Biology Reference
In-Depth Information
Molecules that alter or increase affinity at either of the two sites can have
increased activity.
Despite numerous attempts to identify them, no erythropoietic molecules
suitable for clinical development have been reported that increase biological
effect as a consequence of increased receptor affinity. The reasons are several-
fold. First is the theoretical concern that antibodies, were they to develop in
patients, may be targeted to the changed region of the molecule and would be
neutralizing because they would interfere with the EPO:EPOR interaction.
Should these antibodies cross-react with endogenous EPO, pure red cell apla-
sia would likely result. A second reason is that increased affinity does not
always translate into increased
in vivo
activity because there are additional
requirements beyond receptor binding required to effect a biologic response
in
vivo
. Each receptor-binding event is transient because EPO:EPOR complexes
are rapidly internalized and degraded [9, 10]. In addition, the EPOR-signaling
pathway is down-modulated shortly after activation [10-12]. Thus,
in vivo
ery-
thropoiesis requires continuous stimulation of multiple EPOR through multi-
ple binding events. As a consequence, molecules cleared quickly have low
in
vivo
activity. For example, increased
in vitro
activity and increased receptor
affinity have been observed with EPO analogs that remove sialic acid from
carbohydrates or remove
N
-linked carbohydrates entirely [13, 14]; however,
N
these molecules have reduced
in vivo
activity due to a more rapid clearance
[13, 15]. Increased concentrations can partially compensate for the increased
clearance; however, these compounds must be administered more frequently to
be fully efficacious.
Changes in rHuEPO amino acid sequence can result in increased stability.
These changes can include removal of amino acids that are unstable (trypto-
phan [16]); or are subject to oxidation (methionine), deamidation (asparagine),
or changes that confer increased conformational stability, such as those that
stabilize alpha helices or connecting loops. Such molecules may be more
amenable to long-term storage or suitable for formulations where more stable
EPO molecules are essential, such as in slow-release formulations or automat-
ed delivery systems. Removal of proteolytic cleavage sites by
in vitro
mutage-
nesis can enhance
in vivo
stability. It may be possible to remove antigenic
sites, thereby reducing immunogenicity.
Compounds that bind through the high-affinity site but do not dimerize
because of reduced binding at the low-affinity site can function as antagonists
[8]. Such molecules may have some clinical utility for treatment of EPO
responsive polycythemias, and several such molecules have been described [8,
17].
Molecules with increased serum half-life
A longer duration of action can allow for reduced frequency of administration.
One approach that has been successfully applied to rHuEPO is glycoengineer-
