation induced onto

EPOR by ABT007 in a 2:1 ratio that is different from that caused by EPO. Additional

experimental findings support a distinct EPO binding site and activation mechanism since

ABT007 treatment of EPO-dependent F36E cells results in an altered profile of STAT

proteins compared to that observed upon EPO binding (R.L.S. and E.B.R. unpublished

data).

Discussion

Potent, safe EPO mimics that offer advantages with respect to a more sustained, less

pulsatile elevation of hematocrit may offer both medical benefits and improved patient

convenience. Other EPO mimics, including both peptides30 and activating antibodies 5,6

that activate EPOR by binding to regions outside the EPO binding site have been

described. The potency of previously described antibody mimics is suboptimal compared

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to EPO and unsuitable for therapeutic use. In contrast, our results demonstrate that

ABT007 is a potent, in vivo, stimulator of erythropoiesis that requires less frequent

dosing than NESP, and has a preclinical profile consistent with therapeutic intervention

of anemia associated with kidney failure and cancer31. To our knowledge this is the first

demonstration that an EPO mimic antibody stimulates and sustains erythropoiesis in vivo.

Additionally, ABT007 dosing in transgenic mice results in more efficient and stable

erythropoiesis than that observed following NESP dosing. Fluctuation of hemoglobin

levels is highly correlated with clinical complications in patients with end stage renal

disease32. It is also unlikely that a human antibody against EPOR would induce antibody-

mediated pure red cell aplasia associated with some rHu-EPO therapies4.

Previous results demonstrate that optimal EPOR signaling requires asymmetric EPO as

its ligand7,18,27. In contrast, results presented herein suggest that a symmetric molecule,

ABT007, also effectively activates the EPOR. As a symmetric molecule, ABT007 may

bind and activate the receptor in a manner distinct from EPO. In support of this

hypothesis, crystal structure resolution of the Fab-EPOR complex identified a unique

binding site for ABT007 (Fig. 4). Additionally, based on modeling deduced from the

Fab-EPOR crystal structure, the size of ABT007 precludes it from assuming a

conformation similar to that induced by EPO binding (Fig. 5). Our observation that other

activating human anti-EPOR IgG2 antibodies are less effective in stimulating

erythropoiesis in vivo provides additional support that the unique receptor conformation

imposed by ABT007 plays a critical role in supporting erythropoiesis.

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Other attributes of ABT007, including extended serum half-life, may also contribute to its

in vivo potency. For example, ABT007 has similar pharmacokinetic profiles in mouse

EPOR / /human EPOR transgenic mice + and cynomolgus monkeys (data not shown).

Its serum half-life of ~6 days, significantly longer than that of either rHuEPO or NESP33,

is consistent with in vivo results indicating that ABT007 supports monthly dosing

(Fig. 3). Interspecies scaling has been used successfully for the prediction of clearance

for protein drugs in humans34. Additionally, the in vivo potency of ABT007 is also

influenced by its on-off rate kinetics and binding affinity. Repeated binding and

dissociation from the receptor may permit continuous stimulation of erythropoiesis.

In summary, our findings indicate that ABT007 has several potential dosing and safety

features that make it an attractive alternate for the treatment of anemia. Transgenic mice,

expressing the human EPOR transgene in hematopoietic tissues at levels comparable to

endogenous murine EPOR, provide a relevant model for predicting human dosing and

mitigating the risk associated with overshooting safe hematocrit. The correlation

observed in animals between dose and subsequent increases in hematocrit offers another

attractive clinical feature of ABT007. This characteristic may prove extremely valuable

in predicting human dosing. The value of this therapeutic will ultimately be realized upon

human testing.

Acknowledgments.

We thank Dr. C. Noguchi for providing breeding pairs of mouse EPOR-/-, human EPOR+

transgenic mice and Dr. S. Fesik for comments on the manuscript. We also acknowledge

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the support of Amgen Freemont Inc., Freemont, CA (formerly Abgenix, Inc.) in the

generation of the ABT007 precursor antibody. For crystal structure analysis, data were

collected at beamline 17-BM in the facilities of the Industrial Macromolecular

Crystallography Association Collaborative Access Team at the Advanced Photon Source.

These facilities are supported by the companies of the Industrial Macromolecular

Crystallography Association. Author contribution statement: ZL, RSL, RRL, EBR

designed experiments and analyzed data. ZL, VSS, PD, CGJ, NX, SEL, DAE and JEH

performed experiments and analyzed data. EBR wrote the paper. All the authors are

employees from Abbott Laboratories and have a declared financial interest in a company

whose potential product was studied in the present work.

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Figures.

0

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Figure 1. ABT007 stimulates in vitro erythropoiesis. (A) ABT007 stimulates the

proliferation of EPO-responsive F36E human erythroleukemic cells8. Error bars represent

standard deviation (SD) calculated from the average of duplicate counts. (B) ABT007

supports the formation of erythroid colonies from hematopoietic precursor cells. Typical

colonies, identified microscopically are shown below for (C) the isotype control-, (D)

EPO- and (E) ABT007- treated cells. The colonies, identified microscopically, were red

in color. Error bars represent SD calculated from the average of duplicate counts.

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B C D

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ies

A

0

20

40

60

80

100

120

140

isotype 10ug/ml Epogen 0.05ug/ml A b-12.6 0.5ug/ml A b-12.6 1ug/ml A b-12.6 10ug/ml

Ery

thro

id-d

eriv

ed c

olon

ies

A

0.5 μg/mlABT007

50 ng/mlEPO

IsotypeControl

10 μg/mlABT007

1 μg/mlABT007

0.5 μg/mlABT007

50 ng/mlEPO

IsotypeControl

10 μg/mlABT007

1 μg/mlABT007

Figure 2. ABT007 stimulates formation of transgenic mouse CFU-E colonies. (A)

ABT007 supports the growth of erythroid colonies from bone marrow cells of transgenic

mice. Typical colonies, identified microscopically are shown on the right for (B) the

isotype control, (C) EPO and (D) ABT007 treated cells. The colonies, identified

microscopically, were red in color. Error bars represent standard deviation calculated

from the average of duplicate counts. All images are at the same magnification.

17

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18

-10

-5

0

5

10

15

20

0 7 14 21 28Days

Cha

nge

inhe

mat

ocrit

Vehicle Control3 mg/kg NESP6 mg/kg NESP12 mg/kg NESP20 mg/kg NESP

A

-10

-5

0

5

10

15

20

0 7 14 21 28Days

Cha

nge

inhe

mat

ocrit

1.6 mg/kg Isotype Control

0.2 mg/kg ABT007

0.8 mg/kg ABT007

1.6 mg/kg ABT007

BVehicle Control3 mg/kg NESP6 mg/kg NESP12 mg/kg NESP20 mg/kg NESP

1.6 mg/kg Isotype Control0.2 mg/kg ABT0070.8 mg/kg ABT0071.6 mg/kg ABT007

Q2W Q4W

-10

-5

0

5

10

15

20

0 7 14 21 28Days

Cha

nge

inhe

mat

ocrit

Vehicle Control3 mg/kg NESP6 mg/kg NESP12 mg/kg NESP20 mg/kg NESP

A

-10

-5

0

5

10

15

20

0 7 14 21 28Days

Cha

nge

inhe

mat

ocrit

1.6 mg/kg Isotype Control

0.2 mg/kg ABT007

0.8 mg/kg ABT007

1.6 mg/kg ABT007

BVehicle Control3 mg/kg NESP6 mg/kg NESP12 mg/kg NESP20 mg/kg NESP

1.6 mg/kg Isotype Control0.2 mg/kg ABT0070.8 mg/kg ABT0071.6 mg/kg ABT007

Q2W Q4W

Figure 3. ABT007 dosed every 4 weeks is comparable to NESP dosed every 2 weeks.

Male transgenic mice were dosed subcutaneously either with NESP (A) on days 0 and 14

or antibody (ABT007 or isotype control) (B) on day 0 at the concentrations indicated.

Blood samples were collected weekly and hematocrit measured using a HESKA® Vet

ABC-Diff hematology analyzer. Data represent change in hematocrit mean +/- standard

error of 4-6 mice per treatment group. A typical NESP dose in practice is 3 µg/kg every

two weeks26.

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R30

L26

N31E32 S53

R111P107

F93F205

H110 Y50R99 Y33

Y94

W64

E97

L chainH chain

EPOR

R30

L26

N31E32 S53

R111P107

F93F205

H110 Y50R99 Y33

Y94

W64

E97

L chainH chain

EPOR

Figure 4. Interaction of ABT007 Fab-EPOR. Crystal structure of the binding region of

a single Fab-EPOR monomeric complex. Gray represents the ABT007 Fab light chain

and brown represents the ABT007 Fab heavy chain while green represents EPOR.

Highlighted residues are directly involved in Fab/EPOR binding. Residues F93 and F205

of EPOR, highlighted in purple, are key residues involved in binding EPO and are not

involved in Fab binding.

19

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113 ÅÅ

Fab

Fab

EPO

EPOREPOR

113 ÅÅ

Fab

Fab

EPO

EPOREPOR

Figure 5. Comparison of the Fab-EPOR complex with the EPO-activated EPOR

crystal structure. Two copies of ABT007 Fab (blue) complexed to EPO-activated

EPOR dimer (green) are superimposed onto the EPO (red)-activated EPOR dimer

(brown, 1EER)7 complex. Two independent Fab fragments can be accommodated on the

EPO activated form of EPOR, but the carboxyl termini of the Fab fragments are too

distant (113 ÅÅ) to be derived from a single IgG.

20

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Table 1 EPOR and ABT007 residues involved in interaction

EPOR Heavy chain Type of interaction

R99 Y33 Cation/π stacking

R99 Y50 Cation/π stacking

W64 Y33 π stacking

E97 L100 (main chain) Hydrogen bond

V112 L100 Van der Waals

P107 Y94 Van der Waals

EPOR Light chain Type of interaction

H110 H91 π stacking

P107 Y94 Van der Waals

R111 N31 Hydrogen bond

R111 E32 Hydrogen bond

H114 S53 Hydrogen bond

E25 R30 Hydrogen bond

L26 (main chain) R30 Hydrogen bond

V112 A50 Van der Waals

21

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Erratum

In the article by Liu et al entitled “A potent erythropoietin-mimicking humanantibody interacts through a novel binding site,” which appeared in theOctober 1, 2007, issue of Blood (Volume 110:2408-2413), incorrect unitswere used in the key legend for Figure 3B. The correct unit for all 4 lines inFigure 3B’s key legend is mg/kg.

996 SYRJALA et al BLOOD, 00 MONTH 2008 � VOLUME 111, NUMBER 00

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