Biomedical Engineering Reference
In-Depth Information
FIGURE 38.5 Irreversible fusion of AZD linker with LysH93 in the active site of CVX-2000.
than the CovX-Body itself. More importantly, the release of
the payload due to the reversible enaminone bond could
easily compromise the pharmacokinetic advantage provided
by the carrier antibody scaffold.
The 1,3-diketone CovX-Bodies were found to be stable in
in vitro assay systems and no detectable payload was
released from these CovX-Bodies when subjected to dialy-
sis, thus providing circumstantial proof that the in vitro
activity was mediated by the CovX-Body rather than the free
payloads. However, in vivo PK experiments with 1,3-dike-
tone based CovX-Bodies revealed that the serum half-life of
the fused pharmacophore was significantly lower than the
serum half-life of the CVX-2000 scaffold, likely a result of
the reversible 1,3-diketone linker system. To overcome this
serious limitation of the 1,3-diketone system, an extensive
screening campaign was undertaken to identify electrophilic
linkers that would react selectively and irreversibly with
LysH93. This led to the identification of a phenethyl acyl
azetidinone (AZD) that forms a covalent conjugate with
CVX-2000 (Figure 38.5). The fusion reaction with the AZD
linker is an acylation of LysH93, where in the
-amino group
acts as a nucleophile to selectively react with the electro-
philic azetidinone group. This results in the AZD ring
opening and fusion of the linker with the antibody via a
stable beta alanine peptide bond. Alkyl AZDs alone were
found to be ineffective as selective inhibitors of CVX-2000,
thereby highlighting the importance of the additional phenyl
group in providing a recognition element and enabling an
induced fit of the AZD linker in the active site of CVX-2000.
The effect of the irreversible AZD versus the reversible 1,3-
diketone linker system on the pharmacokinetic profile of the
fused payload is shown in Figure 38.6.
Peptide mapping experiments were conducted to confirm
the site of conjugation between CVX-2000 and AZD linkers.
e
OH
O
O
N
O
S
1000
O
O
N
H
O
O
N
H
N
H
O
CO
2
-
100
O
OH
O
O
O
S
10
O
O
H
O
O
H
H
O
CO
2
-
1
O
0
24
48
72
Time (h)
FIGURE 38.6 Comparison of the PK profile of PEGylated FITC dye when fused with CVX-2000
via the irreversible AZD linker or the reversible diketone linker.
Search WWH ::
Custom Search