Chemistry Reference
In-Depth Information
Anti-CD33
Antibody
hP67.6
n, loading of calicheamicin on
antibody = 0, 1, 2, 3, 4, 5, .....
average n ~ 3
HN
O
O
O
H
3
C
CH
3
O
N
H
HO
S
H
3
C
CH
3
NH
O
O
S
CH
3
I
O
S
OCH
3
O
CH
3
O
O
OCH
3
HN
H
OH
O
HO
OCH
3
O
CH
3
O
CH
3
CH
2
HO
O
OCH
3
N
OH
CH
3
OCH
3
O
n
Figure 5.5
Structure of gemtuzumab ozogamicin. The anti-CD33 antibody is acy-
lated with the bifunctional AcBut linker, which allows for hydrazone
formation with the calicheamicin derivative. This derivative, N-acetyl-g-
calicheamicin dimethylhydrazide (DMH), has a disulfide stabilized by
both the two methyl groups and the calicheamicin bicyclic core, and an
acetylated amino sugar.
Numerous bifunctional linkers were explored, with aliphatic aldehydes and
ketones giving hydrazones that were too unstable (cf. the well-known BR96-
DOX conjugate, which has marginal stability in circulation
16
). Aromatic
aldehydes gave conjugates with reasonable stability and activity, but acet-
ophenones, which release the calicheamicin derivative faster at acidic pH's,
such as found in lysosomes, gave the best activity with an acceptable degree of
stability. The final choice of the bifunctional linker was 4-(4-acetylphenoxy)
butanoic acid (''AcBut''), which resulted in the conjugate shown in Figure 5.5
as the structure of gemtuzumab ozogamicin (hP67.6-NAc-gamma calicheami-
cin DMH AcBut conjugate).
The hydrolytic behavior of the new hydrazone bond in this conjugate was
examined in two ways. First, the carboxylic acid without the antibody, but
containing the calicheamicin derivative attached to the bifunctional AcBut lin-
ker, was subjected to hydrolysis in buffer. At pH7.4, 6% hydrolysis was seen in
24 h at 37 1C, while at pH4.5 (lysosomal pH) 97% hydrolysis was seen. Studies
were also conducted on a related conjugate, inotuzumab ozogamicin (CMC-
544), which is in Phase 3 trials for non-Hodgkin's lymphoma
17
and which has an
anti-CD22 antibody conjugated to the same calicheamicin derivative and
bifunctional AcBut linker that is used in the conjugate gemtuzumab ozogamicin.
This antibody conjugate was studied in human plasma and serum and showed a
slower than expected rate of release of 2-3% per day at pH7.4 (Figure 5.6),
18
which corresponds to an estimated half-life of hydrolysis of 23-34 days.
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