[CANCER RESEARCH 48, 4725-4729, September 1, 1988]
Delivery of Melanoma-associated Immunoglobulin Monoclonal Antibody and Fab
Fragments to Normal Brain Utilizing Osmotic Blood-Brain Barrier Disruption1
Edward A. Neuwelt,2 Peggy A. Burnett, I. Hellström, K. E. Hellström, Paul Beaumier, Christopher I. McCormick,
and Ronald M. Weigel
Oregon Health Sciences University, Division of Neurosurgery, Portland, Oregon 97201 [E. A. N., P. A. B.J; Oncogen, Seattle, Washington 98121 [l. H., K. E. H.J; NeoRx,
Seattle, Washington 98119 [P. B.J; Division of Neurosurgery, Oregon Health Sciences University, Portland, Oregon 97201 [C. l. M.]; and Department of Psychology,
Veterans Administration Hospital, Portland, Oregon 97201 [R. M. W.]
ABSTRACT
lodinated monoclonal antibodies (IgG 96.5 and two monomeric Fab
fragments 96.5 and 48.7) to melanoma-associated antigens were admin
istered after osmotic blood-brain barrier (BBB) opening in normal rats.
Osmotic BBB disruption significantly (/' < 0.0001) increased monoclonal
noma were administered following BBB disruption. The pur
pose was to assess the effect of BBB disruption on antibody
delivery and to determine if the prolonged half-life and NSB to
normal brain observed with a previously described IgM mono
clonal antibody 7 could be decreased when divalent IgG anti
body or monomeric Fab fragments are administered.
antibody delivery to the brain. Following BBB opening and intracarotid
administration, there was no difference in the disrupted brain concentra
tion integral area under the curve between Fab and IgG over the 72-h
experimental period. However, Fab concentration in the disrupted brain
was initially higher than IgG, and the clearance was more rapid (/' <
0.001), decreasing 50% by approximately 4.5 h compared to 25.5 h for
IgG. Plasma clearance was also more rapid for the Fab than IgG. The
levels decreased 50% by 1.5 h for Fab and 15 h for IgG. The route and
timing of antibody infusion had a significant effect on delivery to the
disrupted brain with the Fab fragments but not with the intact IgG.
Antibody recovered from disrupted brain retained its immunological
reactivity as measured by a cell binding assay for at least 24 h. IgG and
Fab delivery to the ipsilateral brain after BBB disruption increased (/' <
0.001) with increasing dose over a more than 3-log dose range. These
data provide information applicable to the therapeutic use of monoclonal
antibodies in brain tumor treatment.
using the lactoperoxidase method as described previously (6, 7). Radi
olabeled antibody was separated from unreacted radioiodine on a pre
packaged disposable 9-ml Sephadex G-25 M column (Pharmacia, I'iscataway, NJ). Following purification, an aliquot of the preparation was
obtained, and protein binding of the radiolabel was determined using
TCA precipitation. The TCA precipitation averaged 92.9 ± 1.2%
among 14 separate iodinations. The specific activity was 2.4 to 7.9 /X '¡/
jig. Preparations were stored at 4°Cin 1% bovine serum albumin. The
INTRODUCTION
TCA precipitability was again determined prior to each study and
averaged 87.9 ±0.4% (range, 80.5 to 97.1%). Animals were given
injections of 17 to 20 x 10' TCA-precipitable cpm (1.2 to 3.4 Mg),
MATERIALS
AND METHODS
Monoclonal Antibody. MAb IgG 96.5, Fab 96.5, and Fab 48.7, which
bind two human melanoma antigens (a M, 97,000 protein and a
proteoglycan) were prepared as previously described (2, 14,15, 20-24).
lodination (' -'M.New England Nuclear) of each antibody was performed
unless otherwise stated.
Electrophoresis of IgG and Fab before and following iodination was
performed to evaluate for high-molecular-weight antibody aggregation.
Nondenaturing gels were run with Triton X-100 to solubilize the
disruption has been shown to increase the delivery of mono
protein (25). Determinations were made from gels at 4% T (acrylamide
clonal antibodies, including IgM M Ah and other high-molecu
plus bisacrylamide). All gels were prepared by dilution of a single stock
lar-weight proteins, to normal brain and cerebrospinal fluid (5solution of 40% (w/v) plus 0.14% (w/v) bisacrylamide and polymerized
9). In a nude rat model of intracerebral human small cell lung into a 0.1- x 8.6- x 10.0-cm slab. The gel was electrophoresed for 3 h
at 12.5 mA in 1% Triton X-100 (40 mM Tris/20 mv sodium acetate,
carcinoma, BBB disruption increased the delivery of both al
bumin and methotrexate to the tumor and brain around the pH 7.4). Gels were stained with Coomassie blue or silver according to
the method of Wray et al. (26). The results of the electrophoresis
tumor (10). Although the BBB is abnormal in most CNS
studies showed the antibodies migrated as a single band with no
tumors, the permeability of the tumor and tumor-infiltrated
apparent antibody aggregation. The iodination procedure did not affect
surrounding brain is highly variable, and BBB disruption may the migration distance of any of the monoclonal antibodies.
be beneficial in obtaining the necessary therapeutic uptake of
In previous studies, these melanoma antibodies were radiolabeled,
larger molecular weight proteins such as monoclonal antibodies
and protein binding was determined using both TCA precipitation and
(5,6,11,12).
electrophoresis (28). TCA precipitation consistently yielded 5% less
When applying the use of tumor-specific MAbs or antibody
iodine-antibody binding than electrophoresis. Thus, the values reported
conjugates (3, 13-19) in brain tumor treatment, the effect of in these studies may be a slight underestimate of protein-bound radioBBB disruption and M Ah delivery, even in the absence of a label.
Osmotic BBB Disruption. Adult Sprague-Dawley rats (200 to 250 g)
target antigen, becomes an important consideration. In the
were anesthetized with sodium pentobarbital (50 mg/kg). BBB disrup
present study in normal rats, an IgG and two monomeric Fab
tion in the rat was performed as previously described (7, 27). Animals
fragments with specificity to antigens present on human mela
were infused with either i.e. saline in control studies or i.e. mannitol
(25%) in the barrier disruption studies (0.12 ml/s for 30 s). Prior to i.e.
Received 7/23/87; revised 1/4/88, 4/22/88; accepted 5/19/88.
infusion of saline or mannitol, Evans blue (2%, 2 ml/kg) was adminis
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
tered i.v. In studies evaluating BBB disruption, delivery of MAb to the
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
brain, and subsequent clearance over time, IgG or Fab was administered
1These studies were supported by the Veterans Administration, USPHS
as an i.e. bolus after i.e. saline or mannitol. Animals were sacrificed at
Grants CA31770 and CA38011, and the Preuss Foundation.
2To whom requests for reprints should be addressed, at The Oregon Health
0.5, 1, 6, 24, or 72 h. One supplemental study compared the effect on
Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97201.
MAb delivery to disrupted brain at 0.5 h when several different methods
3 The abbreviations used are: BBB, blood-brain barrier, i.e., intracarotid; M Ab.
of
MAb administration were used: (a) i.v. bolus immediately after
monoclonal antibody; CSF, cerebrospinal fluid; NSB, nonspecific binding; CBA,
mannitol; (b) i.e. bolus immediately after mannitol; (c) i.v. infusion over
cell binding assay; AUC, area under the curve; CNS, central nervous system;
TCA, trichloroacetic acid; PBS, phosphate-buffered saline.
15 min beginning 10 min before disruption; and (dì
i.e. infusion over
4725
The emergence of monoclonal antibodies with a high degree
of binding and specificity offers a unique immunological ap
proach to brain tumor diagnosis and therapy (1-4). BBB3
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1988 American Association for Cancer Research.
MAb DELIVERY ACROSS THE BBB
15 min beginning immediately after disruption (Fabs only). A second
supplemental study evaluated antibody delivery to disrupted brain at
0.5 h after varying doses of i.e. bolus IgG and Fab were administered.
The dose ranged from 0.049 to 250 tig.
At the time of sacrifice, Evans blue-albumin staining of the brain
was macroscopically graded on a scale from 0 to 3+ as previously
defined (7). All control animals had 0 staining, and all those sacrificed
at 6 h or less postdisruption had 3+ staining. Those sacrificed at 24 or
72 h postdisruption had at least 2+ staining.
Sample Collection and Assay. In all of the animals a plasma sample
was obtained prior to sacrifice. Intravascular radioactivity was elimi
nated by the i.v. infusion of 40 to 60 ml of normal saline warmed to
37"C with concurrent blood withdrawal from the i.e. catheter until
cessation of the heart (in approximately 10 min). A plasma sample was
obtained at the end of the perfusion. This technique resulted in an
average of 96.4 ±0.2% removal of radioactivity from the vascular
compartment among all animals. Brain was divided into contralateral
control (no staining) and ipsilateral disrupted (2 to 3+ staining) hemi
spheres and homogenized in 0.9% sodium chloride (2 to 5:1; v/w).
Plasma and brain samples were counted to obtain cpm/g or ml and to
determine TCA precipitability. All concentrations were corrected to
exclude free '"1 as determined by TCA protein precipitability.
Immunoreactivity of MAb. The ability of iodinated IgG 96.5 and Fab
96.5 contained in disrupted brain tissue homogenate from in vivostudies
to bind to paraformaldehyde-fixed H2669 melanoma cells (28) in vitro
was evaluated. '"l-MAb was administered i.e. following barrier open
ing, and animals were sacrificed by perfusion after 1 or 24 h. Tissue
samples were weighed and homogenized in 0.05 M PBS (2 to 5:1).
Protein-bound 125Iin the tissue homogenates was determined by TCA
precipitation. The mean ±SEM of disrupted brain homogenates was
91.8 ±2.0% for the Fab animals and 96.1 ±0.7% for IgG animals.
Sufficient quantity of brain homogenate to obtain approximately 1
ng of IgG or Fab (7 to 16 x IO6TCA-precipitable cpm) was mixed with
2x10'
cells and incubated at room temperature for 30 min (in vivo
CBA). Four ml of 0.05 M PBS were added, the sample was mixed and
centrifuged, and the first supernatant was separated from the pellet.
This step was repeated to wash the cell pellet, and a second supernatant
was obtained. The three fractions were counted.
% of binding of radiolabeled antibody to cells
cpm in pellet
x 100
total cpm
TCA precipitability of sample
This calculation assumes any free I25Iwas in the supernatant fraction.
Additionally, brain homogenate containing approximately 1 ng of
antibody as described above was added to 4 ml of 0.05 M PBS (no
tumor cells added) and centrifuged. The activity in the pellet fraction
was considered to be the nonspecifically bound antibody to brain
parenchyma tissue.
In concurrent in vitro control studies, 125I-MAb(TCA precipitability
of 90.1% for IgG and 91.5% for Fab) diluted in 1% bovine serum
albumin was used to spike brain homogenate from untreated animals.
The spiked sample was incubated with cells as in the above CBA or
washed with 0.05 M PBS after incubation to determine in vitro NSB.
Values were calculated as described above.
Statistical Analysis. Values are expressed as cpm/g of tissue or cpm/
ml of plasma, or as the percentage of uptake (cpm per g/total cpm
dose). Mean values ±SEM were calculated to summarize the data. All
values are corrected to exclude free I2SI.
RESULTS
Delivery of Monoclonal Antibody to Normal Brain. Regression
analysis showed that there was no significant difference in
delivery to the brain between Fab 48.7 and Fab 96.5. The mean
concentration ±the SEM in disrupted brain was 118,374 ±
24,011 cpm (n = 18) and 121,321 ±26,230 cpm (n = 15), for
these two fragments, respectively. Thus, the two groups were
combined for analysis in which IgG and Fab were compared.
I2SI-MAb concentration in brain and plasma samples was
corrected to exclude any free radiolabel as determined by TCA
precipitation. Table 1 shows the percentage of TCA-precipitable
counts in disrupted brain and plasma at various times after the
infusion of IgG or the Fab fragments. At all time points,
disrupted brain contained greater than 92% protein-bound radioiodine for both IgG and Fab. The radioactivity in plasma
was 97 to 98% bound in the IgG animals but less stable with
the Fabs, decreasing to 53 to 66% at 6 h.
In all experimental designs, paired t tests demonstrated a
significantly higher (P < 0.0001 in all cases) MAb level in the
ipsilateral disrupted hemisphere compared to the contralateral
hemisphere. The results of overall multiple regression analysis
indicate that the delivery of MAb to ipsilateral brain over the
72-h experimental period was increased significantly (P <
0.0001) in BBB-disrupted animals compared to saline-infused
controls. Over 72 h, the mean concentration (cpm/g) in the
infused hemisphere was 112,052 ±11,283 (n = 57) following
i.e. mannitol and was 3,184 ±464 (n = 37) following i.e. saline.
There was an overall pattern of decrease of MAb concentration
in the brain with increasing time that was linear in a log-log
scale (P< 0.0001).
Fig. IA shows that the rate of clearance of radiolabeled IgG
and Fab from the ipsilateral disrupted hemisphere is more rapid
in the Fab group than in the IgG group (P < 0.001). In this
case, the Fab level is initially 2 times higher than the IgG level
but is 4 times lower by the end of the experiment. Fab concen
tration in disrupted brain decreased 50% by approximately 4.5
h, whereas approximately 25.5 h were required for IgG. The
result was an insignificant difference between IgG and Fab
delivery to disrupted brain over time. The mean cpm/g for the
experimental period was 119,714 ±17,436 for Fab (n = 33)
and 101,517 ±12,127 for IgG (n = 24). The brain exposure
integral (area under the curve) was 8.56 x IO6 and 7.92 X IO6
cpm for Fab and IgG, respectively. The ratio of the integrals is
0.92.
The clearance of Fab and IgG from plasma is shown in Fig.
IB. IgG plasma concentration was higher than Fab at all time
points. Fab cleared from plasma at a faster rate than IgG,
decreasing 50% by 1.5 h compared to 15 h for IgG. At 72 h
Table 1 Mean percentage of TCA-precipitable cpm in disrupted brain and
plasma following BBB disruption and monoclonal antibody administration
IMI-IgG or Fab (17 to 20 x 10' cpm) was administered i.e. immediately
following BBB disruption. Animals were sacrificed at the indicated time after
disruption. Brain tissue samples were homogenized, and protein-bound radioac
tivity was determined with trichloroacetic acid.
% of TCA-precipitable cpm
The overall experimental design tested the effects of the following
main factors on MAb levels in the ipsilateral hemisphere: (a) presence
IgG 96.5
Fabs 96.5 and 48.7
or absence of BBB disruption; (b) IgG versus Fab; and (c) time. A
Time
after
Disrupted
Disrupted
multiple regression analysis was conducted using these experimental
disruption (h)
brain
Plasma
n
brain
Plasma
n
factors as independent variables. Logarithmic transformations of MAb
0.3°94.3
±
±96.9
±93.4
0.516247295.7
0.488.9
±
concentrations and time were performed to achieve linearity. First and
±95.2
2.166.4
±
0.897.3
±
±96.7
second order interaction effects among the main experimental factors
0.397.5
±
±96.6
±96.0
3.952.9
±
were also examined. Supplementary analyses were conducted to ex
0.397.5
±
±98.1
±92.5
7.265.9
±
±0.296.7
±0.20.30.30.30.214655692.3
±0.71.00.40.41.292.3
±10.9227767
amine specific relationships among selected variables as indicated below
" Mean ±SEM.
(29).
4726
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MAb DELIVERY ACROSS THE BBB
300,000
1. 000. 000
250,000
100. 000-
200,000
-?
a.
o
0.ooo-
150,000
m
[ti
rfn
100,000
1000-1—r
0.5
1
6
24
72
TUIEAFTERADMINISTRATION
(HRS)
B
50,000
10. 000. 000
Route of
Administration
ic
iv
iv
ic
Bolul Bolu»
Slow Slo>>
1. 000. 000
Fab Melanoma
ic
iv iv
Bolui no>MS]°*
IgG Melanoma
Fig. 2. Concentration (cpm/g) of '"I-MAb (IgG 96.5, Fab 48.7, and Fab 96.5)
in ipsilateral brain 0.5 h after BBB disruption. Antibody was administered either
i.e. bolus immediately after disruption, i.v. bolus immediately after disruption,
i.v. slow infusion over 15 min beginning 10 min before disruption, or i.e. slow
infusion over 15 min beginning immediately after disruption (Fab only). Bars,
SEM. Fab administered i.e. bolus after disruption resulted in the greatest delivery
to disrupted brain. There was no significant difference in IgG delivery to the brain
among the groups.
100. 000-
1000
0.9
TUIE AFTERADMINISTRATION
(HRS)
Fig. I. Concentration (cpm/g) of i.e. administered '"I-MAb (IgG 96.5, Fab
96.5, and Fab 48.7) in ipsilateral brain (A) and plasma (B) measured over 72 h
following BBB disruption, liars. SEM. Fab delivery was initially higher than IgG
and cleared more rapidly (P< 0.0001). The area under the curve for Fab and IgG
is 8.56 x 10' and 7.92 x 10'' cpm, respectively. IgG plasma concentration was
Table 2 Percentage of delivery per gram of brain of the total administered IgG or
Fab dose following BBB disruption
l29I-IgG or Fab, at the indicated dose, was administered i.e. immediately
following BBB disruption. Animals were sacrificed at 0.5 h.
IgG dose 0¿g)
0.049 ±0.003°(4)*
higher than Fab at all time points and was cleared from plasma at a slower rate.
A 50% decrease in concentration was reached at 15 h for IgG and 1.5 h for Fab.
1.34 ±0.13 (5)
1.7(3)Fab
249.5 ±
brain:plasma
hemisphere0.82 ratio0.095
±0.01
0.01 ±0.01
0.010.03
0.03 ±
±0.06
0.67 ±0.07
0.99
±0.101.31
±0.006
0.084 ±0.012
0.1 07
±0.0090.333
dose Oig)
0.075 ±0.004 (5)
±0.01
±0.26
±0.107
2.34 ±0.28 (6)
0.02 ±0.01
1.35 ±0.15
0.288 ±0.031
246.0 ±10.0(3)Contralateral
0.02 ±0.01BBB-disrupted
0.99 ±0.07Disrupted
0.223 ±0.013
' Mean ±SEM.
b Numbers in parentheses, number of animals studied.
postdisruption, the Fab plasma concentration was less than
10% of the IgG level. The plasma integral for the experimental
period was 2.00 x IO7 and 7.85 x IO7 cpm for Fab and IgG,
respectively. Thus, the plasma concentration of Fab is consid
erably lower than the IgG plasma level, while the disrupted
brain integrals are quite similar. The ratio of the brain AUC to
plasma AUC after BBB disruption is 0.43 for Fab and 0.10 for
IgG.
The effect of i.v. or i.e. MAb administration and the effect of
a bolus or slower infusion on antibody delivery to disrupted
brain were evaluated 0.5 h after BBB disruption (Fig. 2). In the
animals in which Fab was given as a bolus infusion immediately
after mannitol, the i.e. route of administration resulted in a 2fold greater delivery than the i.v. route (253,584 ±28,571, «=
6; and 144,125 ±21,406, n = 6, respectively). The i.e. bolus
administration was also better than either of the two slower
infusion protocols evaluated. At the slower infusion rate, i.v.
infusion (105,724 ±14,031, n = 5) was higher than i.e. infusion
(66,818 ±5701, n = 5). In the animals in which IgG was given
in association with barrier disruption, there were no significant
effects of route and duration of infusion on delivery to ipsilateral
brain.
control
hemisphere0.02
A separate analysis considered the effects of varying dose on
MAb delivery to ipsilateral brain (Table 2). Fab or IgG, at
various doses ranging from 0.049 to 250 ng, was administered
i.e. bolus following barrier disruption. Animals were sacrificed
0.5 h after MAb administration. A two-way analysis of variance
indicated an increased (P < 0.001) delivery to disrupted brain
with increasing dose. Even though there was a more than 3-log
dose range, the percentage of the dose/g of brain and the
disrupted brain:plasma ratio were fairly constant, indicating a
linear relationship with the dose. The percentage per g of brain
of the total administered dose was 1.0 to 1.4% for Fab and 0.7
to 1.0% for IgG. These data suggest that the delivery to brain
following barrier opening did not saturate over this dose range.
Immunoreactivity of 12SI-MAbfrom in Vivo Studies. l25I-MAb
(IgG 96.5 and Fab 96.5) from barrier-disrupted brain homogenate obtained from animals sacrificed at 1 and 24 h after
administration was used to demonstrate the immunoreactivity
4727
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MAb DELIVERY ACROSS THE BBB
of the antibody in a cell binding assay (Table 3). NSB was
determined with brain homogenate from untreated animals that
was spiked with iodinated antibody (in vitro) and from brain
homogenate from barrier-disrupted animals (in vivo). These
NSB samples were treated the same as the samples for the CBA
except melanoma cells were not added. The in vitro NSB
averaged 20% for Fab and 16% for IgG. The in vivo NSB was
20% for Fab and 25% for IgG.
In vitro CBA controls were performed in which brain homog
enate from untreated animals was spiked with l2SI-MAb and
incubated with paraformaldehyde-fixed H2669 melanoma cells.
The mean percentage of binding of the controls was 46% for
Fab and 57% for IgG.
l25I-MAb contained in brain homogenate from in vivo studies
had a mean percentage of binding of 47% for Fab and 64% for
IgG. The in vitro NSB was subtracted from the in vitro CBA,
and the in vivo NSB was subtracted from the in vivo CBA to
determine the net specific binding of antibody to melanoma
cells. At 1 and 24 h postdisruption, antigen binding for both
Fab and IgG contained in brain homogenate from the in vivo
studies was essentially identical to the in vitro controls, indicat
ing that the immunoreactivity of the 96.5 IgG and Fab was
maintained in vivo for at least 24 h.
DISCUSSION
The current BBB disruption studies were performed to char
acterize the delivery to brain of IgG and two Fab fragments,
which bind melanoma-associated antigens, and to evaluate non
specific binding and clearance from normal brain. Since mela
noma shares antigens with gliomas, the antibodies in this report
may also be applicable to primary brain tumors (24, 30-35).
These results point to several factors related to the potential of
monoclonal antibodies in the diagnosis and therapy of brain
tumors. One is that BBB disruption markedly enhances inimunologically reactive MAb delivery to the brain, a finding
similar to results obtained with other IgG and IgM MAbs (6,
7). This may be particularly significant in the case of relatively
impermeable tumors or tumor-infiltrated normal brain (8).
Another important finding was that Fab cleared rapidly from
normal brain, decreasing 50% by 4.5 h as compared to 25.5 h
for IgG and more than 3 days for IgM (7). The initial greater
delivery to brain combined with the more rapid clearance of
Fab versus IgG resulted in an overall AUC that was virtually
the same for both antibodies.
The reason for the more rapid clearance of Fab is unclear.
Table 3 Melanoma cell binding of'"I-monoclonal antibody recovered from
brain homogenate following blood-brain barrier disruption
l25I-MAb (IgG or Fab 96.5) was administered i.e. immediately after BBB
disruption. Animals were sacrificed 1 or 24 h after antibody administration.
cellControl
of
(h) after
in vivo
Monoclonal
administration
antibodyIgGFabTime
of antibody1(4)'
24(4)1(3)
CBA minus
in vitro
NSB43.8
39.127.8
of antibody
from disrupted
brain minus
in vivo
NSB37.9
40.530.922.5
24(3)%
24.3binding"CBA
* The percentage of NSB and percentage of CBA were calculated as [(cpm in
pellet/total cpm) x 100J/TCA precipitability of sample. Concurrent in vitro
controls were performed in which brain homogenate from untreated animals was
spiked with '"I-MAb prior to incubation with cells (CBA) or without cells (NSB).
(See "Materials and Methods.")
* Numbers in parentheses, number of animals studied.
One possible explanation is that Fab is smaller and more
diffusible into CSF. However, previous studies of two very
differently sized proteins (M, 68,000 and 1,000,000) showed
delivery to CSF following BBB disruption to behave very sim
ilarly (6). Others have described higher tissue clearance rates
and markedly reduced background of nonspecific and tumorspecific monoclonal immunoglobulin fragments compared with
whole immunoglobulin (21, 36, 37). These studies suggest that
the Fab fragment offers significant pharmacokinetic advan
tages, which may permit increased delivery after BBB opening
to tumor and to tumor-infiltrated surrounding brain, yet is
rapidly cleared from otherwise normal brain. Conversely, the
Fab fragments bound 37% less to the antigen than the intact
immunoglobulin (see Table 3).
In addition, the delivery and percentage of the total dose per
gram of brain were initially higher for Fab than IgG, but the
overall brain exposure was essentially the same. The difference
between Fab and IgG may relate to the Fc portion of the
molecule or molecular size. Evidence that the plasma concen
tration is not entirely responsible for a higher level of Fab in
brain is the 4-fold higher brain:plasma AUC ratio for Fab than
for IgG and the fact that Fab plasma levels were always lower
than IgG.
A second observation relates to the optimal method of MAb
infusion which results in the greatest delivery to disrupted brain.
After evaluation of various methods of Fab administration with
BBB opening, clearly i.e. bolus infusion compared with i.v.
bolus or a slower infusion resulted in the greatest delivery to
brain. Longer infusion times were not evaluated because pre
vious studies have shown a size-dependent decrease in perme
ability with time after BBB opening and thus a theoretical
advantage to administration at times close to the osmotic
procedure (7, 9). There was no such effect with IgG which
correlates with studies by Fenstermacher (38) who predicted no
advantage to intman erial administration when the rate of trans
formation or excretion is very low. These findings correlate
with studies by Bullard et al. in which there was no difference
in the delivery to brain after barrier opening between i.e. or i.v.
administration of a glioma-specific IgG MAb (5).
In a recent pilot study of three patients with melanoma
metastatic to the CNS, there was no uptake of either a nonspe
cific antibody or tumor-specific antibody after i.v. administra
tion (8). After BBB disruption, there was a marked increase in
antibody delivery to the tumor-bearing hemisphere, and serial
brain scans showed that the effective half-life of radiolabeled
Fab antibody in disrupted brain ranged from 12 to 30 h. An
important observation in this initial study was that only one
patient had any evidence of specific increased uptake in the
region of the tumor, and the clearance from the tumor region
was the same as the surrounding apparently normal brain.
However, immunohistological studies indicated that only a
fraction of the antigen binding sites on tumor cells were bound
with antibody, and the lack of persistent localization may have
been the result of giving an inadequate dose of antibody.
Studies are currently under way in nude rats with human
tumors implanted intracerebrally and s.c. (10). These studies
are addressing MAb dose (2, 15); antibody affinity or avidity;
type and dose of radionuclide (4, 20, 39, 40); the delivery of
different tumor-specific IgG, Fab, and F(ab')2 antibodies (14,
21); and other parameters, such as tumor size (41), which may
increase the degree and persistence of localization of mono
clonal antibodies to tumor (42, 43).
4728
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MAb DELIVERY ACROSS THE BBB
REFERENCES
1. Bourdon, M. A., Coleman, R. E., Blasberg, R. G., Groothuis, D. R., and
Bigner, D. D. Monoclonal antibody localization in subcutaneous and intra
cranial human glioma xenografts: paired-label and imaging analysis. Anticancer Res., 4: 133-140, 1984.
2. Goodman, G. !•'..
Beaumier, P., Hellström,I., Fernyhough, B., and Hellström,
K. E. Pilot trial of murine monoclonal antibodies in patients with advanced
melanoma. J. Clin. Oncol., 3: 340-352, 1985.
3. Hellström, L, Beaumier, P. I... and Hellström, K. E. Antitumor effects of
L6, an IgG2a antibody reacting with most human carcinomas. Proc. Nati.
Acad. Sci. USA, 83: 7059-7063, 1986.
4. Lee, Y. S., Bullard, D. E., Friedman, H. S., Wikstrand, C. J., Coleman, R.
I :., Zalutsky, M., Muhlbaier, I.. H., and Bigner, D. D. Experimental radio
therapy of subcutaneous (SC) and intracranial (1C) human glioma (HGL)
xenografts in rodents with '"I monoclonal antibody (Mab) 816C. Proc. Am.
Assoc. Cancer Res., 28: 392, 1987.
5. Bullard, D. E., Bourdon, M., and Bigner, D. D. Comparison of various
methods for delivering radiolabeled monoclonal antibody to normal rat brain.
J. Neurosurg., 61: 901-911, 1984.
6. Neuwelt, E. A., Barnett, P. A., McCormick, C. I., Frenkel, E. P., and Minna,
J. P. Osmotic blood-brain barrier modification: monoclonal antibody, albu
min, and methotrexate delivery to CSF and brain. Neurosurgery, 17: 419423, 1985.
7. Neuwelt, E. A., Minna, J., Frenkel, E., Barnett, P. A., and McCormick, C.
I. Osmotic blood-brain barrier opening to IgM monoclonal antibody in the
rat. Am. J. Physiol., 250: 875-883, 1986.
8. Neuwelt, E. A., Specht, H. D., Barnett, P. A., Dahlborg, S. A., Larson, S.
M., Brown, P., Eckerman, K., Hellström, K. E., and Hellström,I. Increased
delivery of tumor specific monoclonal antibodies to brain after osmotic bloodbrain barrier modification in patients with melanoma metastatic to the CNS.
Neurosurgery, 20: 885-895, 1987.
9. Ziylan, Y. /.., Robinson, P. J., and Rapoport, S. 1. Differential blood-brain
barrier permeabilities to ("Cjsucrose and [3H]inulin after osmotic opening in
the rat. Exp. Neurol., 79: 845-857, 1983.
10. Neuwelt, E. A., Frenkel, E., D'Agostino, A. N., Carney, D., Minna, J.,
Harm-it. P., and McCormick, C. I. Growth of human lung tumor in the brain
of the nude rat as a model to evaluate antitumor agent delivery across the
blood-brain barrier. Cancer Res., 45: 2827-2833, 1985.
11. Bergstrom, M., Collins, P., Ehrin, E., Ericson, K., Eriksson, L., Greitz, T.,
Halldin. C., von Holst, H., Langstrom, B., Lilia, A., Lundqvist, H., and
Hagren, K. Discrepancies in brain tumor extent as shown by computed
tomography and positron emission tomography using [MGa]EDTA, |"C]
glucose, and [' 'Cjmethionine. J. Comp. Assist. Tomogr., 7:1062-1066,1983.
12. Neuwelt, E. A.. Specht, H. D., and Hill, S. A. Permeability of human brain
tumor to "Tc-glucoheptonate
and "Tc-albumin:
implications for mono
clonal antibody therapy. J. Neurosurg., 65: 194-198, 1986.
13. Blythman, H. E., Casellas, P., Gros, O., Gros, P., Jansen, F. K., Paolucci,
F., Pau, B., and Vidal, H. Itnmunotoxins: hybrid molecules of monoclonal
antibodies and a toxin subunit specifically kill tumour cells. Nature (Lond.),
290:145-146, 1981.
14. Carrasquillo, J. A., Krohn, K. A., Beaumier, P., McGuffin, R. W., Brown, J.
P., Hellström,K. E., Hellström,L, and Larson, A. M. Diagnosis or therapy
for solid tumors with radiolabeled antibodies and immune fragments. Cancer
Treat. Rep., 68: 317-328, 1984.
15. Ferens, J. M., Krohn, K. A., Beaumier. P. L., Brown, J. P., Hellström, I.,
Hellström.K. E., Carrasquillo. J. A., and Jarson, S. M. High-level iodination
of monoclonal antibody fragments for radiotherapy. J. NucÃ-.Med., 25:367370, 1984.
16. Franke!, A. E. Antibody-toxin hybrids: a clinical review of their use. J. Biol.
Response Modif., 4:437-446, 1985.
17. Hellström, I., Horn, D., Linsley, P., Brown, J. P., Brankovan, V., and
Hellström, K. E. Monoclonal mouse antibodies raised against human lung
carcinoma. Cancer Res., 46: 3917-3923, 1986.
18. Order, S. E. Editorial comment: molecular missiles and drug delivery. Cancer
Drug Deliv., 2:171, 1985.
19. Schroff, R. W., Woodhouse, C. S., Foon, K. A., Oldham, R. K., Farrell, M.
M., Klein, R. A., and Morgan, A. C., Jr. Intratumor localization of mono
clonal antibody in patients with melanoma treated with antibody to a
250,000-dalton melanoma-associated antigen. J. Nati. Cancer lust.. 74:299306. 1985.
20. Buraggi, G. L., Callegaro. L., Turrin, A., Cascinelli, N., Attui, A., Emaunelli,
H.,
Gasparini,
G., Dovis,
M., Mariani, G., Natali,
P. G.,
Scasellati,M.,G.Deleide.
A., Rosa,G.,l '..Plassio,
and Ferrone,
S. Immunoscintigraphy
with
'"I, "Te,
and '"In-labelled
(F(ab') fragments of monoclonal antibodies to
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
a human high molecular weight melanoma-associated antigen. J. NucÃ-.Med.
Allied Sci., 28: 283-295, 1984.
Buraggi, G. L.. Callegaro, L., Mariani, G., Turrin, A., Cascinelli, N., Aitili,
A., Bombardieri, E., Terno, G., Plassio, G., Dovis, M., Mazzuca, N., Natali,
P. G., Scassettati, G. A., Rosa, U., and Ferrone, S. Imaging with IMI-labeled
monoclonal antibodies to a high molecular weight melanoma-associated
antigen in patients with melanoma: efficacy of whole immunoglobulin and
its F(ab'>2 fragments. Cancer Res., 45:3378-3387, 1985.
Hwang, K. M., Fodstad, O., Oldham, R. K., and Margan, A. C., Jr. Radiolocalization of xenografted human malignant melanoma by a monoclonal
antibody (9.2.27) to a melanoma-associated antigen in nude mice. Cancer
Res., 45:4150-4155, 1985.
Kan-Mitchell, J., Imam, A., Kempf, R. A., Taylor, C. R., and Mitchell, M.
S. Human monoclonal antibodies directed against melanoma tumor-associ
ated antigen. Cancer Res., 46: 2490-2496, 1986.
Woodbury, R. G., Brown, J. P., Yeh, M-Y., Hellström,I., and Hellström, K.
E. Identification of a cell surface protein, p97, in human melanomas and
certain other neoplasms. Proc. Nati. Acad. Sci. USA, 77: 2183-2187, 1980.
Vandlen, R. L., Wu, WC-S., Eisenach, J. C., and Raftery, M. A. Studies of
the composition of purified Torpedo Californien acetylcholine receptor and
of its subunits. Biochemistry, 18: 1845-1854, 1979.
Wray, W., Boulikas, T., Wray, V. P., and Hancock, R. Silver staining of
proteins in polyacrylamide gels. Anal. Biochem., //*: 197-203, 1981.
Ohno, K., Fredericks, W. R., and Rapoport, S. I. Osmotic opening of the
blood-brain barrier to methotrexate in the rat. Surg. Neurol., 12: 323-328,
1979.
Beaumier, P. L., Neuzil, D., Yang, H. M., Noll, E. A., Kishore, R., Eary, J.
F., Krohn, K. A., Nelp, W. B., Hellström, K. E., and Hellström, I. Immunoreactivity assay for labeled anti-melanoma monoclonal antibodies. J. NucÃ-.
Med., 27: 824-828, 1986.
Winer, B. J. Statistical principles in experimental design. In: N. Garmezy,
R. Solomon, L. Jones, and H. Stevenson (eds.), New York: McGraw-Hill,
Inc., 1971.
Carincross, J. G., Mattes, M. J., Beresford, H. R., Albins, A. P., Houghton,
A. N., Lloyd, K. O., and Old, L. F. Cell surface antigens of human astrocyto nia denned by mouse monoclonal antibodies: identification of astrocytoma
subsets. Proc. Nati. Acad. Sci. USA, 79: 5641-5645, 1982.
Piguet, V., Diserens, A-C, Carrel, S., Mach, J-P., and deTribolet, N. The
immunobiology of human gliomas. Springer Semin. Immunopathol., A: I 11
127, 1985.
Schnegg, J. F., Diserens, A. C., Carrel, S., Accolla, R. S., and deTribolet, N.
Human glioma-associated antigens detected by monoclonal antibodies. Can
cer Res., 41: 1209-1213, 1981.
Seeger, R. C., Danon, Y. L., Rayner, S. A., and Hoover, F. Definition of a
tli> I determinant on human neuroblastoma, glioma, sarcoma, and teratoma
cells with a monoclonal antibody. J. Immunol., 128: 983-989, 1982.
Seeger, R. C., Rosenblatt, H. M., Imai, K., and Ferrone, S. Common antigenic
determinants on human melanoma, glioma, neuroblastoma, and sarcoma
cells defined with monoclonal antibodies. Cancer Res., 41:2714-2717,1981.
Wikstrand, C. J., Bourdon, M. A., Pegram, C. N. and Bigner, D. D. Human
fetal brain antigen expression common to tumors of neuroectodermal origin:
gliomas, neuroblastomas, melanomas. J. Neuroimmunol., 5:43-62, 1982
Covell, D. G., Barbet, J., Holton, O. D., Black, C. D. V., Parker, R. J., and
Weinstein, J. N. Pharmacokinetics of monoclonal immunoglobulin Gl,
F(ab')2, and Fab' in mice. Cancer Res., 46: 3969-3978, 1986.
Wahl, R. L., Parker, C. W., and Philpott, G. W. Improved radioimaging and
tumor localization with monoclonal I-(ali').. J. NucÃ-.Med., 24: 316-325,
1983.
38. Fenstermacher, J. D. Intra-arterial infusions of drugs and hyperosmotic
solutions as ways of enhancing CNS chemotherapy. Cancer Treat. Rep., 65:
27-37,1981.
39. limimi,.!. L. Dosimetrie aspects of radiolabeled antibodies for tumor therapy.
J. NucÃ-.Med., 27: 1490-1497, 1986.
40. Leyden, M. J., Thompson, C. H., Lichtenstein, M., Andrews, J. T., Sullivan,
J. R., Zalcberg, J. R., and McKenzie, I. F. C. Visualization of métastases
from colon carcinoma using an iodine 131-radiolabeled monoclonal antibody.
Cancer (Phila.), 57:1129-1135, 1986.
41. Hagan, P. L., Halpern, S. E., Dillman, R. O., Shawler, D. L., Johnson, S.
E., Chen, A., Krishnan, L., Frincke, J., Bartholomew, R. M.. David, G.
S..and Carlo, D. Tumor size: effect on monoclonal antibody uptake in tumor
models. J. NucÃ-.Med., 27:422-427, 1986.
42. Lee, Y., and Bigner, D. D. Aspects of immunobiology and immunotherapy
and uses of monoclonal antibodies and biologic immune modifiers in human
gliomas. Neurol. Clin., 3: 901-917, 1985.
43. Neuwelt, E. A., Barnett, P. A., Bigner, D. D., and Frenkel, E. P. Effects of
adrenal cortical steroids and osmotic blood-brain barrier opening on metho
trexate delivery to gliomas in the rodent: the factor of the blood-brain barrier.
Proc. Nati. Acad. Sci. USA, 79:4420-4423, 1982.
4729
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1988 American Association for Cancer Research.
Delivery of Melanoma-associated Immunoglobulin Monoclonal
Antibody and Fab Fragments to Normal Brain Utilizing Osmotic
Blood-Brain Barrier Disruption
Edward A. Neuwelt, Peggy A. Barnett, I. Hellström, et al.
Cancer Res 1988;48:4725-4729.
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