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conjugates that have been examined), but not the all-or-nothing effect, having a
relative normal distribution of species.
20
With regard to the bimodal distribu-
tion, if one ignores any effects on pharmacokinetics and biodistribution
properties, the average loading alone should determine the potency and
selectivity of the conjugate due to the statistical mixture each cell will inter-
nalize. This means the in vitro studies are likely to be reliable, in spite of a wide
loading distribution, but leads to questions for in vivo studies, as loading can
significantly affect the pharmacokinetics of conjugates. Understanding and
overcoming these effects is still an active area of research in the conjugate
field.
21
Unfortunately, the loading distribution of gemtuzumab ozogamicin was
not appreciated until it was in clinical trials. However, any PK effects are
probably less significant for this anti-leukemia conjugate, as binding to the
antigen occurs rapidly.
i
During the discovery stage of gemtuzumab ozogamicin, its cytotoxic effects
were first examined in vitro on a pair of cell lines: HL-60 acute promyelocytic
leukemia (APL, a sub-type of AML) cells that express CD33 and Raji non-
Hodgkin's lymphoma cells that do not (Table 5.1). Gemtuzumab ozogamicin
showed an IC
50
of 0.31 pM versus the target cell line with 150 000-fold selec-
tivity versus the non-target cell line.
15
It is 20 000-fold more potent than the
calicheamicin derivative that it releases by hydrolysis.
Gemtuzumab ozogamicin was very ecacious against the HL-60 cells when
grown as xenografts in nude mice. Treatment of established 150-200mg sub-
cutaneous tumors with three doses given on days 1, 5, and 9 led to dis-
appearance of the tumor mass in either 4/5 or 5/5 animals at doses of 50-
300 mgkg
1
of calicheamicin, with no regrowth up to 100 days. At a dose of
25 mgkg
1
there was 80% inhibition of tumor growth versus buffer-treated
control animals. A non-binding control conjugate showed a non-significant
20% inhibition at the top dose. See Figure 5.7 for a representative experiment.
Gemtuzumab ozogamicin was also used to treat bone marrow samples from
AML patients in a colony-forming assay and compared to the number of
colonies seen with a non-binding conjugate (Figure 5.8). As indicated by the
bar graph, approximately one-third to one-half of the samples showed positive
responses, depending of the cutoff used.
Table 5.1 Gemtuzumab ozogamicin in vitro activity.
Treatment
IC
50
(nM) Raji Selectivity index
a
IC
50
(nM) HL-60
Gemtuzumab ozogamicin 0.000 31 24 150 000
N-Ac-g-calicheamicin DMH 6.1 3.1 [1]
Ratio 20 000 0.13
a
Ratio of activity corrected for the difference in sensitivity of the two cell lines to calicheamicin.
i
The related conjugate inotuzumab ozogamicin (CMC-544) has little if any free antibody due to its
higher loading.
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