BONE-TARGETED ALKALINE PHOSPHATASE - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 414

fractures and rachitic deformity of the rib cage, has been
reported to be as high as 50% [15]. If neonatal seizures
(generally pyridoxine-responsive) occur, they are a grave
prognostic symptom: a recent review of the literature found
that all reported HPP infants with neonatal seizures had a fatal
outcome by 18 months of age [10]. HPP in children is often
diagnosed when there is premature exfoliation of deciduous
teeth, defined as loss before the age of 5 years. Tooth loss is
due to an absence of mineralized cementum, which is required
for the periodontal ligaments to tether the tooth root to the
surrounding bone. Rickets and its associated bony deformities
are common, resulting in short stature in some patients.
Walking is frequently delayed and may be characterized by
a peculiar waddling gait. Nontraumatic fractures are not
uncommon. HPP may also present later in life, although
upon questioning adults may report a history of early tooth
loss or rickets during childhood. Osteomalacia is the hallmark
of HPP in adults and often presents as bone pain in the feet
resulting from recurrent, poorly healing stress fractures. X-ray
examination may reveal the presence of pseudofractures,
osteopenia, and chondrocalcinosis. Odontohypophosphata-
sia, in which pathology is limited to the teeth, is the mildest
form of the disease.
There is no established medical therapy for HPP [3]. Case
reports of attempted enzyme replacement therapy (ERT)
using intravenous (IV) infusions of ALP-rich plasma from
Paget's bone disease patients, purified human liver ALP, or
purified human placental ALP indicate failure to rescue
affected infants [16-19]. These outcomes suggest that
ALP activity must be increased not merely in the circulation
but in the HPP skeleton itself. This hypothesis has been
supported by favorable outcomes seen in two girls with
infantile HPP following transplant of mesenchyme-derived
cells, which likely contained small numbers of TNAP-
expressing osteoblasts and chondrocytes that became dis-
tributed throughout the skeleton [20,21].
The drug ENB-0040, developed by Enobia Pharma Inc.
(Montr

the C-terminal hydrophobic sequence encoding the GPI-
anchor to create a soluble secreted enzyme (sALP); exten-
sion of the coding sequence of each TNAP subunit with the
human IgG Fc region ( g 1 form) to allow purification
using Protein A chromatography (-Fc); and addition of a
mineral-binding deca-aspartate (D 10 ) sequence [25-27] to
the C-terminus of each Fc fragment. A subsequent publica-
tion [28] has used the name ENB-0040 for this reagent,
while asfotase alfa is the name approved by the International
Nonproprietary Name (INN) Program managed by the
World Health Organization. We will continue to use
sALP-FcD 10 and ENB-0040 throughout this chapter.
The cDNA encoding the fusion protein was inserted into
the pIRES vector (Clontech, San Diego, CA) in the first
multiple cloning site located upstream of the IRES using
NheI and BamHI endonuclease restriction sites. The dihy-
drofolate reductase (DHFR) gene was inserted into the
second multiple cloning site downstream of the IRES using
SmaI and XbaI endonuclease restriction sites. The resulting
vector was transfected into Chinese hamster ovary (CHO-
DG44) cells lacking both DHFR gene alleles [29] using the
Lipofectamine transfection kit (Invitrogen, San Diego, CA).
The purification procedure of sALP-FcD 10 was described in
detail [23] and is only briefly summarized here. The sALP-
FcD 10 concentration in spent media was 3.5mg/L, as

assessed by TNAP enzymatic activity. Culture supernatants
were concentrated, dialyzed against PBS and loaded onto
Protein A-Sepharose columns (Hi-Trap 5 mL, GE Health
Care) equilibrated with PBS. Bound proteins were eluted
with 100mM citrate buffer, pH 4.0. Collected fractions were
immediately adjusted to pH 7.5 with 1M Tris, pH 9.0.
Fractions containing most of the eluted material were dia-
lyzed against 150mM NaCl, 25 mM sodium phosphate
buffer, pH 7.4, containing 0.1 mM MgCl 2 ,20 m MZnCl 2 ,
and filtered through a 0.22 m m (Millipore, Millex-GP)
membrane under sterile conditions. The overall yield of
the purification procedure was 50%, with a purity surpassing
95% as assessed by Sypro ruby-stained SDS-PAGE. Purified
sALP-FcD 10 preparations were stored at 4 C and remained
stable for several months. This enzyme preparation
(research grade) was used in two preclinical studies
[23,24]. The purification of cGMP clinical grade ENB-
0040 (Asfostase alfa) is much more elaborate, with addi-
tional chromatographic and filtration steps [30].
Recombinant ENB-0040 is a soluble glycoprotein of 726
amino acids, which after purification on Protein-A Sephar-
ose and analyzed in reducing SDS-PAGE conditions
migrates as a broad band with an apparent molecular
mass of

eal, Canada), consists of the TNAP polypeptide
sequence devoid of its GPI anchoring domain (for a com-
plete description of the TNAP structure see reference [22]),
which was replaced by a human Fc region to allow rapid
chromatographic purification followed by a deca-aspartate
acid (D 10 ) sequence to confer bone-targeting properties due
to its high affinity of D 10 for bone mineral. The modeled 3D
structure of bone-targeted TNAP is shown in Figure 28.1,
along with in vitro evidence of enhanced binding to
hydroxyapatite and in vivo support of the drug's ability to
home to the mineralizing skeleton.

90,000 Da. ENB-0040 catalytic activity was com-
parable to that of HPLC-purified glycosylation isoforms of
human TNAP using either the artificial colorimetric sub-
strate p-nitrophenylphosphate at pH 9.8 or the physiological
substrates inorganic pyrophosphate and pyridoxal-5 0 -phos-
phate at pH 7.4 [31]. The affinity of purified ENB-0040

28.2 TECHNICAL ASPECTS

Bone-targeted TNAP was initially designated sALP-Fc-D 10
[23,24] due to its chimeric composition: namely, removal of