Biomedical Engineering Reference
In-Depth Information
human carboxypeptidase A [12], human carboxylesterase
[13], and a heat-stabilized human propyl endopeptidase [14].
Nonmammalian enzymes include the widely used
b
-lactamase [15], cytosine deaminase [16], and carboxy-
peptidase G2 (CPG2) [3].
acid mustard drugs, which could be inactivated by a terminal
glutamate, were synthesized and a 4-((chloroethyl)(2-
methoxy))ethyl)amino benzoyl-
L
-glutamic acid commonly
known as CMDA, was selected for further study [25].
23.3.1.1 Preclinical Studies The first ADEPT system
was tested in a drug-resistant choriocarcinoma xenograft
model known as CC3 established in nude mice. CC3
expresses hCG in the tumor. hCG is also secreted in
blood of mice bearing CC tumors with typical values of
>
23.2.4 Abzymes or Catalytic Antibodies
It was proposed early on in ADEPT [17] that catalytic
antibodies could be used to convert prodrugs to drugs
with the main advantage that these could be humanized
thus avoiding immunogenicity issues. Although early stud-
ies met with limited success [18], recent progress shows
more promise [19,20].
40 IU/mL at the time of anti-hCG-CPG2 conjugate admin-
istration resulting in the clearance of enzyme activity from
blood such that the prodrug could be given safely between
56 and 72 h after the conjugate injection. This regimen
resulted in the eradication of established xenografts [26].
The same protocol was applied to a study in the nude
mice bearing the human colon carcinoma xenograft,
LS174T. An anti-CEA antibody-CPG2 conjugate was
injected followed by the prodrug at 56-72 h. This resulted
in toxicity with no effect on the tumor growth. It was
concluded that the difference between the two models
was that the high hCG level in the blood in the CC3 case
resulted in accelerated clearance of the enzyme from blood
because of immune complex formation. However, with the
LS174T xenograft, although CEA was expressed in the
tumor, it is not detectable in circulation. This would result
in higher residual enzyme activity in blood when the prodrug
was given causing toxicity. When prodrug injection was
delayed by 1 week after antibody-enzyme conjugate was
given, there was no toxicity and no growth delay [17]. These
studies confirmed the need for a clearance system to remove
enzyme activity from blood.
23.2.5 Prodrugs
The fundamental requirement for the prodrug is that it
should be a substrate for the selected enzyme only. Other
critical factors include that the prodrug itself should be
nontoxic but should generate a drug that would be highly
toxic but with a short half-life. A series of prodrugs synthe-
sized by Tietze et al. [21] are 4000-6000-fold less toxic than
the generated drugs although there is no indication of the
half-life of these drugs.
The potential advantage of ADEPT is to deliver a much
higher concentration of drug to cancer sites than can be
delivered by conventional chemotherapy. It follows that the
cytotoxic action of drug should be concentration dependent
that is the more that can be delivered, the more cell killing
results. This led to the use of alkylating agents, the cyto-
toxicity of which appears to be dose dependent over a wide
range of concentrations [22]. It was pointed out in the early
publications that if a high concentration of drug is generated
at cancer sites then some of it will leak back into the blood
and access normal cell renewal systems. To minimize
toxicity, it is necessary for the generated drug to have a
very short half-life such that it has enough time to diffuse
and kill tumor cells but decays by the time it enters the
blood. This factor has been ignored in many ADEPT studies
as prodrugs have been made from already licensed drugs
with considerable half-lives [23,24].
23.3.1.2 Removal of Enzyme Activity from Blood It is
clearly essential for this residual conjugate to be removed
from blood before giving prodrug. It is also essential that the
clearing agent should not access tumor sites and inactivate
enzyme there. Monoclonal antibodies to CPG2 had been
produced in anticipation applying accelerated clearance of
the enzyme. One of the monoclonal anti-CPG2 antibodies
(SB43) inactivated the enzyme in vitro and rapidly reduced
enzyme activity in blood (Figure 23.2) [27]. It was necessary
to ensure that this enzyme-inactivating antibody cleared
quickly from blood to prevent its penetration of tumor sites.
This was achieved by glycosylation of the antibody.
Administration of the galactosylated antibody SB43gal by
intravenous infusion reduced enzyme to undetectable levels
within hours without affecting the tumor enzyme levels.
When the LS174T studies were repeated using the three-
phase ADEPT system comprising administration of the anti-
body-enzyme conjugate followed by SB43gal at 19 h, the
prodrug could be given safely at 24 h, resulting in a growth
delay of the tumor. Similar results were obtained in a drug
resistant, CEA expressing ovarian carcinoma model [28,29].
23.3 ADEPT SYSTEMS WITH
CARBOXYPEPTIDASE G2 (CPG2)
23.3.1 System 1: Antibody-Enzyme Conjugates
Antibody-enzyme conjugates were initially constructed
using bifunctional agents, where CPG2 was chemically
conjugated to F(ab
0
)
2
fragments of monoclonal antibodies
directed at hCG and CEA. CPG2 cleaves the terminal
glutamate from folate-type molecules. A series of benzoic
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