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Blood, Vol. 91 No. 2 (January 15), 1998:
pp. 710-716
Absence of Immunogenicity of Diaspirin Cross-Linked Hemoglobin in
Humans
By
Mehul J. Patel,
Edwin J. Webb,
Tina E. Shelbourn,
Cynthia Mattia-Goldberg,
Andrew J.T. George,
Feng Zhang,
Edwin G. Moore, and
Deanna J. Nelson
From Blood Substitutes, Baxter Healthcare Corp, Round Lake, IL; and
the Department of Immunology, Imperial College School of Medicine, The
Hammersmith Hospital, London, UK.
 |
ABSTRACT |
Diaspirin cross-linked hemoglobin (DCLHb) is an intramolecularly
cross-linked hemoglobin-based oxygen carrier being developed as a
therapy for acute blood loss. We report here the absence of
immunogenicity of DCLHb in patients enrolled in phase II and III
clinical trials of DCLHb. Two very sensitive immunoassays, an
enzyme-linked immunosorbent assay (ELISA) and a Western blot assay,
were developed and validated for this assessment. The DCLHb-antibodies used in these assays were raised in monkeys, had similar affinities for
DCLHb and native human hemoglobin (SFHb), and showed cross-reactivity for subunits of DCLHb and SFHb on the Western blot, suggesting that
these antibodies were elicited as a xenogenic response to the protein.
In the ELISA, the optical density of a patient sample exposed to
DCLHb-coated wells was compared with that of the patient sample exposed
to carbonate buffer-coated wells; an optical density ratio of 1.4 was
established for discriminating between a positive (reactive) or
negative DCLHb antibody response. To date, all of the more than 300 patient specimens (preinfusion and postinfusion) from clinical trials
have exhibited a ratio of less than 1.4, confirming the lack of
preexisting antibodies to DCLHb and clearly showing the absence of
DCLHb antibodies after exposure to this new biologic entity. There has
been no requirement for use of the confirmatory Western blot assay.
Taken together, the results from this study indicate DCLHb is not
immunogenic in humans at doses evaluated clinically.
| |
INTRODUCTION |
THE SEARCH FOR A temporary red blood cell
replacement has been pursued for decades.1,2 Historically,
this search has focused on the development of fluids that could be used
in place of blood, primarily to reduce concerns about its availability and safety.2,3 However, in recent years, the objectives of this search have changed, and today, the goal is the development of a
therapy that will restore both oxygen delivery and perfusion after
either global or focal oxygen deficit. This more comprehensive objective has the potential to encompass clinical applications that
range from the more traditional uses of blood for resuscitation after
acute, severe hemorrhage, in which blood transfusion is the current
standard of care, to future applications as a treatment for the
ischemia associated with myocardial infarction or stroke, in which
blood is not indicated.
Hemoglobin, the iron-containing protein that transports oxygen from the
lungs to the tissues, was a natural candidate for use as a red blood
cell substitute. In the red blood cell, hemoglobin reversibly binds and
releases oxygen via a conformational change from a high-affinity to
low-affinity state that is facilitated by the polyanionic effector,
2,3-diphosphoglycerate. However, purified acellular hemoglobin, also
known as stroma-free hemoglobin (SFHb), lacks this capability in the
absence of an effector. In addition, SFHb dissociates into
  -dimers that are filtered in the glomerulus of the kidney; high
concentrations of dimers overwhelm the filtration capacity of this
organ and are toxic. The combination of nephrotoxicity and high
oxygen-binding affinity renders SFHb not clinically
useful.4,5
The shortcomings of SFHb may be corrected by chemical modification of
hemoglobin. For example, covalent cross-linking of purified human
hemoglobin by the cross-linking agent, bis(3,5-dibromosalicyl) fumarate
(DBBF), in the presence of the polyanion tripolyphosphate has been
shown to yield an intramolecularly cross-linked hemoglobin called
diaspirin cross-linked hemoglobin (DCLHb).6,7 DCLHb has an
oxygen-binding capability comparable to hemoglobin in fresh, whole
blood. In addition to its therapeutically useful oxygen-carrying properties, DCLHb functions as an active oncotic and pharmacologic agent. Preclinical and clinical studies indicate that DCLHb is capable
of restoring blood pressure, tissue oxygenation, and circulation after
acute hypotension after insults such as hypovolemic shock.8 Moreover, preclinical studies suggest that DCLHb may be useful in
restoring and maintaining perfusion of key organs after myocardial infarction, stroke, or septicemia.8,9
Proteins administered parenterally to achieve therapeutic benefits may
concomitantly initiate immune responses.10 As a general rule, the potential for immunogenicity is increased when the secondary or tertiary structure of the modified protein or the charge on the
surface of the protein differs from the corresponding properties of the
endogenous entity. Native hemoglobin is generally viewed as a
nonantigenic protein,11 but, because DCLHb is a chemically modified hemoglobin, a novel epitope(s) may have been introduced that
potentially can be immunogenic, either as a direct result of the
chemical modification or due to indirect modification of the protein.
The generation of an antibody response would cause more rapid clearance
of the product, reducing its effectiveness, and may also lead to
hypersensitivity reactions, thereby affecting the outcome of clinical
studies.
The immunogenicity of DCLHb was first evaluated as a part of the
preclinical safety assessment of DCLHb. In one preclinical safety
study, a 500 mg/kg dose of DCLHb was infused into five rhesus monkeys
at monthly intervals for 5 months.12 No immediate or
delayed reactions to the infusions were observed. All serum samples
were negative for anti-DCLHb IgG and IgM. Skin testing with two
concentrations of DCLHb was also negative. Thus, repeated infusions of
DCLHb at this dose did not cause immediate, antibody-mediated, or
delayed cellular immune reactions in these primates.
One of the challenges in testing for the presence of DCLHb antibodies
is the development and validation of sensitive immunoassays that
discriminate between reactive and nonreactive samples. In the present
study, we describe the development of such immunoassays, together with
the characterization of DCLHb-antibodies raised in monkeys. In
addition, we have summarized evaluations of the immunogenicity of DCLHb
using these immunoassays, which were completed as a part of more
comprehensive patient monitoring in phase II and III clinical trials of
DCLHb.
| |
MATERIALS AND METHODS |
Materials.
DCLHb 10% and electrolyte injection (lot no. 94D01AD11) and SFHb were
manufactured by Baxter Healthcare Corp (Deerfield, IL). Biotinylated
molecular weight standards, enhanced chemiluminescence (ECL) Western
blotting detection reagents, and Hyperfilm-ECL were obtained from
Amersham Inc (Arlington Heights, IL). Phosphate-buffered saline (PBS),
Tris-buffered saline (TBS), goat antihuman IgG conjugated with
horseradish peroxidase (IgG-HRP), ImmunoPure (G) IgG Purification Kit,
Emphaze Biosupport medium, Gentle Binding Buffer, p-nitrophenyl phosphate, 5× diethanolamine solution, and Blotto (5% wt/vol) were obtained from Pierce, Inc (Rockford, IL). Goat antihuman IgG-conjugated alkaline phosphatase was obtained from Accurate Chemicals (Westbury, NY). Precast 18% Tris Glycine gels; precut nitrocellulose membranes; Tris-Glycine sodium dodecyl sulfate (SDS)
sample, transfer, and running buffers; and Multimark multicolored standards were obtained from Novex (San Diego, CA). Avidin-HRP and
Tween 20 were obtained from Bio-Rad (Hercules, CA). Purified monkey IgG
was purchased from Sigma (St. Louis, MO). DCLHb antibodies were raised
in rhesus monkeys, as reported previously.12 These antisera
were used as a positive control for DCLHb antibodies. Pooled normal
human serum (NHS) obtained from healthy donors (n=10) served as a
negative control for DCLHb antibodies.
Subjects.
The potential for a human immune response to DCLHb was evaluated using
353 sera samples from both DCLHb-treated and control patients (obtained
3 to 8 weeks postinfusion) and 72 preinfusion samples obtained from
subjects in phase II and III clinical studies of DCLHb. These studies
included both single-dose intravenous infusions of up to 750 mL (75 g)
of DCLHb over 24 hours and repeat dose intravenous infusions of a total
of as much as 1,000 mL (100 g) of DCLHb over 72 hours (12 doses in
total, delivered at 6-hour intervals).
IgG purification.
IgG from monkey sera was purified using the ImmunoPure (G) IgG
Purification Kit. The purity (>99%) of this IgG was confirmed by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) performed under
reducing and nonreducing conditions. The IgG concentration was
determined by UV spectroscopy, using an extinction coefficient of 1.35 (A280 for 1 mg/mL) for IgG molecules.13
Radiolabeling.
The purified IgG from DCLHb-immunized monkey serum and the control
monkey IgG were labeled with 125I using the Iodobeads
method (Pierce Inc). The control IgG was also labeled to determine the
extent of nonspecific binding to the DCLHb affinity column. The
specific activity of the labeled IgG from DCLHb antisera and labeled
IgG from control monkey sera were 3.1 and 3.3 µCi/µg, respectively.
DCLHb-liganded affinity column preparation.
A DCLHb-liganded affinity resin (DCLHb-affinity resin) was prepared by
incubating (overnight at 2°C to 8°C) 1 g of Emphaze Biosupport
medium with 1.2 mL of 10 g/dL DCLHb in 0.8 mol/L sodium citrate/0.1
mol/L sodium bicarbonate solution, pH 8.6 (at 25°C). After
overnight incubation, the resin was pelleted by centrifugation, and
unreacted sites on the Emphaze were quenched with 3 mol/L ethanolamine,
pH 9.0. The affinity resin was isolated by filtration and washed with
10 mmol/L PBS and then 1.0 mol/L NaCl. Affinity columns were prepared
by mixing 0.5 g of the DCLHb affinity resin with 5 mL of Gentle Binding
Buffer. This slurry was added to a 5-mL column (supplied with the
Emphaze kit) and equilibrated with 5 mL of Gentle Binding Buffer.
Active DCLHb antibody concentration.
Estep et al12 have reported raising antibodies against
DCLHb in rhesus monkeys. However, attempts to affinity purify this antibody in its active form were not successful. Therefore, the active
anti-DCLHb antibody concentration in immunized monkey sera was
determined using an adsorption technique wherein approximately 1,000,000 CPM of labeled 125I-IgG from antisera or control
monkey 125I-IgG was applied to DCLHb-affinity columns,
which were prepared in triplicate. Each column was eluted successively
with 5 mL of Gentle Binding Buffer and 6 mL of 1.0 mol/L NaCl. Fraction
(1.0 mL) collection was initiated immediately and also followed all wash steps. The radioactivity in each fraction and in the affinity resin was counted using a gamma counter. As a result of this treatment, 1.95% of the radioactivity in the labeled IgG from the DCLHb antisera bound to the DCLHb affinity resin. After identical treatment, 0.70% of
the radioactivity from the control IgG bound to the resin, corresponding to the nonspecific binding. After correction for this
nonspecific binding, the percentage of active anti-DCLHb antibody was
used to calculate the DCLHb-specific antibody concentration in the IgG
fraction and in the antisera (74.0 µg/mL).
ELISA.
Estep et al12 have reported the development of a
solid-phase ELISA to detect DCLHb-specific antibodies. Although this
assay was able to detect DCLHb antibodies, improvements in the assay were necessary to decrease the nonspecific binding, to increase the
sensitivity, and to facilitate the validation of the ELISA for the
analysis of patient samples from clinical studies. Experimental conditions were optimized with respect to the selection of
antigen-coating buffer, blocking buffer, serum diluent, substrate
diluent, incubation time for the substrate, and substrate quenching
solutions. (See below for details.) As a result of these changes, the
nonspecific binding of serum proteins to the antigen decreased by more
than twofold. Moreover, the sensitivity for detecting DCLHb antibodies increased by more than 10-fold (data not shown).
All incubations were performed at room temperature, except when noted.
All wash steps were performed with PBS-Tween, a 0.05% vol/vol solution
of polyoxyethylene sorbitan monolaurate (Tween 20) in PBS. Carbonate
buffer, pH 9.6, was used to prepare 1 µg/mL stock solutions of DCLHb.
For testing, 100 µL of carbonate buffer (negative control) and each
antigen solution were placed in separate wells of 96-well,
flat-bottomed microtiter plates. After overnight incubation at 4°C,
the ELISA plates were washed and then blocked with 150 µL Blotto for
2 hours at 4°C. Each patient serum sample (diluted 1:100) and
control was diluted in 33% Blotto in TBS. After washing of the plates,
100 µL of a sample or control solution was added; all determinations
were performed in triplicate. The plates were then incubated for 1 hour. The wells were washed, 100 µL of alkaline phosphatase-labeled
antihuman IgG (diluted 1:10,000) was added to each well, and the plates
were incubated for 30 minutes. The plates were washed again, 100 µL
of p-nitrophenyl phosphate was added to each well, and the
plates were incubated for 45 minutes. After 45 minutes, the reaction
was quenched with 50 µL of 2 N NaOH, and the optical density of each
well was read at 405 nm using a Dynatech plate reader.
ELISA validation.
To monitor a large number of patients for an antibody response to DCLHb
and to provide quantitative results, the ELISA was validated. In this
validation, a cutoff value was defined for discriminating between a
positive and a negative antibody response. The validation study design
incorporated both a determination of the sensitivity of the ELISA and a
determination of the precision; the latter considered variability due
to analysis (n = 3), days (n = 5), and ELISA plates (n = 3). The
sensitivity of the assay was defined as the lowest detectable optical
density reading that was higher than the optical density reading of a
normal population before exposure to DCLHb. To identify this value,
serial dilutions from 1:25 to 1:12,800 of the positive control (DCLHb
antisera) and the negative control (pooled NHS) were assayed. The
optical density measurements were normalized as a ratio of the
geometric mean of the value obtained for the positive control relative
to that of the negative control (pooled NHS) and subjected to
statistical analysis.
Western blot.
The Western blot assay comprised the following steps. DCLHb was
separated by SDS-PAGE (18% gel) and was then electrophoretically transferred to a nitrocellulose membrane. The remaining binding sites
on the membrane were blocked with Blotto in PBS overnight at 4°C.
The membrane was then washed with 50% Blotto in PBS with 0.1% Tween.
The membrane was cut into strips exposed to positive control (DCLHb
antisera), negative control (NHS), and patient sera specimens. After
washing of the membrane strips, the antigen-antibody binding on the
membrane was visualized by incubation of the sample strips to IgG-HRP
(diluted 1:5,000). This antibody reacts with monkey IgG. The HRP was
detected with ECL, using short exposure to blue-light sensitive
autoradiography film (Hyperfilm-ECL) to detect the light emitted.
Kinetics.
All kinetic studies were performed using the IAsys resonant mirror
biosensor technique (Affinity Sensors, Cambridge, UK)
described in detail. 14,15 The IAsys was connected to a
circulating waterbath set at 25°C. All samples and buffers were
allowed to equilibrate to 25°C before use. All samples were in PBS-
Tween, and the reaction volume was 200 µL. For kinetic analysis, the
resonant angle was sampled every 0.2 seconds. Data were collected using
the IAsys software and analyzed with FASTfit software (Affinity
Sensors).
The IAsys uses a cuvette system in which individual cuvettes are placed
into and removed from the sensing chamber. Each cuvette incorporates
the resonant mirror device, the sensing surface (in these experiments,
a carboxymethylated dextran hydrogel), and a reaction chamber with a
working volume of 50 to 200 µL. Each cuvette also contains a
propeller that efficiently stirred the contents of the reaction
chamber. (In all experiments described here, the stirring rate was set
at 4,125 rpm.) The temperature in the sensing chamber was maintained at
that of the circulating waterbath. Samples were added directly to the
cuvette using a pipette and evacuated by means of a tube attached to a
peristaltic pump.
DCLHb was coupled to the biosensor surface via the  -amino groups of
lysine residues on the protein using the following procedure. The
cuvette was equilibrated with 200 µL of PBS-Tween for 5 to 10 minutes. The surface was then activated by adding 200 µL of both 0.2 mol/L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.05 mol/L N-hydroxysuccinimide and equilibrating the resulting solution for
8 minutes. The cuvette was washed with PBS-Tween, and 200 µL of 50 µg/mL DCLHb in 10 mmol/L sodium acetate buffer (pH 6.0) was added.
This pH was chosen from preliminary experiments as being optimal for
maximizing the electrostatic interactions of DCLHb with the hydrogel.
After 15 minutes of reaction, the cuvette was washed, 200 µL of 1 mol/L ethanolamine, pH 8.5, was added, and the reaction was continued
for 2 minutes to block activated carboxyl groups that had not reacted
with DCLHb. SFHb was coupled to surface in the same manner except that
the concentration was 5 mg/mL SFHb.
After coupling of protein to the surface, the cuvette was equilibrated
with 180 µL of PBS-Tween for 5 to 10 minutes, and then 20 µL of IgG
preparation was added. IgG association was monitored for 5 minutes
using the IAsys biosensor. The cuvette was washed three times with
PBS-Tween (200 µL), and the IgG dissociation was monitored for 5 minutes. The cuvette was regenerated by equilibrating with 200 µL of
Gentle Elution Buffer for 2 minutes.
| |
RESULTS |
DCLHb antibody characterization.
The interactions of DCLHb antibodies (raised in rhesus monkeys) with
DCLHb and SFHb were investigated using ELISA, Western blot, and
biomolecular interaction analyses. The DCLHb-specific antibodies
recognized both DCLHb and SFHb in the ELISA
(Fig 1) and Western blot
(Fig 2). Western blot results indicated
that DCLHb-specific antibodies recognized epitopes on both the
-cross-linked and -subunits of DCLHb. This Western blot
pattern was consistent with the characteristic pattern for DCLHb on
SDS-PAGE, in which bands at approximately 31 kD (-cross-linked
subunit) and 15 kD (-monomer) were observed. Note that the
antibodies cross-reacted to SFHb but that only a single band was
observed at the approximately 15-kD marker. This result also was
expected, because both the - and -subunits of SFHb have similar
molecular weights (~15 kD).
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| Fig 1.
Specificity of DCLHb antibodies in the ELISA. The
concentrations of DCLHb and SFHb were 10 µg/mL. Monkey serum (MS) and
NHS were diluted 1:100.
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| Fig 2.
Cross-reactivity of DCLHb antibodies to SFHb and DCLHb as
assessed by Western blot. The concentrations of DCLHb and SFHb were 1 µg/mL. Monkey serum was diluted 1:100.
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Association rates were determined using pseudo first order conditions
by addition of antibody at different concentrations. Typical profiles
for the binding of anti-DCLHb antibodies to amine coupled DCLHb are
shown in Fig 3A. All of these curves fitted to the following equation for monophasic interactions (for its derivation, see George et
al16):
|
(1)
|
where Rt is the response at time t,
kobs is the observed rate constant for the binding,
and E is the extent of the reaction (or R at t =  ).
It should be noted that this approach is designed to determine the
kinetics of monoclonal interactions. However, in cases in which a good
fit of the data is obtained, it is reasonable to assume that the
experimental results represent an average affinity, even with
polyclonal antibodies.
Figure 3B shows that kobs has a linear dependence
on antibody concentration, a criterion allowing for the determination
of the association rate constant (ka) from this
plot using equation 2:
![<IT>k<SUB>obs</SUB></IT> = (<IT>k<SUB>a</SUB></IT>[Ab] + <IT>k<SUB>d</SUB></IT>)](/content/vol91/issue2/fulltext/710/img002.gif)
|
(2)
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In theory, the intercept obtained from this equation equals the
dissociation rate constant (kd); however, this
value typically incorporates large errors. Therefore,
kd was measured directly, and this value was fitted
to the equation
below.
|
(3)
|
Average ka and kd values of 2.2 ×104 mol/L 1
sec1 and 5.3 × 104 sec1, respectively, were
obtained for DCLHb, giving an average kd of 2.4 ×108 mol/L. Similar ka
(4.7 × 104 mol/L1
sec1), kd (4.3 × 104 sec1), and an average
Kd (9 × 109 mol/L) were
observed for SFHb.
It should be noted that these figures represent an average, because the
antiserum is polyclonal in nature. However, in this case, the fit to
monophasic binding kinetics was good, suggesting that this approach
provides a reasonable estimate. This average affinity for the DCLHb
antibodies isolated from primate sera suggests that immunoassays
developed using these antibodies provide the sensitivity needed for
detecting antibodies in patient sera. No binding of control monkey IgG
was seen in these experiments (data not shown), and no binding was seen
to an irrelevant antigen (saporin)16 immobilized in the
place of DCLHb.
Establishment of positive/negative cutoff value for ELISA.
To discriminate between a positive and a negative antibody response, a
cutoff value was defined for the DCLHb antibody ELISA. When
establishing a cutoff ratio, it was important to minimize both
false-positives and false-negatives. Therefore, it was appropriate to
consider both the lower tolerance limit for a positive antibody response (using the monkey DCLHb antisera as the positive control) and
the upper tolerance limit for a negative antibody response (using NHS
as the negative control).
Figure 4 shows the results for the DCLHb
antisera at various dilution factors, NHS, and the background expressed
as ratios. Several noteworthy inferences may be drawn from an
examination of Fig 4. First, it was immediately clear that the ratios
of NHS-D (NHS applied to DCLHb-coated wells) to NHS-C (NHS applied to
carbonate buffer-coated wells) was similar to the background ratios,
suggesting that nonspecific binding of serum Ig proteins to DCLHb did
not occur. Second, as expected, higher ratios were observed at each progressively lower dilution factor of the monkey antisera. Third, at a
200-fold dilution of the antisera, a separation between the positive
(monkey antisera) and negative antibody response (NHS) was observed.
Analysis of these data showed that, at a dilution factor of 200-fold
and 400-fold, the average optical density ratio was 2.40 and 1.75, respectively. At these dilutions, the lower tolerance limits for a
positive antibody response (99.5% of the population, 90% confidence)
were 1.4 and 1.17, respectively.
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| Fig 4.
Statistical evaluation of positive to negative cutoff
ratio. The BCKG ratio is the OD ratio of buffer applied to ELISA wells relative to that of NHS (100-fold dilution) applied to carbonate buffer-coated wells. The NHS ratio is the OD ratio of NHS (100-fold dilution) applied to DCLHb-coated wells relative to that of NHS applied
to carbonate buffer-coated wells. The monkey serum ratios are the OD
ratios of monkey serum (at different dilution factors) applied to
DCLHb-coated wells relative to that of NHS applied to carbonate
buffer-coated wells. The lower tolerance limit for the positive DCLHb
antibody (monkey serum) and upper tolerance for the negative DCLHb
antibody responses are shown. The displayed data are the results from
all analyses (including those having multiple analysts, assays, plates,
etc).
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An approach analogous to that described above was used to determine the
upper limit for the negative antibody response (NHS). However, in this
case, a sampling pool of baseline sera samples from 36 patients was
deemed sufficient to account for anticipated variability in the patient
population. An upper tolerance limit of 1.12 on the negative antibody
response was determined using these patient's baseline samples (Fig
4).
The combined use of these confidence values and a consideration of the
precision of the assay enabled the establishment of a positive to
negative cutoff ratio of 1.4 at a 200-fold dilution factor. Under these
conditions, the false-negative and false-positive error rates for the
ELISA were less than 1 per 10,000 and less than 1 per billion,
respectively. Decreasing the dilution factor to 100-fold, the dilution
factor at which patient samples are currently tested, decreased the
false-negative rate to 53 per billion, whereas maintaining the
false-positive error rate at less than 1 per billion.
A standard curve was generated using the active DCLHb antibody
concentration determined earlier (74.0 µg/mL). A limit of
quantitation of 50 ng/mL of DCLHb antibody at the cutoff value of 1.4 was derived from this curve. Comparable results were obtained with the
antisera obtained from a second immunized monkey and, therefore, are
not presented.
Western blot.
It is widely recognized that the Western blot assay is useful in
elucidating the specificity of antibodies to their corresponding antigens. To provide an additional confirmatory assay in the event that
a DCLHb-reactive patient sample was identified, the ECL Western blot
shown in Fig 5 was validated. In this
assay, a response was considered positive if a band was visually
apparent at either the 31-kD or 15-kD molecular weight marker, and its
intensity was similar to that observed for the highest diluted
standard. If no bands were observed at both molecular weight markers,
the response was considered negative. The sensitivity of the assay was
found to be similar to that of the ELISA, because bands were visible to
a 4,000-fold dilution factor of the monkey antisera (18.5 ng/mL of
DCLHb antibodies).
Clinical evaluation.
Figure 6 displays the optical density
ratios observed from analyses of serum samples from three groups: (1)
preinfusion sera samples from subjects receiving DCLHb or the control
infusate, (2) postinfusion sera samples from control patients, and (3)
postinfusion sera samples from DCLHb patients. ELISA analysis of these
samples yielded a combined average optical density ratio of less than 1.4 and average optical density ratios by group of 1.007 ± 0.043, 1.005 ± 0.038, and 1.002 ± 0.032, respectively. These
results clearly show that no DCLHb antibodies were detected in any of these patients.
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| Fig 6.
Antibody response in patients exposed to DCLHb or control
solution. Antibody response ratio is the OD ratio of patient serum applied to DCLHb-coated wells relative to that of patient serum applied
to carbonate buffer-coated wells.
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| |
DISCUSSION |
This study has shown that no antibodies specific to DCLHb were detected
in patient serum samples collected before or after treatment with
DCLHb. To our knowledge, this is the first published summary of
immunogenicity studies conducted in phase II and III clinical trials of
a hemoglobin-based oxygen carrier.
Use of a known titer of a suitable positive control is a prerequisite
of any assay to detect or quantify antibodies. However, human
hemoglobin A0 is generally viewed as a relatively
nonantigenic protein. Moreover, because no patient receiving DCLHb has
exhibited a positive immune response to DCLHb, a true positive control
for these immunoassays was not available. An assay described by Estep et al12 used antisera raised in rhesus monkeys as the
positive control. We also used these antisera as a positive control
when testing patient samples.
For routine monitoring of DCLHb antibodies in patients, both primary
(ELISA) and confirmatory (Western blot) assays were developed and
validated. Many other similar assays that have been described in the
literature use a discriminator such as a twofold to threefold ratio
above background or 2 to 3 standard deviations above the mean
background to differentiate between a positive and a
negative.17 This type of analysis usually leads to high
errors in the false-positive rates. In contrast, in this study, a
cutoff value of 1.4 was based on a statistical evaluation of validation
data that incorporated many different factors contributing to the
variability in the ELISA. As a consequence, both false-positive and
false-negative error rates were very low. Moreover, the ELISA used in
this study had a sensitivity sufficient for the quantitation of
antibodies to DCLHb at low levels (50 ng/mL).
This ELISA, which detects the clinically relevant antibodies to native
(nondenatured) DCLHb, was used in the evaluation of more than 300 patient specimens from clinical trials of DCLHb. These samples included
both preinfusion samples from DCLHb-treated and control patients and,
more importantly, postinfusion samples from DCLHb-treated patients. All
patient sera tested negative for DCLHb-antibodies, confirming the lack
of preexisting antibodies to DCLHb and clearly showing the absence of
DCLHb antibodies after exposure to this biologic product.
These results are not surprising, because DCLHb is not likely to be
immunogenic in humans for several reasons. First, DCLHb is derived from
human hemoglobin, and the fumaryl bis(amide) bridge is buried in the
central water cavity between the two subunits of the protein.
Structural characterization studies by mass spectrometry18 and electronic circular dichroism and fluorescence
spectroscopy19 have shown that DCLHb is structurally very
similar to native human hemoglobin, with the exception of the internal
cross-link; thus, the presence of novel epitopes in DCLHb is not
likely. Biomolecular interaction analysis showed that the affinity of
DCLHb antibodies for DCLHb was characterized by an average antibody
affinity constant in the nanomolar range. A similar affinity constant
was observed for SFHb, suggesting that the antibodies raised in rhesus
monkeys represented a xenogenic response and not one due to the
cross-linker in DCLHb.
In addition, the cross-reactivity of this antibody to SFHb in both the
ELISA and the Western blot further supports the conclusion that the
specificity of these antibodies was not likely to be directed against
the cross-linker or other changes in the structure of molecule caused
by this modification. This cross-reactivity of the DCLHb antibodies to
SFHb was expected, because the antibodies in the monkey sera were more
likely to be polyclonal in nature rather than monoclonal antibodies
specific to DCLHb, confirming the polyclonal nature of the
DCLHb-specific antibodies.
In a preclinical study using primates, a 500 mg/kg dose of DCLHb was
infused at monthly intervals for 5 months; no immediate or delayed
reactions to the infusions were observed. In fact, antibody production
in these primates was only possible after immunization with
adjuvant.12 When these observations are considered together
with the pharmacokinetics of DCLHb after acute infusion of DCLHb (a
circulating half-life of less than 24 hours), it is reasonable to
conclude that DCLHb is not immunogenic in humans.
An earlier in vitro biocompatibility study of DCLHb showed that DCLHb
does not activate the complement system and does not induce
cytokines.20 These same markers were monitored in the phase
I safety study and showed no stimulation of either
cascade.21 Taken together, the results from this study and
the results reported earlier suggest DCLHb is not immunogenic in humans
under conditions in which it would be administered clinically.
| |
FOOTNOTES |
Submitted July 10, 1997;
accepted September 5, 1997.
Address reprint requests to Mehul J. Patel, PhD, Baxter Healthcare
Corp, WG3-2S, 25212 W. State Route 120, Round Lake, IL 60073-9799.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
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Abstract
Introduction
Abstract
