pharmacokinetic features, immunogenicity, and toxicity of ... · vol. 3, 325-337, march 1997...

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.

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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



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



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




AIV

Ki 0

-J

EC

0

Ce

CCe0C00

100

10

0.10 50 100 150

Time (hours)


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-



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

-�‘ .�

- � = �


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



+48 Hr.

1% 39%

16 32 48 64 16 32 48 64


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.



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).


-J

Ea)

.30C)

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a)

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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-



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:� 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

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--1-- F724 . 7.0mg/kg

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Time (Days)


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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).



�, � �

B �

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4 � ‘.l_t � 0,�. ��_�__).__ � .,L..j . C�#.. ‘-‘I.., -.._L.-��’C � .

% � ��*_:� � .. ,

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F585


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,,&’, ..,( ,...

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� ,.�: � �

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: � � � � ,

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±,

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;: - � ‘ :�: . � �‘ . C

, �:‘ ..� � � ..

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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




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.

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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

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