The Genesis of the Antibody Conjugate Gemtuzumab Ozogamicin (Mylotarg) for Acute Myeloid Leukemia - Accounts in Drug Discovery Case Studies in Medicinal Chemistry - page 109

20

15

Rate of hydrolysis ~ 2%/day

10 Human Plasma/Serum

5

0

24 48 72 96

Time (hr)

Figure 5.6 Linker hydrolysis. Inotuzumab ozogamicin was incubated in human
plasma and serum at 37 1C and samples were analyzed at the indicated
time for free N-acetyl-g-calicheamicin DMH.

The difference in hydrolysis between the unconjugated and conjugated forms
(6% vs. 2%) may be due to a lipophilic interaction between the calicheamicin
and the antibody in the conjugate, resulting in an increase in stability; it could
be due to the microenvironment of the hydrazone in the conjugated form; or it
could be the result of different media used (serum/plasma vs. buffer containing
an organic co-solvent). It is not known if the hydrolysis rate differs in vivo from
that seen in plasma and serum. It has been dicult to study this issue because
gemtuzumab ozogamicin, like most conjugates, is a mixture of species. If the
antibody and calicheamicin conjugate clear at different rates in vivo, it is hard to
discern if the linker is showing increased hydrolysis or if the higher loaded
species are clearing faster than the lower loaded species, which would alter the
ratio of calicheamicin to antibody without linker hydrolysis.
Gemtuzumab ozogamicin is a mixture of species due to variation in antibody
glycosylation as well as to the sites and degree of modification, in this case the
identity of the lysines acylated. However, gemtuzumab ozogamicin has two
unusual properties as a conjugate in its constitution. 18 First, the calicheamicin
is not randomly distributed on a large number of lysines scattered around the
antibody, as might be expected and as has been shown for another lysine-based
conjugate, 19 but resides primarily on eight lysines, four on each of the two
heavy chains. Second, the distribution is bimodal; about half of the antibody
carries little if any calicheamicin while the other half has twice the average
loading with very little at lower loadings of 1-3 calicheamicins/antibody. This
all-or-nothing effect might be due to the aggregated state of the highly lipo-
philic calicheamicin, leading to a locally high concentration near the site of any
initial conjugate; or it may be due to a conformational change in the hinge
region where these sites reside, resulting in increased reactivity; or it may be due
to other factors that have yet to be explored.
These two unusual phenomena appear to be unrelated, as a previous cali-
cheamicin conjugate, CMB-401, which lacks the AcBut linker (the amide
conjugate), showed selectivity for the same lysines (as do all the calicheamicin