© 1995 American Heart Association, Inc.
Articles |
A Monoclonal Antibody That Recognizes the GPIIb/IIIa Antagonist DMP 728
Reversal of the Effects of DMP 728 on Platelet Aggregation and Bleeding Time in the Dog
From the DuPont Merck Pharmaceutical Company, Wilmington, Del.
Correspondence to Thomas M. Reilly, PhD, the DuPont Merck Pharmaceutical Company, Cardiovascular Diseases Research, PO Box 80400, Wilmington, DE 19880-0400. E-mail reillytm@a1.lldmpc.umc.dupont.com.
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Abstract |
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Abstract Since hemorrhagic events represent a major safety concern associated with the use of new antithrombotic therapies such as glycoprotein (GP) IIb/IIIa receptor blockade, we evaluated the ability of a monoclonal antibody recognizing DMP 728 (cyclic [D-2-aminobutyryl-N2-methyl-L-argininyl-glycyl-L-aspartyl-3-aminomethyl-benzoic acid] methanesulfonic acid salt), a potent GPIIb/IIIa receptor antagonist, to reverse the pharmacological actions of DMP 728 in the dog. DC11 was chosen for in vivo evaluation based on its ability to inhibit the binding of [3H]DMP 728 to activated platelets and to attenuate the inhibition of ADP-induced aggregation on platelet-rich plasma ex vivo by DMP 728. After anesthesia mongrel dogs were given DMP 728 (20 µg/kg body wt IV) infused into the femoral vein, bleeding times were determined using a Simplate device from incisions on the backside of the tongue, and platelet aggregation was determined ex vivo. Nearly complete inhibition of platelet aggregation was observed for the dogs treated with DMP 728 (20 ug/kg IV) for up to 210 minutes, and bleeding times were prolonged >15 minutes for 2 hours and remained elevated for more than 4 hours. DC11 (0.2 or 1.0 mg/kg body wt IV) given to dogs 10 minutes after DMP 728 resulted in 50% attenuation of the effect of DMP 728 on aggregation at 3 hours. Approximately 34% inhibition of the DMP 728–mediated bleeding time was achieved at 1 hour with the 0.2 mg/kg dose, whereas approximately 50% inhibition of the bleeding time was observed for the 1 mg/kg dose at 1 hour. The bleeding time for the higher dose of DC11 returned to control levels at 2 hours, whereas the lower dose of DC11 returned to control levels at 3 hours. These results suggest that monoclonal antibody DC11 may have potential utility as an antidote to DMP 728 in neutralizing any unexpected bleeding complications associated with its use.
Key Words: glycoprotein IIb/IIIa • DMP 728 • platelet • antibody
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Clinical studies have provided conclusive prospective evidence associating platelet aggregability with the incidence of coronary artery disease.1 2 3 4 5 6 7 8 Platelets adhere at sites of vascular injury or damage, undergo activation, and express functional GPIIb/IIIa receptors, also referred to as
IIb/ß3, for circulating
adhesive ligands, principally fibrinogen.9 10 Formation of
fibrinogen bridges via GPIIb/IIIa receptors on adjacent platelets
results in the formation of platelet aggregates. The GPIIb/IIIa receptor is an attractive therapeutic target for a number of different reasons. It is a platelet-specific protein and represents the most abundant platelet membrane protein, with approximately 50 000 copies expressed per cell.11 The binding of fibrinogen by functional GPIIb/IIIa receptors represents the final common step in platelet activation by all agonists.10 12 Recently, a human-murine chimeric Fab fragment of a monoclonal antibody directed against GPIIb/IIIa, designated 7E3, was evaluated by the EPIC investigators for use in patients.13 The results of the EPIC trial support the concept that GPIIb/IIIa receptor blockade is an effective strategy for the prevention of abrupt vessel closure after high-risk angioplasty and atherectomy. However, the EPIC trial also reinforced that, as with any new antithrombotic therapy, excessive bleeding is the predominant safety concern. The beneficial effect of 7E3 was achieved at the risk of a significant increase in bleeding complications and transfusions.13
DMP 728 is a selective GPIIb/IIIa receptor antagonist with potent antiplatelet and antithrombotic effects in animals14 15 16 that suggest its potential clinical utility in humans. In the present study we have explored the strategy of reversing the pharmacological actions of DMP 728 with the use of a specific antidote. Our approach involved the production of specific monoclonal antibodies recognizing DMP 728, characterization of their ability to neutralize the actions of DMP 728 in vitro, and evaluation of their effects on DMP 728–mediated platelet aggregation inhibition and bleeding time prologation in anesthetized dogs. Our results suggest that one monoclonal antibody, DC11, may represent an appropriate means to neutralize unexpected bleeding complications associated with the use of DMP 728.
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Methods |
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Reagents
DMP 728 and XL085 were synthesized at the DuPont Merck Pharmaceutical Company. RGDS was purchased from Sigma Chemical Co, human fibrinogen from Enzyme Research Laboratories, Inc., and human vitronectin from Calbiochem-Novabiochem Corporation. [3H]DMP 728 (24.3 Ci per mmol/L) was obtained from DuPont NEN.
Monoclonal Antibodies
Monoclonal antibodies were generated using standard hybridoma
technology17 after one intrasplenic and three
intraperitoneal immunizations of female BALB/c mice
with XL085 conjugated to keyhole limpet hemocyanin. XL085 is a closely
related analogue of DMP 728 with lysine residue that allows ready
coupling to large carrier proteins. Following selection in Iscove's
modified Dulbecco's medium (GIBCO) containing HAT media supplement
(Boehringer Mannheim), hybridoma supernatants were screened for
antibody by an ELISA with the use of polystyrene microtiter plates
coated with 500 ng of XL085 conjugated to bovine serum albumin.
Antibody-producing hybridomas were cloned by limiting dilution, and
monoclonal antibodies were purified from culture supernatant by
affinity chromatography over a Protein G Sepharose 4
Fast Flow column (Pharmacia). Class determination was performed with an
isotyping ELISA (Bio-Rad). Cross-reactivity of monoclonal
antibodies with DMP 728, linear RGDS, or the RGD-containing proteins
fibrinogen and vitronectin was determined by preincubating
the antibodies with test compound or protein before the XL085 ELISA, as
previously described.15
Platelet-[3H]DMP 728 Binding
Assays
The binding of [3H]DMP 728 to
activated human platelets at room temperature was evaluated
as follows. Human blood was collected in citrated Vacutainer tubes,
centrifuged for 10 minutes at 100g, and the
platelet-rich plasma (PRP) was carefully removed, making sure
not to disturb the red blood cell pellet. PRP (205 µL;
4x108/mL, platelet concentration) was added to
each well of a 96-well removawell plate (Dynatech), and the
platelets were activated for 10 minutes with a mixture of
agonists containing ADP, arachidonic acid, and
epinephrine (100 µmol/L). Platelets were then incubated
with [3H]DMP 728 (10 nmol/L) for 10 minutes, in the
absence or presence of increasing concentrations of unlabeled DMP 728,
by which time equilibrium binding had been established. To separate
platelet-bound [3H]DMP 728 from free ligand,
samples were centrifuged for 10 minutes at 1800g,
and the supernatant was then removed. Platelet-bound
[3H]DMP 728 was measured in a scintillation counter
(Packard Minaxi Tricarb). Binding was analyzed by the method of
Scatchard18 using nonlinear regression analysis
methodology. To evaluate the effects of antibodies on
[3H]DMP 728 binding, monoclonal antibodies were added to
the activated platelets before [3H]DMP 728
was added. All binding results represent the mean of three
separate experiments.
Platelet Aggregation Assays
The effect of DMP 728 on the aggregation of human platelets
in response to 100 µmol/L ADP was determined as previously
described.14 The ability of different antibodies to
neutralize the inhibitory effects of DMP 728 on
platelet aggregation was evaluated by pretreating the antibodies
(100 nmol/L) with DMP 728 (100 nmol/L) for 10 minutes at 37°C before
the aggregation assays.
Animal Studies
Mongrel dogs of either sex weighing 8 to 12 kg were
anesthetized with pentobarbital (30 mg/kg IV) (Nembutal, Abbott
Laboratories). Additional anesthetic was administered as needed.
Animals were placed on a respiratory pump (model 665, Harvard
Apparatus), and the stroke volume of the respiratory pump
was adjusted to the dog's weight. The femoral artery was cannulated
for blood collection, and the femoral vein was cannulated for compound
administration. DMP 728 (20 ug/kg body wt IV) was administered to a
group of dogs (n=6) followed 10 minutes later with the monoclonal
antibody DC11 (0.2 or 1.0 mg/kg body wt IV). Blood samples were
collected at various time points up to 5 hours for platelet
aggregation determinations as previously described.14
Bleeding times were determined at those various time points using a Simplate device (Organon Teknika). Uniform incisions were made on the middle of the dorsal surface of the tongue, and the blood was blotted with filter paper at 30-second intervals until the bleeding stopped. If the bleeding did not stop within 15 minutes, a bleeding time of >15 minutes was assigned.
Platelet Counts
From each blood sample, 20 µL of PRP was added to 20 mL Isoton
T diluent (Coulter Diagnostics), and platelet number
was determined using a Coulter counter (Coulter Electronics, Inc).
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Monoclonal Antibodies
From two different fusions using spleens from four XL085-immunized mice, 10 monoclonal antibodies were identified that recognized DMP 728 in the ELISA. Since DMP 728 is a cyclic RGD-containing molecule, antibodies were also tested for cross-eactivity against a linear RGD peptide (RGDS) and against the RGD-containing proteins, fibrinogen and vitronectin. None of the antibodies displayed appreciable cross-reactivity for RGDS, fibrinogen, or vitronectin, even at concentrations as high as 100 µg/mL. The antibodies were purified from culture supernatants for further studies.
[3H]DMP 728 Binding to
Activated Platelets
Previous studies14 showed that DMP 728
inhibited platelet aggregation that was induced by ADP to a similar
extent in dog and human platelets (IC50s of 15 and 46
nmol/L, respectively). In view of this similar sensitivity to DMP 728,
we chose to characterize the binding of DMP 728 to human platelets.
Scatchard analysis of [3H]DMP 728 binding to
activated platelets is shown in Fig 1
.
Nonlinear regression analysis of the curvilinear plot suggested
the presence of two classes of binding sites: a high affinity site with
a Kd of 0.07 nmol/L and a lower affinity site
with a Kd of 9.9 nmol/L. This low affinity site
could represent the binding of DMP 728 to
unactivated platelets present in the preparation.
Binding studies in which unactivated platelets were
used support this interpretation, since a Kd of
30 nmol/L was determined. DMP 728 therefore displays a greater than
100-fold selectivity for activated versus
unactivated platelets.
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Once we had established the specific binding of DMP 728 to
activated platelets, the effects of different antibodies
recognizing DMP 728 on such binding were then determined. Six of the
antibodies were effective in blocking DMP 728 binding in
concentration-dependent fashion, with one IgG2a
antibody in particular, designated DC11, identified as a potent
competitor. The concentration-response curve for the DC11-mediated
inhibition of DMP 728 binding to platelets is shown in Fig 2. Under these assay conditions an IC50 of
approximately 0.3 nmol/L was determined for DC11. A control IgG
antibody, KAA8, which recognizes an irrelevant antigen
angiotensin II,19 had no effect on
[3H]DMP 728 binding, supporting the specificity of the
DC11 effect (data not shown).
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Platelet Aggregation
The antibodies were next evaluated for their ability to attenuate
the inhibitory effect of DMP 728 on ex vivo ADP-induced
platelet aggregation. Again, DC11 was a potent
inhibitor of DMP 728 in this assay. Results shown in the
Table indicate that 100 nmol/L DC11 was effective in
blocking DMP 728 at either 20 or 100 nmol/L. As in the binding assay
the KAA8 control antibody had no effect on DMP 728 activity (data not
shown).
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Effects of DC11 in Anesthetized Dogs Treated With DMP
728
The effects of DC11 on the pharmacological actions of DMP 728 were
evaluated in anesthetized dogs. DMP 728, administered as a 20
µg/kg body wt IV bolus, elicited a complete inhibition of
platelet aggregation induced by 100 µmol/L ADP; this effect was
achieved within 5 minutes, and nearly complete inhibition was
maintained for approximately 3 hours (Fig 3). The
effects of DC11, administered 10 minutes after the DMP 728 bolus, are
also shown in Fig 3. A decrease in the
platelet-inhibitory effect of DMP 728 was noted
after administration of DC11, at either a 0.2 or 1 mg/kg dose. This
decrease reached statistical significance at the 140-minute time point.
The higher dose of DC11 was slightly more effective in reversing the
effects of DMP 728 on platelet aggregation, although by 3 hours the
level of platelet inhibition was 
50% for both doses. DC11 alone
had no significant effect on platelet aggregation (data not shown).
No significant effects on platelet counts were observed in any of
the treatment groups (data not shown).
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], IV; n=3) followed at 10 minutes with DC11
(0.2 [
] or 1.0 [
] mg/kg body wt IV; n=6) as described
in "Methods." Platelet aggregation assays were performed on
samples collected at various time points. Data are mean±SEM.
Statistical analysis was performed using a two-way ANOVA
followed by Tukey's multiple comparison test. *Represents a
significant difference from the DMP 728 (20 ug/kg IV) group,
P<.05.
Fig 4 shows the effects of DMP 728 in the absence
and presence of DC11 on the Simplate-determined bleeding time in
these anesthetized dogs. The 20 ug/kg IV bolus of DMP 728
increased bleeding times rapidly to greater than 15 minutes; this level
of bleeding persisted for 2 hours; whereas by 5 hours bleeding time
levels returned to near predosing values (3 minutes). When DC11 was
administered 10 minutes after the DMP 728 bolus, 35% and 50%
inhibitions of bleeding times at 1 hour were noted with the 0.2 and 1.0
mg/kg doses, respectively. These reductions reached statistical
significance at the 60-minute time point. The bleeding time for the
higher dose of DC11 returned to control levels at 2 hours, whereas the
lower dose returned to control levels by 3 hours. A dissociation
between the percent inhibition of platelet aggregation and bleeding
time prolongation was noted. At 120 minutes there was a 50% reduction
in bleeding time prolongation with the 0.2 mg/kg DC11 dose, although
the inhibition of platelet aggregation was 85%. Complete
inhibition of bleeding time prolongation was observed at 120 minutes
for the high-dose DC11 group, in which 60% inhibition of
platelet aggregation was observed. Previous studies have
demonstrated that elevations of bleeding time with DMP 728 are observed
only after levels of platelet aggregation reach 75% to
80%.14 15 16
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The results of the EPIC trial provide strong support for the concept that GPIIb/IIIa receptor blockade is an effective strategy for the prevention of abrupt vessel closure after high-risk angioplasty and atherectomy.13 However, the beneficial effect of 7E3 was achieved at the risk of significant increases in bleeding complications and transfusions. Consequently, it would be desirable to have the appropriate means to neutralize any unexpected bleeding complications from 7E3 or any other pharmacological agent working through the mechanism of GPIIb/IIIa receptor blockade.
In this study, we have evaluated the strategy of the use of a specific antidote to neutralize the pharmacological actions of DMP 728, a potent and selective GP IIb/IIIa receptor antagonist with antiplatelet and antithrombotic effects that have been documented in various animal models.14 15 16 Our approach to the development of an antagonist to DMP 728 involved generating monoclonal antibodies that recognize DMP 728 and evaluating their ability to inhibit the functional activities of DMP 728 in vitro (namely, binding to platelets and inhibition of ADP-induced platelet aggregation). On the basis of its inhibitory effects on DMP 728 in these assays, antibody DC11 was tested to determine its ability to neutralize the pharmacological actions of DMP 728 in anesthetized dogs. DC11, administered 10 minutes after a dose of DMP 728 that completely abolished platelet aggregation and markedly prolonged Simplate-determined bleeding times, promoted a quicker return to predosing levels for both parameters. There was a suggestion of a dose-dependent effect for DC11; however, the rate of return to predosing levels for both parameters was not significantly different for the low-dose (0.2 mg/kg) and the high-dose (1.0 mg/kg) antibody groups. These data suggest that the affinity of the antibody for DMP 728 rather than its concentration is the principal determining factor in reversing the effects of DMP 728. In an earlier study we showed that DMP 728–mediated inhibition of platelet aggregation is related to plasma levels of the compound.14 This type of relationship would be predicted for a compound such as DMP 728, which displays a clear preference for activated rather than unactivated platelets. In addition, the volume of distribution at steady state for intravenous DMP 728 is low (0.8 L/kg), indicating there is very little penetration into the tissues. These observations support the notion that the DC11 mechanism of reversal entails complexing with free DMP 728 and promoting its clearance.
In view of the high affinity of DMP 728 for platelets (Kd=0.07 nmol/L) and its potency in inhibiting platelet aggregation, the ability of DC11 to reverse its pharmacological effects is striking. However, many questions remain concerning the potential clinical use of DC11, or any antibody antidote for a GPIIb/IIIa receptor antagonist. One issue is the rate at which DC11 reversed the effects of DMP 728, and whether this rate is fast enough to avert potentially severe hemorrhagic complications in clinical situations. It would be interesting to determine whether antibodies with higher affinity for DMP 728 could promote a more rapid reversal of the compound's pharmacological effects than those observed with DC11.
A second important issue in this study is our use of Simplate-determined bleeding time as a measure of hemostatic function in the dog, and the relevance of this parameter for predicting bleeding complications in humans. Several investigators have pointed out that template bleeding time measurements in humans do not adequately predict hemorrhagic risks.20 21 22 Clinical evidence has shown that the bleeding time of the skin does not necessarily correlate with bleeding at other anatomic sites or at instrumented intravenous and intra-arterial access sites used for invasive procedures.23 Despite the controversy associated with the predictive risk value of bleeding time prolongations, the observation that DC11 inhibited the antiaggregatory effects of DMP 728 and its bleeding time effects indicate that the antibody is capable of reversing the pharmacological actions of the compound that could lead to unwanted side effects under certain conditions. Furthermore, our data suggest that there is a critical level of platelet inhibition that must be achieved with DMP 728 before prolonging the bleeding time.14 15 16 Full recovery of platelet aggregation is not required to correct the bleeding time.
In summary, these studies are to the best of our knowledge the first example that demonstrates the ability of a specific monoclonal antibody to neutralize in vivo the pharmacological actions of a GPIIb/IIIa receptor antagonist. Despite the fact that development of an antibody means a new drug that would have to be approved for use in patients, the results from this study suggest the clinical potential of antibody antidotes for new antithrombotic agents for which a major safety concern is abnormal bleeding.
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Selected Abbreviations and Acronyms |
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Received June 8, 1995; accepted October 11, 1995.
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References |
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