Post on 21-Sep-2020
Vol. 3, 325-337, March 1997 Clinical Cancer Research 325
Pharmacokinetic Features, Immunogenicity, and Toxicity of
B43(anti-CD19)-Pokeweed Antiviral Protein Immunotoxin
in Cynomolgus Monkeys’
Fatih M. Uckun,2 Yuri Yanishevski,
Nilg#{252}n Turner, Barbara Waurzyniak,
Yoav Messinger, Lisa M. Cheistrorn,
Elizabeth A. Lisowski, Onur Ek, Tamer Zeren,
Heather Wendorf, Mridula-Chandan Langlie,
James D. Irvin, Dorothea E. Myers,
Gene B. Fuller, William Evans, and
Roland GuntherBiotherapy Institute, University of Minnesota Academic HealthCenter, Roseville, Minnesota [F. M. U., Y. M.. 0. E., T. Z., R. G.]:Primate Research Institute, Holloman Air Force Base, New Mexico[G. B. F.]; St. Jude Children’s Research Hospital, Memphis,Tennessee [Y. Y., W. E.]; Alexander & Parker Corporation, Glendale.California [M-C. L., D. E. Mi; Hughes Institute, St. Paul, Minnesota[B. W., L. M. C., E. A. L., H. W.]; Department of Chemistry,Southwest Texas State University, San Marcos, Texas Ii. D. I.]: andAg Biotech Center, Rutgers, The State University of New Jersey,New Brunswick, NJ [N. T.]
ABSTRACT
We studied the pharmacokinetic features, immunoge-
nicity, and toxicity of B43-pokeweed antiviral protein (PAP)
immunotoxin in 13 cynomolgus monkeys. The disposition of
B43-PAP in two monkeys, when administered as a single i.v.
bolus dose, was characterized by a slow clearance (1-2
mi/h/kg) with a very discrete peripheral distribution. B43-
PAP was retained and distributed largely in the blood as the
sole compartment with no significant equilibration with the
extravascular compartment. The circulating B43-PAP im-
munotoxin detected in monkey plasma samples by ELISA
and protein immunoblotting was both immunoreactive with,
and active against, human leukemic cells in vitro. In systemic
immunogenicity and toxicity studies, which involved 11
cynomolgus monkeys, each monkey received a total of seven
Received 9/3/96; revised I 1/20/96; accepted 1 1/25/96.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.
I This work was supported in part by United States Public Health
Service Grants CA-13539, CA-27137, CA-6l549, CA-421 I I,
CA-3640l, CA-20l80, and CA-2l765 from the National Cancer Insti-
tute; Grant RR-08079, NIH, Department of Health and Human Services:special grants from the National Childhood Cancer Foundation, ParkerHughes Trust, and the American Lebanese Syrian Associated Charities;and a State of Tennessee Center of Excellence grant. F. M. U. is aStohlman Scholar of the Leukemia Society of America.2 To whom requests for reprints should be addressed, at Biotherapy
Institute, University of Minnesota, 2625 Patton Road, Roseville, MN55113.
i.v. doses of B43-PAP at a specific dose level of the dose
escalation schedule. B43-PAP-treated monkeys mounted a
dose-dependent humoral immune response against both the
mouse IgG and PAP moieties of the immunotoxin. When
administered i.v. either on an every-day or every-other-day
schedule, B43-PAP was very well tolerated, with no signifi-
cant clinical or laboratory signs of toxicity at total dose
levels ranging from 0.007 to 0.7 mg/kg. A transient episode
of a mild capillary leak with a grade 2 hypoalbuminemia
and 2+ proteinuria was observed at total dose levels equal to
or higher than 0.35 mg/kg. At total dose levels of 3.5 and 7.0
mg/kg, B43-PAP caused dose-limiting renal toxicity due to
severe renal tubular necrosis. The present study completes
the preclinical evaluation of B43-PAP and provides the basis
for its clinical evaluation in children with therapy-refractory
B-lineage acute lymphoblastic leukemia.
INTRODUCTIONALL3 is the most common form of childhood cancer ( I , 2).
Currently, the major challenge in the treatment of childhood
ALL is to cure patients who have relapsed despite intensive
multiagent chemotherapy (I , 2). Consequently, the development
of new potent anti-ALL drugs and the design of combinative
treatment protocols using these new agents have emerged as
exceptional focal points for research in modern therapy of
relapsed ALL.
Immunotoxins are a new class of biotherapeutic agents that
show considerable promise for more effective treatment of re-
fractory ALL (3-5). Immunotoxins have been prepared by co-
valently linking a cell-type-specific monoclonal antibody to a
variety of catalytic ribosome inhibitory protein toxins (3-5).
Several investigators have used different ribosome inhibitory
protein-containing immunotoxins and recombinant immunotox-
ins for in vivo treatment of hematological malignancies, includ-
ing ALL, with mixed results in early clinical trials (6-10). For
an immunotoxin to be optimally effective against ALL, the
target antigen recognized by its monoclonal antibody moiety has
to fulfill at minimum the following essential requirements. (a) It
has to be expressed on leukemic blasts from the majority of
ALL patients. (b) It has to be expressed on the self-renewing
clonogenic ALL blast populations (i.e. , leukemic progenitor
cells). (c) It has to undergo antibody-induced internalization so
that the toxin moiety can be transported into the targeted ALL
blasts. (d� It has to be absent on the peripheral blood myeloid/
3 The abbreviations used are: ALL, acute lymphoblastic leukemia; PAP,pokeweed antiviral protein: BUN, blood urea nitrogen; bR, blockedricin; dgA, deglycosylated ricin A; RTA, recombinant form of theA-chain of ricin; PE, phycoerythrin.
Research. on January 13, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
326 B43-PAP Primate Study
erythroid elements so that the immunotoxin can reach and
effectively kill the small number of clonogenic ALL blasts in
the presence of excess normal cells. (e) It should not be shed
from the surface or circulate in blood in soluble form competing
with surface-bound antigen for the administered immunotoxin
molecules. (f) It has to be absent on the parenchymal cells of the
life-maintaining nonhematopoietic organs. Because the vast ma-
jority of ALLs appear to originate from putative developmental
lesions in normal B-cell precursor clones during early phases of
ontogeny and to express B-lineage lymphoid differentiation
antigens, and because no ALL-specific antigens have yet been
defined, an immunotoxin to be generally applicable in ALL
patients must be directed against a B-lineage associated/re-
stricted surface antigen. CD19 antigen meets all the criteria for
an appropriate target antigen (1 1, 12).
B43-PAP is an anti-CD19 immunotoxin that has been
constructed by covalently linking the anti-CD19 monoclonal
antibody B43 ( 12) to PAP ( 1 3), a Mr 30,000 hemitoxin isolated
from spring leaves of the pokeweed plant (Phytolacca amen-
cana; Refs. 14 and 15). PAP belongs to a family ofenzymes that
inactivate ribosomes by the specific removal of a single adenine
from the conserved loop sequence found near the 3’ terminus of
all larger rRNAs ( 1 3). This specific depurination greatly reduces
the capability of elongation factors to interact with ribosomes
and results in an irreversible shutdown ofprotein synthesis (13).
Importantly, leukemic progenitor cells (i.e, primary clonogenic
blasts) from ALL patients are very sensitive to PAP-containing
immunotoxins targeted to appropriate surface antigens capable
of antibody-induced internalization (14). B43(anti-CD 19)-PAP
proved to be the most effective immunotoxin tested and killed
>99.9% of primary leukemic progenitor cells from B-lineage
ALL patients (14, 16). Furthermore, B43-PAP generated
promising results in preclinical SCID mouse models of hu-
man B-lineage ALL (16-20). As a single agent, B43-PAP
was found to be more potent than cyclophosphamide, yin-
cristine, methylprednisolone, etoposide, topotecan, L-aspara-
ginase, Adriamycin, cytarabine, 1 ,3-bis(2-chloroethyl)-l-ni-
trosourea, or taxol against human B-lineage ALL in the SCID
mouse model system (20).
Currently, very little is known about the safety of this
investigational new biotherapeutic agent. In a recent study in
mice, we found that B43-PAP causes dose-limiting renal and
cardiac toxicity (21). In view of the dose-dependent fatal tox-
icities of B43-PAP in mice and no previous human clinical trials
involving PAP-containing immunotoxins, a primate toxicity
study was warranted. The purpose of the present preclinical
study was to evaluate the pharmacokinetic features, immunoge-
nicity, and toxicity profile of i.v. B43-PAP therapy in cynomol-
gus monkeys. The effects of B43-PAP on humoral immunity of
cynomolgus monkeys could not be examined because B43 does
not react with primate B cells. To our knowledge, this is the first
comprehensive preclinical analysis of an anti-CD19 immuno-
toxin containing PAP in monkeys.
MATERIALS AND METHODSB43(anti-CD19)-PAP Immunotoxin. The procedures
used for the large-scale production and purification of B43-PAP
immunotoxin have been described previously in detail (15). In
brief, PAP amino groups were thiolated using 2-iminothiolane,
and modified PAP was mixed with N-succinimidyl-3-(pyri-
dyldithio)propionate-modified B43 monoclonal antibody using
a 3.5:1 molar ratio of PAP to antibody to generate B43-PAP in
a sulffiydryl-disulfide exchange reaction (I 5). B43-PAP was
initially purified by gel filtration high-performance liquid chro-
matography to remove unreacted PAP (15). Carboxymethyl-
Sepharose ion-exchange chromatography was subsequently
used to purify B43-PAP from unconjugated B43 antibody (15).
Cynomolgus Monkeys. The primate studies, including
the laboratory studies on blood and urine specimens, were
performed in the centralized American Association for Accred-
itation of Laboratory Animal Care-approved and fully accred-
ited Primate Research Facilities of the Biotherapy Institute at the
University of Minnesota and the Primate Research Institute
(Holloman Air Force Base, New Mexico). These studies were
conducted in close collaboration with the Research Animal
Resources department at the University of Minnesota according
to the United States Government Principles for the Utilization
and Care of Vertebrate Animals Used in Testing, Research, and
Training as well as the guidelines of the University of Minne-
sota Animal Care Committee. Monkeys were housed with like
species in an environment in which they could see, hear, and
smell other monkeys according to a cage-enrichment program.
Monkeys were individually caged in suspended stainless steel
cages with squeeze bar attachments. The cages were cleaned,
and the rooms were flushed daily. The temperature and humidity
of the rooms were monitored, and the light cycle was I 2 h on
and 12 h off. All husbandry duties and medical evaluations were
performed by Research Animal Resources personnel and the
staff veterinarian of the Biotherapy Institute or by the staff at the
Primate Research Institute. Monkeys were fed Teklad Monkey
Chow (5 biscuits/kg/day, 1 time/day, 7 days/week). Fruit and
trail mix (cereals, grains, dried fruit, and peanuts) were given in
the morning and afternoon in accordance with a food supple-
ment/diet enrichment plan. Individual laboratory data and ne-
cropsy records were completed for each monkey examined. The
glass slides with affixed tissues were examined by staff veteri-
nary pathologists of the Biotherapy Institute. Tissue slides from
the first study using high B43-PAP doses were also sent to
Colorado Pathology Services, Inc. (Fort Collins, CO) for inde-
pendent histopathological examination and report compilation
by Dr. Donald N. Kitchen, Doctor of Veterinary Medicine, in
accordance with Food and Drug Administration regulations for
Good Laboratory Practice (21 Code of Federal Regulations Part
58), including appropriate standard operating procedures and
designated recording requirements. Histopathological examina-
tion of the following tissues and organs (in alphabetical order)
was performed as required by study protocol: adrenal glands,
bone marrow, brain, cecum, colon, duodenum, heart, ileum,
jejunum, kidney, liver, lung, lymph node, nerve (sciatic), ovary
(in female monkeys), skeletal muscle, skin, spleen, stomach,
testis (in male monkeys), thyroid gland, and uterus (in female
monkeys). We used 1 1 adult male cynomolgus monkeys in the
systemic pharmacology/toxicity/immunogenicity studies. After
admission to the Primate Research Facilities, monkeys were
kept in quarantine for a 6-week period, and several routine tests
were performed, including a tuberculin test. A toxicity grading
system that was adapted from the Children’s Cancer Group
Research. on January 13, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Clinical Cancer Research 327
Clinical Toxicity Criteria was used in the daily evaluation of the
monkeys (Table 1).
Treatment and Clinical/Laboratory Evaluation of
Cynomolgus Monkeys. The pharmacology study was per-
formed on two adult male cynomolgus monkeys that received a
single bolus dose of B43-PAP (F628, 1.0 mg/kg; FR324, 0.5
mg/kg) i.v. The first toxicity study was performed on five male
cynomolgus monkeys. Four of these monkeys received, on 7
consecutive days, daily doses of B43-PAP immunotoxin by i.v.
infusion over 1 h via an antecubital vein after being anesthetized
with 10 mg/kg ketamine. One monkey received unconjugated
B43 monoclonal antibody. Monkeys were monitored twice daily
for treatment-related morbidity and were euthanized either when
found moribund or 30 days after the last day of infusion (that is
day 37 of the study). Vital signs, including heart rate, respiratory
rate, systolic blood pressure, and temperature, were checked
before and 2 h after B43-PAP (or B43) infusion on each of the
treatment days and then twice weekly. Animals were weighed,
and electrocardiograms were performed every other day in the
first week and then twice weekly. The neurological examination
included examination of gait, pelvic limb flexor reflex, thoracic
limb flexor reflex, patellar reflexes, alertness, and overall be-
havior. Blood samples for complete blood counts, including a
differential and platelets, serum samples for total protein, albu-
mm, protein electrophoresis, liver enzymes, bilirubin, creatine
phosphokinase/creatine phosphokinase isoenzymes, lactate de-
hydrogenasellactate dehydrogenase isoenzymes, BUN/creati-
nine, electrolytes, osmolarity, triglycerides/cholesterol, as well
as urine samples for routine analysis, were obtained and ana-
lyzed every other day in the first week and twice weekly
thereafter.
The second toxicity study was performed on six additional
male cynomolgus monkeys. These monkeys received a total of
seven doses of B43-PAP by i.v. infusion over 1 h on an every-
other-day schedule. Monkeys were monitored twice daily for
treatment-related morbidity and euthanized either on day 15
(that is 1 day after the last dose) to examine their organs for
lesions caused by acute toxicity, or on day 30 (that is 16 days
after the last dose) to examine their organs for lesions caused by
subacute toxicity. Laboratory studies were performed according
to the program and schedule that was outlined for the first
toxicitystudy.
Pharmacology Studies and Pharmacokinetic Modeling.
Peripheral blood samples (500 p.1/time point) were collected at
30 mm, 1, 2, 4, 6, 8, 12, 24, 48, 72, 120, and 168 h after the
administration of B43-PAP immunotoxin. Plasma from these
blood samples was then used to determine the in vivo chemical
and functional stability of B43-PAP. Using procedures detailed
in previous publications from our group, the amounts of chem-
ically intact B43-PAP immunotoxin and free B43 monoclonal
antibody in the plasma samples were quantified in three inde-
pendent assays of triplicate samples by solid-phase ELISA and
visualized by protein immunoblot analyses to evaluate the
chemical stability of circulating B43-PAP (15, 22, 23). The
intact B43-PAP immunotoxin and total (free + PAP-conju-
gated) B43 antibody concentrations in the plasma samples were
determined from standard curves that were generated by linear
regression analysis using varying amounts of purified B43-PAP
immunotoxin standard or B43 monoclonal antibody standard.
Free antibody concentrations were determined from the differ-
ence between conjugated (i.e., chemically intact B43-PAP im-
munotoxin) and total (conjugated + free) B43 monoclonal
antibody concentrations.
A linked two-compartment model (see Fig. IA) was used to
simultaneously model both chemically intact B43-PAP immu-
notoxin and the formation of free antibody resulting from the in
vivo degradation of i.v.-administered B43-PAP immunotoxin, as
described previously (20, 22, 23). Maximum likelihood estima-
tion, as implemented in ADAPT II software, was used to fit the
model to the data and estimate the pharmacokinetic parameters
(24). Akaike’s information criterion was used for model selec-
tion (25). The following system of differential equations was
used for the pharmacokinetic model:
dX1-�-=lV-(K12+K13+K10)XX1+K21XX2
dX2
� K12 X X, - K21 X X2
dX3-�-=K13XX1 +K43XX4-(K34+K30)XX3
dX4
� K34 X X3 - K43 x X4
where X12 and X3�4 are the amounts of immunotoxin and free
antibody, respectively, in the model compartments, IV is the
zero-order infusion rate of immunotoxin, K10 is the elimination
rate constant, and K12, K21, and K13 are intercompartment rate
constants for the immunotoxin. Previous studies in rabbits
showed that because of the identical binding properties and
similar sizes of B43 antibody and B43-PAP immunotoxin, the
volume of distribution in the central compartment (Va), elimi-
nation constant from the central compartment (K10), and distri-
bution rate constants (K12 and K21) are not significantly differ-
ent when free B43 and conjugated B43 are compared (i.e. , K10
= 1(30, K12 = 1(34, K21 = K43, and V�1 = V�3). Therefore, our
final model in cynomolgus monkeys assumed the same Vc, K10,
K12, and K21 for both intact B43-PAP and free B43 antibody and
thus included five pharmacokinetic parameters: V�, K10, K,3,
K12, and K2, . The output equations for B43-PAP and free B43
antibody concentrations were as follows:
xiB43-PAP concentration =
(X1 + x3)Total B43 concentration =
ye
Immunotoxin clearance (CL�T) was calculated as V� X (K,0 +
K,3), and free antibody clearance (CLAB) was calculated as V�
x K10. The area under the plasma concentration X time curve
(AUC0�) was estimated as (total dose) divided by CL�T.
The presence of immunoreactive B43-PAP immunotoxin
and free B43 monoclonal antibody in the collected plasma
samples was examined by previously published (15, 22, 23)
direct two-color immunofluorescence staining techniques and
multiparameter flow cytometry using PE-labeled rabbit anti-
PAP IgG to detect cell surface-bound intact B43-PAP immuno-
toxin molecules via their PAP moieties and FITC-labeled goat
Research. on January 13, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Table I Primate toxicity and complications criteria
This primate toxicity grading system was adapted from the Children’s Cancer Group Clinical Toxicity Criteria.
Hematology 4.0-14.0wBcsNeutropeniaLeukocytosis
HemoglobinPlatelets
Feeding Feeding abnormality
Grade
1 2 3 4b
Site Measure WNL’� (Mild) (Moderate) (Severe) (Unaccept.)
Gastrointestinal Diarrhea
Liver
PancreasRenal
>11.5> ISONone
None
�l.3
�6O
�3633.5-5.5
<20
<5.6
Negative
BilirubinAL�AmylasePotassium
HypokalemiaHyperkalemia
Urea nitrogenCreatininePhosphorusUrine
Protein
Blood Negative >10
Negative
1.013-1.035Clear
3.0-3.914.1-20.0lO.O-l .575.0- 150.0
Mild amount of softstool
1.4-1.5
61-ISO364-545
3.1-3.45.6-6.420-391.2-1.55.6-6.9(I or more)
+1
+5 WBCs, <10,000
colonies. ( +)
Wheezing
33-50
33-50Slight
160-195265-300
100-109166-190
I 31-ISOI 16- I 30
80-8970-84Mild weaknessSupportive standing.
minimal paraparesis/ataxia
Infection
Specific gravityPulmonary Clinical
Respiratory rate
Conscious 28-32Anesthetized 20-32
Cardiac Murmur NoneHeart rate
Conscious 195-265Br.tdycardiaTachycardia
Anesthetized I 10-165BradycardiaTachycardia
HypertensionConscious (syst.Y’ 90-130Anesthetized (syst.) 85-I 15
HypotensionConscious (syst.)Anesthetized (syst.)
Neurology MotorExamination of gait
2.0-2.920. 1-30.0
8.0-9.950.0-74.9Decreased intake
Moderate amount of soft stool,diarrhea, minimal bleeding.small amount of mucous instool
1.6-2.0IS 1-300546-726
2.6-3.06.5-7.0
40-591.6-3.07.0-11.1(I or more)
+2 to +3
See blood
Many WBCs (++)
<1.013, >1.035
Crackles
51-70
51-70
Significant
125- I59
30l-335
90-99191-215
1�1-16S
I 31-145
70-79
60-69
Moderate weaknessSupportive standing. stumbles
frequently and falls. mildparaparesis/ataxia
Lethargic. very drowsy
Swelling, hives, itching
Complete local loss, mildgeneral loss
± 10-19.9%1.36-1.5955.0-79.5
0.08-0.1040-45
101-149
111-165
2.0-2.9
294-298
Minimal prodding required
1.0-1.9
30.1-40.06.5-7.9
25.0-49.9
Not eating
Watery diarrhea, excessiveamount of soft stool, largeamount of mucous in stool
2.1-4.0
301-I 200
727-I 815
2.0-2.5
7.1-7.560-793.1-6.0
I 1.2-13.9
(1 or more)
+4
See blood clots
Sheets of WBCs, >10.000colonies, (+ + +) or (4 + + +)
1.008-1.012
Severe respiratory distress
71-80
7 I-SOvery significant
< I 25
>335
80-89
216-240
165-I 80
145-160
55-69
50-59Severe weaknessCan’t stand, when assisted
stumbles and falls frequently,moderate paraparesis/ataxia
Seizures
Generalized swelling. itching.requiring treatment
Severe generalized loss
± �20.O%
1.6-2.1
80.0-99.90.05-0.0730-39
150-500
166-2201.5-1.9
299-303
Strong prodding required
90-I 30
85-115
No change
Normal strength/coordination
<1.0
>40.0<6.5
<25.0Severe dehydration and/or
weight loss
Bloody diarrhea or severedehydration due todiarrhea
>4.0
>1200>1815
<2.0>7.5�80
>6.0
>14
(I or more)Greater than +4 marked
protein loss
Transfusion requiringhematuria
Sepsis
>80
>80
<80
>240
> I 80
>160
<55<SO
ParalysisCan’t stand, slight
movement when held bytail, severe paraparesis
Paraplegic
Comatose
Skin sloughing
Bald
�2.2
a 100.0aO.04
<30
>500>220<1.5>303
Can’t move even withprodding
SevereConsistently > 1O4�F,
consistently <97�F
Life Threatening
Deathly sick
No change DrowsyCentral nervoussystem
Skin Allergic
Alopecia
Weight change From 1st day
Coagulation INRPU
CFIB
Metabolic Glucose
Triglycerides
Albumin
Blood osmolarityActivity Overall activity level
Hunched/Diseomfort
Temperature FeverlHypothermia
Infection
Overall health Not including bloodresults
None
None
±2-4.9%
<1.09
<34.0
>0.15S 1-90
aSSa3.5a288
No symptoms
None
97#{176}F-lOl .5�F
None
Mild rash
Mild localized loss
±5-9.9%
1.09-1.35
34.0-54.9
0.11-0.15
46-SO91-100
56-I 10
3.0-3.49
289-293
Symptoms, able to carryout daily activities
Mild
101 .6�F-lO3�F
Runny eyes/nose. cough.
mild diarrhea
Mild
“ WNL, within normal limits.b Unaccept., unacceptable.
C ALT. alanine aminotransfera.se.d Syst.. systemic.
Moderate Moderate-severe
1O3.l�F-lO4�F >lO4�F. <98.5W conscious,<97�F anesthetized (notinduced)
Localized skin infection, severe Positive culture, with systemiccold, moderate diarrhea. symptomswithout systemic symptoms
Moderate Severe
328 B43-PAP Primate Study
Research. on January 13, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
AIV
Ki 0
-J
EC
0
Ce
CCe0C00
100
10
0.10 50 100 150
Time (hours)
Clinical Cancer Research 329
anti-mouse IgG to detect cell surface-bound B43 antibody mol-
ecules (both in free and PAP-conjugated form).
Plasma samples were also tested for the presence of anti-
leukemic activity against CD19-positive NALM-6 leukemia
cells in a serial dilution clonogenic assay system (26).
Immunogenicity Studies. Monkey IgG responses
against the PAP and mouse IgG moieties of B43-PAP immu-
notoxin were monitored by measuring anti-PAP IgG and anti-
mouse immunoglobulin IgG concentrations in the plasma sam-
ples using solid-phase ELISA, as described previously (22, 23).
Plasma samples with the highest anti-mouse IgG antibody titers
were also assayed for their ability to block the in vitno binding
of B43-PAP immunotoxin to CD19-positive NALM-6 leukemia
cells in blocking experiments. To this end, NALM-6 cells were
incubated for I h on ice in 10-fold diluted plasma samples,
which were (a) obtained either before B43-PAP therapy (neg-
ative control) or at the time of peak humoral immune response
(test sample) and (b) spiked with 1 p.g/ml B43-PAP immuno-
toxin. Cells were subsequently washed twice to remove un-
bound immunotoxin and stained with the rabbit anti-PAP-PEJ
goat anti-mouse-IgG-FITC antibody combination to detect cell
surface-bound B43-PAP molecules via their PAP and IgG moi-
eties, respectively.
RESULTS
Pharmacokinetic Features of B43(anti-CD19)-PAP in
Cynomolgus Monkeys. Plasma samples from two cynomol-
gus monkeys (F628 and F324) treated with a single iv. bolus
dose of B43-PAP were used to determine the in vivo chemical
and functional stability of this anti-CD19 immunotoxin. A
linked, two-compartment first-order pharmacokinetic model
was fit to the ELISA-based data for chemically intact B43-PAP
and free B43 antibody plasma concentrations vensus time (Fig.
lB). In the first monkey (F628) treated with 1 .0 mg/kg B43-
PAP, the central volume of distribution (�c) was 58 mllkg,
which is very similar to the estimated total plasma volume of 49
mi/kg, and the disposition of B43-PAP was characterized by a
slow clearance with a very discrete peripheral distribution.
Thus, B43-PAP is retained and distributed largely in the blood
as the sole compartment with no significant equilibration with
the extravascular compartment. A peak plasma concentration of
18.0 p.g/ml was measured at 1 h postinfusion, and the plasma
half-life was 44.0 h with a clearance of 2 ml/h/kg. The systemic
B43-PAP exposure (i.e. , area under curve) at this dose level was
500 mg X h/liter. The free B43 antibody concentration in-
creased from 0 p.g/ml at 1-2 h to 2 �i.g/ml at 4 h and to 9.7 �g/ml
at 48 h and declined with a plasma half-life of 90.5 h to 3.5
�i.g/ml at 7 days (Fig. 1B). Similar disposition results were
obtained in the second monkey (F324) treated with 0.5 mg/kg
B43-PAP (clearance = 0.8 ml/hlkg). Protein immunoblot anal-
ysis of the plasma samples obtained from both monkeys were
developed with rabbit anti-PAP/alkaline phosphatase-conju-
gated goat anti-rabbit and alkaline phosphatase-conjugated goat
anti-mouse antibodies and provided direct evidence for the
presence ofchemically intact B43-PAP immunotoxin for at least
24 h after infusion with a slow breakdown to release free B43
monoclonal antibody (Fig. 2).
To examine the in vivo functional stability of B43-PAP
Fig. 1 A, pharmacokinetic model. Schematic of linked two-compart-
ment first-order pharmacokinetic models to simultaneously estimatedistribution and elimination rate constants for intact immunotoxin (open
boxes) and for free antibody (shaded boxes). B, pharmacokinetic fea-tures of B43-PAP immunotoxin. Mean plasma concentration versustime plots of B43-PAP immunotoxin (LI) and free antibody (0) in acynomolgus monkey treated with a single iv. 1.0-mg/kg bolus dose ofB43-PAP. A linked two-compartment first-order pharmacokineticmodel was fit to the data for plasma concentrations of chemically intactB43-PAP and free B43 monoclonal antibody versus time. The solid lineis the fitted curve for immunotoxin plasma concentrations, and thedotted line is the fitted curve for free B43 antibody plasma concentra-tions.
immunotoxin, serial plasma samples from both monkeys were
first tested for the presence of immunoreactive B43-PAP im-
munotoxin and B43 monoclonal antibody by immunofluores-
cence staining techniques and flow cytometry using CD 19-
positive NALM-6 B-lineage ALL cells and CD 19-negative
T-lineage ALL cells as in vitno targets. The percentage of
NALM-6 cells showing positive staining with monkey plasma
samples for cell-bound B43 monoclonal antibody (both free and
PAP-conjugated) did not show a significant decrease over the
first 24 h, whereas the percentage of NALM-6 cells showing
positive staining for cell-bound intact B43-PAP immunotoxin
showed a significant decrease over the same time period (Fig.
3). After the first 8 h, the percentage of cells showing positive
staining for cell-bound antibody was �25% higher than the
percentage of cells showing positive staining for cell-bound
chemically intact immunotoxin (Fig. 3). This finding is consist-
ent with the ELISA and protein immunoblotting data on the in
vivo chemical stability and degradation of B43-PAP immuno-
toxin. Unlike the CD19-positive NALM-6 cells, CD 19-negative
CEM T-lineage ALL cells did not show any evidence of cell-
bound intact B43-PAP immunotoxin or B43 monoclonal anti-
body (data not shown). Thus, circulating B43-PAP immuno-
toxin and free B43 monoclonal antibody detected by ELISA and
protein immunoblotting assays were immunoreactive and re-
Research. on January 13, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
kDa
FR 324 Plasma Test Samples Accordingto Time After Infusion of B43-PAP (Hours)
I -24 +1 +2 +4 +8 +12 +24 +48 +72
�� _.�.�I� � �
30- �
A‘ I I
B43 B43-PAP -24
210 -
180 -
150-
B
+1 +2 +4 +8 +12 +24
-�‘ .�
- � = �
330 B43-PAP Primate Study
240 -
210 -
180-
Standards
PAP B43-PAP
tamed their selectivity for CD19-positive leukemia cells. To
further assess the in vivo functional stability of B43-PAP im-
munotoxin, we examined the antileukemic activity of monkey
plasma samples against clonogenic NALM-6 B-lineage ALL
cells. A 4-h exposure of NALM-6 cells to 10-fold diluted
plasma samples obtained from F628 at 1 , 2, 8, 24, and 48 h after
B43-PAP infusion resulted in 98.7, 98.7, 93.1, 93.1, and 22.6%
clonogenic cell death (data not shown). Similarly, a 4-h expo-
sure of NALM-6 cells to 10-fold diluted plasma samples ob-
tamed from F324 at 1 h after B43-PAP infusion resulted in
94.6% clonogenic cell death (data not shown). By comparison,
the clonogenic growth of CDI9-negative CEM cells was not
affected by incubation with plasma samples of B43-PAP-treated
monkeys. Plasma samples obtained before administration of
B43-PAP did not affect the clonogenic growth of NALM-6 or
CEM cells. Thus, B43-PAP immunotoxin present in monkey
plasma samples elicited selective in vitro cytotoxicity against
human CDI9-positive leukemia cells.
Immunogenicity of B43(anti-CD19)-PAP in Cynomol-
gus Monkeys. The immunogenicity of B43-PAP was exam-
med in three cynomolgus monkeys that received daily i.v. doses
of B43-PAP on 7 consecutive days (Fl 165, 0.01 mg/kg/day for
7 days; F585, 0. 1 mg/kg/day for 7 days; Fl 1 1 1, 0.5 mg/kg/day
Fig. 2 In vivo chemical stabil-
ity of B43-PAP immunotoxin.Tenfold diluted plasma samplesfrom FR324, a cynomolgus
monkey treated with a singleiv. 0.5-mg/kg dose of B43-PAP, were examined for thepresence and time-dependent
____________ degradation of B43-PAP immu-
+48 +72 ‘ notoxin by protein immunoblot-ting (Western blot analysis) us-ing rabbit anti-PAP (A) and goat
anti-mouse (B) IgG antibodies.
for 7 days). A control monkey was treated with unconjugated
B43 monoclonal antibody instead (F1083, 1.0 mg/kg/day for 7
days). The kinetics and magnitude of the monkey immune
responses to the murine monoclonal antibody and PAP moieties
of B43-PAP immunotoxin are shown in Fig. 4. In Fl 165, which
was treated at the lowest dose level of B43-PAP (i.e., 0.07
mg/kg total dose), no anti-mouse immunoglobulin or anti-PAP
antibodies were detected by ELISA until day 16. A peak anti-
mouse immunoglobulin IgG concentration of 6.7 p�g/ml was
measured on day 24 (Fig. 4A), and a peak anti-PAP IgG con-
centration of only 1.4 �ig/ml was measured on day 22 (Fig. 4B).
In F585, which was treated at the intermediate dose level (i.e.,
0.7 mg/kg total dose), the anti-mouse immunoglobulin IgG level
became detectable by ELISA on day 10 and showed a gradual
increase until day 31 when a peak concentration of 15.6 p.g/ml
was measured (Fig. 4A). In the same monkey, a peak anti-PAP
IgG concentration of only 0.6 �ig/ml was measured in the day 33
plasma sample (Fig. 4B). In contrast to these two monkeys,
F! 1 1 1 , which was treated with the highest B43-PAP dose of 3.5
mg/kg, developed a marked humoral immune response to both
the murine antibody and PAP toxin moieties of B43-PAP. A
peak anti-mouse immunoglobulin IgG level of 33.0 �g/ml, as
well as a peak anti-PAP IgG level of 72.7 �i.g/ml, were measured
Research. on January 13, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
+48 Hr.
1% 39%
16 32 48 64 16 32 48 64
Clinical Cancer Research 331
Time of Monkey Plasma Collection (Hours After Injection of B43-PAP)
-24 Hr. +1 Hr. ‘-2 Hr. +4 Hr. +6 I-b’.64
0% 0%
w 48
0�
C32_______)(I � I 1
- +81-b’. +12 Hr.
!�� �
16 32 48 64 16 32 48 64
Fig. 3 let vivo functional stability of B43-PAP immunotoxin. Undiluted plasma samples from FR324, a cynomolgus monkey treated with a singleiv. 0.5-mg/kg dose of B43-PAP, were examined for the presence of immunoreactive B43-PAP immunotoxin and free B43 antibody by two-colorimmunofluorescence and flow cytometry. PE-labeled rabbit anti-PAP antibody was used to detect cell surface-bound, chemically intact B43-PAPimmunotoxin via its PAP moiety, whereas FITC-labeled goat anti-mouse IgG served as a probe for both PAP-conjugated (i.e. , intact immunotoxin)and unconjugated free B43 antibody. Fluorescence-activated cell sorting-correlated two-parameter displays of target CD 19-positive NALM-6leukemia cells incubated in vitro with undiluted plasma samples and then stained with PE-anti-PAPIFITC-goat anti-mouse IgG are shown.
in the day 16 plasma sample. Fl083, which was treated with
unconjugated B43 (7.0 mg/kg total dose), developed antibodies
only to mouse immunoglobulin, as expected, with a peak re-
sponse on day 20 (Fig. 4, A and B).
Plasma samples, obtained from the monkeys at time points
corresponding to the peak humoral response to the mouse IgG
moiety of B43-PAP, were also assayed for their ability to block
the in vitro binding of B43-PAP immunotoxin to CD 19-positive
NALM-6 human leukemia cells. As shown in Fig. 4C, monkey
plasma samples were able to block the binding of B43-PAP to
NALM-6 cells, and the extent of blocking correlated with the
presence of anti-mouse immunoglobulin IgG present. Although
minimal inhibition was observed with plasma samples from
Fl 165 or F585, the day 20 plasma sample from F1083 blocked
the binding of B43-PAP to NALM-6 cells by 27%, and the day
I 6 plasma sample from Fl 1 1 1 blocked the binding of B43-PAP
to NALM-6 cells by 57%.
Toxicity of B43(anti-CD19)-PAP in Cynomolgus Mon-
keys when Administered i.v. Daily for 7 Consecutive Days.
In the first toxicity study, involving five cynomolgus monkeys,
four monkeys were treated with B43-PAP immunotoxin at one
of four different dose levels (Fl 165, 0.01 mg/kg/day for 7 days;
F585, 0. 1 mg/kg/day for 7 days; Fl I 1 1 , 0.5 mg/kg/day for 7
days; and F724, 1 .0 mg/kg/day for 7 days). The fifth monkey
(F1083) was treated with 1.0-mg/kg/day doses of unconjugated
B43 monoclonal antibody for 7 consecutive days.
Table 2 details the time of onset, duration, and magnitude
of the maximum toxicities observed. Fl 165 tolerated B43-PAP
treatments very well with no clinical or laboratory signs of
significant toxicity. F585 experienced a transient grade 2 weight
loss, and laboratory studies showed a transient grade 2 normo-
chromic, normocytic anemia (likely due to frequent blood draws
for laboratory tests), grade 2 leukocytosis, grade 4 lipidemia, as
well as a transient grade 2 hypoalbuminemia (Fig. 5).
Fl I 1 1, which received a total of 3.5 mg/kg B43-PAP,
experienced severe renal toxicity after B43-PAP therapy and
was euthanized in moribund condition on day 17. The clinical
toxicity profile included grade 3-4 decrease in overall perform-
ance/activity level, grade 4 weight loss (36.5% decrease from a
6.0-kg baseline weight to 3.8 kg on day 17), which was attrib-
uted to very poor fluid and caloric intake, grade 1 weakness,
grade 1 ataxia, progressive oliguria/anuria, and sudden onset
grade 4 hypothermia on day 17. The laboratory toxicity profile
in order of severity included: (a) uremia (grade 4 increases in
BUN and creatinine, see Fig. 6) with associated alterations in
electrolytes, as well as a grade 2+ proteinuria and grade 2+
hematuria; (b) grade 4 hyperosmolarity; (c) grade 4 lipidemia;
(d) grade 4 leukocytosis; (e) grade 3 hyperglycemia; (f) grade 2
hypoalbuminemia (Fig. 5); and (g) grade 2 elevation of
transaminases (Table 2). Similar to Fl I 1 1, F724, which re-
ceived a total of 7.0 mg/kg B43-PAP, also experienced severe
renal toxicity and was sacrificed in moribund condition on day
8. The combined clinical/laboratory toxicity profile included
progressive oliguria/anuria, uremia (grade 4 increase in BUN,
grade 3 increase in creatinine with associated electrolyte abnor-
malities, as well as a grade 2+ proteinuria and hematuria; Fig.
6), grade 3 tachycardia, grade 4 lipidemia, grade 3 hypoalbu-
minemia (Fig. 5), grade 3 leukocytosis, and grade 4 hypothermia
(Table 2). Unlike Fl I 1 1 and F724, F1083, which was treated
with unconjugated B43 monoclonal antibody at a total dose
level of 7 mg/kg, did not show any clinical or laboratory signs
of toxicity. Thus, B43-PAP caused dose-limiting renal toxicity
in cynomolgus monkeys, and this toxicity was mediated by its
PAP moiety.
Research. on January 13, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
C64
48
Fl 1653%A
D #{149}B43.PAP (0.Olmglkg/d x 7d)0 B43.PAP (O.lOmg/kg/d x 7d)
30 - I a B43-PAP (O.SOmg/kg/d x 7d)1 . B43-MoAb (1.OOmg/kg/d x 7d)
20- / �
r#{176}�/‘ \ I�1\
10� ��/\‘ 0N ‘I
C 10 20 30 40
16
96% 3%� .97%
0% “ 0%
Day+30I 1 � li�J
Day.�1
10 20 30 40
Time After Injection ofB43-PAP/B43 MoAb Therapy (Days)
64FF11116% � � � F 36% � 7%
EF�:�f’0% �Day-11 L_�__J Day+�I6 �
16 32 48 64 16 32 48 64
Intact B43-PAP Immunotoxin + FreeB43 Monoclonal Antibody (FITC)
Fig. 4 Immunogenicity of B43-PAP immunotoxin and unconjugated B43 monoclonal antibody in cynomolgus monkeys. Plasma samples from
cynomolgus monkeys treated with unconjugated B43 antibody or B43-PAP immunotoxin were examined for the presence of anti-mouse (A) andanti-PAP (B) IgG antibodies using solid-phase ELISA. Tenfold diluted plasma samples with the highest anti-mouse IgG antibody concentrations and10-fold diluted pretreatment plasma samples were compared side by side for their ability to block the in vitro binding of B43-PAP immunotoxin (1�i.g/ml) to CD19-positive NALM-6 leukemia cells (C).
332 B43-PAP Primate Study
-J
Ea)
.30C)
C
.a0
a)0C
EE
a)U)
0
C
-J
E
a)
0a)
C
w0�
Cx0
0
C
E� ::F1083 L�1� � L�
�: f2% � 3%� Day-I � � Day+20 �
The macroscopic or microscopic examination of multiple
organs did not reveal any B43-PAP-related histopathological
lesions in Fl 165 and F585. Similarly, no lesions were found in
the organs of Fl083, which was treated with unconjugated B43
monoclonal antibody. At necropsy, Fl 1 1 1 was noted to have no
macroscopic abnormalities other than an enlarged adrenal gland.
F724 was found to have flabby right and left ventricles lacking
reasonable tone. Also noted was a white stippling throughout the
myocardium of both ventricles. The kidneys were tan in color
with prominent structure. The kidneys were soft, and the capsule
was easily removed. Microscopically, kidneys from both mon-
keys showed severe diffuse subacute renal tubular degeneration
and necrosis consistent with the above-detailed clinical and
laboratory signs of severe renal toxicity (Fig. 7). Microscopic
examination of the adrenal gland in Fl 1 1 1 revealed a multifocal
vacuolar degeneration of cortical cells associated with an acute
multifocal hemorrhage, and there were locally extensive lymph-
oid infiltrates in the deep adrenal cortex of F724. Moderate
lymphoid depletion was manifested in both monkeys as small or
absent lymphoid follicles with no germinal centers in the spleen
and lymph nodes. In both monkeys, microscopic examination of
the livers showed an acute mild, multifocal hepatocellular ne-
crosis and hepatitis. Also found in F724 was a minimal-mild,
chronic, nonsuppurative interstitial myocarditis without any
signs of active inflammation or necrosis, which was judged to be
an incidental finding unrelated to B43-PAP therapy.
Toxicity of B43(anti-CD19)-PAP in Cynomolgus Mon-
keys when Administered i.v. Every Other Day for 7 Treat-
ments. The second primate toxicity study was designed as a
preclinical study based on the toxicity and pharmacology data
from the first study and involved six cynomolgus monkeys.
These monkeys received seven i.v. doses of B43-PAP on an
every-other-day schedule. Monkeys were sacrificed either on
day 15 (that is 1 day after the last dose) to examine their organs
for lesions caused by acute toxicity or on day 30 (that is 16 days
after the last dose) to examine their organs for lesions caused by
subacute toxicity. Table 2 details the maximum toxicities ob-
served. FR0491 and F9l 107 received 0.001 mg/kg/dose B43-
Research. on January 13, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
pa
‘C
�
C
aLl
C��
�. aLl�C E
0
c� r-
aLl
Os aLl
C
.C
E
I-
C
aLl
- aLl-E
aLl
� aLl
‘r�E
aLl
sC
Li��
E51
5).�C0E
CE0C
C
C
00C
EE
U)
C5)
‘0C.
C
I-’
N
N
C
.�
5)
l’s
C
E
E1<CS
0C
�00
l’s
C
0
1<CS
CC � �-:::� C’---- CC --CC�C -CC CCC CNC C- �CC
� N � N
C � � R-.--.--- C-CC CC --CCZC -S--C CCC C--C C�.- .�CC
C� .� N N NN
C� CC’-- CC -‘-CC�C --C CCC Ct-C CC CCCCN N N
C sO - RC�-- CC-C CC -CCCC CCC CCC Ct-C -.-C CCC
- N N
C�- CCC- CC -CCC-C CCC CCC C--C CC CCC
C�- CCC- CC CCCCCC -CC CCC CCCC CC CCC
C�- CC-C CC -CCCCC �CC CCC C--C CC -CCC N
� � � � � � ��-�__z CZCC CC ZZZ- .Z ZCC --C CZZC ZC ZCC
�r � N r’� � r� r’� Z N r� r’� N Nr’� C
� � � � ��-?�_,z C--- CC ZZZZZZ ZCC --C CZ-C ZZ ,�1CC
� ��NN N � � .ir’�
�C� C--- CC -CC�C -CC CCC C�C C� �CC
C N N N � N �
C�- C--- CC -CC�CC -CC CCC Cc-C- N N
� I�.2 �
>.‘� .- C) 5)
.� �� � 5)
5)CC
5)C)
E
5)
CS
5)
.-3
ECS
C
C)C
C)5)5)
.C‘O5)C
CCE
CS
E5)
.CC)
C)CS
..0
CSLI
CiCi
.CCC)CS
0.C
�05)CS
�0C
CS�0
C
C).55
C
C
CC
CS
0�0C)
.C!�CiCCCS5)
��;�: CSCC)2CS �
I .� .� �CS >
�E �
CS
- � .C �C)CSCSC
I-. .�_
.-� .�<z 0.CS .5 )L. 0.
C.C0.
CC -CC
5)�E.�CS :�h _ .�i� � �C �
.E2E.E� � �
z�t� �C � �� � � 2 C) � � � #{176}� � 0U) LI tal ri� 0. � � < E- 0 � LI 0. .E � .� .� <
.J Z U)
Clinical Cancer Research 333
Research. on January 13, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
4.5 A Study No. B Study No.2
:� 4.0
E
3.5
E0 � ..
� 3.0 � ‘ � �E � � � �: ..#{149}.. F585 - 5.7mg/kg2 � #{149}. / -o--F1111-3.Smg/kg
(� 2.5 � . ..�. F724 - 7.0mg/kg
-a-. F1083 - 7.0mg/kg (Ab)
2.0 � � I
0 10 20 30 40Time (Days)
Fig. 6 Dose-limiting renal toxicity of B43-PAP
in cynomolgus monkeys. Serum BUN (A) andcreatinine (B) levels of monkeys from the firsttoxicity study are shown as a function of time afteradministration of the first dose of B43-PAP im-munotoxin. Day I first day of treatment.
FR0491 . 5I�7mg/kg
F91107 - 0.007mg/kg
FR0323 . 0.07mg/kg
FR0490 . 0.07mg/kg
..... FR0325 - 0.35mg/kg
-.5-. F89362 - 0.35mg/kg
Time (Days)
8 B
6
-0��- F1165-0.O7mg/kg
..... F585 - 0.7mg/kg
-0- Full -3.5mg/kg
--1-- F724 . 7.0mg/kg
-JVa)E
0)C
C
COa)
0
E0
0)Co
4
2
0 10
Time (Days)
30 40 0 10 20 30 40
Time (Days)
334 B43-PAP Primate Study
�1�0a)E
z:�U)E0
a)C’)
Fig. 5 Hypoalbuminemia in B43-PAP-treated cynomolgus monkeys.Serum albumin levels of monkeysfrom the first toxicity study (A), aswell as the second toxicity study(B), are shown as a function of timeafter administration ofthe first doseof B43-PAP immunotoxin (or B43monoclonal antibody). Day 1 =
first day of treatment
PAP every other day for seven doses. These monkeys showed
no clinical or laboratory signs of significant toxicity. FR0323
and FR0490 received 0.01 mg/kg/dose B43-PAP every other
day for seven doses. FR0323 had borderline grade I and
FR0490 had grade 2 weight loss but no other clinical signs of
significant toxicity. Both monkeys developed grade 2+ protein-
uria but no hypoalbuminemia (Fig. 5). FR0325 and FR89362
were treated with 0.05 mg/kg/dose B43-PAP every other day for
seven doses. FR0325 experienced grade 3 and F89362 experi-
enced grade 4 weight loss, and both developed grade 2+ pro-
teinuria and grade 2 hypoalbuminemia. No significant macro-
scopic or microscopic lesions were found in the organs from any
of these six monkeys.
DISCUSSION
There is limited information about the toxicity profiles of
immunotoxins containing different ribosome inhibitory proteins
(6-10, 27). Grossbard et a!. (27) have used the anti-CD33
immunotoxin My9-bR in patients with refractory myeloid leu-
kemias and the anti-CD19 immunotoxin B4-bR in patients with
refractory B-lineage lymphomas and leukemias. The toxicities
observed in patients treated with these bR-containing immuno-
toxins included dose-limiting hepatotoxicity with grade 3 ele-
vation of serum transaminase levels, mild-moderate capillary
leak syndrome with hypoalbuminemia, thrombocytopenia, fe-
vers, and myalgias (5, 10, 27). Other investigators have used
anti-CD19 and anti-CD22 immunotoxins containing dgA chain
for treatment of B-lineage lymphoma and leukemia patients
(7-9). Side effects included anorexia, fevers, myalgias, and
capillary leak syndrome (7-9). The dose-limiting toxicities were
pulmonary edema/effusion secondary to severe capillary leak,
expressive aphasia, and rhabdomyolysis with renal failure (7-9).
Immunotoxins containing RTA were also used in treatment of
T-lineage leukemia/lymphoma patients (28-30). Toxicities in-
cluded dose-limiting capillary leak syndrome with grade 3 dysp-
nea, myalgias, fever, chills, and fatigue (28-30).
B43-PAP immunotoxin was reported previously to cause
dose-limiting renal and cardiac toxicities in mice (31). We now
provide experimental evidence that B43-PAP causes dose-lim-
iting renal toxicity due to severe and diffuse renal tubular
necrosis in cynomolgus monkeys. In addition, B43-PAP caused
a mild hepatic injury, characterized by transient elevation of
transaminases and a mild multifocal hepatitis detected his-
topathologically. Furthermore, we documented a transient epi-
sode of proteinuna and hypoalbuminemia, which could be due
to a mild capillary leak. The dose-independent grade 1 tachy-
cardia and grade 1 hypertension were most likely due to agita-
tion of monkeys, and a grade 1 hypotension was associated with
daily anesthesia. These borderline changes in vital signs were
probably not due to cardiovascular toxicity. The toxicities as-
sociated with administration of B43-PAP appear to be slightly
different from the toxicity profile of bR-containing immunotox-
ins, which cause significant hepatotoxicity but no renal toxicity
(5, 10, 27), as well as different from the toxicity profile of dgA
or RTA-containing immunotoxins, which cause significant cap-
illary leak but no renal tubular necrosis (7-9, 28-30).
Research. on January 13, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
�, � �
B �
i:’c��’� � � #{149} . ,-
� �.s;L%1�;.: � .�.,s.l c�.1 � ‘t.; ,� ,_ �_\J.’�& ;
4 � ‘.l_t � 0,�. ��_�__).__ � .,L..j . C�#.. ‘-‘I.., -.._L.-��’C � .
% � ��*_:� � .. ,
I., � �I , - � �
� �‘‘�
.��!5�:’�.5C ‘3.L0, � � � � � --
: �: . : - .. �
:� � � 7:’
�; C. #{149}.
gy. . � �
. %�.
� � : .. ‘ �
..
�a �“‘C, �
. C..
....
,�‘ #{149}‘�,#{149}a� f,�c S
�-r:;; ‘��-
�1� �.
�
.� -
� ..
00
F585
Clinical Cancer Research 335
F724
Fig. 7 High-dose B43-PAPcauses renal tubular necrosis incynomolgus monkeys. F585 re-
ceived a total of 0.7 mg/kgB43-PAP, and F724 received atotal of 7.0 mg/kg B43-PAP.No histological lesions werefound in the kidneys of F585
(A-D), whereas the kidneys ofF724 (E-H) showed severe cor-tical tubular necrosis. Many tu-bules were dilated, lined by
flattened epithelium, and filledwith proteinaceous casts (H&Estain; X3OinAandE, X75inB
and F, X150 in C and G, andX300 in D and H).
A. . . ‘C’. � . � .
: �\. ..-.-.‘ � � � ,. . . ,
J,.� , ... t,,&’, ..,( ,...
57 , ) fj�) ?J�:, � �-. � .:�:-:c�%;’�’:’�.:,- . : . �1.. : ‘ . P � i.#{149} s:s,�l �1 ‘ �
..‘\ . . ,. , � , . . I..- . . �,�-.5#{149}_ :��sts’i� I � � -
- . .. I ‘fl: ‘ . . � ‘.
I, �:.‘. � . /, . � . .
� ,.�: � �
-..--.� � � - -�
�Li� �� �L�:21� �
�- � � � ,- .
P/�.?s� � ‘�I. �
.C..� � �
--,� �4f� �_�,#{149}4 �
�L#{149}�
I � I e�
- I
4
.
�b�\;/ -�
.�-p. � � �J��:9” � � �
: � � � � ,
...
±,
.. :. �i�H
� .�
.‘,t.L� �
. .. ...
.. .:�. � ‘: �- � � �y...
;: - � ‘ :�: . � �‘ . C
, �:‘ ..� � � ..
,. . 5#{149}. (5 � : �‘
I�, � � � �
. . 4 . ....., . . , . .. . .
� : . � � ,- . ;
Aron and Irvin (31) demonstrated that PAP nonspecifically
binds to monkey kidney endothelial cells, as well as to the
human epithelial cell line HeLa, resulting in inhibition of cel-
lular protein synthesis and irreversible cell death. Similarly,
Goldmacher et a!. (32) reported that gelonin, another single-
chain ribosome-inactivating protein, enters HeLa cells by pino-
cytosis, which is largely irreversible, and leads to marked cyto-
toxicity. Pinocytosis is a process by which cells indiscriminately
take up substances dissolved in liquid medium (33, 34). Epithe-
hal and endothelial cells have high pinocytotic activity (32).
After formation and internalization of pinosomes, the vast ma-
jority of pinosomes are recycled from the cytoplasm to the
surface where their contents are discharged back into the me-
dium. However, pinocytosis is partially irreversible, because a
Research. on January 13, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
336 B43-PAP Primate Study
fraction of the pinocytosed material does not return to the cell
surface (35, 36). Irreversible pinocytosis of ribosome-inactivat-
ing proteins leads to cytotoxicity to rapidly pinocytosing endo-
thelial, as well as epithelial, cells (3 1 ). We are currently trying
to isolate fully active mutants of recombinant PAP (37) with
reduced ability to bind to and kill endothelial/epithelial cells.
Structural models of these PAP mutants will be generated by
substitution of the mutant residues in the three-dimensional
structure of wild-type PAP and used as rudimentary guides for
understanding mutations abrogating the surface-binding ability
of PAP to endothelial and epithelial cells. We postulate that
B43-PAP’immunotoxins containing such recombinant PAP mu-
tants would cause significantly less renal toxicity. In the interim,
appropriate clinical surveillance and conventional treatment, as
well as prophylactic regimens using low-dose dopamine, pen-
toxifylline, or steroids, may enhance the clinical utility of PAP-
containing immunotoxins (21).
Humoral immune responses to the monoclonal antibody, as
well as toxin portions of bR-, dgA-, or RTA-containing immmu-
notoxins, have contributed to the limited clinical utility of these
biotherapeutic agents (3-10, 28-30). As shown in the present
study, B43-PAP-treated monkeys developed anti-PAP, as well
as anti-mouse, IgG antibodies. Immunogenicity of B43-PAP
might be reduced by replacing the mouse antibody with a
chimeric or humanized version of B43, as well as by attaching
allergens, haptens, or chemical agents such as polyethylene
glycol that suppress immune responses. Antitoxin immune re-
sponses might be alleviated by rotating varieties of the plant
toxin PAP, which may be harvested in three different forms
based on plant structure and maturity or by rotating different
species of toxin (13).
Currently, little is known about the disposition of immu-
notoxins in vivo (3-10, 38-41). In general, plasma disposition
is mono- or biphasic, with elimination half-lives ranging from
hours to a few days (3-10). It has been noted in animals that the
pharmacokinetics of B43-PAP is influenced by the target leu-
kemic burden, such that mice with advanced CD 19-positive
leukemia have a larger volume of distribution and faster clear-
ance than mice without leukemia (38). This was shown to be due
to rapid clearance of the administered immunotoxin by the
CDI9-positive leukemia cells in bone marrow and extramedul-
lary organs such as the spleen and liver (38). The elimination of
some immunotoxins in humans is more rapid than in mice, most
likely due to the development of humoral immune responses or
because of cross-reactivity with normal tissues as seen with
glycosylated ricin immunotoxins (39-41). Although the mech-
anisms of elimination are not well characterized (39), it is
known that the reticuloendothelial system is commonly in-
volved in the elimination of immunotoxins. More recently,
elimination by the targeted cells via internalization through
pinocytosis was also identified as an alternative route of clear-
ance of some immunotoxins (9, 40). This phenomenon has also
been shown with other biotherapeutic agents, such as recombi-
nant human granulocyte colony-stimulating factor, which is
cleared by WBCs (42, 43). The intrinsic stability of immuno-
conjugates may also influence their elimination (40). The favor-
able pharmacokinetic features of B43-PAP immunotoxin, as
documented previously in mice and rabbits (22, 23) and now
also shown in cynomolgus monkeys, are promising from an in
vivo potency standpoint, but may also lead to increased endo-
thelial cell toxicity in clinical trials.
This detailed preinvestigational new drug characterization
of the pharmacokinetic features, immunogenicity, and toxicity
profile of B43-PAP in cynomolgus monkeys extends our pre-
vious studies, which have established the in vitro (14, 15, 26)
and in vivo antileukemic activity of B43-PAP (16-20), as well
as its pharmacodynamic features, immunogenicity (22, 23), and
toxicity (21) in mice. The present study completes the preclin-
ical evaluation of B43-PAP and provides the basis for its Food
and Drug Administration-approved (BB-IND-3864) clinical
evaluation in children with therapy-refractory B-lineage ALL.
Because PAP immunotoxins have not been used previously in
clinical settings, the Phase I trial of B43-PAP will be initiated at
a dose level of 0.001 mg/kg/day for 7 days, which is 100-fold
lower than the highest well-tolerated dose level of 0. 1 mg/kg/
day for 7 days in cynomolgus monkeys. Because of the long
serum half-life of B43-PAP in cynomolgus monkeys, we de-
cided to use a daily I-h infusion schedule in the first Phase I
clinical trial of this immunotoxin.
REFERENCES
1. Trigg, M.. Gaynon, P., and Uckun, F. M. Acute lymphoblasticleukemia in children. In: J. F. Holland, R. C. Bast, D. L. Morton, E. Frei,D. W. Kufe, and R. R. Weichselbaum (eds.), Cancer Medicine, 4th ed.,pp. 2945-2960, 1996.
2. Champlin, C. H., and Gale, R. P. Acute lymphoblastic leukemia:recent advances in biology and therapy. Blood, 73: 2051-2066, 1989.
3. Vitetta, E. S.. Thorpe. P. E., and Uhr, J. W. Immunotoxins. Magicbullets or misguided missiles? Immunol. Today, 14: 252-259, 1993.
4. Uckun, F. M., and Reaman, G. H. Immunotoxins for treatment ofleukemia and lymphoma. Leuk. & Lymphoma, 18: 195, 1995.
5. Grossbard, M. L., and Nadler, L. M. Immunotoxin therapy of lymph-oid neoplasms. Semin. Hematol., 31: 88-97, 1994.
6. Uckun, F. M. Immunotoxins for the treatment of leukemia. Br. J.Haematol., 85: 435-438, 1993.
7. Amlot, P. L.. Stone, M. J.. Cunningham, D., Fay, J., Newman, J.,
Collins, R., May, R., McCarthy, M., Richardson, J., Ghetie, V., Ramilo,0., Thorpe, P. E., Uhr, J. W., and Vitetta, E. S. A Phase I study of ananti-CD22 deglycosylated ricin A chain immunotoxin in the treatmentof B-cell lymphomas resistant to conventional therapy. Blood, 82:
2624-2633, 1993.
8. Vitetta, E. S., Stone, M., Amlot, P., Fay, J., May, R., Till, M.,Newman, J., Clark, P., Collins, R., Cunningham, D., Ghetie, V., Uhr.J. W.. and Thorpe, P. E. Phase I immunotoxin trial in patients withB-cell lymphoma. Cancer Res., 51: 4052-4058, 1991.
9. Sausville, E. A., Headlee, D., Stetler-Stevenson, M. Jaffe, E. S.,Solomon, D., Tigg, W. D., Herdt, J., Kopp, W. C., Rager, H., Steinberg,S. M., and Vitetta, E. S. Continuous infusion of the anti-CD22 immu-notoxin IgG-RFB4-SMPT-dgA in patients with B-cell lymphoma: aPhase I study. Blood, 85: 3457-3465, 1995.
10. Grossbard, M. L., Press, 0. W., Appelbaum, F. R., Bernstein, I. D.,and Nadler, L. M. Monoclonal antibody-based therapies of leukemiaand lymphoma. Blood, 80: 863-878, 1992.
I 1. Uckun, F. M. Regulation of human B-cell ontogeny. Blood, 76:1908-1923. 1990.
12. Uckun, F. M., Jaszcz, W., Ambrus, J. L., Fauci, A. S., Gajl-Peczalska, K. J., Song, C. W., Wick, M. R., Myers, D. E., Waddick,K. G., and Ledbetter, J. A. Detailed studies on expression and functionof CD19 surface determinant using B43 monoclonal antibody. Blood,71: 13-29, 1988.
I 3. Irvin, J. D., and Uckun, F. M. Pokeweed antiviral protein: ribosomeinactivation and therapeutic applications. Pharmacol. Ther., 55: 279-302, 1992.
Research. on January 13, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Clinical Cancer Research 337
14. Uckun, F. M., Gajl-Peczalska, K. J., Kersey, J. H., Houston, L. L.,and Vallera, D. A. Use of a novel colony assay to evaluate the cytotox-icity of an immunotoxin containing pokeweed antiviral protein againstblast progenitor cells freshly obtained from patients with commonB-lineage acute lymphoblastic leukemia. J. Exp. Med., 163: 347-368,1986.
15. Myers, D. E., Irvin, J. D., Smith, R. S., Kuebelbeck, V. M., andUckun, F. M. Production of a pokeweed antiviral protein (PAP)-con-taming immunotoxin, B43-PAP, directed against the CD19 human B-
lineage lymphoid differentiation antigen in highly purified form forhuman clinical trials. J. Immunol. Methods, 36: 221-238, 1991.
16. Waddick, K. G., Gunther, R., Chelstrom, L. M., Chandan-Langlie,M., Myers, D. E., Irvin, J. D., Turner, N., and Uckun, F. M. In vitro andin vivo anti-leukernic activity of B43 (anti-CD19)-pokeweed antiviral
protein imrnunotoxin against radiation resistant human pre-B acutelymphoblastic leukemia cells. Blood, 86: 4228-4233, 1995.
17. Jansen, B., Uckun, F. M., Jaszcz, W. B., and Kersey, J. H. Estab-lishment of a human t(4;ll) leukemia in SCID mice and successfultreatment using anti-CD19 (B43)-pokeweed antiviral protein immuno-toxin. Cancer Res., 52: 406-412, 1992.
18. Uckun, F. M., Manivel, C., Arthur, D., Chelstrom, L., Finnegan,D., Tuel-Ahlgren, L., Irvin, J. D., Myers, D. E., and Gunther, R. Invivo efficacy of B43 (anti-CD19) pokeweed antiviral protein immu-
notoxin against human pre-B cell acute lymphoblastic leukemia inmice with severe combined immunodeficiency. Blood, 79: 2201-2214, 1992.
19. Uckun, F. M., Chelstrom, L. M., Finnegan, D., Tuel-Ahlgren, L.,Irvin, 3. D., Myers, D. E., and Gunther, R. Effective immunochemo-therapy of CALLA+Cp.+ human pre-B acute lymphoblastic leukemiain mice with severe combined immunodeficiency using B43 (anti-CD19) pokeweed antiviral protein (PAP) immunotoxin plus cyclophos-phamide. Blood, 79: 3116-3129, 1992.
20. Uckun, F. M., Evans, W. E., Forsyth, C. J., Waddick, K. G.,T-Ahlgren, L., Chelstrom, L. M., Burkhardt, A., Bolen, J., and Myers,D. E. Biotherapy of B-cell precursor leukemia by targeting genistein toCD19-associated tyrosine kinases. Science (Washington DC), 267:
886-891, 1995.
21. Gunther, R., Chelstrom, L. M., Wendorf, H. R., Schneider, E. A.,Covalciuc, K., Johnson, B., Clementson, D., Irvin, J. D., Myers, D. E.,
and Uckun, F. M. Toxicity profile of the investigational biotherapeuticagent, B43 (anti-CD19)-pokeweed antiviral protein immunotoxin. Leuk.& Lymphoma, 22: 61-70, 1996.
22. Uckun, F. M., Myers, D. E., Irvin, J. D., Kuebelbeck, V. M.,
Finnegan, D., Chelstrom, L. M., and Houston, L. L. Effects of theintermolecular toxin-monoclonal antibody linkage on the in vivo stabil-
ity, immunogenicity, and anti-leukemic activity of B43 (anti-CD19)
pokeweed antiviral protein imrnunotoxin. Leuk. & Lymphoma, 9: 459-476, 1993.
23. Myers, D. E., Yanishevski, Y., Masson, E., Irvin, J. D., Evans,
W. E., and Uckun, F. M. Favorable pharmacodynamic features andsuperior antileukemic activity of B43 (anti-CD19) immunotoxins con-taming two pokeweed antiviral protein molecules covalently linked to
each monoclonal antibody molecule. Leuk. & Lymphoma, 18: 93-102,
1995.
24. D’Argenio, D. Z., and Schumitzky, A. A program package forsimulation and parameter estimation in pharmacokinetics systems.
Cornput. Programs Biomed., 9: 115-134, 1979.
25. Yamaoka, K., Nakagawa, T., and Uno, T. Application of Akaike’s
information criterion (AIC) in the evaluation of linear pharmacokineticequations. J. Pharrnacokinet. Biopharm., 6: 165-175, 1978.
26. Uckun, F. M., Ramakrishnan, S., and Houston, L. L. Increasedefficiency in selective elimination of leukemia cells by a combination of
a stable derivative of cyclophosphamide and a human B cell specificimmunotoxin containing pokeweed antiviral protein. Cancer Res., 45:
69-75, 1985.
27. Grossbard, M. L., and Nadler, L. M. Immunotoxin therapy of
lymphorna. Cancer Treat. Res., 68: 111-131, 1993.
28. Laurent, G., Frankel, A. E., and Hertler, A. A. Treatment of leuke-
mia patients with Tl0l ricin A chain immunotoxins. Cancer Treat. Rep.,37: 483-491, 1988.
29. Ghetie, M. A., and Vitetta, E. S. Recent developments in immuno-toxin therapy. Curr. Opin. Immunol., 6: 707-716, 1994.
30. LeMaistre, C. F., Rosen, S., Frankel, A., Komfeld, S., Saria, E.,Meneghetti, C., Drajesk, J., Fishwild, D., Scannon, P. J., and Byers,V. S. Phase I trial of H65-RTA immunoconjugate in patients withcutaneous T-cell lymphoma. Blood, 78: 1 173-1 182, 1991.
31. Aron, G. M., and Irvin, J. D. Cytotoxicity of pokeweed antiviralprotein. Cytobios, 55: 105-111, 1988.
32. Goldmacher, V. S., Tinnel, N. L., and Nelson, B. C. Evidence that
pinocytosis in lymphoid cells has a low capacity. J. Cell Biol., 102:
1312-1319, 1986.
33. Silverstein, S. C., Steinman, R. M., and Cohn, Z. A. Endocytosis.Annu. Rev. Biochem., 46: 669-722, 1977.
34. Steinman, R. M., Brodie, S. E., and Cohn, Z. A. Membrane flowduring pinocytosis. J. Cell Biol., 68: 665-687, 1976.
35. Besterman, J. M., Airhart, J. A., Woodworth, R. C., and Low, R. B.Exocytosis of pinocytosed fluid in cultured cells: kinetic evidence forrapid turnover and compartmentalization. J. Cell Biol., 9/: 716-727,1981.
36. Brown, M. S., Anderson, R. G., and Goldstein, J. L. Recyclingreceptors: the round trip itinerary of migrant membrane proteins. Cell,32: 663-667, 1983.
37. Hur, Y., Hwang, D. J., Zoubenko, 0., Coetzer, C., Uckun, F. M.,
and Turner, N. Isolation and characterization of pokeweed antiviralprotein mutations in Saccharomyces cerevisiae: identification of resi-
dues important for toxicity. Proc. Natl. Acad. Sci. USA, 92: 8448-8452, 1995.
38. Uckun, F. M., Chelstrom, L. M., Irvin, J. D., Finnegan, D.,Gunther, R., Young, J., Kuebelbeck, V., Myers, D. E., and Houston,L. L. ln vivo efficacy of B43 (anti-CD19)-pokeweed antiviral protein
irnmunotoxin against BCL-l murine B-cell leukemia. Blood, 79:2649-2661, 1992.
39. Henry, R., Begent, J., and Pedley, R. B. Monoclonal antibodyadministration: current clinical pharmacokinetic status and future trends.Clin. Pharmacokinet., 23: 85-89, 1992.
40. Reilly, R. M., Sandhu, J., Alvarez-Diez, T. M., Gallinger, S.,Kirsch, J., and Stern, H. Problems of delivery of monoclonal antibodies:pharmaceutical and pharmacokinetic solutions. Clin. Pharmacokinet.,28: 126-142, 1995.
41. Bocci, V. Metabolism of protein anticancer agents. In: G. Powis
(ed), Anticancer Drugs: Antimetabolite Metabolism and Natural Anti-cancer Agents, pp. 387-436. Pergamon Press, 1994.
42. Stute, N., Santana, V. M., Roclrnan, J. H., Schell, M. J., Ihle, J. N.,
and Evans, W. E. Pharmacokinetics of subcutaneous recombinant hu-
man granulocyte colony-stimulating factor in children. Blood, 79:2849-2854, 1992.
43. Kuwabara, T., Kato, Y., Kobayashi, S., Suzuki, H., and Sugiyama,Y. Nonlinear pharmacokinetics of a recombinant human granulocytecolony-stimulating factor derivative (nartograstim): species differencesamong rats, monkeys, and humans. J. Pharmacol. Exp. Ther., 271:
1535-1543, 1994.
Research. on January 13, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
1997;3:325-337. Clin Cancer Res F M Uckun, Y Yanishevski, N Tumer, et al. cynomolgus monkeys.B43(anti-CD19)-pokeweed antiviral protein immunotoxin in Pharmacokinetic features, immunogenicity, and toxicity of
Updated version
http://clincancerres.aacrjournals.org/content/3/3/325
Access the most recent version of this article at:
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
.pubs@aacr.orgDepartment at
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
.http://clincancerres.aacrjournals.org/content/3/3/325To request permission to re-use all or part of this article, use this link
Research. on January 13, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from